Plasticity of Response Properties of Mouse Visual Cortex Neurons Induced by Optogenetic Tetanization In Vivo.

Heterosynaptic plasticity, along with Hebbian homosynaptic plasticity, is an important mechanism ensuring the stable operation of learning neuronal networks. However, whether heterosynaptic plasticity occurs in the whole brain in vivo, and what role(s) in brain function in vivo it could play, remains unclear. Here, we used an optogenetics approach to apply a model of intracellular tetanization, which was established and employed to study heterosynaptic plasticity in brain slices, to study the plasticity of response properties of neurons in the mouse visual cortex in vivo. We show that optogenetically evoked high-frequency bursts of action potentials (optogenetic tetanization) in the principal neurons of the visual cortex induce long-term changes in the responses to visual stimuli. Optogenetic tetanization had distinct effects on responses to different stimuli, as follows: responses to optimal and orthogonal orientations decreased, responses to null direction did not change, and responses to oblique orientations increased. As a result, direction selectivity of the neurons decreased and orientation tuning became broader. Since optogenetic tetanization was a postsynaptic protocol, applied in the absence of sensory stimulation, and, thus, without association of presynaptic activity with bursts of action potentials, the observed changes were mediated by mechanisms of heterosynaptic plasticity. We conclude that heterosynaptic plasticity can be induced in vivo and propose that it may play important homeostatic roles in operation of neural networks by helping to prevent runaway dynamics of responses to visual stimuli and to keep the tuning of neuronal responses within the range optimized for the encoding of multiple features in population activity.

Understanding Complex Interplay among Different Instabilities in Multiferroic BiMn7O12 Using 57Fe Probe Mössbauer Spectroscopy.

Here, we report the results of a Mössbauer study on hyperfine electrical and magnetic interactions in quadruple perovskite BiMn7O12 doped with 57Fe probes. Measurements were performed in the temperature range of 10 K < T < 670 K, wherein BiMn6.9657Fe0.04O12 undergoes a cascade of structural (T1 ≈ 590 K, T2 ≈ 442 K, and T3 ≈ 240 K) and magnetic (TN1 ≈ 57 K, TN2 ≈ 50 K, and TN3 ≈ 24 K) phase transitions. The analysis of the electric field gradient (EFG) parameters, including the dipole contribution from Bi3+ ions, confirmed the presence of the local dipole moments pBi, which are randomly oriented in the paraelectric cubic phase (T > T1). The unusual behavior of the parameters of hyperfine interactions between T1 and T2 was attributed to the dynamic Jahn-Teller effect that leads to the softening of the orbital mode of Mn3+ ions. The parameters of the hyperfine interactions of 57Fe in the phases with non-zero spontaneous electrical polarization (Ps), including the P1 ↔ Im transition at T3, were analyzed. On the basis of the structural data and the quadrupole splitting Δ(T) derived from the 57Fe Mössbauer spectra, the algorithm, based on the Born effective charge model, is proposed to describe Ps(T) dependence. The Ps(T) dependence around the Im ↔ I2/m phase transition at T2 is analyzed using the effective field approach. Possible reasons for the complex relaxation behavior of the spectra in the magnetically ordered states (T < TN1) are also discussed.

Different vulnerability of fast and slow cortical oscillations to suppressive effect of spreading depolarization: state-dependent features potentially relevant to pathogenesis of migraine aura.

