Compositions containing abiotically-stressed plant-derived exosome-like nanoparticles

Abstract

The present disclosure provides a composition containing a purified population of plant-derived exosome-like nanoparticles isolated from tissue of a vascular plant, wherein the exosome-like nanoparticles comprise a tuned cargo comprising a protein signature and an miRNA signature, wherein the tuned cargo of the plant-derived exosome-like nanoparticles is a result of exposure of the plant to combinations of abiotic stress conditions that cause the plant to modulate its signaling pathways and metabolism to ensure its survival in a challenging environment. The tuned cargo of the plant-derived exosome-like nanoparticles can modulate bioactivities of mammalian cells directly or indirectly. The present disclosure also provides a method for improving appearance of human skin and human hair health, including eyelashes and eyebrows, comprising applying a composition comprising the abiotically stressed plant-derived exosome-like nanoparticles containing the tuned cargo and a carrier; and applying the composition topically.

Description

PCT Application -FINAL COMPOSITIONS CONTAINING ABIOTICALLY-STRESSED PLANT-DERIVED EXOSOME-LIKE NANOPARTICLES CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority to U.S. Provisional Application No: 63 / 672,020 (filed July 16, 2024), which is incorporated by reference herein in its entirety. REFERENCE TO ELECTRONIC SEQUENCE LISTING

[0002] The application contains a Sequence Listing which has been submitted electronically in .XML format and is hereby incorporated by reference in its entirety. Said .XML copy, created on July 16, 2025, is named 2751_00101PCT_SL.xml and is 111,355 bytes in size. The sequence listing contained in this .XML file is part of the specification and is hereby incorporated by reference herein in its entirety. BACKGROUND OF THE INVENTION Mammalian Exosomes: General Principles

[0003] Exosomes are released by most if not all mammalian cell types, including platelets, blood cells, dendritic cells, mast cells, T cells, B cells, epithelial cells, endothelial cells, mesenchymal stem cells, smooth muscle cells, neuronal cells and many tumor cells. [Zhang, J. et al. Genomics Proteomics Bioinformatics (2015) 13: 17-24, citing Liao J., et al. Int J Mol Sci. 2014;15:15530–15551; Kopers-Lalic, D. et al. Adv. Drug delivery rev. (2012) doi: 10.1016 / j.addr.2012.07.006, citing Thery V., et al. Nat. Rev. Immunol. (2002) 2: 569-579].

[0004] Mammalian exosomes, which consist of lipid membranes, are spherical nanovesicles with a diameter of 40–150^nm (approximately 100^nm on average) [Kim, J. et al. Asian J. of Pharmaceutical Sci. (2022) 17: 53-69, citing Sinha, D. et al. Cancers (2021) 13 (2): 326; Villa, F. et al. Pharmaceutics (2019) 11 (11): 557; Zhang, Y. et al. Cell Biosi. (2019) 9 (1): 19; Doyle, LM and Wang, MZ. Cells (2019) 8 (7): 727]. They are constitutively generated by the inward budding of the plasma membrane to form early endosomes. The partial early endosomesintegrate the surrounding lamina to generate intraluminal vesicles (ILVs), which encapsulate exosomes within large intracellular multivesicular bodies (MVBs). The subsequent fusion of MVBs with the plasma membrane leads to the secretion of exosomes from most ILVs into the extracellular space [Id., citing Zhang, Y. et al. Cell Biosci. (2019) 9 (1): 19; Hessvik, NP and Llorente, A. Cell Mol. Life Sci. (2018) 75 (2): 193-208; Farooqi, AA et al. Biotechnol. Adv. (2018) 36 (1): 328-334; Joshi, BS et al. ACS Nano (2020) 14 (4): 4444-4455]. Mammalian exosomes are typically defined by their size, composition and specific exosome marker proteins, such as CD9, CD81, CD63, flotillin and TSG101 [Id., citing Zhang, Y. et al. Cell Biosci. (2019) (1): 19; Yue, B. et al. Cell Prolif. (2020) 53 (7): e12857; Kalluri, R. and LeBleu, VS. Science (2020) 367 (6378): eaau6977]. Mammalian exosome architecture, components, and molecular processing reflect the processes taking place in their cells of origin [Id., citing Hu, Q. et al. Precis. Clin. Med. (2020) 3 (1): 54-66; Gluszko, A. et al. Biomed. Res. Int. (2019) 2019: 1628029].

[0005] Most mammalian EVs comprising exosomes share a core set of proteins and lipids. Mammalian-derived exosomes are characterized by the presence of specific lipids, such as phosphatidylserine, cholesterol, sphingomyelins, and ceramides. There seems to be a conserved protein repertoire in exosomes across cell-types and species [Kopers-Lalic, D. et al. Adv. Drug delivery rev. (2012) doi: 10.1016 / j.addr.2012.07.006, citing Simpson, RJ, et al. Proteomics (2008) 8: 4083-4099]. For example, the endosomal proteins such as Alix and TSG101, which are components of the mammalian ESCRT system, have been identified in the majority of the exosomes studied for their protein content thus far. In addition, heat shock proteins, which are involved in protein trafficking, are frequently found in exosomes [Id., citing van Dommelen, SM et al. J. Control. Release (2011) 161: 635-644]. Mammalian exosomes are further enriched in tetraspanins, like CD9, CD63, CD81 and CD82, which are important molecules for protein- protein interactions in cellular membranes. Tetraspanins bind many proteins, including integrins and MHC molecules [Id., citing Thery, C., et al. Nat. Rev. Immunol. (2009) 9: 581-593; Escola, JM. J. Biol. Chem. (1998) 273: 20121-20127; Keller, S. et al. Immunol. Lett (2006) 107: 102- 108; Stoorvogel, W. et al. Traffic (2002) 3: 321-330]. Specific Rab proteins, a highly conserved family of small GTPases that function as molecular switches and coordinate membrane traffic [Id., citing Stenmark, H. Nat. Rev. Mol. Cell Biol. (2009) 10: 513-525], are often observed in exosomes by mass-spectrometry. Exosomes are also rich in annexins, membrane traffickingproteins that are involved in fusion events. Furthermore, cytoskeletal proteins like myosin, actin, and tubulin are present in exosomes. Finally, metabolic enzymes, antigen presentation molecules, ribosomal proteins and signal transduction molecules have been shown to be present in exosomes [Id., citing Mathivanan, S., et al. Proteomics (2008) 8: 4083-4099]

[0006] Mammalian exosomes may also carry (functional) genetic material, most notably small RNA molecules [Kopers-Lalic, D. et al. Adv. Drug delivery rev. (2012) doi: 10.1016 / j.addr.2012.07.006., citing Zomer, A. et al. Commun. Integr. Biol. (2010) 3: 447-450; Gibbings, D, Voinnet, O. Trends Cell Biol. (2010) 20: 491-501]. Of all RNA molecules detected in exosomes, the class of 22nt long, non-coding miRNAs has received attention since the discovery that miRNAs can be functionally transferred to recipient cells [Id., citing Pegtel, DM et al. Proc. Nat. Acad. Sci. USA (2010) 107: 6328-6333, Valadi, H. et al. Nat. Cell Biol. (2007) 9: 654-659]. MiRNAs regulate gene expression by binding imperfectly to the 3′ untranslated region of the target mRNA that results in translational repression of the mRNA into protein [Id., citing Bartel, DP. Cell (2004) 116: 281-297; Brennecke, J. et al. PLoS Biol. (20: e85 e85]. The term “non-coding RNAs (ncRNAs”) as used herein refers to functional RNA molecules that are transcribed from DNA but not translated into proteins. High-throughput sequencing technology confirmed that over 98% of the human genome is transcribed into ncRNAs, which are divided into two main groups: the small non-coding RNAs (< 200 nucleotides) and the long non-coding RNAs (lncRNAs) (> 200 nucleotides). In general, ncRNAs play a role in heterochromatin formation, histone modification and DNA methylation, leading to regulate gene expression at the transcriptional and post-transcriptional level. Epigenetic related ncRNAs include miRNA, siRNA, piRNA and lncRNA. Non-coding RNA types are summarized in Table 1.

[0007] Table 1 – Non-coding RNA types. RNA Functions Coding Typical Size (nt=nucleotides) microRNA (miRNA) Post-transcriptional No 17-24 nt gene silencing Y RNA Component of Ro60 No ≈ 100 nt ribonucleoprotein particle; initiationRNA Functions Coding Typical Size (nt=nucleotides) factor for DNA replication Signal Recognition Component of SRP No ≈280 nt particle RNA (SRP ribonucleoprotein RNA) complex that directs protein trafficking Transfer RNA Adapter for matching No 76-90 nt (tRNA) amino acid to mRNA Ribosomal RNA RNA component of No 185 (1.9 kb) 28S (5.0 (rRNA) ribosomes kb) Small nuclear RNA RNA processing such No ≈150 nt (snRNA) as mRNA splicing Small nucleolar RNA Guiding chemical No 20-24 nt (snoRNA) modifications of other RNAs Long noncoding Many, including in- No >100 nt RNA (lncRNA) transcription and post-transcription regulation

[0008] mRNA

[0009] mRNAs are a large family of coding RNA molecules that specify protein sequence information. Studies have reported that mammalian EVs contain a substantial proportion of their parent cells’ mRNA pool, many of which are cell type-specific mRNA. [Shao, H. et al. Chem Rev. (2018) 118 (4): 1917-1950, citing Wei, Z. et al. Nat. Commun. (2017) 8: 11:45; Batagov, AO, Kurochkin, IF. Biol. Direct (2013) 8: 12] These mRNA molecules, often in fragmented form, reside within EVs and are protected from RNase degradation. Furthermore, the fraction of polyadenylated mRNA molecules in EVs suggest that some of them (<2 kb) are capable of encoding polypeptides in support of protein synthesis (i.e., functionality in protein translation).This has been confirmed in multiple studies through different translation assays in recipient cells [Id., citing Valadi, H. et al. Nat. Cell Biol. (2007) 9: 654-659; Skog, J. et al. Nat. Cell Biol. (200: 1470-1476; Lai, CP et al. Nat. Commun. (2015) 6: 7029].

[0010] miRNA

[0011] miRNAs are a class of small, noncoding RNAs (typically 17–24 nucleotides) which mediate post-transcriptional gene silencing usually by targeting the 3’ untranslated region of mRNAs. By suppressing protein translation, EV miRNAs are powerful regulators for a wide range of biological processes [Id., citing Mittelbrunn, M. et al. Nat. Commun. (2011) 2: 282; Redzic, JS et al. Semin. Cancer Biol. (2014) 28: 14-23]. miRNAs can also exist in multiple stable forms when circulating in bodily fluids. For example, in addition to being packaged into EVs, circulating miRNAs can also be loaded onto high-density lipoprotein [Id., citing Vickers, KC et al. Nat. Cell Biol. (2011) 13: 423-433; Wagner, J. et al. Arterioscler. Thromb. Vasc. Biol. (2013) 33: 1392-1400] or bound to argonaute 2 (AGO2) protein, a family of proteins that play a role in RNA interference, outside the vesicles [Id.: citing Arroyo, et al. Proc. Natl Acad. Sci. USA (2011) 108: 5003-5008; Turchinovich, A. et al. Methods Mol. Biol. (2013) 1024: 97-107]. The distribution of miRNAs within EVs remains unclear [Id., citing Min, PK & Chan, SY. Eur. J. Clin. Invest. (2015) 45: 860-874; Turchinovich, A. et al. Methods Mol. Biol. (2013): 97: 97- 107; Chevillet, JR et al. Proc. Natl. Acad. Sci. USA (2014) 111: 14888-14893]. As in the case of mRNA, miRNA profiles in EVs reflect their cell of origin but differs somewhat from their parental cells. Some miRNAs have been found preferentially sorted into EVs and remaining functional in recipient cells to regulate protein translation. [Id., citing Villarroya-Beltri, C. et al. nat. Commun. (2013) 4: 2980; Koppers-Lalic, D. et al. Cell Rep. (2014) 8 (6): 1649-1658; Santangelo, L. et al. Cell Rep. (2016) 17: 799-808; Teng, Y. et al. Nat. Commu. (2017) 8: 14448]

[0012] Other RNA Types

[0013] In addition to mRNA and miRNA, many noncoding RNA types have been identified in EVs through next generation sequencing [Id., citing Huang, X. et al. BMC Genomics (2013) 14: 319; Conley, A. et al. RNA Biol. (2017) 14: 305-316]. These RNAs include transfer RNA (tRNA), ribosomal RNA (rRNA), small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), as well as long noncoding RNA (lncRNA) [Id., citing Wei, Z. et al. Nat. Commun.(2017) 8: 1145; Huang, X. et al. BMC Genomics (2013) 14: 319; Crescitelli, R. et al. J. Extracell. Vesicles (2013) 2: 20677].

[0014] Small (about 20-30 nucleotides (nt)) noncoding RNAs regulate eukaryotic genes and genomes (Carthew, RW and Sontheimer, EJ. Cell (2009) 136: 642-55). This regulation can occur at multiple levels of genome function, including chromatin structure, chromosome segregation, transcription, RNA processing, RNA stability, and translation (Id.). The effects of small RNAs on gene expression and control are generally inhibitory, and the corresponding regulatory mechanisms are therefore collectively subsumed under the heading of RNA silencing (Id.). The central theme that runs throughout is that the small RNAs serve as specificity factors that direct bound effector proteins to target nucleic acid molecules via base-pairing interactions (Id.). Invariably, the core component of the effector machinery is a member of the Argonaute protein superfamily (Id.).

[0015] There are three main categories of small RNAs: short interfering RNAs (siRNAs), microRNAs (miRNAs), and piwi-interacting RNAs (piRNAs) (Id.). siRNAs and miRNAs are the most broadly distributed in both phylogenetic and physiological terms and are characterized by the double-stranded nature of their precursors (Id.). In contrast, piRNAs are primarily found in animals, exert their functions most clearly in the germline, and derive from precursors that are poorly understood, but appear to be single stranded (Id.). Where siRNAs and miRNAs bind to members of the Ago clade of Argonaute proteins, piRNAs bind to members of the Piwi clade (Id.).

[0016] The signature components of RNA silencing are Dicers, Agos, and ̴ 21-23 nt duplex- derived RNAs (Id.). Both siRNA and miRNA small RNAs depend on Dicer enzymes to excise them from their precursors, and Ago proteins to support their silencing effector functions (Id.).

[0017] RNase III enzymes, which are dsRNA-specific nucleases, are the source of miRNA / siRNA biogenesis (Id.). One class of large RNase III enzymes has several domains in a specific order from the amino to carboxy terminus: a DEXD / H ATPase domain, a DUF283 domain, a PAZ domain, two tandem RNase III domains, and a dsRNA-binding domain (Id.). Some members of this family differ slightly from this arrangement (Id.).

[0018] The PAZ and RNase III domains play central roles in excising siRNAs preferentially from ends of dsRNA molecules. PAZ domains are shared with Argonaute proteins and arespecialized to bind RNA ends, especially duplex ends with short (̴ 2 nt) 3’ overhangs. An end engages the Dicer PAZ domain, and the substrate dsRNA then extends approximately two helical turns along the surface of the protein before it reaches a single processing center that resides in a cleft of an intramolecular dimer involving the RNase III domains. Each of the two RNase IIII active sites cleaves one of the two strands, leading to staggered duplex scission to generate new ends with̴̴ 2-3’ nt overhangs. The reaction leaves a 5’ monophosphate on the product ends, consistent with a requirement for this group during later stages of silencing. This general model pertains equally to pre-miRNA stem-loop substrates and to long, perfectly base-paired dsRNAs. In some species, different functional categories of small RNAs exhibit slightly different lengths; this appears to be dictated by the distance between the PAZ domain and the processing center in the relevant Dicer enzyme (Id.).

[0019] The roles of the ATPase domain probably vary among different forms of Dicer (Id.). ATP promotes dsRNA processing by Drosophila Dicer 2 and C. elegans Dcr-1, and mutations predicted to cripple ATPase activity in Drosophila Dicer-2 specifically abolish dsRNA processing. In contrast, ATP is dispensable for dsRNA processing by human Dcr (hDcr), and an ATPase defective mutant exhibits no processing defect (Id.).

[0020] Dicers isolated from their natural sources generally are found in a heterodimeric complex with a protein that contains two or three double stranded Ras binding domains (dsRBDs); the Ras-binding domain (RBD) is an independent domain of about 75 residues, which is sufficient for GTP-dependent binding of Ras and other G alpha GTPases. Both hDcr and Drosophila Dcr-2 process dsRNAs effectively in the absence of the heterodimeric partner (TRBP and R2D2, respectively). In at least some cases, the role of Dicer in silencing extends beyond dsRNA processing and into the pathway of RISC assembly; this activity is much more dependent on the dsRBD partner protein (Id.).

[0021] Argonautes

[0022] The Argonaute superfamily can be divided into three separate subgroups: the Piwi clade that binds piRNAs, the Ago clade that associates with miRNAs and siRNAs, and a third clade described in nematodes. All gene regulatory phenomena involving ̴ 20-30 nt RNAs are thought to require one or more Argonaute proteins, which are the central, defining components of an RNA-induced silencing complex (RISC). The double-stranded products of Dicer enter into anRISC assembly pathway that involves duplex unwinding, culminating in the stable association of only one of the two strands with the Ago effector protein. This guide strand directs target recognition by Watson-Crick base pairing; the other strand of the original small RNA duplex (the passenger strand) is discarded (Id.).

[0023] Argonaute proteins are defined by the presence of four domains: the PAZ domain (shared with Dicer enzymes), the PIWI domain that is unique to the Argonaute superfamily, and the N and Mid domains. The overall protein structure is bi-lobed, with one lobe consisting of the PAZ domain and the other lobe consisting of the PIWI domain flanked by N-terminal (N) and middle (Mid) domains. The Argonaute PAZ domain has RNA 3’ terminus binding activity, and the co-crystal structures reveal that this function is used in guide strand binding. The other end of the guide strand engages a 5’ phosphate binding pocket in the Mid domain, and the remainder of the guide tracks along a positively charged surface to which each of the domains contributes. The protein-DNA contacts are dominated by sugar-phosphate backbone interactions. Guide strand nucleotides 2-6, which are especially important for target recognition, are stacked with their Watson-Crick faces exposed and available for base pairing (Id.).

[0024] The PIWI domain adopts an RNase H-like fold that in some cases can catalyze guide strand-dependent endonucleolytic cleavage of a base pair target. This initial cut represents the critical first step in a subset of small RNA silencing events that proceed through RNA destabilization. Not all Argonaute proteins have endonucleolytic activity, and those that lack it usually also lack critical active-site residues that coordinate a presumptive catalytic metal ion (Id.).

[0025] In humans, four of the eight Argonaute proteins are from the Ago clade and associate with both siRNAs and miRNAs (Id.).

[0026] MicroRNA Biogenesis

[0027] MicroRNAs are found in plant and animal branches of Eukaryotes and are encoded by a bewildering array of genes. Transcription of miRNAs is typically performed by RNA polymerase II, and transcripts are capped and polyadenylated. Although some animal miRNAs are individually produced from separate transcription units, many more are produced from transcription units that make more than one product. A transcript may encode clusters of distinct miRNAs, or it may encode miRNA and protein. The latter type of transcript is organized suchthat the miRNA sequence is located within an intron. Many new animal miRNAs are thought to arise from accumulation of nucleotide sequence changes and not from gene duplication (Carthew, RW and Sontheimer, EJ. Cell (2009) 136: 642-55).

[0028] The resulting primary or pri-miRNA transcript extends both 5’ and 3’ from the miRNA sequence, and two sequential processing reactions trim the transcript into the mature miRNA. Processing depends on the miRNA sequence folding into a step-loop structure. A typical animal pri-miRNA consists of an imperfectly paired stem of̴̴ 33 bp, with a terminal loop and flanking segments. The first processing step, which occurs in the nucleus, excises the stem-loop from the remainder of the transcript to create a pre-miRNA product. For most pri-miRNAs, a nuclear member of the RNase III family (Drosha in animals) carries out this cleavage reaction. Although Drosha catalyzes pri-miRNA processing, it depends on a protein cofactor, which contains two dsRBD domains and stably associates with the ribonuclease to form the microprocessor complex (Id.).

[0029] An alternative pathway uses splicing of pri-miRNA transcripts to liberate introns that precisely mimic the structural features of pre-miRNAs. These introns then enter the miRNA processing pathway without the aid of the Microprocessor (Id.).

[0030] The second processing step excises the terminal loop from the pre-miRNA stem to create a mature miRNA duplex of approximately 22 bp length. In animals, the pre-miRNA is exported from the nucleus, and the canonical Dicer enzyme carries out the cleavage reaction in the cytoplasm (Id.).

[0031] MicroRNAs behave like traditional polymeric products of gene activity, such that most species of a miRNA have highly exact ends, although there is a little variation. This feature of miRNAs may allow them to interact with greater specificity on substrate mRNAs without a need for stringent complementarity or large overlap (Id.).

[0032] Consequently, the processing machinery is constructed to produce miRNA duplexes with highly exact ends. The first cut, carried out by Drosha with the aid of its dsRBD domain binding partner protein (called DGCR8), is most critical. DGCR8 directly interacts with the pri-miRNA stem and flanking single-stranded segments. The cleavage site is determined by the distance from the stem-flank junction, which is precisely one turn of a dsRNA helix (11 bp) and is the minimal processing length for an RNase III enzyme. Although Drosha carries out the cleavagereaction, it relies upon DGCR8 to serve as a molecular anchor that properly positions Drosha’s catalytic site the correct distance from the stem-flank junction. Thus, the endpoint of the stem is a critical determinant for one end of the mature miRNA (Id.).

[0033] The second cut performed by Dicer defines the other end of the mature miRNA. Dicer will cleave anywhere along a dsRNA molecule but has a strong preference for the terminus. The PAZ domain of Dicer interacts with the 3’ overhang at the terminus and determines the cleavage site in a ruler-like fashion. The RNase III catalytic sites are positioned two helical turns or 22 bp away from the terminus / PAZ portion of the Dicer-RNA complex (Id.).

[0034] While regulation of miRNA biogenesis has not been extensively studied, a surprising number of miRNA genes are formed under the control of the very targets that they regulate. A rationale behind these double-negative regulatory relationships is that tight regulation of miRNA biogenesis is crucial. Mis-expression of miRNAs frequently mimics loss of function phenotypes for their targets. This would be prevented if biogenesis of a miRNA is strictly controlled by its targets. The restriction would also explain how off-targeting effects by wayward miRNAs are carefully limited (Id.).

[0035] MicroRNA associations

[0036] The mature miRNA duplex is a short-lived entity; it is rapidly unwound when it associates with an Ago protein. Unwinding occurs so rapidly after duplex formation, because the two processes are physically coupled due to Ago2’s presence in a complex with Dicer and TRBP, the double-stranded RNA binding protein that loads siRNA into the RISC (Id.).

[0037] miRNA unwinding is accompanied by differential strand retention, i.e., one strand is retained while the other strand is lost. Strand retention is based on the relative thermodynamic stability of the duplex’s ends. Although the rule is that the 5’ terminus of the retained strand is at the less stably base-paired end of the duplex, this rule is not absolute. The other strand is appreciably detected in Ago complexes, lending ambiguity to the notion of strand asymmetry. Although either strand can become stably associated with Ago proteins, the more commonly associated strand is termed the miRNA strand; the other strand is called the miRNA* strand. miRNA unwinding is not accompanied by cleavage of the ejected strand by the associated Ago (Id.).

[0038] The mammalian Dicer / Ag / miRNA complex is associated with other proteins, e.g., Gemin3, Gemin4, Mov10, and Imp8, as well as the mammalian protein GW182, associates with Ago2. GW182 is both necessary and sufficient for miRNA-bound Ago to silence gene expression. Thus miRNA-bound Ago in association with GW182 can be thought of as the miRISC complex (Id.).

[0039] Post-transcriptional repression by miRNAs

[0040] A miRNA acts as an adaptor for miRISC to specifically recognize and regulate particular mRNAs. If miRISC is tethered to a heterologous RNA recognition factor, the factor enables miRISC to recognize and repress mRNAs that lack miRNA-binding sites. With few exceptions, miRNA-binding sites in animal mRNAs lie in the 3’ untranslated region (UTR) and are usually present in multiple copies. Most animal miRNAs bind with mismatches and bulges, although a key feature of recognition involves Watson-Crick base pairing of miRNA nucleotides 2–8, representing the seed region (Id.).

[0041] While it was thought that perfect complementarity allows Ago-catalyzed cleavage of the mRNA strand, whereas central mismatches exclude cleavage and promote repression of mRNA translation, it appears that translational repression is the default mechanism by which miRNAs repress gene expression, both in animals and plants. Perfectly complementary miRNAs may additionally engage in mRNA cleavage such that their effects are the result of both mechanisms (Id.).

[0042] The mechanisms by which miRISC regulates translation have been subject to ongoing debate. The fundamental issue of whether repression occurs at translation initiation or post- initiation has not yet been resolved. There are three competing models for how miRISC represses initiation. One proposes that there is competition between miRISC and elF4E for binding to the mRNA 5’ cap structure. A second model has proposed that miRISC stimulates de-adenylation of the mRNA tail; translation is repressed because the cap and PABP1-free tail of the deadenylated mRNA are unable to circularize. A third model has proposed that miRISC blocks association of the 60S ribosomal subunit with the 40S preinitiation complex, i.e., the recruitment of eIF6 by miRISC may repress translation by preventing the assembly of translationally competent ribosomes at the start codon (Id.).

[0043] It is unclear why some targets are degraded and others are not (Id.).

[0044] Without being limited by any particular theory, it appears that the mode of regulation of any miRNA (repression vs. activation) in the context of the whole cell and the myriad activities that affect posttranscriptional gene regulation may be context dependent (Id.).

[0045] The cell’s position in the cell cycle is one such context. For example, mammalian miRNA let-7 and an artificial miRNA (CXCR-4) repress translation in proliferating human cells, but change into translational activators when the cell cycle is arrested at the G1 checkpoint by serum starvation. Aphidicollin-induced arrest at G1 also generates translational activation, whereas nocodazole-induced arrest at G2 / M generates translational repression. Lymphocyte growth arrest induces TNFα expression that is required for macrophage maturation; miR-369-3p switches from a repressor to an activator of TNFα translation when cells in culture are growth arrested (Id., citing Vasudevan, S.et al. Science (2007) 318: 1931-1934).

[0046] Binding site position is another context. Interaction of miR-10a with the 5’UTR of certain ribosomal subunit mRNAs leads to their activated translation, whereas interaction with the 3’UTR leads to repression (Id., citing Orom, UA et al. (2008) Mol. Cell 30: 460-471).

[0047] Another context is how small RNA regulation is organized and modulated within the cell. Ago proteins are frequently associated with membrane trafficking compartments, such as the Golgi and ER (Id., citing Cikaluk, D.E. et al. Mol. Biol. Cell (1999) 10: 3357-3372). It has been hypothesized that miRISC factors might become anchored in certain subcellular compartments, e.g., P bodies or GW bodies, two separate pools of sequestered non-translating RNAs (Patel, PH, et al. PLos One (2016) 11(3): e015029). Subunits of miRISC (miRNAs, Ago and GW1821) and their repressed targets also are enriched in GW bodies. While GW bodies are not essential for miRNA repression, GW body formation requires an intact miRNA pathway (Carthew, RW and Sontheimer, EJ. Cell (2009) 136: 642-655).

[0048] miRNA expression

[0049] MicroRNAs regulate gene expression at the post-transcriptional level. The exact functional outcome of an miRNA may be determined by multiple features, including the cell type affected, the inducing signal, and the transcriptomic profile of the cell, which ultimately affect the availability and ability to engage different target mRNAs and bring about its unique responses. Indeed, data suggest that miRNAs may play different roles in diverse biological contexts. [Lee, H-M et al. BMB Rep. (2016) 49 (6): 311-318].

[0050] ESCRT in mammalian cells

[0051] The endosomal sorting complex required for transport (ESCRT) machinery responsible for sorting the cargo proteins of ILVs is divided into four complexes, ESCRT-0, -I, -II, -III, which work cooperatively to generate MVBs with associated proteins VPS4, VTA1, and ALG‑2 interacting protein (ALIX).

[0052] The ESCRT-0 complex is composed of two subunits, hepatocyte growth factor-regulated tyrosine kinase (HRS) and STAM, both of which can engage ubiquitinylated substrates destined for lysosomal degradation. EXCRIT-0 recruits TSG101 of the ESCRT-I complex and isolates the ubiquitinated proteins in the endosomal membrane. The ESCRT-I complex is essential and responsible for cargo sorting in the MVBs, as it induces budding by deforming the plasma membrane. Subsequently, ESCRT-I activates ESCRT-III, which is responsible for the concentration of MVBs’ cargo molecules using the ESCRT-II complex or ALIX, and the ESCRT-III / VPS4 complex triggers the constriction and induces the cleavage of the vesicle buds during abscission (meaning the act of cutting off). Afterwards, MVBs interact with a specific combination of soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) within the plasma membrane, and mammalian-derived exosomes are secreted to the extracellular milieu of the cell [Id., citing Kalluri, R. and LeBleu, VS, Science (2020) 367 (6478): eaau6977; Vietri, M. et al. Nat. Rev. Mol. Cell Biol. (2019) 21 (1): 25-42].

[0053] Depending on the particular composition of their exosomal proteins, the fate and function of mammalian-derived exosomes in their biogenesis, cargo selection, targeting ability, and endocytosis under both physiological and pathological conditions can vary. Among the protein cargoes of mammalian-derived exosomes, those proteins participating in cell adhesion (e.g., integrin, lactadherin, ICAM), intracellular trafficking (e.g., RAB GTPases, annexin), signal transduction (e.g., protein kinases, β-catenin, 14–3–3, G proteins), biogenesis factors (e.g., ALIX, TSG101, syntenin, ubiquitin, clathrin, VPS32, VPS4), and as well as chaperones (e.g., HSP70, HSP90), have been evaluated [Id., citing van Niel, G. et al. Nat. Rev. Mol. Cell Biol. (2018) 19: 213-228]. Generally, mammalian-derived exosomes are characterized by the presence of specific lipids such as phosphatidylserine, cholesterol, sphingomyelins, and ceramides, which are responsible for intercellular signaling as well as being essential in structural stability. In addition, they may also carry genetic material such as messenger RNA (mRNA), microRNAs(miRNAs), and non-coding RNAs. Overall, mammalian-derived exosomes are not only considered to be effective intercellular transporters of proteins, lipids, and nucleic acids, but also novel regulators that can alter the physiological and pathological functions of both recipient and parent cells via their various components.

[0054] Although, the application of mammalian derived exosomes is promising, several major issues limit the clinical use of these exosomes, including (1) their low production yield, (2) the time-consuming and laborious production processes, and (3) the difficulties involved with achieving high-quality and uniform exosomes [Id., citing [Li, P. et al. Theranostics (2017) 7(3): 789-804]; van den Boorn, JG et al. Nat. Biotechnol. (2011) 29 (4): 325-326; Lobb, RJ et al. J. Extracell. Vesicles (2015) 4 (1): 27031; Vader, P. et al. Adv. Drug Deliv. Rev. (2016) 106 (Part A): 148-156; van Deun, J. et al. J. Extracell. Vesicles (2014) 3 (1): 24858]. Plant-derived exosome-like nanovesicles (PELNVs).

[0055] Evidence of the existence of plant extracellular vesicles dates back to 1967. [Halperin, W. and Jensen, WA. J. Ultrastruct. Res. (1967) 18: 428-443]. Notwithstanding that many aspects of plant-derived exosome-like nanovesicles are not fully understood, they may provide therapeutic advantages compared with mammalian-derived exosomes or artificial nanoparticles, including facile large-scale production [Subha, D. et al. Discover Nano 92023) 18: 146, citing Li, Z. et al. Sci. Rep. (2018) 8(1): 14644], low toxicity, reduced immunogenicity [Id., citing Deng, Z. et al. Mol. Ther. (2017) 25 (7): 1641-1654], efficient cellular uptake [Id., citing Wang, Q. et al. Nat. Commun. (2013) 4: 1867] and high biocompatibility and stability [Id., citing Zhang, M. et al. Biomaterials (2016) 101: 321-340].

[0056] WO2020 / 180311 entitled “Plant-based exosome compositions and use thereof for rejuvenating skin” describes isolation of exosomes from leaf flesh of a plant from the Asphodelaceae family, more particularly of the Aloe genus, including Aloe vera, Aloe barbadensis Miller, Aloe aborenscens, or Aloe vera L. conditioned by growing the plant under conditions that included a heat shock of the plant at a temperature of from about 33°C to about 45°C for about 1 hour to about 3 hours. The level of heat shock stress response molecule HSP70 in the heat-shocked plant exosome was from about 10 times to about 20 times higher than the level of heat shock stress response molecule in the exosomes from non-heat-shocked plants.

[0057] Plant-derived exosome-like nanovesicle (PELNV) preparation has been attempted using many common edible plants, such as grapefruit, grape, lemon, broccoli, carrot, apple, coconut, and ginger. [Kim, J. et al. Asian J. of Pharmaceutical Sci. (2022) 17: 53-69, citing Ju, S. et al. Mol. Ther. (2013) 21 (7): 1345-1357; Wang, B. et al. Mol. Ther. (2014) 22 (30: 522-534; Wang, Q. et al. Cancer Res. (2015) 75(12): 2520-2529; Brahmbhatt, M. et al. Nutr. Cancer (2013) 65 (2): 263-272; Raimondo, S. et al. Oncotarget (2015) 6 (23): 19514-19527; Deng, Z. et al. Mol. Ther. (2017) 25 (7): 1641-1654; Mu, J. et al. Mol. Nutr. Food Res. (2014) 58 (7): 1561-1573; Zhao, Z. et al. J. Agric. Food Chem. (2018) 66 (11): 2749-2757; Yu, S. et al. Food Chem. (2019) 272: 372-378; Fujita, D. et al. Mol. Pharm. (2018) 15 (12): 5772-5780; Zhang, M. et al. Mol. Ther. (2016) 24 (10) 1783-1796]. PENLV are known to be similar to mammalian-derived exosomes in terms of such properties as size distribution, surface electric charge, morphology, density, and certain components [Id., citing Ju, S. et al. Mol. Ther. (2013) 21 (7): 1345-1357; Raimondo, S. et al. Oncotarget (2015) : 1951419514-27; Deng, Z. et al. Mol. Ther. (2017) 25 (7): 1641-1654]. Like mammalian-derived exosomes, PELNVs also comprise biomolecules, such as RNAs, proteins, and lipids, that regulate physiological processes [Id., citing Teng, Y. et al. Cell Host Microbe (2018) 24 (5): 637-652].

[0058] Despite many similarities between PELNVs and mammalian-derived exosomes, the two groups of vesicles show some differences. First, the lipid bilayer of mammalian-derived exosomes are mainly composed of cholesterol, glycoshingolipids, ceramides and phosphatidylserine, which provide stability and a unique rigidity [Id., citing Stemersch, S. et al. J. Control Release (2016) 244: 167-83; Nishio, M. et al. Bioscens. Bioelectron (2020) 150: 111918; Mashouri, L. et al. Mol Cancer (2019) 18: 75; Waldenstrom, A. and Ronquist, G. Circ. Res. (2014) 114 (2): 315-324]. In contrast, the exosomal membranes of PELNVs are enriched with phosphatidic acid (PA), phosphatidylcholines (PC), digalactosyldiacylglycerol (DGDG), and monogalactosyldiacylglycerol (MGDG) [Id., citing Teng, Y. et al. Cell Host Microbe (2018) 24 (5): 637-652], which provide inherent mammalian-cell-regulating activities in the intestinal microenvironment.

[0059] Second, there is no canonical ESCRT-0 complex in higher plants; instead, TOM1-like (TOL) proteins with conserved VHS (VPS27, HRS, STAM) domains, which are considered to have a general membrane targeting / cargo recognition role in vesicular trafficking [see Lohi, O. et al. FEBS Lett. (2002) 513 (1): 19-23] act as substitutions for ESCRT-0 as ubiquitin bindingproteins and play a role in the vacuolar sorting of the auxin efflux facilitator PIN-FORMED 2 (PIN2) in the early endosome [Kim, J. et al. Asian J. of Pharmaceutical Sci. (2022) 17: 53-69, citing Gao, C. et al. Trends Plant Sci. (2017) 22 (11): 986-998]. The cargo is subsequently transported to the ESCRT-1 and ESCRT-II complexes via the ubiquitin-binding proteins, the ESCRT-II complex then stimulates and recruits ESCRT-III through an interaction between VPS25 and VSP20. The ESCRT-III complex constricts the plasma membrane and cleaves the necks of the buds that form on the cytosolic face to release ILVs containing cargo into the endosome [Id., citing Yanez-Mo, M. et al. J. Extracell. Vesicles (2015) 4: 27066; Cui, Y. et al. Mol. Plant (2016) 9 (6): 774-786].

[0060] Third, surface markers to differentiate between types of extracellular vesicles in plants are yet to be identified, and the mechanism underlying trafficking of the EVs is unknown.[Subha, D. et al. Discover Nano (2023) 18: 146].

[0061] Fourth, each PELNV has different characteristics and components depending on the cell of origin. For example, Zhuang et al. reported that a 6-shogaol rich in ginger-derived exosome- like nanoparticles (GDENs) activates Nrf2 by regulating TLR4 / TRIF pathway, protecting against alcohol-induced liver damage through the anti-inflammatory actions of this pathway [Id., citing Zhang, X. et al. J. Extracell. Vesicles (2015) 4 (1): 28713]. Ju et al. reported that grape-exosome like nanoparticles induced the recovery of intestinal stem cells through the Wnt / β-catenin signaling pathway, which regulates genes including AXIN-2, Cycline D1, c-MYC, and EGF [Id., citing Ju, S. et al. Mol Ther. (2013) 21 (7): 1345-1357]. In terms of the cellular uptake of grape- derived exosome-like nanoparticles (GELNs), GELNs showed a high selectivity to intestinal stem cells and were significantly inhibited by a cytochalasin-D inhibitor, known as micropinocytosis inhibitor. In contrast, a clathrin-mediated endocytosis inhibitor did not affect their uptake [Id.].

[0062] Fifth, beneficially, unlike mammals, plants do not harbor zoonotic or human pathogens.

[0063] Plants produce EVs in response to numerous biotic and abiotic environmental stresses, including pathogen infection and attack. [Kim, J. et al. Asian J. of Pharmaceutical Sci. (2022) 17: 53-69, citing An, Q. et al. New Phytol. (2006) 172 (3): 563-576; An, Q. et al. Cell Microbiol. (2006) 8 (6): 1009-1019). Qianli et al., who observed the proliferation of intravacuolar MVBs in the cytoplasm and the structural perturbation of the organelle and its membrane by trafficking inbarley leaf cells, provided evidence of the generation and secretion of plant-derived exosome- like nanovesicles [Id., citing Qianli, AJVB, and Huckelhoven, R. Plant Signal Behav. (2007) 2 (1): 4-7]. Additionally, it has been reported that enhancement of fungal infections induces the proliferation of MVBs at the site of infection by stimulating plant innate immune responses in plant defense [Id., An, Q. et al. New Phytol. (2006) 172 (3): 563-576; Wang, F. et al. PLoS Pathog. (2014) 10 (7): e1004243].

