Cellular and Organismal Regulation

Abstract

The present invention relates to medicine, biology, veterinary medicine, pharmacological diagnostics, agriculture, ecology, meteorology, seismology, construction, biotechnology, biomanufacturing, and is provided herein products and methods for managing cellular behavior, cellular memory, and erasure of cellular memory. The present invention describes products and methods that, unlike those known, allow controlling the properties of cells and organisms without the use of mutagens and / or the special introduction of genes and / or the use of specific genetic tools and / or the modification of their environmental conditions.

Description

[Technical field]

[0001] The present invention relates to medical, biological, veterinary, pharmacological diagnostics, agriculture, ecology, meteorology, seismology, construction, biotechnology, biomanufacturing, and herein provided are products and methods for managing cellular behavior, cellular memory, and erasure of cellular memory. [Background technology]

[0002] A known method of introducing new genes, where genes are introduced into cells by various methods such as transformation, transduction, etc. (Chen et al., 1987, Naldini et al., 1996). The introduced genes carry new information or override existing genes.

[0003] There are known methods to modify the properties of cells by editing genomes, cutting out genes, and stitching in genes, when molecules that can artificially change the structure of the genome are introduced into cells (Spicer et al., 2018).

[0004] The present invention describes products and methods which, contrary to what is known, make it possible to control the properties of cells and organisms without the use of mutagenic agents and / or the special introduction of genes and / or the use of specific gene editing tools and / or the modification of their environmental conditions.

[0005] definition Inactivation - destruction; inactivation; cleavage; reduction in number; inhibition of activity; can be done in vitro, in vivo, and / or ex vivo, and in any material.

[0006] Changes - include modification; destruction, inactivation; changes in activity; changes in structure; changes in conformation; changes in nucleic acid components; changes in binding or association with other molecules, i.e. metals, proteins, lipids, and other nucleic acid / non-nucleic acid components; qualitative and / or quantitative changes; changes in signal production, reception, transduction, modification; increases or decreases in number; disposition; changes in activity; recovery after modification; incomplete recovery after modification; changes in production; changes in extracellular secretion; magnetization; these can be performed in vitro, in vivo and / or ex vivo, and in any material).

[0007] Cut-D cells - one treatment with DNA inactivation product.

[0008] Cut-R cells - one treatment with RNA inactivation product.

[0009] One treatment with Cut-DR cells or "Drunk cell" DNA+RNA inactivation products.

[0010] Zero-D Cells Cells are placed in minimal growth conditions (ZD) during treatment with a DNA inactivation product, after 2 or more cycles with a DNA inactivation product.

[0011] Zero-R Cells Cells placed in minimal growth conditions (ZR) during treatment with RNA inactivation products, after 2 or more cycles with RNA inactivation products.

[0012] Zero-DR Cells Cells placed in minimal growth conditions (Z0) during treatment with DNA and RNA inactivation products, after 2 or more cycles with DNA and RNA inactivation products.

[0013] YD cells - 2 or more cycles with DNA inactivation products, placing the cells in the same and / or nutrient rich growth conditions between treatments with the DNA inactivation products.

[0014] YR-2 or more cycles with RNA inactivation products, placing cells in the same and / or highly nutritious growth conditions between treatments with RNA inactivation products.

[0015] Y-DR- Two or more cycles with DNA and RNA inactivation products, placing the cells in the same and / or high nutrient growth conditions between treatments with DNA and RNA inactivation products.

[0016] NAMACS and NAMACS-ANA-cell surface associated nucleic acid molecule(s), and / or other nucleic acids associated with these surface associated nucleic acids.

[0017] TEZRs are nucleic acid molecule(s) that bind to cell surfaces, and / or other nucleic acids that bind to these surface-associated nucleic acids, and can recognize biological, chemical, mechanical and physical agents and produce a cellular response.

[0018] TEZRs can be specific for various cell types and have lengths ranging from 2 to 1,000,000 nucleotides.

[0019] Microorganisms: Bacteria, Archaea, Fungi, Protists, Unicellular Eukaryotes, Unicellular Algae, Viruses.

[0020] To manage: to control, regulate, sense, adjust, change, manipulate, manage, regulate. Summary of the Invention

[0021] In some embodiments, the products can destroy and / or inactivate NAMACS and NAMACS-ANA, reverse transcriptase inhibitors, recombinase inhibitors, e.g., protease inhibitors, integrase inhibitors, recombinases, and cells, organoids, and tissues formed after treatment with these products.

[0022] In one embodiment, the products are used in medicine, veterinary medicine, ecology, meteorology, seismology, agriculture, construction, biotechnology, biomanufacturing to control the function of prokaryotes, eukaryotes including mammals, plants, fungi, animals, organoids, tissues, embryos, organs, single-celled organisms, and multicellular organisms.

[0023] In some embodiments, the products are used to manage relationships with physical, chemical, mechanical, and biological factors.

[0024] In some embodiments, the product may be involved in the generation and / or transduction of a signal within and / or outside the cell.

[0025] In some embodiments, the products are used in the diagnosis, treatment and prevention of diseases caused by protozoa, bacteria, fungi and viruses.

[0026] In some embodiments, the products are used to direct DNA recombination and / or to switch genes on and / or off.

[0027] In some embodiments, the products are used to control bacterial and fungal spore formation.

[0028] In some embodiments, the products are used to direct DNA and / or RNA and / or protein synthesis.

[0029] In some embodiments, the products are used to direct post-synthetic modifications of nucleic acids and / or proteins; DNA methylation.

[0030] In some embodiments, the products are used to manage cell spreading; and re-colonization of bacterial biofilms.

[0031] In some embodiments, the products are used to manage the spread of metastases.

[0032] In some embodiments, the products are used to manage cell properties by placing cells in a "cut" state (such as a "drunk cell"), a "zero" state, or a "Y" state.

[0033] In some embodiments, the modulation of cell properties is due to inactivated TEZR.

[0034] In one embodiment of any of the methods of the invention, the subject is a human.

[0035] In some embodiments, the products are used to direct single stranded DNA, double stranded DNA, single stranded RNA, double stranded RNA, DNA-RNA hybrids, double helical DNA, Pauling triplexes, G-quadruplexes.

[0036] In some embodiments, the products are used to manage organoids, including mitochondria and plastids.

[0037] In some embodiments, the TEZR is on the surface of or within the membrane vesicle.

[0038] In some embodiments, the products are used to regulate a process that is at least in part regulated by type IV secretion.

[0039] In some embodiments, the formation of TEZR is achieved by directing type IV secretion. In some embodiments, the product is used to direct the involvement of reverse transcription, RNA-dependent RNA synthesis, and the formation of cell surface-associated nucleic acid molecule(s) that can lead to the formation of cell surface-associated and / or altered property protein and nucleic acid isoforms.

[0040] In some embodiments, the qualitative and / or quantitative alteration of TEZR occurs within extracellular vesicles.

[0041] In some embodiments, the products are used to regulate the activity of cell surface receptors, including, but not limited to, protein receptors.

[0042] In some embodiments, NAMACS and / or NAMACS-ANA and / or TEZR are artificial.

[0043] In some embodiments, the products are used to regulate the activity of cellular protein kinases.

[0044] In some embodiments, the products are used to manage signaling in mammalian and microbial communities.

[0045] In some embodiments, the products are used to direct viral gene transfer in mammalian and microbial communities.

[0046] In some embodiments, the products are used to regulate cellular activity within any of the components of the microbiota-gut-brain axis.

[0047] In some embodiments, the products are used to manage bacterial colonization and transmigration.

[0048] In some embodiments, the products are used to control mutagenesis and / or cell adhesion to a substrate and / or cell division rate and / or cell division limits.

[0049] In some embodiments, the products are used to direct DNA recombination.

[0050] In some embodiments the products are used to direct the interaction of cellular and extracellular molecules with proteins and / or DNA and / or RNA with prion-like domains of proteins.

[0051] In some embodiments, the products are used to direct processes associated with reverse transcriptases, retroelements, group II introns, CRISPR-Cas systems, diversity generating retroelements, Abi-associated RTs, retrons, multicopy single-stranded DNA (msDNA), and splicing processes.

[0052] In some embodiments, NAMACS and / or NAMACS-ANA and / or TEZR are linked to a receptor having a proteomic structure.

[0053] In some embodiments, the products are used to manage microbial dormancy and persistence.

[0054] In some embodiments, the products are used to increase cell survival in conditions in which untreated cells are killed.

[0055] In some embodiments, the product is used to administer resurrection.

[0056] In some embodiments the products are used to manage the arrest or increase of apoptosis and / or necrosis and / or necroptosis and / or other types of cell death.

[0057] In some embodiments, the products are used to direct the intercellular transport of different genes, which may be encoded in DNA or RNA molecules, and the activity of cellular reverse transcriptase(s) by which the RNA molecules may be transformed into DNA.

[0058] In some embodiments, the products are used to direct delivery to targeted cells.

[0059] In some embodiments, the products are used to regulate the nlrp3 inflammasome, caspase 1 activity and pathway, and the NF-kB pathway.

[0060] In some embodiments, the products are used to manage prokaryote-prokaryote, prokaryote-eukaryote, and eukaryote-eukaryote interactions.

[0061] In some embodiments, the adverse effects of the external environment are ameliorated by wearing clothing that modulates the effects of the geomagnetic field on NAMACS and / or NAMACS-ANA and / or TEZR. In some embodiments, the product is used to manage weather dependency. In some embodiments the products as vaccines against cells NAMACS and / or NAMACS-ANA and / or TEZR and / or DNase and / or RNase are used to treat disease and prolong life.

[0062] In some embodiments, nucleoside and non-nucleoside inhibitors of reverse transcriptase are used alone or in combination with nucleases and / or antibiotics to treat bacterial infections.

[0063] In some embodiments, qualitative and / or quantitative changes of NAMACS and / or NAMACS-ANA and / or TEZR are used to control the function of prokaryotes, eukaryotes, e.g. mammals, plants, fungi, animals, cells, organoids, tissues, embryos, organs, single-celled and multicellular organisms, by antibodies, mini-antibodies, single domain antibodies (nanobodies), antibodies with nuclease activity (abzymes), antibodies conjugated with nucleases and other antibody variants, and / or nucleases, endonucleases, and / or restriction enzymes, and / or exonucleases; non-limiting examples are DNase I, DNase X, DNase gamma, DNase1L1, DNase1L2, DNase 1L3, DNase II (e.g. DNase II alpha, DNase IIβ), caspase-activated DNase (CAD), endonuclease G (ENDOG), AbaSI, AccI, Acc65I, AciI, AclI, AcuI, AfeI, AflII, AflIII, AgeI, AhdI, AleI-v2, AluI, AlwI, AlwNI, ApaI, ApaLI , ApoI, AscI, AseI, AsiSI, AvaI, AvaII, AvrII, BaeGI, BaeI, BamHI, BanI, BanII, BbsI, BbvCI, BbvI, BccI, BceAI, BcgI, BciVI, BclI BfaI BglI BglII BlpI, BmgBI, BmrI, BmtI, BpmI, BpuEI, Bpu10I, BsaAI, BsaBI, BsaHI, BsaI-HF, BsaJI, BsaWI, BsaXI, BseRI, BseYI, BsgI, BsiEI, BsiHKAI, BsiWI, BslI, BsmAI, BcoDI, BsmBI-v2, BsmFI, BsmI, BspCNI, BspEI, BspHI, Bsp1286I, BspMI BfuAI, BsrBI, BsrDI, BsrFI-v2, BsrGI, BsrI, BssHII, BssSI-v2, BstAPI, BstBI, BstEII, BstN I, BstUI, BstXI, BstYI, BstZ17I, Bsu36I, BtgI, BtgZI, BtsCI, BtsIMutI, BtsI-v2, Cac8I, ClaIBspDI、CspCI、CviAII、CviKI-1、CviQI、DdeI、DpnI、DraI、DraIII、 DrdI、EaeI、EagI、EarI、EciI、Eco53kI、EcoNI、EcoO109I、EcoP15I、 EcoRI, EcoRV, Esp3I, FatI, FauI, Fnu4HI, FokI, FseI, FspEI, FspI 、HaeII、HaeIII、HgaI、HhaI、HincII、HindIII、HinfI、HinP1I、HpaI HphI, HpyAV, HpyCH4III, HpyCH4IV, HpyCH4V, Hpy188I, Hpy99I, Hpy166II, Hpy188III, I-CeuI, I-S ceI, KasI, KpnI, LpnPI, MboI, MboII, MfeI, MluCI, MlyI, MmeI, MnlI, MscI, MseI, MslI, MspA1I, MspI HpaII、MspJI、MwoI、NaeI、NarI、Nb.BbvCI、Nb.BsmI Nb.BsrDI、Nb.BssSI、Nb.BtsI、NciI、NcoI NcoI-HF、NdeINgoI、NheI NheI-HF, NlaIII, NlaIV, NmeAIII, NotI NotI-HF, NruI NruI-HF, NsiI NsiI-HF、NspI、Nt.AlwI、Nt.BbvCI、Nt.BsmAI、Nt.BspQI、Nt.BstNBI、Nt.CviPII、PacI、PaqCI、PciI、P flMI, PI-PspI, PI-SceI, PleI, PluTI, PmeI, PmlI, PpuMI, PshAI, PsiI-v2, PspGI, PspOMI, PspXI, PstI PstI-HF、PvuI PvuI-HF、PvuII PvuII-HF、RsaI、RsrII、SacI SacI-HF、SacII、SalI SalI-HF、SapI BspQI、Sau96I、SbfI SbfI-HF, ScaI-HF, ScrFI, SexAI, SfaNI, SfcI, SfiI, SfoI, SgrAI, SmaI, SmlI, SnaBI, SpeI SpeI-HF, SphI-S SspI-HF, StuI, StyD4I, StyI-HF, SwaI, TaqI-v2, TfiI, TseIApeKI, Tsp45I, TspRI, Tth111I PflFI, XbaI, XcmI, XhoI PaeR7I, XmaI TspMI, XmnI, ZraI, granzyme B (GZMB), exonuclease I, exonuclease V, exonuclease VII, exonuclease III, RNaseIf, RNase III, RNase H1, exonuclease I, lambda exonuclease, REC BCD nuclease, REC J nuclease, T6 gene exonuclease, combinations thereof, and mutants or derivatives thereof], phosphodiesterase I, lactoferrin, acetylcholinesterase, engineered nucleases, transferases (i.e. methylases), intercalators, different molecules as adaptors, mitomycin C, bleomycin, metals, oligonucleotides, polysaccharides, aptamers, protection factors against nucleases, reverse transcriptase inhibitors and / or salts of orotic acid, and / or ribavirin and / or acyclovir, and / or conjugated VTL and / or recombinase, protease inhibitors and / or integrase inhibitors, ultrasound and other wave methods, viruses and their components.

[0064] In some embodiments, alterations in NAMACS and / or NAMACS-ANA and / or TEZR include destruction, inactivation; changes in activity; changes in structure; changes in conformation; changes in nucleic acid components; changes in binding or association with other molecules, i.e., metals, proteins, lipids, and other nucleic acid / non-nucleic acid components; qualitative and / or quantitative changes; changes in signal generation, reception, transduction, modification; increases or decreases in number; location; repair after alteration; changes in production; changes in extracellular secretion; magnetization; and these are performed in vitro, in vivo and / or ex vivo, and in any material.

[0065] In some embodiments, the products for controlling the function of cells, tissues, organs, organisms, plants and / or plant seeds may be used before, together with and / or after reverse transcriptase inhibitors and / or recombinase inhibitors and / or protease inhibitors and / or integrase inhibitors and / or proteases and / or salts of orotic acid and / or ribavirin and / or acyclovir, antibodies and / or the compound VTL.

[0066] In some embodiments, a treatment with an integrase inhibitor is used during soaking prior to, simultaneously with, or after treatment with a product to control plant characteristics.

[0067] In some embodiments, the water, soil, membranes in contact with the seed or plant or parts thereof contain and / or are impregnated with various molecules of nucleases, transferases (i.e. methylases), intercalators and / or adapters that bind thereto, mitomycin C, bleomycin, metals, nucleoside reverse transcriptase inhibitors and / or non-nucleoside reverse transcriptase inhibitors and / or salts of orotic acid, and / or ribavirin and / or acyclovir, recombinase and protease inhibitors and / or integrase inhibitors.

[0068] In some embodiments, "cut," "zero," and "Y" state cells are used as antigens.

[0069] In some embodiments, treatment of cells and / or their components with a product alters TLR activity.

[0070] In some embodiments, treatment of cells and / or their components with the product modulates the MyD88-STAT3 or MyD88-NF-κB pathway.

[0071] In some embodiments, TezR is restored using an aptamer.

[0072] In some embodiments, labware (tips, pipettes, dishes, plates, tubes), disposables, liquids (i.e., PBS, water), nutrient media comprise the products for generating cells with new characteristics.

[0073] In one embodiment, "Cut" state, "Zero" state, "Y" state microbial or eukaryotic cells, including "Drunk cells", with unaltered and / or reprogrammed and / or erased memory, are transplanted into individuals, including the same individual from whom these cells were harvested.

[0074] In one embodiment, eukaryotic cells (i.e., stem cells, hematopoietic stem cells, fibroblasts, endothelial cells, renal cells, immune cells, blood cells) are treated with the product to convert them to a "cut" state, a "zero" state, or a "Y" state, including a "drunk cell" state, prior to transplantation into a recipient.

[0075] In one embodiment, cells in the "cut", "zero" and "Y" states, including "drunk cells", are used to transition cells to / from a pluripotent state and are used for the repair and / or regeneration of tissues, organs and parts of the animal or plant body.

[0076] In some embodiments, disease treatment or prevention is effected by disruption of TezR outside or inside a cell.

[0077] In some embodiments, prokaryotic and / or eukaryotic cells that produce agents that inactivate DNA and / or RNA, including representatives of the Bacillaceae family (i.e., Bacillus species), Enterobacteriaceae family (i.e., Escherichia coli, Salmonella species, Klebsiella species), Pseudomonadaceae, Lactococcus, Clostridiaceae, and the fungus Aspergillus species, are added to soil or water for irrigation.

[0078] In some embodiments, the product as an enzyme with nuclease activity is DNase I, and various mutants that weaken actin binding can be used. Specific non-limiting examples of residues of wild-type recombinant human DNase I that can be mutated include, for example, Gln-9, Glu-13, Thr-14, His-44, Asp-53, Tyr-65, Val-66, Val-67, Glu-69, Asn-74, and Ala-114. In various embodiments, the Ala-114 mutation is used. For example, in human DNase I, a high activity mutant containing the sequence of the Ala-114 residue is mutated. Complementary residues of other DNases can also be mutated. Wild-type human recombinant DNAse Specific non-limiting examples of mutations in I include H44C, H44N, L45C, V48C, G49C, L52C, D53C, D53R, D53K, D53Y, D53A, N56C, D58S, D58T, Y65A, Y65E, Y65R, Y65C, V66N, V67E, V67K, V67C, E69R, E69C, A114C, A114R, H44N:T46S, D53R:Y65A, D53R:E69R, H44A:D53R:Y65A, H44A:Y65A:E69R, H64N:V66S, H64N:V66T, Y65N:V67S, Y65N:V67T, V66N:S68T, V67N:E69S, V67N:E69T, S68N:P70S, S68N:P70T, S94N:Y96S, S94N:Y96T. Various DNase mutants for increasing DNase activity can be used. Specific non-limiting examples of mutations in wild-type human recombinant DNAse I include, for example, Gln-9, Glu-13, Thr-14, His-44, Asp-53, Tyr-65, Val-66, Val-67, Glu-69, Asn-74, and Ala-114. Specific non-limiting examples of mutations for increasing the activity of wild-type human recombinant DNAse I include Q9R, E13R, E13K, T14R, T14K, H44R, H44K, N74K, and A114F. For example, a combination of Q9R, E13R, N74K, and A114F mutations can be used.

[0079] In some embodiments, antibiotic resistance expression and overcoming antibiotic resistance using reverse transcriptase inhibitors and pyrimidine series substances, i.e., 2-chloro-5-phenyl-5H-pyrimido[5',4':5,6]pyrano[2,3-d]pyrimidin-4-ol derivatives, are used for cell management for diagnosis, treatment and prevention of disease.

[0080] In some embodiments, the product may be used in combination with drugs, formulations, procedures, medical interventions, non-limiting examples of which include anti-cancer drugs (non-limiting examples of which include chemotherapy, immunotherapy [PD-1, PD-L1, OX-40, CTLA-4 inhibitors], gene therapy, CAR-T, radiation therapy, antibacterial, antiviral drugs, analgesics, anti-stress, anti-aging, regenerative, hormonal, stimulants, antibodies, antipyretics for the prevention and treatment of diseases and conditions of the digestive system, cardiovascular system, central nervous system, musculoskeletal system, trauma, ENT, ophthalmology, respiratory system, endocrine, reproductive, urinary tract, obstetrics and gynecology, and skin systems; immune and autoimmune diseases, immunosuppressants (non-limiting examples of which are TNF blockers), antibiotic therapy, analgesics, siRNA, siDNA, oncolytic viruses, surgery, nutrition, pre-neoplastic and / or neoplastic processes.

[0081] In some embodiments, antibodies are used for the management of prokaryotes or eukaryotes.

[0082] In some embodiments, altering a cell to a "cut" state, a "zero" state, or a "Y" state can cause receptor dysfunction; non-limiting examples of tyrosine kinase-based receptors include EGFR, tumor necrosis factor-related apoptosis-inducing ligand, TLRs, serotonin receptor, CTLA-4, PD-1, and PD-L1, PD-L2, B7 family, VISTA, Tim-3 and LAG-3, TCR, MHC, Gal-9, MHCII, HHLA2, LSECtin, CD80 / 86, CD5, CD7, CD4, CD3, CD28, TIL, estrogen receptor, progesterone receptor, human epidermal growth factor receptor, VEGF, VEGFR, RYK, GDNF, RET, ERBB, INSR, IGF-1R, IRR, PDGFR, CSF-1R, KI T / SCFR, FLK2 / FLT3, FGFR, CCK4, TRKS, TRKB, TRKC, MEN, RON, EPHA, AXL, MER, TYRO, TIE, TEK, DDR, ROS, LTK, ALK, ROR, MUSK, AATYK, RTKINSR group, FGFR group, EGFR group, EPH group, ROR group; those that affect signaling pathways, non-limiting examples of which include those related to WNT, SRC, PI3K, PTEN, AKT, mTOR, PARP, CHK1 / 2, WEE, can be used alone or in combination with other drugs that target such receptors, non-limiting examples of which include monoclonal antibodies (mAbs) that target the extracellular domain and / or receptor catalytic domain and affect aberrant protein phosphorylation.

[0083] In some embodiments, the use of the information recorded in NAMACS and / or NAMACS-ANA and / or TEZR is used for the diagnosis, treatment and prevention of neurodegenerative diseases; pain; cardiovascular diseases; gastrointestinal diseases; urinary system diseases; musculoskeletal diseases; wounds; trauma, cancer; blood diseases; migraines and weather-related conditions; air travel related negative health conditions; poisoning related conditions of various natures; receiving a radiation dose; ultraviolet light exposure related symptoms; overheating related conditions; hypothermia related conditions; wound repair processes and indications for surgical intervention.

[0084] In some embodiments, routes of administration of the present invention include, for example, intracerebral, intraventricular, intraparenchymal injection, intrastriatal, intraspinal, parenteral (e.g., subcutaneous, intramuscular, intravenous, intraarterial, inhalation, intradermal, intrathecal, intracisternal, epidural and infusion), intrathecal injection, enteral (e.g., oral), intramuscular, intraperitoneal, transdermal, rectal, nasal, topical (such as buccal or sublingual), vaginal, intraperitoneal, local, regional, e.g., transdermal, and the like.

[0085] In some embodiments, delivery of DNase and / or RNase to cells is accomplished by using lipid nanoparticle delivery, gold nanocomposite particles, and / or loaded poly(D,L lactide-co-caprolactone) nanocapsules and / or other nanoparticles and / or biohybrid microrobots, and microbes are used to target specific cells in mammals.

[0086] In some embodiments, methods for the treatment and prevention of human disease with therapeutic and prophylactic vaccines against NAMACS and / or NAMACS-ANA and / or TEZR.

[0087] In some embodiments, specificity for product delivery is achieved by delivery of armed antibodies, such as humanized or chimeric antibodies, antigen-targeting antibody fragments, targeted nanomedicines, peptides, antibody drug conjugates against TezR or components thereof.

[0088] In some embodiments, the products are used to treat bacterial / HIV-1 coinfection; a non-limiting example of use in patients is administering reverse transcriptase inhibitors.

[0089] In some embodiments, the regulation or production, activation or action of NAMACS and / or NAMACS-ANA and / or TEZR is regulated by genes associated with retrons, non-limiting examples of genes are msr, msd and RT (msr-msd-RT).

[0090] In some embodiments, the behavior of the cells is modulated by the products or mixtures thereof with: aminoglycosides, annamycins, beta-lactams, carbapenems, cephalosporins, carbapenems, chloramphenicol, fluoroquinolones, glycopeptides, lincosamides, lipopeptides, macrolides, monobactams, nitrofurans, oxazolidinones, penicillins, polypeptides, peptide antimicrobials, quinolones, sulfonamides, tetracyclines, streptogramins, rifamycins, myxopyronines, azoles, polyenes, 5-fluroquinones, benzodiazepines, benzoic acid ... Orocytocin, Echinocandins, Trimethoprim-sulfamethoxazole, Nitrofurantoin, Urinary anti-infectives, Lipopeptides, Sulfonamides, Annamycin, Nitrofurantoin, Nitroimidazoles, Triterpenoids, Azoles, Echinocandins, Nitroimidazoles, Polyene antibiotics, Triterpenoids, Peptide antimicrobials, Bacteriophages, Antiseptics and disinfectants (i.e., alcohols, aldehydes, anilides, biguanides, phenols, diamidines, halogen releasing agents, metal derivatives, peroxygen, quaternary ammonium compounds, gas phase.

[0091] In some embodiments, inactivation and / or alteration, increase and / or decrease and / or modification of activity of tumor cells or the tumor microenvironment is achieved using cells that migrate to tumors and / or metastases (or have tropism for tumors or the tumor environment or can engulf solid tumors) and harbor genes for synthesis and / or excretion of nucleases (including but not limited to DNase, RNase, and combinations thereof) that are delivered directly to the tumor and administered by a variety of methods, including but not limited to oral administration, intravenous injection, intratumoral administration, etc.