BACKGROUND: Spreading depolarization (SD), underlying mechanism of migraine aura and potential activator of pain pathways, is known to elicit transient local silencing cortical activity. Sweeping across the cortex, the electrocorticographic depression is supposed to underlie spreading negative symptoms of migraine aura. Main information about the suppressive effect of SD on cortical oscillations was obtained in anesthetized animals while ictal recordings in conscious patients failed to detect EEG depression during migraine aura. Here, we investigate the suppressive effect of SD on spontaneous cortical activity in awake animals and examine whether the anesthesia modifies the SD effect. METHODS: Spectral and spatiotemporal characteristics of spontaneous cortical activity following a single unilateral SD elicited by amygdala pinprick were analyzed in awake freely behaving rats and after induction of urethane anesthesia. RESULTS: In wakefulness, SD transiently suppressed cortical oscillations in all frequency bands except delta. Slow delta activity did not decline its power during SD and even increased it afterwards; high-frequency gamma oscillations showed the strongest and longest depression under awake conditions. Unexpectedly, gamma power reduced not only during SD invasion the recording cortical sites but also when SD occupied distant subcortical/cortical areas. Contralateral cortex not invaded by SD also showed transient depression of gamma activity in awake animals. Introduction of general anesthesia modified the pattern of SD-induced depression: SD evoked the strongest cessation of slow delta activity, milder suppression of fast oscillations and no distant changes in gamma activity. CONCLUSION: Slow and fast cortical oscillations differ in their vulnerability to SD influence, especially in wakefulness. In the conscious brain, SD produces stronger and spatially broader depression of fast cortical oscillations than slow ones. The frequency-specific effects of SD on cortical activity of awake brain may underlie some previously unexplained clinical features of migraine aura.

Overexpression of KCNN4 channels in principal neurons produces an anti-seizure effect without reducing their coding ability.

Gene therapy offers a potential alternative to the surgical treatment of epilepsy, which affects millions of people and is pharmacoresistant in ~30% of cases. Aimed at reducing the excitability of principal neurons, the engineered expression of K+ channels has been proposed as a treatment due to the outstanding ability of K+ channels to hyperpolarize neurons. However, the effects of K+ channel overexpression on cell physiology remain to be investigated. Here we report an adeno-associated virus (AAV) vector designed to reduce epileptiform activity specifically in excitatory pyramidal neurons by expressing the human Ca2+-gated K+ channel KCNN4 (KCa3.1). Electrophysiological and pharmacological experiments in acute brain slices showed that KCNN4-transduced cells exhibited a Ca2+-dependent slow afterhyperpolarization that significantly decreased the ability of KCNN4-positive neurons to generate high-frequency spike trains without affecting their lower-frequency coding ability and action potential shapes. Antiepileptic activity tests showed potent suppression of pharmacologically induced seizures in vitro at both single cell and local field potential levels with decreased spiking during ictal discharges. Taken together, our findings strongly suggest that the AAV-based expression of the KCNN4 channel in excitatory neurons is a promising therapeutic intervention as gene therapy for epilepsy.

Dissimilar Effect of P-Glycoprotein and Breast Cancer Resistance Protein Inhibition on the Distribution of Erlotinib to the Retina and Brain in Humans and Mice.

P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP) are two ATP-binding cassette efflux transporters that are coexpressed at the human blood-brain barrier (BBB) and blood-retina barrier (BRB). While pharmacological inhibition of P-gp and/or BCRP results in increased brain distribution of dual P-gp/BCRP substrate drugs, such as the tyrosine kinase inhibitor erlotinib, the effect of P-gp and/or BCRP inhibition on the retinal distribution of such drugs has hardly been investigated. In this study, we used positron emission tomography (PET) imaging to assess the effect of transporter inhibition on the distribution of [11C]erlotinib to the human retina and brain. Twenty two healthy volunteers underwent two PET scans after intravenous (i.v.) injection of a microdose (<5 µg) of [11C]erlotinib, a baseline scan, and a second scan either with concurrent i.v. infusion of tariquidar to inhibit P-gp (n = 5) or after oral intake of single ascending doses of erlotinib (300 mg, 650 mg, or 1000 mg, n = 17) to saturate erlotinib transport. In addition, transport of [3H]erlotinib to the retina and brain was assessed in mice by in situ carotid perfusion under various drug transporter inhibition settings. In comparison to the baseline PET scan, coadministration of tariquidar or erlotinib led to a significant decrease of [11C]erlotinib total volume of distribution (VT) in the human retina by -25 ± 8% (p ≤ 0.05) and -41 ± 16% (p ≤ 0.001), respectively. In contrast, erlotinib intake led to a significant increase in [11C]erlotinib VT in the human brain (+20 ± 16%, p ≤ 0.001), while administration of tariquidar did not result in any significant changes. In situ carotid perfusion experiments showed that both P-gp and BCRP significantly limit the distribution of erlotinib to the mouse retina and brain but revealed a similar discordant effect at the mouse BRB and BBB following co-perfusion with tariquidar and erlotinib as in humans. Co-perfusion with prototypical inhibitors of solute carrier transporters did not reveal a significant contribution of organic cation transporters (e.g., OCTs and OCTNs) and organic anion-transporting polypeptides (e.g., OATP2B1) to the retinal and cerebral distribution of erlotinib. In conclusion, we observed a dissimilar effect after P-gp and/or BCRP inhibition on the retinal and cerebral distribution of [11C]erlotinib. The exact mechanism for this discrepancy remains unclear but may be related to the function of an unidentified erlotinib uptake carrier sensitive to tariquidar inhibition at the BRB. Our study highlights the great potential of PET to study drug distribution to the human retina and to assess the functional impact of membrane transporters on ocular drug distribution.