[0064] Evidence indicates that plant extracellular vesicles play a multifaceted role in plant defense [Subha, D. et al. Discover Nano 9202318: 146]. They function as mobile pockets highly enriched with antimicrobials and plant-defense proteins which initiate the plant immune response at the site of pathogen entry. For example, defense-related proteins involved in the myrosinase- glycosinolate system such as penetration resistance protein 3 (PEN3), which provides cell wall defense against pathogens, the nitrate sensor NRT1, the myrosinase epithiospecific modifier 1, and reactive oxygen species (ROS) signaling proteins were identified in the EV proteome [Id. citing Rutter, BD and Innes, RW. Plant Physiol. (2017) 173: 728-41; Rutter, BD, and Innes, RW. Curr. Opin. Plant Biol. (2018) 44: 16-22]. Plant EVs also can hold cell wall remodeling enzymes. On sensing an infection, the plant tries to strengthen its defense by improving cell wall integrity, while the pathogen takes advantage of the cell wall metabolism in the host to establish the infection [Id., citing Vincent, D. et al. Front. Plant Sci. (2020) 10: 1626]. Cell wall remodeling proteins are transported through EVs in several cases. Proteomic studies on EVs from sunflower seedlings revealed that 47% of the identified proteins are cell wall-associated including the enzymes in-charge of polysaccharide reorganization [Id., citing Regente, M. et al. Exp. Bot. (2017) 68: 5485-5495].

[0065] When the proteome of the plant EVs from Arabidopsis was analyzed, a total of 93 RNA binding proteins RBPs) were recognized. Those with ssRNA binding capacity are AGO1, DEAD-box RNA helicase RH11, RH37, RH52, Annexin 1 (ANN1) and annexin 2 (ANN2). These proteins were also easily detected by western blotting in isolated EVs thereby confirming the proteome results. These RBPs co-localize with the TET8 marker and the EV associated small RNAs previously identified (such as TAS1c-SiR483, TAS2-SiR 453 and miR166) were also found associated with this subgroup of exosome like EVs. EV localized RBPs specifically bind with the small RNAs and aid in their selective loading into the EVs. The Annexins (ANN1 andANN2), which were also found in the EVs do not bind with ssRNAs specifically; it has been suggested that they might contribute to the stabilization of the RNA inside the vesicles [Id.]. Plant heat shock proteins counter plant biotic and abiotic stresses

[0066] Plants continuously confront harsh environments like high / low temperatures, salinity, drought, light stresses, flooding, physical wounding and chemical pollutants (e.g., heavy metals), which produce secondary stresses, such as osmotic and oxidative stresses, [al Haq, S. et al. Intl J. Molec. Sci. (2019) 20: 5321, citing Swindell, WR et al. BMC Genom. (2007) 8: 125; Al-Whaibi, MH. J. King Saud Univ. Sci. (2011) 23: 139-150; Guo, M. et al. Front. Plant Sci. (2016) 7: 114; Xu, Y. et al. Int. J. Proteom. (2011) 2011: 529648]. Biotic stresses like pathogens (e.g., viruses, bacteria, and fungi) and pests (e.g., nematodes, insects, and rodents) also restrict plant productivity [Id., citing Dodds, P. and Rathjen, J. Nat. Rev. Genet. (2010) 11: 539; Li, R. et al. PLoS One (2015) 10: e0143261; Jones, JT et al. Mol. Plant Pathol. (2013) 14: 946-561; Rybicki, EP. Arch. Virol. (2015) 160: 17-20; Gorovits, R. et al. Cell Stress Chaperones (2017) 22: 345- 355]. Negative effects of these stresses on plant germination [Id., citing Cheng, L. et al. J. Integr. Plant Biol. (2009) 51: 489-499], include stunted growth [Id., citing Srivastava, S. et al. J. Environ. Biol. (2012) 33: 657; Wahid, A. et al. Environ. Exp. Bot. (2007) 61: 199-223], sunburn and scorching of leaves [Id., citing Rodruguez, M. et al. Plants Biotecnologia (2005) 22: 1-10], loss of photosynthetic pigment, decreased production of photo-assimilates, and depletion of carbohydrate reserves which results in starvation [Id., citing Tan, W. et al. J. Plant Physiol. (2011) 168: 2063-2071; Jiang, C. et al. Plant Cell Environ. (2009) 32: 1046-1059; Demirevska- Kepova, K. et al. Biol. Plant (2005) 49: 521-525; Djanaguiraman, M. et al. J. Agron. Crop Sci. (2009) 195: 213-224]. Abiotic stresses also negatively affect the reproductive characteristics of plants by enhancing male sterility [Id., citing Young, LW et al. J. Exp. Biol. (2004) 55: 485-495] and increasing premature flower and fruit drop [Id., citing Tubiello, FN et al. Proc. Natl Acad. Sci. USA (2007) 104: 19686-19690] which results in significant low yield and quality. It has been reported that an increase in temperature by 1°C results in a 4–10% yield decrease [Id., citing Wang, X. et al. J. Cereal Sci. (2012) 55: 331-6]. As a consequence of these stresses, reactive oxygen species (ROS) are produced which lead to oxidative stress and, ultimately, results in cell death. ROS could be singlet oxygen (1O2), superoxide radical (O2•−), hydrogen peroxide (H2O2) and hydroxyl radical (OH −), which are produced in cell organelles such as mitochondria, peroxisomes and chloroplasts in oxidative stress situations and react with all typesof macromolecules like pigments, proteins, lipids and DNA [Id., citing Karuppanapandian, T. et al. Plant Physiol. Biochem. (2011) 49: 168-177; Moller, IM et al. Anu. Rev. Plant Biol. (2007) 58: 459-481].

[0067] Plants respond morphologically to elevated temperature and light stress by changing their leaf orientation [Id., citing Wahid, A. et al. Environ. Exp. Biol. (2007) 61: 199-223], anatomically by altering stomatal conductance and increased leaf pubescence [Id., citing Zhang, J. et al. Field Crop Res. (2006) 97: 111-119; Banon, S. et al. Sci. Hortic. (Amst.) 2004101: 333- 342], and phenologically by shifting and improvising the developmental stages to escape the abiotic stress condition [Id., citing Sato, S. et al. Ann. Bot. (2006) 97: 731-8]. Plants also change their metabolic processes and physiology to retain root hydraulic conductance [Id., citing Morales, D. et al. Biol. Plant (2003) 47: 203], accumulation of compatible osmolytes, such as sugars, sugar alcohols, proline and phenolic compounds under saline and water-logged conditions, as well as high temperature and water deficit conditions [Id., citing Wahid, A. and Close, TJ. Biol. Plant (2007) 51: 104-109]. Moreover, plants manage to maintain photosynthetic machinery [Id., citing Salvucci, ME et al. Physiol. Plant (2004) 120: 179-186] by changing their assimilate partitioning (e.g., a shift occurs from symplastic, where the symplast pathway involves protoplasts that are found within the living cytoplasm cells, to apoplastic (a non-living route since the water solution moves in the spaces between cells and along the cell wall) [Id., citing Wahid, A. and Ghazanfar, A. J. Plant Physiol. (2006) 163: 723-730]. During the onset of the stress situations, plants also improvise the hormonal balance of abscisic acid (ABA), ethylene, and salicylic acid (SA) as a signaling molecule in the systemic acquired resistance. Similarly, jasmonic acid (JA) and other steroids enhance stress tolerance and resistance [Id., citing Wang, LJ and Li, SH. Plant Sci. (2006) 170: 685-694]. Furthermore, secondary metabolites, such as isopropanioid, carotenoid, flavonoid, anthocyanin, lignin, and isoprenoids [Id., citing Wahid, A. and Ghazanfar, A. J. Plant Physiol. (2006) 163: 723-730; Sharkey, TD. Plant Cell Environ. (2005) 28: 269-277], also are produced and accumulated. Most studies indicate that plant responses to two or more factors are unique and differ from the response to one factor only. [Al- Whabi, MH, J. King Saud University-Science, J. King Saud Univ. – Science (2011) 23: 139- 150].

[0068] Besides these adaptations, plants also have sophisticated adaptive systems at the cellular and molecular levels. During the onset of stress, plants reduce the synthesis of normal proteinproduction and transcribe and translate heat shock proteins (HSPs). Added to transcriptional regulations, plants also have some sophisticated post-transcriptional modifications which help the plant to cope with these stresses, such as alternative splicing and micro RNA (miRNA). Alternative splicing, which generates multiple copies from a single gene, helps the plants to mitigate abiotic stresses [al Haq, S. et al. Intl J. Molec. Sci. (2019) 20: 5321, citing Laloum, T. et al. Trends Plant Sci. (2018) 23: 140-150]. An important plant post-transcriptional modification strategy is miRNA, which binds to the mRNA at any point to repress translation or direct cleavage of the mRNA. Some plant miRNAs also are involved with abiotic stress tolerance [Id., citing Zhang, B. et al. Dev. Biol. (2006) 289: 3-16].

[0069] HSPs are proteins characterized by the presence of a carboxylic terminal called heat- shock domain [Al-Whaibi, MH., J. King Saud Univ. – Science (2011) 23: 139-150, citing Helm, KW et al. Mol. Cell Biol. (1993) 13: 238-247]. The concentration of HSPs dramatically increases when cells are grown at higher temperatures where HSPs help newly synthesized proteins to fold or protect proteins that might misfold and thereby lose their potential functional conformation during a stress event, such as biotic and / or abiotic stress [al Haq, S. et al. Intl J. Molec. Sci. (2019) 20: 5321, citing Weinmann, H. and Ottow, E. In Comprehensive Medicinal Chemistry II, Taylor, JB and Triggle, DJ Eds. Elsevier: Oxford UK (2007); pp. 221-251]. These stress-responsive biomolecules act as molecular chaperones which perform under stress situations [Id., citing Ahuja, I. et al. Trends Plant Sci. (2010) 15: 664-674]. The main functions of HSPs are proper protein folding, unfolding and transport, in conjunction with their localization in the cell, and, subsequently, disposal and degradation of the non-native proteins, [Id., citing Balchin, D. et al. Science (2016) 353: acc4353; Hartl, FU et al. Nature (2011) 475: 324-332; Benesova, M. et al. PLoS ONE (2012) 7: e38017].

[0070] HSPs are important in the plant life cycle as their role extends beyond the protection from biotic and abiotic stresses. Although HSPs (with the exception of ubiquitin) were first characterized due to their response to high temperatures, now many HSPs are found in normal, non-stressed cells, and are produced at particular stages of the cell cycle, or during development in the absence of stress [Id., citing Vierling, E. Annu Rev. Plant Biol. (1991) 42: 579-620]. For example, beside stress-responsive biomolecules, HSPs also are involved in plant growth and development under normal conditions, like the flowers, seeds and fruits set, development [Id., citing Eck, ERHB et al. J. Biosci. (2007) 32: 501-510], tuberization [Id., citing Lehesranta, SJ etal. Proteomics (2006) 6: 6042-6052; Agrawal, L. et al. J. Proteome Res. (2013) 12: 4904-4930; Agrawal, L. et al. J. Proteome Rs. (2008) 7: 3803-17; Ahn, YJ and Zimmerman, JL. Plant Cell Environ. (2006) 29: 95-104] and nutrient uptake [Id., citing Shekhar, S. et al. J. Proteom. (2016) 143: 306-17]. HSPs are found in different compartments of the cells, such as cytoplasm, nucleus, and cell organelles, e.g., mitochondria, chloroplasts and endoplasmic reticulum [Id., citing Water, ER et al. J. Exp. Biol. (1996) 325-338; Boston, RS et al. In Post-Transcriptional Control of Gene Expression in Plants, Springer: Berlin, Germany (1996), pp. 191-222].

[0071] HSP expression is controlled by transcription factors known as the heat shock factors (HSF). Among the HSF classes, HSFA positively regulates plant tolerance to anoxia, heat, osmotic and oxidative stresses [Id., citing Zhuang, L. et al. Intl J. Mol. Sci. (2018) 19: 2702]. HSFA1, found in tomato plants, is considered a master regulator of signal perception, transduction and controlling the expression of stress-responsive genes, including HSPs [Id., citing Guo, M. et al. Front Plant Sci. (2016) 7: 114; Mishra, SK et al. Genes Dev. (2002) 16: 1555-1567], thus, increased expression of HSPs and other stress responsive genes. HSFs play an important and significant role in modifying physiological and biochemical processes, which leads to the development of tolerance to stresses [Id., citing Kotak, S. et al. Curr. Opin. Plant Biol. (2007) 10: 310-316; Scharf, KD et al. Biophys. Acta Gene Regul. Mech. (2012) 1819: 104- 119]. HSPs show a response to biotic and abiotic stress situations by up- or down-regulation, but, sensing signals and transduction, particularly in biotic stress, remains to be explored [Id., citing Singh, RK et al. Sci. Rep. (2016) 6: 32641]. Classification and Nomenclature of HSPs

[0072] Heat shock proteins are conserved in almost all organisms from bacteria, to fungi, plants and animals, including human beings. HSPs are classified and named based on the molecular weight in kilo Dalton (kDa), which ranges from 8–200 kDa [Id]. Based on molecular weight, HSPs generally are classified into the following sub-families: HSP100, HSP90, HSP70, HSP60, and small HSPs (sHsps), which are characterized by a conserved sequence of 80-100 amino acid residues, with a molecular weight ranging from 13 to 43 kDa [Reddy, VS et al. Cell Stress and Chaperones (2018) 23: 441-454; al Haq, S. et al. Intl J. Molec. Sci. (2019) 20: 5321, citing Schoffi, F. et al. Sci. Research (1999) 81-98; Schlesinger, MJ. J. Biol. Chem. (1990) 265: 12111- 4; Kotak, S. et al. Plant Cell (2007) 19: 182-195; Shamovsky, I. and Nudler, E. Cell Mol. LifeSci. (2008) 65: 855-861; Guo, M. et al. Frong. Plant Sci. (2015) 6: 806]. These chaperone families are involved in maintaining cell homeostasis, transportation of newly synthesized proteins across cell organelles, and folding—preventing misfolded, denatured and aggregated proteins caused by stress conditions [Id., citing Balchin, D. et al. Science (2016) 353: acc4353; Ratajczak, E. et al. J. Mol. Biol. (2009) 386: 178-89; Tyedmers, J. et al. Nat. Rev. Mol. Cell Biol. (2010) 11: 777-788].

[0073] The genes which encode different HSPs are found in different cell compartments, such as the nucleus, mitochondria, chloroplast, endoplasmic reticulum and cytosol [Id., citing Liu, D. et al. Plant Physiol. Biochem. (2006) 44: 380-386]. Similarly, the accumulation of these HSPs in different parts of the cell also depends on the intensity of the stress. Nuclear HSPs, for instance, are accumulating in the cytosol at the lower and higher temperatures of 27°C and 43°C, respectively, while the same aggregate in chloroplast is at 37°C [Id., citing Waters, ER et al., J. Exp. Bot. (1996) 47: 325-338].

[0074] Different HSPs are found and differentially expressed in different species and in different genotypes but in the same species, as investigated by Korotaeva et al., (2001) and Nieto-Sotelo et al., (2002) [Korotaeva, NE et al. Russ. J. Plant Physiol. (2001) 48: 798-803; Nieto-Sotelo, J. Plant Cell (2002) 14: 1621-33] in small HSPs where five sHSPs showed a response to a higher temperature (42°C) in maize but only one is expressed in wheat and rice. Likewise, HSP68 is expressed in mitochondria under stress situations in potatoes, tomatoes, and soybeans [Id., citing Neumann, D. et al. Planta (1993) 190: 32-43].

[0075] The most studied species of plant is Arabidopsis thaliana, where the response to heat- shock treatment occurs through the participation of a number of Hsps: 13 HSPS20; 8 Hsp70; 7 Hsp90, 8 Hsp100, and 21 transcription factors (Hsfs) [Al-Whaibi, MH, J. King Saud University – Science (2011) 23: 139-150, citing Swindell, WR et al, BMC Genomics (2007) 8: 125], but in tomato there are at least 15 Hsfs (Id., citing von Koskull-Doring, P. et al Trends Plant Sci. (2007) 12: 452-457).

[0076] Higher plants are characterized by the presence of at least 20 types of sHsps. sHsps are usually undetectable in plant cells under physiological conditions, but are induced upon stress and plant tolerance to stress, including drought, salinity, oxidized species, and low temperatures [Id., citing Low, D. et al Planta (2000) 211: 575-582; Hamilton, EV and Heckathorn, SA PlantPhysiol. (2001) 126: 1266-1274; Scharf, KD et al Cell Stress Chaperones (2001)6: 225-237; Zhang, J-H et al. Scientia Horticulturae (2008) 117: 231-240].

[0077] Furthermore, the sHsps of A. thaliana and Lycopersicon esculenium are divided into three subclasses [Id., citing Scharf, KD et al Cell Stress Chaperones (2001) 5: 225-237; Siddique, M et al, Cell Stress Chaperones (2003) 8: 381-394]. These included: subclass C1, represented by 6 proteins in A. thaliana and five proteins in L. esculenium; subclass C11 represented by two genes in both plants; and subclass CIII represented by one gene in both plants. A study reported the presence of other groups in the cytoplasm of A. thaliana cells, and could be categorized into subclasses: CIV, CV, CVI, and CII. Each subclass has its own distinct characteristics and role.

[0078] There are six groups of genes that encode for the sHsps. The grouping is based on the sequence similarity and the location of these proteins in the cell. There are two classes of proteins (Class I and Class II) in the cytoplasm encoded by two groups of genes. Other locations are chloroplasts, endoplasmic reticulum, mitochondria and membranes [Id., citing Vierling, E. Annu. Rev. Plant Phys. (1991) 42: 579-620; Waters, ER et al. H. Exp. Bot. (1996) 47: 325-338]. The expression of genes for these sHsps is limited in the absence of environmental stress and occurs in some stages of growth and development of plants, such as embryogenesis, germination, development of pollen grains, and fruit ripening [Id., citing Sun, W. et al. Biochim Biophys. Acta (2002) 1577: 1-9].

[0079] Some HSPs showed a tissue-specific response to stress situations; HSP101 was expressed more in reproductive parts like tassels, ear, and endosperm, than in vegetative parts like leaves and roots in maize [al Haq, S. et al. Intl J. Molec. Sci. (2019) 20: 5321, citing Young, TE et al. Plant Physiol. (2001) 127: 777-791]. Some HSPs responded differently to the varying length of stresses. As reported by Heckathorn et al. [Id., citing Heckathorn, SA et al. Int. J. Plant Sci. (1998) 159: 39-45], HSP45, a nuclear protein, accumulated in the chloroplast after a 3 h exposure to heat stress, which returned to its native state after removal of stress. Similarly, HSPs are triggered differently at different development stages. HSP45, for example, showed a response in the whole plant to a stress situation, while HSP64 and HSP72 only showed expression in the reproductive parts i.e., pollens [Id., citing Frova, C. et al. Dev. Genet. (1989) 10: 324-332; Ristic, Z. et al. J. Plant Physiol. (1996) 149: 424-432].

[0080] Regulation of HSPs

[0081] When plants are exposed to stress, the synthesis of normal proteins is decreased while the expression of special genes are up-regulated, and, as a result, synthesis of HSPs is triggered. The transcriptional regulation of HSPs to respond to stresses is called the heat shock response (HSR) [Id., citing Shamovsky, I., Nudler, E. Cell Mol. Life Sci. (2008) 65: 855-861].

[0082] The transcription of HSPs is controlled by regulatory proteins called heat stress transcription factors (HSFs), which are located in the cytoplasm in an inactive state. HSFs are considered as transcriptional activators for heat shock [Al-Whaibi, M. J. King Saud Univ. – Science (2011) 23: 139-150, citing Clos, J. et al Cell (1990) 63: 1085-1097; Baniwal, SK et al. J. Biosci. (2004) 29: 471-487; Hu, W. et al Plant Sci. (2009) 176: 583-590]. Each factor has one carboxylic terminal (C-terminal) and three amino terminals (N-terminal) and has the amino acid leucine [Id., citing Schuetz, TJ et al. Proc. Natl Acad Sci. USA (1991) 88: 6911-15].

[0083] Plants are characterized by a large number of transcriptional factors, at least 21 [Id., citing Nover L. and Baniwal, SK. In Intl Symposium on Environmental Factors, Cellular Stress and Evolution, Varanasi, India, Oct. 13-15 (2006), p. 15]. The factors have been classified into three classes (HsfA, HsfB, and HsfC) according to the structural differences in their aggregation in triples (oligomerization domains). While each factor has its role in the regulatory network, all cooperate in regulating many functions and different stages of response to periodic heat stress (triggering, maintenance, and recovery.

[0084] The HSR is regulated by the heat stress transcription factors (HSFs) in the promoter region, which bind to cis-acting elements known as HSE (Heat Shock Elements) [al Haq, S. et al. Intl J. Molec. Sci. (2019) 20: 5321, citing Akerfelt, M. et al. Nat. Rev. Mol. Cell Biol. (2010) 11: 545-552; Pirkkala, L. et al. FAEB J. (2001) 15: 1118-1131]. HSFs are classified as three types: HSFA, HSFB, and HSFC; the functions of these classes vary from each other. Among the HSFs, HSFA, which is found in the cytosol in a monomeric state, regulates the HSPs cycle. The activity of the HSFA, under normal conditions, is regulated negatively by HSP90s, which are checked in the form of phosphoproteins [Id., citing Ali, A. et al. Mol. Cell Biol. (1998) 18: 4949-4960]. During the onset of stress, this repression is reversed and HSP90 dissociates and changes into a functional trimer state. The HSFA homo-trimer then binds to the Heat Shock Element (HSE) in the promoter region [Sou, J. et al. Cell (1998) 94: 471-480], transcription occurs and HSPs are synthesized [Id., citing Calderwood, SK et al. Sign. Transduct. Insights (2010) 2: 13-24]. Amongthe HSFAs, HSFA1 acts as the master regulator in tomatoes [Id., citing Mishra, SK et al. Genes Dev. (2002) 16: 1555-67]. HSFA2 is structurally and functionally the same as HSFA1 but is expressed only in stressed plants. Under stress situations, HSFA2 forms a super activator hetero- oligomer structure with HSFA1, which is more efficient than the individual HSFs, which not only regulate the down-stream stress related HSP genes, but also the protective enzyme genes such as GST, GR, POX and APX [Id., citing Zhuang, L. et al. Int. J. Mol. Sci. (2018) 19: 2702; Scharf, KD et al. Biochim. Biophys. Acta Gene Regul. Mech. (2012) 1819: 104-119]. Some studies also report that HSP gene expression positively regulates protective enzyme activities. In Arabidopsis, for example, overexpression of HSP17.8 enhanced superoxide dismutase (SOD) activity and, in tobacco, HSP16.9 increased the activities of ROS scavenging by ROS scavenger gene expression, e.g., peroxidase (POD), catalase (CAT) and SOD [Id., citing Driedonks, N. et al. Front. Plant Sci. (2015) 6: 999].

[0085] Post-transcriptional modification, such as alternative splicing, also regulate the HSFs. HSFA2 under heat stress, for instance, binds to its own promoter region and activates its own transcription in a positive auto-regulatory loop. Similarly, HSFA is regulated by DREB2 under stress, which in turn regulates the stress related genes in many plants [Id., citing Laloum, T. et al. Trends Plant Sci. (2018) 23: 140-150] Similarly, miRNAs also play a vital role in the stress response by down-regulation of stress-related genes. For example, some miRNAs are reported to exert positive regulation in drought, cold, salinity, hormones and nutrient starvation stresses, such as miR159, miR319, miR395, miR402 [Id., citing Zhang, B. et al. Dev. Biol. (2006) 289: 3- 16]. Conversely, in Arabidopsis short term heat stress, miR398 negatively regulates the expression of cold shock domain CSD1, CSD2 and capsanthin-capsorubin synthase (CCS), which yield SOD [Id., citing Driedonks, N. et al. Front. Plant Sci. (2015) 6: 999] Biotic Stress Tolerance

[0086] Plant growth, development, yield, and quality are affected adversely by several biotic factors such as pathogenic bacteria, fungi, viruses, and nematodes. Biotic factors directly deprive their host plants of their nutrients, which result in reduced plant vigor, growth, productivity and sometimes leads to death of the host plants. Biotic stresses are a major cause of pre- and post- harvest losses. Animals have an immune system, which helps them to adapt to biotic stresses such as new diseases and memorized the past infections. Although plants lack this adaptiveimmune system, they have evolved several sophisticated strategies to counteract these biotic stresses. These defense mechanisms are stored in the plant’s genome at the genetic level, which encode thousands of stress resistance genes. HSP response to biotic stresses depend on the nature of the causal organisms and plant genotypes, either susceptible or resistant, and the developmental stage [Id., citing Dodds, P. and Rathjen, J. Nat. Rev. Genet. (2010) 11: 539]. Abiotic Stress Tolerance

[0087] Abiotic stresses are extreme environmental conditions like extreme temperatures, water deficit, and ion imbalance due to heavy metals and salinity, which pose a serious threat to plants survival, yield and quality. High Temperature Stress

[0088] Several studies have indicated that many high molecular weight HSPs showed a response under high-temperature stress, such as HSP118, HSP114, HSP110, HSP108, HSP104, HSP103, HSP101, HSP100 and HSP97, respectively, [Id., citing Young, TE et al. Plant Physiol. (2001) 127: 777-91, Singla, SL et al. J. Biosci. (1998) 23: 337-45]. Among the HSP100 class, a significant high temperature response (HTR) was shown by HSP101 [Id., citing Lee, U. et al. Plant J. (2007) 49: 115-127] and also was involved in thermo-tolerance in Arabidopsis [Id., citing Queitsch, C. et al. (2000) 12: 479-492]. It was confirmed further in maize that HSP101 was involved in thermo-tolerance [Id., citing Nieto-Sotelo, J. et al. Plant Cell (2002) 14: 1621- 33]. Merret et al., (2017) and Mcloughlin et al., (2016) [Id., citing Merret, R. et al. Plant Physiol. (2017) 174: 1216-1225; McLoughlin, F. et al. Plant Physiol. (2016) 172: 1221-36] confirmed the role of HSP101 in thermotolerance in Arabidopsis but also established that this played a role in recovery after heat shock. Besides the role of HSP101 as a chaperone, HSP100 also was involved in development [Id., citing Pyatrikas, DV et al. Russ. J. Plant Physiol. (2014) 61: 80- 80]. Low molecular weight HSPs i.e., HSP18.1 and HSP17.9, accumulated in the pea while it was treated for four hours at 42°C. The response and expression of HSPs also were development stage and different tissue specific. In maize subjected to 40°C, HSPs (-101, -70 and -17.6) were induced. Above 36°C, fertilization was reduced, although HSPs were induced in female reproductive parts but, when studied, mature pollens were more sensitive to heat stress [Id., citing Dupuis, I., and Dumas, C. Plant Physiol. (1990) 94: 665-670]. In contrast, HSP70s were expressed more in tomato pollens [Id., citing Duck, NB and Folk, WR. Plant Mol. Biol. (1994)26]: 1031-1039]. In Arabidopsis HSP70 was expressed more in mitochondria under high temperature stress [Id., citing Sung, DY et al. Plant Physiol. (2001) 126: 789-800]. Chloroplast HSP70.1, 70.2 and mitochondrial HSP22 also were involved in seed development besides its role as a chaperone [Id., citing Su, PH and Li, H. Plant Physiol. (2008) 146: 1231-1241; Avelange- Macherel, MH. Plant Cell Environ. (2015) 38: 1299-1311].

[0089] HSP90 also showed increased expression under heat stress situations. HSP90 has been reported in rice and Arabidopsis [Id., citing Hu, W. et al. Plant Sci. (2009) 176: 583-590; Prasad, BD et al. PLoS ONE (2010) 5: e12761] and all classes of HSP90 (A, B, and C) in soybeans [Id., citing Xu, J. et al. PLoS ONE (2013) 8: e69810]. Under normal conditions, HSP90 negatively regulated HSFs and kept the regulation of all HSPs checked [Id., citing Yamada, K. et al. J. Biol. Chem. (2007) 282: 37794-37804].

[0090] HSP70s and HSP60s chaperonin families are the most studied of the chaperones under heat stress, which maintained protein proper folding using ATPs [Id., citing Hartl, FU et al. Nature (2011) 475: 324-332]. Cytosolic HSP70 was involved in heat stress tolerance in Arabidopsis [Id., citing Jungkunz, I. et al. Plant J. (2011) 66: 983-995]. HSP70s have been studied under high temperature stress in a variety of plant crops, such as witch-grass and alfalfa [Id., citing Song, G. et al. Plant Cell Resp. (2018) 37: 1485-1497; Li, Z. et al. J. Plant Res. (107) 130: 387-96]; vegetables like pepper, tomato, cabbage, potato; ornamental plants like chrysanthemum [Id., citing Guo, M. et al. Plant Si. (2016) 252: 246-256; Huang, L. et al. Protoplasma (2019) 256: 39-51; Usman, MG, et al. Cell Stress Chaperones (2018) 23: 223-234; Zhang, S. et al. Plant Cell Physiol. (2017) 59: 58-71; Lee, SS. Et al. Curr. Genom. (2010) 19: 12- 20; Liu, J. et al. Sci. Rep. (2018) 8: 16628; Zhang, Y. et al. Plant Omics (2014) 7: 229]; grain such as wheat [Id., citing Id, HW et al. J. Mol Sci. (2018) 19: 1594]; and tea [Id., citing Chen, J. et al. Int. J. Mol. Sci. (2018) 19: 2633]. Xu et al., (2010) [Id., citing Xu, C. and Huang, B. Crop Sci. (2010) 50: 2543-2552] observed the expression pattern of chloroplast HSP60, not only in normal conditions but also under high-temperature and drought situations. It was responsible for ribulose biphosphate carboxylase / oxygenase (Rubisco, a key enzyme in photosynthesis) assembly, protection and also chloroplast development. Low molecular weight HSPs such as the HSP10, HSP20 and HSP40 families were up-regulated under high-temperature stress situations in various plant crops [Id., citing Xu, Y. et al. Intl J. Prteom. (2011) 2011: 529648; Huang, L. et al. Protoplasma (2019) 256: 39-51; Kumar, N. et al. 3 Biotech (2017) 7: 205; Liao, JL et al. J.Exp. Bot. (2013) 65: 655-671; Lin, CJ et al. J. Agric. Food Chem. (2010) 58: 10545-10552; Majoul, T. et al. Proteomics (2004) 4: 505-13; Wang, X. et al. Genes Genom. (2018) 11: 1-8]. Some small HSPs were also genotype-specific and were up-regulated in resistant cultured varieties bred by humans (“cultivars”), such as foxtail millet, while some small HSPs were down-regulated in sensitive genotypes [Id., citing Singh, RK et al. Sci. Rep. (1016) 6: 32641]. Some co-chaperones were involved in thermo-tolerance as HSP40. Correlation of small HSPs with HSP100, HSP70 and HSP60 suggested their role as holders in disaggregation and protein folding [Zhang, Y. et al. Plant Omics (2014) 7: 229]. Low-Temperature Stress

[0091] Cold stress affects plant enzymes, membrane plasticity, changes physiology and metabolism, sometimes causes water starvation and desiccation, which creates a stress condition for the plant that adversely affects plant growth, development and yield. Low temperature also is associated with protein disfunction and denaturing, which induce the accumulation of HSPs [Id. citing Hashimoto, M. and Komatsu, S. Proteomics (2007) 7: 1293-1202; Hlavackova, I. et al. Int. J. Mol. Sci. (2013) 14: 8000-8024; Kosova, K. et al. J. Proteom. (2011) 74: 1301-1322]. Many HSPs responded to cold stress and were up-regulated in Arabidopsis, tobacco, maize, rapeseed, chicory, poplar, wheat and barley [Id., citing Hlavackova, I. wt al. Intl J. Mol Sci. (2013) 14: 8000-8024; Bae, MS et al. Plant J. (2003) 36: 652-63; Jin, Y. et al. Afr. J. Biotechnol. (2011) 10: 18991-19004; Kollipara, KP et a l. Plant Physiol. (2002) 129: 974-992; Reddy, RK et al. lant Sci. (1998) 131: 131-37; Degand, H. et al. Proteomics (2009) 9: 2903-2907; Ghosh, D. and Xu, J. Front. Plant Sci. (2014) 5: 6; Vitamvas, P. et al. Proteomics (2012) 12: 68-85; Ono, K. et al. Plant Sci. (2001) 160: 455-61]. Under low-temperature stress situations, HSPs were induced and translocated into various cell organelles to protect them from cold stress [Id., citing Bae, MS et al. Plant J. (2003) 36: 652-663]. Bae et al., (2003) investigated this in Arabidopsis treated with cold stress at 40°C for 6 h. HSP70s were up-regulated and their traffic from the cytoplasm to the nucleus was observed. A similar event was observed in the pea mitochondria when treated at 4°C for 36 h [Id., citing Taylor, NL et al. Mol. Cell Proteom. (2005) 4: 1122-1133]. Some HSPs accumulated tissue specifically upon low-temperature exposure, as in poplar, where HSPs were accumulated in leaves [Id., citing Renaut, J. et al. Plant Biol. (2004) 6: 81-90]. Regarding rice, low-temperature stress and a gradual decrease in the temperature from 15°C to 0°C, with an interval of 5°C, up-regulated HSP95 and HSP75, and HSP70 accumulated in the chloroplast, asthis was the part of the plant vulnerable to low temperature [Id., citing Hahn, M. and Walbot, V. Plant Physiol. (1989) 91: 930-938; cui, S. et al. Proteomics (2005) 5: 3162-3172]. Some of the HSPs, like HSP90 in wheat and HSP60 and HSP21 in sunflowers, are down-regulated to cold stress [Id., citing Vitamvas, P. et al. Proteomix (2012) 12: 68-65; Balbuena, TS et al. J. Proteome Res. (2011) 10: 2330-2346]. A similar trend also was reported by Hlavackova et al., (2013) and Rinalducci et al., (2011) [Id., citing Hlavackova, I. et al. Intl J. Mol. Sci. (2013) 14: 8000-8024; Rinalducci, S. et al. J. Proteom. (2011) 74: 643-659] where Rubisco stability was associated with down-regulation of HSP60 and HSP21 in winter wheat. Drought Stress

[0092] Drought stress, in combination with other abiotic stresses such as high light and temperature stress, negatively affects plant morphological, physiological and molecular characteristics, which leads to lowered photosynthesis, hormonal imbalance, mineral nutrient starvation and an ultimate oxidative stress [Id., citing Komatsu, S. et al. J. Proteome Res. (2011) 10: 3993-4004]. Removal of water disrupts the normal structure of the lipid bilayer plasma membrane. This results in the displacement of membrane proteins, denaturation of membrane- based enzymes and, as a result, membrane permeability, physiology and metabolism are adversely affected [Id., citing Salehi-Lisar, SY and Bakhshayeshan-Agdam, H. Drought Stress in Plants: Causes, Consequences, and Tolerance, Physiology and Biochemistry; Springer International Publishing: Cham, Switzerland; Berlin, Germany, 2016; pp. 1–16; Chaves, MM et al. Funct. Plant Biol. (2003) 30: 239-264]. Dehydration stress also affects the quantity and quality of normal plant proteins and, as a result, stress related proteins including HSPs are induced. For example, HSP70 was up-regulated in drought stress in the seedling of upland rice [Id., citing Reddy, PS et al. PLoS ONE (2014) 9: e89125]. Similarly, transgenic Arabidopsis and sugarcane also showed HSP up-regulation and demonstrated drought tolerance [Id, citing Yer, EN et al. Gene (2018) 678: 324-36; Subba, P. et al. J. Proteome Res. (2013) 12: 5025-5047]. The expression pattern of HSPs is also genotype-specific; Burke JJ et al., [Id., citin Burke, JJ et al. Plant Physiol. (1985) 78: 394-398] studied combined drought and heat stress in irrigated and non-irrigated cotton, where more HSPs accumulated in non-irrigated cotton. Maize heat tolerant and sensitive cultivars were studied under high temperature and dehydration situations, where HSP accumulation was more in drought stress conditions [Id., citing Ramanjulu, S. and Bartels, D. Plant Cell Environ. (2002) 25: 141-151]. The same was demonstrated by Benesova et al., [Id.,citing Bensevova, M. et al. PLoS One (2012) 7: e38017], where HSP70 and HSP26 were induced in drought-stressed maize. A study on chickpea HSP70 reported that HSPs were first down-regulated in the early stage of growth in drought-tolerant cultivars (meaning a plant that has been produced in cultivation by selective breeding). In contrast, HSPs were abundant in drought-sensitive cultivars, which indicated that HSPs responded to drought not only in the specific genotypes but, also, during the developmental stage. Similarly, small HSPs expressed highly in drought-tolerant cultivars as compared to those that were sensitive in chickpea [Id., citing Subba, P. et al. J. Proteome Res. (2013) 12: 5025-5047]. The same trend also was observed in poplar and Kentucky bluegrass [Id., citing Xu, C. and Huang, B. Crop Sci. (2010) 50: 2543-2552; Burke, JJ et al. Plant Physiol. (195) 78: 78: 394-398]. HSP17.7 showed drought tolerance in transgenic rice, and other HSPs also were involved in the acclimation of bryophytes to drought stress [Id., citing Agrawal, L. et al. Front. Plant Sci. (2016) 7: 1466; Ristic, Z. et al. Plant Physiol. (1991) 97: 1430-1434]. Proteomics studies revealed that nuclear and HSPs in the extracellular matrix were both up-regulated to drought stress [Id., citing Cruz de Carvalho, R. et al. Plant Cell Environ. (2014) 37: 1499-1515; Pandey, A. et al. Mol. Cell Proteom. (2008) 7: 88- 107; Pandey, A. et al. J. Proteom Res. (2010) 3443-64; Bhushan, D. et al. J. Proteome Res. (2011) 10: 2027-2046]. Salinity Stress

[0093] Studies show that many HSPs are induced and up-regulated in saline stress situations like HSP70 in rice seedlings [Id., citing Ngara, R. et al. Proteomics (2014) 14: 611-621], wheat [Sobhanian, H. et al. J. Proteo. (2011) 74: 1323-37], and poplar HSP70-9, -12 and -33 [Id., citing Manaa, A. et al. J. Exp. Bot. (2011) 62: 2797-2813]. Furthermore, HSP40 in rice [Id., citing Wang, X. et al. Genes Genom. (2018) 11: 1-8] and poplar, HSP100-21 and -75), HSP90-9 and - 12), HSP60-31, -33, -38 and -49), HSP40-113 and -117, and HSP21 were also up-regulated under salt stress [Id., citing Manaa, A. et al. J. Exp. Bot. (2011) 62: 2797-2813]. In wheat hybrid Jinan 177 and its salt-resistant hybrid, protein profiling showed HSPs and chaperones were induced highly under salt stress [Id., citing Wang, M. et al. Proteomics (2008) 8: 1470-89]. HSPs were studied in relation to programmed cell death (PCD) in a rice root at higher salt, where mitochondrial HSP70 were the up-regulated proteins that possibly were involved in PCD regulation [Id., citing Han, F. et al. Biochim. Biophys. Acta Proteins Proteom. (2009) 1794: 1625-1634]. Soybean proteomic studies showed a differential HSPs expression of HSP90,chloroplast HSP70, HSP60 and HSP20 under salt stress [Id., citing Song, H. et al. Plant Mol. Biol. Report (2009) 27: 342-349]. Different HSPs in Arabidopsis like HSP 90 [Id., citing Xu, J. et al. PLoS ONE (2013) 8: e69810; Choudhary, MK, et al. Mol Cell Proteom. (2009) 8: 1579- 98], HSP100, Clp (B1, B2), Clp (D1, D2) and small HSPs in rice [Id., citing Muthusamy, SK et al. Front. Plant Sci. (2016) 7: 929; Song, H. et al. Planta (2009) 229: 955-64] showed tolerance to high salinity stress. The role of HSPs in response to salinity stress is also genotype-specific, as recorded in soybean, where HSPs were induced more in salt resistant cultivars [Id., citing Pi, E. et al. Mol. Cell Proteom. (2016) 15: 266-288]. Light Stress

