Methods for monitoring and treating disorders in the CNS

Abstract

The invention provides series of novel applications of two core methodologies, using Barrier Disrupting Fields (BDF) in conjunction with molecular testing of liquid biopsies in the same patient, and by these means inducing controlled and selective brain barrier opening that can serve various diagnostic and therapeutic purposes in conditions related to the CNS. To that end, the invention provides multiple peripheral biomarkers indicative of selective brain barrier opening, BBBo, BABo and / or BCSFBo, and opening level, and biomarkers enabling the detection and prognosis of various pathological conditions in the CNS, such as CNS tumors, neuropathological and neurodegenerative conditions, cerebrovascular insults and others. Additional therapeutic applications of the invention include monitoring of CNS drug response, controlled CNS drug delivery and clearance of various neurotoxic substances from the CNS.

Description

[0001] METHODS FOR MONITORING AND TREATING DISORDERS IN THE CNS

[0002] TECHNOLOGICAL FIELD

[0003] The invention belongs to the fields of diagnostics and therapeutic management of disorders in the central nervous system (CNS) using barrier disrupting fields (BDF) or low pulse electric fields (L-PEFs) technologies.

[0004] BACKGROUND

[0005] The central nervous system (CNS) is tightly sealed from the peripheral changeable milieu by the blood-brain barrier (BBB), blood-arachnoid barrier (BAB) and blood-cerebrospinal fluid (CSF) barrier (BCSFB), meant to maintain CNS homeostasis and protect neural tissue from pathogens and toxins. Although different by location and governing types of cells, these brain barriers share a general structure of elaborate network of interconnecting junctions, tight and adherens junctions (TJ and AJ) which are responsible fortheir characteristic filtering and paracellular transport obstructing capabilities. BBB is further characterized by the presence of perivascular glia and cerebral pericytes that interact with endothelial capillaries.

[0006] While these barriers are essential for health homeostasis, they impose significant challenges to both drug delivery and biomarker discovery in neurological and neuropsychiatric diseases. Efforts to detect CNS pathology through blood-based biomarkers - commonly named liquid biopsy - have gained momentum in recent years, mainly in the oncology field. However, under physiological conditions, the impermeability of brain barriers often limits the emergence of CNS-derived proteins, nucleic acids and other molecules in peripheral blood. Non-invasive methods for transient and safe opening of brain barriers such as MRI -guided focused ultrasound (MRgFUS) and barrier disrupting fields (BDF) were proposed as a possible solution to this problem by facilitating the release of brain-specific molecules into the circulation.

[0007] BDF, a method based on the application of external low-pulsed electric fields (L-PEFs), was previously described by the inventors as means to induce safe and reversible BBB opening (BBBo) and BAB opening (BABo) [1-3], In addition, BDF treatment was found to increase drug concentrations in the brain, without requiring imaging guidance or skull coupling but only by variation of the applied electrical parameters.

[0008] Notwithstanding, the existing methodology fell short of describing specific conditions for inducing a selective brain barrier opening, i.e., BBBo, BABo and / or BCSFBo, or ways of controlling brain opening duration and level.

[0009] Therefore, one problem to be solved by this invention is how to apply BDF in a controlled and selective manner to be able to monitor and adjust the extent of brain barrier opening and differentiate between different brain barrier openings, both for biomarker discovery and controlled CNS drug delivery. Peripheral CNS-derived biomarkers can be detected at certain levels in blood, saliva or other peripheral fluids, often in connection with CNS injury or disease. These include proteins like tau and S 100B inflammatory markers and subcellular structures like exosomes, which were related to conditions like Alzheimer's, multiple sclerosis (MS), amyotrophic lateral sclerosis (ALS), traumatic brain injury, (TBI), stroke and pre-eclampsia [4-6], Peripheral biomarkers can provide a minimally invasive way to diagnosis and monitoring of neurological and neuropsychiatric disorders, as well as individual’s response to treatment. However, a major challenge is the lack of standardized methods for isolating and quantifying these biomarkers that hinders their translation into routine clinical practice.

[0010] Therefore, another problem to be solved by this invention is how to control BDF in a way that facilitates not only influx of molecules to the brain (drug delivery) but also efflux of CNS-derived molecules into the peripheral system (liquid biopsies) and thereby enhance biomarkers’ detection and discovery.

[0011] In this connection, the brain is typically efficient at regulating and clearing endogenous neurotoxins, which are biproducts of normal brain function once they achieve excessive levels. Some known examples are glutamate, nitric oxide, dopamine and amyloid-beta and tau proteins. The brain is often less efficient in removing exogenous neurotoxins, especially under chronic exposure, such as ethanol (drinking alcohol), lead, mercury and arsenic.

[0012] Therefore, still another problem to be solved by the invention is how to induce efficient efflux of CNS-derived molecules to facilitate clearance of neurotoxic substances from the CNS.

[0013] The invention provides solution to all these problems in the form of a system, methods and uses of BDF in conjunction with liquid biopsies for inducing, controlling and monitoring selective and quantifiable brain barrier opening (BBBo, BABo and / or BCSFBo) to enable more efficient detection and monitoring of peripheral biomarkers of various pathological CNS conditions, and biomarkers of CNS drug response, and more efficient and controlled clearance of neurotoxins and neuro-damaging substances from the CNS, or more efficient and controlled drug delivery into the CNS.

[0014] Several previous disclosures described use of FUS in conjunction with localized brain liquid biopsy [7-8], Previous disclosures by the inventors described certain uses of L-PEFs to modify brain barrier permeability [9-10],

[0015] REFERENCES

[0016] 1. Sharabi S et al 2019. Transient blood-brain barrier disruption is induced by low pulsed electrical fields in vitro: an analysis of permeability and trans -endothelial electric resistivity. Drug Deliv 26: 459-469.

[0017] 2. Sharabi S et al 2021. Non-Invasive Low Pulsed Electrical Fields for Inducing BBB Disruption in Mice-Feasibility Demonstration. Pharmaceutics 13. doi: 10.3390 / pharmaceuticsl3020169. 3. Cooper I et al 2023. BBB opening by low pulsed electric fields, depicted by delayed-contrast MRI, enables efficient delivery of therapeutic doxorubicin doses into mice brains. Fluids Barriers CNS 20: 67. doi: 10.1186 / sl2987-023-00468-7.

[0018] 4. Kapural M et al 2002. Serum S-lOObeta as a possible marker of blood-brain barrier disruption. Brain Res 940(1-2): 102-4. doi: 10.1016 / s0006-8993(02)02586-6.

[0019] 5. Marchi N et al 2003. Serum transthyretin monomer as a possible marker of blood-to-CSF barrier disruption. J Neurosci 23(5): 1949-55. doi-org.rproxy.tau.ac.il / 10.1523 / JNEUROSCI.23-05- 01949.2003

[0020] 6. Tikhonova MA et al 2022. Neurospecific molecules measured in periphery in humans: How do they correlate with brain levels? A systematic review. Int J Mol Sci 23( 16):9193. doi: 10.3390 / ijms23169193.

[0021] 7. Pham -Pacia C et al 2023. Focused ultrasound-mediated liquid biopsy in a Tauopathy mouse model. Radiology 307. / / pubs.rsna.org / doi / 10.1148 / radiol.220869.

[0022] 8. US 11667975B2. Methods and systems for noninvasive and localized brain liquid biopsy using focused ultrasound.

[0023] 9. PCT / IE2019 / 050276. Method for changing brain barrier permeability.

[0024] 10. PCT / IE2024 / 050455. Method and system for increasing permeability of cranial bloodarachnoid barrier.

[0025] GENERAL DESCRIPTION

[0026] In the broadest terms, the invention provides means for a controlled and selective intervention into the brain barriers integrity, BBBo, BABo and / or BCSFBo, for the purpose of both diagnostic and therapeutic applications, with the idea to improve the detection and monitoring of diagnostic CNS- derived biomarkers, facilitate controlled CNS drug delivery, facilitate clearance of neurotoxic substances. The feasibility of achieving these aims was demonstrated in the present disclosure.

[0027] In practical terms, the invention provides an operative framework for inducing non-invasive, transient and selective BBBo, BABo and / or BCSFBo by applying BDF (L-PEFs) to patient’s brain under specified set of conditions, and further, monitoring the outcomes of this operation by parallel analysis of liquid biopsies (e.g., blood, serum, plasma, urine) extracted from the same patient. Apart from being safe, efficient and specific for various types of brain barrier opening, the system and methods of the invention provide an important tool for controlling and monitoring various conditions and interventions into the CNS by measuring CNS-derived biomarkers in liquid biopsies of the same patient who was subjected to selective brain barrier breach.

[0028] This framework and the choice of biomarkers open a wide range of potential applications for the system and methods of the invention. The primary biomarkers of interest are peripheral biomarkers of BBBo, BABo and / or BCSFBo to provide validation of selective brain barrier opening and for controlling and monitoring its duration and level. Additional biomarkers of interest are peripheral biomarkers of various CNS disorders or diseases to permit early diagnosis of specific diseases and follow-up of disease progression. These latter can further serve for monitoring drug response and establishing optimal therapeutic window for controlled drug delivery in the same patient. Still another application is for monitoring peripheral levels and facilitating clearance of various neurotoxins.

[0029] In summary, the invention provides safe and effective means for tracing, monitoring and quantifying a wide range of CNS-derived biomarkers in a specific patient and in real-time, as per specific patient’ s needs; this without significant or endangering interventions compared to the testing of the same markers in the CSF, for example.

[0030] More specifically, the inventors have previously designed an operating system and method for inducing reversible brain barrier breach by L-PEFs, PCT / IL2019 / 050276 (Ref 9) and PCT / IL2024 / 050455 (Ref 10), which are herein incorporated by reference. These tools, however, were still incomplete in terms of the ability to control and quantify the extent of brain barrier opening and to differentiate between various types of openings (BBBo, BABo and / or BCSFBo). Without the monitoring and controlling methods, they were further incomplete in terms of their potential clinical applicability to specific CNS conditions.

[0031] The present invention cured these deficiencies by providing a system and methods for using BDF in a controlled and selective manner to enable the detection and monitoring of a variety of CNS- derived substances in liquid biopsies, and further provided proof concept for this approach by showing that controlled and selective BDF application has led to marked increase in the peripheral levels of CNS- derived proteins across various experimental models.

[0032] Specifically, the inventors presently show that selective opening of BAB only could be achieved by modifying certain BDF parameters, such as voltage intensity for example. In a rat animal model, they could show that the exposure of rats to lower BDF intensities (e .g . <400V applied to the active electrode) has led to preferential BABo by EB staining of rats’ parenchyma (Evans blue), while higher BDF intensities (e.g., 700V) have led to increased BBBo+BABo. Importantly, monitoring the concentrations of Nivolumab antibody (N-Ab, anti-cancer drug) the rats’ CSF in the same experiment has shown that the N-Ab release to the CSF has started under lower BDF intensities (0-200V), suggesting potential involvement of BCSFBo under these conditions. EXAMPLE 1

[0033] Both BAB and BCSFB are similar in that they regulate trafficking of substances between the blood and the CSF, thereby maintaining the unique composition of ions, nutrients and other substances in the CSF and brain tissue. As both BAB and BCSFB present challenges for effective CNS drug delivery, the ability to induce selective BABo and / or BCSFBo is particularly relevant in situations affecting the structures lining of CSF spaces, such as leptomeningeal spread for example.

[0034] The inventors further showed that the experimental groups subjected to selective brain barrier opening were essentially district by qualitative and quantitative parameters of proteomic profiles in peripheral blood. For example, the BABo group (BDF at 400 V) was enriched in proteins related to pathways of cytoplasmic translation, cellular organization, and ubiquitination, while the BBBo+BABo group (BDF at 700 V) was enriched in lipids and membrane -associated proteins, which is consistent with the brain’s lipid-rich composition. Both groups demonstrated enrichment of specific micro-vesicle (MVs) associated proteins after BDF, e.g., Synaptogyrin-1, Gamma-synuclein, S100A16, Claudin-3, and EpCAM. EXAMPLE 2

[0035] More broadly, these findings highlight the differential regulation of CNS compartments and reinforce the utility of BDF for effective and selective barrier modulation. In addition, they support the notion that barrier modulation facilitates MVs efflux from the brain, thus making brain-derived MVs and MVs-derived proteins promising candidate biomarkers for testing in liquid biopsies. More importantly, they demonstrated the potential of BDF as a non -invasive tool for exposing CNS-specific proteins that are otherwise obscured from the detection in the circulation by functioning brain barriers.

[0036] The inventors further validated this approach by exemplifying specific CNS-derived proteins and substances, e.g., sphingomyelin-derived stearic acid and Osteopontin (OPN) protein, and the effect of selective brain barrier opening on their levels in the peripheral blood. Sphingomyelin, and stearic acid as its primary fatty acid constituent, is a critical component of the neuronal myelin and a putative marker of neuronal damage. OPN is a putative biomarker of AD and AD progression, and more recently of traumatic brain injury (TBI). The two makers were tested in rats (sphingomyelin-derived stearic acid) and an analogous mouse model (OPN) after exposure to BDF-mediated BABo, both showing significantly elevated presence in plasma under these conditions. EXAMPLE 3

[0037] Taken together, these findings show that a controlled and reversible BDF application in conjunction with quantitative analysis of biomarkers in liquid biopsies provide a meaningful and non- invasive tool for diagnosing and monitoring specific neuronal conditions, with specific examples of the OPN as an indicator of AD and TBI and sphingomyelin-derived stearic acid of neuronal damage.

[0038] More broadly, these findings demonstrate the applicability of BDF-mediated controlled brain barrier opening and molecular testing of liquid biopsies as a powerful diagnostic and therapeutic tool, in this latter when this scheme is applied together with therapeutic interventions into the CNS (e.g., CNS acting drugs, radiation or brain surgery) or for facilitating clearance of protein and non-protein neurotoxins from the CNS.

[0039] The inventors further showed, using BDF-mediated BABo and proteomic analysis of plasma in an aged mice model, that this scheme is meaningful and conclusive for the detection of proteins and protein pathways indicative of age-related differences and neurodegeneration. Both young and older mice exhibited increases in CNS-related plasma proteins after BDF, with older mice showing a broader and more amplified proteomic shift and high fold changes of PEA- 15, cofilin-2, and ATOX1 proteins as hallmarks of CNS aging and neurodegeneration. On protein pathways level, the older group exhibited enrichment of proteins related to Parkinson’s disease, proteasome activity, prion disease, ALS and tight junctions’ functionality, among others. EXAMPLE 4 In summary, these studies support the concept of using a controlled BDF for increasing the emergence of CNS-derived proteins in peripheral systems (liquid biopsies), with its vast number of derivative applications. Further studies are currently conducted to explore the full scope of clinical applications of this concept in the context of CNS.

[0040] These findings should be further appreciated in view of the known challenges with the detection of brain-derived biomarkers in peripheral systems. Despite recent advances in the identification of such markers, various factors such as their filtering by brain barriers, non-specific release, inter- and intraindividual differences in clearance rate and others make their clinical applicability very limited. As was now demonstrated, controlled BDF application in conjunction with quantitative analysis of CNS-derived biomarkers in liquid biopsies provides detectable levels of known and new biomarkers to enable a more reliable diagnosis, monitoring and treatment of various CNS conditions. Moreover, in the long run, the proposed approach holds the promise of mapping the source of these CNS-derived biomarkers as per their precise location and BBBo, BABo and / or BCSFBo, and related CNS condition.

[0041] The proposed approach is further advantageous in the context of drug delivery. Many drugs, and anticancer drugs in particular, are either blocked or filtered out by brain barriers. The ability to induce controlled BBBo, BABo and / or BCSFBo by BDF can provide a solution to this problem in a targeted and spatially restricted manner. Furthermore, parallel testing, monitoring and quantifying the levels of relevant CNS-derived biomarkers in liquid biopsies can further provide fine tuning and monitoring of drug response during and post treatment as means for optimization of therapeutic time / range windows when the drug is likely to be most effective.

[0042] From practical point of view, liquid biopsies can be obtained from body fluids (e.g., blood, plasma, serum, urine) and tested either directly for peripheral CNS biomarkers of choice or after enrichment by extraction from MVs. Some non-limiting examples of candidate biomarkers can include proteins (e.g., TAU, amyloids, OPN, S 100 proteins, y synuclein), lipids (e.g., stearic acid), various types of DNA (e.g., circulating or mitochondrial DNA) or RNA (e.g., circulating RNA, miRNA), glycoproteins, carbohydrates, minerals, amino acids, neurotransmitters and hormones. Liquid biopsies can be analyzed at various times upon BDF application for monitoring disease progression or establishing therapeutic time / range window for optimization of therapeutic regimens.

[0043] The invention further supports numerous clinical applications, depending on the type of brain barrier opening and the biomarkers of choice. One of the most immediate applications is for early diagnosis and monitoring of various neurologic, cerebrovascular, neuro-developmental, degenerative and psychiatric conditions, CNS infections and injuries, and primary and secondary CNS tumors. Specific examples are neuroinflammatory and neurodegenerative disorders such as MS and AD, brain tumors, stroke, TBI, cerebral ischemia, hypertension, amyloid angiopathies, epilepsy and encephalitis. Another direct application is for monitoring and facilitating clearance of various endogenous or exogenous neurotoxins from the CNS, and eventually from the body as a whole. Still another application is to provide controlled delivery of CNS acting drugs (e.g., neurologic, psychiatric, anticancer, antiviral, vascular and other drugs) , or more broadly, controlled pharmacological or technological CNS acting treatments (e.g., radiation, drug therapy, thermal therapy, brain stimulation, photodynamic therapy (PDT), electroconvulsive therapy (ECT), an ablation, and others).

[0044] In summary, the invention holds promise for early detection and disease monitoring, especially in pre -symptomatic population where pathology-associated proteins are present long before the appearance of clinical symptoms. Reproducibility, safety and non-invasive nature of BDF make it suitable for repeated sampling and longitudinal applications. Beyond diagnostics, the invention can serve as a theragnostic platform, enabling both targeted delivery of drugs and clearance of neurotoxic substances. Its capacity to transiently and selectively open CNS barriers, particularly in conditions like neurodegenerative diseases wherein clearance mechanisms are often compromised, can be exploited to deliver therapeutics directly into the brain while also promoting the removal of toxic proteins such as amyloid-P and other toxins, facilitating their efflux into the bloodstream may reduce neurotoxicity. Additionally, the invention can support real-time therapeutic monitoring and treatment effect via circulating biomarkers. Finally, as has been presently demonstrated, the invention provides access not only to soluble plasma proteins but also to extracellular MVs’ proteins, thus expanding the range of candidate CNS-derived biomarkers that can be tested in liquid biopsies.