Jupiter Science Enabled by ESA's Jupiter Icy Moons Explorer.

ESA's Jupiter Icy Moons Explorer (JUICE) will provide a detailed investigation of the Jovian system in the 2030s, combining a suite of state-of-the-art instruments with an orbital tour tailored to maximise observing opportunities. We review the Jupiter science enabled by the JUICE mission, building on the legacy of discoveries from the Galileo, Cassini, and Juno missions, alongside ground- and space-based observatories. We focus on remote sensing of the climate, meteorology, and chemistry of the atmosphere and auroras from the cloud-forming weather layer, through the upper troposphere, into the stratosphere and ionosphere. The Jupiter orbital tour provides a wealth of opportunities for atmospheric and auroral science: global perspectives with its near-equatorial and inclined phases, sampling all phase angles from dayside to nightside, and investigating phenomena evolving on timescales from minutes to months. The remote sensing payload spans far-UV spectroscopy (50-210 nm), visible imaging (340-1080 nm), visible/near-infrared spectroscopy (0.49-5.56 µm), and sub-millimetre sounding (near 530-625 GHz and 1067-1275 GHz). This is coupled to radio, stellar, and solar occultation opportunities to explore the atmosphere at high vertical resolution; and radio and plasma wave measurements of electric discharges in the Jovian atmosphere and auroras. Cross-disciplinary scientific investigations enable JUICE to explore coupling processes in giant planet atmospheres, to show how the atmosphere is connected to (i) the deep circulation and composition of the hydrogen-dominated interior; and (ii) to the currents and charged particle environments of the external magnetosphere. JUICE will provide a comprehensive characterisation of the atmosphere and auroras of this archetypal giant planet.

Trait Loss in Evolution: What Cavefish Have Taught Us about Mechanisms Underlying Eye Regression.

Reduction or complete loss of traits is a common occurrence throughout evolutionary history. In spite of this, numerous questions remain about why and how trait loss has occurred. Cave animals are an excellent system in which these questions can be answered, as multiple traits, including eyes and pigmentation, have been repeatedly reduced or lost across populations of cave species. This review focuses on how the blind Mexican cavefish, Astyanax mexicanus, has been used as a model system for examining the developmental, genetic, and evolutionary mechanisms that underlie eye regression in cave animals. We focus on multiple aspects of how eye regression evolved in A. mexicanus, including the developmental and genetic pathways that contribute to eye regression, the effects of the evolution of eye regression on other traits that have also evolved in A. mexicanus, and the evolutionary forces contributing to eye regression. We also discuss what is known about the repeated evolution of eye regression, both across populations of A. mexicanus cavefish and across cave animals more generally. Finally, we offer perspectives on how cavefish can be used in the future to further elucidate mechanisms underlying trait loss using tools and resources that have recently become available.

Dynamic Interleukin-1 Receptor Type 1 Signaling Mediates Microglia-Vasculature Interactions Following Repeated Systemic LPS.