[0092] In some embodiments, the cells producing the nuclease are in the "cut" state, the "zero" state, the "Y" state, and are used in combination with surgery, local or systemic chemotherapy, immunotherapy, radiation therapy, and other targeted therapies.

[0093] In some embodiments, Bacillaceae (Bacillus species), Enterobacteriaceae (non-limiting examples include E. coli, Salmonella species, Klebsiella species), Pseudomonadaceae, Bifidobacteriaceae, and Clostridiaceae are used.

[0094] In some embodiments, lytic phages are used against these bacteria or activation of prophage within the bacteria is used to increase the release of nucleases within the tumor, and the bacterial subpopulation that produces the nuclease is then killed due to the release of the nuclease.

[0095] In some embodiments, NAMACS, NAMACS-ANA, and TEZR typing may be used for cell identification.

[0096] In some embodiments, determination of bacterial and fungal NAMACS and / or NAMACS-ANA signatures is used to adjust the effectiveness of sterilization, including pasteurization, to estimate and / or predict the effectiveness of sterilization.

[0097] In some embodiments, the products can regulate the activity of eukaryotic cells, tissues, organs (non-limiting examples include the ability to promote immune responses and kill these cells) for modulation of migration of microbial and / or eukaryotic immune cells and / or viruses (including oncolytic) into these cells, tissues, and organs.

[0098] In some embodiments, qualitative or quantitative analysis of NAMACS and / or NAMACS-ANA and / or TEZR in prokaryotes and eukaryotes may be used as biomarkers for the effectiveness of drug therapy.

[0099] In some embodiments, analysis of NAMACS and NAMACS-ANA, and / or TEZR and / or DNase and RNase genes, presence, expression, levels, and activity of microbial nucleases in cells, tissues, and biological fluids are used to analyze, predict, and modulate bacterial and cell growth, interactions, and susceptibility to antibiotics, immunotherapy, and chemotherapy.

[0100] In some embodiments, the therapeutic effect is achieved by macromicrobial colonization with nuclease-producing microorganisms and eukaryotic organisms.

[0101] In some embodiments, prevention and / or treatment of disease is achieved by reducing DNase and / or RNase activity in cells, human tissues, extracellular spaces, biological fluids of neural tissues, brain, cerebrospinal fluid, including alterations in ion channels, membrane polarization, electrophysiological parameters, neuronal excitability, and synaptic plasticity.

[0102] In some embodiments, the products are used to modulate neuronal activity, memory formation and maintenance.

[0103] In some embodiments, the product is capable of modulating memory in a mammal.

[0104] In some embodiments, the products are used for the regulation of memory of "physiological conditions", non-limiting examples of which include pH, temperature, magnetic field, memory, cell memory, chemotaxis, synergy and antagonism, nutrients, oxygen consumption, gas content.

[0105] In some embodiments the products are used to modulate the memory of antibody forming cells.

[0106] In some embodiments, the product can interfere with the sensing, formation and / or transmission and / or conduction of a signal between molecules, resulting in a response between cells, cell groups, tissues, organs or organisms.

[0107] In some embodiments, use of the product, with or without plating the cells into a new environment, some portion of which must be remembered by the cells, results in the formation of new and / or altered memories.

[0108] In some embodiments, plating cells into a "cut" state, a "zero" state, or a "Y" state, along with plating the cells into new conditions, results in reprogramming of the cells and provides the cells with new properties.

[0109] In some embodiments, the products are used to enhance immune cell memory to improve vaccines.

[0110] In some embodiments, NAMACS and / or NAMACS-ANA and / or TEZR analyses, including those with non-coding genetic information, are used for diagnosis of age, cellular health and disease, cellular origin.

[0111] In some embodiments, the product renders the cells more amenable to reprogramming, thereby making the reprogramming process faster and more efficient.

[0112] In some embodiments, the products for cell reprogramming may be produced together with alterations and modifications of other chaperones (non-limiting example is the CAF-1 histone chaperone).

[0113] In some embodiments, the product is capable of modulating the adaptation, chemotaxis, chemotaxis, or reflexes of eukaryotic or prokaryotic organisms.

[0114] In some embodiments, the product is capable of enhancing cellular cognition and spatial memory.

[0115] In some embodiments, treatment of cells with the products and NAMACS and / or NAMACS-ANA and / or TEZR can enhance the effectiveness of neurotechnology, computer interfaces, brain-machine interfaces, intelligence algorithms, can be used to connect computers to living organisms, and can be used for the development of neuronets.

[0116] In some embodiments, the products are used to regulate the rate and characteristics of fertilization and embryonic development in fish, birds, other animals and humans.

[0117] In some embodiments, the product is used to modulate remote sensing.

[0118] In some embodiments, the products are used to manage epigenetic memory.

[0119] In some embodiments, the product is used for the forgetting of prokaryotic or eukaryotic cells.

[0120] In some embodiments, the products are used to modulate memorization and / or the rate of memorization and / or long-term and / or short-term memory formation.

[0121] In some embodiments, use of the products can alter methylation within the promoter regions of tumor suppressor genes, causing their silencing, and methylation within the genes themselves can induce mutational events.

[0122] In some embodiments, the use of the products can modulate bacterial metabolism, including the metabolism of drugs such as hormones, corticosteroids, anti-cancer drugs, drugs used to treat infectious diseases, drugs used to treat neurodegenerative disorders, etc.

[0123] In some embodiments, the human disease is the result of inactivation and / or alteration of TEZR and / or increased and / or decreased and / or modified activity of prokaryotic and / or eukaryotic cells.

[0124] In some embodiments, the processes of cellular malignancy and / or activation of oncogenes and / or activation of pro-metastasis genes, malignancy of normal cells, epithelial-mesenchymal transition can be modulated by altering NAMACS and / or NAMACS-ANA and / or TEZR.

[0125] In some embodiments, the product is capable of rendering antibiotic resistant bacteria susceptible to the antibiotic.

[0126] In some embodiments, the products can be used to modulate NAMACS and / or NAMACS-ANA disease-related susceptibility genes, including but not limited to: ADAR1, MDA5 (IFIH1), RNaseH subunit, SamHD1, TREX, TBK1, optineurin, P62 (sequestosome 1), progranulin, FUS, VCP, CHMP2B, profilin-1, amyloid beta, Tau, alpha-synuclein, PINK, parkin, LRRK2, DJ-1, GBA, ATPA13. A2, EXOSCIII, TSEN2, TBC1D23, risk factor alleles, PLCG2, TREM2, APOE, TOMM40, IL-33, glucocerebrosidase, ataxin 2 (Ataxin2), C9orf72, SOD1, and FUS, ABL1 (ABL), ABL2 (ABLL, ARG), AKAP13 (HT31, LBC.BRX), ARAF1, ARHGEF5 (TIM), ATF1, AXL, BCL2, BRAF (BRAF1, RAFB1), BRCA1, BRCA2 (FANCD1), BRIP1, CBL (CBL2), CS, F1R (CSF-1, FMS, MCSF), DAPK1 (DAPK), DEK (D6S231E), DUSP6 (MKP3, PYST1), EGF, EGFR (ERBB, ERBB1), ERBB3 (HER3), ERG, ETS1, ETS2, EWSR1 (EWS, ES, P NE), FES(FPS), FGF4(HSTF1, KFGF), FGFR1, FGFR10P(FOP), FLCN, FOS(c-fos), FRAP1, FUS(TLS), HRAS, GLI1, GLI2, GPC3, HER2(ERBB2, TKR1, NEU), HGF (SF), IRF4 (LSIRF, MUM1), JUNB, KIT (SCFR), KRAS2 (RASK2), LCK, LCO, MAP3K8 (TPL2, COT, EST), MCF2 (DBL), MDM2, MET (HGFR, RCCP2), MLH type gene, MMD, MOS (MSV), MRAS (RRAS3), MSH type gene, MYB (AMV), MYC, MYCL1 (LMYC), MYCN, NCOA4 (ELE1, ARA70, PTC3), NF1 type gene, NMYC, NRAS, NTRK1 (TRK, TRKA), NUP214 (CAN,D9S46E), OVC, TP53 (P53), PALB2, PAX3 (HUP2), STAT1, PDGFB (SIS), PIM gene, PML (MYL), PMS (PMSL) gene, PPM1D (WIP1), PTEN (MMAC1), PVT1, RAF1 (CRAF), RB1 (RB), RET, RRAS2 (TC21), ROS1 (ROS, MCF3), SMAD-type genes , SMARCB1(SNF5, INI1), SMURF1, SRC(AVS), STAT1, STAT3, STAT5, TDGF1(CRGF), TGFBR2, THRA(ERBA, EAR 7 etc.), TFG (TRKT3), TIF1 (TRIM24, TIF1A), TNC (TN, HXB), TRK, TUSC3, USP6 (TRE2), WNT1 (INT1), WT1, CCD C26, CDKN2BAS, RTEL1, TERT, ERCC1, ERCC2, ERCC5, BRCA2, IDH1 / 2, NF1, NF2, TSC1, TSC2, PTEN, CASP-9, C AMKK2, P2RX7, MSH6, PDTM25, KDR, VTI1A, ETFA, TMEM127, GSTT1, CHAF1A, RCC1, XRCC1, EME1, ATM, GLTSCR 1, XRCC4, GLM2, PTEN, CDKN2A, CDKN2B, p14 / ARF, XRCC3, MGMT, XRCC4, MMR, IDH1, ERBB2, CDKN2A, CCDC26, SUFU, NPAS2, CCDKN2A, PTCH2, CTNNB1, P21, RIC8A, CASP8, XRCC1, WRN, BRIP1, SMARCE1, MN1, PDGFB, VHL. ,

[0127] In some embodiments, the disease is caused by interaction of the intracellular bacteria NAMACS and / or NAMACS-ANA and / or TEZR with the host cell cytosol.

[0128] In some embodiments, the production is for modulation of microbial interactions in mixed microbial communities, including biofilms, including obtaining a stable mixture of microorganisms, and microbial antagonism.

[0129] In some embodiments, the products are made to alter the properties of cells to prevent complications during aviation / space flight, stays on other planets, therapies and medical interventions in transplantation (engraftment, rejection, transplantation against the host), cancer therapies (chemoradioimmunotherapy, cytokine release syndrome and other side effects of CAR-T therapy).

[0130] In some embodiments, products are made to alter the properties of cell modifications and activity of immune cells and / or cells targeted by components of the immune system are used to modulate the immune response.

[0131] In some embodiments, the product is for altering the properties of cells in fecal microbiome transplants and non-fecal microbiome transplants (comprised of at least one microbial species selected from the group consisting of Actinomycetes, Bifidobacterium, Bacteroidales, Flavobacteriales, Bacillus, Lactobacillales, Firmicutes, Proteobacteria, Spirochaetes, Bacteroidetes, Clostridiales, Erysipelotrichale, Selenomonadales, Fusobacterium, Neisseria, Campylobacteriales, Pasteurella) for the purpose of increasing the efficacy of such microbiome transplants in the treatment of human diseases, non-limiting examples of which include: These include IBD, Crohn's disease, ulcerative colitis, severe, chronic Clostridium difficile infection, colitis, chronic constipation, chronic fatigue syndrome (CFS), collagenous colitis, colonic polyps, constipation predominant FBD, Crohn's disease, functional bowel disorder, irritable bowel syndrome, constipation predominant, alternating diarrhea and constipation in IBS, diarrhea predominant IBS, pain predominant IBS, indeterminate colitis, microscopic colitis, mucous colitis, Non-ulcer dyspepsia, Norwalk viral gastroenteritis, pain-predominant FBD, primary Clostridium difficile infection, primary sclerosing cholangitis, pseudomembranous colitis, small intestinal bacterial overgrowth, NASH, fibrosis, ulcerative colitis, and upper abdominal FBD, autoimmune diseases, neurodegenerative diseases (non-limiting examples: Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, multiple sclerosis, autism), and cancer.

[0132] In some embodiments, the product is used in combination with an antibiotic to modulate spore formation in spore-forming bacteria.

[0133] In some embodiments, the treatment and prevention of human diseases through the use of the products to manage activity within representative microbiota, including skin, gut, brain, lung, vaginal and tumor microbiota.

[0134] In some embodiments, the products are made to modify recipient and / or donor tissues to improve the efficacy of tissue and organ transplants.

[0135] In some embodiments, the products are made to alter the properties of the recipient cells to enhance the effectiveness of CRISPR, TALEN, ZFN, and other gene editing techniques.

[0136] In some embodiments, products are made to prevent NAMACS and / or NAMACS-ANA interactions with proteins.

[0137] In some embodiments, the products are used to produce or modulate ion channels, brain stimulation, cell signaling within the nervous system (e.g., neurons, microglia, etc.), modifying responses to cortical stimulation, cell signaling between neurons and microglia, non-limiting examples of which are synaptic transmission, synaptic connections between neurons, neuronal excitability and synaptic plasticity, brain aging, age-related learning and memory defects, cognitive decline, brain development, neurotoxicity, excitotoxicity, neurodegeneration, neurodevelopment, sleep disorders, epilepsy.

[0138] In some embodiments, increasing or decreasing DNase and / or RNase activity in human tissues, extracellular spaces, and biological fluids (including non-limiting examples of neural tissue, brain, and cerebrospinal fluid) is used to prevent and treat human diseases.

[0139] In some embodiments, the products can be used to modulate the function of Ca, Na, K channels.

[0140] In some embodiments, the products are used to modulate the electrical properties of cell membrane potential, polarization, depolarization and extrapolarization.

[0141] In some embodiments, the reduction of RNase activity in human tissues, extracellular spaces, and biological fluids is used to modulate the electrical properties and depolarization potential of cells (non-limiting examples include neurons), polarization of membrane potential, depolarization, and extrapolarization.

[0142] In some embodiments, the products are used to manage activity within axons and / or dendrites and / or synapses.

[0143] In some embodiments, the products are made to direct viral and / or capsid surface processes of various delivery vehicles, including but not limited to viral vectors (e.g., adeno-associated viral vectors, adenoviral vectors, retroviral vectors [e.g., lentiviral vectors]), to enhance the specificity of gene therapy.

[0144] In some embodiments, the products are used for regulation of miRNA, protein expression.

[0145] In some embodiments, the products are subjected to eukaryotic and prokaryotic cells to alter the evolutionary process.

[0146] In some embodiments, the products are directed to controlling activity in eukaryotic and prokaryotic cells to regulate increased intestinal permeability.

[0147] In some embodiments, the product is administered in conjunction with normal lysosomal function, management of autophagy, control of protein export from neurons, anti-amyloid therapies (including active immunotherapy), drugs targeting protein aggregation, other methods aimed at preventing the accumulation of misfolded proteins, or drugs that have a synergistic effect on these processes.

[0148] In some embodiments, the products are produced within eukaryotic or prokaryotic cells to repair neuronal damage and regenerate neurons and neurological injuries.

[0149] In some embodiments, alterations of NAMACS and / or NAMACS-ANA and / or TEZR are made, including the use of artificial constructs, to form systems for signal transduction and cellular cooperation, and as analogues of the nervous system that provide signals between cells, cell groups, tissues, organs, and their qualitative or quantitative alterations can be used to modify such signal transduction.

[0150] In some embodiments, NAMACS and / or NNAMACS-ANA and / or TEZR analysis are used to evaluate the efficacy of drugs in clinical trials.

[0151] In some embodiments, the products are directed to direct differentiation of stem cells.

[0152] In some embodiments, the products are directed to managing embryonic cells to affect embryogenesis.

[0153] In some embodiments, the products may be used to modulate transmitter formation, release and effectiveness, such as the effects of glutamate, aspartate, D-serine, gamma-aminobutyric acid (GABA), glycine, nitric oxide, carbon monoxide, hydrogen sulfide, dopamine, norepinephrine (noradrenaline), epinephrine (adrenaline), histamine, serotonin, phenethylamine, N-methylphenethylamine, tyramine, 3-iodothyronamine, octopamine, tryptamine, oxytocin, somatostatin, substance P, cocaine and amphetamine regulated transcripts, opioid peptides, adenosine triphosphate (ATP), adenosine, dopamine, acetylcholine, anandamide, and the like.

[0154] In some embodiments the products may be used to modulate the activity of associated nociceptors and / or opioid receptors and / or mechanoreceptors and / or magnetoreceptors and / or chemoreceptors.

[0155] In some embodiments, the product manages the release or effect of neutrophil extracellular traps.

[0156] In some embodiments, the products may be used in vivo or ex vivo to manage surgical outcomes and / or for organ reconditioning prior to transplantation.

[0157] In some embodiments, the products are used to treat drug overdose such as opioids, drug abuse, prevention and treatment of morphine and other drug overdoses, respiratory depression, neuropathic pain, gastrointestinal dysfunction, addiction and substance use disorders.

[0158] In some embodiments, the products are used to modulate interferon-dependent cytoprotection.

[0159] In some embodiments, the products are used to regulate hormone levels, cellular sensitivity to hormones, a non-limiting example being insulin.

[0160] In some embodiments, the product is made to increase and / or decrease and / or alter cellular activity by use of a skin product (cream, tonic, etc.).

[0161] In some embodiments, the products are administered to mammalian cells to affect longevity mechanisms, resulting in a delay in aging due to DNA damage.

[0162] In some embodiments, products that affect lifespan by altering mechanisms that result in the slowing of DNA damage-induced aging activity are used for DNA repair, regulation of DNA recombination, regulation of intragenomic rearrangements, the behavior of prophages, plasmids, transposons and other mobile genetic elements, and regulation of protein synthesis in cells.

[0163] In some embodiments, use of the product may cause receptor dysfunction; non-limiting examples include tyrosine kinase-based receptors such as EGFR, tumor necrosis factor-related apoptosis-inducing ligand, TLRs, serotonin receptors, CTLA-4, PD-1, and PD-L1, PD-L2, B7 family, VISTA, Tim-3 and LAG-3, TCR, MHC, Gal-9, MHCII, HHLA2, LSECtin, CD80 / 86, CD4, CD3, CD28, TIL, estrogen receptor, progesterone receptor, human epidermal growth factor receptor, VEGF, VEGFR, RYK, GDNF, RET, ERBB, INSR, IGF-1R, IRR, PDGFR, CSF-1R, KIT / SCFR, FLK2 / FLT3, FGFR, CCK4, TRKS, TRKB, TRKC, MEN, RON, EPHA, AXL, MER, TYRO, TIE, TEK, DDR, ROS, LTK, ALK, ROR, MUSK, AATYK, RTK, FLT3, JAK3, FAK, BCR, TCR, INSR group, FGFR group, EGFR group, EPH group, ROR group; those that affect signaling pathways, non-limiting examples include those related to WNT, SRC, PI3K, PTEN, AKT, mTOR, PARP, CHK1 / 2, WEE, insulin, opioids, and can be used alone or in combination with other drugs that target receptors (non-limiting examples include monoclonal antibodies (mAbs) targeting the extracellular domain and / or receptor catalytic domain) and / or to overcome drug resistance mutations in such receptors, non-limiting examples include affecting aberrant protein phosphorylation.

[0164] In some embodiments, the alteration of cellular memory by the product is passed on to subsequent generations of cells. In some embodiments,

[0165] In one embodiment, the addition of "cut", "zero" and "Y" state cells to an organism can cause cascade changes in other cells resulting in beneficial effects on health, including rejuvenation, within 24 hours, up to 1 day to 1 week, within 1 month, within 6 months, within 1 year, up to 5 years, up to 10 years, up to 20 years, up to 50 years, up to 80 years, up to 120 years after administration.

[0166] In one embodiment, NAMACS and / or NAMACS-ANA and / or TezR of one cell and / or tissue and / or organism interacts with TezR of another cell and / or tissue and / or organism.

[0167] In one embodiment, NAMACS and / or NAMACS-ANA and / or TezR modulate electrostatic, hydrophobic interactions of cellular components.

[0168] In one embodiment, NAMACS and / or NAMACS-ANA and / or TEZR are used to regulate biological rhythms, such as circadian rhythms.

[0169] In some embodiments, AMACS and / or NAMACS-ANA and / or TEZR can immortalize cells or increase the maximum number of cell divisions.

[0170] In some embodiments, the products are used to create a naive state of cells that is more sensitive or resistant to physical, chemical, mechanical, or biological agents.

[0171] In some embodiments, the products can be used to increase production of cells and / or their metabolic products for use in biotechnology applications.

[0172] In some embodiments, this includes regulating DNA and / or RNA and / or protein synthesis and / or secretion.

[0173] In some embodiments, NAMACS and / or NAMACS-ANA and / or TEZR are used to modulate the function of cellular receptors, including their interaction with ligands.

[0174] In some embodiments, the products are used to increase energy production by cells.

[0175] In some embodiments, the product is used to control regeneration.

[0176] In some embodiments, the products are used to control cell differentiation for the prevention and treatment of disease, and for the creation of organisms with new characteristics.

[0177] In some embodiments the products are used to obtain altered immune system cells and / or stem cells and / or mammalian cells and / or plant cells suitable for embryogenesis and to prevent the occurrence of birth defects and may be used in artificial insemination.

[0178] In some embodiments, treatment of seeds with products of the plant is used in plant breeding and / or selection processes, and / or to modulate plant productivity.

[0179] In some embodiments, eukaryotic and prokaryotic organisms are processed with products to regulate and control the fermentation of foods and beverages.

[0180] In some embodiments, the products are used to increase the productivity of master cell lines containing genes that make desired proteins in eukaryotic and prokaryotic cells and biotechnology (e.g., recombinant DNA and RNA related; amino acids; biopharmaceuticals; cytokines; fusion proteins; growth factors; clotting and coagulation factors; TNF inhibitors; interferons, antibodies; recombinant antibodies; recombinant proteins; AAV, viruses, antibodies; vaccines, vectors, receptors, hormones). In some embodiments,

[0181] In some embodiments, the products are used to alter the activity of plants and / or plant seeds before and / or after planting of agricultural plants.

[0182] In some embodiments, the products can be used for the production of bioenergy.

[0183] In some embodiments, the products are used to manage the energetic, glycemic, or oxidative status of cells, tissues, or organs.

[0184] In some embodiments, the products may be used to increase the transport of external molecules into cells, or the secretion and excretion from cells.

[0185] In some embodiments, the products may be used to modulate the metabolism of bacteria, fungi, mammals, or plants.

[0186] In some embodiments, the products may be used to regulate the energy status of a cell (eg, intracellular ATP content) or to prevent the formation of recurrent, intracellular ATP content.

[0187] In some embodiments, the product can modulate anaerobic survival metabolism in aerobic organisms (both prokaryotes and eukaryotes), non-limiting examples being modulation of microbial colonization of the intestine, anaerobic infection sites, external spaces, and areas with poor vascularization.

[0188] In some embodiments, the product can modulate anaerobic cellular respiration and / or fermentation-generated ATP and / or affect NADH and FADH2 metabolism and / or ion channels and ion trafficking in aerobic and anaerobic environments.

[0189] In some embodiments, the products can be used to correct somatic mosaicism.

[0190] In some embodiments, the products are used for the development of artificial organs and organisms.

[0191] In some embodiments, the products are used for the treatment of human disorders such as migraines, weather-related headaches, and the like.

[0192] In some embodiments, the product is used for the treatment of human diseases such as migraines, weather-related headaches, etc., and is replaced by other microorganisms that do not contain TEZR.