[0045] In summary, the invention provides an effective, safe and versatile tool for selective transient modulation of brain barriers in conjunction with more efficient and broad detection of CNS-derived biomarkers in peripheral samples. Specificity and reproducibility of this approach position it as a promising platform for advancing personalized medicine in the management of neurological neuropsychiatric diseases. Additional work should focus on validation of this approach in clinically valid disease models, optimization of electrical parameters for clinical application, and further exploration of its theragnostic potential.

[0046] BRIEF DECSRIPTION OF DRAWINGS

[0047] To better understand the subject matter, certain embodiments of the invention are now described by way of examples with reference to the following figures.

[0048] Figs 1A-1C illustrate the use of BDF for inducing selective brain barrier opening. Rats were injected intravascularely with 2% Evans blue (EB) and exposed to BDF (L-PEFs) under two voltage intensities (400V and 700V), followed by transcardiac perfusion with saline and brain removal. At BDF 400V, visible staining of the meninges (inset) was observed but not of the brain parenchyma (1 A), which is indicative of BABo only. At BDF 700V, there was staining of the brain parenchyma (arrows) and the meninges (IB), which is indicative of BBBo+BABo. These findings were supported by quantification of the parenchymal staining (1C), with significant differences between groups (0V (Sham), 400V (BABo) and 700V (BABo+BBBo) by OneWay ANOVA with Tukey’s multiple comparison test, *p<0.01, **p<0.007). Figs 2A-2B illustrate the same feature in analogous experiment using BDF under increasing voltage intensities (0-800V in 100V increments), EB brain staining and Nivolumab antibody (N-Ab, anti -cancer drug) administered IV before BDF (mean ± SD, n=2-3 rats per treatment). Figure shows accumulation of EB staining in the brain parenchyma after exposure to BDF at >400V (2A) and a parallel accumulation of N-Ab in the CSF under lower BDF at 200V (3A), which is indicative of potential contribution of BCSFBo.

[0049] Fig. 3 illustrates the applicability of sphingomyelin -derived stearic acid as a meaningful CNS- derived biomarker in liquid biopsies, using rats exposed to BDF at 400V (BABo) and HPLC / GC analysis of rats’ plasma. Figure shows significant increase in the levels of sphingomyelin-derived stearic acid in rats’ plasma after low intensity BDF (p=0.02), suggesting it as a meaningful peripheral marker of BABo and / or BCSFBo and opening level.

[0050] Fig. 4 illustrates the applicability of osteopontin (OPN) protein for the same purpose, using low intensity BDF (200V) in an aging mouse model and quantification by ELISA. Figure shows significant increase in the levels of OPN in mice’s plasma after BDF (p=0.005), suggesting it as a peripheral marker of BABo and / or BCSFBo and further a marker of neurodegeneration and aging CNS.

[0051] Figs 5A-5D illustrate the applicability of molecular testing in liquid biopsies for identification and quantification of CNS-derived biomarkers, using proteomic analysis in an aging mouse model exposed to low intensity BDF (BABo). Figure shows principal component analysis (PCA) of young (5A), old (5B) mice and both groups (5C), with clear distinction between proteome profiles before and after BDF. Venn diagram shows significantly changed proteins (proteins with q<0.05) (5D) and shared proteins in the two groups.

[0052] Figs 6A-6B illustrate the distinction between young and old proteome profiles by protein pathway analysis in the same model. Figure shows the most significant pathways retrieved by Enrichr analysis (KEGG 2021) considering the most elevated proteins (q< 0.05) in plasma of young (6A) and old (6B) mice, all indicative of the prevalence neurodegeneration and CNS aging processes in older mice. Overall, the study supports the conclusiveness of the proposed approach for identification of new peripheral biomarkers of various CNS conditions.

[0053] Fig. 7 illustrates certain embodiments of the system and methods of the invention and the scope of their direct applications in the clinical setting.

[0054] DETAILED DESCRIPTION OF EMBODIMENTS

[0055] The invention can be articulated in terms of systems and methods for the diagnosis and treatment of disorders and conditions in the CNS. The gist of the invention is in providing an authentic and reliable tool for inducing selective and controlled brain barrier opening, effectively evaluating the induction of such opening and opening level in situ, and further in implementing this tool in a general theragnostic scheme for diagnosis and treatment of many CNS conditions. In many embodiments the invention can be broadly articulated as a synergistic approach combining the use of BDF technology (L-PEFs) in combination with molecular or biochemical testing of liquid biopsies in the same individual.

[0056] The terms "Barrier Disrupting Fields (BDF)" or "Dow Pulsed Electric Fields (L-PEFs)” refer herein to low-energy electric or electromagnetic methods allowing to induce brain barrier breach in a controlled, safe and reversible manner. Examples of such methods are provided in PCT / IL2019 / 050276 (Ref 9) and PCT / IL2024 / 050455 (Ref 10) by the inventors, which are herein incorporated by reference. One of the characteristic features of such pulsed electric field (PEFs) methods is that they operate at field intensities below the threshold for cell electroporation (permanent membrane damage), while other PEFs methods operating under higher intensities can perforate cell membranes, inactivate cells and cause cell death.

[0057] The term "liquid biopsy” refers herein to any sample of body fluids, including blood, plasma, serum, urine and CSF.

[0058] In the most basic terms, the invention can be articulated as a liquid biopsy for use in a method of diagnosing, monitoring and / or treating CNS conditions, the distinctive feature of which is measuring levels of one or more CNS-derived biomarkers in one or more liquid biopsies obtained from a subject who is or was subjected to a Barrier Disrupting Fields (BDF) under conditions optimized for inducing a selective brain barrier opening.

[0059] In some embodiments, BDF can be applied under selective conditions to induce blood -arachnoid barrier opening (BABo) or CSF barrier opening (BCSFBo).

[0060] As BABo and BCSFBo are related, in some embodiments, BDF can be applied under selective conditions to induce BABo and BCSFBo

[0061] In some embodiments, the BDF can be applied under selective conditions to induce blood-brain barrier opening (BBBo).

[0062] In some embodiments, BDF can be applied under selective conditions to induce BBBo and BABo and / or BCSFBo.

[0063] Previous studies by inventors have shown that a selective brain barrier opening can be induced by modifying certain BDF (L-PEFs) parameters.

[0064] In some embodiments, BDF can be induced by one or more electric pulse sources.

[0065] In some embodiments, it can be induced by one or more alternating electromagnetic sources.

[0066] In some embodiments, it can be induced by both one or more electric pulse sources and alternating electromagnetic sources.

[0067] In some embodiments, it can be induced by a plurality of single or multiple grounded or energized electrodes connected to the same or multiple electric sources. In some embodiments, it can be induced by units of electrodes that are not pairs, for example by one ground and 2 energized electrodes, one negative and one positive electrode, two grounds and one energized electrode.

[0068] In some embodiments, it can be induced by plurality of electrodes with at least one pair of electrodes delivering similar or different pulse frequency and / or strength.

[0069] In some embodiments, the plurality of electrodes can comprise between 2 to 1200 pairs or at least one of 2-50, 50-100, 100-200, 200-300, 300-400, 400-500, 500-600, 600-700, 700-800, 800-900, 900-1000, 1000-1100, 1100-1200 pairs of electrodes.

[0070] In some embodiments, the electrodes can be surface electrodes, screw electrodes, microelectrodes, implantable electrodes and coil electrodes.

[0071] In some embodiments, the electrodes can have a diameter in the range between about 5 pm to about 10 cm, or in at least one of the following ranges 5-10, 10-20, 20-30, 30-40, 40-50, 50-60, 60-70, 70-80, 80-90, 90-100, 100-200, 200-300, 300-400, 400-500, 500-600, 600-700, 700-800, 700-800, 800- 900, 900-1000 pm or 1 mm, or 1-10, 10-50, 50-100, 100-200, 200-300, 300-400, 400-500, 500-600, 600-700, 700-800, 700-800, 800-900, 900-1000 mm or 1 cm, or 1-2, 2-3, 3-4, 4-5, 5-6, 6-7, 7-8, 8-9 or 9-10 cm.

[0072] In some embodiments, BDF can be induced by at least one of the following conditions: a) in some embodiments, the applied electric current can be in the range between about 1 mA and about 1 A, or in at least one of the following ranges 1-50, 50-100, 100-150, 150-200, 200-250, 250-300, 300-350, 350-400, 400-450, 450-500, 500-550, 550-600, 600-650, 650-700, 700-750, 750-800, 800- 850, 850-900, 900-950 or 950-1000 mA or 1 A, and / or b) in some embodiments, the applied voltage can be in the range between about 10V and about 1000 V, or in at least one of the following ranges 10-50, 50-100, 100-150, 150-200, 200-250, 250-300, 300-350, 350-400, 400-450, 450-500, 500-550, 550-600, 600-650, 650-700, 700-750, 750-800, 800- 850, 850-900, 900-950 or 950-1000 V, and / or c) in some embodiments, the applied pulse duration can be in the range between about 5 ns and about 10 ms, or in at least one of the following ranges 5-10, 10-15, 15-20, 20-25, 25-30, 30-35, 35-40, 40-45, 45-50, 50-55 or 55-60 ns, or 5-10, 10-15, 15-20, 20-25, 25-30, 30-35, 35-40, 40-45, 45-50, SO- 55 or 55-60 ps, or 1-2, 2-3, 3-4, 4-5, 5-6, 6-7, 7-8, 8-9, and 9-10 ms, and / or d) in some embodiments, the applied pulse frequency can be in the range between about 0.5 Hz and about 100 KHz, in at least one of the following ranges 0.5-1, 1-5, 5-10, 10-15, 15-20, 20-25, 25-30, 30-35, 35-40, 40-45, 45-50, 50-55, 55-60, 60-65, 65-70, 70-75, 75-80, 80-85, 85-90, 90-95, 95-100, 100-200, 200-300, 300-400, 400-500, 500-600, 600-700, 700-800, 800-900, 900-1000 Hz, or 1 KHz, or 1-10, 10-220, 20-30, 30-40, 40-50, 50-60, 60-70 70-80, 80-90, or 90-100 KHz, and / or e) in some embodiments, the applied number of pulses can be in the range between about 1 to about 1000, or in at least one of the following ranges 1-10, 10-20, 20-30, 30-40, 40-50, 50-60, 60-70, 70-80, 80-90, 90-100, 100-200, 200-300, 300-400, 400-500, 500-600, 600-700, 700-800, 800-900 or 900-1000 pulses, and / or f) in some embodiments, the applied pulse shape that can be square, triangular, sinus or exponential.

[0073] In some embodiments, BDF can be applied under specific conditions permitting to avoid electroporation.

[0074] To that end, in some embodiments, the BDF conditions can be optimized to include L-PEFs frequency of less than about 75Hz, or a frequency of less than about 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, 5, or 1 Hz, or less than 1, 0.75, 0.5, 0.25, or 0.1 Hz, or a frequency in the range of about 0.5 to about 75 Hz, or in at least one of the following ranges 0.5-1, 1-5, 5-10, 10-15, 15-20, 20-25, 25-30, 30-35, 35-40, 40-45, 45-50, 50-55, 55-60, 60-65, 65-70, 70-75, 75- 80, 80-85, 85-90, 90-95, or 95-100 Hz.

[0075] In the same way, in some embodiments, the BDF conditions can be optimized to include L- PEFs field strength of less than 148 V / cm, or a field strength of less than about 160, 159, 158, 157, 156, 155, 154, 153, 152, 151, 150, 149, 148, 147, 146, 145 V / cm, or less than about 140, 135, 130, 125, 120, 115, 110, 105, 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, 5 or less than 1 V / cm, or a field strength in the range of about 1 to about 150 V / cm, or in at least one of the following ranges 1-10, 10-20, 20-30, 30-40, 40-50, 50-60, 60-70, 70-80, 80-90, 90-100, 100-110, 110-120, 120- 130, 130-140, 140-150 or 150-160 V / cm.

[0076] In some embodiments, BDF can be applied or provided to the subject by a wearable device combining some or all the afore -mentioned structural features, as illustrated in Fig. 7.

[0077] In some embodiments, one or more liquid biopsies can be extracted or obtained from the same patient, such as before and after the application of BDF, or only after the application of BDF, or at different time points upon repeated applications of BDF.

[0078] The liquid biopsies essentially serve the purpose of measuring or quantifying peripheral levels of one or more CNS-derived biomarkers.

[0079] Therefore, in some embodiments, the analysis of liquid biopsies can further involve comparing the levels of biomarkers measured in liquid biopsies obtained from the patient at different points of time, e.g., before and after BDF, or upon repeated BDF applications.

[0080] In some embodiments, the analysis of liquid biopsies can further involve comparing the levels of biomarkers measured in the patient’s liquid biopsies to the levels of the same biomarkers in liquid biopsies obtained from healthy individuals, i.e., those who are unaffected or undiagnosed with a specific CNS condition or disorder.

[0081] The terms "biomarke r". “CNS-derived biomarker" or “peripheral biomarker" encompass herein any biological molecule which can be tested, monitored or quantitated in liquid biopsies and is indicative of a normal or abnormal process, a disease or condition, or drug response to a disease or condition. These terms encompass herein proteins, lipids, DNA and RNA molecules, and derivatives thereof.

[0082] In some embodiments, the biomarker of choice and / or its peripheral levels can be indicative a selective brain barrier opening (BBBo, BABo and / or BCSFBo) and / or opening duration and level, such as S1OOP Ca2+-binding protein, sphingomyelin’s stearic acid, OPN protein and others.

[0083] In some embodiments, the biomarker of choice and its peripheral levels can be indicative of the presence, severity or progression of a pathological CNS condition or disorder such as Amyloid , OPN protein, and others.

[0084] In some embodiments, the elected biomarker can be human Osteopontin (OPN) protein.

[0085] In some embodiments, the elected biomarker can be sphingomyelin-derived stearic acid.

[0086] In some embodiments, the elected biomarker can be selected from sphingomyelin-derived stearic acid, a human Osteopontin (OPN, e.g., Acc. Num. PI045 I), a human SIOOp protein (e.g., Acc. Num. P04271), a human Gelectin (e.g., Acc. Num. P09382, P05162 and P09382 for Gelectins 1, 2 and 3, respectively) a human Annexin (e.g., Acc. Num. P04083 for Annexin Al or NP_001144.1 or P08758 for A4 and A5, respectively), a human Cathepsin (e.g., Acc. Num. P07858 for Cathepsin B and P07711 for Cathepsin L), a human Syntaxin (e.g., Acc. Num. Q16623 for Syntaxin 1A), a human Claudin (e.g., Acc. Num. 095832 for Claudin-1, NP_00I002026 for Claudin-18 and HGNC:8514 for Claudin-11), a human Catenin (e.g., Acc. Num. P35222 for the common Catenin isoform), a Reticulon (e.g., Acc. Num. Q16799 for Reticulon 1 and 095197 for Reticulon 3), ahuman Synaptogyrin (e.g., Acc. Num. 043759), a human Prostaglandin isomerase or reductase (e.g., Acc. Num. P41222 and Q14914, respectively), a human Amyloid P (Ap, e.g., Acc. Num. P05067) or a human Tau protein (e.g., Acc. Num. P10636).

[0087] In some embodiments the elected biomarker can be a human Amyloid P (AP) or Tau proteins.

[0088] In some embodiments, the elected biomarker can be a prognostic CNS tumor biomarker selected from selected from a human 0-6 methylguanine-DNA Methyltransferase (e.g., Acc. Num. P26358 for DNMT1, Q9Y6K1 for DNMT3A, NM_006469.2 for DNMT1 and DNMT3B and related DNMT proteins), a human Epidermal Growth Factor Receptor (EGFR, e.g., Acc Num. P00533), a human Isocitrate Dehydrogenase (e.g., Acc. Num. 075874), ahuman Glial Fibrillary Acidic Protein (e.g., Acc. Num. P14136), a human Telomerase Reverse Transcriptase (TERT, e.g., Acc. Num. 014746), a human Tumor Protein 53 (TP53, e.g., Acc. Num. P04637).

[0089] In some embodiments, the elected biomarker can be selected from a human Synaptogyrin- 1 (SYNGR1, e.g., Acc. Num. 043759), a human Gamma-synuclein (e.g., Acc. Num. 076070), a human S100A16 (e.g., Acc. Num. Q96FQ6), a human Claudin-3 (e.g., Acc. Num. 015551.1) and a human EpCAM 3 (e.g., Acc. Num. P16422).

[0090] In some embodiments, the elected biomarker can be selected from circulating free DNA, circulating cell-free microRNAs, circulating extracellular vesicles, circulating proteins, circulating tumor cells. In some embodiments, the elected biomarker can be a prognostic marker of CNS aging and neurodegeneration, such as a human Amyloid P (A , e.g., Acc. Num. P05067), a human Tau protein (e.g., Acc. Num. P10636), a human PEA-15 3 (e.g., Acc. Num. Q15121), a human Cofilin-2 (e.g., Acc. Num. Q9Y281), or a human AT0X1 (e.g., Acc. Num. 000244).

[0091] In some embodiments, the elected biomarker can be an MVs-derived protein or substance.

[0092] In some embodiments, the elected biomarker can be selected from DNAs, (circulating or mitochondrial DNA), RNAs (circulating RNA or miRNA), glycoproteins, carbohydrates, minerals, amino acids, metabolites, neurotransmitters, amino acids, lipids and hormones.

[0093] Altogether, in some embodiments, the elected biomarkers can be human orthologs of the biomarkers listed in Tables 1-4 or Supplementary Tables 5-8 of this disclosure.

[0094] In some embodiments, these and additional biomarkers and their peripheral levels can be further indicative of response to CNS acting drugs, or more broadly, to response to pharmacological or technological CNS acting treatment, administered to the same patient in conjunction with BDF.

[0095] In some embodiments, these pharmacological or technological CNS acting treatment include radiation, thermal, brain stimulation, drug or genetic therapy, photodynamic therapy (PDT), electroconvulsive therapy (ECT), an ablation, or a combination thereof.

[0096] In some embodiments, the pharmacological CNS acting treatment or the CNS acting drug can be selected anticancer, antiviral, anti-inflammatory, neurologic, psychiatric, vascular or genetic drugs.