[0094] As autotrophs (meaning an organism that is able to form nutritional organic substances from simple inorganic substances such as carbon dioxide), plants require light for photosynthesis. Excess light damages the photosynthetic apparatus and plants undergo a phenomenon known as photorespiration. During this process, toxic chemicals, rather than sugars, along with ROS, are produced. These toxic chemicals in the chloroplast can damage the photosystem II permanently by excessive absorption of light [Id., citing Timperio, AM et al. J. Proteom. (2008) 71: 391-411; Kumar, M. et al. Front. Plant Sci. (2017) 7: 2023]. Rossel et al., [Id. citing Rossel, JB et al. Plant Physiol. (2002) 130: 1109-1120] reported that many HSPs were up-regulated upon high light stress (HLS) in Arabidopsis. A similar over-accumulation of nuclear HSP70 was observed in Chlamydomonas. The thylakoid proteome analysis of Arabidopsis was studied with respect to high light saturation involving isoforms of chloroplast HSP70 along with the accumulation of other osmolytes like anthocyanins and ascorbates. In the marine ecosystem where low light created a stress, HSP70, ClpB1, Sti, and HSP60 were up- regulated [Id., citing Kumar, M. et al. Front. Plant Sci. (2017) 7: 2023]. Under high light saturation, small HSP23 was seen to be involved in the post-transcriptional regulation in a Chenopodium rubrum cell suspension [Id., citing Debel, K. et al. Planta 91997] (201(3): 326- 33). Chemical Pollutant Stress

[0095] Plant productivity is restricted by chemical pollutants in the soil media, such as heavy metals. These pollutants affect plant growth either by displacement of essential cations from specific binding sites or by generation of oxidative stress by the generation of ROS [Al HaQ. S.et al. Intl J. Molec. Sci. (20120: 5321, citing Sharma, SS. And Detz, KJ. Trends Plant Sci. (2009) 14: 43-50)

[0096] HSPs can be induced by heavy metal stress. For example, HSP70s were differentially expressed and accumulated in the roots of tomatoes [Id., citing Rodriguez-Celma, J. et al. J. Proteom. (2010) 73: 1694-1706]. Similarly, the HSP70 sub-family, DnaK (Bip), was up- regulated in rice seedlings [Id., citing Ahsan, N. et al. C.R. Biol. (2007) 330: 735-468]. Arabidopsis exposure to cadmium stress induced many HSPs [Id., citing Sarry, J. et al. Proteomics (2006) 6: 2180-2198]. Similarly, increased expression was reported for HSP80 and HSP17.9 in rice [Id., citing Ahsan, N. et al. CR Biol. (2007) 330: 735-746], HSP 90s in Lotus corniculatus [Id., citing Nacascues, J. et al. New Phytol. (2012) 193: 625-636], HSP17.7 in carrots and HSP26 in soybeans [Id., citing Czmecka, E et al. Mol. Cell Biol. (1988) 8: 1113- 1133] under cadmium, lead and arsenic stresses. Using a comparative proteomic analysis of poplar under cadmium stress, a differential expression pattern of HSP was noted. Similarly, in soybeans, two-folds higher accumulation of HSP was recorded in Cd-accumulating genotypes, while there was less HSP70 expression in lower Cd-accumulating varieties, which showed that HSP expression was also genotype-specific [Id., citing Hossain, Z. et al. Amino Acids (2012) 43: 2393-2416]. When flax was cultured on heavy metal treated media, many heavy metal binding proteins, including HSP70 accumulation, were enhanced, while HSP83 showed down-regulation. HSP90.3 enhanced cadmium stress tolerance by lowering germination potential in Arabidopsis, mediating the antioxidant enzymes [Id., citing Song, HM et al. Biol. Plant (2012) 56: 197-199]. Flooding Stress

[0097] Waterlogging / flooding is also an environmentally limiting factor that hinders plant growth and development. A gradual decrease of redox potential and oxygen in the soil are the ill effects of flooding [Id., citing Hossain, Z. et al. J. Plant Physiol. (2009) 166: 1391-1404]. Studies show that HSPs are involved in plant resistance against flooding stress by up-regulation and higher gene expression, which is organ-specific. As noticed by Chen et al., [Id., citing Chen, Y. et al. Proteome Sci. (2014) 12: 33] in the soybean plasma membrane where HSP70 accumulated more than 10 fold, this occurrence occurred more in cotyledon than the roots of the soybean. Discussed in another proteomic study by the same group of researchers, HSP60 was differentially regulated in soybeans [Id., citing Komatsu, S. et al. PLoS ONE (2013) 8: e65301].In contrast, HSPs were induced in flooding stress, but were not mandatory for resistance in flooding stress and were genotype specific. As for resistant and susceptible cultivars of rice to anoxia and hypoxia conditions, HSPs were more up-regulated in the sensitive cultivars than resistant genotypes [Id., citing Komatsu, S. et al. J. Proteome Res. (2011) 10: 3993-4004]. Proteomics study of flooding stress in relation to PCD in maize revealed that HSP70s were up- regulated [Salehi-Lisar, S.Y.; Bakhshayeshan-Agdam, H. Drought Stress in Plants: Causes, Consequences, and Tolerance, Physiology and Biochemistry; Springer International Publishing: Cham, Switzerland; Berlin, Germany, 2016; pp. 1–16]. The same pattern of flooding tolerance was studied in rice protoplast where ectopic mtHSP70 expression protected H2O2 induced PCD [Id., citing Komatsu, S. et al. J. Proteome Res. (2011) 10: 3993-4004]. Similarly, in Arabidopsis anoxia tolerance was enhanced via HSFA2-mediated production of HSP70 and HSP101. Oxidative / Combined Stress

[0098] Since plants are exposed to many stresses simultaneously, such as light, this creates high temperature stress that leads to dehydration. Such situations lead to oxidative or secondary stress and plants have to adjust their signaling pathways and metabolism to ensure their growth and development [Id., citing Mittler, R. et al. Trends Biochem. Sci. (2012) 37: 118-125; Ngara, R. et al. Proteomics (2014) 14: 611-621; Kilian, J. et al. Plant J. (2007) 50: 347-363; Scarpeci, TE et al. Plant Signal Behav. (2008) 3: 856-857]. Oxidative stress generates ROS which, in high concentrations, are harmful to cellular structures. HSPs respond to multiple stress situations and enable the plants to cope with the challenging environment. For example, HSP70 expression was higher in tobacco to heat stress but was even higher to the combined stress of heat and drought [Id., citing Rizhsky, L. et al. Plant Physiol. (2002) 130: 1143-1151]. Ectopic expression of genes from soybeans in Arabidopsis GmHSP90 showed tolerance to heat, salinity and osmotic stresses, although response in salinity was not as high as to combined stresses [Id., citing Xu, J. et al. PLoS ONE (2013) 8: e69810]. A similar pattern was observed with small HSPs in rice to multiple stresses [Id., citing Zou, J. et al. J. Plant Physiol. (2012) 169: 628-635; Want, A. et al. Plant Breed (2015) 134: 384-393]. Overexpression of HSP17.6 in Arabidopsis enhanced tolerance to salinity combined with dehydration, but no response was noted to high temperature stress only [Id., citing Sun, W. et al. Plant J. 2001] 27: 407-15]. Single or combined stresses led to the production of ROS and oxidative stress which, if not checked timely, are very detrimental to plants [Id., citing Hossain, Z. et al. J. Plant Physiol. (2009) 166: 1391-1404]. Under oxidativestress, overexpression of organelle and cytosolic HSP90 enhanced tolerance in Arabidopsis. Similar results were reported by Nishizawa-Yokoi et al. [Nishizawa-Yokoi, A. et al. Plant Cell Physiol. (2010) 51: 486-496], where HSP90 regulated HSFA2, which enhanced tolerance to oxidative stress. Queitsch et al. [Id., citing Queitsch, C. et al. Plant Cell (2000) 12: 479-492] reported oxidative stress accumulated HSP100 / Clp B, ClpC2 and ClpD1 in rice. HSPs protected vital cellular parts under oxidative stress, as demonstrated by Downs, CA et al. [Downs, CA et al. J. Plant Physiol. (1999) 155: 488-496], where small HSPs protected the photosystem II from oxidative stress and photo-inhibition. Different organelle HSPs also responded to oxidative stress. mtHSP22 accumulation was enhanced in tomatoes under oxidative stress [Id., citing Banzet, N. et al. Plant J. (1998) 13: 519-577]. Small HSPs responded to oxidative stress, as HSP16.4 and HSP17 accumulated in multiple stress situations in Arabidopsis and carrots, respectively [Id., citing Jiang, C. et al. Plant Cell Environ. (2009) 32: 1046-1059; Sarry, J. et al. Proteomics (2006) 6: 2180-98]. Genetic engineering / induction studies

[0099] Al-Whaibi et al [J. King Saud Univ – Science (2011) 23: 39-50] describe a series of field tests to modulate induction of HSPs in plants. In general, the expression of HSPs and their factors Hsfs was induced largely by heat, cold, salinity and osmotic stresses; they emphasized the importance of studying stress combinations to end up with tolerant plants. The response to other stress factors depends on protein class and tissues. For example, under all types of stresses, high expression response for class HSP20 was recorded. Wounding of the roots of the plant stimulated after 12h the expression of several genes from classes HSP20; HSP70 and HSP100. High response of expression of genes for HSPs and Hsfs occurred under UV-B stress in aerial tissues (shoot), but there was no expression in non-aerial tissues [Id., citing Sindell, WR et al. BMC Genomics (2007) 8: 125].

[0100] The response of plants to heat shock results in changes in the level of enzymes, cellular membrane structure, photosynthesis activity and protein metabolism [Id., citing Singla, SL et al. In: Prasad, MNV (Ed), Plant Ecophysiology, John Wiley, New York, (1997) pp. 101-127]. It has been reported that high temperature changed the properties of membranes of the nucleus, ER, mitochondria and chloroplasts of the rice plant O. saiiva [Id., citing Pareek, A. et al. J. Biosci. (1998) 23: 361-367].

[0101] Scientists have modified plant cells to show an increase in cold stress tolerance by increasing gene expression of glycerol 3-phosphate acyltransferase from Cucurbita maxima and A. thaliana in tobacco plant cells, resulting in an increase in the degree of unsaturation of the lipids in the thylakoid membranes of the chloroplast. Therefore, increasing the degree of unsaturation of fatty acids leads to an increase in cold tolerance.

[0102] In an attempt to increase salinity tolerance of the wheat plant, one report disclosed that transgenic plants that contained a gene (CtHSR1) from the yeast Candida tropicalis were subjected to water stress, high salinity and heat stresses under operating greenhouse conditions and in the field. Stress conditions were withholding watering the plant for two weeks (water stress), watering in the presence of 400 mM NaCl (salinity stress), and subjecting the plants to 46°C for 2h followed by a 3 day period recovery at 28°C (heat stress). The results showed improvement of growth under both drought and heat stresses and lesser but still significant to salinity stress [Id., citing Blumwald, E. and Arif, A.]. miRNAs in plants: their role in abiotic stresses

[0103] A growing body of research has demonstrated that miRNAs act on target genes and are involved in various biological functions of plants. Small RNA high-throughput sequencing has been widely used to identify and functionally analyze miRNAs in plants (Zhang, F. et al. Front. Plant Sci. (2022) 13: 919243, citing Sunkar, R. et al. Plant Cell (2005) 17: 1397-1411; Fahlgren, N. et al. PLoS One (2007) 2: e219 et al.; Creighton, CJ et al., Brief Bioinform. (2009) 10: 490- 497). According to the records registered in miRBase (mirbase.org), 38,589 hairpin precursors and 48,860 mature microRNAs have been identified through experimental or computational approaches from 271 organisms, including more than 70 plants, such as Arabidopsis thaliana (326 precursors, 428 mature), Oryza sativa (604 precursors, 738 mature), Zea mays (174 precursors, 325 mature), Triticum aestivum (122 precursors, 125 mature), Glycine max (684 precursors, 756 mature), Solanum tuberosum (224 precursors, 343 mature), Nicotiana tabacum (162 precursors, 164 mature), Solanum lycopersicum (112 precursors, 147 mature), Gossypium raimondii (296 precursors, 296 mature), Medicago truncatula (672 precursors, 756 mature), Populus trichocarpa (352 precursors, 401 mature), Sorghum bicolor (205 precursors, 241 mature), Brassica napus (90 precursors, 92 mature), Vitis vinifera (163 precursors, 186 mature), and so on [Id.].

[0104] SQUAMOSA Promoter-Binding Protein-Like (SPL) genes encode plant-specific transcription factors that play important roles in plant phase transition, flower and fruit development, plant architecture, gibberellins signaling, sporogenesis, and response to copper and fungal toxins. [Chen, X. et al. J. Integr. Plant Biol. (2010) 52 (11): 946-951]. The SQUAMOSA promoter binding protein (SBP)-box proteins are plant-specific transcriptional factors in plants. [Abdulllah, M. et al. Front. Genet. (2018) 9: 64]. MYB proteins are key factors in regulatory networks controlling development, metabolism and responses to biotic and abiotic stresses. [Dubos, C. et al. Trends in PlantSci. (2010) 15 (10): P573-P581].

[0105] A preliminary statistical analysis showed that several miRNAs (miR156, miR159, miR160, miR164, and miR172) are the most studied miRNA families that showed diverse roles in diverse stresses and diverse plant species. MiR156-targeted SQUAMOSA promoter binding protein like (SPL) transcription factors (TFs) modulate plant architecture, including grain size, panicle branching, and higher grain productivity in rice [Id., citing Jiao, Y. et al. Nat. Genet. (2010) 42: 541-544; Miura, K. et al. Nat. Genet. (2010) 42: 545-549; Wang S. et al., Nat. Genet. (2012) 44: 950-954] and modulate plant architecture and tuberization in potato and temporal regulation of shoot development in Arabidopsis thaliana [Id., citing Gruber, AJ and Zavolan, M. Epigenomics (2013) 5: 671-83; Yao, Q. et al. Curr. Opin. Chem. Biol. (2019) 51: 11-17]. The miRNA156-targeted SPL / SBP (SQUAMOSA promoter binding protein)-box protein transcription factors (TFs) regulate tomato ovary and fruit development [Id., citing Ferreira e Silva, GF et al., Plant J. (2014) 78: 604-618]. The miRNA159 regulated MYB TFs in the regulation of programmed cell death in Arabidopsis [Id., citing Alonso-Peral, MM et al., Plant Physiol. (2010) 154: 757-771], floral development and stem elongation in rice [Tsuji, H. et al. Plant J. (2006) 47: 427-444], anther development and heat response in wheat [Id., citing Wang Y. et al., PLoS ONE (2012) 7: e48445], leaf and floral development in tomato [Id., citing Buxdorf, K. et al. Planta (2010) 232: 1009-1022], and targeting of isotrichodermin C-15 hydroxylase and its involvement in immune response of cotton [Id., citing Zhang, T. et al. Nat. Plants (2016) 2: 16153]. The miR160 regulates a group of repressor auxin response factors (ARFs), which are mainly involved in auxin hypersensitivity and regulation of floral organ development, seed germination, and post-germination stages in Arabidopsis thaliana [Id., citing Liu, PP> et al. Plant J. (2007) 52: 133-146; Liu, X. et al. Plant J. (2010) 62: 416-428], ovary patterning, floral organ abscission and lamina outgrowth in tomato [Id., citing Turner, M. et al.Plant Physiol. (2013) 162: 2042-2055], growth and developmental defects of rice [Id., citing Huang, J. et al. Sci. Repts (2016) 6: 29938], and inhibition of symbiotic nodule development in soybean [Id., citing Turner, M. et al. Plant Physiol. (2013) 162: 2042-55]. The miR164-directed cleavage of NACl mRNA affects lateral root development in Arabidopsis [Id., citing Guo, HS et al., Plant Cell (2005) 17: 1376-1386], maize [Id., citing Li, J. et al., BMC Plant Biol. (2012) 12: 220], drought resistance in rice [Id., citing Fang, Y. et al. J. Exp. Bot. (2014) 65: 2119-2135], and boundary specification in tomato [Id., citing Berger, Y. et al. Development (2009) 136: 823- 832] and negatively regulates the resistance of wheat to stripe rust [Id., citing Turner, M. et al. Plant Physiol. (2013) 162: 2042-2055]. miR172 suppressing AP2 genes induce flowering, spikelet determinacy, and floral organ abnormalities in rice (Id., citing Zhu, H. et al., BMC Genomics (2009) 20: 33; Lee, YS et al. Rice (2014) 7: 31), promote vegetative phase change in maize [Id., citing Lauter, N. et al. Proc. Natl Acad. Sci. USA (2005) 102: 9412-9417], regulate soybean nodulation [Id., citing Yan, Z. et al. Mol. Plant Microbe Interact. (2013) 26: 1371- 1377], and affect cleistogamous flowering in barley [Id., citing Nair, SK et al. Proc. Natl Acad. Sci. USA (2010) 107: 490-495] and graft-transmissible induction of potato tuberization [Id., citing Martin, A. et al. Development (2009) 136: 2873-2881]. The expression patterns of miR159, miR160, miR166, miR396, miR393, etc. have significant alterations in drought and salt stress, which suggests that these miRNAs play a vital role in abiotic stress alleviation in chickpea [Id., citing Jatan, R. et al., Genomics (2019) 111: 509-519; Jatan, R. et al. Environ. Exp. Bot. (2019) 157: 217-227]. Moreover, miR164a-CUC1, miR167-NRAMP1, miR393a-5p-TIR1, and miR396a-5p-GRF1 modules might be involved in the regulation of root, leaf, and flower development of Arabidopsis during Pseudomonas putida–inoculation [Id., citing Jatan, R. et al., Int. J. Mol. Sci. (2020) 21: 5468].

[0106] Plant miRNAs also are implicated in abiotic stress response mechanisms with regard to oxidative stress and effects on DNA in different plant species [Id., citing Pagano, L. et al., Environ. Exp. Bot. (2021) 184: 104369]. miRNAs play a key role in responding to unfavorable conditions, such as low temperature stress [Id., citing Aslam, M. et al. Int. J. Mol. Sci. (2020) 21: 8441], high temperature stress [Id., citing Zhang, M. et al. Intl J. Mol Sci. (2019) 20: 1754]; drought stress [Id., citing Ni, Z. et al. Biochem. Biophys. Res. Commun. (2012) 427: 330-35], salt stress [Id., citing Nguyen, DQ et al. Intl J. Mol. Sci. (2020) 21: 7879]; and heavy metal stress[Id., citing Ding, Y. et al. J. Agric. Food Chem. (2020) 68: 1958-1965] through regulating expression of related target genes in plants. Anatomy and physiology of the Skin

[0107] The skin is the largest organ in the body, consists of several layers and plays an important role in biologic homeostasis and is comprised of the epidermis and the dermis. The epidermis, which is composed of several layers beginning with the stratum corneum, is the outermost layer of the skin, and the innermost skin layer is the deep dermis. The skin has multiple functions, including thermal regulation, metabolic function (vitamin D metabolism), and immune functions. FIG. 1 presents a schematic diagram of the anatomy of the skin.

[0108] In humans, the usual thickness of the skin is from 1-2 mm, although there is considerable variation in different parts of the body. The relative proportions of the epidermis and dermis also vary, and a thick skin is found in regions where there is a thickening of either or both layers. For example, on the interscapular (between the shoulder blades) region of the back, where the dermis is particularly thick, the skin may be more than 5 mm thick, whereas on the eyelids it may be less than 0.5 mm. Generally, the skin is thicker on the dorsal or extensor surfaces of the body than on the ventral or flexor surfaces; however, this is not the case for the hands and feet. The skin of the palms and soles is thicker than on any dorsal surface except the intrascapular region. The palms and soles have a characteristically thickened epidermis, in addition to a thick dermis

[0109] The entire skin surface is traversed by numerous fine furrows, which run in definite directions and cross each other to bound small rhomboid or rectangular fields. These furrows correspond to similar ones on the surface of the dermis so that, in section, the boundary line between epidermis and dermis appears wavy. On the thick skin of the palms and soles, the fields form long, narrow ridges separated by parallel coursing furrows, and in the fingertips these ridges are arranged in the complicated loops, whorls (verticil) and spirals that give the fingerprints characteristic for each individual. These ridges are more prominent in those regions where the epidermis is thickest.

[0110] Where there is an epidermal ridge externally there is a corresponding narrower projection, called a “rete peg,” on the dermal surface. Dermal papillae on either side of each rete peg project irregularly into the epidermis. In the palms and soles, and other sensitive parts of the skin, the dermal papillae are numerous, tall and often branched, and vary in height (from 0.05mm to 0.2 mm). Where mechanical demands are slight and the epidermis is thinner, such as on the abdomen and face, the papillae are low and fewer in number. Epidermis

[0111] The epidermis provides the body’s buffer zone against the environment. It provides protection from trauma, excludes toxins and microbial organisms, and provides a semi- permeable membrane, keeping vital body fluids within the protective envelope. Traditionally, the epidermis has been divided into several layers, of which two represent the most significant ones physiologically [See FIG. 2]. The basal-cell layer, or germinative layer, is of importance because it is the primary source of regenerative cells. In the process of wound healing, this is the area that undergoes mitosis in most instances. The upper epidermis, including stratum and granular layer, is the other area of formation of the normal epidermal-barrier function. Stratum Corneum and the Acid Mantle

[0112] Stratum corneum is an avascular, multilayer structure that functions as a barrier to the environment and prevents transepidermal water loss. Recent studies have shown that enzymatic activity is involved in the formation of an acid mantle in the stratum corneum. Together, the acid mantle and stratum corneum make the skin less permeable to water and other polar compounds and indirectly protect the skin from invasion by microorganisms. Normal surface skin pH is between 4 and 6.5 in healthy people; it varies according to area of skin on the body. This low pH forms an acid mantle that enhances the skin barrier function. Other Layers of the Epidermis

[0113] Other layers of the epidermis below the stratum corneum include the stratum lucidum, stratum granulosum, stratum germinativum, and stratum basale. Each contains living cells with specialized functions (FIG. 2). For example, melanin, which is produced by melanocytes in the epidermis, is responsible for the color of the skin. Langerhans cells are involved in immune processing. Dermal Appendages

[0114] Dermal appendages, which include hair follicles, sebaceous and sweat glands, fingernails, and toenails, originate in the epidermis and protrude into the dermis hair follicles and sebaceous and sweat glands contribute epithelial cells for rapid re-epithelialization of woundsthat do not penetrate through the dermis (termed partial-thickness wounds). The sebaceous glands are responsible for secretions that lubricate the skin, keeping it soft and flexible. They are most numerous in the face and sparse in the palm of the hands and soles of the feet. Sweat gland secretions control skin pH to prevent dermal infections. The sweat glands, dermal blood vessels, and small muscles in the skin (responsible for goose pimples) control temperature on the surface of the body. Nerve endings in the skin include receptors for pain, touch, heat, and cold. Loss of these nerve endings increases the risk for skin breakdown by decreasing the tolerance of the tissue to external forces.

[0115] The basement membrane both separates and connects the epidermis and dermis. When epidermal cells in the basement membrane divide, one cell remains, and the other migrates through the granular layer to the surface stratum corneum. At the surface, the cell dies and forms keratin. Dry keratin on the surface is called scale. Hyperkeratosis (thickened layers of keratin) is found often on the heels and indicates loss of sebaceous gland and sweat gland functions if the patient is diabetic. The basement membrane atrophies with aging; separation between the basement membrane and dermis is one cause for skin tears in the elderly. Dermis

[0116] The dermis, or the true skin, is a vascular structure that supports and nourishes the epidermis. In addition, there are sensory nerve endings in the dermis that transmit signals regarding pain, pressure, heat, and cold. The dermis is divided into two layers: the superficial dermis and the deep dermis.

[0117] The superficial dermis consists of extracellular matrix (collagen, elastin, and ground substances) and contains blood vessels, lymphatics, epithelial cells, connective tissue, muscle, fat, and nerve tissue. The vascular supply of the dermis is responsible for nourishing the epidermis and regulating body temperature. Fibroblasts are responsible for producing the collagen and elastin components of the skin that give it turgor. Fibronectin and hyaluronic acid are secreted by the fibroblasts. The structural integrity of the dermis plays a role in the normal function and youthful appearance of the skin.

[0118] The deep dermis is located over the subcutaneous fat; it contains larger networks of blood vessels and collagen fibers to provide tensile strength. It also consists of fibroelastic connective tissue, which is yellow and composed mainly of collagen. Fibroblasts are also presentin this tissue layer. The well-vascularized dermis withstands pressure for longer periods of time than subcutaneous tissue or muscle. The collagen in the skin gives the skin its toughness. Dermal wounds, e.g., cracks or pustules, involve the epidermis, basal membrane, and dermis. Typically, dermal injuries heal rapidly. Hair

[0119] Hair is a filamentous outgrowth of protein found only on mammals. The hair of non- human mammal species is commonly referred to as fur. In some species, hair is absent at certain stages of life.

[0120] Hair grows from hair follicles deep in the dermis and projects from the epidermis of the skin.

[0121] Human skin has two types of hair: vellus hair and terminal hair. Much of human hair is short, under-pigmented vellus hair rather than terminal hair. The most noticeable part of human hair is the hair on the head, which can grow longer than on most mammals and is denser than most hair found elsewhere on the body. The term “scalp” refers to the integument of the upper part of the head, usually including the associated subcutaneous structures. The scalp is the anatomical area bordered by the face anteriorly and the neck to the sides and posteriorly. A healthy scalp is characterized by clean, hydrated skin and good blood circulation, balanced oil production, and the absence of inflammation, itching and flaking.

[0122] Vellus hair is short, fine, “peach fuzz” body hair. It is a very soft, generally pale, and short hair that grows in most places on the human body in both sexes. It is usually less than two cm long and the follicles are not connected to sebaceous glands. It is most easily observed in women and children, as they have less terminal hair to obscure it. It is also found in pre- adolescents and in male pattern baldness.

[0123] Terminal hair is developed hair, which is generally longer, coarser, thicker and darker than the shorter and finer vellus hair. Phases of growth in terminal hair are more apparent than in vellus hair; it generally has a longer anagen phase. It has associated sebaceous glands, whereas a vellus hair may not. Under certain conditions, such as puberty, some vellus hair may become androgenic hair. Under other conditions, such as male pattern baldness, it may revert to a vellus- like state.

[0124] Each hair comprises two structures: the follicle in the skin and the shaft we see. The follicle contains several layers. At the base of the follicle is a projection called a papilla, which contains capillaries, or tiny blood vessels, that feed the cells. The dermal papilla consists of an egg-shaped accumulation of mesenchymal stem cells (MSCs) surrounded by ground substance that is rich in acid mucopolysaccharides (AMPs). The living part of the hair, the area surrounding the papilla called the bulb, is the only part fed by the capillaries. The bulb encompasses the dermal papilla and the hair matrix. The hair matrix surrounds the top and sides of the dermal papilla. It is the actively growing portion of the follicle, which consists of a collection of epidermal cells that rapidly divide, move upward, and give rise to the hair shaft and the internal root sheath. The cells in the bulb divide every 23 to 72 hours, faster than any other cells in the body.

[0125] The hair follicle contains stem cells from different developmental origins, such as epithelial stem cells, melanocyte stem cells, and mesenchymal stem cells (MSCs) [ Wang, B. et al. World J. Stem Cells (2020) 12(6): 462-470, citing Kiani, MT et al. ACS Biomater. Sci. Eng. (2018) 4: 1193-1207]. These stem cells continuously self-renew, differentiate, regulate hair follicle development, and contribute to hair follicle cycles which consist of the growth phase (anagen), regression phase (catagen), and rest phase (telogen) throughout adult life [Id., citing Agabalyan, NA et al. Exp. Dermatol. (2017) 26: 505-509]. During catagen and telogen, follicles prepare their stem cells for the next anagen. During anagen, bulge stem cells are activated by induction signals from the dermal papilla and migrate downward to the bulb region, where they proliferate and differentiate to regenerate the inner and outer root sheath, matrix, and hair shaft. Human hair follicle-derived MSCs are dermal papilla or sheath cells from human hair follicles that express the MSC immunophenotype and possess multi-lineage differentiation potential [Id., citing Liu, JY et al. Tissue Eng. Part A (2010) 16: 2553-2564].

[0126] The follicle is surrounded by two sheaths—an inner and outer sheath. These sheaths protect and mold the growing hair shaft. The inner sheath follows the hair shaft and ends below the opening of a sebaceous (oil) gland, which produces sebum, a natural conditioner and sometimes an apocrine (scent) gland. The outer sheath continues all the way up to the gland. An erector pili muscle attaches below the gland to a fibrous layer around the outer sheath. When this muscle contracts, it causes the hair to stand up.

[0127] The primary component of the hair fiber is keratin. Keratins are proteins, long chains (polymers) of amino acids. The hair shaft contains three layers of keratin. The inner layer, which is called the medulla, may not be present. The next layer is the cortex, which makes up the majority of the hair shaft. The outer layer is the cuticle, which is formed by tightly packed scales in an overlapping structure similar to roof shingles. Most hair conditioning products attempt to affect the cuticle. Pigment cells are distributed throughout the cortex and medulla giving the hair its characteristic color. Eyelashes and Eyebrows

[0128] The term “eyebrow” refers to an area of coarse skin hairs above the eye that follows the shape of the brow ridges. The main function of the eyebrow is to prevent moisture, mostly salty sweat and rain, from flowing into the eye, an organ critical to sight. The typical curved shape of the eyebrow (with a slant on the side) and the direction in which eyebrow hairs are pointed, make sure that moisture has a tendency to flow sideways around the eyes, along the side of the head and along the nose. Eyebrows also prevent debris such as dandruff and other small objects from falling into the eyes, as well as providing a more sensitive sense for detecting objects being near the eye, like small insects. Eyebrows also have an important facilitative function in communication, strengthening facial expressions such as surprise, confusion, or anger.

[0129] The terms “eyelash” and “lash” are used interchangeably to refer to one of the hairs that grow at the edge of the eyelid. The eyelashes consist of curved sensory hairs originating from the eyelid margins. Compared to scalp skin, the skin of the eyelids contains a thinner epidermis and no hypodermis [Nguyen, B. et al. Am. J. Clin. Dermatol. (2023) 24: 55-67, citing Thibaut, S. et al. Br. J. Dermatol. (2010) 162 (2): 304-310]. Eyelashes are rooted approximately 2 mm deep into the dermis and lack the arrector pili muscles associated with most other hair follicles [Id., citing Thibaut, S. et al. Br. J. Dermatol. (2010) 162 (2): 304-310; Aumond, S. and Bitton, E. J. Optom. (2018) 11(4): 211-22]. Humans typically have about 90-160 eyelashes on the lower lids, spread across 5-6 rows, and 75-80 eyelashes on the lower lids, dispersed between 3-4 rows [Id., citing Aumond, S. and Bitton, E. J. Optom. (2018) 11(4): 211-222]. Eyelashes protect the eye from debris and provide a warning that an object (such as an insect or dust mite) is near the eye (which then is closed reflexively).

[0130] The inside of the nose contains small hairs called cilia. These cilia and nasal mucus clean the air drawn into the nose of the microscopic particles we inhale, including dust, pollen, and pollutants, for ultimate passage to the lungs. Hair Growth

[0131] There are three stages of hair growth: catagen, telogen, and anagen.

[0132] Anagen is the active growth phase of the hair during which the cells in the root of the hair are dividing rapidly. Anagen hairs are anchored deeply into the subcutaneous fat and cannot be pulled out easily. When a new hair is formed, it pushes the club hair up the follicle and eventually out. During this phase, the hair grows about 1 cm every 28 days. Scalp hair stays in this active phase of growth for 2-6 years. Human subjects that have difficulty growing their hair beyond a certain length have a short active phase of growth. Human subjects that have very long hair have a long active phase of growth. The hair on the arms, legs, eyelashes, and eyebrows have a very short active growth phase of about 30-45 days, which is why they are so much shorter than scalp hair.

[0133] The anagen phase is followed by a catagen phase. The catagen phase is a transitional stage that lasts for about 2-3 weeks. About 3% of all hairs are in this phase at any time. During this time growth stops and the outer root sheath shrinks and attaches to the root of the hair. This is the formation of what is known as a club hair.

[0134] After catagen, the hair goes into a telogen phase. Telogen is the resting phase, which accounts for 10-15% of all hairs. It lasts for about 100 days for hairs on the scalp and much longer for hairs on the eyebrow, eyelash, arm and leg. During this phase, the hair follicle is completely at rest and the club hair is completely formed. As compared with anagen hair, telogen hair is located higher in the skin and can be pulled out relatively easily. Pulling out a hair in this phase will reveal a solid, hard, dry, white material at the root. Normally, about 25-100 telogen hairs are shed each day.

[0135] In the normal scalp, approximately 80 to 90 percent of follicles are growing (anagen), about 5 to 10 percent are resting (telogen), and 1 to 3 percent are undergoing involution (catagen) Each day up to 75 hairs in telogen are shed from the scalp and about the same number of follicles enter anagen.Hair Loss

[0136] The term “alopecia” is a medical term for the absence or loss of hair including eyelashes, eyebrows, and scalp hair, as a result of illness, functional disorder, or hereditary disposition. For example, the term “Alopecia adnata” refers to underdevelopment of the eyelashes. Alopecia frequently occurs in patients undergoing treatment for cancer or suffering from other diseases, such as AIDS, where cytotoxic drugs are used.

[0137] Hair loss typically is categorized as scarring or nonscarring. Scarring alopecia, also known as “cicatricial alopecia”, refers to a collection of hair loss disorders that may be diagnosed in up to 3% of hair loss patients. It occurs worldwide in otherwise healthy men and women of all ages. While there are many forms of scarring alopecia, the common theme is a potentially permanent and irreversible destruction of hair follicles and their replacement with scar tissue. Examples include bullous diseases, chemical alopecia, discoid lupus erythematosus, folliculitis (severe), lichen planopilaris, dissecting cellulitis, and tumors.

[0138] The term “nonscarring alopecia” refers to hair loss without permanent destruction of the hair follicle. Examples include anagen effluvium, androgenetic alopecia, chemical alopecia, folliculitis (mild), inherited disorders of the hair shaft, telogen effluvium, alopecia areata, and traumatic alopecia.

[0139] The term “anagen effluvium” refers to the hair loss associated with chemotherapeutic agents that cause immediate destruction and release of anagen hair.

[0140] The term “androgenic alopecia” refers to a gradual decrease of scalp hair density in adults with transformation of terminal to vellus hairs, which become lost as a result of familial increased susceptibility of hair follicles to androgen secretion following puberty. The most common form of androgenic alopecia is male pattern baldness. The most common form of androgenic alopecia in women is female pattern alopecia, a diffuse partial hair loss in the centroparietal area of the scalp, with preservation of the frontal and temporal hairlines. When it occurs in females, it is associated with other evidence of excessive androgen activity, such as hirsuitism.

[0141] The term “telogen effluvium” refers to a condition resulting from an abrupt shift of large numbers of anagen hairs to telogen hairs on the scalp, with a corresponding change in the ratio ofanagen hair to telogen hair from the normal ratio of 90:10 to 70:30. This form of alopecia generally begins approximately 3 months after a major illness or other stress (e.g., surgery, parturition, rapid weight loss, nutritional deficiency, high fever, or hemorrhage) or hormonal derangement; it also has been reported after the initiation of treatment with certain medications.

[0142] The term “alopecia areata” refers to a common condition of undetermined etiology characterized by circumscribed, nonscarring, usually asymmetrical areas of baldness on the scalp, eyebrows, and beaded portion of the face. Hairy skin anywhere on the body may be affected. It is thought to be an autoimmune disease occurring on areas of the body (most commonly the scalp) where the person's immune system attacks hair follicles, thereby suppressing and arresting hair growth.

[0143] Those suffering from hair loss often experience embarrassment and the fear being ridiculed by others because they look different. Some may take to wearing oversized eyeglasses in an attempt to hide the absence of eyelashes and / or eyebrows. Loss of nasal cilia may render some more susceptible to respiratory illnesses.

[0144] Therapies for hair loss are designed primarily for scalp applications. These include topical minoxidil (Rogain®), antiandrogen agents, including the androgen-receptor blockers sprionolactone, cyproterone acetate, and flutamide, and the 5α-reductase inhibitor finasteride (Propecia®, Merck & Co.®). Latanoprost, bimatoprost, and travoprost ophthalmic solution are prostaglandin F2 alpha analogues, marketed for the treatment of ocular hypertension that have the common side effect of eyelash hypertrichosis and trichomegaly. Bimatoprost (0.03%) is the only one approved for hypotrichosis of the eyelashes. However, topical prostaglandins may cause unwanted side effects, such as darkening of the iris color, periocular pigmentation; uveitis, deepening of the superior sulcus and fat atrophy; enhancement of the eyelid crease; and a decrease in proptosis. [Starace, M. et al. Dermatol. Ther. (2023) 13: 1243-1253]. Heat Shock Proteins in Cutaneous Biology

[0145] Heat shock proteins are of fundamental importance in cutaneous biology, from protection against UV-induced damage to wound healing and repair. They play important regulatory roles in the control of apoptosis, regulation of steroid aporeceptors, kinases, and other protein remodeling events. They are also implicated in the control of cell growth, and as such, arepotential targets for cancer diagnosis and treatment. Currently, emphasis is being placed on the potential use of these proteins in the prevention and treatment of disease.

[0146] Cells are repeatedly exposed to environmental or endogenous stresses that can alter normal cell behavior and increase cell vulnerability. In order to ensure tissue integrity and function, cells cope with cellular injuries by adapting their metabolism, protecting essential intracellular constituents, inhibiting cell death signaling pathways and activating those devoted to damage repair.

[0147] The molecular chaperones of the heat-shock protein (HSP) family are critical effectors of this adaptive response. They protect intracellular proteins from misfolding or aggregation, inhibit cell death signaling cascades and preserve the intracellular signaling pathways that are essential for cell survival. Most HSPs are rapidly overexpressed in response to cellular injuries including genotoxic stress. DNA damage can dramatically alter cell behavior and contribute to a number of diseases including developmental defects, neurodegenerative disorders, and cancer. Thus, the ability of cells to repair DNA damage is essential for preserving cell integrity. DNA damage activates a coordinated response that includes detecting DNA lesions before their transmission to daughter cells, blocking cell cycle progression and DNA replication and repairing the damage. Although the role of HSPs in proteins homeostasis and cell death, especially apoptosis, has been widely reported, much less is known about their function in DNA repair.