[0193] In some embodiments, products that can be used to target pathways include KRAS / ERK / MEK, PI3K / AKT / mTOR, JAK-STAT, and FAK / SRC, WNT signaling, heat shock regulation, glycogen synthase kinase 3 (GSK-3), and transforming growth factor beta (TGFβ). [Brief description of the drawings]

[0194] [Figure 1] The effects of the tested compounds on swarm motility, biofilm formation and biofilm size are shown. [Diagram 2] (A) Shows the effect of the products on swimming motility, chemotaxis and control of bacterial growth; (B) Shows the use of 2,8-dichloro-5-(4-nitrophenyl)-5,9-dihydro-4H-pyrimido[5',4':5,6]pyrano[2,3-d]pyrimidine-4,6(1H)-dione (compound VTL) to mediate cell migration; (C) Shows the use of Raltegravir added to the medium together with RNase A to mediate cell growth. [Diagram 3] 1 shows the absence of RNase A internalization in B. pumilus. [Figure 4] 4 shows the regulation of cell size by the compounds tested. [Diagram 5] The effect of the products on the growth of microorganisms (A) Gram-positive bacteria and (B) Gram-negative bacteria is shown. [Figure 6]The effect of the products used on enhancing bacterial growth is shown: (A) control Bacillus VT1200, grown for 24 hours at 37°C; (B) Bacillus grown in medium supplemented with DNase I, grown for 24 hours at 37°C. [Figure 7] The effect of the products used on enhancing the virulence of the bacteria is shown. [Figure 8] The effect of the products used on the interaction of bacteria with phages is shown. [Figure 9] Values ​​represent the mean of three independent experiments. (A) Heatmap summarizing the effect of nucleases on survival after heating S. aureus cultures for 10 min at various temperatures. Color intensity represents the mean log10 CFU / mL, ranging from white (minimum) to blue (maximum). Values ​​represent the mean of three independent experiments. [Figure 10] The effect of the tested compounds on sporulation is shown. [Figure 11] The role of TezR in magnetic reception is shown. [Figure 12] 1 shows the effect of various compounds on cellular adaptation to gas composition. [Figure 13] The effects of the tested compounds on bacterial chemotaxis and substrate recognition are shown. [Figure 14] The effects of the tested compounds on cellular memory and forgetting are shown. [Figure 15] The effect of the tested compounds (DNase and RNase) on the generation of cells with novel biochemical characteristics is shown. The biochemical characteristics of (A) B. pulilus and (b) C. albicans after the use of the test products were tested using the Vitek-2 system. The test reaction data are displayed as "positive", marked in blue, or "negative", marked in white. Data are representative of three independent experiments. [Figure 16]The effect of treatment with reverse transcriptase inhibitors is shown. Heat map representation of growth by control S. aureus or S. aureus after treatment with nuclease and with reverse transcriptase inhibitors (RTI). OD600 is labeled with a color scale from white (minimum) to red (maximum). Values ​​show representative results of three independent experiments. [Figure 17] Figure 1 shows the effect of the tested compounds on signal transport. Figure 2 shows a heat map showing the effect of recombinase on signal transduction in relation to temperature tolerance. CFUs are labeled with a color scale from white (minimum) to blue (maximum). Values ​​show representative results of three independent experiments. [Figure 18] Figure 2 shows transcriptome analysis of Staphylococcus aureus after treatment with the products tested. [Figure 19] The morphology of cells after the use of DNase and RNase compounds is shown (microscope at 40x magnification). [Figure 20] 1 shows the role of surface-associated nucleic acids in tumor cell survival. [Figure 21] The effect of the products on non-tumor cell survival is shown. [Figure 22] The effect of the tested products on the cell cycle is shown. [Figure 23] Quantitative analysis of the distribution or percentage of cells in each stage is shown. [Figure 24] The effect of the tested products on plant growth is shown. [Diagram 25] Figure 1 shows the role of the tested products on plant growth. Probes: 1–3 control, 4–6 raltegravir; 7–9 DNase; 10–12 raltegravir and DNase. [Figure 26] 1 shows the role of RNases in regulating plant and seed development. [Figure 27] The effects of the "cut", "zero" and "Y" conditions on germination are shown. [Figure 28] The effects of the “cut” condition, the “zero” condition, and the “Y” condition on plant traits are shown. [Figure 29]The role of the tested products in modulating various stages of virus-host interaction is shown. Morphological changes indicated a reduction in cytopathological effect (CPE) in Vero cells after the use of the tested products captured at 48 h.pi (magnification, 10x). The scale bar represents 100 μm. [Diagram 30] It shows that the tested products can improve viral infection. Viruses in the supernatant were harvested at 48 hpi and subjected to titration. Data are expressed as mean ± SD (n=3). *p<0.05 compared to control Vero cells infected with HSV-1. [Diagram 31] A heat map of amyloid production is shown, using a color gradient to mark high amyloid production in dark blue and absence of amyloid production in white. [Diagram 32] Modulation of remote signal distribution by the compounds tested is shown. [Diagram 33] Demonstrates bacterial motility. [Diagram 34] Illustrates signal generation and spreading, as well as regulation of intergenerational memory formation. [Diagram 35] The use of the tested products to mediate directional cell migration is shown (sector 4 is supplemented with RNaseA 100 μg / mL). [Diagram 36] Figure 2 shows the effect of the products on the protein-based insulin receptor. [Figure 37] FIG. 2 shows the effect of the tested products on the protein-based insulin receptor. [Figure 38] The effect of the tested products on neuronal excitability is shown. [Figure 39] The effect of various products on the cellular response to light is shown. [Diagram 40] 4 shows the effect of various products on the cellular response to blue light. [Diagram 41] 1 shows the effect of various products on managing the cellular response to visible light in mammalian cells. [Diagram 42] The use of the tested products on cellular response to electrical stimulation is shown. [Diagram 43] 1 shows the effect of TezR on regulating microbial growth under various geomagnetic conditions. [Diagram 44] 1 illustrates the use of the μ-metal test system to modulate cellular activity. [Diagram 45] 1 shows the microbial response of healthy and weather-dependent individuals. [Figure 46] 1 shows an increase in RNase activity by isolated bacteria in response to geomagnetic conditions. [Figure 47] Y190 (n is 1-3; m is 4-14; z is 1-6; X is an acid). [Figure 48] FIG. 1 shows the effect of tested compound-induced cellular memory loss on the modulation of proinflammatory cytokine production by immune cells. [Figure 49] The effect of the products on the cellular response to their stimulation by proinflammatory factors is shown. [Figure 50] The effect of the tested products on telomere shortening is shown. [Figure 51] The effect of the products on the cellular response is shown. [Figure 52] 1 shows the effect of surface disruption on E-cadherin mRNA in various cell types. [Diagram 53] Protection of cell surface nucleic acids from nucleases. [Figure 54] 1 shows changes in immune memory in cells. [Figure 55] 1 shows the role of TezR inactivation in a wound healing cell model. [Figure 56] The effect of the tested products on intracellular tramadol sensitivity is shown. [Figure 57] The effect of the tested products on the opioid receptors is shown. [Figure 58] 4 shows the effect of the use of the tested products on cell resistance to UV exposure. [Figure 59] The specificity of the antibody for NAMACS and / or NAMACS-ANA and / or TEZR is shown. [Figure 60] 4 shows the change in sex of fish due to the product. [Figure 61] The effect of the products on the blood is shown.

[0195] Working Example The present invention is also described and demonstrated by the following examples. However, the use of these and other examples anywhere in this specification is merely illustrative and in no way limits the scope and meaning of the invention or any of the exemplified terms. Similarly, the present invention is not limited to any of the specific preferred embodiments described herein. Indeed, many modifications and variations of the present invention may be apparent to those skilled in the art upon reading this specification, and such variations can be made without departing from the spirit or scope of the present invention. Therefore, the present invention is to be limited only by the terms of the appended claims and the full scope of equivalents to which those claims are entitled.

[0196] Example 1: Methods for the synthesis of products and compounds for directing cell and organism behavior In order to reduce the penetration of low molecular weight compounds (proteins) into cells, the following modification method was used. 1. Quaternized (quaternary) aminoalkyl derivatives. Modifications were performed by introducing highly basic ionogenic groups, such as quaternary amino groups or guanidine groups, into the molecule. Aminoalkyl groups were introduced using aminomethylation reactions at aromatic nuclei of the substrate (1), or aminoalkylation at oxygen, nitrogen atoms, or other nucleophilic centers (2), and reductive amination of carbonyl groups (3). (1)ArH→Ar-CH2NMe2→Ar-CH2N+Me3 (2)X-OH→X-OCH2CH2NMe2→X-OCH2CH2N+Me3 (3)XC=O→X-CH-NHR→X-CH-N+Me2R

[0197] 2. Guanidino derivatives. To obtain these, a nitrile group is introduced into the substrate followed by amination (4) or an aminoalkyl group is introduced followed by replacement of the amino group by a guanidine group (5). (4)X-OHorX-Hal→X-CN→X-CH2NH2→X-CH2-N=C(NH2)2 (5)ArH→Ar-CH2NH2→Ar-CH2-N=C(NH2)2

[0198] 2. In order to reduce the permeation of macromolecular compounds (proteins) into cells, the following modification method was used.

[0199] Modification of proteins with hydrophobic residues at the sulfur atom of cysteine ​​fragments. The modification is carried out by alkylation to introduce residues such as alkyl groups with six or more carbon atoms (6), aryl ketone groups (7), and perfluoroalkyl groups (8). (6)A-SH+CH3(CH2)10Hal→AS-(CH2)10CH3 (7)A-SH+PhCOCH2-Hal→AS-CH2COPh (8)A-SH+C6F5CH2Cl→AS-CH2C6F5

[0200] 2. Modification of terminal amino or OH groups by: - combination of proteins with aldehydes and subsequent reduction of the alkylimines to alkylamines (9), -Acylation of proteins with acid anhydrides (10), -Thiocarbamoylation of Proteins with Alkyl Isothiocyanates (11) (9)A-NH2+C6H13CH=O→A-NH-CH2C6H13 (10)A-NH2+(C6H13CO)2O→A-NH-COC6H13 (11)A-NH2+C6H5N=C=S→A-NH-CSNH2

[0201] 2. Modification of terminal amino or OH groups by: To prevent the organic compound from penetrating into cells, it is desirable to associate the organic compound with an amino acid, preferably asparagine, glutamine, or lysine. Furthermore, carbohydrate fragments or structural analogs thereof can be introduced into the substance molecule.

[0202] Example 2: Development of vaccines and antibodies to control cellular activity Prepared NAMACS and NAMACS-ANA were isolated from bacteria or eukaryotic cells by QIAamp DNA Mini Kit according to the manufacturer's instructions. In some vaccines, mouse DNaseI or RNase was used together with methylated bovine serum albumin (Sigma). A mixture consisting of 0.5 volumes of complete Freund's adjuvant and 0.5 volumes of antigen solution was used. To obtain antibodies, animals (white rabbits, 4 months old) were immunized intravenously with a mixture consisting of 0.5 volumes of complete Freund's adjuvant and 0.5 volumes of antigen solution. Two reimmunizations were performed 21 and 28 days later with a mixture of Freund's incomplete adjuvant. The obtained antibodies interacted with the DNA used for immunization. In follow-up experiments, each vaccination included 1 to 3 doses of 1.0 μg / dose to 1.0 g / dose of nucleic acid or protein and adjuvant (e.g., Freund's adjuvant) administered by enteral, topical, intramuscular or intravenous or subcutaneous injection.

[0203] Example 3: Products and methods for managing microbial community motility, biofilm formation, and biofilm size To test the effect of compounds on the management of swarming bacterial biofilms, glass Petri dishes containing a mixture of Columbia and Nutrient Agar media supplemented or not with the compound to be tested were prepared.

[0204] Different compounds were used, taken at various concentrations from 0.1 to 1000 μg / mL, some of them used directly (Table 1) and some were modified as described in Example 1 to avoid any penetration inside the cells (Table 2). Then, 25 μL of the suspension containing 5.5 log10 cells was inoculated in the center of the agar and the dishes were incubated at 37°C for various times. Biofilms were photographed with a digital camera (Canon 6; Canon, Tokyo, Japan) and analyzed with Fiji / ImageJ software. The effect of the tested compounds was analyzed by the change in swarming motility, confirmed by the formation of larger colonies on the agar with an irregular swarming pattern. All the tested products have similar effects on the bacteria (Figure 1, Tables 1-2).

[0205] For data in Fig. 1, bacteria were harvested by centrifugation at 4000 rpm for 15 min (Microfuge 20R; Beckman Coulter, La Brea, CA, USA) and pellets were washed twice with phosphate-buffered saline (PBS, pH 7.2) (Sigma-Aldrich) or nutrient medium to an optical density at 600 nm (OD600) of 0.003–0.5. Bacteria were treated with nuclease (DNase I) for 30 min at 37°C, washed three times with PBS or broth unless otherwise stated, centrifuged at 4000xg for 15 min after each wash, and resuspended in PBS or broth.

[0206] [Table 1] TIFF2024515541000003.tif251159TIFF2024515541000004.tif255159TIFF2024515541000005.tif255159TIFF2024515541000006.tif95159

[0207] Compounds were also used that were modified as described above to prevent their penetration into cells.

[0208] [Table 2]

[0209] The results clearly show that the tested compounds can be used to control bacterial growth, biofilm formation and bacterial swarming motility and that this occurs by addition of the tested products to the culture medium.

[0210] Interestingly, a combined single treatment of cells with the test product together with the addition of cells to the medium resulted in a significant difference in swarming motility compared to the large biofilms formed by B. pumilus with only the test product (nuclease) added to the medium. Biofilms of B. pumilus cultured on agar containing DNase I and pretreated with DNase I were characterized by the absence of swarming motility. These data clearly show that treatment of cells with the tested product results in a different biological effect compared to the addition of the nuclease under test to the medium.

[0211] Example 4: Products and methods for managing bacterial spreading and chemotaxis. To test the effect of the tested products / compounds on bacterial dispersion and chemotaxis, assay plates containing Columbia agar (supplemented with test compounds) were prepared by adding 250 μL of fresh human plasma to a sector constituting 1 / 6 of the plate. Different compounds were used at various concentrations from 0.1 to 1000 μg / mL, some of which were used directly (Table 3) and some were modified as described in Example 1 to avoid any penetration inside the cells (Table 4). Plasma was filtered through a 0.22 μm pore size filter (Millipore Corp., Bedford, MA, USA) immediately before use. Written informed consent was obtained from all patients to use their blood samples for research purposes and the study was approved by the Institutional Review Board of the Human Microbiology Institute (#VB-021420).

[0212] An aliquot containing 5.5 log10 of B. pumilus VT1200 in 25 μL was placed in the center of the plate, which was then incubated at 37°C for 24 hours and photographed with a Canon 6 digital camera. Swimming motility and chemotaxis were assessed by measuring the movement of the central colony towards the plate sector containing plasma. Colony dispersion was assessed based on the appearance of small colonies on the agar surface. Data are shown in Figures 2a, b, 3 and Tables 3 and 4.

[0213] The internalization of RNase A in cells was also controlled. To do so, B. pumilus (5.5 log10 cells / ml) in PBS was incubated with fluorescein isothiocyanate (FITC)-labeled RNase A for 15 or 60 min at 37°C. Bacteria were washed three times with PBS to remove any unbound proteins. After washing, bacteria were cultured for 2 h in LB broth, washed to remove residual medium components, and placed on a microscope slide for visualization. Fluorescence was monitored using a fluorescence microscope (Axio Imager Z1, Carl Zeiss, Germany). To visualize the internalization of RNase A, biofilms of B. pumilus incubated with 100 μg / mL of fluorescein-labeled RNase A were obtained as described above. After growth at 37°C for 24 h, bacteria were washed three times with PBS to remove unbound proteins and placed under a microscope to monitor fluorescence using a fluorescence microscope (Axio ImagerZ1, Carl Zeiss, Germany).

[0214] Control B. pumilus grew as rounded biofilms on the agar surface. However, the addition of human plasma as a chemoattractant triggered swimming motility and directional migration towards the plasma. Visual inspection of the biofilms revealed that the use of compounds that inactivate or destroy DNA bound to the cell surface resulted in the loss of their chemotaxis and swimming ability. The use of RNase for a single treatment of the cells, or the addition of RNase to the nutrient medium, triggered swimming motility towards the chemoattractant and dispersal of the biofilm, accompanied by the formation of multiple separate colonies in the agar zone where plasma was added (Figure 2a-b). The combined use of nucleases, used to treat the cells and added to the medium, stimulated active sporulation and negative chemotaxis in the center of the colonies.

[0215] We further tested whether the compound 2,8-dichloro-5-(4-nitrophenyl)-5,9-dihydro-4H-pyrimido[5',4':5,6]pyrano[2,3-d]pyrimidine-4,6(1H)-dione added to the agar could induce cell migration towards the chemoattractant (Figure 2c).Under the conditions used, RNase A was not internalized by B. pumilus (Figure 3).

[0216] [Table 3] TIFF2024515541000009.tif235159

[0217] These results clearly show that the tested compounds regulate swimming motility and chemotaxis. Moreover, various products that inactivate or inhibit RNA molecules in these environments may contribute to the same biological effect.

[0218] [Table 4]

[0219] The results summarized in Tables 3-4 clearly show that the tested modifying compounds control swimming motility and chemotaxis.

[0220] Example 5: Products and methods for controlling cell morphology B. pumilus 1200 were treated with nuclease as previously described or cultured on TGV agar medium supplemented with nuclease and cell size was analyzed after 24 h (Figure 4) (Tetz et al., 2018). Cells treated with DNase and RNase resulted in increased cell size (p<0.001), and the same trend of increased cell size was seen for cells cultured in medium containing DNase, or all treated with DNase+RNase and cultured in medium containing DNase+RNase. On the other hand, culture in medium containing RNase significantly reduced cell size (P<0.05). These findings indicate that cell morphology can also be modified and regulated by the test compounds.

[0221] Example 6: Products and methods for managing cell characteristics The effect of the test products on the growth and / or developmental activity characteristic of various cell lines was investigated. Cells were separated from the extracellular matrix and left untreated or treated with the compounds to be tested. Changes in monolayer formation in wells of 96-well plates containing appropriate nutrient medium and supplementary additives were investigated. Cell monolayers were analyzed at 6, 12, 24 and 48 hours to monitor differences in growth or cell behavior characteristics.

[0222] The following parameters were analyzed: (1) cell size, (2) cell morphology, (3) presence of multinucleated cells, and (4) rate of monolayer formation. Control cultures had one point for each of these parameters and were designated as "++++." Alterations in any of these parameters were marked with the exception of a "+." The data are shown in Table 5. [Table 5]

[0223] These data clearly demonstrate that the tested products can be used to control cell characteristics and growth.

[0224] Example 7: Products and methods for managing proteins associated with the development of neurodegenerative and autoimmune diseases Products were tested to manage proteins involved in the formation of neurodegenerative and autoimmune diseases. It was investigated whether prion misfolding and the formation of aggregated fibrils could be inhibited by the test products used at 10 μg / mL.

[0225] For these tests, full-length tau, β-amyloid, α-synuclein, SOD1, TDP-43, and IAPP (protein associated with Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, and diabetes) were used as examples of prion-like proteins to prepare aggregated tau for seeding experiments. To do so, they were used in monomeric aggregate-free conditions at concentrations between 10 and 100 μM with or without 25 μM heparin in the buffer and incubated at 37 °C for different times. Protein aggregation was followed by the protein misfolding cyclic amplification (PMCA) method by monitoring Thioflavin T (ThT) fluorescence levels over time from samples taken from replicate tubes and subjected to cyclic agitation. At various time points, ThT fluorescence was measured in the plate using a plate spectrofluorometer.

[0226] Leukocytes and E. coli VT27 cells, either untreated or treated with test compounds, were used. Data are shown in Tables 6, 7, and 8. [Table 6] [Table 7] The results of accelerated protein misfolding relative to untreated and positive controls with all cell surface DNA and / or RNA molecules are listed in Table 8. [Table 8]

[0227] It was unexpectedly discovered that the test products significantly inhibited protein misfolding. Disruption of cell surface-bound DNA and RNA resulted in significant inhibition of protein misfolding.

[0228] Example 8: Products and methods for controlling microbial growth. S. aureus and E. coli were treated with different compounds as described above, after which the compounds were washed off and the bacteria were plated in LB broth. Growth curves are shown as OD600 values ​​and bacterial counts as a function of time in Figure 5a, b, respectively. Treatment of cells with the test products resulted in alterations in bacterial growth of both Gram-positive and Gram-negative bacteria. Surprisingly, these data show that the various products can affect both synthetic activity (reduction in OD600) and CFU counts.

[0229] Example 9: Products and methods for managing accelerated microbial growth. Bacillus VT1200 was grown on TGV agar supplemented with DNase I (1 μg / ml), histone 5 (100 μg / ml), or TATA box-binding family (5 μg / ml). Control bacteria were grown on regular agar.

[0230] 100 μL of bronchoalveolar lavage fluid (BAL) from pneumonia patients was dissolved in 200 μL of sterile water and separated into two parts, one of which was treated with up to 100 μg / mL DNaseI (Sigma, 2000 Kunitz units / mL) for 1.0 min up to 120 min, while the other part was supplemented with the same amount of buffer. Bacteria and BAL were then washed from the test products with PBS, followed by centrifugation at 4000xg (Microfuge® 20R, Beckman Coulter) for 5 min and resuspended in PBS (final concentration was 6 log10 cells / mL for bacteria). 20 μL of bacteria or BAL suspension was added to the center of a 24-well plate containing LB agar. Plates were incubated at 37°C and monitored every hour for the presence of bacterial growth. Data are shown in Table 9 and Figure 6. [Table 9]

[0231] It is confirmed that the products used result in a significant acceleration of microbial growth. The various products can be used for diagnosis, antibiotic selection, antimicrobial susceptibility testing, acceleration of microbial growth of importance for biomanufacturing and early detection of bacterial growth.

[0232] Example 10: Products and methods for managing microbial toxicity To obtain oligonucleotides, a mixture of Gram-positive and Gram-negative bacteria was lysed and DNA was isolated according to standard methods or standard eukaryotic DNA was used (salmon sperm DNA, Thermofisher). 5 μl of 1 M CaCl2 and 1 M MgCl2 solution was added to 10 mg of DNA obtained in 10 ml of sterile water. 2.5 mg of DNase was added to the reaction mass and left at room temperature overnight (8-12 h) or at 37 °C for 5 h. To inactivate DNase at the end of the DNA depolymerization reaction, the reaction mass was placed in a boiling water bath for 5-10 min until the liquid in the tube boils. After inactivation of the enzyme, the reaction mass was poured into a Millipore centrifugal concentrator equipped with a 10 kDa membrane and centrifuged at 3000 rpm for the time required to completely separate the low molecular weight fraction and concentrate the high molecular weight fraction. The low molecular weight fraction was collected and its optical density was measured against water at λ = 260 nm.

[0233] The S. aureus SA58-1 groups were as follows; group 1, left untreated (control), treated with DNase1L3 1 pg / mL (group 2) or histone H5 1 μg / mL (group 3), pseudouridine synthase (0.1 μg / mL) (group 4), RNase II (1 pg / mL) (group 5), group 6, treated with DNase1L3 + RNase II, group 7, treated with histone H5 + pseudouridine synthase, group 8, DNase1L3 added to agar, group 9, RNase II added to agar, group 10, DNase1L3 and RNase II added to agar, group 11, treated with DNase1L3 and RNase II and also DNase1L3 and RNase II added to agar, group 12, cells treated with oligonucleotides derived from bacterial DNA, group 13, cells treated with oligonucleotides derived from eukaryotic DNA.

[0234] Hemolysis tests were performed as previously described with minor modifications (Manukumar et al., 2017). Briefly, 15 μl of 5x10e5 bacterial cells were seeded in the center of Columbia agar plates supplemented with 5% sheep red blood cells and incubated at 37°C for 24 h. A greenish zone around the colony signified the activity of α-hemolysin; whereas the activities of β-hemolysin (positive) and γ-hemolysin (negative) were indicated by the presence or absence of a clear area around the colony. The size (mm) of the hemolysis zone was measured (Figure 7).

[0235] Lecithinase activity was measured by plating cells on egg yolk agar and incubating for 48 h at 37° C. The presence of precipitation zones and their diameter were assessed (Bennett et al., 2003).

[0236] S. aureus SA58-1 was obtained as described above and further grown on agar supplemented with reverse transcription inhibitors, acyclovir, ribavirin, potassium orotate, and lithium orotate at concentrations ranging from 0.1 to 1000 μg / mL on Columbia agar supplemented with 5% red blood cells.

[0237] The hemolytic activity of control cells or cells treated with the test products and grown in medium not supplemented with reverse transcription inhibitors, acyclovir, ribavirin, or potassium was used as separate controls and considered as 100% (Table 10). [Table 10]

[0238] This result suggests that the tested products can be used to modulate the virulence of bacteria.

[0239] Example 11. Products and methods for directing cell differentiation The effect of the various products on cell differentiation and persister formation was tested. Stationary phase cultures of E. coli were separated from the extracellular matrix and left untreated (control) or after 15 min pretreatment with the test products. Probes were normalized by CFU, diluted in LB broth supplemented with ampicillin (150 μg / ml) and incubated for 6 h. Samples were taken before the addition of ampicillin and 6 h after ampicillin treatment by plating on LB agar without antibiotics to measure the number of colony forming units. The frequency of persisters was calculated as the ratio of the number of persisters in the sample to the initial total cell number before antibiotic treatment for each probe (Table 11).

[0240] [Table 11]

[0241] As expected, in the control E. coli, 1 / 1304 of the original cells were resistant to ampicillin. However, the number of persisters increased significantly after the use of the tested products. Thus, the tested products can be used to modulate the formation of persisters and can be used in healing, prevention of the spread of infectious diseases, and industry.

[0242] Example 12. Products and methods for directing mutagenesis We next investigated how the different test products could manage the spontaneous mutagenesis rate. In these experiments, we measured the spontaneous mutation frequency in E. coli ATCC25922 to rifampicin by counting viable RifR mutants after incubation on agar plates supplemented with rifampicin (Table 12). Spontaneous mutagenesis was inhibited by products that inactivated surface-bound DNA molecules (DNaseI, Cas9), meaning that they blocked the occurrence of replication errors. Surprisingly, the use of products that affected both surface-bound DNA and RNA molecules (DNaseI+RNase;Cas9+ILF3) caused spontaneous mutagenesis and led to a significant increase in the number of RifR mutants. [Table 12]

[0243] Values ​​represent the mean of at least three independent experiments.

[0244] The data obtained clearly show that the products used allow for controlled mutagenesis.

[0245] Example 13: Products and methods for DNA recombination management To determine the role of the products under test in bacterial recombination, control E. coli LE392 were incubated with λ phage (carrying the Ampr and Kanr genes) for a time sufficient to cause phage adsorption and DNA injection. The cells were then treated with nuclease (10 μg / mL), propidium iodide (1 μg / mL), or a combination of modified short hairpin RNA (250 μg / mL) and modified T6 gene exonuclease (0.1 μg / mL).

[0246] Control E. coli LE392 was incubated with lambda phage but not treated with nuclease. Treatment of the cells with either test compound increased recombination frequency, as indicated by an increase in the rate at which the phage lysogenized sensitive bacteria, resulting in an increase in the number of antibiotic-resistant mutants (Figure 8). The highest increase was observed in bacteria treated with compounds that inactivate both DNA and RNA. Taken together, these findings indicate that the tested compounds can be used to modulate recombination frequency.

[0247] The effects of potassium orotate, ribavirin, acyclovir, azidothymidine, lamividine, tenofovir, nevirapine, etravirine (all added at 50 μg / mL) on 24 hour growth at 37° C. were also examined. The data are shown in Table 13. [Table 13]

[0248] The results indicate the possibility of controlling DNA recombination by the tested compounds.

[0249] Example 14: Products and methods for managing host-virus interactions The products for managing host-virus interactions were tested on overnight cultures of Staphylococcus aureus ATCC29213, Pseudomonas aeruginosa VT-16-20B. Bacteriophages used: Staphylococcus phage VTSA-29213, Pseudomonas aeruginosa VTPA-20B phage. Bacteria were separated from the extracellular matrix and pretreated for 15 min with nuclease (10 μg / mL) as described above, pretreated with histone H2B (1000 μg / mL) and ribosomal protein L22 and cold shock protein A (100 μg / mL), the phages were plated on the medium by the agar layer method and the number of negative colonies was determined after 48 h of incubation at 27° C. The results are shown in Table 14. [Table 14]

[0250] The data obtained indicate that the product acts to control virus-cell interactions, including increasing viral output, and can modulate various steps of pathogen-host interactions, including virus-host integration, blocking viral cell recognition, and viral replication.

[0251] Example 15. Products and methods for managing cellular temperature sensitivity Evaluation of whether the test products can modulate the thermal resistance of bacteria revealed that the control S. aureus VT209 showed maximum resistance up to 50°C, while S. aureus after using the products examined was viable at higher temperatures (Figure 9, Table 15). S. aureus VT209 grown overnight in LB broth was detached from the extracellular matrix by washing with PBS and then diluted with PBS to an OD600 of 0.5. Bacteria were detached from the extracellular matrix and left untreated, treated with nuclease (10 μg / mL) or treated with the proteins listed in Table 14 for 5 min, and 5.5 log10 CFU / mL was placed in a 2 mL microcentrifuge tube (Axygen Scientific Inc., Union City, CA, USA). Each tube was heated for 15 min in a dry bath (LS™ Digital Dry Bath; Corning, Corning, NY, USA) at 37°C, 40°C, 45°C, 50°C, 55°C, 60°C, 65°C, 70°C, or 75°C. After heating, control S. aureus were immediately treated with nuclease to remove primary TezR, washed three times to remove nuclease, serially diluted, plated on LB agar, and the number of CFUs was determined within 24 h. [Table 15]

[0252] The data obtained clearly show that the tested products can be used for the modulation of responses to temperature, thermosensitivity and thermotolerance.