Introduction: Lipopolysaccharide (LPS) preconditioning involves repeated, systemic, and sub-threshold doses of LPS, which induces a neuroprotective state within the CNS, thus preventing neuronal death and functional losses. Recently, proinflammatory cytokine, Interleukin-1 (IL-1), and its primary signaling partner, interleukin-1 receptor type 1 (IL-1R1), have been associated with neuroprotection in the CNS. However, it is still unknown how IL-1/IL-1R1 signaling impacts the processes associated with neuroprotection. Methods: Using our IL-1R1 restore genetic mouse model, mouse lines were generated to restrict IL-1R1 expression either to endothelia (Tie2-Cre-Il1r1r/r) or microglia (Cx3Cr1-Cre-Il1r1 r/r), in addition to either global ablation (Il1r1 r/r) or global restoration of IL-1R1 (Il1r1 GR/GR). The LPS preconditioning paradigm consisted of four daily i.p. injections of LPS at 1 mg/kg (4d LPS). 24 hrs following the final i.p. LPS injection, tissue was collected for qPCR analysis, immunohistochemistry, or FAC sorting. Results: Following 4d LPS, we found multiple phenotypes that are dependent on IL-1R1 signaling such as microglia morphology alterations, increased microglial M2-like gene expression, and clustering of microglia onto the brain vasculature. We determined that 4d LPS induces microglial morphological changes, clustering at the vasculature, and gene expression changes are dependent on endothelial IL-1R1, but not microglial IL-1R1. A novel observation was the induction of microglial IL-1R1 (mIL-1R1) following 4d LPS. The induced mIL-1R1 permits a unique response to central IL-1ß: the mIL-1R1 dependent induction of IL-1R1 antagonist (IL-1RA) and IL-1ß gene expression. Analysis of RNA sequencing datasets revealed that mIL-1R1 is also induced in neurodegenerative diseases. Discussion: Here, we have identified cell type-specific IL-1R1 mediated mechanisms, which may contribute to the neuroprotection observed in LPS preconditioning. These findings identify key cellular and molecular contributors in LPS-induced neuroprotection.

A New Approach to the Formation of Nanosized Gold and Beryllium Films by Ion-Beam Sputtering Deposition.

Thin films of beryllium and gold that are several tens of nanometers thick were obtained, for the first time, on silicon and quartz substrates by the ion-beam method with tenfold alternation of deposition and partial sputtering of the nanosized metal layer. Scanning electron and atomic force microscopy indicate the predominant lateral growth of nanosized metal layers along the substrate surface. Optical spectra indicate the suppression of the localized plasmon resonance. The growth of the film occurs under the influence of the high-energy component of the sputtered metal atoms' flux. The main role in the formation of the nanosized metal film is played by the processes of the elastic collision of incident metal atoms with the atoms of a substrate and a growing metal film. Metal films that are obtained by the tenfold application of the deposition-sputtering of a nanoscale metal layer are characterized by stronger adhesion to the substrate and have better morphological, electrical, and optical characteristics than those that are obtained by means of direct single deposition.

Pharmacophore-Based Discovery of Substrates of a Novel Drug/Proton-Antiporter in the Human Brain Endothelial hCMEC/D3 Cell Line.

A drug/proton-antiporter, whose the molecular structure is still unknown, was previously evidenced at the blood-brain barrier (BBB) by functional experiments. The computational method could help in the identification of substrates of this solute carrier (SLC) transporter. Two pharmacophore models for substrates of this transporter using the FLAPpharm approach were developed. The trans-stimulation potency of 40 selected compounds for already known specific substrates ([3H]-clonidine) were determined and compared in the human brain endothelial cell line hCMEC/D3. Results. The two pharmacophore models obtained were used as templates to screen xenobiotic and endogenous compounds from four databases (e.g., Specs), and 45 hypothetical new candidates were tested to determine their substrate capacity. Psychoactive drugs such as antidepressants (e.g., imipramine, desipramine), antipsychotics/neuroleptics such as phenothiazine derivatives (chlorpromazine), sedatives anti-histamine-H1 drugs (promazine, promethazine, triprolidine, pheniramine), opiates/opioids (e.g., hydrocodone), trihexyphenidyl and sibutramine were correctly predicted as proton-antiporter substrates. The best performing pharmacophore model for the proton-antiporter substrates appeared as a good predictor of known substrates and allowed the identification of new substrate compounds. This model marks a new step in the characterization of this drug/proton-antiporter and will be of great use in uncovering its substrates and designing chemical entities with an improved influx capability to cross the BBB.