[0097] In some embodiments, the elected biomarker can be an exogenous or endogenous neurotoxin, released from the CNS by BDF and monitored by direct testing in liquid biopsies. Examples of exogenous neurotoxins include, but are not limited to, lead, ethanol, glutamate, nitric oxide, botulinum toxin (Botox), tetanus toxin, and tetrodotoxin. Endogenous neurotoxins can the neurotransmitters such as nitric oxide and glutamate, Amyloid P (AP) and Tau protein.

[0098] The choices of biomarkers and the choices of BDF conditions for inducing selective brain barrier opening are directly relevant for the main applications of the invention.

[0099] In some embodiments, the molecular testing of liquid biopsies can serve for validating selective brain barrier opening, opening type and level. In such cases, the elected and quantified biomarkers with selected from those related to BBBo, BABo and / or BCSFBo and opening duration and level.

[0100] In some embodiments, the molecular testing of liquid biopsies can serve for diagnosing and monitoring the progression of specific CNS disorders or conditions. In such cases, the elected and quantified biomarkers will be selected from those related to a specific CNS disorder or disease and disease progression and / or severity.

[0101] The terms “conditions or disorders in the CNS” or “CNS conditions or disorders'" encompass herein any type of clinical and subclinical condition involving the CNS, the brain and the spinal cord, including a primary disease or condition wherein the CNS is the primary source of illness and a secondary disease wherein the CNS is a sequela or complication of a disease starting in another part of the body. In clinical terms, the invention is directed to a wide range of conditions and disorders in the CNS, including neurologic, neurodevelopmental, neurodegenerative, cerebrovascular, psychiatric disorders, infections and traumatic injuries in the CNS, benign and malignant CNS tumors and secondary CNS tumors.

[0102] In some embodiments, the molecular testing of liquid biopsies can serve for diagnosing and monitoring the progression of CNS disorders or diseases selected from neurologic, neurodevelopmental, neurodegenerative, cerebrovascular, psychiatric disorders or diseases, infections or traumatic injury in the CNS, and benign or malignant tumors in the CNS, primary or secondary.

[0103] In some embodiments, the CNS disorder or disease can be selected from stroke, a brain tumor, a traumatic brain injury, cerebral ischemia, hypertension, an amyloid angiopathy, Alzheimer's disease (AD), Parkinson's disease (PD), Huntington disease (HD), Amyotrophic lateral sclerosis (ALS), multiple sclerosis (MS), a muscular dystrophy, essential tremor, schizophrenia, major depression, meningitis, epilepsy and encephalitis.

[0104] In some embodiments, the molecular testing of liquid biopsies can serve for monitoring and facilitating clearance of CNS from neurotoxins or neuro -damaging substances. In such cases, the elected and quantified biomarkers would be those related neurotoxicity.

[0105] The terms “neurotoxicity” and “neurotoxin” encompass herein any physical, chemical, and biological agent directly or indirectly responsible for structural and functional changes in the CNS. Examples of such agents were referred to above.

[0106] In summary the advantages of the invention are 4-fold:

[0107] 1. in allowing to induce a selective brain barrier opening, BBBo, BABo and / or BCSFB, in a controlled, safe and reversible manner,

[0108] 2. in allowing to detect and monitor abnormalities related to various CNS conditions and disorders,

[0109] 3. in allowing to monitor the effect of CNS acting therapies, and

[0110] 4. in facilitating the elimination and clearance of neurotoxins from the CNS, all these in real-time and without damaging interventions.

[0111] The invention can further be articulated in a similar way in terms of methods, systems and uses.

[0112] The methods of the invention can be generally articulated as methods for diagnosing, monitoring and / or treating at least one condition in the CNS, with the main steps of: i. obtaining at least one liquid biopsy from a subject who is or was subjected to a BDF under conditions optimized for inducing a selective brain barrier opening, and ii. measuring levels of at least one CNS -derived biomarker in said at least one liquid biopsy of said subject, and optionally, iii. repeating steps (i) and (ii) in the same subject, i.e., obtaining liquid biopsies from the same subject before and after BDF, or upon repeated BDF exposures. In some embodiments, the methods of the invention can be articulated as: i. administering a BDF to the subject under conditions optimized for inducing a selective brain barrier opening, BBBo, BABo and / or BCSFBo, ii. measuring levels of at least one CNS-derived biomarker in at least one liquid biopsy obtained from the subject before and after BDF, and iii. comparing the level of said at least one biomarker in said liquid biopsies, and optionally iv. repeating steps (i) to (iii) in the same subject at various time points of disease progression or treatment.

[0113] The specifics of the BDF applications and the markers of choice, as well as the potential applications of the methods of the invention have been previously discussed.

[0114] The systems of the invention can be articulated as a system comprising a plurality of single or multiple grounded or energized electrodes configured and operable to provide a BDF to a subject in need of being diagnosed, monitored and / or treated for at least one condition in the CNS, wherein the BDF is provided under conditions optimized for inducing selective brain barrier opening, BBBo, BABo and / or BCSFBo, and wherein the subject has provided at least one liquid biopsy for measuring levels of at least one CNS-derived biomarker.

[0115] In some embodiments, the uses of the invention can be articulated as systems and methods for diagnosing and monitoring the progression of disorders or diseases in the CNS.

[0116] In some embodiments, the uses of the invention can be articulated as systems and methods for monitoring response to a pharmacological or technological CNS acting treatments.

[0117] In some embodiments, the uses of the invention can be articulated as systems and methods for inducing or facilitating neurotoxicity clearance.

[0118] The terms “about” or “approximately” are meant to encompass herein a deviation of up to ±10% from the specified values or ranges of values, and more specifically up to ±1%, ±2%, ±3%, ±4%, ±5%, ±6%, ±7%, ±8%, ±9% or ± 10% deviation therefrom.

[0119] EXAMPLES

[0120] The figures and examples provided herein serve only illustrative purposes and are not intended to be limiting in terms of scope. As the final scope of the claimed matter will be determined by the scope of the claims.

[0121] EXAMPLE 1 : BDF-mediated induction of selective brain barrier opening

[0122] This study explored the potential of BDF (L-PEFs) to induce selective brain barrier opening, BBBo, BABo and / or BCSFBo. Previous studies exploring the effect of different BDF parameters (e.g., electric current, voltage, pulse duration and frequency, number pulses, pulse shape, etc., and avoidance of electroporation) on EB brain staining suggested that voltage intensity can be a determining factor to induce selective brain barrier opening. Two independent in vivo experiments were conducted for selective induction of BABo vs BBBo+BABo by BDF in rats. In both, naive male Sprague Dawley rats (250-300 g) were treated using three different protocols: i. 100 pulses at 700 V, 50 psec pulse duration, frequency of 4 Hz, n=3, ii. 100 pulses at 400 V, 50 psec pulse duration, frequency of 4 Hz, n=3, and iii. Sham (n=2). Evans Blue dye (EB, 0.4ml, 2%.

[0123] After filtration with 0.2 pm membrane) was injected to the tail vein 1 min post treatment and brain extraction performed after transcranial perfusion with saline. EB concentration in the brain was calculated against a calibration curve, using a fluorescence plate reader at ex / em of 620 / 680 nm.

[0124] Materials and Methods

[0125] BDF Procedure

[0126] Animals were anesthetized using a mixture of ketamine and xylazine during the procedure. BDF were delivered using an electroporator (BTX 830; Harvard Apparatus, Holliston, MA) connected to two custom designed stainless steel round electrodes with 11 mm diameter. After shaving the head using a shaving cream, electrodes were covered with conductive gel (Abralyt Hi CL, EASYCAP GmbH, Germany) and were pressed against the sides of the head. The electrodes with conductive gel were pressed against the skull sides.

[0127] Evans blue extravasation into brain and meninges

[0128] Evans blue (EB) was prepared as a 2% stock solution. 200 mg EB powder was dissolved in 10ml saline. 400 pl of the 2% EB solution was injected intravascularly per rat. For quantification, EB was extracted from the tissue as follows: Brain samples were placed (~50 mg) in a 50% Trichloroacetic Acid (TCA) solution, at a 1:3 ratio (W:V). Next, they were homogenized for 5 min at max speed using a cooled (4°C) metal -bead homogenizer (Bullet Blender BBx24). Following homogenization, the TCA extracts from the tissue were centrifuged at 10,000 g for 20 min, in 4 °C, to remove precipitates and tissue debris. Finally, 150 pl of supernatants from each sample was added to a 96-well plate. An EB calibration curve was added to quantify the amount of EB in each sample. The plate was read, using a plate reader, at a fluorescence ex / em of 620 / 680 nm. Data analysis for EB concentrations was performed with One-Way ANOVA with Tukey’s multiple comparison test using GraphPad Prism v 10.0.

[0129] Results and Conclusions

[0130] In the first experiment, after exposure to BDF at 400V, brain meninges were stained blue but no visible staining was observed in the brain parenchyma (Fig. 1A). After exposure to BDF at 700V, a visible staining of both meninges and parenchyma was observed (Fig. IB). Quantification of EB concentrations in brain parenchyma in 0V (Sham), 400V (BABo) and 700V (BBBo+BABo) groups yielded 1.3+0.7, 1.2+0.4 and 19. 1+6.2 ng / mg, respectively, with significant differences between groups (F(2,5)=18.36, p<0.005, post hoc Tuckey test 0V-400V; p=0.99, 0V-700V; p<0.0I, 400V-700V; p<0.007). (Fig. 1C). These findings were essentially reproduced in the second experiment, further measuring the concentrations of Nivolumab antibody (N-Ab, anti -cancer drug) administered IV before BDF exposure (n=2-3 per treatment). After BDF application, an increasing accumulation of EB stain in the brain parenchyma (BBBo+BABo) was observed with BDF >400V (Fig. 2A) and a parallel accumulation of N-Ab in the CSF under these conditions (Fig. 2B). Importantly, the release of N-Ab to the CSF has started at much lower BDF intensities (0-200V), suggesting potential involvement of BCSFBo under these conditions.

[0131] Both studies demonstrated the potential of inducing selective brain barrier opening by modification of specific BDF parameters, with the examples of inducing BABo or BCSFBo under lower voltage intensities and BBBo+BABo / BCSFBo under higher voltage intensities. Selective opening of BAB and / or BCSFB is particularly relevant for drug delivery in situations affecting the structures lining of CSF spaces, such as leptomeningeal spread.

[0132] EXAMPLE 2: Identification of peripheral biomarkers of brain barrier opening

[0133] The same experimental framework was used to study the effect of selective brain barrier opening on plasma or blood proteome profiles, and more broadly, to permit identification of peripheral biomarkers differentiating between different types of opening and opening level.

[0134] To that end, Sprague Dawley rats (250-300 g) were exposed to the two validated BDF (L-PEFs) protocols (for detailed BDF parameters see EXAMPLE 1): i. inducing BABo by exposure to BDF at 400V (n=5), ii. inducing BABo+BBBo by BDF at 700V (n=4), respectively,

[0135] Blood was collected one week before BDF treatment via retro-orbital bleed and 15-40 min after treatment via cardiac puncture.

[0136] Materials and Methods

[0137] Isolation of plasma microvesicles in rats

[0138] Microvesicles (MVs) isolation was performed using previously described methods (e.g., Harel M et al 2015 in Mol Cell Proteomics 14: 1127-1136). Briefly, 250 pl plasma samples were collected and stored at -80 °C. Samples were thawed on ice, centrifuged at 1000 x g for 20 min to remove cell debris, followed by MVs precipitation by centrifugation at 20,000 x g for 60 min at 4 °C. Pellets were then washed twice with 1 ml ice-cold PBS and centrifuged again at 20,000 x g for 60 min at 4 °C.

[0139] Proteomics study

[0140] For in-solution digestion and liquid chromatography-mass spectrometry (LC-MS / MS) analysis, MVs pellets were solubilized in 6M urea / 2M Thiourea in 100 mM Tris-HCl (pH 8.5), reduced with ImM dithiothreitol (DTT) at RT for 30 min and alkylated with 5mM iodoacetamide (IAA) for 30 min in the dark. The lysates were diluted 4-fold with 50mM ammonium bicarbonate, followed by overnight digestion with Trypsin / Lys-C mix (MS grade Promega, 1: 100 enzyme to protein ratio) and sequencing grade modified trypsin (Promega, 1:50 enzyme to protein ratio). Resulting peptides were acidified with trifluoroacetic acid (TFA), purified on Cis StageTips (Pierce) and vacuum dried. The dried peptides were resuspended in 2% acetonitrile / 0. 1% TFA before the LC-MS / MS analysis.

[0141] For mass-spectrometry protein analysis, purified peptides were separated using the EASYnLC1200 nano-HPLC on EasySpray columns (Pep Map 50 cm, 0.75 pm i.d) packed with 2 pm, Cis material with lOOA pore size using a water-acetonitrile gradient and were injected to the Q-Exactive HF mass spectrometer (Thermo Scientific) via the EasySpray ionization source. The peptides were loaded with Buffer A (0.1% formic acid) and eluted with a gradient of 5-30% Buffer B (80% acetonitrile / 0.1% formic acid), at a flow rate of 300 nl / min, over a gradient of 100 min.

[0142] Mass spectrometric acquisition: MS acquisition was performed in a data-independent (DIA) manner at positive-ion mode. The DIA method consisted of a survey scan at 60,000 resolution from 400 to 1000 m / z, with automatic gain control target of 3E6 and 100 ms injection time. Then, 25 DIA windows, spanning 400-975 m / z, were acquired at 30,000 resolution with automatic gain control target of 1E6 and 50 ms injection time. Normalized collision energy was set to 27.

[0143] Mass spectrometric raw data analysis: Raw MS files were processed using DIA -NN 1.8.1 in high-precision mode with global cross-run normalization enabled. The output was at 1% precursor and protein group levels. Protein quantification was performed using the MaxLFQ algorithm as implemented in the diann R package. Database search was performed using the Rat Uniprot database in a library -free mode.

[0144] Identified proteins were filtered based on valid values to retain only proteins present in all samples in at least one of the experimental groups (pre- or post- BDF). After log2 transformation the missing values were imputed with values that form a normal distribution with a width of 0.3 and downshift of 1.8 standard deviations of the overall protein intensity distribution. A two-sample Student’s t test was performed comparing pre- and post BDF samples (FDR<0.05, S0=0. 1).

[0145] Results and Conclusions

[0146] In the BABo group (BDF at 400 V), 4,064 proteins were identified in plasma-derived MVs, and 3,045 proteins after filtering for at least five valid values in at least one group. In the BBBo+BABo group (BDF at 700 V), 3,235 proteins were identified, and 2,382 after filtering for at least four valid values in at least one group. LC-MS analysis for differentially changed proteins, identified 983 proteins in the BBBo+BABo group and 1032 proteins in the BABo group (FDR q value < 0.05, SO = 0.1, no restriction to fold change), the majority of which were up-regulated in both groups.

[0147] Table 1 shows a selection of CNS-derived proteins that were up-regulated in both BBBo+BABo and BABo groups, with shaded proteins enriched after BABo vs. non-shaded proteins enriched after BBBo+BABo. Table 1. CNS-derived proteins elevated in plasma after selective BDF-induced brain barrier opening.

[0148] BABo group BBBo+BABo group

[0149] Protein fold increase1fold increase1CNS relevance

[0150] (p-value) (p-value)

[0151] Prostaglandin Expressed in brain tissue and linked reductase 1 to brain prostaglandin metabolism.

[0152] Expressed in various CNS cell types, e.g., cerebellar inhibitory neurons

[0153] Annexin A8 13.4 (0.01) 44.4 (0.0003) and choroid plexus epithelial cells.

[0154] Other Annexins were related to the maintenance of BBB integrity.

[0155] Used as a biomarker for cancer-

[0156] ErpCAM T 7.4 / i z (n0.m03A) u 31.s8 / (n0.n0i0m3) associ .a, ted E .Vs in , . the ., CSF, a . nd . mcore

[0157] ' ' ' recently explored in the context ot neurodegenerative disorders.

[0158] One of the four brain-enriched

[0159] Synaptogyrin-1 7. 1 (0 05) 29 2 (0 0004) s nnp.i c vesicle proteins along with synaptogyrm 3, synaptophysin and synaptoporin..

[0160] Highly and selectively expressed in the choroid plexus epithelial cells,

[0161] Claudin-3 4.3 (0.04) 19.3 (0.0004) with minimal expression in brain parenchyma microvessels, suggesting a specific role in BCSFB.

[0162] 1Fold increase calculated from proteins intensities in post BDF samples / pre-BDF samples.

[0163] Significant differences were observed between plasma proteomic profiles in BABo and BBBo+BABo groups, before and after BDF exposure, supporting the applicability of BDF for the induction of a selective brain barrier opening, and further for the identification of peripheral biomarkers indicative of the opening per se and of other CNS conditions.

[0164] In line with the above, principal component analysis (PCA) showed clear distinction between proteins profiles before and after BDF by both protocols. Hierarchical clustering of significantly changing proteins divided them into two clusters: (1) including 881 and 950 proteins up-regulated by BDF in the two treatment groups (BBBo+BABo and BABo), respectively, and (2) including 102 and 82 proteins down-regulated by BDF in the two groups, respectively.

[0165] Subsequent gene ontology (gene pathway) analysis was performed on the subset of proteins (n=100) most elevated before and after BDF in the two treatment groups, using Enrichr method (Chen EY et al 2013 in BMC Bioinformatics; 14: 128; Kuleshov MV et al 2016 in Nucleic Acids Res 44:W90- 7). The results are shown in Tables 2-3.

[0166] Table 2. Gene ontology analysis of proteins elevated in plasma of BBBo+BABo rats.

[0167] Term p-value q-value1

[0168] Endoplasmic Reticulum Tubular Network Organization 1.18097 le-08 0.000004

[0169] (G0:0071786)

[0170] Membrane Lipid Biosynthetic Process 1.360361e-08 0.000004

[0171] (G0:0046467)

[0172] Sphingolipid Metabolic Process 2.296427e-08 0.000005

[0173] (G0:0006665)

[0174] Sphingolipid Biosynthetic Process 1.140599e-07 0.000017

[0175] (G0:0030148)

[0176] Monoacylglycerol Metabolic Process 4.063219e-07 0.000049

[0177] (G0:0046462)

[0178] Monoacylglycerol Catabolic Process 2.399297e-06 0.000239

[0179] (G0:0052651)

[0180] Organonitrogen Compound Biosynthetic Process 2.793729e-06 0.000239

[0181] (GO: 1901566)

[0182] Unsaturated Fatty Acid Metabolic Process 4.524523e-06 0.000322

[0183] (G0:0033559)

[0184] Long-Chain fatty-acyl-CoA Metabolic Process 4.842629e-06 0.000322

[0185] (G0:0035336)

[0186] Endoplasmic Reticulum Organization 7.356942e-06 0.000441

[0187] (G0:0007029)

[0188] Shown are most significant ontologies terms in GO Biological Process 2023 analysis.