[0253] Example 16: Products and methods for managing sporulation and treating diseases associated with sporulating bacteria. Using B. pumilus VT1200, it was first confirmed whether the test product modulated sporulation. For the analysis of sporulation, B. pumilus was separated from the extracellular matrix and left untreated (control) or incubated with the test product (10 μg / mL) for 60 min. 5.5 log10 and 100 μl of the bacterial culture were plated on Columbia agar medium as a lawn and the number of spores was evaluated within 24 h under a microscope by counting cells and spores in 20 microscopic fields and three replicates. For each image, the number of spores and the number of cells were calculated. The ratio of spores to the total number of cells and spores in each bin was then plotted (Figure 10). The data obtained showed that the tested products could be used for the control of sporulation and for the treatment of diseases associated with sporulating bacteria.

[0254] Example 17: Products and methods for managing cellular sensitivity to environmental factors The products for controlling the sensitivity of cells to pH were tested using a model of E. coli VT-267 cultured at different levels of pH, which were separated from the extracellular matrix, pretreated with the test compounds for 30 min and plated in LB broth (Oxoid) with pH values ​​adjusted from 3 to 9 (Table 16). [Table 16]

[0255] The data obtained clearly show that the test product can be used for the management of the response to environmental conditions.

[0256] Example 18: Products and methods for managing magnetic susceptibility The effect of the test compounds on magnetic susceptibility was performed using a model of the growth of B. pumilus VT1200 when exposed to normal magnetic fields and to a shielded geomagnetic field. B. pumilus treated with the test products were obtained as described above. A final inoculum of 5.5 log10 CFU / mL in 25 μL was dropped into the center of agar-filled Petri dishes. Magnetic exposure conditions were adjusted by placing the Petri dishes in a custom-made box with 2-5 layers of 10 μm thick μ-metal (to shield the geomagnetic field) at 37°C for 24 hours (Table 17). In a second experiment, control B. pumilus were separated from the extracellular matrix, treated with RNase, and exposed to normal magnetic conditions or to a shielded geomagnetic field as described above, and colony morphology was analyzed after 8 and 24 hours. Images of the plates were taken using a Canon 6 digital camera (Figure 12). [Table 17]

[0257] The data obtained clearly show that the test product can be used for the management of magnetic susceptibility.

[0258] Example 19: Products and methods for managing growth in different gas compositions The potential of the test products to modulate the cellular response to changes in gas composition was analyzed. P. putida was isolated from the extracellular matrix, left untreated (control) or treated with the test compounds for 15 min, placed on agar and cultured under anoxic conditions. Control P. putida was unable to grow under anaerobic conditions, whereas treatment with RNase and the other test compounds allowed anaerobic growth of P. putida (Figure 12, Table 18). Taken together, these findings indicate that the tested compounds can be used for adaptation to changes in gas composition. [Table 18]

[0259] These results also show that the products used are capable of controlling the cellular response to gas composition.

[0260] Example 20: Products and methods for chemical sensing and managing nutrient and xenobiotic utilization. To investigate the role of test compounds in xenobiotic utilization, B. pumilus and E. coli were isolated from the extracellular matrix, left untreated (control) or pretreated with test compounds, and inoculated onto M9 minimal medium supplemented with the xenobiotics dexamethasone (100 μg / mL) or lactose (100 μg / mL) as the sole source of carbon and energy. The effect of test compounds on the lag phase, consisting of the time required to sense and begin to utilize these nutrients, was compared.

[0261] The time lags following treatment with the test compounds (FIG. 13a) showed a delay in dexamethasone uptake and consumption by 3 and 2 hours (p<0.05) compared to the time lags for the control bacteria (Table 18).

[0262] We hypothesized that the long time required by bacteria after treatment with the test product to start using dexamethasone was due to a disruption in sensing and altered control nutrient consumption, rather than a change in transcriptional activity. To test this hypothesis, we performed experiments in which E. coli were pretreated with dexamethasone, then treated with the test product, and cultured in M9 supplemented with dexamethasone, resulting in the same time lag as control E. coli in the same M9 medium. In other words, the presence of nucleic acids bound to the cell surface is a prerequisite for cells to sense and utilize nutrients, and once the control cells sense dexamethasone, they will continue to utilize dexamethasone, even if they are subsequently treated with the test product.

[0263] Consistent with this hypothesis, control E. coli exposed to dexamethasone for at least 20 min and then treated with test products and inoculated into M9 supplemented with dexamethasone showed similar growth and time lags as control E. coli (Figure 13B).

[0264] The universal effect of the test products on regulating the interaction of cells with exogenous nutrients was evaluated by culturing lac-positive strain E. coli in M9 medium supplemented with lactose as the sole source of carbon and energy. Treatment of cells with the test compounds increased the time lag by 2 h compared to control E. coli, indicating that these tested products can control lactose utilization (Figure 13C, Table 18). As with dexamethasone, when control E. coli were pre-exposed to lactose for 20 min and then treated with the test products and then cultured in M9 medium supplemented with lactose, their behavior and time lag were similar to that of control E. coli (Figure 13D). This finding further supported the controlled role of the test products that can regulate lactose metabolism more than the lac-operon and the effectiveness of the test compounds to control these processes. [Table 19]

[0265] Example 21: Products and methods for managing cellular memory and forgetting We tested whether the test products could modulate cellular memory formation and examined this possibility using an "adaptive" memory experiment. We found that control B. pumilus "remembered" the first exposure to dexamethasone, as indicated by a shortening of the lag period from 5 hours after the first exposure to 2 hours after the second exposure (Figure 14A).

[0266] Dexamethasone-susceptible B. pumilus maintained a time lag of less than 2 hours after treatment with the product and subsequent recovery (Figure 14B). Several repetitions of treatment and recovery with the test product used at concentrations between 10 μg / mL and 1000 μg / mL resulted in "forgetting" of any previous exposure to dexamethasone, and the behavior of the corresponding B. pumilus became similar to that of the control B. pumilus upon the first exposure to dexamethasone (lag period of 5 hours).

[0267] We found that after one or two treatments with the test products, the cells continued to respond faster to the substrate than they did upon the first contact (Figure 15B). However, after three cycles of inactivation with the test products, B. pumilus required the same contact time as naive cells to sense and trigger substrate utilization. We reasoned that multiple cycles of treatment of cells with the tested products resulted in the disruption of some kind of retained "memory" that allows them to maintain and lose past interaction history (reducing the time required to initiate substrate utilization).

[0268] Example 22: Products and methods for directing the generation of cells with novel properties Next, we studied how the test compounds could be used to develop cells with unique properties. Zero cells were generated using the products as described above (treated for several cycles from 10 pg / mL to 100 μg / mL and the biochemical properties of the resulting zero cells were analyzed). Biochemical tests were performed using the colorimetric reagent cards GN (gram-negative bacteria) and BCL (gram-positive spore-forming bacilli) of a VITEK® 2 Compact30 system (BioMerieux, Marcyl'Etoile, France) according to the manufacturer's instructions. The generated data were analyzed using the VITEK® 2 software version 7.01 according to the manufacturer's instructions. We also used eukaryotic Candida cells to generate cells with unique properties by using the test products once. The results clearly show that cells with unique biochemical properties can be generated by putting the cells in a "zero state" or treating them once with the test products, and that they are able to metabolize and degrade products that cannot be metabolized by the control cells (Figure 15A, B).

[0269] Example 23: Products and methods for managing cell growth characteristics Reverse transcriptase inhibitors (used at concentrations 2-100x lower than the MIC) were used against control S. aureus and S. aureus after treatment with different nucleases (10 μg / mL). Zidovudine (AZT), tenofovir (TNF), nevirapine (NVP), and etravirine (ETR) at 5 μg / mL were added to the broth and OD600 was monitored hourly for 6 hours at 37°C. The data (Figure 16) show the unique signature of the combined use of nuclease and reverse transcriptase inhibitors on cellular characteristics.

[0270] Example 24. Products and methods for directing intracellular signal trafficking. We next found that the initiation of the signaling cascade after interaction between cells and ligands is dependent on recombinase (an HIV integrase inhibitor). Considering that we previously showed that treatment with the test products improved survival at high temperatures, we hypothesized that raltegravir may also block signaling and result in higher thermostability in control bacteria not treated with nucleases. S. aureus treated with raltegravir, dolutegravir, elvitegravir, and bictegravir at non-toxic concentrations of 0.01 to 10 μg / mL or untreated were gradually heated to 65 °C and analyzed for the presence or absence of viable bacteria. S. aureus treated with recombinase was able to survive at temperatures 15 °C higher than cells not treated with recombinase (Figure 17). These results clearly show that recombinase blocks signaling from the ligand to the cells.

[0271] Example 25: Products and methods for managing bacterial susceptibility to antibiotics. Standard NCCLS disk diffusion tests were performed on the isolates using supplemented mixed Columbia and peptided meat agar, using standard ampicillin 10 μg, gentamicin 10 μg, azithromycin 15 μg, clindamycin 10 μg, cotrimoxazole 25 μg test disks (Hardy diagnostics). S. aureus VT213, either isolated or not from the extracellular matrix, were treated (2-180 min) with the test products. After 24 h incubation at 37°C, the diameter of the zone was measured in the usual way; significant ingrowth within the zone to the edge of the disk was considered as constitutive resistance. The data are shown in Table 20. [Table 20]

[0272] The effect of protease or integrase inhibitors (used at concentrations below the MIC) on cells treated with the test products was also examined. The data are shown in Tables 21 and 22. [Table 21]

[0273] The use of the test products alone or in combination with integrase inhibitors or protease inhibitors makes it possible to modulate the susceptibility of the bacteria to antibiotics. [Table 22]

[0274] The use of the test products alone or in combination with integrase inhibitors or protease inhibitors made it possible to modulate the sensitivity of the microorganisms to antibiotics, their effect being more pronounced on cells that do not have an extracellular matrix.

[0275] These data clearly show that potassium orotate products increased the susceptibility of bacteria to antibiotics. The antibacterial effect of potassium orotate was more pronounced in cells following treatment with the products tested.

[0276] Example 26: Products and methods for controlling gene activity and epigenetic processes in prokaryotes. Next, we tested how the test products could be used to modulate gene expression. To isolate RNA, the cell suspension obtained after 2.5 hours of nuclease treatment was washed three times with PBS, pH 7.2 (Sigma), centrifuged each time at 4000xg for 15 minutes (Microfuge20R, BeckmanCoulter), and then resuspended in PBS. RNA was purified using the RNeasy Mini Kit (Qiagen) according to the manufacturer's protocol. RNA quantity and quality were assessed spectrophotometrically by measuring UV absorbance at 230 / 260 / 280 nm with a Nano Drop OneC spectrophotometer (ThermoFisher Scientific). Transcriptome sequencing (RNA-Seq) libraries were prepared using the Illumina TruSeq Stranded Total RNA Library Prep kit. RNA was ribo-depleted using the Epicentre Ribo-Zero magnetic gold kit (catalog no. RZE1224) according to the manufacturer's guidelines. Libraries were pooled in equimolar amounts and sequenced on an Illumina NextSeq 500 (Illumona, San Diego Calif.) platform using paired 150 nucleotide reads (maximum 130 MM reads).

[0277] Cells were separated from the extracellular matrix and treated with the test products, which resulted in significant changes in bacterial gene expression with a large number of differentially expressed proteins (|log2 fold change|>0.5 and p-values ​​<0.05) (Figure 18, Tables 23-25). There were major changes in the regulation of genes responsible for ATP production, secretion systems, virulence factors, efflux pumps, and synthetic activity. [Table 23] TIFF2024515541000030.tif231159TIFF2024515541000031.tif32159 [Table 24] TIFF2024515541000033.tif35159 [Table 25]

[0278] The use of the tested products can regulate gene activity and epigenetic processes in prokaryotes.

[0279] Example 27: Products and methods for managing eukaryotic gene activity and epigenetic processes. We next found that the use of the test products had a global impact on eukaryotic gene expression using Vero cells. RNA extraction and transcriptome sequencing were performed as described above. Treatment of cells after isolation from the extracellular matrix with the products led to significant changes in the expression of several important genes with the expression of many different proteins (|log2 fold change|>0.5 and p-value<0.05) (Tables 26-28). There were major changes in the regulation of genes responsible for ATP production, secretion systems, virulence factors, efflux pumps, and synthetic activity. [Table 26] TIFF2024515541000036.tif121159 [Table 27] [Table 28] TIFF2024515541000039.tif36159

[0280] Treatment with the products can cooperate to regulate various cellular processes and pathways, some of which are shown in Table 29. [Table 29] TIFF2024515541000041.tif243159TIFF2024515541000042.tif249159TIFF2024515541000043.tif25215 9TIFF2024515541000044.tif251159TIFF2024515541000045.tif249159TIFF2024515541000046.tif76159

[0281] These data clearly demonstrate that the products used are able to direct gene activity without interfering with any number of cellular processes, including the KRAS / BRAF / MEK pathway.

[0282] Example 28: Products and methods for managing eukaryotic cell behavior In this test, Ehrlich ascites carcinoma cells were used as tumor cell cultures and mouse fibroblasts as non-tumor. Cells were cultured in RPMI1640 medium containing 10% heat-inactivated fetal bovine serum (FBS) (Sigma), 100 g / mL streptomycin and 100 U / mL penicillin G at 37°C in a humidified atmosphere of 5% CO2 in air (all Sigma). Prior to the use of the compounds to be tested, DMEM was removed from the cell monolayer and cells were treated with the test products at 37°C for 15-60 min in fresh DMEM without FBS. The cell monolayer was then washed three times with PBS to remove any remaining test products. Negative control - H202.

[0283] For flow cytometry, subconfluent cell cultures were harvested, washed twice with DMEM without FBS, and resuspended in DMEM supplemented with FBS. Products were added at final concentrations of 1.0-100 μg / ml for 1.0-120 min as described above. Cells were washed from nucleases and incubated for an additional 2.5 h in fresh DMEM with FBS at 37°C as described above. Cells were suspended in PBS containing 0.2 μM YO-PRO-1 (Invitrogen, Y3603) and 1.5 μM PI (Invitrogen P3566). In total, 10,000 cells were analyzed for each measurement. The percentage of apoptotic cells was determined by flow cytometry using a CytoFLEX flow cytometer (Beckman Coulter, Brea, CA, USA). Apoptotic cells were stained with YO-PRO-1 but impermeable to PI. Dead and late apoptotic cells were permeable to both dyes. Results were expressed as the percentage of permeabilized cells. Experiments were performed in triplicate. Data were analyzed using FlowJo10 software (Treestar Inc., Ashland, US). Data are shown in Table 30 and Figures 19-21. [Table 30]

[0284] The data surprisingly clearly show that, whilst the tested products increase the viability of non-tumour cells, they have the opposite effect on tumour cells, decreasing their viability.

[0285] Example 29: Products and methods for controlling the eukaryotic cell cycle The effect of the test products on cell cycle phases was analyzed using flow cytometry in Vero cells (Figure 22). Quantitative analysis of the distribution or percentage of cells in each phase was performed from at least 10,000 cells per sample. Each bar represents the mean ± SD of data from three independent experiments. ****p<0.0001

[0286] Quantitative data revealed that Vero cells accelerated S-phase progression after separation from the extracellular matrix and treatment with the products (Figure 23). Thus, Vero cells treated with DNase 25 μg / mL alone or in combination with RNase 25 μg / mL had approximately a 2.4-fold reduced distribution in S-phase and a 7.6-fold increased distribution in G2-phase (p<0.0001). Similarly, Vero cells treated with DNase and RNase showed a 2.7-fold lower percentage in S-phase and a 7.2-fold increase in G2-phase (p<0.0001). In both cases, the number of cells in G1-phase remained the same as that seen in the controls. These results indicate that these products control the length of S-phase and regulate DNA replication, the p53-DREAM pathway, and S-phase checkpoint kinases.

[0287] Example 30: Products and methods for managing cancer therapy and enhancing chemotherapy efficacy The product was tested for the treatment of brain tumors. 3.0x10e4 cells / well were seeded in 24-well plates (Corning) and detached from the extracellular matrix. Acute growth inhibition / cytotoxicity assays were observed after exposure of U87-MG human glioblastoma cells treated with the test products (used at concentrations of 0.01-250 μg / mL for 5-60 min treatment in the presence or absence of temozolomide (200 mM) for 72 h). Cells were counted using a Z2 Coulter particle counting and size analyzer (Beckman Coulter).

[0288] U87-MG human glioblastoma cells were maintained in DMEM medium supplemented with 10% FBS (Sigma), L-glutamine and antibiotics (all Sigma).

[0289] Significant differences were observed between tumors treated with temozolomide and tumors after product and temozolomide. The data are shown in Table 31. [Table 31]

[0290] The data presented show that the tested products unexpectedly affected the viability of glioma cells and enhanced the efficacy of chemotherapy.

[0291] Example 31: Products and methods for managing chemotherapy-resistant eukaryotic cells Cells A549 (wild type EGFR / mutant K-Ras) were maintained in RPMI-1640 medium (Sigma, USA) supplemented with 10% heat-inactivated fetal bovine serum (Thermo Fisher), penicillin (100 U / ml), streptomycin (100 μg / ml) and L-glutamine (2 mM) at 37°C under 5% CO2 atmosphere, and harvested with trypsin-EDTA when the cells reached exponential growth. Cells were cultured in 96-well plates, in which the number of A549 was 6,000 cells / well. Before treatment with the test products, some cells were detached from the extracellular matrix. Then, cells were exposed to gemcitabine at various concentrations for 72 h in 96-well plates, and IC50 was measured. The IC50 value of gemcitabine was determined by MTT (MTT solution was added to each well). The optical density (OD) of each well was measured at 490 nm after 4 hours of incubation. The percentage of cell growth inhibition by v was calculated as follows: [(OD490 control cells-OD490 treated cells) / OD490 control cells]x100.

[0292] Table IC50, the concentration that results in 50% inhibition of maximum cell growth based on the type of product used. The data are shown in Table 32. [Table 32]

[0293] The results obtained show that the different products modify the sensitivity of cells to chemotherapeutic agents, an effect that is more pronounced when the extracellular matrix is ​​removed and the nucleic acids bound to the cell surface are affected.

[0294] Example 32: Products and methods for managing neoplastic transformation. The role of the test products in preventing neoplastic transformation was evaluated. To this end, serum-supplemented medium of RWPE-1 cells was removed, and the cell monolayer was washed once with PBS and once with serum-free medium. Cells were then treated with the test compounds and exposed to phorbol 12 myristate (PMA) 50 ng / mL, and MMP9 expression was monitored. Data are shown in Table 33. [Table 33]

[0295] It is clearly confirmed that the test product inhibits cancer transformation and modulates anti-cancer responses.

[0296] Example 33: Products and methods for directing the growth of eukaryotic cells. In this study, Ehrlich ascites carcinoma cells were used as tumor cell cultures and mouse fibroblast cells as non-tumor. Cells were cultured in RPMI1640 medium containing 10% heat-inactivated fetal bovine serum (FBS) (Sigma), 100 μg / mL streptomycin, and 100 U / mL penicillin G (all Sigma) at 37°C in a humidified atmosphere of 5% CO2 in air.

[0297] Cells were treated with ribavirin, abacavir, azidothymidine, tenofovir, etravirine, lamividine, potassium orotate, all at concentrations between 0.1 μg / ml and 100 μg / ml. Optical density OD600 (Microtiter plate reader (Epoch2-BioTek), 37°C, hourly). Data are shown in Table 34. [Table 34]

[0298] The data obtained unexpectedly showed that the use of the tested products altered the characteristics of cancer cells: potassium orotate and tenofovir also altered cell size.

[0299] Example 34. Products and methods for managing eukaryotic cell memory An "adaptation" memory experiment was used to test the effect of the test products on memory formation in eukaryotic cells. Ten different Candida albicans strains of clinical isolates were used. C. albicans were cultured for 24 hours on Sabouraud medium, washed from the extracellular matrix and either left untreated (control) or treated with the test products as described above. The time required for the cells to start utilizing maltose was expressed as the duration of the lag phase during the first and second exposure to this xenobiotic. To adjust for the second maltose exposure, control cells grown for 18 hours in M9 broth supplemented with 50 μg / mL maltose (corresponding to the first exposure) were collected, treated or not with the test products, the OD600 was adjusted and then replated in M9 broth supplemented with maltose for the second maltose exposure. The data are shown in Table 35. [Table 35]

[0300] It was clearly confirmed that the control C. albicans "remembered" the first exposure and the second exposure to maltose shortened the lag phase by 3 hours, which means that the bacteria "remembered" the first exposure and can start to utilize maltose sooner. The inventors found that the tested products can prevent the memory by the cells, and therefore the cells cannot recognize the second exposure to maltose.

[0301] Example 35: Products and methods for management of cellular memory An "adaptation" memory experiment was used to produce C. albicans with a "memory" of maltose as described above. In this study, ten different strains of C. albicans were used, cultivated as described above. "Maltose-sensitive" C. albicans treated with test products at concentrations ranging from 1 μg / ml up to 10 mg / ml were then exposed for 30 seconds to 24 hours. Cells were treated either once with the test products or with multiple rounds of treatment followed by a washing period in nutrient-free minimal medium (i.e., M9 medium without maltose). As shown in Tables 36 and 37, treatment of cells for one cycle and recovery for 24 hours did not affect the memory of maltose-sensitive cells, and the time lag of such cells between the second maltose exposure was shortened compared to the time lag in maltose-naive cells. However, more than two rounds of treatment with nucleases and other test products with the formation of the so-called "zero" state resulted in forgetting of the previous exposure to maltose for some cells, and more than three rounds for all cells. Thus, the behavior of "zero state" C. albicans upon a second contact with maltose was similar to that of control C. albicans upon a first maltose exposure, with a minimum time of contact to result in maltose utilization of 3 h.

[0302] Data was obtained that show that the use of the tested products and putting cells into a "zero state" can be used to modulate cellular memory and forgetting. [Table 36] [Table 37]

[0303] Example 36: Products and methods for managing cellular forgetting Considering the wide range of cellular memories that can be managed and erased by test compounds, we next decided to induce cellular forgetting of its resistance to a specific treatment.To this end, human breast cancer cells MCF-7 resistant to adriamycin (ADR) (MCF-7 / ADR) were cultured in RPMI1640 medium supplemented with 10% FBS, 0.1 mg / mL streptomycin and 100 units / mL penicillin at 37°C and 5% CO2.

[0304] Cells were washed from the extracellular matrix or not separated from the matrix and treated with the test product used at 25 μg / mL for 15 minutes. Some cells were treated with the test product to generate "zero cells" as described above. Then, the test compound was washed off and cells were seeded in 96-well plates (8000 cells / well) and then treated with different concentrations of ADR. The forgetting ability of cells was determined as the cells that were able to tolerate the treatment, which was determined using the MTT assay described above. The data is shown in Table 38. [Table 38]

[0305] The data obtained clearly show that the cells after treatment with the tested compounds were able to forget the pattern of ADR resistance and become sensitive to it.

[0306] Example 37: Products and methods for treating tumors with product-antibody conjugates. The human adenocarcinoma alveolar epithelial cell line A549 cell line was grown in DMEM medium (Sigma) supplemented with 10% fetal bovine serum (Gibco) and 1% streptomycin (Sigma).

[0307] A549 cells were seeded at a density of 5x10e5 cells / well in 6-well plates (Coring) for 24 hours at 37° C. The culture medium was then replaced with fresh medium, washed from the extracellular matrix containing extracellular TezR, and then placed into fresh medium supplemented with the monoclonal antibody cetuximab (IMC-C225), a recombinant chimeric monoclonal antibody that binds to the extracellular domain of the epidermal growth factor receptor, or without cetuximab.

[0308] The product was conjugated with cysteamine hydrochloride (7.0 ng, 60 pmol in 2.2 μL PBS, pH 8.8) for 1 h at room temperature. The reaction solution was transferred to a tube containing p-SCN-Bz-DOTA (35 μg, 49.0 nmol) and reacted at room temperature for 1 h. The reaction mixture was centrifuged at 1000×g for 40 min and the pellet was resuspended in deionized water. C225 (1.0 mg, 6.58 nmol, 2 mg mL-1) was modified with N-succinimidyl S-acetylthioacetate (15.5 μg, 66. nmol) for 1 h at room temperature and applied to a Sephadex G50 ultrafine column. DNA abzymes were obtained from HMI lab (prof. V. Tets know-how).

[0309] SATA-modified C225 (1 mL, 400 μg mL-1) was treated with hydroxylamine (200 μL, 0.5 M) for 2 h at room temperature and applied to a Sephadex G50 ultrafine column. C225-SH (10 μg mL-1) was conjugated with DOTA-DNase, DOTA-RNase, suspension (10x1010 particles mL-1).

[0310] Probes: Probes were incubated for 24 hours, the medium was changed and cells were counted the next day using a cell counter after removing the cells from the plate with a pre-made trypsin-EDTA solution (Sigma).

[0311] Cells grown on cover slips were stained with propidium iodide (Sigma) according to the protocol: 50 μl of 15 μM propidium iodide was added to each well, followed by an additional 15 min incubation on an orbital shaker in the dark, and the fluorescence intensity was measured with the same filter set. The presence of specific receptors was determined by measuring the fluorescence intensity with a microplate reader (Synergy Neo2, BioTek, VT, USA) using a 488 / 20 nm excitation filter and a 645 / 40 nm emission filter. The data are shown in Tables 39 and 40. [Table 39] [Table 40]

[0312] As can be seen, delivery of products that disrupt cell surface-bound nucleic acids resulted in significant anti-tumor effects, either alone or in combination with targeted anti-tumor therapy.

[0313] Example 38: Products and methods for managing various side effects of treatments SCID beige mice were used for 4-6 weeks. Raji tumor cells (ATCC® CCL-86) were injected intraperitoneally and allowed to grow for 21 days. 7.3 log10 of human 1928zCAR T cells were used to target B leukemia cells and induce cytokine release syndrome.