A Single Episode of Cortical Spreading Depolarization Increases mRNA Levels of Proinflammatory Cytokines, Calcitonin Gene-Related Peptide and Pannexin-1 Channels in the Cerebral Cortex.

Cortical spreading depolarization (CSD) is the neuronal correlate of migraine aura and the reliable consequence of acute brain injury. The role of CSD in triggering headaches that follow migraine aura and brain injury remains to be uncertain. We examined whether a single CSD occurring in awake animals modified the expression of proinflammatory cytokines (Il1b, TNF, and Il6) and endogenous mediators of nociception/neuroinflammation-pannexin 1 (Panx1) channel and calcitonin gene-related peptide (CGRP), transforming growth factor beta (TGFb) in the cortex. Unilateral microinjury of the somatosensory cortex triggering a single CSD was produced in awake Wistar rats. Three hours later, tissue samples from the lesioned cortex, intact ipsilesional cortex invaded by CSD, and homologous areas of the contralateral sham-treated cortex were harvested and analyzed using qPCR. Three hours post-injury, intact CSD-exposed cortexes increased TNF, Il1b, Panx1, and CGRP mRNA levels. The strongest upregulation of proinflammatory cytokines was observed at the injury site, while CGRP and Panx1 were upregulated more strongly in the intact cortexes invaded by CSD. A single CSD is sufficient to produce low-grade parenchymal neuroinflammation with simultaneous overexpression of Panx1 and CGRP. The CSD-induced molecular changes may contribute to pathogenic mechanisms of migraine pain and post-injury headache.

One-Year Demographical and Clinical Indices of Patients with Chronic Disorders of Consciousness.

This work aims to evaluate the prognostic value of the demographical and clinical data on long-term outcomes (up to 12 months) in patients with severe acquired brain injury with vegetative state/unresponsive wakefulness syndrome (VS/UWS/UWS) or a minimally conscious state (MCS). Patients (n = 211) with VS/UWS/UWS (n = 123) and MCS (n = 88) were admitted to the Federal Research and Clinical Center of Intensive Care Medicine and Rehabilitology after anoxic brain injury (n = 53), vascular lesions (n = 59), traumatic brain injury (n = 93), and other causes (n = 6). At the beginning of the 12-month study, younger age and a higher score by the Coma Recovery Scale-Revised (CRS-R) predicted a survival. However, no reliable markers of significant positive dynamics of consciousness were found. Based on the etiology, anoxic brain injury has the most unfavorable prognosis. For patients with vascular lesions, the first three months after injury have the most important prognostic value. No correlations were found between survival, increased consciousness, and gender. The demographic and clinical characteristics of patients with chronic DOC can be used to predict long-term mortality in patients with chronic disorders of consciousness. Further research should be devoted to finding reliable predictors of recovery of consciousness.

Remotely Self-Healable, Shapeable and pH-Sensitive Dual Cross-Linked Polysaccharide Hydrogels with Fast Response to Magnetic Field.

The development of actuators with remote control is important for the construction of devices for soft robotics. The present paper describes a responsive hydrogel of nontoxic, biocompatible, and biodegradable polymer carboxymethyl hydroxypropyl guar with dynamic covalent cross-links and embedded cobalt ferrite nanoparticles. The nanoparticles significantly enhance the mechanical properties of the gel, acting as additional multifunctional non-covalent linkages between the polymer chains. High magnetization of the cobalt ferrite nanoparticles provides to the gel a strong responsiveness to the magnetic field, even at rather small content of nanoparticles. It is demonstrated that labile cross-links in the polymer matrix impart to the hydrogel the ability of self-healing and reshaping as well as a fast response to the magnetic field. In addition, the gel shows pronounced pH sensitivity due to pH-cleavable cross-links. The possibility to use the multiresponsive gel as a magnetic-field-triggered actuator is demonstrated.