[0148] Hsp72 and hsp27 are among the best investigated stress proteins in skin biology. The hsp70 family consists of two major cytoplasmatic members, namely hsp72 (hsp70) that shows strong stress inducibility and hsp73 (hsc70) that is constitutively expressed in all investigated cells and tissues. Proteins of the hsp70 family have an 18-kD peptide-binding domain at the carboxy-terminal end and a 45-kD ATP-binding domain at the amino-terminal end [Jonak, C. et al. Intl J. Cosmetic Sci. (2006) 28: 233-242, citing Osipiuk, J. et al. Acta Crystallogr. D. Biol. Crystallogr. (1999) 55: 1105-1107; Wu, B. et al. Mol. Cell Biol. (1985) 5: 330-341.

[0149] Hsp27 is a member of the ‘small heat shock family’. It can be found in various cells and tissues without prior stress stimulation: breast, uterus, cervix, placenta, platelets, epidermis and adnexal structures [Id., citing Cocca, DR et al. J. Natl Cancer Instit. (1993) 85: 1558-70]. Hsp27 is expressed in different tumor tissues and cell lines. As an intracellular protein, it can be found perinuclearly and is translocated to the nucleus after stimulation. Hsp27 and hsp25 (the murineanalogue of hsp27) provide their chaperone function as large oligomer complexes [Id., citing Jakob, U. et al. J. Biol. Chem. (1993) 268: 1517-1520; Rogalla, T. et al. J. Biol. Chem. (1999) 274: 18947-18956]. Phosphorylation of hsp27 leads to the formation of tetramers resulting in a decrease of their protective functions. Since tumor necrosis factor alpha and interleukin 1 can mediate hsp27 activities, it is speculated that this stress protein is involved in the pathogenesis of inflammatory skin diseases [Id., citing Kaur, P et al. FEBS Letters (1989) 227: 175-178; Arrigo, AP. Mol. Cell Biol. (1990) 10: 1276-1280; Guesdon, F. et al. J. Biol. Chem. (1993) 268: 4236- 4243]. It has been proven experimentally that hsp27 expression in situ and in vitro correlates with human keratinocyte differentiation [Id., citing Kindas-Muggee, I. and Trautinger, F. Cell Growth Differ. (1994) 5: 777-781; Trautinger, F. et al. Brit. J. Dermatol. (1995) 133: 194-202]. Epidermal HSPs

[0150] Like in all other investigated tissues, the expression of HSPs in human skin is stimulated under stress conditions. In contrast to other cells, keratinocytes show significant basal Hsp72 expression without prior stress [Id., citing Trautinger, F. et al. J. Invest. Dermatol. (1993) 101: 334-338]. Hsp72 is expressed throughout all layers of the epidermis including adnexal structures (hair follicle and sweat gland). Melanocytes, fibroblasts and other (epi)dermal cells are negative for Hsp72 in immunohistochemical staining. Heat shock ex vivo and in vitro results in ‘superinduction’ of Hsp72 in keratinocytes and de novo expression in dermal cells. The expression of Hsp27 correlates with keratinocyte differentiation and increases continuously from the basal layer to the stratum granulosum [Id., citing Kindas-Mugge, I. and Trautinger, F. Cell Growth Differ. (1994) 5: 777-81; Trautinger, F. et al. Brit. J. Dermatol. (1995) 133: 194-202]. Hsp27 is not detectable immunohistochemically in the stratum corneum. It has been suggested that this finding could be explained either by degradation of hsp27 in corneocytes or by its incorporation into the cornified cell envelope matrix making the antigen inaccessible for antibody staining.

[0151] In basal cell carcinomas and squamous cell carcinomas, Hsp27 is absent or sparsely expressed, despite in well-differentiated areas of the tumors [Id., citing Trautinger, F. et al. Brit. J. Dermatol. (1995) 133: 194-202]. Hsp27 overexpression in a transfected squamous carcinoma cell line (A431) results in a delay of tumor development and reduction of tumorigenicity after injection into nude mice [Id., citing Kindas-Mugge, L. et al. Cell Growth Differ. (1996) 7: 1167-1174]. Although Hsp27 has also been described to have a role in thermotolerance, this findings support the hypothesis that in the epidermis Hsp27 is involved mainly in the regulation of cell growth, differentiation and tumorigenicity [Id., citing Welsh, MJ and Gaestel, M. Ann. N.Y. Acad. Sci. (1998) 851: 28-35]. Heat Shock Proteins and UV-Induced Cell Death

[0152] Heat-induced inhibition of UVB-induced cell death has been described for in vitro and in situ settings in murine and human skin [Id., citing Mattin, EV., J. Biol. Chem. (1992) 267: 23189-23196; Maytin EV et al. (1993) Cancer Res.(1993) 53: 4952-4959; Maytin, EV et al. J. Invest. Dermatol. (1994) 103: 547-553; Kane, KS, and Maytin, EV. J. Invest. Dermatol. (1995) 104: 62-67; Trautinger, F. et al. J. Invest. Dermatol. (1995) 105: 160-162]. In cell culture using several different assay systems to assess cell viability and function, it has been demonstrated that hyperthermia can reduce damage from UVB. Mild heat conditions have been used and maximal effects were observed when the recovery period between heat and UVB was 6 h. The protective effect disappeared 12 h after heat treatment [Id., citing Trautinger, F. et al. J. Invst. Dermatol. (1995) 105: 160-162]. Investigators have addressed the question of whether hsps are responsible for this effect. Inhibition of protein and mRNA synthesis as well as specific inhibition of Hsp72 block the development of heat-induced UVB tolerance [Id., citing Trautinger, F. et al. J. Invest. Dermatol. (1995) 105: 1600-1662; Simon, MM et al. J. Clin. Invest. (1995) 95: 926-933]. Formation of sunburn cells (SBC), resembling UV-induced apoptotic epidermal keratinocytes, was used as an endpoint in a mouse study [Id., citing Kane, KS and Maytin, EV. J. Invst. Dermatol. (1995) 104: 62-67]. The number of SBC in heat-treated skin (41°C, 3h) was significantly reduced compared with a sham control. These results were later also confirmed in human skin [Id., citing Trautinger, F. et al. J. Invest. Dermatol. (1996) 107: 442-443]. Hsp72 is among the gene products responsible for this effect. Indeed, constitutive expression of Hsp72 is an inherent protective mechanism in epidermal cells, and overexpression of Hsp72 is at least in part involved in heat-induced UVB resistance.

[0153] Since UV radiation is one of the most abundant and potentially harmful environmental factors for human skin Roh et al. [Ann. Dermatol. (Seoul) 20(4): 184-189] asked whether sun light exposure induces HSP. The expression of HSP was examined in human epidermal cell lines (normal human keratinocyte (NHK), epidermoid carcinoma A431cells, normal humanmelanocytes, and SK30 malignant melanoma cells), in human dermal fibroblasts (HDF), which are the main cellular components of the dermis at baseline, and after heat treatment, UVA irradiation, and UVA + UVB irradiation. HSP70 was detected by immunoblotting using a monoclonal mouse anti-human HSP70 antibody (Santa Cruz Biotechnology, Santa Cruz, CA) detected by peroxidase-conjugated goat anti-mouse immunoglobulins (Tagoimmunologicals®, Camarillo CA). Each of the examined cell types constitutively expressed high amounts of HSP70 without stress conditions. In summary, epidermal cells (NHK, A431 cells, NHMs and SK30 cells) showed high baseline HSP70 expression and no additional upregulation after UV irradiation, whereas HDF showed very low baseline HSP70 expression and high up-regulation of expression after UV irradiation. Role of Transient Receptor Potential (TRP) Channels in Skin

[0154] TRP channels in skin are crucial for achieving temperature sensitivity to maintain internal temperature balance and thermal homeostasis, as well as to protect skin cells from environmental stresses such as infrared (IR) or near-infrared (NIR) radiation via Hsp production. [Hsu, W-L and Yoshioka, T. Biophysics (2015) 11: 25-32].

[0155] Many kinds of thermosensitive TRP channels are activated by heat, potentially inducing hsps to respond to a wide variety of physiological and environmental insults. Mammalian TRP channels consist of six-transmembrane cation-permeable channels that are classified into six subfamilies based on amino acid sequence homology: TRPC, TRPV, TRPM, TRPP, TRPA, and TRPML [Id., citing Venkatachalam, K. and Montell, C. Ann. Rev. Biochem. (2007) 76: 387- 417]. The functional properties of these six subfamilies of TRP channels are listed and exhibited in Zheng, J. Compr. Physiol. (2013) 3(1): 221-242. Four heat-activated channels, TRPV1-4, and two cold-activated channels, TRPM8 and TRPA1, belong to temperature-sensitive transient receptor potential channels, and are expressed in dorsal root ganglion sensory neurons, skin, and other cells [Id., citing Clapham, DE. Nature (2003) 426: 517-524]. Temperature sensitivity is crucial to protect against skin damage as well as to maintain internal temperature balance or thermal homeostasis [Id., citing Toth, BI, et al. Br. J. Pharmacol. (2014) 171: 2568-2581]. Thermo TRP channels, TRPV1,3,4, TRPM8, and TRPA1, are known to be expressed in human keratinocytes (KCs); two of them, TRPV1 and TRPA1, are expressed in human skin fibroblasts (HDFs) [Id., citing Venkatachalam, K. and Montell, C. Ann. Rev. Biochem. (2007) 76: 387-417;Toth, BI et al. Br. J. Pharmacol. (2014) 171: 2568-2581], which are major regulators of skin cell proliferation and differentiation, in the skin barrier, and in immune functions responding to skin injury [Id., citing Toth, BI et al. Br. J. Pharmacol. (2015) 171: 2568-2581].

[0156] Although the TRPC family does not belong to thermosensitive TRP channels, it controls calcium entry under epidermal receptor stimulation, which is an important part of a functional system for maintaining skin homeostasis [I., citing Beck, B. et al. J. Invest. Dermatol. (2006) 126: 1982-1993]. TRPV Subfamily

[0157] Among the six subfamilies of TRPV channels, TRPV1, TRPV3, and TRPV4 are known to be found in skin cells, especially in keratinocytes (KCs). TRPV1-type channels are activated by heat, low pH, and pro-inflammation [Id., citing Venkatachalam, K. and Montell C. Ann. Rev. Biochem. (2007) 76: 387-417]. It was also reported that TRPV1-type channels are sensitive to some physical stresses such as high (>42°C) temperature, membrane stretching, and several chemicals (ethanol, lidocaine, mono-acylglycerols, and 2-Aminoethoxydiphenyl borate, 2APB) [Id., citing Vay, L. et al. Br. J. Pharmacol. (2012) 165: 787-801]. Ion selectivity, which is expressed as PCa / PNa, is approximately 10 for chemical stimulation and 4 for physical stimulation [Id., citing Venkatachalam, K. and Montell C. Ann. Rev. Biochem. (2007) 76: 387- 417]. TRPV1 is also expressed in dermal fibroblasts [Id., citing Lan, CC et al. J. Dermatol. Sci. (2013) 72: 290-295]. The TRPV1 channel in skin was found to be essential for the control of skin growth as well as for barrier functions, cutaneous immunological functions, skin pathology and several cutaneous diseases [Id., citing Toth, BI et al. Br. J. Pharmacol. (2014) 171: 2568- 2581], and skin aging [Id., citing Lan, CC. et al. J. Dermatol. Sci. (2013) 72: 290-295]. TRPV1 can be activated directly by OAG, a membrane-permeable diacylglycerol (DAG) analog, although the 1-oleoyl-2-acetyl-sn-glycerol (OAG)-induced calcium response is one-fifth of the capsaicin-induced signal [Id., citing Woo, DH et al. Mol. Pain (2008) 4: 42]. The activation of TRPV1 by membrane-permeable OAG does not mean it is activated by G protein-coupled receptor (GPCR), because DAG produced by the hydrolysis of phosphatidylinositol 4,5- bisphosphate (PIP2) by phospholipase c (PLC) β is phosphorylated to phosphatidic acid (PA) by DAG kinase immediately [Id., citing Sandal, M. et al. Curr. Protein Pept. Sci. (2013) 14: 650- 657]. PIP2 appears to bind to the TRPV1 channel directly, causing channel inhibition that isrelieved by PLC β–catalyzed PIP2 hydrolysis. The C-terminal of TRPV1 transcription disrupts the PIP2 binding site and impairs thermal responsiveness. Moreover, TRPV3 is sensitive to warm temperature (33°C–39°C), and PCa / PNa is around 3. Heat-activated TRPV3 current displays strong outward rectification, striking thermal hysteresis, and sensitization following repeated activation [Id., citing Ramsey, IS et al. Ann. Rev. Physiol. (2006) 68: 619-647]. The TRPV3 channel’s roles in the skin include nociception, skin integrity, wound healing, hair growth, and sebocyte functions. Similar to the TRPV3 channel, the TRPV4 channel covers a temperature range (27°C–34°C), and the ratio of PCa / PNa is close to 6. Unlike TRPV1–TRPV3, TRPV4 is apparently insensitive to activation by 2APB [Id., citing Chung, MK et al. J. Neurosci. (2004) 24: 5177-5182; Mergler, S. et al. J. Cell Physiol. (2011) 226: 1828-2842]. The TRPV4 channel is involved in mechano-sensation, osmo-sensation, nociception, modulation of cell migration, the shear stress sensor, and control adherence junction in the skin cochlea [Id., citing Nilius, B. and Owsianik, G. Genome Biol. (2011) 12: 218]. TRPA1

[0158] The physiological functions of TRPA1 are summarized as follows: thermo-sensation, the most versatile chemo-sensor, mechanical sensation, nociception and, olfactory transduction [Id., citing Nilius, B. and Owsianik, G. Genome Biol. (2011) 12: 218].

[0159] TRPA1, well known as a noxious cold pain sensor, is also expressed highly in KCs [Id., citing Donnerer, J. et al. Pharmacology (2012) 89: 7-12] and in HDFs [Id., citing Nilius, B. and Owsianik, G. Genome Biol. (2011) 12: 218]. The role of this channel may be essentially different from those of other TRPV channels because it is directly activated by intracellular Ca2+ through binding to the N-terminal EF-hand domain of TRPA1 [Id., citing Zayats, V. et al. J. Mol. Model. (2013) 19: 4689-4700]. The mechanism underlying this action was suggested to be activation and / or sensitization of TRPA1 channels by GPCR coupled to PLC β signaling, such as bradykinin receptors [Id., citing Wilson, SR et al. Nat. Neurosci. (2011) 14: 595-602]. In addition, since TRPA1 is involved in inflammatory pain (increased sensitivity to painful stimuli), TRPA1 may underlie some components of inflammatory hyperalgesia (increased sensory neurons for cold hyperalgesia). Thus, TRPA1 is most likely a chemical sensor for injury and inflammation [Id., citing Obata, K. et al. J. Clin. Invest. (2005) 115: 2393-2401]. Many TRPA1 ligands, such as mustard oil, acrolein, formalin, and iodoacetamide, can activate TRPA1extracellularly, while acetaldehyde, H2O2, 15d-prostaglandin J2, and prostaglandin A2 (PGA2), all of which are called reactive electrophilic species (RES), work intracellularly [Id., citing Bang, S. and Hwang, SW. J. Gen. Physiol. (2009) 133: 257-62]. These RESs are generated under oxidative stress with the chemical reactivity being transferred from ROS to RES [Id., citing Bang, S. and Hwang, SW. J. Gen. Physiol. (2009) 133: 257-262]. It is likely that these ROS promote disulfide formation between vicinal thiol residues, resulting in TRPA1 dysfunction [Id., citing Bang, S. and Hwang, SW. J. Gen. Physiol. (2009) 133: 257-262]. However, TRPA1 activation is recovered by the application of dithiothreitol (DTT), which reduces the disulfide bond to the –SH group of cysteine in TRPA1 [Bang, D. et al. Mol. Biosyst. (2009) 5: 750-756].

[0160] The sensitivity of TRPA1 to intracellular Ca2+and ROS accelerates cell depolarization, which will activate voltage-dependent Ca2+channels, leading to skin aging, carcinogenesis, and apoptosis [Id., citing Nagata, K. et al. J. Neurosi. (2005) 25: 4052-4061]. Thus, TRPA1 may help regulate the proliferation and differentiation of KCs and skin inflammation [Id., citing Andrade, EL et al. Pharmacol. Ther. (2012) 133: 189-204]. Some functional properties of TRPA1 depend on its ability to interact with TRPV1. The coexistence of TRPA1 with TRPV1 seems to be important for the action of an endogenous mediator, such as bradykinin or calcium [Id., citing Bessac, BF and Jordt, SE. Physiology (Bethesday) 200823: 360-370]. Akopian et al. also found that TRPV1 can regulate the mediator of TRPA1 by Ca2+through the PIP2 signaling pathway [Id., citing Akopian, AN et al. J. Physiol. (2007) 583: 175-193]. TRPM8

[0161] The TRPM channel controls certain skin functions, especially those related to melanocyte biology. TRPM8 displays a cold receptor of the body, and its activation can be modulated by many cooling compounds and many other odorant agents isolated from plants, e.g., linalool, geraniol, and hydroxycitronellal [Id., citing Behrendt, HJ et al. Br. J. Pharmcol. (2004) 141: 737-745; Harteneck, C. Naunyn Schmiedebergs Arch. Pharmacol. (2005) 371: 307- 314]; and the selectivity of Ca2+is about three times higher than that of Na+[Id., citing Venkatachalam, K. and Montell C. Ann. Rev. Biochem. (2007) 76: 387-417]. These results were confirmed electrophysiologically using whole-cell patch clamp experiments [Id., citing Voets, T. et al. Nature (2004) 430: 748-753]. It was also confirmed that the TRPM8 channel was activated by depolarization, which means that the TRPM8 channel is very sensitive to changes in the cell’sionic balance. Thus, it is natural to consider that the TRPM8 channel is involved in body temperature regulation [Id., citing Gavva, NR et al. Mol. Pain (2012) 8: 36]. TRPC6 and TRPC7

[0162] The TRPC subfamily was established as the first recognized mammalian TRP channel [Id., citing Hardie, RC. J. Physiol. (2007) 578: 9-24]. According to the results obtained by Sakura et al., the TRPC subfamily is divided into three groups on the basis of sequence alignments: TRPC1 / 4 / 5, TRPC3 / 6 / 7, and TRPC2 [Id., citing Sakura, H. and Ashcroft, FM. Diaetologia (1997) 40: 528-532]. TRPC1 / 4 / 5 / 7 channels are expressed in HaCaT, an immortal human keratinocyte line [Id., citing Beck, B. et al. Cell Calcium (2008) 43: 492-505], while Cai et al. detected TRPC1 / C5 / C6 / C7 in gingival KCs [Id., citing Cai, S. et al. J. Dermatol. Sci. (2005) 40: 21-28]. TRPC7 is also a receptor-operated, DAG-activated channel in KCs, mediating a PLC β4-activated transducer current in intrinsic photosensitivity [Id., citing Beck, B. et al. J. Invest. Dermatol. (2006) 126: 1982-1993; Hardie, RC. Handb. Exp. Pharmacol. (2014) 223: 795- 826]. As was previously described, most of the TRPC subfamily shows similar characteristics for activation by DAG and inhibition by SKF96365. Thus, it is reasonable to discuss the characteristics of TRPC6 and TRPC7 as representative of the TRPC subfamily in KCs, since these channels are activated by membrane-permeable DAG [Id., citing Beck, B. et al. Cell Calcium (2008) 43: 492-505]. Muller et al. suggested that TRPC6 is a specific channel for inducing KCs differentiation [Id., citing Muller, M. et al. J. Biol. Chem. (2008) 283: 33942- 33954]. Eventually, several members of the canonical TRPC subfamily were identified in the skin, where they mostly control the growth and differentiation of KCs under physiological and pathological conditions [Id., citing toth, BI et al. Br. J. Pharmacol. (2014) 171: 2568-2581. These TRPC channels in the skin are considered to be significantly involved in skin cell growth, barrier function, and cutaneous diseases. Based on the above discussion, as TRP channels open through oxidative stress, intracellular calcium elevation induces ROS and ATP production in mitochondria resulting in disulfide bonds formation to protein dysfunction. The Skin as an Immune Organ

[0163] The skin harbors a highly specialized immunological niche crucial for the maintenance of tissue homeostasis, defense, and repair. It protects the host from invasion by employing physical barriers, biomolecules, and an intricate network of resident immune and non-immunecells and skin structures. In the absence of a challenge, resident immune cells promote skin physiological functions. Physical Barriers

[0164] The barrier function of the epidermis is mainly mediated by corneocytes in the stratum corneum. These cells are organized in a “bricks and mortar” manner, interspersed by lipids such as ceramides, cholesterol, and free fatty acids [Nguyen, AV and Soulika, AM, Intl J. Molec. Sci. (2019) 20 (8): 1811, citing Mnon, GK et al. Int. J. pharm. (2012) 435: 3-9]. Each corneocyte contains a lipid envelope linked to keratin filament bundles that fill the intracellular compartments of the corneocyte, thus strengthening its rigidity [Id., citing Madison, KC. J. Investig. Dermatol. (2003) 121: 231-241]. The stratum corneum is composed of three layers and it is both an outside‒in barrier to prevent the entry of foreign substances and microorganisms, and an inside‒out barrier to prevent water loss [Id., citing Kubo, A. et al. Sci. Rep. (2013) 3: 1731].

[0165] The formation of the physical barrier of the skin function depends on junction adhesion molecules and tight junction proteins. Junctional adhesion molecules (JAMs), claudins, zonula occludens-1 (ZO-1), and occludins are found in epidermal layers. Biomolecules of the Skin

[0166] The main classes of biomolecules that participate in skin defense by disrupting bacterial membranes are antimicrobial peptides (AMPs) and lipids [Id., citing Niyonsaba, F. et al. Exp. Dermatol. (2017) 26: 989-998; Niyonsaba, F. et al. Crit. Rev. Immunol. (2006) 26: 545-576; Van Smeden, J. and Bouwstra, JA. Curr. Probl. Dermatol. (206) 49: 8-26]

[0167] AMPs are amphipathic peptides that are expressed constitutively or induced after cell activation in response to inflammatory or homeostatic stimulation. The most thoroughly studied AMP families in human skin are the defensins and the cathelicidins, which are produced by a variety of cells in the skin such as keratinocytes, fibroblasts, dendritic cells, monocytes, and macrophages, and sweat and sebaceous glands [Id., citing Niyonsaba, F. et al. Exp. Dermatol. (2017) 26: 989-998; Niyonsaba, F. et al. Crit. Rev. Immunol. (2006) 26: 545-576]. AMPs are produced as propeptides and become active after proteolytic cleavage. In contrast to defensin family members, which are produced by distinct genes, there is only one gene associated withcathelicidins (cathelicidin antimicrobial peptide—CAMP) [Id., citing Pfalzgraff, A. et al. Front. Pharmacol. (2018) 9: 281]. A number of active peptides can be generated via proteolytic cleavage of the inactive CAMP product, but the most studied cathelicidin is LL-37 [Id., citing Ann. Dermatol. (2012) 24: 126-135]. AMPs may act synergistically and have broad activity against microbial species [Id., citing Clausen, ML and Agner, T. Curr. Probl. Dermatol. (2016) 49: 38-46; Hanson, MA et al. eLife (2018) 493817].

[0168] AMPs have roles in modulating host immune responses. Human LL-37 was shown to induce differentiation of monocyte-derived dendritic cells, subsequent cytokine production, and expression of the co-stimulatory molecule CD86 [Id., citing Davidson, DJ et al. J. Immunol. (2004) 172: 1146-1156]. LL-37 and β-defensins can also serve as alarmins for keratinocytes by inducing their proliferation and migration [Id., citing Tokumaru, S. et al. J. Immunol. (2005) 175: 4662-4668]. Furthermore, human LL-37 exerts its alarmin effects on immune cells in a synergistic manner with other inflammatory mediators, such as IL-1β [Id., citing Yu, J. et al. J. Immunol. (2007) 179: 7684-7691]. Human α- and β-defensins serve as chemoattractants for activated neutrophils, memory and naïve T cells and immature dendritic cells [Id., citing Zhang, L-J and Gallo, RL. Curr. Biol. (2016) 26: R14-R19], while β-defensins 3 and 4 can recruit monocytes and macrophages [Id., citing Oppenheim, JJ and Yang, DJC. Curr. Opin. Immunol. (2005) 17: 359-365].

[0169] In addition, AMP functions have been associated with the processes of aging and memory [Id., citing Reinholz, M. et al. An. Dermatol. (2012) 24: 126-135; Zhang, L-J. and Gallo, RL. Curr. Biol. (2016) 26: R14-R19; Lezi, E. et al. Neuron (2018) 97: 125-138]. LL-37 has been shown to exert proangiogenic effects and may also play a role in tissue repair [Id., citing Koczulla, R. et al. J. Clin. Invest. (2003) 111: 1665-1672].

[0170] Lipids such as sphingomyelin, glucosylceramides, and phospholipids are intermediate molecules, readily converted into sphingosine and dihydrosphingosine, which exert antimicrobial activity against certain bacterial strains such as Staphylococcus aureus, Streptococcus pyogenes, Micrococcus leutus, and Proprionibacterium acnes [Id., citing Bibel, DJ et al. J. Investig. Dermatol. (1992) 98: 269-273]. These lipids are stored in lamellar bodies found in corneocytes in the stratum corneum [Id., citing Van Smeden, J. Biochim. Biophys. Acta (2014) 1841: 295-313].

[0171] Sebocytes residing in the sebaceous glands produce sebum, which is rich in lipids such as triacylglycerol, wax esters, non-esterized fatty acids, and squalene. There is a consensus that sebum serves as a “seal” for the hair follicles, thus preventing entry of microbes into the deeper layers of the skin. The AMP dermcidin is expressed by sebocytes, suggesting that sebum exerts defensive functions [Id., citing Dahlhoff, M. et al. J. Dermatol. Sci. (2016) 81: 124-126]. Furthermore, sebum can be further processed into free fatty acids by skin commensal bacteria [Id., citing Kendall, AC et al. Prog. Lipid Res. (2013) 52: 141-164; Bibel, DJ et al. J. Invst. Dermatol. (1989) 92: 632-638], and in humans, sebum-derived free fatty acids induce β-defensin 2 expression by sebocytes, further suggesting that sebaceous glands serve an innate defensive role [Id., citing Nakatsuji, T. et al. J. Investig. Dermatol. (2010) 130: 985-994].

[0172] The pH of human skin is 5.4‒5.9, which makes the skin an inhospitable environment for potential pathogens [Id., citing Schmid-Wendtner, MH and Korting, HC. Skin Pharmacol. Physiol. (2006) 19: 296-302; Fluhr, JW et al. J. Investig. Dermatol. (2001) 117: 44-51]. Furthermore, the dramatic difference in pH levels between the skin (pH 5.4-5.9) and the blood (pH = 7.4) serves as a secondary defensive mechanism in the event that microbes breach the skin tissue and enter the circulation.

[0173] There are various ways that the skin maintains a low pH.

[0174] Filaggrin, a filament-associated protein that binds keratin fibers, is broken down into histidine, which is further processed by histidase, expressed by corneocytes into the acidic metabolite trans-urocanic acid [Id., citing Scott, IR. Biochem. J. (1981) 194: 829-838]; this has been implicated in the acidification of the stratum corneum [Id., citing Krien, PM and Kermici, M. J. Investig. Dermatol. (2000) 115: 414-420]. Fatty acids produced in the stratum corneum also alter the acidity of the skin [Id., citing Bibel, DJ et al. J. Investig. Dermatol. (1989) 92: 632- 638; Fluhr, JW et al. J. Investig. Dermatol. (2001) 117: 44-51]. In addition, sweat glands produce acidic electrolytes and lactic acid, which lowers the pH of the skin [Id., citing Wilke, K. et al. Int. J. Cosmet. Sci. (2007) 29: 169-179] and promotes epidermal turnover [Id., citing Thueson, DO et al. Dermatol. Surg. (1998) 24: 641-645].

[0175] The physiological pH of the skin is hospitable for commensal bacteria such as Staphylococcus epidermidis, preventing pathogenic strains such as Staphylococcus aureus fromestablishing infections in the host [Id., citing Korting, HC et al. Acta Derm. Venereol. (1990) 70: 429-431; Elias, PM. Semin. Immunopathol. (2007) 29: 3-14]. Immune Cells of the Skin

[0176] Skin-resident immune cells promote tissue function in homeostasis and act as sentinels by actively sampling environmental antigens. Both myeloid and lymphoid cell subsets are found in the skin in steady state. Some of these resident immune cells migrate to lymph nodes to either induce peripheral tolerance to tissue self-antigens or initiate robust immune responses. In the event of a challenge, such as infections or tissue injury, immune cells resident in the skin and those infiltrating from the periphery interact to create an intricate defense network to resolve the insult and restore the tissue to its original state. Myeloid Cells

[0177] Skin-resident myeloid cells include Langerhans cells, dermal dendritic cells, macrophages, mast cells, and eosinophils. Neutrophils are rarely found in healthy skin and thus are not “skin-resident cells.” However, neutrophils populate the skin in inflammatory conditions and after a wound.

[0178] Skin-resident myeloid cells contribute to skin homeostasis by secreting growth factors needed for the survival of keratinocytes, fibroblasts, and endothelial cells. In addition, they maintain optimal tissue function by phagocytosing debris and apoptotic cells, supporting vasculature integrity, and promoting tolerance.

[0179] In inflammatory conditions, myeloid cells respond immediately and produce pro- inflammatory mediators that drive the activation of cells in the local vicinity and infiltration of the affected site by peripheral immune cells. Skin myeloid cells also serve as a liaison between the innate and adaptive immune system. Langerhans Cells

[0180] Langerhans cells (LCs) are the sole myeloid cell type in the epidermis. Phenotypically, LCs are characterized by high expression of MHC class II and the presence of langerin+ Birbeck granules [Id., citing Deckers, J. et al. Front. Immunol. (2018) 9: 93; Kaplan, DH. Nat. Immunol. (2017) 18: 1068-1075]. LC maintenance depends on keratinocyte-derived IL-34 [Id., citing Kaplan, DH. Nat. Immunol. (2017) 18: 1068-1075; Ginhoux, F. et al. Science (2010) 330: 841-5;Greter, M. et al. Immunity (2012) 37: 1050-1060; Wang, Y. et al. Nat. Immunol. (2012) 13: 753- 760], one of the ligands for the colony stimulating factor-1 receptor (CSF-1R). CSF-1R is constitutively expressed on LCs and global deletion of this receptor results in a total lack of LCs [Id., citing Wang, Y. et al. Nat. Immunol. (2012) 13: 753-760; Wang, Y. and Colonna, M. Eur. J. Immunol. (2014) 44: 1575-1581; Ginhoux, F. et al. Nat. Immunol. (2006) 7: 265-273].

[0181] LCs are derived from two sources, the extra-embryonic yolk sac and fetal liver monocytes. LCs renew from a local progenitor cell [Id., citing Merad, M. et al. Nat. Immunol. (2002) 3: 1135-1141]. In homeostasis, LCs anchor themselves within the epidermis through interactions between epithelial cell adhesion molecules (EpCAM) or E-cadherin expressed on LCs and E-cadherin expressed by keratinocytes [Id., citing Deckers, J. et al. Front. Immunol. (2018) 9: 93; Choi, HW et al. Clin. Exp. Dermatol. (2018) 43: 291-295; Mayumi, N. et al. Eur. J. Immunol. (2013) 43: 270-280]. Furthermore, autocrine and paracrine TGFβ signaling restricts LCs in the epidermis by regulating the expression of E-cadherin on LCs [Id., citing Bobr, A. et al. Proc. Natl Acad. Sci. USA (2012) 109: 10492-10497; Kel, JM et al. J. Immunol. (2010) 185: 3248-3255] and increases phagocytic behavior in LCs during steady state [Id., citing Bauer, T. et al. J. Exp. Med. (2012) 209: 2033-2047]. While anchored, LCs sample antigens and, upon activation, they can extend their processes from the cell body outward to the stratum corneum or below toward the stratum basale [Id., citing Deckers, J. et al. Front. Immunol. (2018) 9: 93]. LCs participate in tight junction formation and thus can sample the microenvironment without damaging the barrier [Id., citing Decker, J. et al. Front. Immunol. (2018) 9: 93].

[0182] LCs are migratory cells and continually travel to the skin draining lymph nodes to promote tolerance in homeostasis [Id., citing Ghigo, C. et al. J. Exp. Med. (2013) 210: 1657- 1664] or to initiate adaptive immune responses [Id., citing Romani, N. et al. J. Exp. Med. (1989) 169: 1169-1178; Romani, N. et al. J. Exp. Med. (1989) 93: 600-609; Schuler, G. and Steinman, RM. J. Exp. Med. (1985) 161: 526-546].

[0183] The migration of LCs (and also of dermal dendritic cells) through the dermis is mediated via CXCR4 signaling after binding to its cognate chemokine CXCL12, which is produced by dermal fibroblasts [Id., citing Kabashima, K. et al. Am. J. Pathol. (2007) 171: 1249-1257].

[0184] In the presence of inflammatory mediators and other activators such as pathogen- associated molecular patterns (PAMPs) and danger-associated molecular patterns (DAMPs), LCsupregulate co-stimulatory molecules and migrate from the epidermis en masse to the draining lymph nodes, where they prime adaptive immune responses in a manner equivalent to that of conventional dendritic cells [Id., citing Deckers, J. et al. Front. Immunol. (2018) 9: 93; Wang, B. et al. Immunology (1996) 88: 284-288; Griffiths, CE et al. Cytokine (2005) 32: 67-70; Kashem, SW et al. Immunity (2015) 42: 356-366; Nakajima, S. et al. J. Allergy Clin. Immunol. (2012) 129: 1048-1055]. TGFβ signaling is disrupted during inflammation, thus also promoting LC migration from the epidermis [Id., citing Bobr, A. et al. Proc. Natl Acad. Sci. USA (2012) 109: 10492-10497]. LCs are professional antigen-presenting cells and activate both CD8+ cytotoxic T lymphocytes and CD4+ helper T lymphocytes. In humans, efficient antigen presentation by LCs is dependent upon caveolin-1, a scaffold protein that serves a multitude of functions including transport of lipids, signal transduction, and membrane trafficking [Id., citing Polak, ME et al. J. Investig. Dermatol. (2014) 134: 695-703; Liu, P. et al. J. Biol. Chem. (2002) 277: 41295-41298]. LCs can also exert immunoregulatory and tolerogenic functions [Id., citing Deckers, J. et al. Front. Immunol. (2018) 9: 93; Flacher, V. et al. EMBO Mol. Med. (2014) 6: 1191-1204; Shklovskaya, E. et al. Proc. Natl Acad. Sci. USA (2011) 108: 18049-18054]. Dermal Dendritic Cells

[0185] Dendritic cells that reside in the dermis are known as dermal dendritic cells (dDCs). In a similar manner to LCs, dDCs migrate to the lymph nodes, and are professional antigen- presenting cells efficient at priming adaptive immune responses [Id., citing Hain, T. et al. J. Investig. Dermatol. (2018) doi: 10.1016 / j.jid / 2018.08.022; Tomura, M. et al. Sci. Rep. (2014) 4: 6030]. dDCs can induce tolerance to topically applied antigens encountered in the hair follicles [Id., citing Tordesillas, L. et al. Nat. Commun. (2018) 9: 5238].

[0186] Unlike LCs, all dDCs are derived from progenitors originating from the bone marrow, with replenishment occurring roughly every seven days [Id., citing Kim, TG et al. Intl J. Mol. Sci. (2017) 19: 42; Ober-Blobaum, JL, et al. Methods Mol Biol. (2017) 1559: 37-52]. The two main subsets of dDCs are the conventional dendritic cell type 1 (cDC1) and the conventional dendritic cell type 2 (cDC2) [Id., citing Ober-Blobaum, JL, et al. Methods Mol Biol. (2017) 1559: 37-52; Kashem, SW et al. Annu. Rev. Immunol. (2017) 35: 466-499].

[0187] dDCs are implicated in maintaining homeostatic interactions between the host and skin- resident commensal bacteria. Plasmacytoid DCs (pDCs) are a DC subset, found in the skinexclusively under inflammatory conditions. pDCs are mass producers of IFNα, which is essential for viral defense [Id., citing Kashem, SW et al. Annu. Rev. Immunol. (2017) 35: 466-993].

[0188] Human dDCs are subdivided into cDC1 (CD141+), cDC2 (CD1c+), and CD14+ dDCs

[0123] . cDC1s co-express CD304 (neuropilin-1), XCR1, and CD370 (CLEC9A). Unlike murine dDCs, human dDCs do not express langerin [Id., citing Kashem, SW et al. Annu. Rev. Immunol. (2017) 35: 466-499; Haniffa, M. et al. Immunity (2012) 37: 60-73]. Human cDC1s can cross- present in a similar manner to murine cDC1s [Jongbloed, SL et al. J. Exp. Med. (2010) 207: 1247-1260] and are potent in inducing Th1 responses [Id., citing Segura, E. et al. J. Exp. Med. (2012) 209: 653-660]. cDC2s and CD14+ dDCs co-express CD11b and CX3CR1. Both human cDC1s and cDC2s are capable of inducing Th2 responses [Id., citing Segura, E. et al. J. Exp. Med. (2012) 209: 653-660]. Human pDCs express CD304, CD303 and CD123 and like their murine counterparts, they are only found in inflamed skin. Human peripheral blood pDCs upregulate the expression of B cell maturation antigens upon activation via TLR9 signaling, suggesting that pDCs play a role in the maintenance of plasma cells, in addition to their inflammatory functions [Id., citing Schuh, E. et al. J. Immunol. (2017) 198: 3081-3088].

[0189] Macrophages

[0190] Macrophages are found in the dermal layer of the skin and require IL-34 for survival [Id., citing Wang, Y. et al. Eur. J. Immunol. (2014) 44: 1575-1581; Ginhoux, F. and Jung, S. Nat. Rev. Immunol. (2014) 14: 392-404]. Two sources of dermal macrophages have been identified so far. The first source is embryo-derived progenitors that seed the skin prenatally and are self- renewing in a similar fashion to LCs [Id., citing Guilliams, M. et al. Nat. Rev. Immunol. (2014) 14: 571-578]. The second and major source of dermal macrophages is circulating monocytes (monocyte-derived macrophages) that mature once they reach the skin. This population replenishes roughly every 10 days [Id., citing Sheng, J. et al. Immunity (2015) 43: 382-393; Baranska, A. et al. J. Exp. Med. (2018) 215: 1115-33]. Monocytes that give rise to dermal macrophages express lymphocyte antigen 6C (Ly6C), and home to the skin in a CCR2- dependent manner [Id., citing Malissen, B. et al. Nat. Rev. Immunol. (2014) 14: 417-428]. As monocytes mature into skin-resident macrophages, the expression of CCR2 is downregulated [Id., citing Tamoutounour, S. et al. Immunity (2013) 39: 925-938]. CD64 expression is prominent on dermal macrophages and is used as a marker to differentiate them from the dDCs[Id., citing Baranska, A. et al. J. Exp. Med. (2018) 215: 1115-1133; Tamoutounour, S. et al. Immunity (2013): 39: 925-938]. CD36, DC-SIGN, and IL-10 are highly expressed by macrophages isolated from healthy skin, suggesting that they adapt an immunoregulatory phenotype [Id., citing Tamoutounour, S. et al. Immunity (2013): 39: 925-938; Lonati, A. et al. J. Investig. Dermatol. (1996) 106: 96-101]. In steady state, macrophages remove cellular debris [Id., citing Malissen, B. et al. Nat. Rev. Immunol. (2014) 14: 417-428, Tamoutounour, S. et al. Immunity (2013): 39: 925-938], and have also been implicated in homeostatic hair regeneration [Id., citing Yanez, DA et al. Pflugers Arch. (2017) 469: 455-463; Eichmullelr, S. et al. J. Histochem. Cytochem. (1998) 46: 361-370].