[0189] 1q-value is an adjusted p-value calculated using Benjamini-Hochberg correction method for multiple hypotheses testing.

[0190] Table 3. Gene ontology of proteins elevated in plasma of BABo rstas.

[0191] Term p-value q-value1

[0192] Cytoplasmic Translation 2.109607e-08 0.000027

[0193] (G0:0002181) Positive Regulation of Cellular Component Organization 9.766629e-08 0.000063

[0194] (G0:0051130)

[0195] Positive Regulation Of Protein-Containing Complex Assembly 6.024138e-07 0.000257

[0196] (G0:0031334)

[0197] Regulation Of Viral Entry into Host Cell 2.628256e-06 0.000841

[0198] (G0:0046596)

[0199] Peptide Biosynthetic Process 1.454193e-05 0.003723

[0200] (G0:0043043)

[0201] Gene Expression 1.767264e-05 0.003770

[0202] (G0:0010467)

[0203] Translation 2.29818 le-05 0.004202

[0204] (G0:0006412)

[0205] Regulation of Protein-Containing Complex Assembly 2.954030e-05 0.004726

[0206] (G0:0043254)

[0207] Macromolecule Biosynthetic Process 3.742646e-05 0.005323

[0208] (G0:0009059)

[0209] Positive Regulation of Protein Ubiquitination 6.379648e-05 0.008166

[0210] (G0:0031398)

[0211] Shown are most significant ontologies terms in GO Biological Process 2023 analysis.

[0212] 1q-value is an adjusted p-value calculated using Benjamini-Hochberg correction method for multiple hypotheses testing.

[0213] Gene ontology data revealed that under the selective induction of BBBo+BABo, the most prominent proteins in plasma were CNS-derived proteins related to lipid metabolism, in line with the notion of a brain being the second most lipid enriched body tissue. In contrast, the selective induction of BBBo revealed a more heterogeneous picture, with top scores for proteins related to translation and cellular organization processes.

[0214] Overall, the results supported the applicability of the BDF method as a tool for inducing selective brain barrier opening, as well as the prospect of using this tool for identification of peripheral biomarkers indicative, in real-time, of a selective brain barrier opening, opening type, duration and level. Candidate biomarkers are listed in Tables 1-3, either by protein name or reference to gene ontologies, additional markers are listed in Supplemental Tables 5-6.

[0215] In a broader sense, the results further suggest that the same approach, using controlled and selective induction of brain barrier opening by BDF in conjunction with molecular testing of liquid biopsies, can be applied for diagnosis and monitoring of many conditions related to CNS function and disease, such as early diagnosis of CNS disorders, monitoring of CNS drug response, or removal of CNS-derived neurotoxin and other neuro -damaging substances. EXAMPLE 3: Testing for specific CNS-derived biomarkers in liquid biopsies

[0216] Two candidate biomarkers were tested in vivo in animal models exposed to low intensity BDF (BABo and / or BCSFBo).

[0217] The first elected candidate biomarker was sphingomyelin-derived stearic acid - a breakdown product of sphingomyelin and a putative non-protein biomarker of CNS conditions involving myelin and lipid metabolism disturbances. It was tested in plasma of rats exposed to BDF (L-PEFs) at 400 V to rats (for detailed BDF parameters see EXAMPLE 1).

[0218] The second candidate biomarker was Osteopontin (OPN) protein, elected based on previous studies reporting its overproduction in brain microglia in connection with histo-pathological phenotypes of Alzheimer’s (AD) in the brain of animal models and humans (Qiu et al 2023 in PNAS 120(6) e2218915120). This marker was tested in an aging mouse model exposed to BDF at 100 pulses, 4Hz, 200V (equivalent to 400 V in rats).

[0219] Materials and Methods

[0220] Analysis of the lipid biomarker

[0221] Samples of 300 pl plasma were obtained from rats before and after BDF exposure.

[0222] For sphingomyelin separation by TLC (thin layer chromatography): 300pl plasma was processed to extract total lipids after the addition of 5 pg heptadecanoic acid as internal standard. The lipids extracted with organic solvents (Chloroform: Methanol, 2: 1, v: v) were applied to Silica gel-G plates which were immersed in a solvent mixture containing Chloroform: Methanol: Acetic acid: DDW (80:20: 1:3, v:v:v:v). Following the lipid separation, the silica spots containing SM, identified according to a standard applied in the same plate, were scrapped and processed for esterification and analysis of the fatty acids by GC-FID.

[0223] For fatty acid identification by GC-FID (gas chromatography with flame ignition detection): after addition of 1 ml reagent B3F, the tubes containing SM in the scraped silica, were gassed with nitrogen, closed tightly and heated at 85°C for 45 min with occasional shaking. After cooling, 1 ml of hexane was added, the tubes’ content was mixed and, after a short centrifugation, the hexane layer containing methyl esters fatty acids was transferred into a new tube. Before GC-FID analysis, the hexane extracts were concentrated by evaporation under nitrogen. One-twentieth of the final suspension was applied in 1 pl hexane into the chromatograph. The fatty acid profiles were compared to known mixtures of animal fatty acids, PUFA2 (Supelco, USA) for identification. Stearic acid in the samples was compared between samples and quantified based on the included heptadecanoic acid as internal standard.

[0224] Analysis of the OPN biomarker

[0225] Blood samples were collected from aged mice (11 months old) before and 30 min after BDF (at 100 pulses, 4Hz, 200V). Plasma was prepared and diluted 1: 1000. OPN quantification was performed with ELISA. Statistical analysis used paired t-test (1 tail, since we expected elevation, p =0.005 and r=0.99 for effectiveness of pairing).

[0226] Results and Conclusions

[0227] The testing for sphingomyelin-derived stearic acid concentrations in rats’ plasma before and after exposure to low intensity BDF revealed significant increase in the stearic acid levels after BDF (Fig. 3), Analogous increase (by 25%) was detected for the OPN biomarker after exposure of mice to the same conditions (Fig. 4). Both findings suggest that testing for levels of sphingomyelin-derived stearic acid or OPN markers in liquid biopsies can provide a meaningful and informative tool for the detection and quantification of BABo and / or BCSFBo.

[0228] More broadly, as OPN is also a putative biomarker of AD and AD progression, and more recently of traumatic brain injury (TBI), these findings suggest that BDF-mediated controlled and selective brain barrier opening and testing for OPN in liquid biopsies can be used in the early diagnosis and monitoring of AD, and by extension, testing for additional peripheral biomarkers for diagnosis and monitoring other CNS conditions and disorders.

[0229] Both examples of use of OPN and sphingomyelin -derived stearic acid in the presently developed scheme and method demonstrate the applicability of the same approach for the detection, monitoring and clearance of protein and non-protein neurotoxins from the body.

[0230] EXAMPLE 4: Identification of peripheral biomarkers related to age differences

[0231] The study applied the same experimental approach for exploring proteomic changes in liquid biopsies (blood) of young and aging mice after exposure to BDF, aiming at identification of peripheral biomarkers related to age differences.

[0232] To that end, six male c57bl / 6 mice age 4 months (young adults) and four male mice age 11 months (older adults) were treated with BDF previously related to BBBo, i.e., 100 pulses at 200V with 50 psec pulse duration at a frequency of 4Hz (see EXAMPLE 3). Blood was collected 30 min (old) or 5 days (young) before BDF and 30 min after BDF via retro-orbital bleed. Aliquots of plasma were stored at -80 °C for downstream analysis.

[0233] Materials and Methods

[0234] Preparation of MVs was not feasible in mice due to the limited plasma volume. Additionally, this study provided an opportunity to analyze crude plasma (enriched in soluble proteins) as opposed to the previous analysis of MVs (contain various types of proteins). Samples of mouse plasma were processed using the ProteoSpin kit (Norgen, Canada) to deplete abundant proteins and enhance the detection of a broader range of proteins by LC-MS.

[0235] Sample Preparation: Plasma samples were depleted using the ProteoSpin kit to remove high- abundance proteins. Protein digestion was subsequently performed with trypsin followed by S-trap to ensure efficient peptide generation. Liquid Chromatography-Mass Spectrometry: The resulting peptides were analyzed using nanoflow liquid chromatography (nanoElute2), with mobile phase of A) H2O+0.1% formic acid; B) acetonitrile + 0. 1% formic acid. Separation was done using a gradient of 2% B to 35% in 60 min using a C18 nano column 0.075x250mm (lonOpticks, Australia). The column was coupled to high-resolution, high-mass-accuracy mass spectrometry (timsTOF Pro) via the Captive Spray electrospray ionization (Bruker). Each sample was analyzed individually in a randomized order using Data-independent Acquisition Parallel Isolation Serial Fragmentation (DIA-PASEF) discovery mode to ensure robust and unbiased peptide detection.

[0236] Data Processing: Raw mass spectrometry data were processed using Spectronaut vl8.1 software. Peptide identification was performed by searching against the UniProt / SP mouse reference database, supplemented with a list of common contaminant proteins to account for potential background signals.

[0237] Results and Conclusions

[0238] Proteomics analysis was performed on crude plasma depleted from protein abundance. Comparing proteomic profiles before and after BDF exposure, a total of 1127 and 1077 proteins were identified for young and old groups, respectively, including a substantial number of increased CNS- derived proteins in both young and old groups. The old group generally showed a broader proteomic shift.

[0239] PCA showed clear distinction between proteome profiles in plasma of both young and older mice before and after BABo (Figs 3A-3B). PCA plot combining the data of both young and aged mice indicated clustering at time points before vs. after BDF (Fig. 3C). Venn diagram showed certain amount of shared elevated proteins after BDF in both groups (Fig. 3D).

[0240] Taken together, the results suggested that the application of BDF-mediated controlled brain barrier opening and molecular testing of liquid biopsies succeeded in revealing significant changes on proteomic level in crude plasma. Further analysis revealed that many of the detected changes involved proteins with relevance to the CNS, which in this case were also indicative of CNS aging. Table 4 lists the most prominent CNS-derived proteins that were elevated in plasma of young and old mice after BDF exposure, additional markers are listed in Supplemental Tables 7-8.

[0241] Table 4. CNS-enriched proteins elevated in plasma after BDF in young and old mice.

[0242] Young group Old group fold

[0243] Protein fold increase increase CNS relevance

[0244] (p-value) (p-value)

[0245] Enriched in CNS astrocytes and

[0246] PF A 1 ^ t vT considered a biomarker of astroglial

[0247] , ,as rocytic5.3 (0.02)** 49.2 (0.00004) damage Was found upregulated in the phosphoprotein , . ,r, brain in neurodegenerative and TB1, and in serum after CNS injury. Enriched in brain astrocytes and neurons and considered a biomarker in neurodegenerative conditions.

[0248] S100A6 2.7 (0.0008) 1.8 (0.02) Upregulated in early AD and

[0249] Parkinson’s disease the brain and correlates with neuroinflammation and neuronal stress responses.

[0250] Pro-inflammatory CNS protein and enriched during pathological conditions.

[0251] S100A9 4.3 (0.04)** 2 7 (0.009) Was val.dated as a CSF biomarker of early stages AD and AD progression, and related to the neuroinflammatory cascades in TBL

[0252] Actin-binding proteins with important

[0253] Cofilin-1 3.3 (0.006) 4.8 (0.0006) roles in CNS cytoskeletal dynamics.

[0254] Cofilin-2 24 0 (0 04)** 76.0 (0 02) Cofflin-2 proposed as a serum biomarker

[0255] ^a enulari<le174 (0 002) 20.6 (0 0002) Suggested to play crucial role in AD mu dictiuidi microglial polarization and pathology. channel protein

[0256] „ Plays critical role in copper homeostasis opper ranspor 32.1 (0.0001) 27.5 (0.003) in the brain and has emerging relevance protein ATOX1*v vin neurodegenerative disease&.

[0257] Expressed in the brain and plays critical cAMP- depen dent role in neuronal signaling, synaptic protein kinase i i i in otm plasticity, and neuroprotection, catalytic subunit • ( • ) Upregulated in AD mouse models, and alpha (Prkaca)* its modulation is linked to memory function and neuroinflammation.

[0258] * Not detected in blood in pre- BDF samples.

[0259] ** In these proteins p value < 0.05 and q value (FDR) >0.05.

[0260] Gene pathway analysis (Enrichr) was conducted on significantly elevated proteins, including 39 and 226 proteins in young and old groups, respectively. Many of the identified pathways (KEGG 2021) were CNS related, especially for the old group, such as pathways related to Parkinson disease, proteasome, tight junctions’ functionality, prion disease, amyotrophic lateral sclerosis, pathways of neurodegeneration, and others (Figs 6A-6B).

[0261] Taking together, these studies showed that the application of BDF under specific predefined conditions has led to measurable and reproducible increase in in the levels of CNS-derived proteins in peripheral plasma across multiple experimental models. More broadly, they provided proof of concept for the general applicability of a method using BDF-mediated controlled and selective brain barrier breach in conjunction with molecular testing of liquid biopsies for management of various CNS conditions, including early diagnosis and monitoring of CNS diseases, monitoring of CNS drug response, and monitoring and clearance of various CNS damaging substances (see also Fig. 7). Suppl Table 5. Proteins elevated in plasma of BABo rats (>10-fold change)

[0262] Protein Acc Gene Organism Protein description n-value1Fold change before vs. after BDF

[0263] Q6IFW1_RAT Krt33a Rat RCG34348, isofor 0.0003265 77.47

[0264] A0A0G2K126_RKrt86 Rat Keratin 86 0.0002453 76.43

[0265] LRC15_RAT Lrrcl5 Rat Leucine -rich repe 0.0003218 50.11

[0266] K1C13_RAT Krtl3 Rat Keratin, type I cy 0.0009526 35.24

[0267] CP2DQ_RAT Cyp2d26 Rat Cytochrome P45 0.0040596 28.76

[0268] PEF1_RAT Pefl Rat Peflin 0.0003706 25.81

[0269] B5DFK8_RATGPdxdcl Rat Pdxdcl protein 0.0001025 24.75

[0270] TACD2_RAT Tacstd2 Rat Tumor-associate 0.0008378 23.12

[0271] GFPT1_RAT Gfptl Rat Glutamine— fruct 0.0008453 22.73

[0272] D3Z898_RAT Samhdl Rat SAM and HD dom 0.0202135 20.19

[0273] H15_RAT Histlhlb Rat Histone Hl.5 0.0284552 19.77

[0274] UPK1B_RAT Upklb Rat Uroplakin-lb 0.0119257 19.69

[0275] RS15_RAT Rpsl5 Rat 40S ribosomal pr 0.0072704 18.79

[0276] D3ZK86_RAT;D3ZK88_RA Rat Uncharacterized 0.0114865 17.30

[0277] Q6IFW0_RAT Krt33b Rat RCG32608 0.0125734 16.16

[0278] Q5U213 RAT Parp4 Rat Parp4 protein 0.0049482 16.00

[0279] MBNL2_RAT Mbnl2 Rat Muscleblind-like 0.0007408 15.49

[0280] D4ABY7 RAT Rat Uncharacterized 0.0024949 15.28

[0281] HAIR_RAT Hr Rat Lysine -specific de 0.0506441 14.42

[0282] D3ZQI1_RAT Gpx7 Rat Glutathione pero 0.0063725 14.13

[0283] ANXA8_RAT Anxa8 Rat Annexin A8 0.0145716 13.43

[0284] Q5U362 RAT Anxa4 Rat Annexin 0.0362646 13.29

[0285] Q5U2Vl_RATFkbplO Rat Peptidylprolyl iso 0.0276847 12.77

[0286] B0BMX3 RAT Rat S100 calcium bin 0.0046077 12.58

[0287] A0A0G2K0T6_R Sncg Rat Gamma-synuclei 0.0023625 12.55

[0288] AOAOU1RRV7_ Srsf3 Rat RCG61099, isofor 0.0006506 12.38

[0289] VKOR1_RAT Vkorcl Rat Vitamin K epoxid 0.0325628 12.01

[0290] M0R5G7_RATKrtap22-2 Rat Keratin-associate 0.0193500 11.81

[0291] MBP_RAT Mbp Rat Myelin basic prot 0.0032937 11.76

[0292] A0A0G2JXE0_R Histlh Rat Histone H2B 0.0376794 11.68

[0293] SPB5_RAT Serpinb5 Rat Serpin B5 0.0043104 11.49

[0294] D3ZG43_RAT Ndufs3 Rat NADH dehydroge 0.0282627 11.28

[0295] D3ZPW7 RAT Gpx8 Rat Glutathione pero 0.0039322 11.23

[0296] TEP1_RAT Tepl Rat Telomerase prot 0.0340439 10.98

[0297] A0A096MJ98_RAT Rat Uncharacterized 0.0002780 10.98

[0298] A0A096MJR6 RAT Rat Poly [ADP-ribose 0.0062184 10.91

[0299] D3ZBR0 RAT D Krtap3 - Rat Keratin-associate 0.0183416 10.87

[0300] TF_RAT F3 Rat Tissue factor 0.0002730 10.87

[0301] MAP4_RAT Map4 Rat Microtubule -asso 0.0006378 10.87

[0302] A0A0G2JSU6_RAndpro Rat Cystatin 0.0037338 10.72

[0303] FKBP9_RAT Fkbp9 Rat Peptidyl -prolyl ci 0.013608 10.34

[0304] P4HA1_RAT P4hal Rat Prolyl 4-hydroxyl 0.0027964 10.24

[0305] S10AB_RAT SI 00a 11 Rat Protein S100-A 11 0.0014702 10.22

[0306] D4A006_RATKrtap8-l Rat Keratin-associate 0.0257819 10.19

[0307] KCRM RAT Ckm Rat Creatine kinase 0.0728107 10.13 AL3A1 RAT Aldh3al Rat Aldehyde dehydr 0.0026905 10.12

[0308] K2C4 RAT Krt4 Rat Keratin, type II cy 0.0363640 10.10

[0309] 22P1 RAT Andpro Rat Cystatin-related 0.0032202 10.09

[0310] ^-values by Student’s t-test before and after BDF

[0311] Suppl Table 6. Proteins elevated in plasma of BBBo+BABo rats (>10-fold change)