Molecular and Functional Study of Transient Receptor Potential Vanilloid 1-4 at the Rat and Human Blood-Brain Barrier Reveals Interspecies Differences.

Transient receptor potential vanilloid 1-4 (TRPV1-4) expression and functionality were investigated in brain microvessel endothelial cells (BMEC) forming the blood-brain barrier (BBB) from rat and human origins. In rat, Trpv1-4 were detected by qRT-PCR in the brain cortex, brain microvessels, and in primary cultures of brain microvessel endothelial cells [rat brain microvessel endothelial cells (rPBMEC)]. A similar Trpv1-4 expression profile in isolated brain microvessels and rPBMEC was found with the following order: Trpv4 > Trpv2 > Trpv3 > Trpv1. In human, TRPV1-4 were detected in the BBB cell line human cerebral microvessel endothelial cells D3 cells (hCMEC/D3) and in primary cultures of BMEC isolated from human adult and children brain resections [human brain microvascular endothelial cells (hPBMEC)], showing a similar TRPV1-4 expression profile in both hCMEC/D3 cells and hPBMECs as follow: TRPV2 > > TRPV4 > TRPV1 > TRPV3. Western blotting and immunofluorescence experiments confirmed that TRPV2 and TRPV4 are the most expressed TRPV isoforms in hCMEC/D3 cells with a clear staining at the plasma membrane. A fluorescent dye Fluo-4 AM ester was applied to record intracellular Ca2+ levels. TRPV4 functional activity was demonstrated in mediating Ca2+ influx under stimulation with the specific agonist GSK1016790A (ranging from 3 to 1000 nM, EC50 of 16.2 ± 4.5 nM), which was inhibited by the specific TRPV4 antagonist, RN1734 (30 µM). In contrast, TRPV1 was slightly activated in hCMEC/D3 cells as shown by the weak Ca2+ influx induced by capsaicin at a high concentration (3 µM), a highly potent and specific TRPV1 agonist. Heat-induced Ca2+ influx was not altered by co-treatment with a selective potent TRPV1 antagonist capsazepine (20 µM), in agreement with the low expression of TRPV1 as assessed by qRT-PCR. Our present study reveals an interspecies difference between Rat and Human. Functional contributions of TRPV1-4 subtype expression were not identical in rat and human tissues reflective of BBB integrity. TRPV2 was predominant in the human whereas TRPV4 had a larger role in the rat. This interspecies difference from a gene expression point of view should be taken into consideration when modulators of TRPV2 or TRPV4 are investigated in rat models of brain disorders.

Caprine Bactenecins as Promising Tools for Developing New Antimicrobial and Antitumor Drugs.

Proline-rich antimicrobial peptides (PR-AMPs) having a potent antimicrobial activity predominantly toward Gram-negative bacteria and negligible toxicity toward host cells, are attracting attention as new templates for developing antibiotic drugs. We have previously isolated and characterized several bactenecins that are promising in this respect, from the leukocytes of the domestic goat Capra hircus: ChBac5, miniChBac7.5N-α, and -ß, as well as ChBac3.4. Unlike the others, ChBac3.4 shows a somewhat unusual pattern of activities for a mammalian PR-AMP: it is more active against bacterial membranes as well as tumor and, to the lesser extent, normal cells. Here we describe a SAR study of ChBac3.4 (RFRLPFRRPPIRIHPPPFYPPFRRFL-NH2) which elucidates its peculiarities and evaluates its potential as a lead for antimicrobial or anticancer drugs based on this peptide. A set of designed structural analogues of ChBac3.4 was explored for antibacterial activity toward drug-resistant clinical isolates and antitumor properties. The N-terminal region was found to be important for the antimicrobial action, but not responsible for the toxicity toward mammalian cells. A shortened variant with the best selectivity index toward bacteria demonstrated a pronounced synergy in combination with antibiotics against Gram-negative strains, albeit with a somewhat reduced ability to inhibit biofilm formation compared to native peptide. C-terminal amidation was examined for some analogues, which did not affect antimicrobial activity, but somewhat altered the cytotoxicity toward host cells. Interestingly, non-amidated peptides showed a slight delay in their impact on bacterial membrane integrity. Peptides with enhanced hydrophobicity showed increased toxicity, but in most cases their selectivity toward tumor cells also improved. While most analogues lacked hemolytic properties, a ChBac3.4 variant with two additional tryptophan residues demonstrated an appreciable activity toward human erythrocytes. The variant demonstrating the best tumor/nontumor cell selectivity was found to more actively initiate apoptosis in target cells, though its action was slower than that of the native ChBac3.4. Its antitumor effectiveness was successfully verified in vivo in a murine Ehrlich ascites carcinoma model. The obtained results demonstrate the potential of structural modification to manage caprine bactenecins' selectivity and activity spectrum and confirm that they are promising prototypes for antimicrobial and anticancer drugs design.