[0191] Macrophages are plastic, and one way to categorize their effector functions is as pro- inflammatory “M1” or anti-inflammatory / pro-repair “M2.” M1 macrophages express inducible nitric oxide synthase (iNOS), and secrete inflammatory cytokines such as TNFα, IL-1β, and IL-6 [Id., citing Mills, CD. Front. Immunol. (2015) 6: 212; Mills, CD and Ley, K. J. Innate Immun. (2014) 6: 716-726]. M2, or “alternatively activated” macrophages, adopt an anti-inflammatory and / or pro-repair phenotype. M2 macrophages can be further subdivided into M2a, M2b, M2c, and M2d. M2a macrophages are known to be pro-fibrotic due to TGFβ production [Id., citing Ohji, M. et al. Curr. Eye Res. (1993) 12: 703-709]. M2b macrophages express the co-stimulatory molecule CD86 and as such they are the most pro-inflammatory of the M2 subsets. M2c macrophages are induced by IL-10 or TGFβ, express the Mer tyrosine kinase (MerTK) and promote neovascularization [Id., citing Martinez, FO et al. Front. Biosci. (2008) 13: 453-461] and have high scavenging and debris clearing activity. M2d macrophages are responsive to IL-6 and exhibit a few of the properties of tumor-associated macrophages such as secretion of IL-12 and IL-10 [Id., citing Martinez, FO et al. Front. Biosci. (2008) 13: 453-461, Ferrante, CJ and Leibovich, SJ. Ad. Wound Care (New Rochelle) (2012) 1: 10-16; Arora, S. et al. Immunobiology (2018) 223: 383-396; Wang, Q. et al. Cell Res. (2010) 20: 701-712]. M2d cells are also known to express high levels of the adenosine receptor A2AR in the presence of LPS [Id., citing Ferrante, CJ et al. Inflammation (2013) 36: 921-931]. A2AR signaling has been shown to attenuate pro-inflammatory cytokine production [Id., citing Koroskenyi, K. et al. Biochim. Biophys. Acta (2016) 1863: 1461-1471], suggesting that A2AR-expressing M2d cells may be involved in the resolution of the inflammatory response. Mast Cells

[0192] Mast cells are commonly found in the dermal layer. In humans, mast cells are found in all areas of the skin but are most numerous in the arms and the legs [Id., citing Janssens, AS et al. J. Clin. Pathol. (2005) 58: 285-289]. The density of mast cells in the papillary dermis increases with age and they are most often localized in the proximity of PGP9.5+ nerve fibers expressing vasoactive intestinal peptide (VIP), which was shown to suppress mast cell degranulation [Id., citing Pilkington, SM et al. Br. J. Dermatol. (2018) doi: 10.111.bjd.17268]. This has been associated with the reduction of the amount of extracellular matrix remodeling in the skin observed during the later stages of life.

[0193] Mast cells contain granules containing preformed mediators such as histamine, sulfated proteoglycans, serotonin, and tryptase and / or chymase. In both humans and mice, mast cells resident in the skin express both tryptase and chymase, whereas other tissue-resident mast cells express only tryptase [Id., citing Olivera, A. et al. J. Allergy Cli. Immunol. (2018) 142: 381- 383].

[0194] Mast cells are classically known for their involvement in allergic reactions as they produce and release copious amounts of histamine when their Fcε receptors are crosslinked via IgE-antigen complexes [Id., citing Shi, LB et al. Pestic. Biochem. Physiol. (2018) 148: 159-165; Hirano, T. et al. Sci. Rep. (2018) 8: 14237]. They also make large amounts of prostaglandin D2 (PGD2), a lipid-derived inflammatory mediator.

[0195] Mast cells are mass producers of leukotrienes (LTs), which are short-lived lipid inflammatory mediators synthesized via the 5-lipoxygenase (5-LO) pathway.

[0196] A variety of cytokines and growth factors are produced by mast cells either constitutively or in response to a stimulus [Id., citing Mukai, K. et al. Immunol. Rev. (2018) 282: 121-150]. Many of these cytokines and growth factors such as TNFα and vascular endothelial growth factor (VEGF) may be pre-formed and packaged in mature mast cell granules [Id., citing Wernersson, S. and Pejler, G. Nat. Rev. Immunol. (2014) 14: 478-494; Gordon, JR, Galli, SJJN. Nature (1990) 346: 274; Grutzkau, A. et al. Mol. Biol. Cell (1998) 9: 875-884]. Proper formation of mast cell granules is mediated mostly by proteoglycan serglycin [Id., citing Braga, T. et al. Biochem. J. (2007) 403: 49-57].

[0197] Dermal mast cells can prime adaptive responses during cutaneous infections. Mast cell- derived IL-1β induces production of histamine and IL-8 in human mast cells, suggesting that IL-1β is part of a positive feedback loop for mast cell activation [Id., citing Subramanian, N. and Bray, MA. J. Immunol. (1987) 138: 271-275; Kim, GY et al. J. Immunol. (2010) 184: 3946- 3954]. TNFα produced by mast cells is known to drive migration of dermal dendritic cells to draining lymph nodes in a murine model of hapten-induced contact hypersensitivity [Id., citing Suto, H. et al. J. Immunol. (2006) 176: 4102-4112]. Mast cell-derived TNFα is also crucial for maintenance of tolerance toward allogeneic skin grafts in mice [Id., citing De Vries, VC et al. Immunity (2011) 35: 550-561]. Studies revealed that mast cells are able to form immunological synapses with γδ T lymphocytes when challenged with dengue virus [Id., citing Mantri, CK and St. John , AL. J. Clin. Investig. (2018) doi: 10.1172 / JCI122520197] and can acquire MHC class II expression via vesicle transfer from dendritic cells after administration with dinitrofluorobenzene [Id., citing Dudeck, J. et al. J. Exp. Med. (2017) 214: 3791-3811]. Eosinophils

[0198] Eosinophils are skin-resident cells [Id., citing Yu, YR et al. PLoS ONE (2016) 11: e0150606; Ramirez, GA et al. Biomed. Res. Int. (2018) 99095275], but not much is known about their role in tissue homeostasis. Eosinophilic granules are loaded with potent and toxic proteins: major basic protein (MBP), eosinophil peroxidase (EPO), eosinophil protein X / eosinophil- derived neurotoxin (EPX / EDN), and eosinophil cationic protein (ECP) [Id., citing Long, H. et al. Clin. Rev. Allergy Immunol. (2016) 50: 189-213].

[0199] Like mast cells, eosinophils produce inflammatory lipid mediator leukotrienes (LTs) and prostaglandin D2 (PGD2) [Id., citing Luna-Gomes, T. et al. J. Immunol. (2011) 187: 6518- 6526], with the latter being crucial for eosinophilic infiltration of the skin in hypersensitivity reactions such as atopic dermatitis [Id., citing He, R. et al. J. Allergy Clin. Immunol. (2010) 126: 784-790]. In addition, eosinophils generate extracellular DNA traps (ETs) containing eosinophil granules [Id., citing Ueki, S. et al. Blood (2013) 121: 2074-2083; Yousefi, S. et al. Nat. Med. (2008) 14: 949-953]. These traps are believed to play a role in antibacterial defense.

[0200] Eosinophils are classically known to promote host defense against parasitic infections [Id., citing Jacobesen, EA et al. Blood (2012) 120: 3882-3890]. The roles of eosinophils in dermatoses, or skin diseases associated with eosinophilia such as allergic contact dermatitis and urticaria, is emerging [Id., citing Long, H. et al. Clin. Rev. Allergy Immunol. (2016) 50: 189- 213; Foster, EL et al. PLoS ONE (2011) 6: e22029; Oyoshi, MK et al. Proc. Natl Acad. Sci.USA (2012) 109: 4992-7; Wang, SF et al. Med. Inflamm. (2016) 2016: 5032051]. In eosinophilic dermatoses, extensive eosinophilic degranulation in the skin results in local tissue damage. Lymphoid Immune Cells

[0201] The skin harbors different types of lymphoid cells, all of which are important in both steady state and inflammatory responses. Both human and murine skin contain γδ T lymphocytes and αβ T lymphocytes, along with natural killer T cells. γδ T cells are the dominant T cell population in murine skin, while αβ T cells are the dominant T cell population in human skin [Id., citing Mestas, J. and Hughes, CC. J. Immunol. (2004) 172: 2731-8; Elbe, A. et al. Semin. Immunol. (1996) 8: 341-349]. αβ T Lymphocytes

[0202] In both mice and humans, αβ T lymphocytes are found in the epidermis and dermis [Id., citing 224], and traffic to the skin from the periphery via cutaneous lymphocyte antigen (CLA) interactions with E-selectin (expressed on endothelial cells), which is upregulated under inflammatory conditions [Id., citing Kantele, A. et al. J. Immunol. (1999) 162: 5173-5177; Groves, RW et al. Br. J. Dermatol. (1991) 124: 117-123]. αβ T lymphocytes in the skin are resident memory T cells (TRM), which are long-lived and distinct from their circulating counterparts [Id., citing Seidel, JA et al. Clin. Exp. Immunol. (2018) 194: 79-92].

[0203] Most TRM in the skin are derived from antigen-specific effector T cells, which previously infiltrated the tissue as a result of an infection. After resolution, these TRMcells seed all areas of the skin but are denser in areas of antecedent infection [Id., citing Clark, RA. Sci. Trans. Med. (2015) 7: 269rv261]. Following a skin infection, TRM cells are also found in distal organs such as the lung and gastrointestinal tract [Id., citing Clark, RA. Sci. Transl. Med. (2015) 7:269rv261].

[0204] TRMexpress lower levels of CD28 than effector memory T cells, but can mount robust local recall responses [Id., citing Seidel, JA et al. Clin. Exp. Immunol. (2018) 194: 79-92] without emigrating from the tissue to do so [Id., citing Gebhardt, T. et al. Nat. Immunol. (2009) 10: 524-530]. TRMalso exert sentinel-like functions by promoting recruitment of other memory T cells from the periphery to sites of infection [Id., citing Ariotti, S. et al. Science (2014) 346: 101- 105; Schenkel, JM et al. Nat. Immunol. (2013) 14: 508-513].

[0205] The most studied skin TRMare CD8+ T cells. CD8+ TRMcells are usually found in the epidermis and may dislocate dendritic epidermal T cells [Id., citing Mackay, LK et al. Nat. Immunol. (2013) 14: 1294-1301]. All CD8+ TRM express CD69, an early marker of lymphocyte activation, and a high proportion also express CD103 [Id., citing Mackay, LK et al. Nat. Immunol. (2013) 14: 1294-1301]. CD103 is required for the development of CD8+ TRM cells in the skin [Id., citing Mackay, LK et al. Nat. Immunol. (2013) 14: 1294-1301] and mediates adhesion interactions with keratinocytes via an E-cadherin-independent manner [Id., citing Jenkinson, SE et al. Immunology (2011) 132: 188-96].

[0206] CD4+ TRM cells also make up a significant portion of the skin-resident lymphocyte population and are found in both the epidermis and dermis [Id., citing Mackay, LK et al. Nat. Immunol. (2013) 14: 1294-1301]. Although the function of CD4+ T cells in the skin is not studied to the same extent as CD8+ TRM, they are often the dominant αβ T cell subset. During steady state, clusters of antigen-presenting cells with CD4+ memory T cells are found around the hair follicles in both murine and human skin, and cells in these clusters circulate between the skin and periphery [Id., citing Collins, N. et al. Nat. Commun. (2016) 7: 11514]. These clusters are formed because keratinocytes in the hair follicles produce IL-7 and IL-15, which are required for homeostatic maintenance of the T cell populations [Id., citing Adachi, T. et al. Nat. Med. (2015) 21: 1272-1279]. γδ T Lymphocytes:

[0207] Unlike the αβ T lymphocytes, γδ T cells do not undergo the same stringent negative selection process during development and are released from the thymus in waves, with the first wave of γδ T cells seeding the dermis of the skin [Id., citing Sutoh, Y. et al. Front. Immunol. (2018) 9: 1059; Xiong, N. et al. Immunity (2004) 21: 121-131; Xiong, N. and Raulet, DH. Immunol. Rev. (2007) 215: 15-31].

[0208] The functions of dermal γδ T cells in inflammatory skin conditions are well documented. This population of dermal T cells is inclined to produce IL-17 [Id., citing Gray, EE. Et al. J. Immunol. (2011) 186: 6091-6095], indicating their significance in cutaneous diseases such as psoriasis [Id., citing Van der Fits, L. et al J. Immunol. (2009) 182: 5836-5845; Mabuchi, T. et al. J. Investig. Dermatol. 2(2009) 182: 5836-5845].

[0209] γδ T cells in the skin can also play a protective role in cutaneous defense; a previous study showed that γδ T cell-deficient mice exhibited larger lesions and reduced IL-17 production in response to Staphylococcus aureus compared to wild type (WT) control mice [Id., citing Cho, JS et al. J. Clin. Investig. (2010) 120: 1762-73].

[0210] γδ T cells are not MHC-restricted [Id., citing Champagne, E. Arch. Immunol. Ther. Exp. (2011) 59: 117-37] and can recognize soluble antigens [Id., citing Champagne, E. Arch. Immunol. Ther. Exp. (2011) 59: 117-137; Born, WK and O’Brien, RL. Arch. Immuol. Ther. Exp. (2009) 57: 129-135], antigens derived from damaged or stressed cells [Id., citing O’Brien, RL and Born, W. Semin. Immunol. (1991) 3: 81-87; O’Brien, RI et al. Proc. Nat. Acad. Sci. USA (1992) 89: 4348-4352; Heng, MK. Madame Curie Bioscience Database, Landes Bioscience: Austin TX, SA (2013); pp. 2000-2013], or antigens complexed with non-classical MHC molecules such as CD1b, CD1c, and CD1d [Id., citing Cui, Y. et al. Biol. Direct. (2009) 4: 47; Luoma, AM et al. Trends Immunol (2014) 35: 613-621] or MHCI-related chain A / B (MICA / MICB) [Id., citing Champsaur, M. and Lanier, LL. Immunol. Rev. (2010) 235: 267-285]. One study suggested that γδ T cells also recognize butyrophilin-like Btnl / BTNL proteins, which are part of the B7 superfamily that regulates immune responses via costimulatory or coinhibitory signals [Id., citing Melandri, D. et al. Nat. Immunol. (2018) 19: 1352-1365]. B Lymphocytes

[0211] B cells are rather sparse in the skin in steady state and it is unclear whether they are indeed resident to the skin [Id., citing Nihal, M. et al. J. Mol. Diagn. (2000) 2: 5-20; Geherin, SA et al. J. Immunol. (2012) 188: 6027-6035; Egbuniwe, IU, et al. Trends Immunol. (2015) 36: 102- 111]. However, the roles of B lymphocytes in skin inflammatory conditions are well documented. In humans, B cells are found in elevated levels in cutaneous diseases such as atopic eczema, cutaneous leishmaniasis, and cutaneous sclerosis [Id., citing Simon, D. et al. J. Allergy Clin. Immunol. (2008) 121: 122-128; Geiger, B. et al. Br. J. Dermatol. (2010) 162: 870-874; Lafyatis, R. et al. Arthritis Rheum. (2009) 60: 578-583]. B cells are found in the reticular dermis over the course of these diseases and are associated with increased levels of IgM, IgE, and IgG. In a similar manner to T lymphocytes, B lymphocytes traffic to the skin tissue via cutaneous lymphoid antigen (CLA) interactions [Id., citing Postigo, AA et al. J. Clin. Investig. (1994) 94: 1585-1596]. B cells also play a role in delayed-type hypersensitivity reactions in the skin.Non-Immune Cells

[0212] Pattern recognition receptors (PRR) are expressed by most cells of the skin and have been characterized on keratinocytes, fibroblasts, adipocytes, melanocytes, and endothelial cells [Id., citing Chen, L. and DiPietro, LA. Adv. Wound Care (2017) 6: 344-355]. Activation of these receptors results in the production of cytokines and chemokines by non-immune skin cells, thus participating in the local immune response [Id., citing Ahn, JH et al. Exp. Dermatol. (2008) 17: 412-417; Faure, E. et al. J. Biol. Chem. (2000) 275: 11058-11063; Taylor, KR et al. J. Biol. Chem. (2004) 279: 17079-17084; Song, MJ et al. Biochem. Biophys. Res. Commun. (2006) 346: 739-745].

[0213] Keratinocyte-derived inflammatory responses have been extensively studied. These cells express almost all intracellular and extracellular PRRs and produce a variety of cytokines, chemokine and AMPs to protect the host against infection [Id., citing Bitschar, K. et al. J. Dermatol. Sci. (2017) 87: 215-220; Pasparakis, M. etal. Nat. Rev. Immunol. (2014) 14: 289; Schittek, B. Curr. Probl. Dermatol. (2011) 41: 54-67]. Keratinocytes both in the epidermis and skin elements are in constant interaction with local immune cells and produce factors crucial in homeostasis and in tissue repair [Id., citing Wang, Y. et al. Nat. Immunol. (2012) 13: 753-760; Adachi, T. et al. Nat. Med. (2015) 21: 1272-1279; Chodaczek, G. et al. Nat. Immunol. (2012) 13: 272-282; Takashima, A. et al. J. Investig. Dermatol. (1995) 105: 505-535]. Other studies demonstrated that keratinocytes produce IL-33 in response to hypo-osmotic stress [Id., citing Pietka, W. et al. J. Invest. Dermatol. (2018 doi 10.1016 / j.jId.2018.07.023], and that human and murine keratinocytes produce IL-6 and IL-1β mediated by NFκB signaling in response to UVB irradiation [Id., citing Tang, SC et al. J. Dermatol. Sci. (2017) 86: 238-248].

[0214] Fibroblasts immunomodulatory functions have also been well delineated. They express PRRs, synthesize many cytokines, and were shown to produce AMPs [Id., citing Bautista- Hernandez, LA et al. Eur. J. Microbiol. Immunol. (2017) 7: 151-157]. Dermal fibroblasts and keratinocytes produce serum amyloid A in response to PRR signaling [Id., citing Morizane, S. et al. Clin. Exp. Dermatol. (2018) doi: 10.111 / ced.13604], which is believed to induce the production of pro-inflammatory cytokines from various immune cells [Id., citing Eklund, KK et al. Crit. Rev. Immunol. (2012) 32: 335-348]. A recent investigation showed that human fibroblasts cultured in vitro produced massive amounts of TNFα, IL-1β, IL-6, IL-8, and IL-25when subjected to thermal stress [Id., citing Jiang, L. et al. Biomed. Pharmacother. (2018) 107: 24-33]. Influence of Circadian Oscillations in Skin on Immune Response, Skin Homeostasis, Stress Mediation, and Aging

[0215] The circadian clock, an intrinsic autonomous clock that controls the body’s divergent functions during day and night, affects the expression of multiple genes that mediate skin stem cell metabolism and proliferation, DNA repair, stimulus response and immunity. [Duan, J. et al. FEBS Lett (2021) 595 (19): 2413-2436] Circadian rhythm is closely linked to immunological processes and skin homeostasis, and its dysynchrony can be linked to the perturbation of the skin. [Salazar, A. and von Hagen, J. Intl Jm Molec. Sci. (2023) 24: 5635].

[0216] The mammalian circadian clock at a cellular level consists of at least 3 overlapping feedback loops (FIG. 3) [Id., citing Sherratt, MJ et al. Matrix Bio. (2019) 84: 97-110; Lyons, AB et al. J. Clin. Aesthet. Dermatol. (2019) 12: 42-45; Curtis, AM et al. Immunity (2014) 40: 178- 186].

[0217] In the first loop, the core circadian clock proteins BMAL1 (basic helix–loop–helix ARNT like 1; also called ARTNL, aryl hydrocarbon receptor nuclear translocator-like) dimerizes either with CLOCK (circadian locomotor output cycles kaput) or with NPAS2 (neuronal PAS domain protein 2) and then triggers the expression of PER (period circadian regulator; PER1-3), CRY (cryptochrome circadian regulator; CRY1-2), ROR (or RAR, related orphan receptor), NR1D1 (nuclear receptor subfamily 1 group D member 1; also called REV-ERE), DBP (D-box binding PAR Bzip transcription factor), and other clock controlled genes by binding to their E- box elements (5′-CACGTG-3′) in the promoter region. On reaching a critical concentration, the proteins PER and CRY dimerize to inhibit their own expression by preventing the binding of BMAL1: CLOCK to DNA, which results in an oscillation of these proteins [Id., citing Sherratt, MJ et al. Matrix Biol. (2019) 84: 97-110; Lyons, AB et al. J. Clin. Aesthet. Dermatol. (2019) 12: 42-45; Curtis, AM et al. Immunity (2014) 40: 178-186; Richards, J. and Gumz, ML. Am. J. Physiol. Regul. Integr. Comp. Physiol. (2013) 304: R1053-R1064].

[0218] In the second loop, the protein ROR binds to the RORE element 5′-(A / G)GGTCA-3′ in the promoter of BMAL1, CLOCK, and NFIL3 (nuclear factor, interleukin 3 regulated), resultingin their transcription. The binding of ROR to the RORE element is inhibited by NR1D1, and potentially also by related proteins from this family. This completes the second loop.

[0219] The protein DBP, whose expression is under the control of BMAL1:CLOCK from the first loop binds to the D box (5′-TTATG(T / C)AA-3′) in the promoter region of PER. This binding is negatively regulated by NFIL3 from the second loop. Taken together, this is considered the third loop.

[0220] Similar to PER1 / 2, CRY1 is regulated by a combinatorial mechanism involving both E- box and RORE, giving rise to a phase distinct from DBP and REV-ERB. The DEC loop is ancillary to the core circadian loops, and is characterized by the expression of DEC and other circadian controlled genes, which are under the control of BMAL1: CLOCK. DEC, in turn, inhibits the binding of BMAL1: CLOCK to the E-box element, thereby regulating its own expression [Id., citing Ono, D. et al. Sci. Rep. (2021) 11: 19240; Honma, S. et al. Nature (2002) 419: 841-844].

[0221] Posttranslational modifications (PTMs) and proteasomal degradation of these core components of the molecular clock machinery are essential to maintain the oscillatory nature of these proteins and the proteins they regulate. These PTMs include phosphorylation, glycosylation, ubiquitination, acetylation, and SUMOylation, as reviewed by Hirano et al. [Id., citing Hirano, A. et al. Nat. Struct. Mol. Biol. (2016) 23: 1053-1060]. In some cases, a further level of complexity is introduced in the form of crosstalk between these mechanisms; for instance, O-linked β-N-acetylglucosamine (O-GlcNAc) competing for the same serine and threonine residues as kinases for phosphorylation [Id., citing Hirano, A. et al. Nat. Struct. Mol. Biol. (2016) 23: 1053-1060; Hardin, PE and Panda, S. Curr. Opin. Neurobiol. (2013) 23: 724- 731] Zeitgebers and the Circadian Clock in Mammalian Skin

[0222] The intracellular molecular clock oscillates in response to environmental signals known as ‘Zeitgebers’, derived from German and directly translating to ‘time giver’. As a result, in the study of circadian rhythm, time in days is often divided into ‘zeitgeber time’. Light is the primary zeitgeber. It is detected via the optical nerve, which then transmits signals of perceived light to the hypothalamic suprachiasmatic nucleus (SCN) [Id., citing Curtis, AM et al. Immunity (2014) 40: 178-186]. This information is used to entrain the functional molecular clocks inperipheral tissues via the autonomic nervous system and the hypothalamus pituitary adrenal axis via hormones including glucocorticoids and catecholamines (epinephrine and norepinephrine) [Id., citing Kalsbeek, A. et al. Mol. Cell Endocrinol. (2012) 349: 20-29]. The hormones prolactin, growth hormone, and melatonin have been implicated in circadian signaling. Other zeitgebers that have been exploited in in vivo studies include sleep wake cycles, feeding and fasting regimes, and temperature. These zeitgebers are linked to the presence of light, as well as circulating levels of melatonin [Id., citing Sato, K. et al. J. Pineal. Res. (2020) 688: e12639; Slominski, AT et al. J. Investig. Dermatol. (2018) 138: 490-499; Kleszcynski, K. et al. Dermato- endocrinology (2011) 3: 27-31].

[0223] It has been shown that light can entrain the circadian clock in these peripheral tissues even after SCN ablation or scarring, although oscillations were found to be lower in these cases [Id., citing Buhr, ED et al. Curr. Biol. (2019) 29: 3478-3487; Welz, PS et al. Cell (2019) 177: 1436-1447; Koronowski, KB et al. Cell (2019) 177: 1448-1462]. The peripheral clocks also can communicate with each other, achieving entrainment independent of the SCN. The potential of UV, visible, and infra-red light to cause DNA damage, oxidative stress, lipid peroxidation, etc., in skin cells has been well characterized. Furthermore, UV light is also able to stimulate the production of melanin and melanocyte stimulating hormone (MSH) in the skin. The knowledge that skin cells have been known to interact with light makes it even more plausible that they can be directly entrained by light exposure. Influence of the Circadian Clock on the Immune Response of the Skin

[0224] The components of the circadian clock machinery are crucial to the development and functioning of a robust immune system. Indeed, most immune cell lineages have intrinsic clocks that govern their maturation, migration, differentiation, and function. An example is NFIL3, which is responsible for the development and maintenance of a population of interferon-gamma (IFN-γ) producing group 1 innate lymphoid cells and NK cells [Id., citing Greenberg, EN et al. Proc. Natl Acad. Sci. USA (2020) 117: 5761-5771]. This, in turn, can be linked to the rhythmic activation of IFN-sensitive gene pathways in the skin, including a key transcription factor IFN regulatory factor 7 (Irf7) via Toll-like receptor 7 (TLR7). Similarly, ROR-α expression is thought to be increased in activated phenotype Treg cells in mouse skin [Id., citing Malhotra, N. et al. Sci. Immunol. (2018) 3: eaao6923]. Activated Treg cells expressing ROR-α have beenshown to attenuate the function of group 2 innate lymphoid cells that reside in the skin. This has been shown to limit allergic skin inflammation in models of atopic dermatitis mediated by type II cytokines including IL-4, IL-5, and IL-13.

[0225] The circadian clock determines the rhythm with which immune cells circulate or migrate into tissues. The adhesion molecules ICAM-1 and VCAM-1 on endothelial cells vary based on the degree of inflammation, as well as rhythmicity, and act as homing signals for leukocytes in homeostasis, as well as inflammation [Id., citing He, W. et al. Immunity (2018) 49: 1175-1190]. Moreover, in skin, CD44 appears to be the adhesion molecule that varies in a circadian manner and acts as a honing signal for leucocytes in endothelial cells that constitute the capillaries of the dermis [Id., citing He, W. et al. Immunity (2018) 49: 1175-1190; Zoller, M. et al. J. Leukoc. Biol. (2007) 82: 57-71]. This circadian rhythmic variation prevents overactivation of the immune system when an external challenge is unlikely, and preparation of the immune system in more active phases of the day when a host is more likely to be faced with a challenge of a pathogen [Id., citing He, W. et al. Immunity (2018) 49: 1175-1190].

[0226] Although the immune system remains constantly vigilant and primed to mount a response to antigens, research into the influence of circadian rhythm on the immune system suggests an existence of a partition of the day into two phases. The first phase is one of heightened vigilance during waking hours where most activity occurs and an immune onslaught is most likely. This is followed by a recovery phase where resolution of inflammation and tissue repair occurs in the entire organism including the skin [Id., citing Curtis, AM et al. Immunity (2014) 40: 178-186]. The modulation of the two immunological pathways in skin has been shown to be influenced by glucocorticoids and nutrient intake, which act as zeitgebers. Of these, glucocorticoids have been studied in greater depth and have been linked to the central clock in the SCN [Id., citing San Phan, T. et al. J. Sci. Adv. (2021) 7: eabe0337; Palomino-Segura, M. and Hidalgo, A. J. Exp. Med. (2021) 218: e20200798; Waggoner, SN, Curr. Allergy Asthma Rep. (2020) 20: 2; Oster, H. et al. Endocr. Rev. (2016) 38: 3-45]. The secretion of adrenocorticotropin (ACTH) from the anterior pituitary gland is under the control of the SCN [Id., citing Curtis, AM et al. Immunity (2014) 40: 178-186]. However, ablation of the adrenal glands does not lead to loss of circadian oscillation in the skin and other peripheral tissue. In addition to the fact that skin can be directly entrained, this retention of circadian rhythm could be explained by the fact that keratinocytes of the epidermal layer in the skin are capable ofregulating immune function by de novo synthesis of glucocorticoids via 11ß-hydroxylase (Cyp11b1), in addition to reactivation of inactive glucocorticoids via the enzyme 11ß- hydroxysteroid dehydrogenase type 1 (HSD11B1) [Id., citing San Phan, T. et al. J. Sci. Adv. (2021) 7: eabe0337; Buhr, ED et al. Curr. Biol. (2019) 29: 3478-3487].

[0227] Inflammation can disrupt the local, peripheral circadian clocks, as well as the central clock in the SCN. Recent reports implicate the NF-kB pathway in playing a central role in causing these perturbations [Id., citing Shen, Y. et al. PLoS Genet. (2021) 17: e1009933; Hong, HK et al. Genes Dev. (2018) 32: 1367-1379; Haspel, JA, et al. Nat. Commun. (2014) 5: 4753]. In addition to this pathway, researchers have shown that TNF-α, IFN-γ, IL-1, and LPS are capable of disrupting the oscillations of a core clock gene and the genes that they control [Id., citing Curtis, AM et al. Immunity (2014) 40: 178-186; Waggoner, SN. Curr. Allergy Asthma Rep. (2020) 20: 2; Abreu, M. et al. Sci. Rep. (2018) 8: 11474; Yoshida, K. et al. Scand. J. Rheumatol. (2013) 42: 276-280; Bashir, MM et al. Arch. Dermatol. Res. (2009) 301: 87-91; Cavadini, G. et al. Proc. Natl Acad. Sci. USA (2007) 104: 12843-12848; Cermakian, N., et al. Chronobiol. Int. (2013) 30: 870-888; Castanon-Cervantes, O. et al. J. Immunol. (2010) 185: 5796-5805; Okada, K. et al. J. Surg. Res. (2008) 145: 5-12]. In humans, epidemiological studies have also associated shift work, where the circadian clock is assumed to be disrupted, with higher risk of psoriasis [Id., citing Paganelli, R. et al. Clin. Mol. Allergy (2018) 16: 1; Wu, G. et al. Proc. Natl Acad. Sci. USA (2018) 115: 12313-12318]. These inflammatory reactions brought on by circadian disruptions are also likely to compromise the integrity of the skin, since, in human keratinocytes, TIMP3, which is a broad spectrum inhibitor of extracellular matrix (ECM)- degrading enzymes (MMPs, ADAM, ADAMTS), is likely to be under CLOCK control [Id., citing Sherratt, MJ et al. Matrix Biol. (2019) 84: 97-110; Palomino-Segura, M. and Hidalgo, A. J. Exp. Med. (2021) 218: e20200798; Waggoner, SN. Curr. Allergy Asthma Rep. (2020) 20: 2; Scheiermann, C. et al. Nat. Rev. Immunol. (2018) 18: 423-437; Matsui, MS et al. Int. J. Mol. Sci. (2016) 17: 801; Fan, D. and Kassiri, Z. Front. Physiol. (2020) 11: 661; Park, S. et al. FASEB J. (2018) 32: 1510-23; Yeom, M. et al. Molecules (2018) 23: 745]. Thus, the magnitude of immune responses in the skin are profoundly impacted by the circadian system. In turn, disruption of the circadian rhythm of the skin leads to immune hyperactivity or an aberrant immune response that can manifest as pathologies such as dermatitis or psoriasis. Influence of the Circadian Clock on Skin Homeostasis and Stress Mediation

[0228] Circadian oscillations are observed in keratinocytes and melanocytes of the epidermis and the fibroblasts of the dermis [Id., citing Sandu, C. et al. Cell. Mol. Life Sci. (2015) 72: 2237- 2248; Sandu, C. et al. Cell Mol. Life Sci. (2012) 69: 3329-3339]. The circadian clock machinery responsible for oscillations impact the metabolic processes of these cells and has an impact of tissue homeostasis. The epidermis is generated from epidermal stem cells in the basal layer that undergo asymmetric cell division, giving rise to either daughter stem cells or keratinocytes that will undergo a process of differentiation and desquamation to form the horny layer of the stratum corneum. It takes approximately 14 days for epidermal stem cells to end up as part of the stratum corneum. During this 2-week period, the process of differentiation does not occur continuously, but instead appears to occur in five sequential 24-h cyclic phases coordinated by the circadian clock. When studied via gene expression, each phase lasts for 4–5 h [Id., citing Sherratt, MJ et a. Matrix Biol. (2019) 84: 97-110; Janich, P. et al. Cell Stem Cell (2013) 13: 745-753; Eckhart, L. et al. Biochim. Biophys. Acta (2013) 1833: 3471-3480]. In differentiated keratinocytes, the genes upregulated in the first three phases under circadian control remained similar to their undifferentiated counterparts, but included genes associated with DNA damage protection and repair, indicating constant vigilance against assault to the genetic code. In the next two phases, differentiated keratinocytes seem to shift their focus to building a defensive barrier with genes for differentiation and keratin organization being upregulated. This is likely to include the surface lipids of the skin that are under clock control and contribute to the skin barrier [Id., citing Janich, P. et al. Cell Stem Cell (2013) 13: 745-753; Jia, Y et al. Exp. Dermatol. (20019) 28: 858- 862]. The differentiation process is not only dependent on the expression of the clock genes, but also their amplitude.

[0229] Melanocytes and dermal fibroblasts have also shown to possess functioning circadian clock machinery, but the amplitude of oscillations appear smaller than that of keratinocytes. Despite this, the circadian clock plays a functional role in melanocytes by controlling the abundance of melanosomes, as well as the expression of melanin synthesis enzyme, Tyrosinase and the phosphorylation of melanocyte inducing transcription factor (MITF), which increases when BMAL1 or PER1 are silenced [Id., citing Hardman, JA et al. J. Investig. Dermatol. (2015) 135: 1053-1064; Slominski, AT et al. J. Investig. Dermatol. (2015) 135: 943-945]. The protein OPN4 (Melanopsin) has been shown to affect the molecular clock components and their responsiveness to classical clock activators in melanocytes. Knocking out OPN4 in melanocytesresulted in rapid cell cycle progression and increased cellular proliferation, which correlated with the altered gene expression of MITF and the core circadian clock components [Id., citing De Assis, LVM et al. Curr. Issues Mol. Biol. (2021) 43: 1436-1450]. The impact of the function of the circadian clock on dermal fibroblasts is yet to be characterized. However, the influence of circadian rhythm on the synthesis and secretion of Type I collagen, a major component of the ECM of the dermis, is already known [Id., citing Chang, J. et al. Nat. Cell Biol. (2020) 22: 74- 86]. Furthermore, the efficiency of migration and adhesion of fibroblasts modulated via actin dynamics was found to be circadian regulated [Id., citing Hoyle, NP et al. Sci. Transl. Med. (2017) 9: eaal2774].

[0230] Not only is normal function of skin impacted by circadian oscillations, but also the ability of skin to deal with stress is influenced by the cellular clock. As described above, the genes involved in DNA damage protection and repair in the epidermis are under clock control, as well as genes that mediate oxidative stress responses, in particular NRF2, the peroxiredoxins, glutathione peroxidase, and sestrins [Id., citing Ishii, T. et al. Free Radic. Biol. Med. (2018) 119: 34-44; Ndiaye, MA et al. Antioxid. Redox. Signal. (2014) 20: 2982-2996; Edgar, RS et al. Nature 92012] 485: 459-64; Avitabile, D. et al. Intl J. Biochem. Cell Biol. (2014) 53: 24-34; Kolinjivadi, AM et al. J. Endocr. Re.at. Cancer (2021) 28: R55-R66; Kondratov, RV et al. Aging (2009) 1: 979-987]. In human skin, the activity of the DNA repair enzyme 8-oxoguanine DNA glycosylase (OGG1) was higher at night [Id., citing Manzella, N. et al. Sci. Rep. (2015) 5: 13752]. This is particularly important in the case of melanocytes, since they can accumulate DNA damage long after UV exposure, via melanin excitation [Id., citing Lyons, AB et al. J. Clin. Aesthet. Dermatol. (2019) 12: 42-45; Premi, S. et al. Science (2015) 347: 842-847].

[0231] The evolutionary conserved hormone melatonin is also responsible for combating DNA damage and oxidative stress, as well as maintaining skin homeostasis [Id., citing Slominski, AT et al. J. Investig. Dermatol. (2018) 138: 490-499]. This correlates with the finding that the circadian rhythm of melanin secretion is disrupted in psoriatic patients [Id., citing Mozzanica, N. et al. Acta Derm. Venereol. (1988) 68: 312-16]. Since this molecule and its related metabolites are free radicle scavengers, it is capable of stress mediation [Id., citing Slominski, AT et al. J. Investig. Dermatol. (2018) 138: 490-499; Ndiaye, MA et al. Antiox. Redox Signal. (2014) 20: 2982-2996]. Skin pigmentation and hair growth are also controlled by melatonin; its activity may also be influenced by the skin’s circadian clock [Id., citing Hardman, JA et al. J. Investig.Dermatol. (2015) 135: 1053-1064; Slominski, AT et al. J. Investig. Dermatol. (2015) 135: 943- 45; Al-Nuaimi, Y. et al. J. Investig. Dermatol. (2014) 134: 610-619]. Melatonin has also been implicated in the control of skin and body temperature in a circadian manner. In rat skin, the circadian clock machinery dermal fibroblasts is capable of using melatonin as an internal signal to fine-tune its oscillations, with temperature being used as an external queue [Id., citing Cuesta, M. et al. J. Biol. Rhythms (2017) 32: 257-273]. The circadian clock and seasonality

[0232] Physiological changes in human skin are a consequence of seasonality triggered by several parameters such as light irradiation of various wavelengths with changing intensities over the year, temperature, temperature shifts between indoors and outdoors, humidity, and sweat resulting in, for example, a decrease in pH due to acidification and wind accelerated evaporation, thus modulating trans-epidermal water loss (TEWL). The seasonal temperature changes modulate blood microcirculation and thus the accessibility to nutrients, which contributes to physiological skin changes. Intersection of Circadian rhythms and aging

[0233] The role of circadian rhythm and its relationship with skin aging is only now beginning to be understood. The rhythmicity of multiple features are altered with age. This includes sleep, body temperature cycles, and locomotor activity [Id., citing Duan, J. et al. FEBS Lett. (2021) 595: 2413-2436; Hood, S. and Amir, S. J. Clin. Investig. (2017) 127: 437-446]. For circadian rhythm, the amplitude of oscillation of clock controlled genes decreases with age in peripheral tissue, but not in the SCN, particularly when Per2 is being tracked [Id., citing Buijink, MR et al. J. Biol. Rhythms (2020) 35: 167-179]. This indicates that age-related circadian alteration in peripheral clocks are independent of the SCN clock. In aged skin, this problem is further compounded by the presence of senescent cells. Senescent cells show dampened circadian rhythmicity and are less efficient in the transmission of circadian signals to their clocks [Id., citing Kunieda, T. et al. Circ. Res. (2006) 98: 532-539]. Therefore, senescence is implicated as the mechanism by which aging impairs entrainment of peripheral circadian clocks [Id., citing Kunieda, T. et al. Cir. Res. (2006) 98: 532-539; Ahmed, R. et al. Front. Neurosci. (2021) 15: 638122]. Furthermore, aged dermal fibroblasts secrete a unique aging-associated set of proteins, distinct from the canonical senescence-associated secretory phenotype. Among these includemultiple candidates involved in inflammatory signaling and maintenance or alteration of the tissue microenvironment [Id., citing Waldera Lupa, DM et al. J. Investig. Dermatol. (2015) 135: 195419-68]. Thus, there is a high likelihood that aging via senescence is directly capable of disrupting the above mentioned immunological and stress mediatory pathways in skin. Since the rigidity of the tissue microenvironment is also impacted, aging also likely dampens the circadian clock of keratinocytes (which prefer a softer matrix) and fibroblasts (which prefer a firmer matrix) [Id., citing Williams, J. et al. J. Cell Sci. (2018) 131: jcs208223; Yang, N. et al. Nat. Commun. (2017) 8: 14287].