[0312] Fold change

[0313] Protein Acc Gene Organism Protein description p-value1before vs. after

[0314] A0A0G2K7Z Mogatl Rat Monoacylglycero 0.000420091 1675.60 AAAD RAT Aadac Rat Arylacetamide de 0.001623837 1183.59 D3Z9Pl_RATKdsr Rat 3-ketodihydrosp 0.001044625 1158.59 D4A2H2 RA Sptlcl Rat Serine palmitoylt 0.00026447 947.08 TECR_RAT Tecr Rat Very-long-chain 6.40822E-05 937.92

[0315] CATE_RATCtse Rat Cathepsin E 0.002793566 826.27

[0316] G3V8V4 RA Cers2 Rat Ceramide syntha 0.000198313 752.19 D3ZN35 RA Sdrl6c6 Rat Short chain dehy 0.000284594 546.86 Z4YNJ9 RATLOC68145 Rat Similar to stearo 0.000359282 445.49 DHB12 RAT HsdI7bI2 Rat Very-long-chain 0.001459701 417.10 PXL2A RAT Prxl2a Rat Peroxiredoxin-lik 0.000261742 400.37 D4A0T8 RA Dhrs7 Rat Dehydrogenase / 0.00137637 398.22 D3ZXB2 RA RGD 15644 Rat Similar to Mdes p 0.000111146 390.22 Q5I0N0_RATAadac Rat Arylacetamide de 0.000284431 356.16 RTN1 RAT Rtnl Rat Reticulon-1 0.000947697 346.03

[0317] UD2B7 RAT Ugt2b7 Rat UDP-glucuronosy 0.000145466 304.23

[0318] UD11 RAT Ugtlal Rat UDP-glucuronosy 0.001904286 282.64

[0319] B0BNL8 RA Agpat3 Rat Agpat3 protein (F 7.72657E-05 261.49

[0320] ERMP 1 RAT Ermp 1 Rat Endoplasmic reti 0.000110824 255.27

[0321] DRS7B RAT Dhrs7b Rat Dehydrogenase / 0.034910342 251.18

[0322] RTN4 RAT Rtn4 Rat Isoform 3 of Reti 0.000728807 237.35

[0323] FACRI RAT Farl Rat Fatty acyl-CoA re 0.001349443 218.59

[0324] D3ZBJ1 RATRGD15596 Rat Similar to novel p 1.291 IE-05 207.88 Q6AY 58_RA Bcap31 Rat B-cell receptor-a 0.002049018 199.66

[0325] FAAH1 RAT Faah Rat Fatty-acid amide 0.00051427 177.33

[0326] SGPPI RAT Sgppl Rat Sphingosine-l-ph 0.001042628 173.14

[0327] D4A9V0 RA Sptlc3 Rat Serine palmitoylt 0.02510934 167.95

[0328] ATLA3 RAT Atl3 Rat Atlastin-3 0.009976606 167.55

[0329] D3ZJZ0 RAT Tmem205 Rat RGD 1563250 (Pr 0.000238064 163.83 CYB5B RAT Cyb5b Rat Cytochrome b5 t 0.000373248 151.29

[0330] D3ZTU9 RA Aadacl4 Rat Arylacetamide de 0.012214555 146.75

[0331] PTGDS RAT Ptgds Rat Prostaglandin-H2 0.001357888 143.66

[0332] DHCR7 RAT Dhcr7 Rat 7-dehydrocholes 0.00170164 138.88

[0333] RDH7 RAT Rdh7 Rat Retinol dehydrog 0.000895096 133.75

[0334] UD18 RAT Ugtla8 Rat UDP-glucuronosy 0.000131067 133.44

[0335] A0A0G2JSS9Atl3 Rat Atlastin-3 0.002289324 128.96 MGSTI RATMgstI Rat Microsomal gluta 0.011116516 127.79 D3ZBA8 RA RGD13088 Rat Similar to arylace 0.000459647 123.37 Q5U2Z8 RA Elovll Rat Elongation of ver 0.000130035 121.24 GGLO RAT Gulo Rat L-gulonolactone 0.00018777 111.98 P0N2 RAT Pon2 Rat Serum paraoxon 0.000328831 109.79

[0336] MBOA5_RATLpcat3 Rat Lysophospholipid 0.003023595 106.25

[0337] ABHD6 RAT Abhd6 Rat Monoacylglycero 2.35327E-05 103.77

[0338] D3ZIP8 RAT Rat Uncharacterized 0.001018351 100.52

[0339] DHB4 RAT Hsdl7b4 Rat Peroxisomal mul 0.00631797 92.40

[0340] D3ZF54_RATAnolO Rat Anoctamin 0.000602465 90.39

[0341] NLTP_RAT Scp2 Rat Non-specific lipid 0.005603967 90.17

[0342] DGAT1 RAT Dgatl Rat Diacylglycerol O- 3.70328E-05 90.05

[0343] ABCD3 RAT Abcd3 Rat ATP-binding cass 0.001942303 89.10

[0344] SCRB2 RAT Scarb2 Rat Lysosome memb 0.010515274 87.68

[0345] D3ZHG0 RA Hacd2 Rat Very-long-chain ( 0.001235051 87.59

[0346] Q6T5E7 RA Ugtla8 Rat UDP-glucuronosy 0.0002585 83.66

[0347] TM 109 RAT Tmem 109 Rat Transmembrane 0.002811487 81.27

[0348] PGRC2 RAT Pgrmc2 Rat Membrane -assoc 0.000206053 81.14

[0349] Q6IFW1 RA Krt33a Rat RCG34348, isofor 0.008855238 77.65

[0350] ACSL6 RAT Acsl6 Rat Long-chain-fatty- 0.001531826 73.60

[0351] TMM33 RA Tmem33 Rat Transmembrane 0.000845015 71.91

[0352] D4A9Z0 RA Far2 Rat Fatty acyl-CoA re 0.001334234 71.28

[0353] D3ZN47 RA Gk5 Rat Glycerol kinase 5 0.036953395 69.84

[0354] NB5R3 RAT Cyb5r3 Rat NADH-cytochrom 0.001089125 68.94

[0355] REEP6 RAT Reep6 Rat Receptor express 0.002203455 67.62

[0356] AMACR RATAmacr Rat Alpha-methylacy 0.000698139 67.35

[0357] AL3A2 RAT Aldh3a2 Rat Fatty aldehyde d 0.000161292 66.52

[0358] ERLN2 RAT Erlin2 Rat Erlin-2 0.006633194 64.65

[0359] DPP2 RAT Dpp7 Rat Dipeptidyl peptid 0.000290184 63.74

[0360] Q6IFV9_RATKrt34 Rat Keratin 34 0.00987569 62.64

[0361] GDPDI RATGdpdl Rat Lysophospholipa 0.001469496 62.48

[0362] RTN3 RAT Rtn3 Rat Isoform 2 of Reti 0.000203105 61.56

[0363] PXMP4 RAT Pxmp4 Rat Peroxisomal me 0.000857715 60.77

[0364] PLCD_RAT Agpat4 Rat 1-acyl-sn-glycero 0.048642454 59.96

[0365] D3Z865_RATMpdul Rat Mannose-P-dolic 0.005274216 59.68

[0366] ACSL5 RAT Acsl5 Rat Long-chain-fatty- 0.063605574 58.35

[0367] Q5XIF0_RATTex264 Rat Testis expressed 0.000332472 56.97

[0368] A0A0G2K12 Krt86 Rat Keratin 86 0.011824607 56.68

[0369] D3ZI62 RAT Tmem86a Rat Transmembrane 0.001384486 54.87

[0370] RER1_RAT Rerl Rat Protein RER1 0.000433677 53.90

[0371] AOXD RAT Aox4 Rat Aldehyde oxidas 0.002877911 53.73

[0372] TM135_RAT Tmeml35 Rat Transmembrane 0.000706912 53.58

[0373] SPHK1 RAT Sphkl Rat Sphingosine kina 0.001462058 50.44

[0374] MLEC RAT Mlec Rat Malectin 0.010933675 50.43

[0375] DHC24 RAT Dhcr24 Rat Delta(24) -sterol r 0.00021083 49.68

[0376] HYEP_RATEphxl Rat Epoxide hydrolas 0.006922489 49.62

[0377] RDH2 RAT Rdh2 Rat Retinol dehydrog 0.002879619 49.13

[0378] D3ZIJ5 RAT Dpm3 Rat Dolichyl -phospha 0.002017768 47.85

[0379] A0A0G2JVC8Lss Rat Terpene cyclase / 0.002651845 47.02

[0380] D4ACG2 RA Ilvbl Rat IlvB (Bacterial ac 0.018400034 46.22

[0381] ABD 12 RAT Abhd 12 Rat Monoacylglycero 0.000540692 46.19

[0382] D4A3G7 RA Krtap7- 1 Rat Keratin-associate 0.041529494 45.90

[0383] D3ZFXl_RATTmeml64 Rat Similar to Expres 0.002449539 45.78 ACOX 1 RAT Acox 1 Rat Peroxisomal acyl 0.003321757 45.28

[0384] VAPA RAT Vapa Rat Vesicle-associate 0.002928311 44.90

[0385] ANXA8 RAT Anxa8 Rat Annexin A8 0.000331008 44.38

[0386] A0A0G2JU1 Mgst2 Rat Microsomal gluta 0.00019088 44.02

[0387] D4ABI7_RAT Hacd3 Rat Very-long-chain ( 0.000373314 43.03

[0388] D3ZUB0 RA Rent Rat Reticulocalbin 1 0.002614245 42.22

[0389] EI24_RAT Ei24 Rat Etoposide -induce 0.001534613 41.91

[0390] ACACA RAT Acaca Rat Acetyl-CoA carbo 0.005295084 41.62

[0391] A0A0G2K8D Mospd2 Rat Motile sperm do 0.002430287 40.84

[0392] B5DFA3 RA Acsml Rat Bucs 1 protein 0.008897848 40.24

[0393] B0BMZl_RAFam241b Rat Family with sequ 0.005526796 40.12

[0394] A0A0G2JWX0_RAT;A0A Rat Uncharacterized 0.014719893 39.85

[0395] FADS1 RAT Fadsl Rat Acyl-CoA (8-3)-de 0.002562905 37.80

[0396] ECHP_RAT Ehhadh Rat Peroxisomal bifu 0.001043511 37.25

[0397] D4A994 RA Emcl Rat ER membrane pr 0.00681839 37.20

[0398] Q8BHI5_RATDgatl Rat O-acyltransferas 8.66553E-05 36.98

[0399] A0A1N6HSS SAMN054 Rat Protein required 0.043184222 36.88

[0400] AMRP RAT Lrpap 1 Rat Alpha-2 -macrogl 0.005765731 36.73

[0401] BI1_RAT Tmbim6 Rat Bax inhibitor 1 0.001063192 36.39

[0402] HM0X2 RA Hmox2 Rat Heme oxygenase 0.000495329 36.34

[0403] A0A0G2K1Z Hml3 Rat Histocompatibilit 0.000565472 35.33

[0404] ELOV5 RAT Elovl5 Rat Elongation of ver 0.008183723 34.52

[0405] AOXB RAT Aox2 Rat Aldehyde oxidas 0.00081751 34.29

[0406] TMCOl RATTmcol Rat Calcium load-act 0.0101493 33.54

[0407] ACSL1 RAT Acsll Rat Long-chain-fatty- 2.94286E-05 33.03

[0408] ERO1A RAT Erol a Rat ERO 1 -like protein 0.00092856 32.40

[0409] UD2B2 RAT Ugt2b Rat UDP-glucuronosy 0.013243068 32.20

[0410] EPCAM RATEpcam Rat Epithelial cell adh 0.002756448 31.79

[0411] A0A0G2QC0 Tf Rat Serotransferrin 0.004158012 31.75

[0412] D3ZTR5_RATChchd2;LO Rat RCG21454 0.003257444 31.57

[0413] G3V8M6_RAFolrl Rat Folate receptor 1 0.00169081 31.40

[0414] D4A5K6 RA Zmpste24 Rat Zinc metallopept 0.02583272 31.30

[0415] ERP29 RAT Erp29 Rat Endoplasmic reti 0.000724096 31.00

[0416] D3ZKR8 RA Tmeml67 Rat Protein kish 0.006330779 30.72

[0417] B2RYD7 RA Stt3b Rat RCG25591, isofor 0.025512155 30.08

[0418] MBOA2_RATMboat2 Rat Membrane -boun 0.001715591 29.99

[0419] F1M495 RA Pla2rl Rat Mannose recept 0.006897525 29.95

[0420] M0R3V4_RAMydgf Rat Myeloid-derived 0.001747215 29.68

[0421] SNG1 RAT Syngrl Rat Synaptogyrin-1 0.000425427 29.17

[0422] M0R5D4_RAAadacl2 Rat Arylacetamide de 0.052085442 29.02

[0423] Q7TMZ5_RAArl6ipl Rat ADP-ribosylation 0.001891826 28.99

[0424] D4A006 RA Krtap8-1 Rat Keratin-associate 0.007910531 28.95

[0425] MGLL RAT Mgll Rat Monoglyceride li 0.01187693 28.03

[0426] ERG7 RAT Lss Rat Lanosterol synth 0.131374763 27.95

[0427] D3ZF12_RATSpcs3 Rat Signal peptidase 0.002902298 27.35

[0428] DECR2 RAT Decr2 Rat Peroxisomal 2,4- 0.000655946 27.12

[0429] ABHGA_RATAbhdl6a Rat Protein ABHD 16A 5.28545E-05 27.08

[0430] UGDH RAT Ugdh Rat UDP-glucose 6-d 0.000148141 26.55 IR3IP_RAT Ier3ipl Rat Immediate early 0.002182479 26.30

[0431] UD16 RAT Ugtla6 Rat UDP-glucuronosy 0.003031087 26.24

[0432] PTGR1 RAT Ptgrl Rat Prostaglandin re 0.002353746 25.86

[0433] CBPD RAT Cpd Rat Carboxypeptidas 0.010875099 25.78

[0434] G3V6C4 RA Ugdh Rat UDP-glucose 6-d 0.004807357 25.52

[0435] ADAS RAT Agps Rat Alkyldihydroxyac 0.029607255 25.47

[0436] NCLN RAT Ncln Rat Nicalin 0.001898373 25.32

[0437] G3V6N5_RA Pqlc3 Rat PQ loop repeat-c 0.005909119 24.80

[0438] D3ZWT2_RAFitm2 Rat Fat storage-indu 0.002026208 24.69

[0439] CEPT1 RAT Ceptl Rat Choline / ethanola 0.003609158 24.64

[0440] VKORL RAT Vkorclll Rat Vitamin K epoxid 0.000284934 24.52

[0441] PGRC1 RAT Pgrmcl Rat Membrane -assoc 0.019657327 24.51

[0442] D4A8Nl_RA Dpml Rat Dolichyl -phospha 0.00160218 24.33

[0443] K1C13 RAT Krtl3 Rat Keratin, type I cy 0.030888266 24.03

[0444] LAMP 1 RAT Lamp 1 Rat Lysosome-associ 0.002090775 23.94

[0445] D3ZD1 I RA Spcs2 Rat Signal peptidase 0.009347364 23.76

[0446] A0JN30_RATCnpy2 Rat Canopy 2 homol 0.004204187 23.67

[0447] D3ZSA9_RATNomol Rat Nodal modulator 0.001923911 23.62

[0448] D4A 1N8 RA Gdpd3 Rat Glycerophospho 0.012953126 23.55

[0449] B2RYP0_RATRhoc Rat Ras homolog fam 0.065279943 23.49

[0450] FADS3 RAT Fads3 Rat Fatty acid desatu 0.007626575 23.31

[0451] CDS1_RAT Cdsl Rat Phosphatidate cy 0.000127801 22.99

[0452] A0A0G2JWHStt3a Rat STT3A, catalytic s 0.000436899 22.61

[0453] FlLQ09_RATAtl2 Rat Atlastin GTPase 2 0.003277441 22.55

[0454] CCD47 RAT Ccdc47 Rat Coiled-coil doma 0.001112091 22.51

[0455] ABHD5 RAT Abhd5 Rat l-acylglycerol-3- 0.070651725 22.45

[0456] ODP2 RAT Dlat Rat Dihydrolipoyllysi 0.06353018 21.75

[0457] RTN4 RAT Rtn4 Rat Isoform 2 of Reti 0.013924949 21.67

[0458] M0R5G7_RAKrtap22-2 Rat Keratin-associate 0.026258394 21.62

[0459] LAMP2 RAT Lamp2 Rat Lysosome-associ 0.002701028 21.43

[0460] CDIPT RAT Cdipt Rat CDP-diacylglycer 0.000549912 21.10

[0461] RETST RAT Retsat Rat All-trans-retinol 0.003106509 20.97

[0462] Q5XIU4_RATBcap29 Rat B-cell receptor-a 0.006544382 20.90

[0463] B2RZD1 RA Sec61b Rat Protein transport 0.006353661 20.88

[0464] MSPDI RATMospdl Rat Motile sperm do 0.000682041 20.88

[0465] NSDHL RAT Nsdhl Rat Sterol-4-alpha-ca 0.006756468 20.80

[0466] FCGRN RAT Fcgrt Rat IgG receptor FcR 0.009423191 20.75

[0467] TXD12 RAT Txndcl2 Rat Thioredoxin dom 0.001435865 20.46

[0468] A0A0G2K7G Agfg2 Rat ArfGAP with FG r 0.005333822 20.19

[0469] D3ZBR0 RA Krtap3-1;K Rat Keratin-associate 0.045664196 20.16 SC11A RAT Seel la Rat Signal peptidase 0.004622933 20.13

[0470] B2RYF6_RATClptml Rat CLPTM 1 , transm 0.010786058 20.08

[0471] D3ZK72_RATKrtapl6-5 Rat Keratin-associate 0.015559477 19.83

[0472] G3V7K5 RA Npcl Rat NPC intracellular 0.00478316 19.82

[0473] G3V7V3 RA Slc27a4 Rat Solute carrier fa 0.021970927 19.81

[0474] GFPT1 RAT Gfptl Rat Glutamine — firuct 0.002181849 19.77

[0475] CYB5_RAT Cyb5a Rat Cytochrome b5 0.000651959 19.66

[0476] PRAF3 RAT Arl6ip5 Rat PRA1 family prot 0.000151493 19.58 D3ZEE9_RATFads6 Rat Fatty acid desatu 0.012143211 19.54