[0314] group: 1. Control - Untreated 2. Antibody against Pseudomonas aeruginosa DNA (1 μg / mL, once every 7 days) 3. Antibody against Pseudomonas aeruginosa DNA 1000 μg / mL twice daily 4. Antibody to Pseudomonas aeruginosa DNA 1 μg / mL once every 3 days + nevirapine 7.5 mg / kg once a day 5. Antibody to Pseudomonas aeruginosa DNA 1000 μg / mL twice daily + nevirapine 7.5 mg / kg once daily 6. Antibody against E. coli RNA 1 μg / mL once every 5 days 7. Antibody against E. coli RNA 1000 μg / mL twice a day 8. Anti-D8 conjugated antibody and DNase I administered twice daily 9. Antibody to Pseudomonas aeruginosa DNA 1 μg / mL twice daily + lamivudine 10 μg / mL 10. Antibody to Pseudomonas aeruginosa DNA 1 μg / mL twice daily + tenofovir 10 μg / mL 11. Antibody to Pseudomonas aeruginosa DNA 1 μg / mL twice daily + etravirine 10 μg / mL 12. Antibody to Pseudomonas aeruginosa DNA 1 μg / mL twice daily + abacavir 10 μg / mL

[0315] Survival data is presented in Table 41 below. [Table 41]

[0316] The data obtained show that the product, alone and in combination with nucleoside inhibitors, resulted in significant improvement of cytokine release syndrome and other side effects of CAR-T therapy.

[0317] Example 39: Products and methods for controlling disease-related receptor activity Panc-1 cancer cells were grown in DMEM medium (Sigma) supplemented with 10% fetal bovine serum (Gibco) and 1% streptomycin (Sigma) at 37° C. in a humidified atmosphere containing 5% CO 2 .

[0318] Migration of Panc-1 cells through BD-Matrigel Invasion Chambers (24 transwell, pore size 8 μm) was analyzed. Cells were treated with the test products in the concentration range of 1-1000 μg / mL as described above, some cells were additionally treated with recombinant human EGF 20 ng / ml (Sigma-Aldrich), washed with PBS, resuspended in DMEM (serum-free) and added to the upper compartment of the invasion chamber (1x10e5 cells / well). The lower compartment of the chamber contained conditioned medium. After 24 h of incubation at 37°C, cells on the upper surface were completely removed by wiping with a cotton swab.

[0319] After incubation, cells remaining on the top surface of the membrane were removed by wiping with a cotton swab. Cells that migrated from the top to the bottom of the filter were fixed with methanol, stained with crystal violet solution, and counted under a light microscope (40 fields per filter) (Table 42). [Table 42]

[0320] These data clearly demonstrate that the use of the tested compounds, including the formation of null cells, can be used to inhibit disease-related receptors, including EGFR phosphorylation and inactivation of the EGFR signaling pathway.

[0321] Example 40: Products and methods for managing fungal susceptibility to antifungal drugs Nystatin-resistant strains of Candida albicans F4 were isolated from the extracellular matrix as described above and treated with the test products. The resulting fungi were plated on Sabouraud dextrose agar supplemented with 5 μg / mL nystatin (Sigma) and incubated at 37° C. for 24 hours, and the number of colony forming units was determined (Table 43). [Table 43]

[0322] The products tested make it possible to increase the sensitivity of fungi to antifungal antibiotics and to overcome antibiotic resistance.

[0323] Example 41: Products and methods for the diagnosis of disease The composition of nucleic acids bound to the cell surface of normal and malignant cells was investigated using needle biopsies of colon cancer (stage III) established from histologically confirmed adenocarcinoma or biopsies of normal tumor tissue, PDX cells from BXPc3 (pancreatic cancer), BL0293 (bladder cancer), LG1049F (lung cancer), MC38 (colon cancer), and BR1126F (breast cancer), and PDX from patients without malignant tumors.

[0324] Needle biopsies of primary tumors / controls were collected under sterile conditions in specimen bottles containing RPMI1640 medium supplemented with 5% penicillin-streptomycin-neomycin mixture (GIBCO). 20 mg specimens were placed in each well of a 12-well plate on a shaker in a refrigerator at 4°C for 16-20 hours, supplemented with trypsin, and then carefully transferred to fresh RPMI1640 supplemented with 10% horse serum and penicillin-streptomycin to 1% of the total solution. The plate was then placed in a 37°C warm water bath for 20 minutes, then the tissue was transferred to a 20 ml vial containing Hank's balanced salt solution, gently shaken, and then 0.1% collagenase solution was added for 45 minutes at 37°C. After dissociation of the tissue, the probes were centrifuged at 100xg for 10 minutes at room temperature. The supernatant was removed and the cell homogenate was resuspended in 2.5 ml of RPMI1640 medium.

[0325] Nucleic acids bound to the cell surface were visualized with DAPI, SYTOX green (excitation: 504, emission 523), and propidium iodine (excitation: 493, emission 636) using an ECHO (ECHO San Diego CA) Revolve microscope and a Synergy Neo2 multimode microplate reader (Biotek).

[0326] To isolate DNA and / or RNA bound to the cell surface, tumor and control cells were washed from the nutrient medium matrix in PBS and then centrifuged at 3000gx10 min. The cells were then placed in 0.9% NaCl supplemented with EchoR1 and HindIII nuclease plus Mg buffer at 37°C for 1 h. The cells were separated by centrifugation at 3000gx10 min and the supernatant was filtered through a 0.22μM filter (Millipore). DNA was isolated from the supernatant using the QIAamp DNA MiniKit (Qiagen). RNA was isolated using the Quick-RNA kit (Zymo Research).

[0327] Immune cells were obtained by Ficoll centrifugation as described below.

[0328] Whole genome sequences were obtained using the Illumina HiSeq 2500 sequencing platform (Illumina GAIIx, Illumina, San Diego, CA, USA). Library preparation, sequencing reactions, and runs were performed according to the manufacturer's instructions. *The amount of nucleic acid bound to the cell surface of each type of untreated control cells was indicated as "normal."

[0329] Some cells were also stained with Sytox as described above and the corresponding changes in surface green fluorescence were analyzed as an indication of changes in cell surface bound nucleic acid. The data are shown in Tables 44 and 45. [Table 44] [Table 45]

[0330] Thus, qualitative and quantitative changes in cell surface bound nucleic acid occur in mammalian diseases, which can be used for diagnostic purposes of mammalian diseases.

[0331] Example 42: Products and methods for the treatment of psychiatric disorders. The study included 25 volunteers suffering from schizophrenia with severe agitation. To reduce exacerbations, the volunteers received the drugs administered in conjunction with basic therapy. Efficacy was analyzed based on the change in the Total Positive and Negative Syndrome Scale (PANSS) score within a 2-week time frame. Potassium orotate, etinavir, ribavirin, abacavir, tobramycin were administered at regular doses; DNase, RNase were administered orally at 50 mg x BID. The data are shown in Table 46. [Table 46]

[0332] The obtained data shows that the use of the tested products can be beneficial for psychiatric and neurological disorders. The products can cause autoreprogramming and restoration of proper function. We also found that the combined use of products that inhibit the formation of nucleic acids bound to cell surface and reverse inhibitors that destroy them is highly effective in the treatment of psychiatric and psychiatric disorders.

[0333] Example 43: Products and methods for managing plant growth Plant germination and product yield were measured for a variety of plants, including Arabidopsis. Dried vernalized seeds were sterilized by 70% (v / v) ethanol washes in microcentrifuge tubes and then treated with a solution of 50% (v / v) bleach and approximately 0.5% (v / v) Tween 20 for 10 min. The bleach solution was removed using a sterile transfer pipette in a laminar flow hood, and the seeds were then rinsed 8–10 times with sterile water. Seeds were incubated in aqueous solutions containing different compounds previously shown to bind to or inactivate cell surface-bound nucleic acids, used at concentrations ranging from 0.01 μg / ml to 1000 μg / ml.

[0334] Control seeds were soaked in water without adding test compounds. Seeds were then sown 5 cm deep in clay loam soil that had been plowed and thinned with a disk plow. The soil for some probes was supplemented with fertilizer according to the manufacture's instructions. Emergence, shoot length, root length, and chlorophyll were measured on the fifth or seventh day. Leaf chlorophyll content was measured 7 days after planting the seeds. Fresh leaf material (50 mg) was homogenized in 10 ml of 95% ethanol. The homogenate was centrifuged at 1500xg for 20 min, and the supernatant was collected and measured using a NanoDrop OneC spectrophotometer (ThermoFisher Scientific, Waltham, MA, USA) at 649 and 665 nm. Concentrations of chlorophyll-α, chlorophyll-β, and total chlorophyll (α+β) were calculated using the formula: Total chlorophyll content was determined using the formula:

[0335] Chlorophyll α = 13.95xA665-6.68xA649(1)

[0336] Chlorophyll β = 24.96xA649-7.32xA649(2)

[0337] Total chlorophyll = (Chlorophyll-α + Chlorophyll-β) x final volume of sample (ml) x dilution factor / fresh weight of sample taken (3)

[0338] Concentrations were expressed as mg chlorophyll g-1 fresh weight by using the following formula:

[0339] Total chlorophyll (mg g-1FW) = [20.2(D645) + 8.02(D663)] x [V / (1000xW)],

[0340] where V = volume of 80% aqueous acetone solution (ml), W = weight of fresh leaves (g), D645 = absorbance at 645 nm wavelength, and D663 = absorbance at 663 nm wavelength.

[0341] The products tested had a significant effect on seedling emergence and plant germination rate.

[0342] As can be seen, the use of the tested products affected various plant characteristics and significantly increased plant growth.

[0343] The effect of the test products on regulating plant and seed growth under optimal and stressful conditions was also examined (Tables 47, 48, 49, Figures 24, 25, 26). [Table 47]

[0344] As can be seen, tomatoes grown after treatment with RNase exhibited very robust growth. [Table 48] [Table 49] TIFF2024515541000067.tif147159

[0345] The effect of the tested products on the plant characteristics was also evaluated in terms of chlorophyll content (Tables 50, 51). [Table 50]

[0346] It is clearly confirmed that the tested products modulate the chlorophyll content. [Table 51]

[0347] The products tested have a significant impact on the plants and on the yield of the products. Moreover, the use of these products makes it possible to overcome stressful conditions for the plants.

[0348] Example 44: Products and methods for managing plant growth To test the effect of nuclease use on plants, tomato seeds were pretreated with DNaseI or RNaseA at concentrations between 10 and 10,000 μg / mL for 60 minutes, washed from nuclease, sown in plastic trays and transplanted with one seedling into a 3 liter plastic pot filled with compost. The experiment was carried out in a greenhouse at a moderate temperature of 22°C and humidity of 34. The data are shown in Table 52. [Table 52]

[0349] These data clearly show that the tested compounds significantly improved the plant characteristics.

[0350] Example 45: Products and methods for controlling plant traits The effects of different products on different plant characteristics were measured using non-limiting examples of plants such as spring wheat, soybean, tomato, rice, potato, barley, corn, oat, corn, cotton, and cassava seeds. Dry vernalized seeds were treated as above and pretreated with test compounds. Data are shown in Table 53. [Table 53]

[0351] It can be clearly seen that the seeds treated with the tested products have specific growth characteristics.

[0352] Example 46: Products and methods for multi-generational seed treatment and storage management. Seeds of Dianthus amurensis were obtained after one treatment with the test products (DNaseI or / and RNaseA) as described above. Seeds of the second generation were obtained from plants grown after treatment with the test products (without any additional nuclease treatment). Flowers were cultivated according to the recommendations at https: / / plantcaretoday.com / dianthus-care.html.

[0353] Seeds were transplanted into plastic seedling pots (LxWxD 3.25''x2,75''x2,75'') containing a mixture of soil and sphagnum moss (3:1, v / v) with organic fertilizer. The greenhouse temperature was maintained at 25±2°C and 10±2°C during day and night, respectively. Each treatment consisted of three replicates, with 1 / 100 plants planted per plastic pot. At harvest and after treatment, plant growth parameters were measured, including plant height, leaf area, flower weight, dry weight of leaves, stems, and roots (Tables 54, 55). Plant height was determined by measuring the height from the base of the stem to the first leaf. Leaf length was measured using a ruler. After measuring the fresh weight, the plant material was dried at 70°C for 2 days to measure the corresponding dry weight (Kwon et al., 2019). The effect of treatments on chlorophyll stability was estimated by measuring the chlorophyll content after treatment. Chlorophyll was extracted from fresh leaf samples of both treated and untreated plants as described above. Values ​​presented are shown as the mean ± SE of a minimum of three independent replicates (n = 3). Results were considered statistically significant at p < 0.05. [Table 54] [Table 55]

[0354] The nuclease treated seeds showed a significant advantage over the control plants, especially in growth rate: the seeds harvested from the first generation plants preserved the growth characteristics and therefore the second generation plants grown from these seeds retained all the vegetative characteristics of the first generation plants.

[0355] Example 47: Products and methods for controlling seed characteristics Seeds of A. trilicaire were treated with nucleases (DNase and / or RNase) as described above. The characteristics of plants obtained from these seeds are listed in Table 56 in comparison with plants grown from control untreated seeds. [Table 56]

[0356] Seeds treated with nucleases to produce "cut" and "zero" seeds showed significant advantages over control plants in a variety of aspects.

[0357] Example 48: Products and methods for growing plants and seeds under non-optimal conditions We investigated how sowing seeds in the "cut", "zero" and "Y" states affects plant growth at higher soil salinity. To do so, triticale (xTriticosecale Wittmack) seeds were sown and grown on potato dextrose agar containing 0 (deionized water as control) and 250 mM salt (MgSO4) in 9 cm diameter Petri dishes. Seeds were pretreated once or three times with 0.1-5000 μg / ml nuclease to produce the "Y" or "zero" states. The nuclease was washed off and the cells were placed in a growth chamber at 25 ± 1 °C with 12 h daylight. The number of seeds that grew and germinated were observed and counted daily for up to 7 days. Grown seeds were referred to as seeds that had reached the ability to produce at least one noticeable shoot or radicle. Seeds were considered germinated when a radicle of at least 2 mm emerged from the seed coat. After 7 days of treatment application, parameters were measured and calculated.

[0358] Seeds were planted in plastic seedling pots (LxWxD 3.25''x2,75''x2,75'') filled with a mixture of soil and sphagnum moss (3:1, v / v) containing organic fertilizer. The greenhouse temperature was maintained at 25±2°C and 10±2°C during day and night, respectively. Each treatment consisted of three replicates, with 1 / 100 plants planted per plastic pot. Plant growth parameters were measured at harvest and after treatment. Values ​​presented are shown as mean±SE with a minimum of three independent replicates (n=3). Data are shown in Figures 28 and 28.

[0359] The data obtained clearly show that the treatment of seeds with the products tested protects the growing plants against the negative effects of non-optimal growing conditions.

[0360] Example 49: Products and methods for managing DNA virus-cell interactions Vero cells were cultured in RPMI1640 medium containing 10% heat-inactivated fetal bovine serum (FBS) (Sigma), 100 g / mL streptomycin and 100 U / mL penicillin G in a humidified atmosphere of 5% CO2 in air at 37°C in 96-well plates (2x10e4 cells / well) for 22 hours. The medium was replaced with fresh medium, supplemented with nuclease (0.01 μg / mL) or nucleic acid binding protein (100 mg / mL) or a combination thereof, and incubated at 37°C for 1 hour. The medium was removed, the cells were washed with PBS, HSV-1 was added, and incubated at 37°C for 1.5 hours. The medium was then replaced with fresh medium, and the cells were incubated for an additional 48 hours. The virus titer in the cell medium was determined by standard plaque assay using 10-fold serial dilutions of cell supernatants of Vero cells incubated for 48 hours, after which the cells were fixed, stained and plaques were counted. The data are shown in Figures 29 and 30.

[0361] It is clearly confirmed that cells treated with the test compound exhibit a lower cytotoxic effect after viral infection and can be used to manage viral infection.

[0362] Example 50: Products and methods for managing tumor progression Lewis carcinoma cells were separated from the extracellular matrix and either left untreated or treated with the test products for 30 minutes as described above. After treatment, the cells were washed to avoid further contact of the cells with the test products and injected subcutaneously into C57BL / 6 mice (12 weeks old, 20 mice) weighing approximately 18 g. The disruptive effect of the test products on carcinogenesis is shown in Table 57. [Table 57]

[0363] As can be seen from the presented data, the use of the tested products leads to a reduction in their invasive activity and can be used as an antitumor strategy.

[0364] Example 51: Products and methods for managing metastasis MC38 control cells or MC38 cells after treatment with test products were examined for their ability to develop metastases. To induce colorectal liver metastases, 5x10e4 MC38 were injected into the spleen of 8-10 week old C57BL / 6JWT mice using a 23ga needle through a 1cm midline laparotomy. Tumor cells were allowed to circulate for 30 minutes before the spleen was removed and closed (to prevent splenic tumor formation). The presence of liver metastases, calculated as metastatic rate (%), was calculated on day 21. The data are shown in Table 58. [Table 58]

[0365] It is clearly shown that the use of the tested products reduced the metastatic activity of tumor cells.

[0366] Example 52: Products and Methods for the Treatment of Diabetes and Diabetic Retinopathy Patients Fifteen patients (5 men, 10 women) with type 1 and type 2 diabetes and confirmed severe nonproliferative / proliferative diabetic retinopathy were enrolled in the study.

[0367] Each patient had been on individualized insulin therapy for at least three years. Blood glucose levels were measured by placing a drop of finger blood onto a "test strip," which was then inserted into an electronic blood glucose meter.

[0368] Patients received Group 1-DNaseI (bovine), Group 2-RNase (bovine), or Group 3-DNase+RNase (bovine) combination BID 200mg / capsule. Group 4-Riboflavin 800mg x times daily. n=3 in each treatment group. Group 5 2 patients-Modified bleomycin. Each patient signed a comprehensive informed consent form prior to administration of the drug.

[0369] Compared with the pretreatment period, there was a significant improvement in blood glucose normalization in all treatment arms of the study (Table 59). [Table 59]

[0370] Compared to pretreatment, patients in all treatment groups in this study experienced significant improvements in visual acuity. The data are shown in Table 60. [Table 60]

[0371] As can be seen, the tested product significantly improved vision and retinal detachment. The use of the tested product also reduced blood sugar levels, including the patient's resistance to insulin. Furthermore, the patient was able to wean from insulin therapy.

[0372] Example 53: Products and methods for the prevention and treatment of diseases associated with protein misfolding E. coli 25922 following treatment with the test products used at concentrations between 0.1 μg / ml and 100 mg / ml activity were obtained as described above and plated on Columbia agar (Oxoid) supplemented with or without reverse transcriptase inhibitor (100 mg / ml).

[0373] The bacteria were then washed with PBS and the supernatant was filtered through a 0.2 uM filter and measured at OD500 using a microtiter plate reader (Epoch2-BioTek). The amount of amyloid was recalculated as total OD600. The data is shown in Figure 31.

[0374] The products tested significantly reduced the amount of amyloid production by bacteria within the biofilm.

[0375] Some of the reverse transcription inhibitors also reduced amyloid production, a change that was dependent on pretreatment of the cells with nucleases. Reduction of amyloid production by cells can be exploited for their antimicrobial potential, as well as for the prevention and / or treatment of infectious and neurodegenerative diseases.

[0376] Example 54: Products and methods for managing cellular interactions Bacillus VT1200 was washed from the extracellular matrix and treated with nuclease as described above. 10 μL of 10e7 bacteria was plated on Columbia agar in various combinations. Analysis of microbial growth was evaluated within 24 hours. Data is shown in Figure 32 and Table 61. [Table 61]

[0377] It was clearly confirmed that the tested products could induce distant changes in other untreated cells, which means that treated cells can be used to manage cellular interactions.

[0378] Example 55: Products and methods for controlling cell motility Bacillus VT1200 was grown overnight on Columbia agar (Oxoid). The cells were washed with PBS buffer and the cells were separated from the extracellular matrix by two sets of centrifugation (Microfuge® 20R, BeckmanCoulter) at 4000xg for 5 min. Two 90 mm Petri dishes were then filled with Columbia agar (Oxoid), one of which was supplemented with 0.1-1000 μg / mL of the test product. The agar was then cut into two identical pieces and the two halves of the agar (supplemented and not supplemented with the product) were placed on the same Petri dish, separated by a foil or plastic bridge. The washed bacteria were then standardized to 6 log10 cells / ml and plated as a line through the "bridge" from the agar not supplemented with the test product to the part of the agar supplemented with the test product. The same lines were made on two control Petri dishes: agar not supplemented with any product affecting the cells (Fig. 33A) and agar supplemented with the test product (DNase I) (Fig. 33B).An experimental dish containing two halves of agar without any supplementation (top of Fig. 33C,D) and agar supplemented with the test product (bottom of Fig. 33C,D).

[0379] To control for limited penetration of the test products from supplemented to unsupplemented areas, identical composite plates were made with the addition of blue dye (Figure 33E) - there was no sign of the paint penetrating from one area of ​​the agar to another. Figure 33 shows the data for DNase I and Table 62 summarizes the results obtained for the other products. [Table 62]

[0380] As can be seen, the tested products are able to induce the formation of identical changes in very distant parts of the whole system, which means that the products are able to manage the same changes that induce cell migration.

[0381] Example 56: Products and methods for managing intergenerational memory Bacillus VT1200 was cultivated in medium supplemented with the test products as described above. The control probe (Figure 33A) showed regular growth, while cells grown in medium supplemented with DNaseI (Figure 33B) grown on plain agar showed aberrant, enlarged bacterial growth. Cells grown in medium supplemented with DNaseI were also cultured on agar with defects on its surface to accommodate the change in electric / magnetic field (Figure 33C). When bacteria were taken from medium supplemented with DNaseI (from 33B) and cultured on control agar medium not supplemented with the product (Figure 33D), the biofilm still had an altered morphology similar to that observed in medium supplemented with the product. (Table 63). [Table 63]

[0382] The data obtained show that the product can direct cellular changes that can be corrected in cellular memory and that these changes can be passed on to other generations. Furthermore, the data obtained show that signal transduction depends on electrical and / or magnetic conditions, which in turn can be modulated by test compounds.

[0383] Example 57: Products and methods for directing directional cell migration and colony formation Bacillus VT1200 was grown overnight on 90 mm Petri dishes filled with Columbia agar (Oxoid) and divided into four sectors, each of which was treated as follows.

[0384] Sector #1 - Control

[0385] Sector #2-agar was supplemented with the products to be tested in the concentration range from 0.01 μg / ml up to 100 mg / ml.

[0386] Sector #3-agar was supplemented with human plasma from volunteers.

[0387] Sector #4-agar was supplemented with human plasma form volunteers pretreated with the product RNaseA at 100 μg / mL.

[0388] The data is shown in Figure 35 and Table 64.

[0389] It is clearly confirmed that the tested product (sector #4) induced cell migration towards the chemoattractant (plasma), whereas the case containing intact cells (blood in sector #3) had no such inducing effect. [Table 64]

[0390] This experiment demonstrates that the products and methods tested can be used to modulate cell migration, directional colonization, invasion and infection processes, dispersion, migration, directional chemotaxis, and can be utilized in biomanufacturing, infection treatment, and microbiome transplantation.

[0391] Example 58: Products and Methods for the Prevention and Treatment of Autoimmune Conditions Serum antibodies against P. aeruginosa DNA, E. coli RNA, and antibodies conjugated with DNase I were obtained as described above.

[0392] To model GVHD, a xenogeneic model of MHC class I and II, C57BL / 6 (H-2b) to BALB / c (H-2d), was used. Recipient animals were 8-week-old females. To prepare cell suspensions from euthanized donor mice, CD8 T cells were isolated from the spleen using a CD8 purification kit (Miltenyi Biotec) according to the manufacturer's instructions. The yield was 6.7 log10 cells, pellets resuspended in 1640 RPMI with 5% FBS (all Gibco). Bone marrow and splenocyte suspensions were prepared in saline for injection.

[0393] Mice were then irradiated with two isodose doses of 4.5 cGy each, after which they were injected with 6.5 log10 bone marrow cells and 7 log10 splenocytes. Beginning on the same day as the BMT, mice were randomly assigned to receive the following treatments of the products tested at concentrations ranging from 1 μg / ml to 1000 μg / ml:

[0394] group:

[0395] 1. Control - Untreated

[0396] 2. Antibody against Pseudomonas aeruginosa DNA 1μg / ml twice a day

[0397] 3. Antibody to Pseudomonas aeruginosa DNA 100μg / ml twice a day

[0398] 4. Antibody against Pseudomonas aeruginosa DNA 10 μg / ml twice daily + nevirapine 0.1 to 50.0 mg / kg once daily

[0399] 5. Antibody against E. coli RNA 0.1 μg / mL once a day

[0400] 6. Antibody against E. coli RNA 1000 μg / mL once a day

[0401] 7. Anti-D8 conjugate antibody and DNase I administered twice daily

[0402] 8. Before injection, cells were treated with 0.1 μg / mL DNase once every 48 hours.

[0403] 9. Before injection, cells were treated with 1000 μg / mL DNase once every 48 hours.

[0404] 10. Administer antibodies against surface-bound DNA once a day

[0405] Survival data is presented in Table 65 below. [Table 65]

[0406] The data obtained indicates that the products and methods tested resulted in a significant improvement in the severity of the autoimmune process and GVHD symptoms, and improved survival rates.

[0407] Example 59: Products and methods for the treatment of cancer. Vaccines from intracellular DNA of P. aeruginosa or E. coli biofilms or DNA of NAMACS and NAMACS-ANA or a mixture of microorganisms isolated from mammalian (mouse) feces were obtained as described previously. Mice (C57BL / 6.8 weeks old, 6 mice / group) were subcutaneously injected with H59. Mice were divided into untreated, mice intravenously injected once or twice with the vaccine.

[0408] Livers were excised from mice when the flank tumor size reached 2.5 cm3, and liver metastatic nodules were analyzed (Table 66). [Table 66]

[0409] The data clearly show that vaccines having bacterial DNA and NAMACS and NAMACS-ANA among their components have high anti-cancer activity.

[0410] Example 60: Products and methods for modulating protein-based receptors CHO cells were first serum-starved for 24 hours and plated at a density of 4.2 log10 cells / well in 48-well culture plates. Cells were detached from the extracellular matrix as described above and either treated with PBS to generate CHO control or treated with the test products described above and ITS complexes (insulin, 5 μg, transferrin, 5 μg, selenium, 5 ng / ml) in DMEM according to the manufacturer's instructions (Sigma-Aldrich). The number of attached cells was measured after 24 hours of growth according to previously established methods. The results are shown in Figures 36, 37 and Table 67. [Table 67]

[0411] As can be seen, the use of the tested products makes it possible to monitor and control protein receptors.