[0234] The circadian oscillations of epidermal cells remain robust even under aged conditions, but they are rewired to adapt to the stressors associated with an aged environment and remain committed to development and maintenance of the skin barrier through daily rhythmic cell division, in spite of DNA damage they many have incurred [Id., citing Solanas, G. et al. Cell (2017) 170: 678-692]. Plant-derived exosomes affect a human keratinocyte cell line in vitro

[0235] Plant-derived secondary metabolites are natural substances including alkaloids, flavonoids, polyphenols, terpenoids, and quinones; these are widely used as cosmeceuticals (cosmetic products that contain bioactive ingredients with potential therapeutic benefits for the skin) because they exert beneficial effects on the human skin, such as antiaging, moisturizing, whitening, regeneration, and nutritional supply. [Cho, JH et al. Applied Biological Chemistry (2022) 65: 8]. Typical examples are ginseng (Panax ginseng) and green tea (Camellia sinensis). Ginseng has several ginsenosides as representative active ingredients and exerts antiaging, anti- inflammatory, and antioxidative effects [Id., citing Kang, TH et al. J. Ethnopharmacol. (2009) 123: 446-451; Hong, CE and Lyu, SY. Immune Netw. (2011) 11: 42-49; Park, HJ et al. J. Ginseng Res. (2012) 36: 225-414-415]. Green tea has reportedly been effective in antioxidation, photoprotection, and improvement in skin-related conditions owing to the active component of flavonoids including catechin and polyphenols [Id., citing Yarnell, E. and Abascal, K. Altern. Compl. Ther. (2012) 18: 141-144; Meetham, P. et al. Rev. Bras. Farmacogn. (2018) 28: 214-217; Koch, W. et al. Molecules (2019) 24: 4277].

[0236] To assess whether plant exosomes exert effects on human cells, extracts and exosomes from ginseng and green tea were used to treat keratinocytes in vitro and the change intranscriptosomes was compared to analyze the differential effect on cells. To prepare plant extracts, the leaf part of Green tea (1 kg) and the root part of Ginseng plants were hot air dried at 45°C for 24 h, finely ground using a blender and 25g then extracted in 25 L of distilled water at 80°C for 3hr and filtered through a 0.45 um mesh filter. Exosomes were prepared by ultracentrifugation or an aqueous two-phase extraction system (ATPS).

[0237] A Human Epidermal Keratinocyte (HaCaT) cell line was cultured in Dulbecco Modified Eagles Medium supplemented with 10% fetal bovine serum (FBS), 1% penicillin and 1% streptomycin at 37 C, 5% CO2. Keratinocytes were seeded at a density of 1 x 105in a 35 mm cell culture plate and incubated for 24h. After incubation, cells were treated with fresh media containing 2% of a water extract or 1 x 108 / mL plant exosomes and mock-treated with distilled water as a control for 6 h. Extracts are normally treated for^>^24 h, so this 6 hr treatment window was shorter than normally required. Experiments were conducted when cell density reached 70- 80% confluency. Exosomes showed a Gaussian distribution of nanoparticles within the range of 87-226 nm, inclusive, for ginseng and 108-234 nm, inclusive, for green tea.

[0238] The exosome treatment group had significantly higher numbers of expressed genes than the extract treatment or control groups. In the extract treatment group, 386 genes and 420 genes were upregulated for ginseng and green tea, respectively and 390 and 460 genes were downregulated. In the exosome treatment group, 877 and 843 genes were upregulated and 658 and 620 genes were downregulated for ginseng and green tea, respectively. This result implies that exosomes were effectively absorbed to keratinocytes. A previous study had reported that cabbage-derived exosomes were effectively absorbed to human cells by penetrating into them [Id., citing You, JY et al. Bioact. Mat. (2021) 6: 4321-4332].

[0239] Expression patterns of genes related to skin aging, regenerating, barriers, and moisturizing were analyzed. Gene expression level of MMP12, MMP13, and NOTCH3, which are associated with skin aging, reportedly increase when exposed to light stress such as UVB and cause decomposition of collagen and skin aging [Id., citing Tewari, A. et al. J. Invest. Dermatol. (2014) 134: 2598-2609; Pittayapruek, P. et al. Int. J. Mol. Sci. (2016) 17: 868; Rossi, M. and Abdelmohsen, K. Cells (2021) 10: 1740]. Expression of these genes significantly decreased in the exosome treatment groups of ginseng and green tea, with no notable changes in the extract treatment groups. However, a previous study reported that the gene expression was decreasedby^>^twofold change when keratinocytes were treated with compound K, obtained from ginseng extract, for 24 h [Id., citing Kim, S. et al. Biochem. Biophys. Res. Commun. (2004) 316: 348- 55]. Analyzing the expression of genes related to skin regeneration revealed that fibroblast growth factor 12 (FGF12), which suppresses radiation damage to skin tissue, increased specifically in the ginseng exosome treatment group [Id., citing Nakayama, F. et al. J. Biol. Chem. (2011) 286: 25823-34]. A gene encoding a heparin sulfate sulfotransferase enzyme (HS3ST3A1), which facilitates skin regeneration by affecting skin regeneration signal delivery, and a lysyl oxidase (LOX) gene, which considerably influences healing of skin wounds by facilitating ECM stabilization through ECM formation, development, maturation and remodeling showed an increase in the exosome treatment group [Id., citing Patel, VN et al. Dev. Cell (2014) 29: 662-673; Cai, L. et al. Tissue Eng. Regen. Med. (2017) 14: 15-30]. A vimentin (VIM) gene, which affects the differentiation and migration of keratinocytes; ELOVL3, whose protein product belonged to a highly conserved family of microsomal enzymes involved in the formation of very long chain fatty acids (VLCFA), which affects lipid acid biosynthesis; and a keratin 1 (KRT1) gene whose protein product is a fibrillary protein that form skin, hair, and nails, are all recognized as genes that construct skin barrier and prevent water loss [Id., citing Velez-delValle, C. et al. Sci. Rep. (2016) 6: 24389; Cheng, F. et al. Proc. Nat. Acad. Sci. USA (2016) E4320- E4327; Westerberg, R. et al. J. Biol. Chem. (2004) 279: 5621-5629; Roth, W. et al. J. Cell Sci. (2012) 125: 5269-5279]. These genes responded particularly to the exosome treatment group and showed the highest increase in response to ginseng exosomes. The medicinal plant Aloe Vera: pharmacological properties

[0240] Aloe vera (Aloe barbadensis Miller, family Xanthorrhoeaceae) is a perennial green herb with bright yellow tubular flowers that is extensively distributed in hot and dry areas of North Africa, the Middle East of Asia, the Southern Mediterranean, and the Canary Islands. The colorless mucilaginous gel from Aloe vera leaves has been extensively used for pharmacological and cosmetic applications. Traditionally, this medicinal plant has been employed to treat skin problems (burns, wounds, and anti-inflammatory processes). Moreover, Aloe vera has shown other therapeutic properties including anticancer, antioxidant, antidiabetic, and antihyperlipidemic effects. Aloe vera contains more than 75 different compounds, including vitamins (vitamin A, C, E, and B12), enzymes (i.e., amylase, catalase, and peroxidase), minerals (i.e., zinc, copper, selenium, and calcium), sugars (monosaccharides such as mannose-6-phosphate and polysaccharides such as glucomannans), anthraquinones (aloin and emodin), fatty acids (i.e., lupeol and campesterol), hormones (auxins and gibberellins), and others (i.e., salicylic acid, lignin, and saponins) [Sanchez, M. et al. Molecules (2020) 25: 1324, citing Surjushe, A. et al. Indian J. Dermatol. (2008) 53: 163-166; Malik, I and Zarnigar, HN. Int. Res. J. Phar. (2003) 4: 75-79; Maan, AA et al. J. Herb. Med. (2018) 12: 1-10]. Skin protection

[0241] Most in vitro studies on skin protection study the ability of Aloe vera and active compounds in wound healing. The immortalized human keratinocyte HaCaT cell line, the primary normal human epidermal keratinocytes HEKa cell line, and fibroblast cell lines are the most used. These studies have revealed that Aloe vera and its major compounds (aloesin, aloin, and emodin) exert their protective action mainly through antioxidant and anti-inflammatory mechanisms.

[0242] The most common models for in vivo studies are genetically modified animals (BALB / c mice, HR-1 hairless mice and SKH-1 hairless mice) and UV and X-ray skin damage in animals. Most of these in vivo studies have been done with Aloe vera extracts and gel. Application of topical Aloe vera favored wound healing in animal models with dermal incisions by reducing inflammatory cell infiltration, increasing CD4+ / CD8+ ratio lymphocytes, and improving epidermal thickness and collagen deposition [Id., citing Brandao, ML et al. Acta Cir. Bras. (2016) 31: 570-577; Oryan, A. et al. Annals Plast. Sug. (2016) 77: 37-46; Takzaree, N. t al. J. Physiol. Pharm. (2016) 94: 1285-90; Yos Adi Prakoso, K. J. Trop. Med. (2018) 2018: 6218303]. Clinical trials have demonstrated that Aloe vera facilitated rapid tissue epithelialization and granulation in burns [Id., citing Irani, PS and Varaie, S. Iran. J. Med. Sci. (2016) 41: S3], promoted healing of cesarean wounds [Id., citing Molazem, Z. et al. Global J. Health Sci. (2015) 7: 203], and accelerated wound healing of split-thickness skin graft donor sites [Id., citing Burusapat, C. et al. Plast. Reconstr. Surg. (2018) 142: 217-236]. Furthermore, Aloe vera has been investigated in randomized, double-blind, placebo-controlled studies for its benefits to maintain healthy skin. The daily oral intake of 40 µg of Aloe sterol (cycloartenol and lophenol) for at least 12 weeks improved skin elasticity in men under 46 years exposed to the sunlight but who do not use sunscreen to protect themselves [Id., citing Tanaka, M. et al. Clin. Cosmet. Invest. Dermat. (2016) 9: 435-442], reduced facial wrinkles in Japanese women over 40 yearsold by stimulating hyaluronic acid and collagen production [Id., citing Tanaka, M. et al. Clinical Cosmet. Invest. Dermat. (2015) 8: 95-104], and increased gross elasticity, net elasticity, and biological elasticity in women aged 30–59 [Id., citing Tanaka, M. et al. Skin Pharmacol. Physiol. (2016) 29: 309-317]. Anti-inflammatory and anti-oxidant activity

[0243] Most recent studies on anti-inflammatory activity of Aloe vera are focused on the action mechanism of isolated compounds in murine macrophage RAW264.7 cells and mice stimulated with LPS. Hence, the potential anti-inflammatory effect of aloin was reported to be related to its ability to inhibit cytokines, ROS production, and the JAK1-STAT1 / 3 signaling pathway [Id., citing Ma, Y. et al. Int. J. Mol. Med. (2018) 42: 1925-1934; Jiang, K. et al. Int. Immunopharm. (2018) 64: 140-50]. Moreover, aloe-emodin sulfates / glucuronides (0.5 μM), rhein sulfates / glucuronides (1.0 μM), aloe-emodin (0.1 μM), and rhein (0.3 μM) inhibited pro- inflammatory cytokines and nitric oxide production, iNOS expression, and MAPKs phosphorylation [Id., citing Li, CY et al. Am. J. Chin. Med. (2017) 45: 847-861].

[0244] The antioxidant activity of Aloe vera has been attributed, at least in part, to anthraquinones and related compounds (10 µM) which possess peroxyl radical scavenging activity and reducing capacity [Id., citing Sun, YN et al. Nat. Prod. Res. (2017) 31: 2810-2813].

[0245] The present disclosure provides compositions containing exosome-like nanoparticles derived from plants that can be formulated for delivery to human skin. The plant exosomes are derived from plants of the family Asphodelaceae comprising the genus Aloe; or the plant is from Family Papaveraceae and is a Celandine plant; or the plant is from Family Passifloraceae and is a Passiflora ligularis or a Passiflora edulis plant. Plants can be subjected to two or more abiotic environmental stresses selected from high / low temperature, salinity, drought, light stresses, flooding, physical wounding, or heavy metal pollutants, which produce secondary plant stresses comprising oxidative stress and osmotic stress. The plants’ response to these abiotic stresses is to generate exosomes comprising cargo comprising nucleic acids (e.g., miRNAs), plant proteins including heat shock proteins, and lipids that in vivo would enable adjustments of its signaling pathways and metabolism to ensure its growth and development in a challenging environment. The present disclosure provides that instead, the plant exosomes will be harvested for cosmetic, cosmeceutical or therapeutic applications in human subjects. We have demonstrated that plantexosomes can bind and release a signature cargo into human skin keratinocytes. The released cargo can promote survival of keratinocytes, fibroblasts and endothelial cells resident in the skin; reduce oxidative stress, modulate inflammation, modulate perturbations of the circadian clock of the skin comprising immune responses comprising inflammation; restore youthful appearance to the skin by producing collagen and elastin components that give skin its turgor, and maintain the integrity of the statum corneum and prevent water loss. The nanoparticles offer advantageous properties stemming from the tunable delivery of the signature cargo comprising natural biochemicals derived from the origin plants with no evidence of plant-induced inflammation or toxicity. SUMMARY OF THE INVENTION

[0246] According to one aspect, the present disclosure provides a composition comprising a purified population of plant-derived exosome-like nanoparticles isolated from tissue of a vascular plant, wherein size of the exosome-like nanoparticles is about 50 nm-500 nm inclusive; wherein the exosome-like nanoparticles comprise a tuned cargo comprising a signature of miRNAs selected from ath-miR166a-3p; ath-miR166b-3p; ath-miR166e-3p; ath-miR396a-5p; ath-miR396b-5p; ath-miR396b-5p; ath-miR156ff-5p; ath-miR168b-5p; ath-miR156c-5p; ath- miR162a-3p; ath-miR162b-3p; ath-miR396a-3p; ath-miR168a-5p; ath-miR156b-5p; ath-mi156a- 5p; ath-miR156d-5p; ath-miR164c-5p; ath-miR408-3p; ath-miR165a-3p; ath-miR160a-5p; ath- miR157b-5p; ath-miR157a-5p; ath-miR164b-5p; ath-miR5016; ath-miR5998b; ath-miR5020a; ath-miR836; ath-miR158a-5p; ath-miR395e; ath-miR402; ath-miR5662; ath-miR5630a; ath- miR5630b; ath-miR3933; ath-miR5998a; ath-miR172e-3p; ath-miR5024-3p; ath-miR447a-3p; ath-miR414; ath-miR167a-3p; ath-miR172b-5p; ath-miR5636; ath-miR824-3p; ath-miR172e-5p; ath-miR404; ath-miR447b; ath-miR826b; ath-miR169g-5p; ath-miR868-3p; ath-miR830-3p; ath- miR169f-5p; ath-miR828; ath-miR8182; ath-miR160a-3p; ath-miR5635d; ath-miR399c-3p; ath- miR5635c; ath-miR398a-3p; ath-miR391-5p; ath-miR781a; ath-miR157c-3p; ath-miR399b; ath- miR5014b; ath-miR5635b; ath-miR779.2; ath-miR390b-5p; ath-miR833a-3p; ath-miR849; ath- miR5635a; ath-miR841b-3p; ath-miR390a-5p; ath-miR447c-3p; ath-miR835-3p; ath-miR5638b; ath-miR2112-3p; ath-miR5653; ath-miR166a-5p; ath-miR159b-5p; ath-miR166b-5p; ath- miR843; ath-miR5015; ath-miR781b; ath-miR4245; ath-miR169b-5p; ath-miR5013; ath- miR864-5p; ath-miR866-5p; ath-miR5595a; ath-miR403-3p; ath-miR164c-3p; ath-miR835-5p; ath-miR165b; ath-miR3434-5p; ath-miR8176; ath-miR5631; ath-miR399a; ath-miR4227; ath-miR5666; ath-miR778; ath-miR851-3p; ath-miR5663-3p; ath-miR832-3p; ath-miR5646; ath- miR856; ath-miR837-5p; ath-miR846-3p; ath-miR827; ath-miR5633; ath-miR413; ath-miR838; ath-miR5654-5p; ath-miR172d-5p; ath-miR5642a; ath-miR420; ath-miR831-3p; ath-miR156d- 3p; ath-miR5018; ath-miR8168; ath-miR866-3p; ath-miR8170-3p; ath-miR395b; ath-miR395c; ath-miR780.2; ath-miR167c-5p; ath-miR393a-3p; ath-miR395f; or a combination thereof; and a signature of proteins selected from the group consisting of heat shock protein (HSP) chaperones; heat shock transcription factors (Hsfs); a ribulose 1,5-bisphosphate carboxylase / oxygenase subunit; a calcium dependent protein kinase, a PLA8 family protein; a glutathione transferase, or a combination thereof; wherein the tuned cargo of the plant-derived exosome-like nanoparticles is produced by exposure of the plant to combinations of abiotic stress conditions that cause the plant to modulate its signaling pathways and metabolism to ensure its survival in a challenging environment.

[0247] According to some embodiments of the composition, the plant is of Family Asphodelaceae or is an aloe vera plant. According to some embodiment, the plant is from Family Papaveraceae and is a Celandine plant. According to some embodiments, the plant is from Family Passifloraceae and is a Passiflora ligularis or a Passiflora edulis plant. According to some embodiments, the plant is from the Family Rubiaceae and is a Morinda citrifolia plant.

[0248] According to some embodiments, the plant tissue includes roots, stems, leaves, flowers, seeds, fruits, a liquid extract of the plant tissue, a nut milk, or a combination thereof.

[0249] According to some embodiments, the HSP chaperones induced under the abiotic stress conditions include HSP100, HSP90, HSP70, HSP60 a small HSP; or a combination thereof; and the Hsfs induced under the abiotic stress conditions include HsfA, HsfB or HsfC, or a combination thereof.

[0250] According to some embodiments, primary abiotic stress conditions, which include high / low temperature; salinity; drought; dehydration; flooding; heavy metal chemical pollutants; light stresses or physical wounding, produce secondary stresses comprising oxidative stress and osmotic stress.

[0251] According to some embodiments, the tuned cargo of the plant-derived exosomes is a result of exposure of the plant to two high temperature abiotic stress conditions.

[0252] According to some embodiments, the tuned protein cargo of the plant-derived exosomes correlates to a protein signature comprising human proteins including a keratin; semaphorin receptor plexin-B1 mitogen-activated protein kinase kinase 2 (MEKK2), diacylglycerol kinase; T cell receptor beta chain; a fez family zinc finger protein or a combination thereof.

[0253] According to some embodiments, the tuned protein cargo of the plant-derived exosomes can modulate bioactivities of mammalian cells directly or indirectly.

[0254] According to some embodiments, the mammalian cells are human cells; and the bioactivities comprise one or more correlated signaling pathways in the human cells. According to some embodiments, the human mammalian cells are cells of human skin.

[0255] According to some embodiments, the correlated human signaling pathways includes PI3K signaling, ERK / MAPK signaling; insulin growth factor 1 receptor (IGF1R) signaling, VEGFA / VEGFR2 signaling; leptin signaling; cytokine signaling; interleukin signaling, semaphorin signaling; sirtuin signaling; LRP1 signaling, or a combination thereof.

[0256] According to some embodiments, administration of the composition comprising the exosome-like nanoparticles comprising the tuned cargo modulates: collagen production in human dermal fibroblasts in vitro; or elastin production in human dermal fibroblasts in vitro; or hyaluronic acid production in human dermal fibroblasts in vitro; or interferon a2 production in mammalian PBMCs exposed to a microbial agent in vitro; or VEGFA production in human dermal fibroblasts in vitro; or a combination thereof.

[0257] According to some embodiments, the composition is a nutraceutical composition comprising a dietary amount of the purified plant-derived exosomes comprising the tuned cargo; or the composition is a cosmetic composition comprising a cosmetic amount of the purified plant-derived exosomes and a cosmetically acceptable carrier; or the composition is a cosmeceutical composition comprising a cosmeceutical amount of the purified plant-derived exosomes and a cosmeceutically acceptable carrier; or the composition is a therapeutic composition; comprising a therapeutic amount of the purified plant-derived exosomes and a pharmaceutically acceptable carrier.

[0258] According to another aspect, the present disclosure provides a method for improving appearance of human skin comprising: exposing a vascular plant to combinations of abioticstress conditions that cause the plant to modulate its signaling pathways and metabolism to ensure its survival in a challenging environment; purifying from tissue of the vascular plant exposed to the combinations of abiotic conditions a population of plant-derived exosome-like nanoparticles (plant-derived exosomes) comprising a tuned cargo, wherein size of the plant- derived exosomes is about 50 nm-500 nm inclusive; preparing a composition comprising about 1x10E8 to about 1x10E12, inclusive, abiotically stressed plant-derived exosome-like nanoparticles containing the tuned cargo and a cosmetically acceptable carrier; and applying the composition topically to human skin; wherein the tuned cargo of the plant-derived exosomes comprises: a signature of miRNAs selected from ath-miR166a-3p; ath-miR166b-3p; ath- miR166e-3p; ath-miR396a-5p; ath-miR396b-5p; ath-miR396b-5p; ath-miR156ff-5p; ath- miR168b-5p; ath-miR156c-5p; ath-miR162a-3p; ath-miR162b-3p; ath-miR396a-3p; ath- miR168a-5p; ath-miR156b-5p; ath-mi156a-5p; ath-miR156d-5p; ath-miR164c-5p; ath-miR408- 3p; ath-miR165a-3p; ath-miR160a-5p; ath-miR157b-5p; ath-miR157a-5p; ath-miR164b-5p; ath- miR5016; ath-miR5998b; ath-miR5020a; ath-miR836; ath-miR158a-5p; ath-miR395e; ath- miR402; ath-miR5662; ath-miR5630a; ath-miR5630b; ath-miR3933; ath-miR5998a; ath- miR172e-3p; ath-miR5024-3p; ath-miR447a-3p; ath-miR414; ath-miR167a-3p; ath-miR172b- 5p; ath-miR5636; ath-miR824-3p; ath-miR172e-5p; ath-miR404; ath-miR447b; ath-miR826b; ath-miR169g-5p; ath-miR868-3p; ath-miR830-3p; ath-miR169f-5p; ath-miR828; ath-miR8182; ath-miR160a-3p; ath-miR5635d; ath-miR399c-3p; ath-miR5635c; ath-miR398a-3p; ath-miR391- 5p; ath-miR781a; ath-miR157c-3p; ath-miR399b; ath-miR5014b; ath-miR5635b; ath-miR779.2; ath-miR390b-5p; ath-miR833a-3p; ath-miR849; ath-miR5635a; ath-miR841b-3p; ath-miR390a- 5p; ath-miR447c-3p; ath-miR835-3p; ath-miR5638b; ath-miR2112-3p; ath-miR5653; ath- miR166a-5p; ath-miR159b-5p; ath-miR166b-5p; ath-miR843; ath-miR5015; ath-miR781b; ath- miR4245; ath-miR169b-5p; ath-miR5013; ath-miR864-5p; ath-miR866-5p; ath-miR5595a; ath- miR403-3p; ath-miR164c-3p; ath-miR835-5p; ath-miR165b; ath-miR3434-5p; ath-miR8176; ath-miR5631; ath-miR399a; ath-miR4227; ath-miR5666; ath-miR778; ath-miR851-3p; ath- miR5663-3p; ath-miR832-3p; ath-miR5646; ath-miR856; ath-miR837-5p; ath-miR846-3p; ath- miR827; ath-miR5633; ath-miR413; ath-miR838; ath-miR5654-5p; ath-miR172d-5p; ath- miR5642a; ath-miR420; ath-miR831-3p; ath-miR156d-3p; ath-miR5018; ath-miR8168; ath- miR866-3p; ath-miR8170-3p; ath-miR395b; ath-miR395c; ath-miR780.2; ath-miR167c-5p; ath- miR393a-3p; ath-miR395f; or a combination thereof; and a signature of proteins selected fromthe group consisting of heat shock protein (HSP) chaperones; heat shock transcription factors (Hsfs); a ribulose 1,5-bisphosphate carboxylase / oxygenase subunit; a calcium dependent protein kinase, a PLA8 family protein; a glutathione transferase; or a combination thereof.

[0259] According to some embodiments of the method, the plant is a plant of Family Asphodelaceae or an aloe vera plant; or the plant is from Family Papaveraceae and is a Celandine plant; or the plant is from Family Passifloraceae and is a Passiflora ligularis or a Passiflora edulis plant. According to some embodiments, the plant is from the Family Rubiaceae and is a Morinda citrifolia plant.

[0260] According to some embodiments, the plant tissue includes roots, stems, leaves, flowers, seeds, fruits, a liquid extract of the plant tissue, a nut milk or a combination thereof.

[0261] According to some embodiments, the HSP chaperone induced under the abiotic stress conditions include HSP100, HSP90, HSP70, HSP60, a small HSP or a combination thereof; and the Hsf induced under the abiotic stress conditions include HsfA, HsfB or HsfC, or a combination thereof.

[0262] According to some embodiments, primary abiotic stress conditions including high / low temperature; salinity; drought; dehydration; flooding; heavy metal chemical pollutants; light stresses or physical wounding produce secondary stresses comprising oxidative stress and osmotic stress.

[0263] According to some embodiments, the tuned protein cargo of the plant-derived exosome- like nanoparticles correlates to a protein signature comprising human proteins including a keratin; semaphorin receptor plexin-B1 mitogen-activated protein kinase kinase 2 (MEKK2), diacylglycerol kinase; T cell receptor beta chain; fez family zinc finger protein or a combination thereof.

[0264] According to some embodiments, the tuned protein cargo of the plant-derived exosomes can modulate bioactivities of mammalian cells directly or indirectly.

[0265] According to some embodiments, the bioactivities comprise one or more correlated signaling pathways in the human cells.

[0266] According to some embodiments, the correlated signaling pathways in the human cells include PI3K signaling, ERK / MAPK signaling; insulin growth factor 1 receptor (IGF1R)signaling, VEGFA / VEGFR2 signaling; leptin signaling; cytokine signaling; interleukin signaling, semaphorin signaling; sirtuin signaling; LRP1 signaling, or a combination thereof.

[0267] According to some embodiments, the composition comprising the tuned cargo of the exosome-like plant nanoparticles when applied to the skin may modulate gene expression in immune cells, keratinocytes, melanocytes or fibroblasts in the skin; modulate a signaling pathway that contributes to inflammation, immune dysfunction or both in the skin; modulate circadian rhythms of the skin and its components; rejuvenate appearance of the skin by improving youthful appearance of skin; reducing appearance of wrinkles by stimulating hyaluronic acid and collagen production; improving skin clarity; improving skin texture; improving skin luminosity; improving skin radiance; or a combination thereof.

[0268] According to some embodiments, the tuned protein cargo of the plant-derived exosome- like nanoparticles correlates to a protein signature comprising a huma protein in a signaling pathway in human skin, wherein the signaling pathway is a PI3K / AKT / mTOR pathway, an MAPK pathway, an IGF-1R pathway, a sirtuin pathway, an LRP1 pathway, or a combination thereof.

[0269] According to another aspect, the present disclosure provides a method for promoting hair health, comprising: exposing a vascular plant to combinations of abiotic stress conditions that cause the plant to modulate its signaling pathways and metabolism to ensure its survival in a challenging environment; purifying from tissue of the vascular plant exposed to the combinations of abiotic conditions a population of plant-derived exosome-like nanoparticles (plant-derived exosomes), comprising a tuned cargo wherein size of the plant-derived exosomes is about 50 nm-500 nm inclusive; preparing a composition comprising about 1x10E8 to about 1x10E12, inclusive, abiotically stressed plant-derived exosome-like nanoparticles containing the tuned cargo and a cosmetically acceptable carrier; and applying the composition topically to a subject in need thereof; wherein the tuned cargo comprises: a signature of miRNAs selected from ath- miR166a-3p; ath-miR166b-3p; ath-miR166e-3p; ath-miR396a-5p; ath-miR396b-5p; ath- miR396b-5p; ath-miR156ff-5p; ath-miR168b-5p; ath-miR156c-5p; ath-miR162a-3p; ath- miR162b-3p; ath-miR396a-3p; ath-miR168a-5p; ath-miR156b-5p; ath-mi156a-5p; ath-miR156d- 5p; ath-miR164c-5p; ath-miR408-3p; ath-miR165a-3p; ath-miR160a-5p; ath-miR157b-5p; ath- miR157a-5p; ath-miR164b-5p; ath-miR5016; ath-miR5998b; ath-miR5020a; ath-miR836; ath-miR158a-5p; ath-miR395e; ath-miR402; ath-miR5662; ath-miR5630a; ath-miR5630b; ath- miR3933; ath-miR5998a; ath-miR172e-3p; ath-miR5024-3p; ath-miR447a-3p; ath-miR414; ath- miR167a-3p; ath-miR172b-5p; ath-miR5636; ath-miR824-3p; ath-miR172e-5p; ath-miR404; ath-miR447b; ath-miR826b; ath-miR169g-5p; ath-miR868-3p; ath-miR830-3p; ath-miR169f-5p; ath-miR828; ath-miR8182; ath-miR160a-3p; ath-miR5635d; ath-miR399c-3p; ath-miR5635c; ath-miR398a-3p; ath-miR391-5p; ath-miR781a; ath-miR157c-3p; ath-miR399b; ath-miR5014b; ath-miR5635b; ath-miR779.2; ath-miR390b-5p; ath-miR833a-3p; ath-miR849; ath-miR5635a; ath-miR841b-3p; ath-miR390a-5p; ath-miR447c-3p; ath-miR835-3p; ath-miR5638b; ath- miR2112-3p; ath-miR5653; ath-miR166a-5p; ath-miR159b-5p; ath-miR166b-5p; ath-miR843; ath-miR5015; ath-miR781b; ath-miR4245; ath-miR169b-5p; ath-miR5013; ath-miR864-5p; ath- miR866-5p; ath-miR5595a; ath-miR403-3p; ath-miR164c-3p; ath-miR835-5p; ath-miR165b; ath-miR3434-5p; ath-miR8176; ath-miR5631; ath-miR399a; ath-miR4227; ath-miR5666; ath- miR778; ath-miR851-3p; ath-miR5663-3p; ath-miR832-3p; ath-miR5646; ath-miR856; ath- miR837-5p; ath-miR846-3p; ath-miR827; ath-miR5633; ath-miR413; ath-miR838; ath- miR5654-5p; ath-miR172d-5p; ath-miR5642a; ath-miR420; ath-miR831-3p; ath-miR156d-3p; ath-miR5018; ath-miR8168; ath-miR866-3p; ath-miR8170-3p; ath-miR395b; ath-miR395c; ath- miR780.2; ath-miR167c-5p; ath-miR393a-3p; ath-miR395f; or a combination thereof; and a signature of proteins selected from the group consisting of heat shock protein (HSP) chaperones; heat shock transcription factors (Hsfs); a ribulose 1,5-bisphosphate carboxylase / oxygenase subunit; a calcium dependent protein kinase, a PLA8 family protein; a glutathione transferase; or a combination thereof, wherein the composition increases proliferation of dermal papillae cells and hair follicle stem cells and increases hair growth.

[0270] According to some embodiments, applying topically includes applying to scalp, eyebrows, eyelashes or a combination thereof of the subject.

[0271] According to some embodiments, the method decreases hair loss; increases hair density; increases appearance of hair thickness; improves scalp health; improves hair shine; improves hair volume or body; or a combination thereof.

[0272] According to some embodiments, the composition comprising the tuned cargo of the exosome-like plant nanoparticles, when applied topically, modulates gene expression in the dermal papillae cells, hair follicle stem cells, or a combination thereof.

[0273] According to some embodiments, the composition comprising the tuned cargo of the exosome-like plant nanoparticles, when applied topically, modulates gene expression in human follicle dermal papilla cells (HFDPCs).

[0274] According to some embodiments, the composition comprising the tuned cargo of the exosome-like plant nanoparticles, when applied topically, modulates gene expression of IL-6 in HFDPCs 24 hours after application.

[0275] According to some embodiments, the composition comprising the tuned cargo of the exosome-like plant nanoparticles, when applied topically, modulates gene expression of CORIN, LEP, IL1B, IL-6, SRD5A2, BMP4, TGFB1, IGF1, HEY1, or a combination thereof in HFDPCs 72 hours after application. BRIEF DESCRIPTION OF THE DRAWINGS

[0276] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0277] FIG. 1 is a schematic diagram of skin anatomy.

[0278] FIG. 2 is a schematic of cell types in the layers of the skin from the skin surface to the dermis, including the stratum corneum, the stratum lucidum, stratum granulosum, stratum germinativum, and stratum basale. Each contains living cells with specialized functions.

[0279] FIG. 3 is a schematic of the molecular structure of the mammalian circadian clock. The circadian clock consists of three core feedback loops. The newly described feedback loop has also been depicted. Loop 1: basic helix loop helix ARNT like 1 (BMAL1) gene dimerizes with Clock Circadian Regulator (CLOCK) gene or Neuronal PAS Domain Protein 2 (NPAS2) gene. These dimers bind to the promoter region E-box elements (5′-CACGTG-3′), triggering Period 1- 3 (PER1-3), crystalline (CRY), retinoic acid-related orphan receptor (ROR), nuclear receptor subfamily 1, group D, member 1(NR1D1), and transcription factor albumin D site-binding protein (DBP) gene expression. In mammals, DBP rhythmically activates transcription of various genes through a DNA cis-element, D-box. PER and CRY dimerize on reaching a critical concentration, inhibiting their own expression, causing oscillation of the expression of theseproteins. Loop 2: The transcription of BMAL1, CLOCK, and NFIL3 (a rhythmically expressed transcription factor) is triggered when ROR binds to the RORE element 5′-(A / G)GGTCA-3′ in their promoter region. This binding of ROR to RORE is inhibited by NR1D1. Loop 3: PER transcription is initiated when the D box element (5′-TTATG(T / C)AA-3′) in its promoter region is bound to by DBP (Loop1). This binding is negatively regulated by NFIL3 (Loop 2). DEC loop: The DEC protein that gives this loop its name, is responsible for its own oscillatory expression by inhibiting the binding of BMAL1: CLOCK to E-box elements (5′-CACGTG-3′) in its promoter. (taken from Salazar, A. and von Hagen, J., Intl J. Molec. Sci. (2023) 24: 5635)

[0280] FIG. 4 is a schematic of the mitogen activated protein kinase (MAPK) signaling pathways [taken from Soares-Silva, M. et al. Front. Microbiol. (2016) 7: 183]. The major MAPK pathways involved in inflammatory diseases are extracellular regulating kinase (ERK), p38 MAPK, and c-Jun NH2-terminal kinase (JNK). All three MAPK pathways may be activated by TGF-β, and signaling through these cascades can further regulate the expression of Smad proteins and mediate Smad-independent TGF-β responses. Smaroteins are key signal transducers for the TGF-β superfamily. These three MAPK pathways are all involved in TGF-β-induced fibrosis. [He, W. and Dai, C. Curr. Pathobiol. Rep. (2015) 3: 183-92, citing Tsou, PS et al. Am. J. Physiol. Cell Physiol. (22014) 307: C2-13; Kamato, D. et al. Cell Signal (2013) 25: 2017-24; Pannu, J. et al. J. Biol. Chem. (2007) 282: 10405-13; Yu, L. et al. J. Biol. Chem. (2002) EMBO J. 21: 3749-59]. Upstream kinases include TGFβ-activated kinase-1 (TAK1) and apoptosis signal-regulating kinase-1 (ASK1). Downstream of p38 MAPK is MAPK activated protein kinase 2 (MAPKAPK2 or MK2). TGF-β can signal in a noncanonical manner via the MAPK family.

[0281] FIG. 5 is a schematic of the PI3K / Akt / mTOR pathway (Taken from Porta, et al. Front. Oncol. (2014) 4: art. 64). Activation of growth factor receptor protein tyrosine kinases results in autophosphorylation on tyrosine residues. P13K is then recruited to the membrane by directly binding to phosphotyrosine consensus residues of growth factor receptors or adaptors through one of the two SH2 domains in the adaptor subunit. This leads to allosteric activation of the catalytic (CAT) subunit of PI3K. PI3K activation leads to the production of the second messenger phosphatidylinositol-4,4-bisphosphate (PI3,4,5-P3) from the substrate phosphatidylinositol-4,4-bisphosphate (PI-4,5-P2). PI3,4,5-P3 then recruits a subset of signaling proteins with pleckstrin homology (PH) domains to the membrane, including proteinserine / threonine kinase-3’-phosphoinositide-dependent kinase I (PDK1) and Akt / protein kinase B (PKB) [Id., citing Fruman, DA et al., Phosphoinositide kinases. Annu. Rev. Biochem. (1998) 67: 481-507, Fresno-Vara, JA, et al., PI3K / Akt signaling pathway and cancer. Cancer Treat. Rev. (2004) 30: 193-204]. Akt (also known as protein kinase B) is activated in response to stimulation of tyrosine kinase receptors, such as platelet-derived growth factor (PDGF), insulin- like growth factor, and nerve growth factor. Stimulation of Akt has been shown to be dependent on phosphatidylinositol 3-kinase (PI3-kinase) activity. Mammalian target of rapamycin (mTOR) is an atypical serine / threonine kinase that is present in two distinct complexes. The first, mTOR complex 1 (mTORC1), is composed of mTOR, regulatory protein associated with mTOR (Raptor), GβL (also known as mammalian lethal with Sec13 protein 8 or mLST8), and DEP domain containing m-TOR Interacting Protein (DEPTOR) and is inhibited by rapamycin. It is a master growth regulator that senses and integrates diverse nutritional and environmental cues, including growth factors, energy levels, cellular stress, and amino acids. It couples these signals to the promotion of cellular growth by phosphorylating substrates that potentiate anabolic processes such as mRNA translation and lipid synthesis or limit catabolic processes such as autophagy. The small GTPase Rheb, in its GTP-bound state, is a necessary and potent stimulator of mTORC1 kinase activity, which is negatively regulated by its GTPase-activating protein (GAP), the tuberous sclerosis heterodimer TSC1 / 2. TSC1 and TSC2 are the tumor-suppressor genes mutated in the tumor syndrome TSC (tuberous sclerosis complex). Their gene products form a complex (the TSC1–TSC2 (hamartin–tuberin) complex), which, through its GAP activity towards the small G-protein Rheb (Ras homologue enriched in brain), is a critical negative regulator of mTORC1. Most upstream inputs are funneled through Akt and TSC1 / 2 to regulate the nucleotide-loading state of Rheb. The second mTOR complex (mTORC2) is sensitive to growth factors, not nutrients, and is associated with rapamycin-insensitivity mTORC2 is thought to modulate growth factor signaling by phosphorylating the C-terminal hydrophobic motif of some cAMP-dependent, cGMP-dependent and protein kinase C (AGC) kinases such as Akt and SGK [Jhanwar-Uniyal, M. et al. Adv. Biol. Regul. (2015) 57: 64-74].