[0477] RCN2 RAT Rcn2 Rat Reticulocalbin-2 0.004468621 19.46

[0478] CLD3_RAT Cldn3 Rat Claudin-3 0.000349531 19.33

[0479] TMED2_RATTmed2 Rat Transmembrane 0.000678789 18.90

[0480] S61A1 RAT Sec61al Rat Protein transport 0.004178991 18.81

[0481] DHB7 RAT Hsdl7b7 Rat 3 -keto-steroid re 0.004898381 18.58

[0482] F2Z3 S3 RAT St6gal 1 Rat Beta-galactoside 0.001010007 18.20

[0483] Q642E2 RA Rpl28 Rat 60S ribosomal pr 0.00223762 18.00

[0484] F7FJQ3_RATNpc2;rel Rat NPC intracellular 0.016028776 17.90

[0485] D3ZXQ0 RA Ces2g Rat Carboxylic ester 0.018961877 17.70

[0486] Q4QQS6_RA Alg5 Rat ALG5, dolichyl-ph 0.001058606 17.64

[0487] TMM19 RA Tmeml9 Rat Transmembrane 0.009271083 17.56

[0488] CD166 RAT Alcam Rat CD 166 antigen 0.014544373 17.47

[0489] Q6P7A7 RA Rpnl Rat Dolichyl-diphosp 0.003901496 17.33

[0490] D3ZZR9_RATFkbp2 Rat Peptidylprolyl iso 0.032766696 17.30

[0491] OST48 RAT Ddost Rat Dolichyl-diphosp 0.003266844 17.16

[0492] CATC_RATCtsc Rat Dipeptidyl peptid 0.017587836 17.07

[0493] PX11A RAT Pexl la Rat Peroxisomal me 0.000868545 16.98

[0494] D3ZBC7 RA Dhrsl3 Rat Dehydrogenase / 0.009607312 16.97

[0495] RL27_RAT Rpl27 Rat 60S ribosomal pr 0.005823637 16.57

[0496] MAGTI RATMagtl Rat Magnesium tran 0.001109151 16.41

[0497] MANF RAT Manf Rat Mesencephalic a 0.032331079 16.38

[0498] REEP5 RAT Reep5 Rat Receptor express 0.024369117 15.61

[0499] A0A140TAI6 Rpl36 Rat 60S ribosomal pr 0.008727868 15.49

[0500] AACS_RATAacs Rat Acetoacetyl -Co A 0.011553149 15.19

[0501] A0A0G2JW56_RAT;D3Z Rat Uncharacterized 0.010626349 14.95

[0502] CISDI RAT Cisdl Rat CDGSH iron-sulfu 0.008288484 14.94

[0503] B2GV15 RA Dbt Rat Dihydrolipoamid 0.003255012 14.93

[0504] TXTP_RAT Slc25al Rat Tricarboxylate tr 0.005347 14.84

[0505] GNPAT RAT Gnpat Rat Dihydroxy aceton 0.001364415 14.77

[0506] FMO3 RAT Fmo3 Rat Dimethylaniline 0.00326272 14.63

[0507] CALL3 RAT Calml3 Rat Calmodulin-like p 0.013441058 14.60

[0508] ERG1 RAT Sqle Rat Squalene monoo 0.001890887 14.56

[0509] MARC2_RATMarc2 Rat Mitochondrial am 0.007288499 14.52

[0510] SSRD_RAT Ssr4 Rat Translocon-assoc 0.012596213 14.44

[0511] A0A0G2JUJ9 Sec63 Rat SEC63 homolog, 4.84548E-05 14.02

[0512] D3ZJ32_RATEsyt2 Rat Extended synapt 0.000371932 13.96

[0513] BDH_RAT Bdhl Rat D-beta-hydroxyb 0.004759955 13.94

[0514] D4A340 RA LOC69119 Rat Similar to arylace 0.001149747 13.88

[0515] D3ZXT5_RATUpk3bll Rat RCG21672 0.049001683 13.86

[0516] F7FNS3_RATDerll Rat Derlin 0.003614696 13.84

[0517] GLNA RAT Glul Rat Glutamine synth 0.008732626 13.81

[0518] OSTC_RAT Ostc Rat Oligosaccharyltra 0.007013732 13.80

[0519] Q0D2L6 RA Rragc Rat Ras-related GTP- 0.002948626 13.62

[0520] EMC2 RAT Emc2 Rat ER membrane pr 0.002516351 13.52

[0521] M0R402 RA Tmx3 Rat Thioredoxin-rela 0.051871079 13.49

[0522] A0A0G2K2F Prr33 Rat RCG48030 0.046287225 13.49

[0523] SPB5_RAT Serpinb5 Rat Serpin B5 0.064594253 13.47

[0524] Q5U362 RA Anxa4 Rat Annexin 0.026181372 13.43 A0A0G2JXJ3 Fam3d Rat Family with sequ 0.002542848 13.40

[0525] VAMP2_RATVamp2 Rat Vesicle-associate 0.009776341 13.35

[0526] CP2DQ RAT Cyp2d26 Rat Cytochrome P45 0.041753694 13.30

[0527] CDS2_RAT Cds2 Rat Phosphatidate cy 0.020714393 13.29

[0528] B0BN20 RA Tspan6 Rat Tetraspanin 0.016469254 13.08

[0529] DPEP1 RAT Dpepl Rat Dipeptidase 1 0.011771432 13.00

[0530] IVD_RAT Ivd Rat Isovaleryl-CoA de 0.00397369 12.93

[0531] 3BHS5 RAT Hsd3b5 Rat NADPH-depende 0.002203733 12.80

[0532] A0A0G2JYD6Mfsd8 Rat Maj or facilitator 0.003108269 12.67

[0533] VTI1B RAT Vtilb Rat Vesicle transport 0.020278396 12.59

[0534] RPN1 RAT Rpnl Rat Dolichyl-diphosp 0.002607969 12.48

[0535] ERAP 1 RAT Erapl Rat Endoplasmic reti 0.001732364 12.46

[0536] APMAP RATApmap Rat Adipocyte plasm 0.001852959 12.42

[0537] GSTT2 RAT Gstt2 Rat Glutathione S-tra 0.010737756 12.41

[0538] HABP2 RAT Habp2 Rat Hyaluronan-bind 0.001047102 12.31

[0539] Q5XI86_RATPtrh2 Rat Peptidyl-tRNA hy 0.00010263 12.28

[0540] PREB_RAT Preb Rat Prolactin regulat 0.005389601 12.22

[0541] B2RYS8_RATNdufb8 Rat NADH dehydroge 0.034616882 12.18

[0542] CALX_RATCanx Rat Calnexin 0.000514717 12.16

[0543] HS71A_RAT;Hspala;Hs Rat Heat shock 70 kD 0.045823249 12.04

[0544] AT1B1 RAT Atplbl Rat Sodium / potassiu 0.10442398 11.98

[0545] B 1 WC34 RA Prkcsh Rat Protein kinase C 0.000972791 11.96

[0546] CP51A RAT Cyp51al Rat Lanosterol 14-alp 0.091068594 11.66

[0547] VPS45 RAT Vps45 Rat Vacuolar protein 0.037790512 11.62

[0548] EST1E RAT Cesle Rat Carboxylesterase 0.004938117 11.62

[0549] RS26_RAT Rps26 Rat 40S ribosomal pr 0.00415157 11.59

[0550] TMED7_RATTmed7 Rat Transmembrane 0.00448116 11.57

[0551] BCAT2 RAT Bcat2 Rat Branched-chain- 0.001847582 11.56

[0552] RL23A RAT Rpl23a Rat 60S ribosomal pr 0.010039753 11.56

[0553] F1M9Q8_RA Agps Rat Alkylglycerone -p 0.000767052 11.52

[0554] S39A4 RAT Slc39a4 Rat Zinc transporter 0.014916149 11.48

[0555] NDUAA_RATNdufalO Rat NADH dehydroge 0.017058744 11.45

[0556] Q7TP42 RA Sec62 Rat Ab2-292 0.019032913 11.43

[0557] FAS_RAT Fasn Rat Fatty acid syntha 0.002731282 11.38

[0558] SDHA RAT Sdha Rat Succinate dehydr 0.047589296 11.35

[0559] NENF RAT Nenf Rat Neudesin 0.005961799 11.30

[0560] STX6_RAT Stx6 Rat Syntaxin -6 0.026876897 11.28

[0561] F7EQ81_RATGns Rat N -acetylglucosam 0.007763294 11.13

[0562] THRSP RAT Thrsp Rat Thyroid hormone 0.004154136 11.11

[0563] D3ZKN1 RA Bpifa6 Rat BPI fold-containi 0.035946144 11.02

[0564] RABP1 RAT Crabpl Rat Cellular retinoic a 0.00793591 10.94

[0565] Q52KJ9_RATTmxl Rat Thioredoxin dom 0.013988997 10.91

[0566] NCPR RAT Por Rat NADPH— cytochr 0.006456756 10.85

[0567] DAD1 RAT Dadi Rat Dolichyl-diphosp 0.04398314 10.83

[0568] UPK1B RAT Upklb Rat Uroplakin-lb 0.014082899 10.73

[0569] VMP1 RAT Vmpl Rat Vacuole membra 0.001725969 10.68

[0570] D3ZV63 RA Odr4 Rat Odr-4 GPCR local 0.005760992 10.67

[0571] SCAM4_RATScamp4 Rat Secretory carrier 0.078592936 10.64

[0572] ACLY_RATAcly Rat ATP -citrate synth 0.012439715 10.64 A0A096MJA Asph Rat Aspartate-beta-h 0.042443445 10.63 A0A0G2K8MTmed4 Rat Transmembrane 0.001832335 10.57 D4A465 RA Lamtor2 Rat Late endosomal / 0.035464054 10.53 D3ZZZ9_RATCtnndl Rat Catenin (Cadheri 0.031247967 10.47 G3V6U3 RA Alg2 Rat ALG2, alpha- 1,3 / 0.011890514 10.41 RS24_RAT Rps24 Rat 40S ribosomal pr 0.089801758 10.38 AQP5 RAT Aqp5 Rat Aquaporin-5 0.077556844 10.36 RRAGA RAT RragB Rat Ras-related GTP 0.054516222 10.32 CLCB_RATCltb Rat Isoform Non-brai 0.002716743 10.09 TXD15 RAT Txndcl5 Rat Thioredoxin dom 0.009639207 10.09

[0573] MCAT RAT Slc25a20 Rat Mitochondrial ca 0.028867672 10.06 A0A0G2K37 Sphkl Rat Sphingosine kina 0.006583058 10.02

[0574] ^-values by Student’s t-test before and after BDF

[0575] Suppl Table 7. Proteins elevated in plasma of young mice

[0576] Protein Acc Gene Organism / Protein description p-value Fold change before vs. after BDF

[0577] 008997 Atoxl Mus muscul Copper transport protein 0.000103037 32.1

[0578] P14206 Rpsa Mus muscul Small ribosomal subunit 0.000118869 27.1

[0579] P63323 Rpsl2 Mus muscul Small ribosomal subunit 0.00447769 18.5

[0580] Q9Z1Q5 Clicl Mus muscul Chloride intracell, channel 0.001657322 17.4

[0581] Q05816 Fabp5 Mus muscul Fatty acid-binding proten 0.003764429 16.8

[0582] Q9DBP5 Cmpkl Mus muscul UMP-CMP kinase 0.006087659 16.5

[0583] Q8BP47 NARS1 Mus muscul Asparagine — tRNA ligase 0.00401667 13.9

[0584] Q8BGQ7 Aarsl Mus muscul Alanine— tRNA ligase 0.000987674 12.9

[0585] Q9JKF1 Iqgapl Mus muscul Ras GTPase -activating- 0.005564783 12.7

[0586] P54227 Stmnl Mus muscul Stathmin 0.006944672 12.5

[0587] Q64674 Srm Mus muscul Spermidine synthase 0.003024158 12.2

[0588] P14131 Rpsl6 Mus muscul Small ribosomal subunit 0.002535124 11.9

[0589] Q9JHW9 Aldhla3 Mus muscul Retinaldehyde dehydro 0.006365691 11.7

[0590] Q91V12 Acot7 Mus muscul Cytosolic acyl coenzyme 0.000357164 11.4

[0591] P62245 Rpsl5a Mus muscul Small ribosomal subunit 0.000632069 8.4

[0592] P30416 Fkbp4 Mus muscul Peptidyl-prolyl cis-trans 0.003787666 6.7

[0593] Q8R1F1 Niban2 Mus muscul Protein Niban 2 0.007668032 6.2

[0594] Q9D3H2 Obpla Mus muscul Odorant-binding protein 0.003285067 5.9

[0595] Q6UGQ3 Scgb2b2 Mus muscul Secretoglobin family 0.004927707 5.4

[0596] P47754 Capza2 Mus muscul F-actin-capping protein 0.004949653 5.3

[0597] P07743 Bpifa2 Mus muscul BPI fold-containing family 0.000697022 3.9

[0598] Q5XJY5 Arcnl Mus muscul Coatomer subunit delta 0.003453881 3.9

[0599] P10126 Eeflal Mus muscul Elongation factor 1-alph 0.004393118 3.5

[0600] Q61598 Gdi2 Mus muscul Rab GDP dissociation in 0.00426562 3.4

[0601] P18760 Cfll Mus muscul Cofilin- 1 0.006200531 3.3

[0602] Q99PT1 Arhgdia Mus muscul Rho GDP -dissociation 0.003924595 2.9

[0603] P17182 Enol Mus muscul Alpha-enolase 0.005094278 2.9 P14069 S100a6 Mus muscul Protein SI 00-A6 0.000836869 2.7

[0604] P34884 Mif Mus muscul Macrophage migration 0.005741581 2.6

[0605] P58252 Eef2 Mus muscul Elongation factor 2 0.006328908 2.6

[0606] Pl 7742 Ppia Mus muscul Peptidyl-prolyl cis-trans 0.002125367 2.6

[0607] P26041 Msn Mus muscul Moesin 0.003370028 2.6

[0608] P16110 Lgals3 Mus muscul Galectin-3 0.004300016 2.4

[0609] P68037 Ube213 Mus muscul Ubiquitin-conjugating 0.000966483 2.3

[0610] P62962 Pfiil Mus muscul Profilin- 1 0.007897169 2.3

[0611] P62821 RablA Mus muscul Ras-related protein Rab 0.00256085 2.3

[0612] 009131 Gstol Mus muscul Glutathione S -transferase 0.007622338 2.3

[0613] P16858 Gapdh Mus muscul Glyceraldehyde-3-phos 0.002972466 2.3

[0614] P40142 Tkt Mus muscul Transketolase 0.001751069 2.2

[0615] Suppl Table 8. Proteins elevated in plasma of old mice

[0616] Protein Acc Gene Organism / Protein description p-value Fold change before vs. after BDF

[0617] P45591 Cfl2 Mus muCofilin-2 0.02142697 76.0

[0618] Q91XF0 Pnpo Mus muPyridoxine-5'-phosphate oxidase 0.00013275 61.0

[0619] Q62048 Peal5 Mus muAstrocytic phosphoprotein PEA- 15 4.28E-05 49.2

[0620] Q9D9V3 Echdcl Mus muEthyhnalonyl-CoA decarboxylase 7.98E-05 40.4

[0621] 035887 Calu Mus muCalumenin 0.00023524 40.3

[0622] D3Z6Q9 Bin2 Mus muBridging integrator 2 0.00076166 34.2 Q9JKS4 Ldb3 Mus muLIM domain-binding protein 3 0.00012586 33.8 Q9D0I9 Rars 1 Mus muArginine— tRNA ligase, cytoplasmic 0.00053013 32.2 Q9CQV8 Ywhab Mus mul4-3-3 protein beta / alpha 0.00244683 32.1 008997 Atoxl Mus muCopper transport protein ATOX1 0.00313069 27.5 Q8BK64 Ahsal Mus muActivator of 90 kDa heat shock prote 0.00076137 27.4 Q60854 Serpinb6 Mus muSerpin B6 0.00050206 26.1 Q8CI15 Sphkl Mus muSphingosine kinase 1 0.00185134 25.7 P62631 Eefla2 Mus muElongation factor 1 -alpha 2 0.01461744 24.9 Q63918 Cavin2 Mus muCaveolae-associated protein 2 0.00018369 24.5 Q9JII6 Akrlal Mus muAldo-keto reductase family 1 membeO.01141495 24.4 P46471 Psmc2 Mus mu26S proteasome regulatory subunit 7 0.00049455 24.3 Q8C1B7 Septinl l Mus muSeptin- 11 0.00215761 23.7 Q99LS3 Psph Mus muPhosphoserine phosphatase 0.00205877 23.6 P13707 Gpdl Mus muGlycerol-3 -phosphate dehydrogenas 0.00161915 22.1 P04247 Mb Mus muMyoglobin 0.02441012 21.7 Q9R0Q7 Ptges3 Mus muProstaglandin E synthase 3 0.02260945 21.6 Q99J08 Secl412 Mus muSEC14-like protein 2 3.01E-05 20.7 Q9ES46 Parvb Mus muBeta-parvin 0.00061759 20.7 Q9Z1Q5 Clicl Mus muChloride intracellular channel protei 2.44E-06 20.6 Q8QZR5 Gpt Mus muAlanine aminotransferase 1 0.00027145 20.3 Q8VED9 Lgalsl Mus muGalectin-related protein 0.00467786 20.2 088569 Hnmpa2b Mus muHeterogeneous nuclear ribonucleopr 0.007759 19.9 Q08093 Cnn2 Mus muCalponin-2 4.75E-05 19.6 P45376 Akrlbl Mus muAldo-keto reductase family 1 membeO.00721108 19.3 Q01730 Rsul Mus muRas suppressor protein 1 0.01339656 18.3 Q9WVL0 Gstzl Mus muMaleylacetoacetate isomerase 0.00068946 17.2