[0412] Example 61: Products and methods for managing wound healing The effect of the test products on the management of stem cells used in wound healing was investigated. Mouse embryonic stem cells (CGR8, Sigma) (MESCs) were cultured on GMEM + 2 mM glutamine + 0.05 mM 2-mercaptoethanol (2ME) + 1000 units / ml DIA / LIF + 10% fetal bovine serum (FBS). MESCs were treated with the different test products at concentrations ranging from 1 μg / ml to 1000 μg / ml for 1.0-60.0 minutes prior to application to the wound. Eight-week-old C57BL / 6 mice (n=30) were anesthetized with ketamine and xylazine.

[0413] A full-thickness skin defect of 1 cm in diameter was created in the neck region of each animal after surgical preparation with hair removal and alcohol scrub of selected areas. Full-thickness burns were established under general anesthesia on both sides of the dorsal trunk.

[0414] 5x10e4 cells / ml MESC were transferred to each wound and covered with a sterile dressing. Fresh cells were added every 4 days. Control animals were left untreated but covered with a sterile dressing. Data are shown in Table 68. [Table 68]

[0415] As can be seen, the products and methods exhibit significantly higher rates of wound healing.

[0416] Example 62: Products and methods for managing memory and cognitive processes Male BL6 mice (approximately 3–4 weeks and P12–P21 for paired synaptic transmission studies) were sacrificed by cervical dislocation and decapitated.

[0417] Parasagittal hippocampal and neocortical slices (350 mM) were cut using a Microm HM650V microslicer in cold (2–4°C) high Mg2, low Ca2aCSF consisting of the following: 127 mM NaCl, 1.9 mM KCl, 8 mM MgCl2, 0.5 mM CaCl2, 1.2 mM KH2PO4, 26 mM NaHCO3, and 10 mM D-glucose (pH 7.4 bubbled with 95% O2 and 5% CO2, 300 mOsm).

[0418] Neocortical slices were cut at a 15° angle, with the blade starting to cut from the surface of the neocortex (layer 1) towards the caudal border of the neocortex [to ensure the integrity of layer V pyramidal cell (layer VPC) dendrites]. Slices were stored at 34°C in standard aCSF (1 mM Mg2 and 2 mM Ca2) for 1-8 h. Statistical analysis was performed with 2-way ANOVA.

[0419] Control probes were left untreated and experimentally treated with the test products as described above. The results are shown in Figure 38 and Tables 69, 70, 71. It is clear that the use of the tested products reduces neuronal excitability. [Table 69] [Table 70] [Table 71]

[0420] The obtained data clearly show the effect of the tested product on neuronal excitability, which is a key component of synaptic plasticity, learning and memory, as well as of aging, and whose dysfunction is associated with age-related learning and memory deficits. Moreover, the tested product can be used to enhance the cognitive capabilities of the human brain and restore memory, speech and movement by managing the transmission and reception of electrical signals to and from machines via the brain.

[0421] Example 63: Products and methods for managing cellular responses to light. The effect of the tested products in the concentration range of 1 μg / ml to 1000 μg / ml with exposure times of 1 to 60 minutes on the control of cellular characteristics by light was investigated. Bacillus VT1200 was isolated from the extracellular matrix as described above and treated with the test products. The cells were cultured on Columbia agar for 24 hours at 37°C in an incubator in either (i) the dark or (ii) the light (visible or blue). The data are shown in Figures 39 and 40 and Table 72. [Table 72]

[0422] It was clearly observed that the control bacterial colonies grown under light showed changes in morphology, while the morphology of the colonies formed after treatment with the tested products remained almost unchanged, which means that after treatment with the tested products, the cells are unable to respond to the appearance of light and have different regulation towards physical factors.

[0423] Additionally, various test products were analyzed for the response of eukaryotic cells to light.

[0424] For light exposure, 4.0 log10 aliquots of Vero cells were placed into wells of a 24-well plate. Cells were allowed to attach for 3.5 hours at 37°C in DMEM containing 10% FBS, the medium was replaced with DMEM, and cells were treated with nuclease as described above. The medium was replaced for 30 minutes, cells were washed with DMEM, and fresh DMEM containing FBS was added. Plates were exposed to a visible light source equipped with a 150W (840lm) halogen lamp (Philips, Shanghai, China) for 24 hours at 37°C. Cell status was observed and photographed under a Zeiss Axiovert 40C microscope (10x magnification). Results are presented in Table 41. It was observed that within 24 hours after 30 minutes of exposure to RNase, most cells had the tri-radioactive morphotype, whereas all Vero controls were unable to grow due to phototoxicity. Taken together, these results suggest that the tested products are able to manage the cellular response to light and play an important role in photoprotection against light-induced cytotoxicity.

[0425] Example 64: Products and methods for managing cellular responses to electrical stimulation The effect of the test products in the concentration range of 1 μg / ml to 1000 μg / ml with exposure for 1 to 60 minutes on the control of cell characteristics in response to electrical stimulation was investigated. Bacillus VT1200 was isolated from the extracellular matrix as described above and left untreated or treated with the test products and cultured on Columbia agar for 10.0 to 48 hours at 37°C in an incubator (i) in the dark or (ii) under 1 mA electrical stimulation. The data are shown in Figure 42 and Table 73. [Table 73]

[0426] It is clearly confirmed that the tested products control the behaviour of bacteria in response to electrical stimulation.

[0427] Example 65: Products and methods for monitoring environmental conditions, radiation, and biology TezR was used to monitor environmental, meteorological, and geomagnetic conditions. To do so, B. pumilus VT1200, isolated from the extracellular matrix and treated with DNase as described above, were seeded daily on the surface of Columbia and PeptedMeat 90 mm Petri dishes, placed in Mu-metal boxes, and incubated at 37°C for 24 hours. Changes in biofilm morphology were analyzed and these changes were correlated with geomagnetic storms. The data are shown in Figure 43. As will be seen, by culturing microorganisms treated with the product in Mu-metal, it is possible to detect geomagnetic storms as well as other changes and disturbances in the magnetosphere, as well as other environmental factors such as geological exploration, water conditions, radiation and magnetic conditions, exposure to solar radiation, floods, earthquakes, etc.

[0428] Example 66: Products and methods for controlling magnetically dependent cellular activity We found that certain bacteria in the human microbiome respond to changes in the geomagnetic field. Pure bacterial cultures were obtained by dissolving 1 ml of saliva samples from individuals suffering from magnetotoxicity at 1:10,000 in PBS, plating on Columbia and Pepted Meat agar supplemented with 10% red blood cells on 90 mm Petri dishes, and incubating at 37°C for 10-72 hours.

[0429] These pure bacterial cultures were then subcultured in normal or altered geomagnetic fields (μ-metal as above) and we found that the growth and activity of some bacteria was altered when grown in the altered geomagnetic field (Figure 44).

[0430] Since the growth and activity of this bacterium can be regulated by changes in the geomagnetic field, we thought that this phenomenon could be exploited to switch on or off the activity of a specific gene. One such bacterium was B. pumilus VT1200, which naturally produces RNaseI. The DNA fragment was purified and ligated into a pET-15b vector (Novagen, Madison, WI) to construct the expression pETDNaseI plasmid. This plasmid was transformed into B. pumilus VT1200 (also shown to have high tropism for tumors) for initial cloning. The PstI and SacI sites in the DNaseI gene were used to select positive clones. Next, the T7 RNA polymerase gene was added into B. pumilus VT1200 to turn on the pET-15b protein expression system for the production of DNaseI (SEQ ID NO:1).

[0431] SEQ ID NO:1 MRGMKLLGALLALAALLQGAVSLKIAAFNIRTFGRTKMSNATLVSYIVQILSRYDIALVQEVRDSHLTAVGKLLDNLNQDAPDTYHYVVSEPLGRKSYKERYLFVYRPDQVSAVDSYYYDDGCEPCGNDTFNREPFIVRFF SRFTEVREFAIVPLHAAPGDAVAEIDALYDVYLDVQEKWGLEDVMLMGDFNAGCSYVRPSQWSSIRLWTSPTFQWLIPDSADTTATPTHCAYDRIVVAGMLLRGAVVPDSALPFNFQAAYGLSDQLAQAISDHYBVEVMLK

[0432] Colonies of B. pumilus VT1200 were grown at 37°C in Columbia agar supplemented with 50 mg / mL ampicillin. DNaseI activity was measured using the method described by Kunitz. Overnight colonies were washed with sterile PBS, and bacteria were centrifuged (3000xg) and washed three times with sterile PBS (Ginco) before being injected into 8-week-old BALB / C mice (N8 / group). Intravenous (into the tail vein) injections of bacteria were performed at a concentration of 7.0 log10 in 50 μl of PBS.

[0433] We investigated whether the change in geomagnetic field could cause an increase in the activity of B. pumilus VT1200. To do so, the animals were placed in a 4-layer μ metal envelope for 120 minutes, and the DNase and RNase activities in the blood were measured. The DNase and RNase activities of B. pumilus at each time point in normal geomagnetic field were taken as 100%. The data are shown in Table 74. [Table 74]

[0434] The data presented showed that the activity of specific genes can be switched on and off in the macroorganism, as well as in cells cultured ex vivo and injected into the macroorganism.

[0435] Example 67: Diagnostic and therapeutic methods related to geomagnetic activity and weather-dependent changes. Saliva samples from five healthy individuals and five subjects with weather-dependent headaches, migraines, and airplane headaches were dissolved 10,000-fold in PBS and plated on Columbia agar supplemented with red blood cells (5%). Probes were incubated in normal, altered, or inhibited geomagnetic fields (μ-metal) at 37°C for 24 hours. Representative images of patient subjects and probes are shown in Figure 45.

[0436] It is clearly confirmed that some microorganisms from the oral cavity of patients with weather dependency changed their growth after plating on altered magnetic conditions, which can be used to identify bacterial strains with weather dependency status, diagnose patients with weather dependency and underlying diseases, and target therapeutic interventions.

[0437] Two bacterial strains whose growth was enhanced in the inhibited geomagnetic field were isolated and, using the method described above, were found to produce large amounts of RNase specifically in response to altered geomagnetic conditions. To do so, bacterial cultures of these bacteria were maintained at 37°C on agar plates supplemented with 10 μg / ml RNA as described above, and RNase activity was assessed as a clear zone around the colony. Every day for 30 days, 25 μl of bacterial culture (1x10e5 bacteria / ml) was plated in the center of a 90 mm glass Petri dish, and the zone around the colony was analyzed. At the end of the observation period, the bacterial RNase activity was compared between days with normal solar activity and days with a solar storm (Figure 46).

[0438] We then modified the bacteria as described above to develop Zero-D, Zero-R, and Zero-DR cells to induce weather-dependent forgetting in the cells, and found that the bacteria then had reduced expression of RNase (measured as described above) caused by sunlight storms compared to the untreated control (which was designated as 100%). The data are shown in Table 75. [Table 75]

[0439] Next, 40 patients who experienced weather-dependent headaches of various kinds, such as headaches, migraines, and airplane headaches, more than four times a year were retrospectively enrolled. These patients were treated with: (1) oral rinses twice a week with 10e5 / ml of Zero-D, Zero-R, and Zero-DR bacteria from the same patient, (2) oral rinses twice a week with 10e5 / ml of Zero-D, Zero-R, and Zero-DR bacteria from another patient, (3) some patients received antibiotics (penicillins, tetracyclines, and macrolides at 1 / 2 the recommended dose) during the airplane travel or during the aura before the migraine began, or (4) oral rinses with 0.01% compound Y190 (Figure 47), or (5) potassium orotate, etinavir, ribavirin, and abacavir at normal doses. All patients were monitored for an additional 12 months (Table 76). [Table 76]

[0440] Therefore, the use of the tested products can reduce weather dependency and the occurrence of migraines.Furthermore, the inventors have previously shown that higher expression of RNase is associated with a shortened life span, so that uses or products and methods that inhibit the RNase activity of the microbiota can be used to extend life span.

[0441] Example 68: Products and methods for the management of autoimmune diseases by management of cellular memory Peripheral venous blood was collected from patients with type 1 diabetes (t1D), systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), atopic dermatitis (AD), asthma (A) or from healthy controls (age-matched). Monocytes were obtained using density centrifugation on Ficoll with follow-up negative selection using magnetic beads and further sorted with specific antibodies (preserving the CD14+CD16- fraction). Monocytes at 5x10e5 cells / well were seeded in 96-well plates containing HL-1 medium with 2mM L-glutamine, 100U / ml penicillin and streptomycin mixture, non-essential amino acids and heat-inactivated serum. Cells were separated from the extracellular matrix as described above and either left untreated or treated with products in the concentration range of 1μg / ml up to 1000μg / ml with 1-60 min exposure under control. The number of IL-6 secreting cells was counted. Data are shown in Figure 48.

[0442] It is clearly observed that patients with various autoimmune diseases have a higher number of IL-6 monocytes compared to controls. The use of the tested products may regulate and inhibit IL production by cells and modulate the autoimmune behavior of immune cells.

[0443] To investigate how various test products can protect cells by being targeted by components of the immune system, memory T cells treated with or untreated with test products were co-cultured with monocytes from control, T1D, SLE, RA, AD and patients in the presence of anti-CD3 antibody for 120 hours to alter their memory. Memory T cells were then supplemented with IL-2 and grown for an additional 144 hours. The number of cells producing IL-17 was counted. The data are shown in Figures 48, 49 and Table 77. [Table 77]

[0444] The erasure of the cellular memory of T cells by the tested products was clearly confirmed to have inhibited the activation of T cells by IL-17 by monocytes from patients with autoimmune diseases; thus preventing these cells from being targeted by components of the immune system.

[0445] Example 69: Products and methods for directing the synthesis and transport of products from cells. The rat INS-1 cell line was used, which is capable of producing and releasing insulin-releasing hormone after glucose stimulation. The cells were maintained in RPMI1640 serum-free medium supplemented with D-glucose and supplemented with trophic and antimicrobial factors as described above under a humidified atmosphere. The cells were either untreated (control) or treated with the test products at concentrations ranging from 1 μg / ml to 1000 μg / ml for different periods of time ranging from 3 min to 24 h. The culture medium was collected and stored at -80°C until use in the insulin release assay. Insulin release was detected by using a rodent insulin ELISA.

[0446] Comparison of insulin release and content between mBMDS and INS-1 cells. Data is shown in Table 78. [Table 78]

[0447] It is clearly established that the product can be used to direct the production and secretion of various products by cells, including hormones.

[0448] Example 70. Products and methods for managing the memory of eukaryotic cells to generate new sensors and sensing systems. The use of the test products to reprogram cells in adaptive memory experiments was investigated. As a control, C. albicans or those after treatment with the test products were placed in M9 supplemented with dexamethasone and monitored for the onset of growth. After each passage, cells were placed in Sabouraud broth for 1.0 up to 72 hours, then washed and placed in M9 supplemented with dexamethasone for 4 hours, after which the extracellular matrix was removed and cells were treated with test products in the concentration range of 1 μg / ml to 1000 μg / ml, and the fungus was again placed in M9 with dexamethasone for the next 20 hours of growth. The data are shown in Table 79. [Table 79]

[0449] As can be seen, the formation of cells using multiple cycles of the tested products, followed by washout periods after each cycle, allowed these cells to start sensing and fermenting new products without any artificial genome modification. Such management of the genome information of cells allows them to recognize and inactivate xenobiotics; to form cells that sense new factors, inactivate, utilize and synthesize programmed products, non-limiting examples being the use of such organisms for environmental pollution control, waste management, construction, food preparation (i.e., fermentation of products, acting as probiotics), biotechnology.

[0450] Example 71: Products and methods for managing stem cells and extending lifespan. To evaluate the effect of the test products on lifespan and stem cell differentiation, the antioxidant and anti-aging activity of mesenchymal stromal cell conditioned medium (MSCM) was administered and evaluated using umbilical cord-derived mesenchymal stromal cells treated or untreated with the products at concentrations ranging from 1 μg / ml up to 1000 μg / ml with 1-240 min exposure. Briefly, mesenchymal stromal cells were isolated from umbilical cord. Fibroblasts were isolated from human foreskin as previously described, incubated in collagenase for 90 min, and incubated in DMEM supplemented with 10% FBS and antibiotics. Control probes were cultured at normal glucose (6 mmol / L) levels. To adjust for stress, cells were placed at hyperglycemic levels of 30 mmol / L. To induce fibroblast differentiation, cells were separated from the extracellular matrix and either left untreated or treated with the test products and further incubated with recombinant human TGF-β1 (5 ng / mL, 40 h). Intracellular ROS was measured by DCFH-DA fluorescence. For this purpose, cells were incubated with 10 μmol / L DCFH-DA. The regulatory role of MSC-CM (treated or not treated with nuclease) was evaluated by pretreating fibroblasts with 2.5% MSC-CM grown and plated in high glucose environment (Table 80). [Table 80]

[0451] Results shown here are from three experiments. *P<0.05 for MSCM vs. cells altered by the products tested.

[0452] These results demonstrate that the effects of the tested products on cells, including stem cells, can manage oxidative stress associated with cellular aging. Furthermore, we demonstrate that the effects of the tested products can be used to manage the upregulation of genes associated with cellular aging, such as p16 and p21.

[0453] We also analyzed how the tested products could regulate cell differentiation (Tables 81, 82). [Table 81] [Table 82]

[0454] At normoglycemic levels, in the presence of TGF-β1, 62±6% of fibroblasts differentiate into myofibroblasts in 72 hours. These data clearly show that product treatments differentially affected cell differentiation and pluripotent cell behavior.

[0455] Example 72: Products and methods for aging management Normal human dermal fibroblasts were isolated from juvenile foreskin and cultured according to standard procedures for several passages. Cells were separated from the extracellular matrix and either untreated or treated with test products ranging in concentration from 1 μg / ml to 1000 μg / ml and incubated for 30 seconds to 60 minutes. Some cells were subjected to multiple cycles of nuclease treatment followed by a washout period to generate "zero cells". Mean telomere length was measured from total genomic DNA. DNA was extracted using a Qiagen DNA kit. Mean telomere length was measured by using the following previously described qPCR method [Salpea KD, Nicaud V, Tiret L, Talmud PJ, Humphries SE (2008) The association of telomere length with paternal history of premature myocardial infarction in the European Atherosclerosis Research Study II. J Mol Med 86:815-824]. 815-824]. Relative telomere length, known to correlate with chronological age, was calculated as the ratio of telomere repeats to single-copy gene copies (T / S ratio) determined by quantitative PCR and adjusted for cumulative population doubling. Cumulative population doubling was estimated as the number of population doublings, gradually adding population doublings at each passage (population doubling = [ln(number of cells harvested)-ln(number of cells seeded)] / ln2). Results are shown in Figure 50.

[0456] As can be seen, the use of the tested products and shifting the cells to the "zero" state inhibited telomere shortening (all p<0.05). This effect was most pronounced in the cells in the "zero" state.

[0457] Example 78: Products and methods for controlling cell characteristics ex vivo and subsequent allogeneic transplantation. Female NOD SCID (CB17-Prkdcscid / NcrCrl) mice weighing 18-20 g were used. Subcutaneous tumors were established by injection of 7.0 log10 Raji cells. CD8 T cells were harvested. Some CD8 T cells were treated with products ranging in concentration from 1 μg / ml up to 1000 μg / ml, some were transduced with lentiviral vectors encoding CD19 CAR and then treated with nuclease. Some cells were treated with multiple cycles (2-10) of test products followed by a washout period to generate "zero cells" or continuous treatment for 48 hours. Some cells were pretreated with a combination of reverse transcriptase and integrase inhibitor (0.1-1000.0 μg / mL). Cells were implanted back into animals 8 days after tumor implantation. Tumor volumes were measured 60 days after tumor implantation and rounded to the nearest "5" (Table 83). [Table 83]

[0458] These data clearly indicate that the products tested can be used to manage the genetic information of cells to reprogram them and subsequently result in altered function of these cells upon transplantation. Combinations of products may further enhance this effect.

[0459] Example 79: Products and methods for managing tumor resistance. ATCC cell line E0771 was maintained in DMEM supplemented with 10% FBS and 1% penicillin / streptomycin at 37° C. in a 5% CO 2 atmosphere.

[0460] Control E0771 or treatment with products ranging in concentration from 1 μg / ml to 10 mg / ml were used alone or in combination with reverse transcriptase and integrase inhibitors (0.1-1000 μg / mL). Stimulation of PD-L1 expression was performed by treating cells with IFN-γ. The level of PD-L1 expression was assessed with an anti-PD-L1 antibody and rounded to the nearest "1". The data are shown in Table 84. [Table 84]

[0461] These data clearly indicate that the tested products can be used for modulating PD-L1 expression, proto-oncogene expression and crosstalk between cancer cells and immune cells.

[0462] Example 80. Products and methods for managing life span. C. elegans (Carolina biosciences) were maintained using standard methods in nematode growth medium. Synchronized samples were prepared by egg-laying method by placing young adults on plates seeded with E. coli for 4 hours and then removing them. Eggs (#100) were pretreated with products in the concentration range of 0.1 μg / ml to 1 mg / ml. All life span analyses were performed at 22°C and rounded to the nearest "0.1". Viability was assessed every 2 days and nematodes were considered dead if they did not respond to gentle touch with a sterile wire. Some cells were pretreated with a combination of reverse transcriptase and integrase inhibitors (0.1-1000.0 μg / mL). Data are shown in Table 85. [Table 85]

[0463] These data clearly show that the use of the products tested can be used to extend life.

[0464] Example 81. Products and methods for controlling product yields in biomanufacturing. To study the effect of the test products on insulin precursor (IP) production, the pPIC9K expression vector construct was used to transform the Pichia yeast strain GS115his-. The cells were then pretreated or not with concentrations ranging from 1 μg / ml to 1000 μg / ml. Some cells were pretreated with a combination of reverse transcriptase and integrase inhibitor (0.1-1000.0 μg / mL). The transformants were plated in 500 mL Mini Bioreactors (in normal or altered geomagnetic conditions by placing them in a microtissue) filled with 250 mL of autoclaved growth medium adjusted to pH 5.0 with 25% NH4OH. The stirrer speed was controlled between 200 and 800 rpm at 30 °C. After the glycerol-depleted growth phase, a glycerol-enrichment phase was initiated using a glycerol solution (50% glycerol (w / w), biotin and PTM1). After 6 hours, production of IP was initiated by addition of 99% methanol, biotin 0.2 and PTM1. Quantification of IP was performed by HPLC. Data is shown in Table 86. [Table 86]

[0465] The data obtained indicates that the use of test products in normal and altered magnetic fields can be used for the control of biomanufacturing, including increasing product yield.

[0466] Example 82: Cell protection products and methods for products to manage cell behavior. Antibody against RNase at 10 μg / ml was added to the agar of 90 mm Petri dishes filled with a mixture of Columbia and Pepted meat agar containing 1 / 6 sectors from 50 μL of fresh human volunteer plasma filtered through a 0.22 μM filter. Control plates contained no antibody. 25 μL of overnight B. pumilus VT1200 was placed in the center of the plate, and the plate was incubated at 37° C. for 24 hours and photographed with a Canon6D (Canon, Japan). The data are shown in FIG. 51.

[0467] It is clearly confirmed that the products as anti-RNase antibodies can be used for the management of cellular responses.

[0468] Example 83: Products for controlling eukaryotic and prokaryotic pathogenicity and for the diagnosis of diseases associated with NAMACS and / or NAMACS-ANA capable of recognizing biological, chemical and physical agents. Surgical cells from pancreatic cancer patients were trypsin-treated, left untreated or treated with test products at concentrations ranging from 1 μg / ml to 10 mg / ml.

[0469] The oral microbiota of healthy individuals was left untreated or treated with test products at concentrations ranging from 1 μg / ml to 10 mg / ml.

[0470] Pooled blood from healthy volunteers (n=5, mean age 43.4 years) was left untreated or treated with (i) 10e2-10e8 cells / ml of isolated cancer cells or (ii) 10e2-10e9 bacteria / ml of oral microbiota and incubated at 37°C for 1.0-up to 360 minutes and then heated to 100°C for 10 seconds-up to 60 minutes. LC / MS was performed. Table 87 below shows the effect of the products as generated found in the plasma of healthy volunteers and cancer patients. [Table 87] TIFF2024515541000106.tif95159

[0471] The products can be used for the prevention and treatment of diseases associated with and / or related to NAMACS and NAMACS-ANA of eukaryotic and microbiota cells.These nucleic acid molecules and the proteins formed in the test plasma of healthy individuals after their addition can be used for the diagnosis of various diseases.

[0472] Example 84: Analysis of Cell Surface Bound Nucleic Acids as Indicators of Health and Disease in Conjunction with Other Diagnostic Tests Twelve patients were suspected by routine analyses (screening tests such as colonoscopy, prostate-specific antigen, mammography, cytology, circulating tumor DNA, biomarker detection) to have a specific malignancy (pancreatic, lung, colorectal, prostate, liver, mesothelioma) but the diagnosis had not yet been established and required other confirmatory analyses. The composition of nucleic acids bound to the cell surface of cells taken from needle biopsies of cancer or sputum (in the case of lung cancer patients) was investigated. Cells from the same locations were obtained from surgical material of non-tumor patients.

[0473] Nucleic acids bound to the cell surface were visualized with DAPI, SYTOX green (excitation: 504, emission 523), and propidium iodide (excitation: 493, emission 636) using an ECHO (ECHO San Diego CA) Revolve microscope and a Synergy Neo2 multimode microplate reader (Biotek).

[0474] To isolate cell surface-bound nucleic acids from tissues of suspected tumor-bearing or control patients, tissues were homogenized and collagenase was added. Cells were gently washed and filtered at 0.22uM to pass debris and any intracellular nucleic acids that may be present in the material. Cells were then placed in 0.9% NaCl supplemented with BamHI and HindIII BbvCI, BgII, FokI, AcuI nucleases, plus Mg buffer, for 1 hour at 37°C. Cells were separated by centrifugation at 3000gx10 minutes and the supernatant was filtered through a 0.22μM filter (Millipore). DNA was isolated from the supernatant using the QIAamp DNA MiniKit (Qiagen). RNA was isolated using the Quick-RNA kit (Zymo Research).

[0475] Whole genome sequences were obtained using an Illumina HiSeq 2500 sequencing platform (Illumina GAIIx, Illumina, San Diego, CA, USA). Library preparation, sequencing reactions, and runs were performed according to the manufacturer's instructions. *The amount of nucleic acid bound to the cell surface of each type of unaltered cells was suggested as "normal". Some cells were also stained with Sytox as described above, and the corresponding changes in surface green fluorescence were analyzed as an indication of cell surface-bound nucleic acid changes. The data are shown in Table 88. [Table 88]

[0476] These data clearly demonstrate that cell surface bound nucleic acids can be used in conjunction with other diagnostic methods for highly accurate diagnosis of mammalian disease.