In Vivo Antimicrobial and Wound-Healing Activity of Resveratrol, Dihydroquercetin, and Dihydromyricetin against Staphylococcus aureus, Pseudomonas aeruginosa, and Candida albicans.

An increase in the spread of antibiotic-resistant opportunistic microorganisms causes serious problems in the treatment of purulent infections, burns, and trophic ulcers. We tested the antimicrobial activity in vivo of three polyphenols, Resveratrol, Dihydroquercetin (Taxifolin), and Dihydromyricetin (Ampelopsin) from Norway spruce bark to promote the elimination of Staphylococcus aureus, Pseudomonas aeruginosa, and Candida albicans from wounds. Purulent infection was modelled on wounds in rats infected with suspensions containing 109 CFU (colony-forming unit)/mL of pathogens. The wound area was treated daily with solutions of the polyphenols or placebo for 14 days after the beginning of the treatment. The animals were examined daily, and each stage of the wound healing (inflammation, granulation, and maturation (marginal epithelialisation) was documented. The planimetric analysis of the wound recovery percentage was performed on the 3rd, 10th, and 14th day after the start of curing. Then, one echelon (three or four animals from each subgroup) was withdrawn from the experiment on days 3 (three animals), 10 (three animals), and 14 (four animals) for microscopy analysis of cytological composition of their wound defects by microscopy and microbiological analysis of their contamination with pathogens. Our results show that they are also able to suppress mast cell infiltration and stimulate lymphocyte and macrophage (monocyte) infiltration into the wound. Resveratrol stimulated the replacement of the scar with normal tissue (with a clear boundary between the dermis and epidermis) and the restoration of hair follicles. Resveratrol turned out to be significantly better than some commercial antimicrobial (Levomecol) and antifungal (Clotrimazole) ointments and can be proposed as a promising drug for topical use for the treatment of trophic ulcers and burns.

An Interspecies Molecular and Functional Study of Organic Cation Transporters at the Blood-Brain Barrier: From Rodents to Humans.

Organic cation transporters (OCTs) participate in the handling of compounds in kidneys and at the synaptic cleft. Their role at the blood-brain barrier (BBB) in brain drug delivery is still unclear. The presence of OCT1,2,3 (SLC22A1-3) in mouse, rat and human isolated brain microvessels was investigated by either qRT-PCR, quantitative proteomics and/or functional studies. BBB transport of the prototypical substrate [3H]-1-methyl-4-phenylpyridinium ([3H]-MPP+) was measured by in situ brain perfusion in six mouse strains and in Sprague Dawley rats, in primary human brain microvascular endothelial cells seeded on inserts, in the presence or absence of OCTs and a MATE1 (SLC49A1) inhibitor. The results show negligible OCT1 (SLC22A1) and OCT2 (SLC22A2) expression in either mice, rat or human brain microvessels, while OCT3 expression was identified in rat microvessels by qRT-PCR. The in vitro human cellular uptake of [3H]-MPP+ was not modified by OCTs/MATE-inhibitor. Brain transport of [3H]-MPP+ remains unchanged between 2- and 6-month old mice, and no alteration was observed in mice and rats with inhibitors. In conclusion, the evidenced lack of expression and/or functional OCTs and MATE at the BBB allows the maintenance of the brain homeostasis and function as it prevents an easy access of their neurotoxicant substrates to the brain parenchyma.