[0282] FIG. 6 is a schematic of the IGF-1 signaling pathway [taken from Iams, WT and Lovly, CM, Clin. Cancer Res. (2015) 21 (19): 42770-77]. Insulin-like growth factors (IGFs) bind specifically to the IGF1 receptor on the cell surface of targeted tissues. Ligand binding to the α subunit of the receptor leads to a conformational change in the β subunit, resulting in theactivation of receptor tyrosine kinase activity. Activated receptor phosphorylates several substrates, including insulin receptor substrates (IRSs) and Src homology collagen (SHC). Phosphotyrosine residues in these substrates are recognized by certain Src homology 2 (SH2) domain-containing signaling molecules. These include an 85 kDa regulatory subunit (p85) of phosphatidylinositol 3-kinase (PI 3-kinase), growth factor receptor-bound 2 (GRB2) and SH2- containing protein tyrosine phosphatase 2 (SHP2 / Syp), which lead to the activation of downstream signaling pathways, PI 3-kinase pathway and Ras-mitogen-activated protein kinase (MAP kinase) pathway.

[0283] FIG.7, taken from Su, J. et al. Front. Immunol. (2021) 11: 593564, is a schematic representation of leptin and leptin receptor signal transduction pathways and functions. Stimulation of the leptin receptor by leptin can activate Janus (JAK2) kinase, resulting in tyrosine phosphorylation of the receptor and downstream proteins, including Signal transducer and Activator of Transcription 3 (STAT3), a member of the signal transducers and activators of transcription (STAT) transcription factor family, which functions together with the janus kinases (JAK) in JAK-STAT signaling networks commonly activated by cytokines, protein tyrosine phosphatase 2 (SHP-2), an SH2 domain-containing protein tyrosine phosphatase, Insulin Receptor Substrate 2 (IRS2, an adaptor molecule that plays a fundamental role in insulin and IL- 4, IL-7 and IL-9 signaling), and phosphoinositide 3-kinase (PI3K) that play a role in regulating transcription of genes essential for energy intake and lipid metabolism. ACC (acetyl-CoA carboxylase); AMPK (adenosine monophosphate kinase); CPTI (carnitine palmitoyltransferase 1); ERK (extracellular signaling-regulated kinase); FAS (fatty acid synthase); SCD1 (stearoyl- coenzyme A desaturace 1, enzyme in fatty acid metabolism); TG (triglycerides); PL (phospholipids); CE (cholesterol esters)

[0284] FIG. 8 is a bar graph showing HSP70 gene expression in purified aloe-derived exosomes before treatment and after heat stress treatment compared to the whole plant and a pre-treatment control.

[0285] FIG. 9A is a bar graph of collagen 1 (ng / mL) in human dermal fibroblasts against a media control; treatment samples (Aloe 1, Aloe 2, and adipose stromal stem cells (ASC)); and a TGFb positive control showing that Aloe-derived purified exosomes can induce collagen Isynthesis in human dermal fibroblasts. FIG. 9B is a bar graph showing percent change of collagen in human dermal fibroblasts treated as in FIG. 9A.

[0286] FIG. 10A is a bar graph of VEGF-A concentration (ng / mL) in human dermal fibroblasts treated with 5% FBS (positive control), Aloe exosomes (1x10E9) Aloe Exosomes (1x10E8), and Aloe exosomes (1x10E7). FIG. 10B shows VEGFA concentration fold change; vs. 5% FBS positive control, Aloe exosomes (1x10E9) Aloe Exosomes (1x10E8), and Aloe exosomes (1x10E7). FIG. 10C shows VEGFA concentration (ng / mL) versus Il-8 (1000 ng / mL). IL-8 (100 mg / mL), IL-8 (10 ng / mL), IL-8 (1 ng / mL), IL8 (0.1 ng / mL), and 10% FBS positive control

[0287] FIG. 11A, FIG. 11B, and FIG. 11C show bar graphs of fold change in production of elastin (blue), hyaluronic acid (orange) and collagen 1 (gray) in human skin dermal fibroblasts treated with Aloe exosomes FIG. 11A Prep 1 [1x10E8, 3.33x10E7, 1,11x10E7], and Prep 2 (1.00x10E8, 3.33E7, 1.11x10E7), FIG. 11B Prep 3 [1x10E8, 3.33x10E7, 1,11x10E7], and Prep 4 (1.00x10E8, 3.33x10E7, 1.11x10E7), and FIG. 11C Prep 5 [1x10E8, 3.33x10E7, 1,11x10E7] compared to a media control.

[0288] FIG. 12 shows average cytokine production (pg / mL interferon a2 (INFa2) in PBMCs in vitro in response to bacterial exposure. Treatment groups from left to right are: media controls (dark green= media only, black=media plus dextran) -Media + P. acnes; adipose stromal stem cells (ASC) (yellow) Aloe exosomes (light green=prep 1; and light blue=prep II).

[0289] FIG. 13A, FIG. 13B, FIG. 13C, FIG. 13D, FIG. 13E and FIG. 13F show delivery of exosomes containing fluorescent-labeled RNA cargo into cell cytoplasm of a monolayer of human skin fibroblasts in vitro at 4 degrees C (TOP ROW) and at 37 degrees C (BOTTOM row). Top Row, incubation at 4°C: FIG. 13A (cells alone (no addition of labeled exosomes) incubated at 4°C), FIG. 13B (addition of Aloe exosomes at 4°C), FIG. 13C (addition of adipose MSC exosomes at 4° C). At 4° C, minimal Aloe and MSC exosome fluorescence were expected, since 4° C impedes exosome binding and internalization. Bottom Row, incubation at 37°C: FIG. 13D (cells alone incubated, at 37° C, no addition of labeled exosomes), FIG. 13E (addition of aloe exosomes at 37° C) and FIG. 13F (addition of adipose MSC exosomes at 37ºC). At 37° C, MSC exosome fluorescence is readily visible (blue arrows) when compared to the negative control at 4° C shown in FIG. 13C, indicating successful labeled RNA cargo delivery.

[0290] FIG. 14A, FIG. 14B, and FIG. 14C show exosome attachment to human dermal fibroblast cells in suspension culture. Exosomes comprised fluorescent-labeled RNA. FIG. 14A, Cells alone (negative control), green; HS aloe exosomes (red). FIG. 14B cells plus labeled heat shock MSC exosomes. FIG. 14C, merging of left and right panels, showing overlay of labeled HS aloe and HS MSC peaks.

[0291] FIG. 15A and FIG. 15B, show bar graphs from two experiments showing effect of heat- shocked (HS) human adipose stromal stem cell (ASC) exosomes and HS aloe exosomes on cell proliferation of hair dermal papillae, compared to media controls (control media, test media).

[0292] FIG. 16 is a bar graph showing cell counts (x 1000) vs. exosome preparation (left to right, control media; test media; Aloe-exosomes (1.5 x 10E9; 3.0 x 10E9; 6.0x10E9); adipose stromal stem cells (ASCs) (1.5 x10E8; 3.0x10E8; 6.0 x 10E8); amniotic fluid (AF) (1.5 x 10E8; 3.0x10E8; 6.0x10E8). Overall, all test concentrations of exosomes showed greater cell proliferation than the Test Media alone. All but 1.5 and 3.0 for ASC and 6.0 for AF showed equal to or greater cell proliferation than the Control growth media.

[0293] FIG. 17A and FIG. 17B are bar graphs showing in vitro antioxidant activity of Aloe- derived exosomes and human ASC exosomes by 2,2′-azino-bis-(3-ethylbenzothiazoline-6- sulfonic) acid (ABTS) (FIG.17A) and oxygen radical absorbance capacity (ORAC) (FIG. 17B) assays.

[0294] FIG. 18, taken from Revollo, JR and Li, X. Trends Biochem. Sci. (2013) 38 (3): 160-67, schematically depicts the enzymatic reaction catalyzed by Sirt1. Sirt1 catalyzes the deacetylation of several proteins by consuming nicotinamide adenine dinucleotide (NAD+), generating nicotinamide (NAM) and 2′-O-Acetyl-ADP-Ribose. NAM is recycled back into NAD+ by the enzymes nicotinamide phosphoribosyltransferase (NAMPT), nicotinamide mononucleotide adenylyl transferase (NMNAT), and the nicotinamide mononucleotide (NMN) intermediate.

[0295] FIG. 19A is a photograph of the scalp of Subject 1, a female with hair loss from salon coloring treatments every two weeks, showing hair loss before treatment, FIG. 19B is a photograph of the same area of the scalp after 5 weeks of treatment.

[0296] FIG. 20A is a photograph of the scalp of Subject 2, a female with hair loss due to androgenetic alopecia, showing hair loss before treatment, FIG. 20B is a photograph of the same area of the scalp after 8 weeks of treatment.

[0297] FIG. 21A is a photograph of the scalp of Subject 3, a male with hair loss post COVID- 19, showing hair loss before treatment, FIG. 21B is a photograph of the same area of the scalp after 15 weeks of treatment.

[0298] FIG. 22A is a photograph of the scalp of Subject 4, a male with hair loss of unknown pathology, showing hair loss before treatment, FIG. 22B is a photograph of the same area of the scalp after 20 weeks of treatment.

[0299] FIG. 23A a photograph of the scalp of Subject 5, a male with alopecia areata, showing hair loss before treatment, FIG.23B is a photograph of the same area of the scalp after 12 weeks of treatment.

[0300] FIG. 24A a photograph of the scalp of Subject 6, a 58 year old male with androgenetic alopecia and stress, showing hair loss before treatment, FIG. 24B is a photograph of the same area of the scalp after 20 weeks of treatment.

[0301] FIG. 25 is a bar graph of a lactate dehydrogenase (LDH) cytotoxicity assessment of human follicle dermal papilla cells (HFDPCs) 24 hours after treatment with 1.5 billion native, not heat shocked, aloe-derived exosomes (TM1), 1.5 billion engineered, heat shocked, aloe- derived exosomes (TM2), 5 billion engineered, heat shocked, aloe-derived exosomes (TM3), and 5 billion engineered, heat shocked) human adipose stromal stem cell (ASC)-derived exosomes (TM4) compared to an untreated negative control (UNT), a media + serum positive control (Positive Ctrl), a Triton X-100® treated positive control (Triton), and a PBS treated vehicle control (Vehicle Ctrl).

[0302] FIG. 26 is a qPCR amplification curve for the selected endogenous control gene, peptidylprolyl isomerase A (PPIA) for gene expression analysis of human follicle dermal papilla cells (HFDPCs) 24 hours after treatment with 1.5 billion native, not heat shocked, aloe-derived exosomes, 1.5 billion engineered, heat shocked, aloe-derived exosomes, 5 billion engineered, heat shocked, aloe-derived exosomes, and 5 billion engineered, heat shocked, human adipose stromal stem cell (ASC)-derived exosomes, compared to an untreated negative control, a media+ serum positive control, and a PBS treated vehicle control. Each line in the amplification curve represents a different sample across treatment groups.

[0303] FIG. 27A, FIG. 27B, and FIG.27C are qPCR amplification curves showing poor quality data for different genes expressed in human follicle dermal papilla cells (HFDPCs) 24 hours after treatment with 1.5 billion native, not heat shocked, aloe-derived exosomes, 1.5 billion engineered, heat shocked, aloe-derived exosomes, 5 billion engineered, heat shocked, aloe- derived exosomes, and 5 billion engineered, heat shocked, human adipose stromal stem cell (ASC)-derived exosomes, compared to an untreated negative control, a media + serum positive control, and a PBS treated vehicle control. Each line in the amplification curve represents a different sample across treatment groups. FIG. 27A shows a poor quality amplification curve for the WIF1 gene. FIG. 27B shows a poor quality amplification curve for the WNT3A gene. FIG. 27C shows a poor quality amplification curve for the PROM1 gene.

[0304] FIG. 28A and FIG. 28B are exemplary qPCR amplification curves showing high quality data for different genes expressed in human follicle dermal papilla cells (HFDPCs) 24 hours after treatment with 1.5 billion native, not heat shocked, aloe-derived exosomes, 1.5 billion engineered, heat shocked, aloe-derived exosomes, 5 billion engineered, heat shocked, aloe- derived exosomes, and 5 billion engineered, heat shocked, human adipose stromal stem cell (ASC)-derived exosomes, compared to an untreated negative control, a media + serum positive control, and a PBS treated vehicle control. Each line in the amplification curve represents a different sample across treatment groups. FIG. 28A shows a high quality amplification curve for the BMP6 gene. FIG. 28B shows a high quality amplification curve for the SRD5A2 gene.

[0305] FIG. 29A, FIG. 29B, and FIG. 29C are exemplary qPCR amplification curves showing a high fold-change for different genes expressed in human follicle dermal papilla cells (HFDPCs) 24 hours after treatment with 1.5 billion native, not heat shocked, aloe-derived exosomes, 1.5 billion engineered, heat shocked, aloe-derived exosomes, 5 billion engineered, heat shocked, aloe-derived exosomes, and 5 billion engineered, heat shocked, human adipose stromal stem cell (ASC)-derived exosomes when compared to control groups (vehicle or untreated control groups). Each line in the amplification curve represents a different sample across treatment groups. FIG. 29A shows a high fold-change amplification curve for the DKK1gene. FIG. 29B shows a high-fold change amplification curve for the IL1B gene. FIG. 29C shows a high fold-change amplification curve for the MKI67 gene.

[0306] FIG. 30 is a bar graph of a lactate dehydrogenase (LDH) cytotoxicity assessment of human follicle dermal papilla cells (HFDPCs) 72 hours after treatment with 5 billion engineered (heat shocked) aloe exosomes (TM 1), 5 billion native (not heat shocked) aloe-derived exosomes (TM 2), 5 billion engineered (heat shocked) human adipose stromal stem cell (ASC)-derived exosomes (TM 3), and 5 billion native (not heat shocked) human ASC-derived exosomes (TM 4), compared to an untreated control (UNT), a media + serum positive control (Positive Ctrl), a Triton X-100® treated positive control (Triton), and a PBS treated vehicle control (Vehicle Ctrl).

[0307] FIG. 31 is a qPCR amplification curve for the selected endogenous control gene, Hypoxanthine Phosphoribosyltransferase 1 (HPRT1) for gene expression analysis of human follicle dermal papilla cells (HFDPCs) 72 hours after treatment with 5 billion engineered (heat shocked) aloe exosomes, 5 billion native (not heat shocked) aloe-derived exosomes, 5 billion engineered (heat shocked) human adipose stromal stem cell (ASC)-derived exosomes, and 5 billion native (not heat shocked) human ASC-derived exosomes, compared to an untreated control, a positive control, negative control, and a PBS treated vehicle control. Each line in the amplification curve represents a different sample across treatment groups.

[0308] FIG. 32A, FIG. 32B, and FIG. 32C are qPCR amplification curves showing poor quality data for different genes expressed in human follicle dermal papilla cells (HFDPCs) 72 hours after treatment with 5 billion engineered (heat shocked) aloe exosomes, 5 billion native (not heat shocked) aloe-derived exosomes, 5 billion engineered (heat shocked) human adipose stromal stem cell (ASC)-derived exosomes, and 5 billion native (not heat shocked) human ASC- derived exosomes, compared to an untreated control, a positive control, negative control, and a PBS treated vehicle control. Each line in the amplification curve represents a different sample across treatment groups. FIG. 32A shows a poor quality amplification curve for the PROM1 gene. FIG. 32B shows a poor quality amplification curve for the WIF1 gene. FIG. 32C shows a poor quality amplification curve for the WNT3A gene.

[0309] FIG. 33A, FIG. 33B, FIG. 33C, and FIG. 33D are exemplary qPCR amplification curves showing high quality data for different genes expressed in human follicle dermal papilla cells (HFDPCs) 72 hours after treatment with 5 billion engineered (heat shocked) aloe exosomes,5 billion native (not heat shocked) aloe-derived exosomes, 5 billion engineered (heat shocked) human adipose stromal stem cell (ASC)-derived exosomes, and 5 billion native (not heat shocked) human ASC-derived exosomes, compared to an untreated control, a positive control, negative control, and a PBS treated vehicle control. Each line in the amplification curve represents a different sample across treatment groups. FIG. 33A shows a high quality amplification curve for the LEF1 gene. FIG. 33B shows a high quality amplification curve for the TCF4 gene. FIG. 33C shows a high quality amplification curve for the TGFB1 gene. FIG. 33D shows a high quality amplification curve for the WNT5A gene.

[0310] FIG. 34A, FIG. 34B, and FIG. 34C are exemplary qPCR amplification curves showing a high fold-change for different genes expressed in human follicle dermal papilla cells (HFDPCs) 72 hours after treatment with 5 billion engineered (heat shocked) aloe exosomes, 5 billion native (not heat shocked) aloe-derived exosomes, 5 billion engineered (heat shocked) human adipose stromal stem cell (ASC)-derived exosomes, and 5 billion native (not heat shocked) human ASC-derived exosomes, compared to an untreated control, a positive control, negative control, and a PBS treated vehicle control when compared to control groups (vehicle or untreated control groups). Each line in the amplification curve represents a different sample across treatment groups. FIG. 34A shows a high fold-change amplification curve for the BMP2 gene. FIG. 34B shows a high-fold change amplification curve for the IL6 gene. FIG. 34C shows a high fold-change amplification curve for the LEP gene. DETAILED DESCRIPTION OF THE INVENTION Definitions:

[0311] As used herein and in the appended claims, the singular forms “a”, “an”, and “the” include plural reference unless the context clearly dictates otherwise. Thus, for example, reference to a “peptide” is a reference to one or more peptides and equivalents thereof known to those skilled in the art, and so forth.

[0312] As used herein, the term “about” means plus or minus 20% of the numerical value of the number with which it is being used. Therefore, about 50% means in the range of 40%-60%.

[0313] In general, terminology may be understood at least in part from usage in context. For example, terms, such as “and”, “or”, or “and / or,” as used herein may include a variety of meanings that may depend at least in part upon the context in which such terms are used. Typically, “or” if used to associate a list, such as A, B, or C, is intended to mean A, B, and C, here used in the inclusive sense, as well as A, B, or C, here used in the exclusive sense. In addition, the term “one or more” as used herein, depending at least in part upon context, may be used to describe any feature, structure, or characteristic in a singular sense or may be used to describe combinations of features, structures or characteristics in a plural sense. Similarly, terms, such as “a,” “an,” or “the,” again, may be understood to convey a singular usage or to convey a plural usage, depending at least in part upon context. In addition, the term “based on” may be understood as not necessarily intended to convey an exclusive set of factors and may, instead, allow for existence of additional factors not necessarily expressly described, again, depending at least in part on context.

[0314] The term “activation” or “lymphocyte activation” refers to stimulation of lymphocytes by specific antigens, nonspecific mitogens, or allogeneic cells resulting in synthesis of RNA, protein and DNA and production of lymphokines; it is followed by proliferation and differentiation of various effector and memory cells. T-cell activation is dependent on the interaction of the TCR / CD3 complex with its cognate ligand, a peptide bound in the groove of a class I or class II MHC molecule. The molecular events set in motion by receptor engagement are complex. Full responsiveness of a T cell requires, in addition to receptor engagement, an accessory cell-delivered costimulatory activity, e.g., engagement of CD28 on the T cell by CD80 and / or CD86 on the antigen presenting cell (APC).

[0315] The term “active” refers to the ingredient, component or constituent of the compositions of the described invention responsible for the intended cosmetic, cosmeceutical or therapeutic effect.

[0316] The term “adaptive immunity” as used herein refers to a specific, delayed and longer- lasting response by various types of cells that create long-term immunological memory against a specific antigen. It can be further subdivided into cellular and humoral branches, the former largely mediated by T cells and the latter by B cells. This arm further encompasses cell lineagemembers of the adaptive arm that have effector functions in the innate arm, thereby bridging the gap between the innate and adaptive immune response.

[0317] “Administering” when used in conjunction with a cosmetic, cosmeceutical or therapeutic means to give or apply a cosmetic, cosmeceutical or therapeutic directly into or onto a target organ, tissue or cell, or to administer the cosmetic, cosmeceutical or therapeutic to a subject, whereby the cosmetic, cosmeceutical or therapeutic positively impacts the organ, tissue, cell, or subject to which it is targeted. Thus, as used herein, the term “administering”, when used in conjunction with EVs or compositions thereof, can include, but is not limited to, providing EVs into or onto the target organ, tissue or cell; or providing EVs systemically to a subject, whereby the therapeutic reaches the target organ, tissue or cell. “Administering” may be accomplished by oral, parenteral or topical administration or by such methods in combination with other known techniques.

[0318] The term “allogeneic” as used herein refers to being genetically different although belonging to or obtained from the same species.

[0319] The term “allograft” as used herein refers to a transplant of tissue from an allogeneic donor of the same species.

[0320] The term “allograft immunity” as used herein refers to any immune response by the host against a transplanted tissue.

[0321] The term "amino acid” is used to refer to an organic molecule containing both an amino group and a carboxyl group; those that serve as the building blocks of naturally occurring proteins are alpha amino acids, in which both the amino and carboxyl groups are linked to the same carbon atom. The terms “amino acid residue” or “residue” are used interchangeably to refer to an amino acid that is incorporated into a protein, a polypeptide, or a peptide, including, but not limited to, a naturally occurring amino acid and known analogs of natural amino acids that can function in a similar manner as naturally occurring amino acids.

[0322] The abbreviations used herein for amino acids are those abbreviations which are conventionally used: A=Ala=Alanine; R=Arg=Arginine; N=Asn=Asparagine; D=Asp=Aspartic acid; C=Cys=Cysteine; Q=G1n=Glutamine; E=Glu=Glutamic acid; G=Gly=Glycine; H=His=Histidine; I=Ile=lsoleucine; L=Leu=Leucine; K=Lys=Lysine; M=Met=Methionine;F=Phe=Phenyalanine; P=Pro=Proline; S=Ser=Serine; T=Thr=Threonine; W=Trp=Tryptophan; Y=Tyr=Tyrosine; V=Val=Valine. The amino acids may be L- or D-amino acids. An amino acid may be replaced by a synthetic amino acid which is altered so as to increase the half-life of the peptide or to increase the potency of the peptide, or to increase the bioavailability of the peptide.

[0323] The following represent groups of amino acids that are conservative substitutions for one another:

[0324] Alanine (A), Serine (S), Threonine (T);

[0325] Aspartic Acid (D), Glutamic Acid (E);

[0326] Asparagine (N), Glutamine (Q);

[0327] Arginine (R), Lysine (K);

[0328] Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and

[0329] Phenylalanine (F), Tyrosine (Y), Tryptophan (W).

[0330] The term "anesthetic agents" as used herein refers to agents that resulting in a reduction or loss of sensation. Non-limiting examples of anesthetic drugs that are suitable for use in the context of the described invention include pharmaceutically acceptable salts of lidocaine, bupivacaine, chlorprocaine, dibucaine, etidocaine, mepivacaine, tetracaine, dyclonine, hexylcaine, procaine, cocaine, ketamine, pramoxine and phenol.

[0331] The term "anti-acne" as used herein refers to agents that alleviate the symptoms of acne. The term "acne" as used herein refers to an inflammatory disease of the sebaceous glands, characterized by comedones and pimples. Examples of anti-acne agents include, without limitation, keratolyses, such as salicylic acid, sulfur, glycolic, pyruvic acid, resorcinol, and N- acetylcysteine; and retinoids such as retinoic acid and its derivatives (e.g., cis and trans, esters).

[0332] The term “angiogenesis” as used herein refers to the formation of new blood vessels from pre-existing vasculature. Angiogenesis is central to a number of physiological conditions, from embryogenesis to wound healing, and is a hallmark of pathological conditions, such as tumorigenesis. [Abhinand, CS et al. J. Cell Commun. Signal. (2016) 10 (4): 347-54].

[0333] The terms “animal,” “patient,” and “subject” as used herein include, but are not limited to, humans and non-human vertebrates such as wild, domestic and farm animals. According tosome embodiments, the terms “animal,” “patient,” and “subject” may refer to humans. According to some embodiments, the terms “animal,” “patient,” and “subject” may refer to non- human mammals.

[0334] The term “annexins” as used herein refers to Ca+2-dependent proteins that bind to membrane phospholipids in plants. [Laohavisit, A. and Davies, JM. New Phytologist (2011) 189: 40-53]. They have been found to be stimulated by abiotic stress, including light, salinity, heat, cold, drought; oxidative and mechanic stress. [Saad, RB et al. Plant Signaling & Behavior (2020) 15 (1): e1699264; Baucher, M. et al. Plant Signaling & Behavior (2012) 7 (4): 524-8] Annexin expression also has been shown to be upregulated during pathogen attack or symbiotic interaction [Baucher, M. et al. Plant Signaling & Behavior (2012) 7 (4): 524-8, citing de Carvadho, NF, et al. Mol. Plant Microbe Interact. (1998) 11: 504-13; Manthey, K. et al. Mol. Plant Microbe Interact (2004) 17: 1063-77; Vandeputte, O. et al. Mol. Plant Pathol. (2007) 8: 185-94]. The dynamic properties of plant annexins together with their expression patterns suggest that they are involved in cell signaling processes and therefore in plant development al adaptation to environmental changes.

[0335] The term "antibiotic agent" as used herein means any of a group of chemical substances having the capacity to inhibit the growth of, or to destroy bacteria, and other microorganisms, used chiefly in the treatment of infectious diseases. Examples of antibiotic agents include, but are not limited to, Penicillin G; Methicillin; Nafcillin; Oxacillin; Cloxacillin;Dicloxacillin; Ampicillin; Amoxicillin; Ticarcillin; Carbenicillin; Mezlocillin; Azlocillin; Piperacillin; Imipenem; Aztreonam; Cephalothin; Cefaclor; Cefoxitin; Cefuroxime; Cefonicid; Cefmetazole; Cefotetan; Cefprozil; Loracarbef; Cefetatnet; Cefoperazone; Cefotaxime; Ceftizoxime; Ceftriaxone; Ceftazidime; Cefepime; Cefixime; Cefpodoxime; Cefsulodin; Fleroxacin; Nalidixic acid; Norfloxacin; Ciprofloxacin; Ofloxacin; Enoxacin; Lomefloxacin; Cinoxacin; Doxycycline; Minocycline; Tetracycline; Amikacin; Gentamicin; Kanamycin; Netilmicin; Tobramycin; Streptomycin; Azithromycin; Clarithromycin; Erythromycin; Erythromycin estolate ; Erythromycin ethyl succinate; Erythromycin glucoheptonate; Erythromycin lactobionate; Erythromycin stearate; Vancomycin; Teicoplanin; Chloramphenicol; Clindamycin; Trimethoprim; Sulfamethoxazole; Nitrofurantoin; Rifampin; Mupirocin; Metronidazole; Cephalexin; Roxithromycin; Co-amoxiclavuanate; combinations of Piperacillin and Tazobactam; and their various salts, acids, bases, and other derivatives. Anti-bacterial antibiotic agentsinclude, but are not limited to, penicillins, cephalosporins, carbacephems, cephamycins, carbapenems, monobactams, aminoglycosides, glycopeptides, quinolones, tetracyclines, macrolides, and fluoroquinolones.

[0336] The term "anti-dandruff agents" as used herein refers to agents that reduce, eliminate or prevent a scurf from forming on skin, especially of the scalp, that comes off in small white or grayish scales. Exemplary anti-dandruff ingredients usable in context of the described invention include, without limitation, zinc pyrithione, shale oil and derivatives thereof such as sulfonated shale oil, selenium sulfide, sulfur; salicylic acid, coal tar, povidone-iodine, imidazoles such as ketoconazole, dichlorophenyl imidazolodioxalan, clotrimazole, itraconazole, miconazole, climbazole, tioconazole, sulconazole, butoconazole, fluconazole, miconazole nitrate and any possible stereo isomers and derivatives thereof such as anthralin, piroctone olamine (Octopirox), selenium sulfide, and ciclopiroxolamine, and mixtures thereof.

[0337] The term "antihistamine agent" as used herein refers to any of various compounds that counteract histamine in the body and that are used for treating allergic reactions (such as hay fever) and cold symptoms. Non-limiting examples of antihistamines usable in context of the described invention include chlorpheniramine, brompheniramine, dexchlorpheniramine, tripolidine, clemastine, diphenhydramine, promethazine, piperazines, piperidines, astemizole, loratadine and terfenadine.

[0338] The term "anti-irritant" as used herein refers to an agent that prevents or reduces soreness, roughness, or inflammation of a bodily part.

[0339] The term "anti-oxidant agent" as used herein refers to a substance that inhibits oxidation or reactions promoted by oxygen or peroxides. Non-limiting examples of antioxidants that are usable in the context of the described invention include ascorbic acid (vitamin C) and its salts, ascorbyl esters of fatty acids, ascorbic acid derivatives (e.g., magnesium ascorbyl phosphate, sodium ascorbyl phosphate, ascorbyl sorbate), tocopherol (vitamin E), tocopherol sorbate, tocopherol acetate, other esters of tocopherol, butylated hydroxy benzoic acids and their salts, ό- hydroxy-ZjSjTjδ-tetramethylchroman-l-carboxylic acid (commercially available under the tradename Trolox®), gallic acid and its alkyl esters, especially propyl gallate, uric acid and its salts and alkyl esters, sorbic acid and its salts, lipoic acid, amines (e.g., N5N- diethylhydroxylamine, amino-guanidine), sulfhydryl compounds (e.g., glutathione), dihydroxyfumaric acid and its salts, glycine pidolate, arginine pilolate, nordihydroguaiaretic acid, bioflavonoids, curcumin, lysine, methionine, proline, superoxide dismutase, silymarin, tea extracts, grape skin / seed extracts, melanin, and rosemary extracts.

[0340] The term "anti-protozoal agent" as used herein means any of a group of chemical substances having the capacity to inhibit the growth of or to destroy protozoans used chiefly in the treatment of protozoal diseases. Examples of antiprotozoal agents, without limitation, include pyrimethamine (Daraprim®), sulfadiazine, and Leucovorin.

[0341] The term "antipruritic agents" as used herein refers to those substances that reduce, eliminate or prevent itching. Suitable antipruritic agents include, without limitation, pharmaceutically acceptable salts of methdilazine and trimeprazine.

[0342] The term "anti-skin atrophy actives" refers to substances effective in replenishing or rejuvenating the epidermal layer by promoting or maintaining the natural process of desquamation. The term “anti-wrinkle agent” as used herein an agent intended to reduce the appearance of wrinkles in the skin, e.g., retinols, Bakuchiol, vitamin C (ascorbic acid), niacinamide; tranexamic acid; azelaic acid.

[0343] Non-limiting examples of antiwrinkle and antiskin atrophy actives which can be used in context of the described invention include retinoic acid, its prodrugs and its derivatives (e.g., cis and trans) and analogues; salicylic acid and derivatives thereof, sulfur-containing D and L amino acids and their derivatives and salts, particularly the N-acetyl derivatives, an example of which is N-acetyl L-cysteine; thiols, e.g. ethane thiol; alpha-hydroxy acids, e.g. glycolic acid, and lactic acid; phytic acid, lipoic acid; lysophosphatidic acid, and skin peel agents (e.g., phenol and the like).

[0344] The term “antigen” as used herein, is meant to refer to a molecule containing one or more antigenic determinants or epitopes (either linear, conformational or both) that will stimulate a host’s immune-system to make a humoral and / or cellular antigen-specific response. The term is used interchangeably with the term “immunogen.” Normally, a B-cell epitope will include at least about 5 amino acids but can be as small as 3-4 amino acids. A T-cell epitope, such as a CTL epitope, will include at least about 7-9 amino acids, and a helper T-cell epitope at least about 12- 20 amino acids. Normally, an epitope will include between about 7 and 15 amino acids, such as, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids. The term includes polypeptides which includemodifications, such as deletions, additions and substitutions (generally conservative in nature) as compared to a native sequence, as long as the protein maintains the ability to elicit an immunological response. These modifications may be deliberate, as through site-directed mutagenesis, or may be accidental, such as through mutations of the host that produces the antigens.

[0345] The term “antigen presentation” as used herein, generally refers to the display of antigen on the surface of a cell, e.g., in the form of peptide fragments bound to MHC molecules.

[0346] As used herein, the term “antigen presenting cell (APC)” refers to a class of cells capable of displaying on its surface (“presenting”) one or more antigens in the form of peptide-MHC complex recognizable by specific effector cells of the immune system and thereby inducing an effective cellular immune response against the antigen or antigens being presented. Examples of professional APCs are dendritic cells and macrophages, though any cell expressing MHC Class I or II molecules can potentially present peptide antigen. An APC can be an irradiated population of PBMCs. An APC can be an “artificial APC,” meaning a cell that is engineered to present one or more antigens. Before a T cell can recognize a foreign protein, the protein has to be processed inside an antigen presenting cell or target cell so that it can be displayed as peptide-MHC complexes on the cell surface.

[0347] As used herein the term “antigen processing” refers to the intracellular degradation of foreign proteins into peptides that can bind to MHC molecules for presentation to T cells.

[0348] The term “Argonaute 2” or “AGO2” as used herein refers to an RNA binding protein that can shuttle between the cytoplasm and nucleus in a context-dependent fashion [Sharma, NR et al. J. Biol. Chem. (2016) 291: 2302-9] and is a key effector of RNA-silencing pathways. It is a major component of the RNA-induced silencing complex (RISC).

[0349] The term "astringents" are generally protein precipitants that have such low cell penetrability that the action essentially is limited to the cell surface and interstitial spaces. Astringents are locally applied. The astringent action is accompanied by contraction and wrinkling of the tissue and by blanching. Astringents are used therapeutically to arrest hemorrhage by coagulating the blood, to promote healing, to toughen the skin or to decrease sweating. The principal components of astringents are salts of aluminum, zinc, manganese, iron or bismuth.

[0350] The term “binding” and its other grammatical forms as used herein means a lasting attraction between chemical substances. Binding specificity involves both binding to a specific partner and not binding to other molecules. Functionally important binding may occur at a range of affinities from low to high, and design elements may suppress undesired cross-interactions. Post-translational modifications also can alter the chemistry and structure of interactions. “Promiscuous binding” may involve degrees of structural plasticity, which may result in different subsets of residues being important for binding to different partners. “Relative binding specificity” is a characteristic whereby in a biochemical system a molecule interacts with its targets or partners differentially, thereby impacting them distinctively depending on the identity of individual targets or partners.

[0351] The term “biomarker” (or "biosignature") as used herein refers to a peptide, a protein, a nucleic acid, an antibody, a gene, a metabolite, or any other substance used as an indicator of a biologic state. It is a characteristic that is measured objectively and evaluated as a cellular or molecular indicator of normal biologic processes, pathogenic processes, or pharmacologic responses to a therapeutic intervention. The term "indicator" as used herein refers to any substance, number or ratio derived from a series of observed facts that may reveal relative changes as a function of time; or a signal, sign, mark, note or symptom that is visible or evidence of the existence or presence thereof. Once a proposed biomarker has been validated, it may be used to diagnose disease risk, presence of disease in an individual, or to tailor treatments for the disease in an individual (choices of drug treatment or administration regimes). In evaluating potential therapies, a biomarker may be used as a surrogate for a natural endpoint, such as survival or irreversible morbidity. If a treatment alters the biomarker, and that alteration has a direct connection to improved health, the biomarker may serve as a surrogate endpoint for evaluating clinical benefit. Clinical endpoints are variables that can be used to measure how patients feel, function or survive. Surrogate endpoints are biomarkers that are intended to substitute for a clinical endpoint; these biomarkers are demonstrated to predict a clinical endpoint with a confidence level acceptable to regulators and the clinical community.

[0352] The term “cargo” as used herein refers to a load or that which is conveyed. With respect to exosomes and / or extracellular vesicles, the term cargo refers to a substance encapsulated in the exosome and or extracellular vesicle. The compound or substance can be, e.g., a nucleic acid(e.g., nucleotides, DNA, RNA), a polypeptide, a lipid, a protein, or a metabolite, or any other substance that can be encapsulated in an exosome and / or an extracellular vesicle.

[0353] The term “cargo profile” as used herein refers to measurements of cargo components that characterize a population of extracellular vesicles.

[0354] The term "carrier" as used herein describes a material that does not cause significant irritation to a mammal and does not abrogate the biological activity and properties of the actives of the composition. Carriers must be of sufficiently high purity and of sufficiently low toxicity to render them suitable for administration to the mammal being treated. The carrier can be inert, or it can possess pharmaceutical benefits, cosmetic benefits or both.

[0355] The term "caustic agents" as used herein refers to substances capable of destroying or eating away epithelial tissue by chemical action. Caustic agents can be used to remove dead skin cells. For example, beta-hydroxy acids, naturally derived acids with a strong kerolytic effect, are useful for problem skin, acne or peeling.

[0356] “Cluster of Differentiation” or “cluster of designation” (CD) molecules are utilized in cell sorting using various methods, including flow cytometry. Cell populations usually are defined using a "+" or a "-" symbol to indicate whether a certain cell fraction expresses or lacks a particular CD molecule.

[0357] The term "comparison window" refers to a contiguous and specified segment of a polynucleotide sequence, wherein the polynucleotide sequence may be compared to a reference sequence and wherein the portion of the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences.

[0358] The term "compatible" as used herein means that the components of a composition are capable of being combined with each other in a manner such that there is no interaction that would substantially reduce the efficacy of the composition under ordinary use conditions.

[0359] The term “complementary DNA” or “cDNA” as used herein refers to synthetic DNA that has been transcribed from a specific mRNA through a reaction using the enzyme reverse transcriptase. While DNA is composed of both coding and non-coding sequences, cDNA contains only coding sequences.

[0360] The term "condition" as used herein includes a variety of conditions related to skin or mucosal membranes. This term is meant to include disorders or diseases, the promotion of healthy epithelium; dry skin; and inflammation caused by any underlying mechanism or disorder.

[0361] The term “conditioned medium” (or plural, media), as used herein refers to spent culture medium harvested from cultured cells containing metabolites, growth factors, RNA and proteins released into the medium by the cultured cells.

[0362] The term “contact” and its various grammatical forms as used herein refers to a state or condition of touching or of immediate or local proximity.

[0363] The term “cosmeceutical” as used herein refers to a topical preparation sold as a cosmetic that has performance characteristics that affect the skin positively beyond the time of its application.