[0623] P68510 Ywhah Mus mu 14-3 -3 protein eta 0.00020676 17.2

[0624] Q9CZU6 Cs Mus muCitrate synthase, mitochondrial 0.00408224 16.4

[0625] P70670 Naca Mus muNascent polypeptide -associated com 0.00014157 16.2

[0626] Q64105 Spr Mus muSepiapterin reductase 0.0021243 16.1

[0627] P62334 Psmc6 Mus mu26S proteasome regulatory subunit 1 0.0013696 16.0

[0628] Q9EQP2 Ehd4 Mus muEH domain-containing protein 4 0.00276482 16.0

[0629] Q9CQI6 Cotll Mus muCoactosin-like protein 0.00513322 15.7

[0630] Q76MZ3 Ppp2rla Mus muSerine / threonine-protein phosphata 0.00225101 15.6

[0631] Q9D819 Ppal Mus mulnorganic pyrophosphatase 0.00906192 15.6

[0632] P56376 Acypl Mus muAcylphosphatase-1 0.00181474 15.5

[0633] P62814 Atp6vlb2 Mus muV-type proton ATPase subunit B, braiO.00143405 15.5

[0634] Q8K1M6 Dnmll Mus muDynamin-l-like protein 0.00167443 15.4

[0635] P18654 Rps6ka3 Mus muRibosomal protein S6 kinase alpha-3 0.00224852 15.0

[0636] Q9DB16 Cab391 Mus muCalcium-binding protein 39-like 0.01648592 14.9

[0637] Q61166 Mapre 1 Mus muMicrotubule-associated protein RP / E 0.01011246 14.7

[0638] Q9WUM4 Corolc Mus muCoronin-lC 0.00180007 14.6

[0639] Q6PDN3 Mylk Mus muMyosin light chain kinase, smooth m 0.01317549 13.9

[0640] P63323 Rpsl2 Mus muSmall ribosomal subunit protein eSl 0.00027856 13.8

[0641] A2AQ07 Tubbl Mus muTubulin beta- 1 chain 0.01715021 13.6

[0642] P14602 Hspbl Mus muHeat shock protein beta-1 0.00732362 13.5

[0643] P97447 Fhll Mus muFour and a half LIM domains proteinO.01014179 13.5

[0644] Pl 1404 Fabp3 Mus muFatty acid-binding protein, heart 0.01814751 13.4

[0645] Q62393 Tpd52 Mus muTumor protein D52 0.0043165 12.7

[0646] P10518 Alad Mus muDelta-aminolevulinic acid dehydratasO.02489843 12.7

[0647] Q91ZJ5 Ugp2 Mus muUTP— glucose-l-phosphate uridylyltrO.01721816 12.6

[0648] P61750 Arf4 Mus muADP-ribosylation factor 4 0.02123195 12.4

[0649] Pl 6045 Lgalsl Mus muGalectin-1 0.00591472 12.2

[0650] Q99JW4 Limsl Mus muLIM and senescent cell antigen-like -cO.02587476 12.1

[0651] Q62446 Fkbp3 Mus muPeptidyl-prolyl cis-trans isomerase F 0.00237982 11.9

[0652] Q9WV80 Snxl Mus muSorting nexin-1 0.00251395 11.7

[0653] Q9Z0P5 Twf2 Mus muTwinfilin-2 0.00279021 11.6

[0654] P19096 Fasn Mus muFatty acid synthase 0.02155899 11.6

[0655] P05977 Myll Mus muMyosin light chain 1 / 3, skeletal muscO.00766941 11.4

[0656] P40936 Inmt Mus mulndolethylamine N-methyltransferas 0.01326541 11.3

[0657] P05132 Prkaca Mus mucAMP -dependent protein kinase cata 0.00232957 11.1 035226 Psmd4 Mus mu26S proteasome non-ATPase regulat 0.02169098 11.0 P62196 Psmc5 Mus mu26S proteasome regulatory subunit 8 0.00953515 10.8

[0658] Q5FWK3 Arhgap 1 Mus muRho GTPase -activating protein 1 0.00448283 10.8

[0659] Q9WVJ2 Psmdl3 Mus mu26S proteasome non-ATPase regulat 0.00018027 10.6

[0660] P62141 Ppplcb Mus muSerine / threonine-protein phosphata 0.01053264 10.6

[0661] Q9D0J8 Ptms Mus muParathymosin 0.0101104 10.6

[0662] Q9DCN2 Cyb5r3 Mus muNADH-cytochrome b5 reductase 3 0.00038405 10.1 009172 Gclm Mus muGlutamate— cysteine ligase regulator 0.02232165 10.1

[0663] Q8VDD5 Myh9 Mus muMyosin-9 0.01762189 10.1

[0664] Q91V41 Rabl4 Mus muRas-related protein Rab-14 0.00114187 10.1 088342 Wdrl Mus muWD repeat-containing protein 1 0.01138468 9.8 035685 Nude Mus muNuclear migration protein nudC 0.01849426 9.6

[0665] Q9CPX6 Atg3 Mus muUbiquitin-like-conjugating enzyme A0.00778026 9.6 P26516 Psmd7 Mus mu26S proteasome non-ATPase regulat 0.01627165 9.6

[0666] P62192 Psmcl Mus mu26S proteasome regulatory subunit 4 0.00504422 9.5

[0667] Q9D1E6 Tbcb Mus muTubulin-folding cofactor B 0.00088286 9.5

[0668] Q9JHK5 Pick Mus muPleckstrin 0.00131825 9.5

[0669] Q6IRU2 Tpm4 Mus muTropomyosin alpha-4 chain 0.0122718 9.4

[0670] Q9JLJ2 Aldh9al Mus mu4-trimethylaminobutyraldehyde deh 0.0097558 9.4

[0671] B2RPV6 Mmml Mus muMultimerin-1 0.02547029 9.3

[0672] Q9Z0N1 Eif2s3x Mus muEukaryotic translation initiation factoO.02314075 9.0 P48774 Gstm5 Mus muGlutathione S -transferase Mu 5 0.00088432 8.9

[0673] Q99J77 Nans Mus muSialic acid synthase 0.01207472 8.8

[0674] Q9Z126 Pf4 Mus muPlatelet factor 4 0.01057129 8.6

[0675] Q8CG03 Pde5a Mus mucGMP-specific 3',5'-cyclic phosphodiO.00029294 8.5

[0676] Q5SWU9 Acaca Mus muAcetyl-CoA carboxylase 1 0.02040444 7.9

[0677] Q9R0P3 Esd Mus muS-formylglutathione hydrolase 0.00320859 7.9

[0678] Q60605 Myl6 Mus muMyosin light polypeptide 6 0.00103544 7.6

[0679] P16546 Sptanl Mus muSpectrin alpha chain, non-erythrocyt 0.00067112 7.5

[0680] Pl 1983 Tcpl Mus muT -complex protein 1 subunit alpha 0.01213721 7.5

[0681] Q9CR00 Psmd9 Mus mu26S proteasome non-ATPase regulat 0.01729875 7.3

[0682] Q8CIN4 Pak2 Mus muSerine / threonine-protein kinase PAK 0.01873512 7.3

[0683] Q9WTX6 Cull Mus muCullin-1 0.00179458 7.3

[0684] P68368 Tuba4a Mus muTubulin alpha-4A chain 0.00054066 7.3

[0685] P63158 Hmgbl Mus muHigh mobility group protein B 1 0.01707695 7.2

[0686] P32848 Pvalb Mus muParvalbumin alpha 0.01476691 7.0

[0687] P26039 Tlnl Mus muTalin-1 0.00028497 7.0

[0688] Q9CQM9 Glrx3 Mus muGlutaredoxin-3 0.02162811 7.0

[0689] Q99LC5 Etfa Mus muElectron transfer flavoprotein subuni 0.00526247 7.0

[0690] P28653 Bgn Mus muBiglycan 0.01211377 6.8

[0691] P21107 Tpm3 Mus muTropomyosin alpha-3 chain 0.01739909 6.7

[0692] Q61792 Laspi Mus muLIM and SH3 domain protein 1 0.02106544 6.6

[0693] P51859 Hdgf Mus muHepatoma-derived growth factor 0.01053802 6.6

[0694] 089053 Coro la Mus muCoronin-lA 0.00984734 6.5

[0695] P48758 Cbrl Mus muCarbonyl reductase [NADPH] 1 0.01954586 6.4

[0696] P34884 Mif Mus muMacrophage migration inhibitory fac 0.00865439 6.4

[0697] P24472 Gsta4 Mus muGlutathione S-transferase A4 0.01965064 6.3

[0698] Q62523 Zyx Mus muZyxin 0.01906011 6.3

[0699] P53810 Pitpna Mus muPhosphatidylinositol transfer protein 0.0169157 6.2

[0700] Q99JI6 Rap lb Mus muRas-related protein Rap-lb 0.00132191 6.2

[0701] P07743 Bpifa2 Mus muBPI fold-containing family A membeiO.00167783 6.1

[0702] P80315 Cct4 Mus muT-complex protein 1 subunit delta 0.01828387 5.9

[0703] Q8VCT4 Cesld Mus muCarboxylesterase ID 0.00158398 5.8

[0704] Q8BJY1 Psmd5 Mus mu26S proteasome non-ATPase regulat 0.01122299 5.8

[0705] P62962 Pfiil Mus muProfilin-1 0.00024177 5.5

[0706] Q9JJZ2 Tuba8 Mus muTubulin alpha-8 chain 0.02305408 5.4

[0707] P99024 Tubb5 Mus muTubulin beta-5 chain 0.0026773 5.4

[0708] P63328 Ppp3ca Mus muProtein phosphatase 3 catalytic subu 0.02506402 5.3

[0709] P14152 Mdhl Mus muMalate dehydrogenase, cytoplasmic 0.01135565 5.3

[0710] Q61425 Hadh Mus muHydroxyacyl-coenzyme A dehydrogeO.01335914 5.3

[0711] Q9JHU4 Dynclhl Mus muCytoplasmic dynein 1 heavy chain 1 0.00723808 5.3 008638 Myhl l Mus muMyosin- 11 0.01995795 5.2 Q8BTM8 Flna Mus muFilamin-A 0.00166087 5.1

[0712] P68372 Tubb4b Mus muTubulin beta-4B chain 0.00098192 5.1

[0713] Q8R050 Gsptl Mus muEukaryotic peptide chain release fact 0.02259382 5.0

[0714] P05213 Tuba lb Mus muTubulin alpha-lB chain 0.00106878 5.0

[0715] P40124 Capl Mus muAdenylyl cyclase-associated protein 0.00114962 4.9

[0716] P18760 Cfll Mus muCofilin-1 5.75E-05 4.8

[0717] P40142 Tkt Mus muTransketolase 0.00709982 4.7

[0718] 035945 Aldhla7 Mus muAldehyde dehydrogenase, cytosolic 10.002761 4.6

[0719] Q7TMM9 Tubb2a Mus muTubulin beta-2A chain 0.00244239 4.6

[0720] Q8K1B8 Fermt3 Mus muFermitin family homolog 3 0.00246264 4.6

[0721] P61205;P84Arf3;Arfl Mus muADP-ribosylation factor 3 ;ADP-ribos0.00165921 4.4

[0722] P70296 Pebpl Mus muPhosphatidylethanolamine-binding p 0.00455453 4.4

[0723] Q07235 Serpine2 Mus muGlia-derived nexin 0.0120445 4.4

[0724] P35441 Thbsl Mus muThrombospondin-1 0.01649555 4.3

[0725] Q99PT1 Arhgdia Mus muRho GDP-dissociation inhibitor 1 0.00194496 4.3

[0726] P39876 Timp3 Mus muMetalloproteinase inhibitor 3 0.02579739 4.3

[0727] Q9WVA4 Tagln2 Mus muTransgelin-2 0.00097313 4.2

[0728] P06151 Ldha Mus muL-lactate dehydrogenase A chain 0.00842914 4.1

[0729] Q60864 Stipl Mus muStress-induced-phosphoprotein 1 0.0084096 4.0

[0730] Pl 7742 Ppia Mus muPeptidyl-prolyl cis-trans isomerase A0.00073721 4.0

[0731] P59325 Eif5 Mus muEukaryotic translation initiation facto0.02182834 4.0

[0732] P70349 Hintl Mus muAdenosine 5'-monophosphoramidas 0.01165878 3.9

[0733] P35700 Prdxl Mus muPeroxiredoxin-1 0.0052211 3.9

[0734] Q61171 Prdx2 Mus muPeroxiredoxin-2 0.00532327 3.8

[0735] Q923D2 Blvrb Mus muFlavin reductase (NADPH) 0.0058172 3.8

[0736] P17182 Enol Mus muAlpha-enolase 0.00226632 3.8

[0737] P62821 RablA Mus muRas-related protein Rab-1 A 0.00707815 3.8

[0738] Q9D154 Serpinb laMus muLeukocyte elastase inhibitor A 0.02443952 3.8

[0739] Q9R0P5 Dstn Mus muDestrin 0.00472875 3.7 055042 Snca Mus muAlpha-synuclein 0.02143285 3.7 P68037 Ube213 Mus muUbiquitin-conjugating enzyme E2 L30.00787305 3.6

[0740] Pl 1499 Hsp90abl Mus muHeat shock protein HSP 90-beta 0.00584296 3.6

[0741] Q3THE2 Myll2b Mus muMyosin regulatory light chain 12B 0.00467085 3.6

[0742] P10639 Txn Mus muThioredoxin 0.00755301 3.6

[0743] P10126 Eef 1 a 1 Mus muElongation factor 1 -alpha 1 0.0110753 3.6

[0744] Q64339 Isgl5 Mus muUbiquitin-like protein ISG 15 0.01002557 3.6

[0745] Q01853 Vcp Mus muTransitional endoplasmic reticulum A).01547615 3.5

[0746] P26043 Rdx Mus muRadixin 0.00239556 3.5

[0747] P13634 Ca 1 Mus muCarbonic anhydrase 1 0.01113182 3.5

[0748] P80316 Cct5 Mus muT-complex protein 1 subunit epsilon 0.00790257 3.5

[0749] P05064 Aldoa Mus muFructose-bisphosphate aldolase A 0.01349327 3.4

[0750] P24549 Aldhlal Mus muAldehy de dehydrogenase 1 Al 0.00457021 3.4

[0751] Q9QUI0 Rhoa Mus muTransforming protein RhoA 0.0050859 3.4

[0752] P60843 Eif4al Mus muEukaryotic initiation factor 4A-I 0.02050522 3.3

[0753] Q9D8N0 Eeflg Mus muElongation factor 1 -gamma 0.01310448 3.3

[0754] Q99LX0 Park7 Mus muParkinson disease protein 7 homolog 0.012527 3.3 P16858 Gapdh Mus muGlyceraldehyde-3-phosphate dehydr 0.01128231 3.3 P62827 Ran Mus muGTP -binding nuclear protein Ran 0.00958221 3.3

[0755] Q93092 Taldol Mus muTransaldolase 0.00549548 3.3 P07901 Hsp90aal Mus muHeat shock protein HSP 90-alpha 0.01469409 3.3

[0756] P63242 Eif5a Mus muEukaryotic translation initiation factoO.00641494 3.2

[0757] P24270 Cat Mus muCatalase 0.00957007 3.2

[0758] P12399 Ctla2a Mus muProtein CTLA-2-alpha 0.01881119 3.2

[0759] P63017 Hspa8 Mus muHeat shock cognate 71 kDa protein 0.0022095 3.1

[0760] P15327 Bpgm Mus muBisphosphoglycerate mutase 0.00809606 3.1

[0761] P61982 Ywhag Mus mul4-3-3 protein gamma 0.00590948 3.1

[0762] P46638;P62 Rabi lb;Ra Mus muRas-related protein Rab-1 lB;Ras-rela 0.00441159 3.0

[0763] Q99KB8 Hagh Mus muHydroxyacylglutathione hydrolase, m 0.02314717 3.0 P27005 S100a8 Mus muProtein S100-A8 0.01687817 3.0 P23492 Pnp Mus muPurine nucleoside phosphorylase 0.01082397 2.9 Q9Z2M7 Pmm2 Mus muPhosphomannomutase 2 0.02579495 2.9

[0764] P0CG49;P0CUbb;Ubc;RMus muPolyubiquitin-B;Polyubiquitin-C;Ubiq 0.00456974 Pl 0649 Gstml Mus muGlutathione S -transferase Mu 1 0.01069432 2.8 P58252 Eef2 Mus muElongation factor 2 0.01661808 2.8 Q9D1A2 Cndp2 Mus muCytosolic non-specific dipeptidase 0.01270203 2.8 P58774 Tpm2 Mus muTropomyosin beta chain 0.00870383 2.8 P31725 S100a9 Mus muProtein S100-A9 0.00864324 2.7 P63001 Rael Mus muRas-related C3 botulinum toxin subst 0.00283042 2.7 Q01768 Nme2 Mus muNucleoside diphosphate kinase B 0.01094361 2.7 P61089 Ube2n Mus muUbiquitin-conjugating enzyme E2 N 0.00503612 2.7 P60766 Cdc42 Mus muCell division control protein 42 homo 0.01328459 2.7 P00920 Ca2 Mus muCarbonic anhydrase 2 0.00190212 2.6 P54822 Adsl Mus muAdenylosuccinate lyase 0.02354117 2.5 P97371 Psmel Mus muProteasome activator complex subu 0.00187555 2.5 Q8K274 Fn3krp Mus muKetosamine-3-kinase 0.02160293 2.5 Q8CIZ8 Vwf Mus muvon Willebrand factor 0.02010486 2.3 Q64727 Vcl Mus muVinculin 0.00136268 2.2 P27773 Pdia3 Mus muProtein disulfide-isomerase A3 0.00666321 2.2 P63028 Tptl Mus muTranslationally-controlled tumor pro 0.02504335 2.2 P26041 Msn Mus muMoesin 0.02043352 2.1 P59999 Arpc4 Mus muActin-related protein 2 / 3 complex su 0.01526526 2.1 Q61316 Hspa4 Mus muHeat shock 70 kDa protein 4 0.00155815 2.1 P62737 Acta2 Mus muActin, aortic smooth muscle 0.01343218 2.1 Q6P069 Sri Mus muSorcin 0.02077547 2.0 Q99JY9 Actr3 Mus muActin-related protein 3 0.01241861 2.0 P52480 Pkm Mus muPyruvate kinase PKM 0.02240104 2.0 Q9JM76 Arpc3 Mus muActin-related protein 2 / 3 complex su 0.0010925 2.0

[0765] Q9CVB6 Arpc2 Mus muActin-related protein 2 / 3 complex su 0.00713909 1.8 035744 Chil3 Mus muChitinase-like protein 3 0.00711558 1.8 P14069 S100a6 Mus muProtein S100-A6 0.02278302 1.8 P08905 Lyz2 Mus muLysozyme C-2 0.02035164 1.8 P60710 Actb Mus muActin, cytoplasmic 1 0.00195487 1.8 P70195 Psmb7 Mus muProteasome subunit beta type -7 0.00675302 1.7 Q64442 Sord Mus muSorbitol dehydrogenase 0.01528036 1.6 Q9R1P3 Psmb2 Mus muProteasome subunit beta type -2 0.00074471 1.6 Q9Z2U0 Psma7 Mus muProteasome subunit alpha type -7 0.00213876 1.6 070435 Psma3 Mus muProteasome subunit alpha type -3 0.00083265 1.5

Claims

CLAIMS1. A liquid biopsy for use in a method of diagnosing, monitoring and / or treating at least one condition in the CNS, the method comprises measuring levels of at least one CNS-derived biomarker in at least one liquid biopsy obtained from a subject who is or was subjected to a Barrier Disrupting Fields (BDF) under conditions optimized for inducing a selective brain barrier opening.