[0477] Example 85: Products and methods for managing product yields in biomanufacturing. To investigate the effect of the test products on product yield in biomanufacturing, a cell line producing insulin precursor (IP) was used. To this end, the E. coli expression vector construct was used to transform E. coli ATCC25922 strain. The cells were then pretreated or not pretreated with the test compound. The transformants were plated in flasks modeling 500 mL bioreactors filled with 250 mL of growth medium (either in normal or altered geomagnetic conditions by placing them in a μ-tissue).

[0478] Nuclease-treated or untreated recombinant IgG-producing suspension CHO cell lines were seeded at 2x10e cells / ml in 30 ml of nutrient medium. Recombinant mouse IgG production yields were assayed 1-6 days after transfection using a Protein G biosensor (forteBIO® Octet RED96 system).

[0479] Quantification of IP was performed by HPLC. Yields of IgG production were measured by day 5. Data are shown in Table 89. [Table 89]

[0480] The data obtained show that the tested product controls product yield in both normal and altered magnetic fields, with or without shaking, and can be used for controlling biomanufacturing and increasing product yield.

[0481] Example 86: Products and methods for managing neoplastic transformation. The effect of the products on the prevention of neoplastic transformation was evaluated. To do so, serum-supplemented medium of RWPE-1 cells was removed and the cell monolayer was washed once with PBS and once with serum-free medium. Cells were then detached from the extracellular matrix, treated with test compounds in the concentration range of 1 μg / ml to 10 mg / ml, exposed to phorbol 12 myristate (PMA) 50 ng / mL, and the expression of MMP9 as a signature of neoplastic transformation was monitored. The data are shown in Figure 90. [Table 90]

[0482] It is clearly confirmed that the tested products are capable of inhibiting cancer transformation.

[0483] Example 87: Products and methods for controlling the synthesis and production of active modulators by cells. Five obese patients (group OB-CONTROL) collected samples intact using plastic fecal collection containers. The probes were frozen. Prior to analysis, the probes were thawed in an anaerobic chamber, the extracellular matrix was removed, and the probes were dissolved in PBS, treated or not with products ranging in concentration from 1 μg / ml up to 1000 μg / ml, left at 37° C. for 6 hours, and filtered through a 0.33 mm pore size filter. The liquid fraction was removed for analysis of SCFAs using an Agilent 7890b gas chromatograph. The data are shown in Table 91. [Table 91]

[0484] It is clearly confirmed that the tested products are capable of modulating the production of metabolites including short chain fatty acids.

[0485] Example 88: Products and methods for managing cellular responses. CAR-T cells and mock T cells were obtained as previously described [7], dissociated from the extracellular matrix, treated with test compounds at concentrations ranging from 1 μg / ml to a maximum of 10 mg / ml for 1-240 min, and resuspended in RPMI+IL-2 / RPMI. Some CAR-T cells and mock T cells were treated with multiple rounds of nuclease to generate zero-D, zero-R, or zero-DR cells as previously described. Raji and Jeko cells were also dissociated from the extracellular matrix, treated with test compounds at concentrations ranging from 1 μg / ml to a maximum of 1 mg / ml for 1-240 min, and resuspended in RPMI+IL-2 / RPMI. The effect of the tested products was evaluated by monitoring specific lysis.

[0486] The results are shown in Tables 92 and 93. [Table 92]

[0487] These data indicate that compounds used in these settings sensitize cells to anti-cancer cell therapies and immune cells. [Table 93]

[0488] These data indicate that treatment of immune cells with the tested compounds increases their antitumor activity.

[0489] Example 89: Products and methods for modulating pathological diseases when administered systemically or locally. The goal was to show that the test product, when used systemically and locally, can be used for the modulation of fibrosis and NASH formation.

[0490] The STAM mouse model of NASH was used. The STAM model was created in C57BL / 6 mice by combining chemical treatment (200 μg streptozotocin) and a high-fat diet (60% of energy from fat). NASH develops at 7-8 weeks and progresses to fibrosis at 10-12 weeks. Animals were treated intravenously (twice a week) with nuclease inhibitors (mouse actin and recombinant mouse RNase inhibitor). Some animals received two intrahepatic injections of these products at 2 and 4 weeks after the start of the experiment. Comparison of NAS from mouse liver specimens of 10-week-old mice included steatosis and fibrosis.

[0491] The results are shown in Tables 94 and 95. [Table 94]

[0492] These data indicate that inhibition of nucleases can be used for the treatment of mammalian diseases, including the control of steatosis. [Table 95]

[0493] It is clearly confirmed that the use of the tested compounds reduces NAS and fibrosis.

[0494] Example 90: Products and methods for modulating pathological diseases when administered systemically or locally. The goal was to show that the test product, when used systemically and locally, can be used for the modulation of fibrosis and NASH formation.

[0495] The STAM mouse model of NASH was used. The STAM model was created in C57BL / 6 mice by combining chemical treatment (200 μg streptozotocin) and a high-fat diet (60% of energy from fat). NASH develops at 7-8 weeks and progresses to fibrosis at 10-12 weeks. Animals were treated intravenously (twice a week) with nuclease inhibitors (mouse actin and recombinant mouse RNase inhibitor). Some animals received two intrahepatic injections of these products at 2 and 4 weeks after the start of the experiment. Comparison of NAS from mouse liver specimens of 10-week-old mice included steatosis and fibrosis.

[0496] The results are shown in Tables 96 and 97. [Table 96]

[0497] These data indicate that inhibition of nucleases can be used for the treatment of mammalian diseases, including the control of steatosis. [Table 97]

[0498] It is clearly confirmed that the use of the tested compounds reduces NAS and fibrosis.

[0499] Example 91: Products and methods for controlling cellular activity. The effect of disruption of cell surface nucleic acid and protection from disruption of cell surface nucleic acid was examined as a therapeutic intervention to treat and prevent disease progression. Cell surface nucleic acid was protected by inhibiting DNase with actin and by inhibiting RNase with RNase-binding proteins or oligomers of vinylsulfonic acid (OVS) derivatives as previously described.

[0500] Primary lung cancer cells were isolated as previously described (Zheng et al., 2011) and maintained for over 25 passages, and H1299 cells were used. To determine whether loss of surface nucleic acid breaks could lead to a disease-inducing state or vice versa, we examined the expression of E-cadherin (Loh et al., 2019), a known key factor in epithelial-mesenchymal transition after loss of surface nucleic acid breaks.

[0501] RNA extraction and transcriptome analysis were performed as described above. As shown in Figure 52, disruption of different surface nucleic acids had different effects on up- and down-regulation of E-cadherin expression in different cells.

[0502] These data indicate that both the destruction / inactivation and prevention of destruction of surface nucleic acids in some cells has therapeutic potential.

[0503] Example 96: Development of products and methods for protecting primary cell surface nucleic acids. We investigated whether protection of extracellular nucleic acids from nucleases could be used to prevent cellular changes typical of cells following destruction of cell surface nucleic acids. This was done using a dispersion model of B. pumilus VT1200. Control bacteria were treated with RNase to induce their dispersion, and experimental bacteria were treated with RNase together with ribonuclease inhibitors or with anti-RNase antibodies for 30 min at 37° C. The data are shown in FIG. 53.

[0504] As can be seen, protection of cell surface nucleic acid resulted in a significant inhibition of changes in cellular characteristics following loss of cell surface nucleic acid.

[0505] Example 97: Modulation of RNase expression in organisms to control health and longevity. Given the wide range of features regulated by cell surface-bound nucleic acids, we suggest that alterations in RNase expression may be associated with the development of various diseases and longevity.

[0506] The wild-type E. coli strain VT-9 and an isogenic mutant strain carrying the RNase gene (E. coli VT-9RNase+) were obtained from the laboratory of Human Microbiology Institute. Expression of RNase was also confirmed by RNA destruction in the medium as described above.

[0507] C. elegans were grown under standard conditions on Nematode Growth Medium. Plates were seeded with E. coli VT-9WT or E. coli VT-9RNase+ at 20° C. Some animals were left untreated and some recombinant mice had RNase inhibitor (40 U / ml) added. Data are shown in Table 98. [Table 98]

[0508] These data indicate that longevity and health can be regulated by altering RNase expression levels.

[0509] Pyrosequencing (454 platform; Roche) was used to identify genome-wide base substitution mutations in worms fed control and RNase-producing E. coli strains. We found that the average μbs of E. coli fed E. coli strain VT-9WT was 2.9x10e-9 per site per generation. The average μbs of E. coli fed E. coli strain VT-9RNase+ was 1.3x10e-8 per site per generation. However, E. coli strain VT-9RNase+ grown in medium supplemented with recombinant mouse RNase inhibitor had 9.7x10e-8 μbs per site per generation.

[0510] These data surprisingly show that the level of RNase production in an organism regulates the frequency of spontaneous mutations and genome stability, and that modulation of RNase activity can control these processes.

[0511] Example 98: Development of products and methods for erasure of autoimmune memory. Given that the role of NAMACS, NAMACS-ANA, and TEZR in memory formation was discovered here for the first time, we investigated the use of the various products to erase memory in immune cells.

[0512] Blood was collected from 10 HLA-A2 positive type 1 diabetes patients. Peripheral blood mononuclear cells were isolated using Ficoll density gradient centrifugation, and CD8+ T cells were isolated using StemCell Isolation Kit (STEMCELL Technologies). CD8+ T cells were left untreated or disrupted multiple times with DNase and RNase to a "zero state" as described above, and cultured in 96-well round-bottom plates at 3x10e3 cells / well in DMEM medium. 5x10e5 CD8+ T cells were then co-cultured with HLA-A*0201 transfected K562 cells (2.5e10x5 cells) that were untreated or treated with a set of islet antigen peptides (IA-2797-805ZnT8186-194, IGRP228-236, PPI2-10, PPI34-42) for 4 hours. Cytokine levels in the supernatants were then measured by Luminex. The results are shown in Figure 54.

[0513] The data obtained clearly demonstrate that the proposed products and methods can be used to modulate cytokine production, the FOXP3 pathway, and to erase cellular and immunological memory.

[0514] Example 99: Use of products and methods to modulate cell migration and metastasis The A549 cell line was grown as a monolayer in RPMI-1640 medium supplemented with 10% fetal calf serum (FCS) and L-glutamine (2 mM). The cell line was maintained in an incubator with a humidified atmosphere (37° C., 5% CO2 in air).

[0515] A549 cells were seeded (10,000 cells / well) in 24-well plates and allowed to attach to the surface under standard incubation conditions (RPMI-1640 medium supplemented with 10% fetal bovine serum and L-glutamine (2 mM), 5% CO2, 37°C) for 24 hours. Confluent cell monolayers were scratched in a straight line using a sterile plastic pipette tip. The detached cells were then carefully rinsed with RPMI-1640 medium to remove debris and floating cells. The medium was removed and the cells were treated with nuclease.

[0516] Fresh medium and cells were then added. The scratch zones were photographed every hour with a Zeiss Axiovert 40C (Carl Zeiss AG, Germany). The results are shown in Figure 55 and Table 99. [Table 99]

[0517] As can be seen, the use of the tested products, when used in cell therapy, significantly affected the actin cytoskeleton and increased cell migration, which is essential for many physiological processes, including wound repair, embryonic development, wound repair, tumor invasion, angiogenesis, and metastasis.

[0518] Example 100: Products and methods for modulating cellular sensitivity to opioids Subconfluent cultures of T98G human glioblastoma cells, which highly express opioid receptors, were harvested, washed twice with FBS-free DMEM, and resuspended in DMEM supplemented with FBS. Cells were treated with nuclease at a final concentration of 100 μg / ml for 30 minutes as described above. After removal of nuclease, cells were seeded in 96-well plates at a density of 4.0 log10 cells / well and exposed to freshly prepared tramadol (Sigma-Aldrich) at a concentration of 200 μM for 3 hours at 37° C. and 5% CO2.

[0519] The resulting T98G cells were plated in media supplemented with tramadol and their growth was compared to the growth of the same cells in media without tramadol. Tramadol treatment showed an inhibitory effect on cell adhesion of control cells but not on cells treated with nuclease (Figure 54a, b). These results suggest that cells after treatment with the test products are not sensitive to tramadol-induced inhibition of cell adhesion, suggesting that the use of products that disrupt or inactivate TezR may be used to monitor the function of protein receptors, including through the PI3K / AKT pathway and in response to opioid compounds (Xia et al. 2016). The data are shown in Figures 56 and 57.

[0520] Example 101: Products and methods for life extension. Autologous vaccines against genomic DNA of P. aeruginosa and / or vaccines from NAMACS and NAMACS-ANA or mixtures of microorganisms isolated from feces of mammals (mice) were obtained as described above. Mice (c57bl / 6, 6 mice / group) were treated with different regimens of these vaccines and their life span was monitored. Some mice were vaccinated at a young age (approximately 200 days) and some mice were vaccinated at an older age (approximately 400 days). Data are shown in Table 100 (rounded to 1). [Table 100]

[0521] The data clearly show that vaccines that contain bacterial DNA or bacterial TezR in their composition significantly extend life span.

[0522] Example 102: Products and methods for managing antibiotic resistance We investigated how treatment with the test products affected the susceptibility of microorganisms to antimicrobial agents. Zero-D, Zero-R, and Zero-DR cells were obtained as described above after treatment with test products ranging in concentration from 1 μg / ml to a maximum of 1 mg / ml for 30 seconds to 2 hours.

[0523] Antibiotic susceptibility was estimated by standard disk diffusion method using SIR (susceptible, intermediate, or resistant) according to the Clinical and Laboratory Standards Institute (CLSI) recommendations (Tables 101, 102). [Table 101] TIFF2024515541000121.tif50159

[0524] S-susceptible, I-intermediate, R-resistant [Table 102]

[0525] These data clearly demonstrate that the product is capable of altering bacterial susceptibility to antimicrobial agents and can render antibiotic resistant cells susceptible to antibiotics.

[0526] Example 103: Products and methods for managing genome rearrangements B. pumilus 1278 and C. albicans VT-9 were treated once or through multiple cycles with products that inactivate cell surface-bound nucleic acids as described above to generate zero cells. B. pumilus 1278 were then plated on Sabouraud Dextrose Broth (SDB) and Potato Dextrose Broth (PDB). These broths are commonly used for fungi but not for bacteria and are not "memorized" by bacteria. For bacteria to grow on these media, significant genome rearrangements must be achieved. (Table 103) [Table 103]

[0527] C. albicans VT-9 was then plated on Columbia Broth (CB) and Mueller-Hinton Broth (MHB). These broths are commonly used for bacteria, but not for fungi, and are not "memorized" by fungi. For C. albicans to grow on these media, significant genome rearrangements must be achieved. (Table 104) [Table 104]

[0528] These data clearly demonstrate that the products and regimes of their use tested can be used to control genome rearrangements in prokaryotes and eukaryotes.

[0529] Example 104: Products and methods for controlling cellular synthetic activity and aging. Primary human fibroblasts from mice were obtained as described previously and used at passage 5 (http: / / www.jove.com / video / 53565). Confluent dermal fibroblasts cultured in 24-well plates were maintained in standard DMEM supplemented with 0.1% fetal bovine serum, washed from the extracellular matrix and then treated once or several times with the test products at various concentrations ranging from 0.1 to 100 μg / mL as described previously, after which the tested products were washed off with nutrient medium. Collagen production was measured after pulsing the cells with 3 μCi / ml of [3H]proline, followed by the incorporation of [3H]proline into collagenous proteins. Three aliquots of 250 μL each of conditioned medium were mixed with 25 μg BSA containing 2 mM CaCl2, 1 mM phenylmethylsulfonyl fluoride, 4 mM N-ethylmaleimide, and 100 U / ml collagenase (or sterile water) and then incubated for 4 hours at 37° C. The remaining proteins were precipitated with 10% trichloroacetic acid for 45 minutes at +4° C., centrifuged, and the resulting pellet was washed again with 10% trichloroacetic acid and then solubilized in 0.3 N NaOH / 1% sodium dodecyl sulfate. Radioactivity in the resulting protein pellet was then measured and collagenase-resistant uptake subtracted from total uptake. The data (rounded to 5) are shown in Table 105. [Table 105]

[0530] The data obtained clearly demonstrate that the products and methods tested can be used to modulate cell synthetic activity, collagen production, aging, and joint repair.

[0531] These cells were then injected into mouse skin and collagen expression was analyzed. To this end, the hair on the backs of 8-week-old C57BL / 6 mice was shaved and approximately 10,000 cells were injected into the skin with a distance of 5 mm between injection sites. To examine the increase in collagen levels, the hydroxyproline content of the skin areas after injection of the different types of cells was quantified after 8 weeks. Hydroxyproline levels were elevated in all sites injected with treated cells compared to skin zones injected with control cells. The data are shown in Table 106. [Table 106]

[0532] The data obtained clearly show that the use of the tested products or the cells obtained after the use of the tested products can significantly modulate the characteristics of the macro-microbe, including its regeneration and rejuvenation, after transplantation into the macro-microbe.

[0533] Example 105: Products and methods for managing wound healing. The use of cells following treatment with the tested products in wound healing was investigated. Mouse fibroblasts were obtained as described above, washed from extracellular matrix and treated with the test products as described above at 50 μg / ml for 30 minutes, after which the test products were washed off with nutrient medium and applied to full thickness 1 cm diameter skin defects in 8 week old C57BL / 6 mice as described above. The data are shown in Table 107. [Table 107]

[0534] These data clearly indicate that the use of the tested products or cells after treatment with the tested products can be used for the treatment of various diseases including burns, ulcers and wounds.

[0535] Example 106: Products and methods for managing cellular memory To isolate cancer-associated fibroblasts (CAFs), 5x10e5 4T1 cells were injected into the mammary fat pad of BALB / c mice (8 weeks old, female). After 24 days of growth, the primary tumors were excised, then homogenized and digested with 1 mL of L-15 medium containing 0.25% trypsin and collagenase (2 mg / mL) and incubated for 60 min at 37°C using Red Blood Cell Lysis Buffer. Immune cells were excluded using superparamagnetic beads with rat anti-mouse CD45 and CD24 antibodies and affinity polyclonal sheep anti-rat IgG binding to the bead surface. Isolation of CAFs was performed by fluorescence-activated cell sorting using FITC+ / RFP- / DAPI- to exclude dead and cancer cells. The resulting CAFs were washed from the extracellular matrix and treated with the test product (one or more times) as described above, and after treatment, the test product was washed off with nutrient medium. The modified CAFs were then injected into the tumor site of 4T1-bearing tumor-bearing BALB / c mice (14 days after tumor cell implantation). Tumor volumes (rounded to the nearest 5) were measured on day 28 (Table 108). [Table 108]

[0536] These data clearly show that the use of the listed products or the transfer of cells obtained after treatment with the different regimens of the tested products can cause anti-cancer effects.

[0537] Example 107: Products and methods for erasing the memory of cancer cells PANC1 cells were maintained in the recommended growth medium containing 10% fetal bovine serum at 37°C, 5% CO2. The cells were then separated from the extracellular matrix and treated with test compounds, after which the test products were washed off with nutrient medium. Expression of KRAS was analyzed by subsequent RT-PCR using KRAS primers (FW5-CAGGAAGCAAGTAGTAATTGATGG-3; REV5-TTATGGCAAATACACAAAGAAAGC-3) and normalization to 18srRNA after isolating RNA as described above. Data are shown in Table 109. [Table 109]

[0538] The data obtained clearly show that the proposed methods and products can be used for reversing the pro-tumorigenic state of cells, for cellular reprogramming and for erasing oncology-related memory.

[0539] Example 108: Products and methods for the treatment of trauma and the regeneration or repair of nerve tissue and cells Eight-week-old NOD-SCID mice were anesthetized as described above, then a laminectomy was performed between vertebrae 10 and 9, and spinal cord injury (moderate injury) was induced using a special device at 70 kdyn. The wound was closed and the mice were treated daily with gentamicin (6 mg / kg) with daily bladder drainage. On day 6, several days after the spinal cord injury, the animals were anesthetized again and neuronal stem cells (pretreated with test compound and then washed out of the test product) were microinjected at 1x10e2 to 5x10e9 cells into the center of the spinal cord injury. Motor function was analyzed once a week using the Basso Mouse Scale (BMS) measurement system. The data are shown in Table 110. [Table 110]

[0540] The data obtained indicates that the modified cells may be used for the treatment of neural tissue and cell regrowth or repair, including that caused by trauma.

[0541] Example 109: Products and methods for managing cartilage regeneration Destabilization of the medial meniscus of the right knee was performed in fully anesthetized 16-week-old C57BL / 6 mice as previously described ( Christiansen et al., 2015 ) and treated daily with gentamicin (8 mg / kg) for 3 days.

[0542] Mesenchymal cells were treated with the products to be tested as described above and injected intra-articularly into the same knee of anesthetized animals on the 7th day after injury. Some animals received weekly intra-articular injections of 10 μl DNase (2000 Kunitz units / mg) and / or 10 μl RNase (100 units / mg). OARSI scores were assessed weekly. [Table 111]

[0543] The data obtained show that cartilage repair was significantly greater in animals treated with the experimental cells and therapeutics.

[0544] Example 111: Products and Methods for Managing Pain Primary keratinocytes were isolated from human foreskin as described above and cultured in serum-free medium supplemented with 4 ng / ml recombinant epidermal growth factor and 40 μg / ml bovine pituitary extract (all from Invitrogen Life Technologies). The cells were then washed from the extracellular matrix and then treated once or several times with the test products in the concentration range of 0.1 to 100 μg / mL as described above, after which the test products were washed off with nutrient medium. Expression of IL-1α was quantified by real-time RT-PCR using the following primers: (forward) 5'-ATCAGTACCTCACGGCTGCT-3', and (reverse) 5'-TGGGTATCTCAGGCATCTCC-3'. The data are shown in Table 112. [Table 112]

[0545] The data obtained show that the use of the test compounds reduces the expression of IL-α and IL-1α-NF-κB-CCL2 signaling pathways (Paish et al., 2018), which are known to induce monocyte activation and migration that contribute to pain-like sensitivity. Thus, the tested products can be used for pain management.

[0546] Example 112: Products and methods for auto-mesotherapy Forty patients with progressive alopecia (grade III-IV) participated in the study. Patients were excluded if they had any of the following known causes of alopecia (anemia, malignant tumors, malnutrition, connective tissue disease, treatment for oncological diseases, SARS-CoV-2 infection), use of medications affecting hair growth in the past 6 months, pregnancy, or psychiatric disorders. Patients were randomly assigned to different groups and treated with mesotherapy once a month for 6 months. PRP was prepared using a Plateletex Kit (DCare, Chicago, Illinois, USA). 10 ml of whole blood was collected from a vein in a tube covered with EDTA, transferred to a siliconized glass tube, and spun at 3500 rpm x 10 min. This step allowed the whole blood to separate into three layers. The blood was separated, and the two upper layers containing platelets and white blood cells were collected and centrifuged at 1500 rpm for 15 min. The lower platelet rich area was harvested and either treated with nuclease or not. The nuclease was either washed away with another set of centrifugation or left in the PRP suspension. The PRP suspension was injected using a Mesotherapy hypodermic needle 30gX4mm. The data is shown in Table 113. [Table 113]

[0547] The data obtained show that the use of the tested products significantly enhances the effectiveness of mesotherapy products.

[0548] Example 113: Products and methods for overcoming efflux system-dependent antibiotic resistance Achromobacter xylosoxidans, carrying multidrug resistance genes encoding efflux pumps (Bador et al., 2011, Berra et al., 2014, Adewoye et al., 2016, Isler et al., 2020), was treated with reverse transcriptase and integrase inhibitors to overcome bacterial resistance to fluoroquinolone macrolides, rifamycin, tetracycline, chloramphenicol, sulfanilamide, and trimethoprim. Nevirapine and etravirine were used at concentrations between 0.1 and 100 μg / mL as effectors of the intracellular part of the Tetz receptor system. The minimum inhibitory concentrations were evaluated according to CLSI guidelines. The data are presented in Tables 114 and 115. [Table 114] [Table 115] TIFF2024515541000136.tif103159

[0549] Surprisingly, the products tested increased the sensitivity of cells to chemotherapeutic agents.

[0550] Example 114: Products and methods for erasing cellular memory Primary cancer cells with confirmed EGFR expression were either washed with PBS and centrifuged at 200gx5 min to remove extracellular matrix or proceeded to the next treatment without removing extracellular matrix. All cells were then treated once or three times with nuclease 50μg / mL for 20 min (and then passaged in medium without fetal serum) and then grown in DMEM medium (Sigma) supplemented with 10% fetal bovine serum (Gibco) and 1% streptomycin (Sigma) at 37°C in a humidified atmosphere containing 5% CO2.

[0551] Between cycles of DNase use, some Zero-D, Zero-R, and Zero-DR0 cells were further treated with an integrase inhibitor (raltegravir, 2.5 μg / ml).

[0552] EGFR expression was assessed after 5 passages in regular medium without or after any additional treatment. Data are shown in Table 116. [Table 116]

[0553] Surprisingly, the data show that multiple cycles of treatment with the tested products result in the cells forgetting their pro-tumorigenic phenotype.

[0554] Example 115. Products and methods for modulating immune cell activity Neutrophils were isolated from EDTA-anticoagulated whole blood of two healthy volunteers by Ficoll density gradient centrifugation using Lymphoprep™ (Stemcell Technologies). After centrifugation at 750xg for 30 min, the lower cell fraction containing neutrophils was collected and the remaining red blood cells were lysed. Neutrophils were adjusted to 1x10e6 cells / ml in DMEM (serum-free). Cells were treated with test compounds as described above. NETosis was then induced by seeding purified neutrophils (5x10e5 cells / cm2) and stimulating with a mixture of bacterial LPS isolated from Pseudomonas aeruginosa and Escherichia coli for 3 h at 37°C. Neutrophils and NETs were then washed twice with PBS. Extracellular DNA in the supernatant was stained with 100 nM Sytox Orange and quantified by fluorimetry (530 / 640 nm). The data are shown in Table 117. [Table 117]

[0555] These data clearly demonstrate that the use of the tested compounds, including the formation of zero cells, can be used to inhibit neutrophil activation and the formation of neutrophil extracellular traps.