[0364] The term "cosmetic composition' as used herein refers to a composition that is intended to be rubbed, poured, sprinkled, or sprayed on, introduced into, or otherwise applied to a subject or any part thereof for cleansing, beautifying, promoting attractiveness, or altering the appearance, or an article intended for use as a component of any such article, except that such term does not include soap.

[0365] The terms “cosmetic signature”, cosmeceutical signature” and “therapeutic signature” as used herein respectively refer to a specific and complex combination of biomarkers that reflect a biological state that leads to a specific cosmetic, cosmeceutical or therapeutic effect.

[0366] The term "cosmetically acceptable carrier" as used herein refers to a substantially non- toxic carrier, conventionally useable for the topical administration of cosmetics, with which compounds will remain stable and bioavailable.

[0367] The terms "cosmetic amount”, cosmeceutical amount” or “pharmaceutical amount” as used herein refer to the amount of any of the compositions of the invention that result in a beneficial effect following administration to a subject. The cosmetic, cosmeceutical, or pharmaceutical effect can improve physical appearance and aesthetics, treating, a condition, disease or disorder, or any other beneficial effect. The concentration of the active(s) is selected so as to exert its cosmetic, cosmeceutical or pharmaceutical effect, but low enough to avoidsignificant side effects within the scope and sound judgment of the skilled artisan. The effective amount of the composition may vary with the particular epithelial tissue being treated, the age and physical condition of the biological subject being treated, the severity of the condition, the duration of the treatment, the nature of concurrent therapy, the specific compound(s), composition or other active ingredient(s) employed, the particular carrier utilized, and like factors. A skilled artisan can determine an effective amount of the inventive compositions by determining the unit dose. As used herein, a "unit dose" refers to the amount of inventive composition required to produce a response of 50% of maximal effect (i.e. ED50). The unit dose can be assessed by extrapolating from dose-response curves derived from in vitro or animal model test systems.

[0368] The term “cross-dressing” as used herein refers to a third pathway for cross-presentation. In cross-dressing, dendritic cells acquire preformed MHC class I molecules in complex with antigens from other cells by the process of trogocytotis (meaning the transfer of cell membrane patches or individual proteins between cells [Yewdell, JW and Dolan, BP, Nature (2011) 471 (7340): 581-582, citing Joly, E. and Hudrisier, D. Nature Immunol. (2003) 4: 815; Herrera OB et al. J. Imunol. (2004) 173: 4828-4837] or through gap junctions. This allows antigen presentation by acceptor dendritic cells to occur immediately, without any processing. Cross-dressing is used to activate memory T cells, but not naïve T cells, in response to viral infection [Id., citing Wakins, LM and Bevan, MJ. Nature (2011) 471: 629-632].

[0369] The term “cross-presentation” as used herein refers to a process by which proteins taken up by dendritic cells from the extracellular milieu can give rise to peptides presented by MHC class I molecules. It enables antigens from extracellular sources to be presented by MHC class I molecules and to activate CD8 T cells.

[0370] The term “cross-priming” as used herein refers to activation of CD8 T cells by dendritic cells in which the antigenic peptide presented by MHC class I molecules is derived from an exogenous protein (i.e., by cross-presentation), rather than produced within the dendritic cells directly (compare direct presentation).

[0371] The term “culture medium” (or plural, media), as used herein refers to a substance containing nutrients in which cells or tissues are cultivated for controlled growth.

[0372] The term “cytokine” as used herein refers to small soluble protein substances secreted bycells, which have a variety of effects on other cells. Cytokines mediate many important physiological functions, including growth, development, wound healing, and the immune response. They act by binding to their cell-specific receptors located in the cell membrane, which allows a distinct signal transduction cascade to start in the cell, which eventually will lead to biochemical and phenotypic changes in target cells. Generally, cytokines act locally. They include type I cytokines, which encompass many of the interleukins, as well as several hematopoietic growth factors; type II cytokines, including the interferons and interleukin-10; tumor necrosis factor (TNF)-related molecules, including TNFα and lymphotoxin; immunoglobulin super-family members, including interleukin 1 (IL-1); and the chemokines, a family of molecules that play a critical role in a wide variety of immune and inflammatory functions. The same cytokine can have different effects on a cell depending on the state of the cell. Cytokines often regulate the expression of, and trigger cascades of, other cytokines.

[0373] The term damage-associated molecular patterns” or “DAMPS” as used herein refers to molecules released by stressed or dying cells that bind to pattern recognition molecules (PRMs) and induce inflammation.

[0374] The term “dendritic cells (DC)” as used herein refers to professional antigen presenting cells, which induce naïve T cell activation and effector differentiation. [Patente, TA, et al., Frontiers Immunol. (2019) doi.org / 10.3389 / fimmu.2018.03176]. Human DC are identified by their high expression of major histocompatibility complex (MHC) class II molecules (MHC-II) and of CD11c, both of which are found on other cells, like lymphocytes, monocytes and macrophages [Id., citing Carlens J, et al. J Immunol. (2009) 183:5600–5607; Drutman SB, et al. J Immunol. (2012) 188:3603–3610; Hochweller K, S et al. Eur J Immunol. (2008) 38:2776– 2783; Huleatt JW, Lefrançois L. J Immunol. (1995) 154:5684–5693; Rubtsov AV, et al. Blood (2011) 118:1305–15; Probst HC, et al. Clin Exp Immunol. (2005) 141:398–404; Vermaelen K, Pauwels R. Cytometry (2004) 61A:170–177].

[0375] As used herein, the term “derived from” is meant to encompass any method for receiving, obtaining, or modifying something from a source of origin.

[0376] As used herein, the terms “detecting”, “determining”, and their other grammatical forms, are used to refer to methods performed for the identification or quantification of a biomarker, such as, for example, the presence or level of miRNA, or for the presence or absence of acondition in a biological sample. The amount of biomarker expression or activity detected in the sample can be none or below the level of detection of the assay or method.

[0377] The term “differentiation” as used herein refers to a process of development with an increase in the level of organization or complexity of a cell or tissue, accompanied by a more specialized function.

[0378] The term “direct presentation” as used herein refers to a process by which proteins produced within a given cell give rise to peptides presented by MHC class I molecules. This may refer to APCs (such as dendritic cells), or to nonimmune cells that will become the targets of CTLs.

[0379] When used to describe the expression of a gene or polynucleotide sequence, the terms “down-regulation”, “disruption”, “inhibition”, “inactivation”, and “silencing” are used interchangeably herein to refer to instances when the transcription of the polynucleotide sequence is reduced or eliminated. This results in the reduction or elimination of RNA transcripts from the polynucleotide sequence, which results in a reduction or elimination of protein expression derived from the polynucleotide sequence (if the gene comprised an ORF). Alternatively, down-regulation can refer to instances where protein translation from transcripts produced by the polynucleotide sequence is reduced or eliminated. Alternatively, still, down- regulation can refer to instances where a protein expressed by the polynucleotide sequence has reduced activity. The reduction in any of the above processes (transcription, translation, protein activity) in a cell can be by about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100% relative to the transcription, translation, or protein activity of a suitable control cell. Down- regulation can be the result of a targeting event as disclosed herein (e.g., indel, knock-out), for example.

[0380] The term “Drosha” as used herein refers to a nuclear RNase III that cleaves primary miRNAs to release hairpin-shaped pre-miRNAs that are subsequently cut by the cytoplasmic RNase III Dicer to generate mature miRNAs.

[0381] The terms "drying agent" or "desiccant" as used herein refers to a substance that has an affinity for water such that it will extract the water from other materials.

[0382] The term “emollient” as used herein refers to fats or oils in a two-phase system (meaning one liquid is dispersed in the form of small droplets throughout another liquid). Emollients soften the skin by forming an occlusive oil film on the stratum corneum, preventing drying from evaporation in the deeper layers of skin. Thus, emollients are employed as protectives and as agents for softening the skin, rendering it more pliable. Emollients also serve as vehicles for delivery of hydrophobic compounds. Common emollients used in the manufacture of cosmetics include, but are not limited to, butters, such as Aloe Butter, Almond Butter, Avocado Butter, Cocoa Butter, Coffee Butter, Hemp Seed Butter, Kokum Butter, Mango Butter, Mowrah Butter, Olive Butter, Sal Butter, Shea Butter, glycerin, and oils, such as Almond Oil, Aloe Vera Oil, Apricot Kernel Oil, Avocado Oil, Babassu Oil, Black Cumin Seed Oil, Borage Seed Oil, Brazil Nut Oil, Camellia Oil, Castor Oil, Coconut Oil, Emu Oil, Evening Primrose Seed Oil, Flaxseed Oil, Grape Seed Oil, Hazelnut Oil, Hemp Seed Oil, Jojoba Oil, Kukui Nut Oil, Macadamia Nut Oil, Meadowfoam Seed Oil, Mineral Oil, Neem Seed Oil, Olive Oil, Palm Oil, Palm Kernel Oil, Peach Kernel Oil, Peanut Oil, Plum Kernel Oil, Pomegranate Seed Oil, Poppy Seed Oil, Pumpkin Seed Oil, Rice Bran Oil, Rosehip Seed Oil, Safflower Oil, Sea Buckthorn Oil, Sesame Seed Oil, Shea Nut Oil, Soybean Oil, Sunflower Oil, Tamanu Oil, Turkey Red Oil, Walnut Oil, Wheatgerm Oil

[0383] As used herein “emulsion” refers to a colloid system in which both the dispersed phase and the dispersion medium are immiscible liquids where the dispersed liquid is distributed in small globules throughout the body of the dispersion medium liquid. A stable basic emulsion contains at least the two liquids and an emulsifying agent. Common types of emulsions are oil- in-water, where oil is the dispersed liquid and an aqueous solution, such as water, is the dispersion medium, and water-in-oil, where, conversely, an aqueous solution is the dispersed phase. It also is possible to prepare emulsions that are nonaqueous. Creams of the oil-in-water type include hand creams and foundation creams. Water-in-oil creams include cold creams and emollient creams.

[0384] Creams may be diluted only with suitable diluents specified in the appropriate entries, and diluted creams must be freshly prepared without the application of heat. Creams should be stored in a cool place and supplied in well-closed containers that prevent evaporation and contamination of the contents. When making a natural cream, however, butters first are melted.The vessel is removed from the heat and the oils are added. When the solution is 100 degrees F, the balance of the liquid portion of the formula then is slowly added while continuously stirred.

[0385] The term “endogenous” as used herein refers to that which is naturally occurring, incorporated within, housed within, adherent to, attached to, or resident in.

[0386] As used herein, the term “enrich” is meant to refer to increasing the proportion of a desired substance, for example, to increase the relative frequency of a subtype of cell or cell component compared to its natural frequency in a cell population. Positive selection, negative selection, or both are generally considered necessary to any enrichment scheme. Selection methods include, without limitation, magnetic separation and fluorescence-activated cell sorting (FACS).

[0387] The term “entrainment” as used herein refers to a process of coordinating the internal circadian clock to external rhythmic time-cues (Zeitgeber), mainly light. The organization of circadian phases is dependent on the geographical and seasonal context and individual clock properties (personal chronotypes).[Schmal, C. et al. Front. Physiol. (2020) 11: 272].

[0388] The term “ESCRT machinery” as used herein refers to an evolutionarily conserved, multi-subunit membrane remodeling complex originally identified in yeast for its essential role in the biogenesis of intraluminal vesicles (ILVs) upon a class of endosome called the multivesicular body (MVB), whose role in mammalian cells includes a number of topologically equivalent membrane modeling events, see Olmos, Y., Oarlton, JG. Curr. Opin. Cell Biol. (2016) 38: 1-11, citing Katzmann, DJ., et al. Cell (2001) 106: 145-155; Babst, M., et al. Dev. Cell (2002) 3: 283-289; Babst, M., et al., Dev. Cell (2002) 3: 272-282]

[0389] The term “exogenous” as used herein refers to that which is non-naturally occurring, or that is originating or produced outside of a specific EV, cell, organism, or species.

[0390] The term “expand” and its various grammatical forms as used herein refers to a process by which dispersed living cells propagate in vitro in a culture medium that results in an increase in the number or the amount of viable cells.

[0391] As used herein, the term “expression” and its various grammatical forms refers to the process by which a polynucleotide is transcribed from a DNA template (such as into an mRNA or other RNA transcript) and / or the process by which a transcribed mRNA is subsequentlytranslated into peptides, polypeptides, or proteins. Transcripts and encoded polypeptides may be collectively referred to as “gene product.” If the polynucleotide is derived from genomic DNA, expression may include splicing of the mRNA in a eukaryotic cell. Expression may also refer to the post-translational modification of a polypeptide or protein.

[0392] The term “extracellular matrix” as used herein refers to a scaffold in a cell’s external environment with which the cell interacts via specific cell surface receptors. The extracellular matrix serves many functions, including, but not limited to, providing support and anchorage for cells, segregating one tissue from another tissue, and regulating intracellular communication. The extracellular matrix is composed of an interlocking mesh of fibrous proteins and glycosaminoglycans (GAGs). Examples of fibrous proteins found in the extracellular matrix include collagen, elastin, fibronectin, and laminin. Examples of GAGs found in the extracellular matrix include proteoglycans (e.g., heparin sulfate), chondroitin sulfate, keratin sulfate, and non- proteoglycan polysaccharide (e.g., hyaluronic acid). The term “proteoglycan” refers to a group of glycoproteins that contain a core protein to which is attached one or more glycosaminoglycans.

[0393] The term “extracellular vesicles (EVs)” as used herein refers to nanosized, membrane- bound vesicles released from mammalian cells that can transport cargo—including DNA, RNA, and proteins—between cells as a form of intercellular communication. Different EV types, including microvesicles (MVs), exosomes, oncosomes, and apoptotic bodies, have been characterized on the basis of their biogenesis or release pathways. Microvesicles bud directly from the plasma membrane, are 100 nanometers (nm) to 1 micrometer (μm) in size, and contain cytoplasmic cargo (Zaborowski, MP., et al. BioScience (2015) 65 (8): 783-797, citing Heijnen, HF., et al. Blood (1999) 94: 3791-3799). Another EV subtype, exosomes, is formed by the fusion between multivesicular bodies and the plasma membrane, by which multivesicular bodies release smaller vesicles (exosomes) whose diameters range from 40 to 120 nm (Id., citing El Andaloussi, S., et al. Nature Reviews Drug Discovery (2013) 12: 347-357; Cocucci, E. and Meldolesi J. Trends in Cell Biology (2015) 25: 364-372). Dying cells, release vesicular apoptotic bodies (50 nm–2 μm) that can be more abundant than exosomes and or extracellular vesicles or MVs under specific conditions and can vary in content between biofluids (Id., citing Thery, C., et al. J. Immunology (2001) 1666: 7309-7318; El Andaloussi, S., et al. Nature Reviews Drug Discovery (2013) 12: 347-357). Membrane protrusions can also give rise to large EVs, termed oncosomes (1–10 μm), which are produced primarily by malignant cells in contrast totheir nontransformed counterparts (Id., citing Di Vizio, D., et al. Am. J. Pathol. (2012) 181: 1573-1584; Morello, M., et al. Cell Cycle (2013) 12: 3526-3536).

[0394] The terms "formulation" and "composition" are used interchangeably herein to refer to a product of the described invention that comprises all active and inert ingredients.

[0395] As used herein the term “fragment” and its other grammatical forms are meant to refer to portions of a nucleic acid, polynucleotide or oligonucleotide shorter than the full sequence of a reference molecule. The sequence of bases in a fragment is unaltered from the sequence of the corresponding portion in the molecule from which it arose; there are no insertions or deletions in a fragment in comparison with the corresponding portion of the molecule from which it arose. As contemplated herein, a fragment of a nucleic acid or polynucleotide, such as an oligonucleotide, is 15 or more bases in length, or 16 or more, 17 or more, 18 or more, or 19 or more, or 20 or more, or 21 or more, or 22 or more, or 23 or more, or 24 or more, or 25 or more, or 26 or more, or 27 or more, or 28 or more, or 29 or more, 30 or more, 50 or more, 75 or more, 100 or more bases in length, up to a length that is one base shorter than the full length sequence.

[0396] The term “free radical” as used herein refers to a highly reactive and usually short-lived molecular fragment with one or more unpaired electrons. Free radicals are highly chemically reactive molecules. Because a free radical needs to extract a second electron from a neighboring molecule to pair its single electron, it often reacts with other molecules, which initiates the formation of many more free radical species in a self-propagating chain reaction. This ability to be self-propagating makes free radicals highly toxic to living organisms. Oxidative injury may lead to widespread biochemical damage within the cell. The molecular mechanisms responsible for this damage are complex. For example, free radicals may damage intracellular macromolecules, such as nucleic acids (e.g., DNA and RNA), proteins, and lipids. Free radical damage to cellular proteins may lead to loss of enzymatic function and cell death. Free radical damage to DNA may cause problems in replication or transcription, leading to cell death or uncontrolled cell growth. Free radical damage to cell membrane lipids may cause the damaged membranes to lose their ability to transport oxygen, nutrients or water to cells.

[0397] The term “gene” as used herein refers to a DNA polynucleotide sequence that expresses an RNA (RNA is transcribed from the DNA polynucleotide sequence) from a coding region, which RNA can be a messenger RNA (encoding a protein) or a non-protein-coding RNA. A“gene” can refer to the coding region alone or may include regulatory sequences upstream and / or downstream to the coding region (e.g., promoters, 5′-untranslated regions, 3′-transcription terminator regions). A coding region encoding a protein can alternatively be referred to herein as an “open reading frame” (ORF). A gene that is “native” or “endogenous” refers to a gene as found in nature with its own regulatory sequences; such a gene is located in its natural location in the genome of a host cell. A “chimeric” gene refers to any gene that is not a native gene, comprising regulatory and coding sequences that are not found together in nature (i.e., the regulatory and coding regions are heterologous with each other). Accordingly, a chimeric gene may comprise regulatory sequences and coding sequences that are derived from different sources, or regulatory sequences and coding sequences derived from the same source but arranged in a manner different than that found in nature. A “foreign” or “heterologous” gene refers to a gene that is introduced into the host organism by gene transfer. Foreign / heterologous genes can comprise native genes inserted into a non-native organism, native genes introduced into a new location within the native host, or chimeric genes. A homologous gene is a type of gene that is inherited by two different species that evolved from the same ancestor. A paralogue gene is one of a set of homologous genes that have diverged from each other as a consequence of genetic duplication. A “transgene” is a gene that has been introduced into the genome by a gene delivery procedure (e.g., transformation). A “codon-optimized” open reading frame has its frequency of codon usage designed to mimic the frequency of preferred codon usage of the host cell.

[0398] The term “growth factor” as used herein refers to extracellular polypeptide molecules that bind to a cell-surface receptor triggering an intracellular signaling pathway, leading to proliferation, differentiation, or other cellular response. These pathways stimulate the accumulation of proteins and other macromolecules, e.g., by increasing their rate of synthesis, decreasing their rate of degradation, or both.

[0399] The term “hair health” as used herein refers to shiny hair with a smooth texture and clean-cut ends or tapered tips. Hair consists of an outer hydrophobic lipid epicuticle, a layer of flattened overlapping cuticle cells surrounding the elongated polyhedral cortical cells. The normal cuticle, which has a smooth appearance, allowing light reflection and limiting friction between the hair shafts, is responsible for the luster and texture of the hair [Sinclair, RD. J. Investig. Dermatology Symposium Proceedings (2007) 12: 2-5, citing Draelos, ZD. Dermatol.Clin. (1991) 9: 199-227]. The cortical layer determines many of the mechanical properties of the hair. It consists of closely packed spindle-shaped cortical cells filled with keratin filaments that are orientated parallel to the longitudinal axis of the hair shaft, and an amorphous matrix of high sulfur proteins [Id., citing Dawber, R. Clin. Dermatol. (1996) 4: 105-112]. The outer, intensely hydrophobic layer and the cortex confer the physical properties of luster (shine) and volume (body) that contribute to the appearance of hair health.

[0400] The term “heat shock proteins” or “HSPs” as used herein refers to highly conserved multimolecular complexes expressed constitutively under normal growth conditions in cells that act as molecular chaperones, which play a regulatory role in the folding of proteins, intracellular transport of proteins in cytosol, endoplasmic reticulum and mitochondria, repair or degradation of proteins and refolding of misfolded proteins. In addition to being constitutively expressed, these proteins can be induced by a range of environmental, pathological, or physiological stimuli.

[0401] The term “homolog” as used herein refers to being s...

Claims

CLAIMS What is claimed is:

1. A composition comprising a purified population of plant-derived exosome-like nanoparticles isolated from tissue of a vascular plant, wherein: size of the exosome-like nanoparticles is about 50 nm-500 nm inclusive the exosome-like nanoparticles comprise a tuned cargo comprising, a signature of miRNAs selected from ath-miR166a-3p; ath-miR166b-3p; ath-miR166e- 3p; ath-miR396a-5p; ath-miR396b-5p; ath-miR396b-5p; ath-miR156ff-5p; ath-miR168b-5p; ath- miR156c-5p; ath-miR162a-3p; ath-miR162b-3p; ath-miR396a-3p; ath-miR168a-5p; ath- miR156b-5p; ath-mi156a-5p; ath-miR156d-5p; ath-miR164c-5p; ath-miR408-3p; ath-miR165a- 3p; ath-miR160a-5p; ath-miR157b-5p; ath-miR157a-5p; ath-miR164b-5p; ath-miR5016; ath- miR5998b; ath-miR5020a; ath-miR836; ath-miR158a-5p; ath-miR395e; ath-miR402; ath- miR5662; ath-miR5630a; ath-miR5630b; ath-miR3933; ath-miR5998a; ath-miR172e-3p; ath- miR5024-3p; ath-miR447a-3p; ath-miR414; ath-miR167a-3p; ath-miR172b-5p; ath-miR5636; ath-miR824-3p; ath-miR172e-5p; ath-miR404; ath-miR447b; ath-miR826b; ath-miR169g-5p; ath-miR868-3p; ath-miR830-3p; ath-miR169f-5p; ath-miR828; ath-miR8182; ath-miR160a-3p; ath-miR5635d; ath-miR399c-3p; ath-miR5635c; ath-miR398a-3p; ath-miR391-5p; ath-miR781a; ath-miR157c-3p; ath-miR399b; ath-miR5014b; ath-miR5635b; ath-miR779.2; ath-miR390b-5p; ath-miR833a-3p; ath-miR849; ath-miR5635a; ath-miR841b-3p; ath-miR390a-5p; ath-miR447c- 3p; ath-miR835-3p; ath-miR5638b; ath-miR2112-3p; ath-miR5653; ath-miR166a-5p; ath- miR159b-5p; ath-miR166b-5p; ath-miR843; ath-miR5015; ath-miR781b; ath-miR4245; ath- miR169b-5p; ath-miR5013; ath-miR864-5p; ath-miR866-5p; ath-miR5595a; ath-miR403-3p; ath-miR164c-3p; ath-miR835-5p; ath-miR165b; ath-miR3434-5p; ath-miR8176; ath-miR5631; ath-miR399a; ath-miR4227; ath-miR5666; ath-miR778; ath-miR851-3p; ath-miR5663-3p; ath- miR832-3p; ath-miR5646; ath-miR856; ath-miR837-5p; ath-miR846-3p; ath-miR827; ath- miR5633; ath-miR413; ath-miR838; ath-miR5654-5p; ath-miR172d-5p; ath-miR5642a; ath- miR420; ath-miR831-3p; ath-miR156d-3p; ath-miR5018; ath-miR8168; ath-miR866-3p; ath- miR8170-3p; ath-miR395b; ath-miR395c; ath-miR780.2; ath-miR167c-5p; ath-miR393a-3p; ath- miR395f; or a combination thereof; anda signature of proteins selected from the group consisting of heat shock protein (HSP) chaperones; heat shock transcription factors (Hsfs); a ribulose 1,5-bisphosphate carboxylase / oxygenase subunit; a calcium dependent protein kinase, a PLA8 family protein; a glutathione transferase, or a combination thereof; wherein the tuned cargo of the plant-derived exosome-like nanoparticles is produced by exposure of the plant to combinations of abiotic stress conditions that cause the plant to modulate its signaling pathways and metabolism to ensure its survival in a challenging environment.

2. The composition of claim 1, wherein a. the plant is from Family Asphodelaceae or is an aloe vera plant, or b. the plant is from Family Papaveraceae and is a Celandine plant; or c. the plant is from Family Passifloraceae and is a Passiflora ligularis or a Passiflora edulis plant; or d. the plant is from Family Rubiaceae and is a Morinda citrifolia plant.

3. The composition of claim 1, wherein the plant tissue includes roots, stems, leaves, flowers, seeds, fruits, a liquid extract of the plant tissue, a nut milk, or a combination thereof.

4. The composition of claim 1, wherein a. the HSP chaperones induced under the abiotic stress conditions include HSP100, HSP90, HSP70, HSP60, a small HSP or a combination thereof; and b. the Hsfs induced under the abiotic stress conditions include HsfA, HsfB, HsfC or a combination thereof.

5. The composition of claim 1, wherein primary abiotic stress conditions, which include high / low temperature; salinity; drought; dehydration; flooding; heavy metal chemical pollutants;light stresses or physical wounding, produce secondary stresses comprising oxidative stress and osmotic stress.

6. The composition of claim 5, wherein the tuned cargo of the plant-derived exosomes is a result of exposure of the plant to two high temperature abiotic stress conditions.

7. The composition of claim 1, wherein the tuned protein cargo of the plant-derived exosomes correlates to a protein signature comprising human proteins including a keratin; semaphorin receptor plexin-B1 mitogen-activated protein kinase kinase 2 (MEKK2), diacylglycerol kinase; T cell receptor beta chain; a fez family zinc finger protein or a combination thereof.

8. The composition of claim 1, wherein the tuned cargo of the plant-derived exosome-like nanoparticles can modulate bioactivities of mammalian cells directly or indirectly.

9. The composition of claim 8, wherein a. the mammalian cells are human cells; and b. the bioactivities comprise a correlated signaling pathway in the human cells.

10. The composition of claim 9, wherein the human mammalian cells are cells of human skin.

11. The composition of claim 9, wherein the correlated human signaling pathways includes PI3K signaling, ERK / MAPK signaling; insulin growth factor 1 receptor (IGF1R) signaling, VEGFA / VEGFR2 signaling; leptin signaling; cytokine signaling; interleukin signaling, semaphorin signaling; sirtuin signaling; LRP1 signaling, or a combination thereof.

12. The composition of claim 6, wherein administration of the composition comprising the exosome-like nanoparticles comprising the tuned cargo modulates: collagen production in human dermal fibroblasts in vitro; or elastin production in human dermal fibroblasts in vitro; or hyaluronic acid production in human dermal fibroblasts in vitro; or interferon a2 production in mammalian PBMCs exposed to a microbial agent in vitro; or VEGFA production in human dermal fibroblasts in vitro; or a combination thereof.

13. The composition of claim 1, wherein the composition: is a nutraceutical composition comprising a dietary amount of the purified plant- derived exosomes comprising the tuned cargo; is a cosmetic composition comprising a cosmetic amount of the purified plant- derived exosomes and a cosmetically acceptable carrier; or is a cosmeceutical composition comprising a cosmeceutical amount of the purified plant-derived exosomes comprising the tuned cargo and a cosmeceutically acceptable carrier; or is a therapeutic composition; comprising a therapeutic amount of the purified plant-derived exosomes comprising the tuned cargo and a pharmaceutically acceptable carrier.

14. A method for improving appearance of human skin comprising: exposing a vascular plant to combinations of abiotic stress conditions that cause the plant to modulate its signaling pathways and metabolism to ensure its survival in a challenging environment; purifying from tissue of the vascular plant exposed to the combinations of abiotic conditions a population of plant-derived exosome-like nanoparticles (plant-derived exosomes)comprising a tuned cargo, wherein size of the plant-derived exosomes is about 50 nm-500 nm inclusive; preparing a composition comprising about 1x10E8 to about 1x10E12, inclusive, abiotically stressed plant-derived exosome-like nanoparticles containing the tuned cargo and a cosmetically acceptable carrier; and applying the composition topically to human skin; wherein the tuned cargo of the plant-derived exosomes comprises: a signature of miRNAs selected from ath-miR166a-3p; ath-miR166b-3p; ath- miR166e-3p; ath-miR396a-5p; ath-miR396b-5p; ath-miR396b-5p; ath-miR156ff-5p; ath- miR168b-5p; ath-miR156c-5p; ath-miR162a-3p; ath-miR162b-3p; ath-miR396a-3p; ath- miR168a-5p; ath-miR156b-5p; ath-mi156a-5p; ath-miR156d-5p; ath-miR164c-5p; ath-miR408- 3p; ath-miR165a-3p; ath-miR160a-5p; ath-miR157b-5p; ath-miR157a-5p; ath-miR164b-5p; ath- miR5016; ath-miR5998b; ath-miR5020a; ath-miR836; ath-miR158a-5p; ath-miR395e; ath- miR402; ath-miR5662; ath-miR5630a; ath-miR5630b; ath-miR3933; ath-miR5998a; ath- miR172e-3p; ath-miR5024-3p; ath-miR447a-3p; ath-miR414; ath-miR167a-3p; ath-miR172b- 5p; ath-miR5636; ath-miR824-3p; ath-miR172e-5p; ath-miR404; ath-miR447b; ath-miR826b; ath-miR169g-5p; ath-miR868-3p; ath-miR830-3p; ath-miR169f-5p; ath-miR828; ath-miR8182; ath-miR160a-3p; ath-miR5635d; ath-miR399c-3p; ath-miR5635c; ath-miR398a-3p; ath-miR391- 5p; ath-miR781a; ath-miR157c-3p; ath-miR399b; ath-miR5014b; ath-miR5635b; ath-miR779.2; ath-miR390b-5p; ath-miR833a-3p; ath-miR849; ath-miR5635a; ath-miR841b-3p; ath-miR390a- 5p; ath-miR447c-3p; ath-miR835-3p; ath-miR5638b; ath-miR2112-3p; ath-miR5653; ath- miR166a-5p; ath-miR159b-5p; ath-miR166b-5p; ath-miR843; ath-miR5015; ath-miR781b; ath- miR4245; ath-miR169b-5p; ath-miR5013; ath-miR864-5p; ath-miR866-5p; ath-miR5595a; ath- miR403-3p; ath-miR164c-3p; ath-miR835-5p; ath-miR165b; ath-miR3434-5p; ath-miR8176; ath-miR5631; ath-miR399a; ath-miR4227; ath-miR5666; ath-miR778; ath-miR851-3p; ath- miR5663-3p; ath-miR832-3p; ath-miR5646; ath-miR856; ath-miR837-5p; ath-miR846-3p; ath- miR827; ath-miR5633; ath-miR413; ath-miR838; ath-miR5654-5p; ath-miR172d-5p; ath- miR5642a; ath-miR420; ath-miR831-3p; ath-miR156d-3p; ath-miR5018; ath-miR8168; ath- miR866-3p; ath-miR8170-3p; ath-miR395b; ath-miR395c; ath-miR780.2; ath-miR167c-5p; ath- miR393a-3p; ath-miR395f; or a combination thereof; anda signature of proteins selected from the group consisting of heat shock protein (HSP) chaperones; heat shock transcription factors (Hsfs); a ribulose 1,5-bisphosphate carboxylase / oxygenase subunit; a calcium dependent protein kinase, a PLA8 family protein; a glutathione transferase; or a combination thereof.

15. The method of claim 14, wherein a. the plant is from Family Asphodelaceae or is an aloe vera plant, or b. the plant is from Family Papaveraceae and is a Celandine plant; or c. the plant is from Family Passifloraceae and is a Passiflora ligularis or a Passiflora edulis plant; or d. the plant is from Family Rubiaceae and is a Morinda citrifolia plant.

16. The method of claim 14, wherein the plant tissue includes roots, stems, leaves, flowers, seeds, fruits, a liquid extract of the plant tissue, a nut milk or a combination thereof.

17. The method of claim 14, wherein a. the HSP chaperone induced under the abiotic stress conditions comprises HSP100, HSP90, HSP70, HSP60, a small HSP, or a combination thereof; and b. the Hsf induced under the abiotic stress conditions comprises HsfA, HsfB, HsfC or a combination thereof.

18. The method of claim 14, wherein primary abiotic stress conditions including high / low temperature; salinity; drought; dehydration; flooding; heavy metal chemical pollutants; light stresses or physical wounding produce secondary stresses comprising oxidative stress and osmotic stress.

19. The method of claim 14, wherein the tuned protein cargo of the plant-derived exosome- like nanoparticles correlates to a protein signature comprising human proteins including a keratin; semaphorin receptor plexin-B1 mitogen-activated protein kinase kinase 2 (MEKK2), diacylglycerol kinase; T cell receptor beta chain; a fez family zinc finger protein or a combination thereof.

20. The method of claim 14, wherein the tuned cargo of the plant-derived exosomes can modulate bioactivities of mammalian cells directly or indirectly.

21. The method of claim 20, wherein the bioactivities comprise a correlated signaling pathway in the human cells.

22. The method of claim 20, wherein the correlated signaling pathways in the human cells include PI3K signaling, ERK / MAPK signaling; insulin growth factor 1 receptor (IGF1R) signaling, VEGFA / VEGFR2 signaling; leptin signaling; cytokine signaling; interleukin signaling, semaphorin signaling; sirtuin signaling; LRP1 signaling, or a combination thereof.

23. The method of claim 14, wherein the composition comprising the tuned cargo of the exosome-like plant nanoparticles when applied to the skin may a. modulate gene expression in immune cells, keratinocytes, melanocytes or fibroblasts in the skin; b. modulate a signaling pathway that contributes to inflammation, immune dysfunction or both in the skin; c. modulate circadian rhythms of the skin and its components; d. rejuvenate appearance of the skin by: i. improving youthful appearance of skin;ii. reducing appearance of wrinkles by stimulating hyaluronic acid and collagen production; iii. improving skin clarity; iv. improving skin texture; v. improving skin luminosity; vi. improving skin radiance; vii. or a combination thereof.

24. The method of claim 14, wherein the pathway is a PI3K / AKT / mTOR pathway, an MAPK pathway, an IGF-1R pathway, a sirtuin pathway, an LRP1 pathway, or a combination thereof.

25. A method for promoting hair health comprising: exposing a vascular plant to combinations of abiotic stress conditions that cause the plant to modulate its signaling pathways and metabolism to ensure its survival in a challenging environment; purifying from tissue of the vascular plant exposed to the combinations of abiotic conditions a population of plant-derived exosome-like nanoparticles (plant-derived exosomes), wherein size of the plant-derived exosomes is about 50 nm-500 nm inclusive; preparing a composition comprising about 1x10E8 to about 1x10E12, inclusive, abiotically stressed plant-derived exosome-like nanoparticles containing the tuned cargo and a cosmetically acceptable carrier; and applying the composition topically to a subject in need thereof; wherein the tuned cargo comprises: a signature of miRNAs selected from ath-miR166a-3p; ath-miR166b-3p; ath- miR166e-3p; ath-miR396a-5p; ath-miR396b-5p; ath-miR396b-5p; ath-miR156ff-5p; ath- miR168b-5p; ath-miR156c-5p; ath-miR162a-3p; ath-miR162b-3p; ath-miR396a-3p; ath-miR168a-5p; ath-miR156b-5p; ath-mi156a-5p; ath-miR156d-5p; ath-miR164c-5p; ath-miR408- 3p; ath-miR165a-3p; ath-miR160a-5p; ath-miR157b-5p; ath-miR157a-5p; ath-miR164b-5p; ath- miR5016; ath-miR5998b; ath-miR5020a; ath-miR836; ath-miR158a-5p; ath-miR395e; ath- miR402; ath-miR5662; ath-miR5630a; ath-miR5630b; ath-miR3933; ath-miR5998a; ath- miR172e-3p; ath-miR5024-3p; ath-miR447a-3p; ath-miR414; ath-miR167a-3p; ath-miR172b- 5p; ath-miR5636; ath-miR824-3p; ath-miR172e-5p; ath-miR404; ath-miR447b; ath-miR826b; ath-miR169g-5p; ath-miR868-3p; ath-miR830-3p; ath-miR169f-5p; ath-miR828; ath-miR8182; ath-miR160a-3p; ath-miR5635d; ath-miR399c-3p; ath-miR5635c; ath-miR398a-3p; ath-miR391- 5p; ath-miR781a; ath-miR157c-3p; ath-miR399b; ath-miR5014b; ath-miR5635b; ath-miR779.2; ath-miR390b-5p; ath-miR833a-3p; ath-miR849; ath-miR5635a; ath-miR841b-3p; ath-miR390a- 5p; ath-miR447c-3p; ath-miR835-3p; ath-miR5638b; ath-miR2112-3p; ath-miR5653; ath- miR166a-5p; ath-miR159b-5p; ath-miR166b-5p; ath-miR843; ath-miR5015; ath-miR781b; ath- miR4245; ath-miR169b-5p; ath-miR5013; ath-miR864-5p; ath-miR866-5p; ath-miR5595a; ath- miR403-3p; ath-miR164c-3p; ath-miR835-5p; ath-miR165b; ath-miR3434-5p; ath-miR8176; ath-miR5631; ath-miR399a; ath-miR4227; ath-miR5666; ath-miR778; ath-miR851-3p; ath- miR5663-3p; ath-miR832-3p; ath-miR5646; ath-miR856; ath-miR837-5p; ath-miR846-3p; ath- miR827; ath-miR5633; ath-miR413; ath-miR838; ath-miR5654-5p; ath-miR172d-5p; ath- miR5642a; ath-miR420; ath-miR831-3p; ath-miR156d-3p; ath-miR5018; ath-miR8168; ath- miR866-3p; ath-miR8170-3p; ath-miR395b; ath-miR395c; ath-miR780.2; ath-miR167c-5p; ath- miR393a-3p; ath-miR395f; or a combination thereof; and a signature of proteins selected from the group consisting of heat shock protein (HSP) chaperones; heat shock transcription factors (Hsfs); a ribulose 1,5-bisphosphate carboxylase / oxygenase subunit; a calcium dependent protein kinase, a PLA8 family protein; a glutathione transferase, or a combination thereof; wherein the composition increases proliferation of dermal papillae cells and hair follicle stem cells and increases hair growth.

26. The method of claim 25, wherein applying topically includes applying to scalp, eyebrows, eyelashes or a combination thereof of the subject.

27. The method of claim 26, wherein the method decreases hair loss; increases hair density; increases appearance of hair thickness; improves scalp health; improves hair shine; improves hair volume or body; or a combination thereof.

28. The method of claim 25, wherein the composition comprising the tuned cargo of the exosome-like plant nanoparticles, when applied topically, modulates gene expression in the dermal papillae cells, hair follicle stem cells, or a combination thereof.

29. The method of claim 28, wherein the composition comprising the tuned cargo of the exosome-like plant nanoparticles, when applied topically, modulates gene expression in human follicle dermal papilla cells (HFDPCs).

30. The method of claim 29, wherein the composition comprising the tuned cargo of the exosome-like plant nanoparticles, when applied topically, modulates gene expression of IL-6 in HFDPCs 24 hours after application.

31. The method of claim 28, wherein the composition comprising the tuned cargo of the exosome-like plant nanoparticles, when applied topically, modulates gene expression of CORIN, LEP, IL1B, IL-6, SRD5A2, BMP4, TGFB1, IGF1, HEY1, or a combination thereof in HFDPCs 72 hours after application.

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