2. The liquid biopsy for use according to claim 1, wherein the selective brain barrier opening comprises at least one of blood-brain barrier opening (BBBo), blood-arachnoid barrier opening (BABo) and / or blood-cerebrospinal-fluid (CSF) barrier opening (BCSFBo).

3. The liquid biopsy for use according to claim 1 to 2, wherein said method is repeated on more than one liquid biopsy obtained from the subject before and after BDF, or liquid biopsies obtained from the subject upon repeated BDF exposures.

4. The liquid biopsy for use according to any one of claims 1 to 3, wherein the method further comprises comparing the levels of the at least one CNS-derived biomarker measured in liquid biopsies of the subject, or comparing said levels to the level of the same biomarker in liquid biopsies obtained from healthy individuals.

5. The liquid biopsy for use according to any one of the preceding claims, wherein the at least one liquid biopsy is a sample of blood, plasma, serum, CSF or urine.

6. The liquid biopsy of any one of the preceding claims, wherein the BDF is a Low Pulsed Electric Fields (L-PEFs).

7. The liquid biopsy of any one of the preceding claims, wherein the least one measured CNS- derived biomarker is selected from the group of protein, lipid or DNA or RNA molecules, or any derivative thereof.

8. The liquid biopsy for use according to any one of the preceding claims, wherein the at least one measured CNS-derived biomarker is related to the opening of BBB, BAB and / or BCSFB, and the level of said biomarker in liquid biopsies is related to the opening level.

9. The liquid biopsy for use according to any one of the preceding claims, wherein the at least one measured CNS-derived biomarker is related to a CNS disorder or disease, and the level of said biomarker in liquid biopsies is related to the disorder or disease progression and / or severity.

10. The liquid biopsy for use according to claim 9, wherein said CNS disorder or disease is selected from the groups of neurologic, neurodevelopmental, neurodegenerative, cerebrovascular, psychiatric disorders or diseases, infections or traumatic injury in the CNS, and benign or malignant tumors in the CNS, primary or secondary.

11. The liquid biopsy for use according to claim 10, wherein said CNS disorder or disease is selected from the group of stroke, a brain tumor, a traumatic brain injury, cerebral ischemia, hypertension, an amyloid angiopathy, Alzheimer's disease (AD), Parkinson's disease (PD), Huntington disease (HD),Amyotrophic lateral sclerosis (ALS), multiple sclerosis (MS), a muscular dystrophy, essential tremor, schizophrenia, major depression, meningitis, epilepsy and encephalitis.

12. The liquid biopsy for use according to any one of the preceding claims, wherein the subject is or was further subjected to at least one pharmacological or technological CNS acting treatment, and the level of the at least one CNS-derived biomarker measured in liquid biopsies is related to the level of treatment response.

13. The liquid biopsy for use according to claim 12, wherein said at least one pharmacological or technological CNS acting treatment is selected from the groups of radiation, thermal, brain stimulation, drug or genetic therapy, photodynamic therapy (PDT), electroconvulsive therapy (ECT), an ablation, or a combination thereof.

14. The liquid biopsy for use according to claim 13, said CNS acting drug therapy comprises at least one drug selected from the groups of anticancer, antiviral, anti-inflammatory, neurologic, psychiatric, vascular or genetic drugs.

15. The liquid biopsy for use according to any one of the preceding claims, wherein the at least one measured CNS-derived biomarker is a neurotoxin or neuro -damaging substance, and the level of said biomarker in liquid biopsies is related to the level of neurotoxicity clearance .

16. The liquid biopsy for use according to any one of the preceding claims, wherein the at least one measured CNS-derived biomarker is selected from the group of sphingomyelin-derived stearic acid, Osteopontin (OPN), SlOOp protein, a Gelectin, an Annexin, a Cathepsin, a Syntaxin, a Claudin, a Catenin, a Reticulon, Synaptogyrin, a Prostaglandin isomerase or reductase, Amyloid P (AP) or Tau protein.

17. The liquid biopsy for use according to any one of the preceding claims, wherein the at least one measured CNS-derived biomarker is selected from the group of 0-6 methylguanine-DNA methyltransferase, Epidermal growth factor receptor, Isocitrate dehydrogenase, Glial fibrillary acidic protein, Telomerase reverse transcriptase (TERT), Tumor protein 53 (TP53).

18. The liquid biopsy for use according to any one of the preceding claims, wherein the at least one measured CNS-derived biomarker is selected from the groups of circulating free DNA, circulating cell- free microRNAs, circulating extracellular vesicles, circulating proteins, circulating tumor cells.

19. The liquid biopsy for use according to any one of the preceding claims, wherein the BDF is induced by one or more electric pulse sources and / or alternating electromagnetic sources.

20. The liquid biopsy for use according to any one of the preceding claims, wherein the BDF is induced by a plurality of single or multiple grounded or energized electrodes connected to the same or multiple electric sources.

21. The liquid biopsy for use according to claim 20, wherein said plurality of electrodes comprises between 2 to 1200 pairs of electrodes with at least one pair of electrodes delivering similar or different pulse frequency and / or strength.

22. The liquid biopsy for use according to claim 20 or 21, wherein said electrodes are selected from surface electrodes, screw electrodes, microelectrodes, implantable electrodes and coil electrodes, optionally of a diameter in the range of 5 pm to about 10 cm.

23. The liquid biopsy for use according to any one of the preceding claims, wherein the BDF conditions optimized for inducing a selective brain barrier opening are selected from at least one of:— electric current in the range between about 1 mA and about 1 A,— voltage in the range between about 10 V and about 1000 V,— pulse duration in the range between about 5 ns and about 10 ms,— pulse frequency in the range between about 0.5 Hz and about 100 KHz,— number of pulses in the range between about 1 to about 1000, and— pulse shape selected from square, triangular, sinus or exponential.

24. The liquid biopsy for use according to any one of the preceding claims, wherein the BDF conditions optimized for inducing a selective brain barrier opening comprise a pulsed electric field at a frequency of less than about 75 Hz in a brain tissue.

25. The liquid biopsy for use according to any one of the preceding claims, wherein the BDF conditions optimized for inducing a selective brain barrier opening comprise a pulsed electric field strength of less than about 148 V / cm in a brain tissue.

26. The liquid biopsy for use according to any one of the preceding claims, wherein BDF is provided to the subject by a wearable device.

27. A method for diagnosing, monitoring and / or treating at least one condition in the CNS, the method comprising: i. obtaining at least one liquid biopsy from a subject who is or was subjected to a BDF under conditions optimized for inducing a selective brain barrier opening, and ii. measuring levels of at least one CNS-derived biomarker in said at least one liquid biopsy of said subject.

28. The method of claim 27, wherein the selective brain barrier opening comprises at least one of BBBo, BABo and / or BCSFBo.

29. The method of claim 27 to 28, wherein said method is repeated on more than one liquid biopsy obtained from the subject before and after BDF, or liquid biopsies obtained from the subject upon repeated BDF exposures.

30. The method of any one of claims 27 to 29, further comprising:iii. comparing the levels of the at least one CNS-derived biomarker measured in liquid biopsies of the subject, or comparing said levels to the level of the same biomarker in liquid biopsies obtained from healthy individuals.

31. A method for diagnosing, monitoring and / or treating at least one condition in the CNS, in a subject in need thereof, the method comprising: i. administering a BDF to the subject under conditions optimized for inducing a selective brain barrier opening, BBBo, BABo and / or BCSFBo, ii. measuring levels of at least one CNS-derived biomarker in at least one liquid biopsy obtained from the subject before and after BDF, and iii. comparing the level of said at least one biomarker in said liquid biopsies, and optionally iv. repeating steps (i) to (iii) in the same subject in the course of time.

32. The method of any one of claims 27 to 31, wherein the at least one liquid biopsy is a sample of blood, plasma, serum, CSF or urine.

33. The method of any one of claims 27 to 32, wherein the BDF is a L-PEFs.

34. The method of any one of claims 27 to 33, wherein the least one measured CNS-derived biomarker is selected from the groups of protein, lipid or DNA or RNA molecules, or any derivative thereof.

35. The method of any one of claims 27 to 34, wherein the at least one measured CNS-derived biomarker is related to the opening of BBB, BAB and / or BCSFB, and the level of said biomarker in liquid biopsies is related to the opening level.

36. The method of any one of claims 27 to 35, wherein the at least one measured CNS-derived biomarker is related to a CNS disorder or disease, and the level of said biomarker in liquid biopsies is related to the disorder or disease progression and / or severity.

37. The method of claim 36, wherein said CNS disorder or disease is selected from the groups of neurologic, neurodevelopmental, neurodegenerative, cerebrovascular, psychiatric disorders or diseases, infections or traumatic injury in the CNS, and benign or malignant tumors in the CNS, primary or secondary.

38. The method of claim 37, wherein said CNS disorder or disease is selected from the group of stroke, a brain tumor, atraumatic brain injury, cerebral ischemia, hypertension, an amyloid angiopathy, Alzheimer's disease (AD), Parkinson's disease (PD), Huntington disease (HD), Amyotrophic lateral sclerosis (ALS), multiple sclerosis (MS), a muscular dystrophy, essential tremor, schizophrenia, major depression, meningitis, epilepsy and encephalitis.

39. The method of any one of claims 27 to 38, further comprising administering to the subject at least one pharmacological or technological CNS acting treatment, and the level of the at least one CNS- derived biomarker measured in liquid biopsies is related to the level of treatment response.

40. The method of claim 39, wherein said at least one pharmacological or technological CNS acting treatment is selected from the groups of radiation, thermal, brain stimulation, drug or genetic therapy, photodynamic therapy (PDT), electroconvulsive therapy (ECT), an ablation, or a combination thereof.

41. The method of claim 40, said CNS acting drug therapy comprises at least one drug selected from the groups of anticancer, antiviral, anti-inflammatory, neurologic, psychiatric, vascular or genetic drugs.

42. The method of any one of claims 27 to 41, wherein the at least one measured CNS-derived biomarker is a neurotoxin or neuro-damaging substance, and the level of said biomarker in liquid biopsies is related to the level of neurotoxicity clearance.

43. The method of any one of claims 27 to 42, wherein the at least one measured CNS-derived biomarker is selected from the group of sphingomyelin -derived stearic acid, Osteopontin (OPN), SlOOp protein, a Gelectin, an Annexin, a Cathepsin, a Syntaxin, a Claudin, a Catenin, a Reticulon, Synaptogyrin, a Prostaglandin isomerase or reductase, Amyloid P (AP) or Tau protein.

44. The method of any one of claims 27 to 43, wherein the at least one measured CNS-derived biomarker is selected from the group of 0-6 methylguanine-DNA methyltransferase, Epidermal growth factor receptor, Isocitrate dehydrogenase, Glial fibrillary acidic protein, Telomerase reverse transcriptase (TERT), Tumor protein 53 (TP53).

45. The method of any one of claims 27 to 44, wherein the at least one measured CNS-derived biomarker is selected from the groups of circulating free DNA, circulating cell-free microRNAs, circulating extracellular vesicles, circulating proteins, circulating tumor cells.

46. The method of any one of claims 27 to 45, wherein the BDF is administered by one or more electric pulse sources and / or alternating electromagnetic sources.

47. The method of any one of claims 27 to 46, wherein the BDF is administered by a plurality of single or multiple grounded or energized electrodes connected to the same or multiple electric sources.

48. The method of claim 47, wherein said plurality of electrodes comprises between 2 to 1200 pairs of electrodes with at least one pair of electrodes delivering similar or different pulse frequency and / or strength.

49. The method of claim 47 or 48, wherein said electrodes are selected from surface electrodes, screw electrodes, microelectrodes, implantable electrodes and coil electrodes, optionally of a diameter in the range of 5 pm to about 10 cm.

50. The method of any one of claims 27 to 49, wherein the BDF conditions optimized for inducing a selective brain barrier opening are selected from at least one of:— electric current in the range between about 1 mA and about 1 A,— voltage in the range between about 10 V and about 1000 V,— pulse duration in the range between about 5 ns and about 10 ms,— pulse frequency in the range between about 0.5 Hz and about 100 KHz,— number of pulses in the range between about 1 to about 1000, and— pulse shape selected from square, triangular, sinus or exponential.

51. The method of any one of claims 27 to 50, wherein the BDF conditions optimized for inducing a selective brain barrier opening comprise a pulsed electric field at a frequency of less than about 75 Hz in a brain tissue.

52. The method of any one of claims 27 to 51, wherein the BDF conditions optimized for inducing a selective brain barrier opening comprise a pulsed electric field strength of less than about 148 V / cm in a brain tissue.

53. The method of any one of claims 27 to 52, wherein BDF is administered to the subject by a wearable device.

54. A system comprising a plurality of single or multiple grounded or energized electrodes configured and operable to provide a BDF to a subject in need of being diagnosed, monitored and / or treated for at least one condition in the CNS, wherein the BDF is provided under conditions optimized for inducing selective brain barrier opening, BBBo, BABo and / or BCSFBo, and wherein the subject has provided at least one liquid biopsy for measuring levels of at least one CNS-derived biomarker.

55. The system of claim 54, wherein the BDF is provided to the subject by a wearable device.

56. The system of claim 54 or 55, wherein said at least one liquid biopsy was provided before and / or after the BDF.

57. The system of ant one of claims 54 to 56, wherein said at least one liquid biopsy is selected from a sample of blood, plasma, serum, CSF or urine.

58. The system of any one of claims 54 to 57, wherein the BDF is a L-PEFs.

59. The system of any one of claims 54 to 58, wherein the BDF is induced by one or more electric pulse sources and / or alternating electromagnetic sources.

60. The system of any one of claims 54 or 59, wherein said the BDF is induced by a plurality of single or multiple grounded or energized electrodes connected to the same or multiple electric sources.

61. The system of claim 60, wherein said plurality of electrodes comprises between 2 to 1200 pairs of electrodes with at least one pair of electrodes delivering similar or different pulse frequency and / or strength.

62. The system of claims 60 or 61, wherein said electrodes are selected from surface electrodes, screw electrodes, microelectrodes, implantable electrodes and coil electrodes, optionally of a diameter in the range of 5 pm to about 10 cm.

63. The system of any one of claims 54 or 62, wherein the BDF conditions optimized for inducing a selective brain barrier opening are selected from at least one of:— electric current in the range between about 1 mA and about 1 A,— voltage in the range between about 10 V and about 1000 V,— pulse duration in the range between about 5 ns and about 10 ms,— pulse frequency in the range between about 0.5 Hz and about 100 KHz,— number of pulses in the range between about 1 to about 1000, and— pulse shape selected from square, triangular, sinus or exponential.

64. The system of any one of claims 54 or 63, wherein the BDF conditions optimized for inducing a selective brain barrier opening comprise a pulsed electric field at a frequency of less than about 75 Hz in a brain tissue.

65. The system of any one of claims 54 or 63, wherein the BDF conditions optimized for inducing a selective brain barrier opening comprise a pulsed electric field strength of less than about 148 V / cm in a brain tissue.

66. The system of any one of claims 54 to 65, wherein the at least one measured CNS-derived biomarker is related to the opening of BBB, BAB and / or BCSFB, and the level of said biomarker in liquid biopsies is related to the opening level.

67. The system of any one of claims 54 to 66, wherein the at least one measured CNS-derived biomarker is related to a CNS disorder or disease, and the level of said biomarker in liquid biopsies is related to the disorder or disease progression and / or severity.

68. The system of claim 67, wherein said at least one CNS disorder or disease is defined as in any one of claims 9 to 11.

69. The system of any one of claims 54 to 66, wherein said the at least one measured CNS-derived biomarker is related to response to at least one pharmacological or technological CNS acting treatment, and the level of said biomarker in liquid biopsies is related to the level of treatment response.

70. The system of claim 69, wherein said at least one pharmacological or technological CNS acting treatment is selected from the groups of radiation, thermal, brain stimulation, drug or genetic therapy, photodynamic therapy (PDT), electroconvulsive therapy (ECT), an ablation, or a combination thereof.

71. The system of claim 70, wherein said CNS acting drug therapy comprises at least one drug selected from the groups of anticancer, antiviral, anti-inflammatory, neurologic, psychiatric, vascular or genetic drugs.

72. The system of any one of claims 54 to 71, wherein the at least one measured CNS-derived biomarker is a neurotoxin or neuro-damaging substance, and the level of said biomarker in liquid biopsies is related to the level of neurotoxicity clearance.

73. The system of any one of claims 54 to 72, wherein the at least one measured CNS-derived biomarker is defined as in any one of claims 7 to 18.

74. A system for use in a method for diagnosing and monitoring the progression of a disorder or a disease in the CNS, the system being defined as in any one of claims 54 to 73.

75. A system for monitoring response to a pharmacological or technological CNS acting treatment, the system being defined as in any one of claims 54 to 73.

76. A system for inducing neurotoxicity clearance, the system being defined as in any one of claims 54 to 73.

77. A method for diagnosing and monitoring the progression of a disorder or a disease in the CNS in a subject suffering therefrom, the method being defined as in any one of claims 27 to 53.

78. A method for monitoring response to a pharmacological or technological CNS acting treatment in a subject subjected thereto, the method being defined as in any one of claims 27 to 53.

79. A method for inducing neurotoxicity clearance in a subject in need thereof, the method being defined as in any one of claims 27 to 53.

80. A method of inducing neurotoxicity clearance in a subject in need thereof, the method comprises applying to the subject a BDF under conditions optimized for inducing a selective brain barrier opening and measuring levels of at least one neurotoxin or neuro -damaging substance in at least one liquid biopsy obtained from the subject before and after BDF.

Images