[0556] Example 116: Products and methods for altering cellular settings and cellular memory formation For the formation of cells with new memory, we either treated the cells with 3 rounds of RNaseA with or without DNase (50 μg / mL each, 30 min exposure time at 37°C) followed by a washout period with nutrient-free minimal medium (i.e., M9 medium without maltose) or put the cells into a "Y" state by treating them with 3 rounds of RNaseA with or without DNase (50 μg / mL each, 30 min exposure time at 37°C) followed by a washout period with normal nutrient-rich medium to form zero-state C. albiclas. Some cells in minimal medium were supplemented with unusual composition of nucleic acids (1 μg / mL DNA and 1 μg / mL RNA) isolated from human feces by QIAamp DNA Stool Mini Kit and QIAgen RNAmini kit. The lag phase (minimum contact time to trigger maltose utilization) of these cells was then measured, as well as the next generation of these cells, which were obtained by maintaining them in M9 medium containing maltose for an additional 24 hours to restore nucleic acids bound to the cell surface (Table 118). [Table 118]

[0557] These data indicate that the null state can be used to change cellular settings and reprogram cells. Putting cells into the "Y" state can increase the cells' response to the external environment.

[0558] Example 117: Products and methods for controlling UV resistance To determine whether the test products could be involved in UV resistance, S. aureus VT209 was treated with the test products. The control probe was left untreated. 8.5 log10 CFU / mL of bacteria in PBS was added to a 9 cm Petri dish and placed under a light holder with a new 254 nm UV light tube (TUV30W / G30T8; Philips, Amsterdam, The Netherlands) and irradiated for different times at a distance of 50 cm. After treatment, the bacteria were serially diluted and plated on nutrient agar plates, incubated for 24 hours, and CFU was measured. Notably, the use of the tested products protected the bacteria from UV-induced death, resulting in significantly higher viable counts compared to the control S. aureus after UV irradiation (p<0.05) (Figure 58).

[0559] The data obtained surprisingly show that the use of the tested products may provide significant protection against UV-induced damage and UV-induced cytotoxicity.

[0560] Example 118: Products and methods for targeted cell delivery, development of antibodies against NAMACS and / or NAMACS-ANA and / or TEZR. Maltose-naive and maltose-sensitive C. albicans were obtained as described above. Rabbits were vaccinated with NAMACS and NAMACS-ANA of maltose-sensitive C. albicans using Freund's adjuvant as described above. Maltose-naive C. albicans was then added at a concentration of 10e12 cells / ml to 10 ml of rabbit serum obtained after immunization, the probe was incubated at 37°C for 3-6 hours, and the fungus was removed by centrifugation at 4200 g / min. This procedure was repeated three times and the serum was purified from any remaining fungus by filtration through a 0.22 μm filter. As a result, the serum was depleted and only had antibodies against NAMACS and NAMACS-ANA and / or TEZER involved in the sensing of maltose by maltose-sensitive Candida.

[0561] Next, the growth inhibitory activity of the serum against maltose-sensitive and maltose-naive C. albicans was analyzed as previously discussed (Magliani et al., 1997). To this end, 3x10e2 cells of maltose-sensitive or maltose-naive C. albicans in 10 μl of PBS were incubated with 100 μl of serum for 22 h at 37° C. Inhibition was assessed by the number of CFU that resulted in growth after plating the yeast on potato dextrose agar (Sigma-Aldrich) and incubating for 48 h at 37° C. The data are shown in FIG. 59.

[0562] These data clearly show that the proposed method allows the selection of highly specific antibodies against NAMACS and NAMACS-ANA and / or TEZR.

[0563] Example 119: Sex control in fish. Arctic char eggs (7-10 eggs / mL) were treated with the products (DNase, RNase, Histone 5, Ribosomal Protein S40 at concentrations of 1-1000 μg / mL for 1 min-24 h) to bring the cells to the "cut" and "zero" states. After each treatment, the products were removed by washing with water. Artificial insemination was performed as previously described (Bellard et al., 1988). PCR for rapid sexing was then performed as previously described (Rud et al., 2015). The data are shown in Figure 60.

[0564] The data obtained shows that after treating fish eggs to Cut-R, Zero-R and Zero-DR conditions, there is a 15% increase in female fish.

[0565] Example 120: Effect of products on cell surface-associated electrophysiological dysfunction. To investigate the effect of the test products on the electrophysiological properties of cardiomyocytes, hiPSC-CMs obtained as described above were used and treated with 0.1-100 μg / ml of the test products for 30 min. Some probes were further treated with a nuclease inhibitor (recombinant RNase inhibitor). The data are shown in Table 119. [Table 119]

[0566] It is clearly confirmed that the use of the tested compounds can be used for the regulation of cardiac cell repolarization and arrhythmias.

[0567] Example 121: Product management of natural antibodies (NAbs) Blood from a 32-year-old male in group B was treated with DNase and RNase at concentrations of 0.1 μg / ml to 50 μg / ml for 1 min to 6 h. After treatment, the blood was examined using the ORTHO AutoVue Innova System. Treatment with DNase and RNase significantly reduced NAbs against B cells by 2-3 fold (Figure 61). References Chen C, Okayama H. ​​High-efficiency transformation of mammalian cells by plasmid DNA.Molecular and cellular biology.1987 Aug;7(8):2745-52. Naldini L, Blomer U, Gallay P, Ory D, Mulligan R, Gage FH, Verma IM, Trono D. In vivo gene delivery and stable transduction of nondividing cells by a lentiviral vector.Science.1996 Apr 12;272(5259):263-7. Spicer A, Molnar A. Gene editing of microalgae: scientific progress and regulatory challenges in Europe.Biology.2018 Mar;7(1):21. Tetz, G. and Tetz, V., 2021.Evaluation of a New Culture-Based AtbFinder Test-System Employing a Novel Nutrient Medium for the Selection of Optimal Antibiotics for Critically Ill Patients with Polymicrobial Infections within 4 h. Microorganisms, 9(5), p.990. Manukumar, H. M. & Umesha, S. MALDI-TOF-MS based identification and molecular characterization of food associated methicillin-resistant Staphylococcus aureus.Sci.Rep.7, (2017). Bennett, R. W. & Monday, S. R. S.aureus in International handbook of foodborne pathogens. in (ed. Miliotis MD, B. J.) 41-59 (2003). Kwon OK, Mekapogu M, Kim KS.Effect of salinity stress on photosynthesis and related physiological responses in carnation (Dianthus caryophyllus).Horticulture, Environment, and Biotechnology.2019 Dec;60(6):831-9. Zheng C, Sun YH, Ye XL, Chen HQ, Ji HB.Establishment and characterization of primary lung cancer cell lines from Chinese population.Acta Pharmacologica Sinica.2011 Mar;32(3):385-92. Loh CY, Chai JY, Tang TF, Wong WF, Sethi G, Shanmugam MK, Chong PP, Looi CY.The E-cadherin and N-cadherin switch in epithelial-to-mesenchymal transition: signaling, therapeutic implications, and challenges.Cells.2019 Oct;8(10):1118. Xia M, Tong JH, Ji NN, Duan ML, Tan YH, Xu JG.Tramadol regulates proliferation, migration and invasion via PTEN / PI3K / AKT signaling in lung adenocarcinoma cells.Eur Rev Med Pharmacol Sci.2016 Jun 1;20(12):2573-80. Christiansen BA, Guilak F, Lockwood KA, Olson SA, Pitsillides AA, Sandell LJ, Silva MJ, van der Meulen MC, Haudenschild DR.Non-invasive mouse models of post-traumatic osteoarthritis.Osteoarthritis and cartilage.2015 Oct 1;23(10):1627-38. Paish HL, Kalson NS, Smith GR, del Carpio Pons A, Baldock TE, Smith N, Swist-Szulik K, Weir DJ, Bardgett M, Deehan DJ, Mann DA.Fibroblasts promote inflammation and pain via IL-1α induction of the monocyte chemoattractant chemokine (CC Motif) ligand 2.The American journal of pathology.2018 Mar 1;188(3):696-714. Isler B, Kidd TJ, Stewart AG, Harris P, Paterson DL.Achromobacter infections and treatment options.Antimicrobial Agents and Chemotherapy.2020 Oct 20;64(11):e01025-20. Bador J, Amoureux L, Duez JM, Drabowicz A, Siebor E, Llanes C, Neuwirth C. First description of an RND-type multidrug efflux pump in Achromobacter xylosoxidans, AxyABM.Antimicrobial agents and chemotherapy.2011 Oct;55(10):4912-4. Adewoye L, Topp E, Li XZ.Antimicrobial drug efflux genes and pumps in bacteria of animal and environmental origin.InEfflux-mediated antimicrobial resistance in bacteria 2016 (pp. 561-593).Adis, Cham. Berra, S., Ayachi, S. and Ramotar, D., 2014.Upregulation of the Saccharomyces cerevisiae efflux pump Tpo1 rescues an Imp2 transcription factor-deficient mutant from bleomycin toxicity.Environmental and Molecular Mutagenesis, 55(6), pp.518-524. Magliani W, Conti S, Bernardis FD, Gerloni M, Bertolotti D, Mozzoni P, Cassone A, Polonelli L. Therapeutic potential of antiidiotypic single chain antibodies with yeast killer toxin activity.Nature biotechnology.1997 Feb;15(2):155-8. Bellard R. Artificial insemination and gamete management in fish.Marine & Freshwater Behavior & Phy.1988 Oct 1;14(1):3-21. Rud YP, Maistrenko MI, Buchatskii LP.[Sex identification of the rainbow trout Oncorhynchus mykiss by polymerase chain reaction].Ontogenez.2015 Mar-Apr;46(2):87-93..PMID: 26021121. * * *

[0568] The present invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description. Such modifications are intended to be included within the scope of the appended claims.

[0569] All patents, applications, publications, test methods, literature, and other materials cited herein are incorporated by reference in their entirety as if physically present herein.

Claims

1. White blood cells that have been treated with DNase and RNase after isolation from an individual, which white blood cells are capable of reducing the size of a tumor.

2. The white blood cells described in claim 1 administered to a patient having platelets treated with DNase and RNase.

3. The leukocytes described in claim 1, administered to a patient having hematopoietic stem cells treated with DNase and RNase.

4. The leukocytes described in claim 1 administered to a patient having fibroblasts treated with DNase and RNase.

5. The white blood cells of claim 1 administered to a patient having platelets treated with DNase and RNase and fibroblasts treated with DNase and RNase.

6. The leukocytes and platelets of claim 2, wherein the leukocytes and platelets are further treated with LPS.

7. The method of claim 1, wherein the white blood cells include leucocytes, lymphocytes (T cells, B cells, NK cells, neutrophils, eosinophils, monocytes, basophils, macrophages).

8. The leukocytes of claim 1, treated with DNase and RNase in two or more rounds.

9. A method for preparing one or more of the leukocytes, fibroblasts, platelets, and hematopoietic stem cells according to claim 1, comprising: (a) isolating one or more of white blood cells, fibroblasts, platelets, and hematopoietic stem cells, each of which is isolated from an individual; (b) treating one or more of white blood cells, fibroblasts, platelets, and hematopoietic stem cells with DNase and RNase; (c) washing the treated one or more of white blood cells, fibroblasts, platelets, and hematopoietic stem cells to remove the DNases and RNases of step (b); (d) administering one or more of said treated white blood cells, said treated fibroblasts, said treated platelets, and said treated hematopoietic stem cells into a patient to treat a solid tumor; A method comprising:

10. The leukocytes of claim 9 administered to a patient having platelets treated with DNase and RNase.

11. The leukocytes described in claim 9, administered to a patient having hematopoietic stem cells treated with DNase and RNase.

12. The leukocytes described in claim 9, administered to a patient having fibroblasts treated with DNase and RNase.

13. The leukocytes of claim 9 administered to a patient having platelets treated with DNase and RNase and fibroblasts treated with DNase and RNase.

14. The method of claim 9, wherein tumor size is reduced following administration of one or more of the treated white blood cells, the treated platelets, the treated fibroblasts, and the treated hematopoietic stem cells to the patient.

15. A method for producing a method of treating a subject, comprising: (a) isolating one or more of white blood cells, fibroblasts, platelets, and hematopoietic stem cells, each of which is obtained from an individual; (b) treating one or more of white blood cells, fibroblasts, platelets, and hematopoietic stem cells with an antibody that binds DNA and an antibody that binds RNA; (c) washing the treated one or more white blood cells, fibroblasts, platelets, and hematopoietic stem cells to remove the antibodies of step (b); (d) administering one or more of the treated leukocytes, treated fibroblasts, treated platelets, and treated hematopoietic stem cells into the patient to treat said solid tumor; 23. A method for preparing one or more of white blood cells, fibroblasts, platelets, and hematopoietic stem cells, comprising:

16. The method of claim 15, wherein one or more of the white blood cells, platelets, fibroblasts, and hematopoietic stem cells are treated with an antibody that binds DNA and an antibody that binds RNA, and then one or more of the white blood cells, cells, platelets, fibroblasts, and hematopoietic stem cells are treated with DNase and RNase prior to administration to the patient.

17. The method of claim 15, wherein one or more of the treated white blood cells, the treated platelets, the treated fibroblasts, and the treated hematopoietic stem cells reduce the size of a solid tumor following administration to the patient.

18. The method of claim 15, wherein one or more of the white blood cells, the platelets, the fibroblasts, and the hematopoietic stem cells are treated in two or more rounds with an antibody that binds DNA and an antibody that binds RNA.

19. The method of claim 15, wherein one or more of white blood cells, platelets, fibroblasts, and hematopoietic stem cells are treated with DNase and RNase in two or more rounds.

20. The method of claim 1, wherein one or more of the treated white blood cells, treated platelets, treated fibroblasts, and treated hematopoietic stem cells are administered to the patient by transdermal injection, enterally, parenterally, topically, subcutaneously, intramuscularly, intravenously, intraarterially, intraperitoneally, intradermally, intrathecally, intracerebral, intraventricular, intraparenchymal injection, intrastriatal, intraspinal, intraventricular, intracisternally, intraventricular, intraventricular, intracardiac, intraarticular, intraumbilical, amniocentesis, topically, rectally or vaginally, sublingually or buccally and by inhalation, and as eye and / or nasal drops.

21. A method for producing an improved plant, comprising treating plant cells with DNase and RNase.

22. The method of claim 21, wherein the treated plant cells have improved biological activity compared to plant cells not treated with DNase and RNase.

23. The improved biological activity is one of the following: accelerated / enhanced growth rate, increased proliferation, enhanced reproduction, increased productivity, increased germination rate, accelerated time to flowering, regulating flowering in plants (accelerating the time to flowering or delaying it), increasing or decreasing the duration of flowering, increased organ size, increased plant vigor, increased photosynthetic area, increased leaf number, increased flower number, increased root length, increased pod number, improved tillering, improved flowability and settling, increased flowering, increased plant height, increased cation content, 23. The method of claim 22, comprising one or more of: increased plant biomass, increased plant weight, increased fiber length, accelerated shoot growth, increased grain yield, accelerated germination and fruit set; increased and altered content of oil, starch, protein, nutrients, vitamins, fatty acids, amino acids, sugars in seeds, plants, flowers, roots, fruits; adjusting senescence, increasing plant numbers, ameliorating the negative effects of hypoxia, darkness, drought, flooding, low temperature, soil salinity, nutrient or mineral or nitrogen deficiency, improving stress tolerance to other negative biological, chemical and physical effects.

24. The method of claim 21, wherein the treated plant cells are used to grow plants on a farm.

25. The method of claim 21, wherein the treated plant cells are used to grow plants in outer space.

26. The method of claim 21, wherein the treated plant cells are from wheat, soybean, rice, sugarcane, potato, barley, corn, oats, rice, sorghum, tomato, banana, coffee, hybrid plants, new plant sugarcane, corn, cotton, grapes, bananas, cassava, beans, nuts, oil crops, agricultural and ornamental grasses, flowers, trees and shrubs.

27. A method for producing an improved plant, comprising treating plant cells with an antibody that binds DNA and an antibody that binds RNA.

28. The method of claim 27, wherein the treated plant cells have improved biological activity compared to plant cells that are not treated with an antibody that binds to DNA and an antibody that binds to RNA.

29. A method for producing an improved plant, comprising treating plant cells with cells that produce a nuclease.

30. The method of claim 29, wherein the nucleus inactivates one or both of DNA and RNA.

31. The method of claim 29, wherein the treated plant cells are used to grow plants on a farm.

32. The method of claim 29, wherein the treated plant cells are used to grow plants in outer space.

33. The method of claim 21, wherein the treated plant cells are from wheat, soybean, rice, sugarcane, potato, barley, corn, oats, rice, sorghum, tomato, banana, coffee, hybrid plants, new plant sugarcane, corn, cotton, grapes, bananas, cassava, beans, nuts, oil crops, agricultural and ornamental grasses, flowers, trees and shrubs.

34. The method of claim 21, wherein the treated plant cells are treated with DNase and RNase two or more times.

35. The method of claim 26, wherein the treated plant cells are treated with an antibody that binds to DNA and an antibody that binds to RNA more than once.

36. A plant cell which has been treated with the DNase and the RNase and has, after further treatment with the DNase and the RNase, improved growth, higher synthetic processes, lower nutritional requirements, increased germination, higher product yield, increased fruit weight, increased number of fruits, increased number of flowers, increased resistance to infections, increased growth under stressful environmental conditions, increased resistance to flooding, increased shoot length and root length, increased number of leaves, increased number of root nodules, increased number of pods, increased plant weight, increased shoot weight, increased root weight, increased number of leaves, increased leaf length, increased leaf width, increased number of buds, increased number of fruits per plant, increased fruit size, increased fruit weight, increased number of seeds, increased total seed weight, increased stem diameter, decreased days to flowering, decreased days to fruiting.

37. The method of claim 21, wherein the treated plant cells have the ability to grow under low light conditions when compared to plant cells that have not been treated with DNase or RNase.

38. The method of claim 21, wherein the treated plant cells include plant cells, plant cell cultures, plant tissues, organ seeds, grains, plant spores, cuttings, layering, and grafting material.

39. The plant cell of claim 21, wherein the treated plant cell is further treated with an antibody that binds to DNA, an antibody that binds to RNA, or a prokaryotic and / or eukaryotic cell that produces a nuclease.

40. The plant cell of claim 21, wherein the treated plant cell is capable of growing in soil having increased salinity compared to the soil in which the plant is normally grown.

41. (a) isolating one or more of a cell, a cell culture, an organoid, a tissue, an embryo, an organ, a unicellular organism, or a multicellular organism; (b) treating one or more of the cells, the cell cultures, the organoids, the tissues, the embryos, the organs, the single-cell organisms, the multicellular organisms with DNAse and RNase; (c) washing one or more of the treated cells, the treated cell culture, the treated organoid, the treated tissue, the treated embryo, the treated organ, the treated single cell organism, the treated multicellular organism after treatment with the DNase and RNase; (d) biomanufacturing a protein using one or more of the treated cells, the treated cell cultures, the treated organoids, the treated tissues, the treated embryos, the treated organs, the treated single cell organisms, and the treated multicellular organisms; The method of claim 1, further comprising:

42. The method of claim 41, wherein one or more of the treated cells, the treated cell cultures, the treated organoids, the treated tissues, the treated embryos, the treated organs, the treated unicellular organisms and the treated multicellular organisms are treated with two or more rounds of DNAse and RNAse.

43. 42. The method of claim 41, wherein the treated cells are CHO cells.

44. The method of claim 43, wherein treatment of the CHO cells with DNase and RNase increases the amount of protein produced by the CHO cells compared to CHO cells that are not treated with DNase and RNase.

45. The method of claim 41, wherein said treatment of the cells, cell cultures, organoids, tissues, embryos, organs, unicellular organisms, and multicellular organisms increases the amount of antibodies, cytokines, growth factors, viral vectors, antigens, vaccines, engineered antibody complexes, trivalent T cell engagers, checkpoint modulators, naive proteins, recombinant proteins, vitamins, hormones, vaccines, and antibody-cytokine fusions produced when compared to cells, cell cultures, organoids, tissues, embryos, organs, unicellular organisms, and multicellular organisms that are not treated with DNase and RNase.

46. ​​The method of claim 41, wherein the cell is a fungal cell.

47. The method of claim 43, wherein the treatment of the fungal cells with DNase and RNase increases the amount of protein produced by the CHO cells compared to fungal cells that are not treated with DNase and RNase.

48. The fungal cell of claim 47, wherein said treatment of the fungal cells increases the yield of antibodies, cytokines, growth factors, viral vectors, antigens, vaccines, engineered antibody conjugates, trivalent T cell engagers, checkpoint modulators, naive proteins, recombinant proteins, vitamins, hormones, vaccines, and antibody-cytokine fusions compared to fungal cells not treated with DNase and RNase.

49. The method of claim 41, wherein the cell is a human cell.

50. The method of claim 43, wherein treatment of the human cells with DNase and RNase increases the amount of protein produced by the human cells compared to CHO cells that are not treated with DNase and RNase.

51. (a) isolating one or more of a cell, a cell culture, an organoid, a tissue, an embryo, an organ, a unicellular organism, or a multicellular organism; (b) treating one or more of the cells, the cell cultures, the organoids, the tissues, the embryos, the organs, the single-cell organisms, and the multicellular organisms with an antibody that binds DNA and an antibody that binds RNA; (c) washing one or more of the treated cells, the treated cell cultures, the treated organoids, the treated tissues, the treated embryos, the treated organs, the treated single cell organisms, the treated multicellular organisms after treatment with the antibodies that bind DNA and the antibodies that bind RNA; (d) biomanufacturing a protein using one or more of the treated cells, the treated cell cultures, the treated organoids, the treated tissues, the treated embryos, the treated organs, the treated single cell organisms, and the treated multicellular organisms; The method of claim 1, further comprising:

52. The method of claim 51, wherein one or more of the treated cells, treated cell cultures, treated organoids, treated tissues, treated embryos, treated organs, treated unicellular organisms and treated multicellular organisms are treated with two or more rounds of antibodies against DNA and RNA.

53. (a) isolating one or more of a cell, a cell culture, an organoid, a tissue, an embryo, an organ, a unicellular organism, or a multicellular organism; (b) treating one or more of the cell, the cell culture, the organoid, the tissue, the embryo, the organ, the single-cell organism, and the multicellular organism with an organism that produces a nuclease; (c) washing one or more of the treated cells, the treated cell culture, the treated organoid, the treated tissue, the treated embryo, the treated organ, the treated single cell organism, the treated multicellular organism after treatment with the nuclease-producing organism; (d) biomanufacturing a protein using one or more of the treated cells, the treated cell cultures, the treated organoids, the treated tissues, the treated embryos, the treated organs, the treated single cell organisms, and the treated multicellular organisms; The method of claim 1, further comprising: (e) The method of claim 5, wherein the organism that produces the nuclease is a prokaryote or a eukaryote.

54. The method of claim 53, wherein one or more of the treated cells, treated cell cultures, treated organoids, treated tissues, treated embryos, treated organs, treated unicellular organisms, treated multicellular organisms are further treated prior to, during, or after treatment with a nuclease-producing organism by one or more of RNase, DNase, DNase-producing cells, or RNase-producing cells.

55. The method of claim 53, wherein the treated cells are CHO cells.

56. The method of claim 55, wherein treatment of the CHO cells with DNase and RNase increases the amount of protein produced by the CHO cells compared to CHO cells that are not treated with DNase and RNase.

57. The method of claim 53, wherein said treatment of the cells, cell cultures, organoids, tissues, embryos, organs, unicellular organisms, and multicellular organisms increases the amount of antibodies, cytokines, growth factors, viral vectors, antigens, vaccines, engineered antibody complexes, trivalent T cell engagers, checkpoint modulators, naive proteins, recombinant proteins, vitamins, hormones, vaccines, and antibody-cytokine fusions produced when compared to cells, cell cultures, organoids, tissues, embryos, organs, unicellular organisms, and multicellular organisms that are not treated with DNase and RNase.

58. The method of claim 53, wherein the cell is a fungal cell.

59. The method of claim 58, wherein the treatment of the fungal cells with DNase and RNase increases the amount of protein produced by the CHO cells compared to fungal cells not treated with DNase and RNase.

60. The fungal cell of claim 59, wherein said treatment of the fungal cells increases the yield of antibodies, cytokines, growth factors, viral vectors, antigens, vaccines, engineered antibody conjugates, trivalent T cell engagers, checkpoint modulators, naive proteins, recombinant proteins, vitamins, hormones, vaccines, and antibody-cytokine fusions compared to fungal cells not treated with DNase and RNase.

61. A growth medium supplemented with one or more antibiotics, wherein the antibiotics are administered at a concentration lower than the maximum concentration achievable in the tissue or organ from which the biological sample is derived. [0023] Claim 62: Pairwise comparisons of microbial growth in the same well for different time periods where the effectiveness of the antibiotic is assessed by monitoring the appearance and progression or absence of signs of microbial growth within a particular time period or within a combination of the following non-limiting examples of time periods listed below: 0-1 hr, 0-2 hr, 1-2 hr, 0-3 hr, 1-3 hr, 2-3 hr, 0-4 hr, 1-4 hr, 2-4 hr, 0-5 hr, 1-5 hr, 2-5 hr, 3-5 hr, 0-6 hr, 1-6 hr, 2-6 hr, 3-6 hr, 4-6 hr, 0-5 hr, 8 hours, 1 hour to 8 hours, 2 hours to 8 hours, 3 hours to 8 hours, 4 hours to 8 hours, 0 hours to 9 hours, 1 hour to 9 hours, 2 hours to 9 hours, 3 hours to 9 hours, 4 hours to 9 hours, 5 hours to 9 hours, 0 hours to 12 hours, 1 hour to 12 hours, 2 hours to 12 hours, 4 hours to 12 hours, 6 hours to 12 hours, 8 hours to 12 hours, 0 hours to 18 hours, 1 hour to 18 hours, 2 hours to 18 hours, 4 hours to 18 hours, 6 hours to 18 hours, 8 hours to 18 hours, 10 hours to 18 hours, 0 hours to 24 hours, 1 hour to 24 hours, 2 hours to 24 hours, 4 hours to 24 hours, 12 hours to 36 hours, 24 hours to 36 hours.

63. A growth medium for culturing fungi comprising one or more of the following: potato decoction 200 g / l, corn flour tincture 50 ml / l, oat flour decoction 350 ml / l, potato carrot decoction 300 ml / l, sucrose 30 g / l, cellobiose 20 g / l, yeast extract 4 g / l, maltose 40 g / l, NaNO3-2.0 g / l, K2HPO4-1.0 g / l, MgSO4-0.5 g / l, KCl-0.5 g / l, FeSO4-1 g / l, ZnSO4-0.1 g / l, MnCl2-0.1 g / l, and Twin 80-10 ml / lm.

64. A device for automatic and semi-automatic preparation of test systems containing various media and antibiotic compositions, characterized by its ability to prepare sets of media and antibiotics at various concentrations thereby preparing them immediately prior to use.

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