Cannabidiol Increases Proliferation, Migration, Tubulogenesis, and Integrity of Human Brain Endothelial Cells through TRPV2 Activation.

The effect of cannabidiol (CBD), a high-affinity agonist of the transient receptor potential vanilloid-2 (TRPV2) channel, has been poorly investigated in human brain microvessel endothelial cells (BMEC) forming the blood-brain barrier (BBB). TRPV2 expression and its role on Ca2+ cellular dynamics, trans-endothelial electrical resistance (TEER), cell viability and growth, migration, and tubulogenesis were evaluated in human primary cultures of BMEC (hPBMEC) or in the human cerebral microvessel endothelial hCMEC/D3 cell line. Abundant TRPV2 expression was measured in hCMEC/D3 and hPBMEC by qRT-PCR, Western blotting, nontargeted proteomics, and cellular immunofluorescence studies. Intracellular Ca2+ levels were increased by heat and CBD and blocked by the nonspecific TRP antagonist ruthenium red (RR) and the selective TRPV2 inhibitor tranilast (TNL) or by silencing cells with TRPV2 siRNA. CBD dose-dependently induced the hCMEC/D3 cell number (EC50 0.3 ± 0.1 µM), and this effect was fully abolished by TNL or TRPV2 siRNA. A wound healing assay showed that CBD induced cell migration, which was also inhibited by TNL or TRPV2 siRNA. Tubulogenesis of hCMEC/D3 cells in 3D matrigel cultures was significantly increased by 41 and 73% after a 7 or 24 h CBD treatment, respectively, and abolished by TNL. CBD also increased the TEER of hPBMEC monolayers cultured in transwell, and this was blocked by TNL. Our results show that CBD, at extracellular concentrations close to those observed in plasma of patients treated by CBD, induces proliferation, migration, tubulogenesis, and TEER increase in human brain endothelial cells, suggesting CBD might be a potent target for modulating the human BBB.

LC-MS/MS-based quantification of efflux transporter proteins at the BBB.

Targeted protein quantification using tandem mass spectrometry coupled to high performance chromatography (LC-MS/MS) has been used to quantify proteins involved in the absorption, distribution, metabolism and excretion (ADME) of xenobiotics to better understand these processes. At the blood-brain barrier (BBB), these proteins are particularly important for the maintenance of brain homeostasis, but also regulate the distribution of therapeutic drugs. Absolute quantification (AQUA) is achieved by using stable isotope labeled surrogate peptides specific to the target protein and analyzing the digested proteins in a triple-quadrupole mass spectrometer in multiple reaction monitoring (MRM) mode to achieve a high specificity, sensitivity, accuracy and reproducibility. The main objective in this work was to develop and validate an UHPLC-MS/MS method for quantification of the ATP-binding cassette (ABC) transporter proteins Bcrp and P-gp and Na+/K + ATPase pump at the BBB. Three isoforms of the α-subunit from this pump (Atp1a 1, 2 and 3) were quantified to evaluate the presence of non-endothelial cells in the BBB using one common and three isoform-specific peptides; while Bcrp ad P-gp were quantified using 2 and 3 peptides, respectively, to improve the confidence on their quantification. The protein digestion was optimized, and the analytical method was comprehensively validated according to the American Food and Drug Administration Bioanalytical Method Validation Guidance published in 2018. Linearity across four magnitude orders (0.125 to 510 pmol·mL-1) sub-pmol·mL-1 LOD and LOQ, accuracy and precision (deviation < 15% and CV < 15%) were proven for most of the peptides by analyzing calibration curves and four levels of quality controls in both a pure solution and a complex matrix of digested yeast proteins, to mimic the matrix effect. In addition, digestion performance and stability of the peptides was shown using standard peptides spiked in a yeast digest or mouse kidney plasma membrane proteins as a study case. The validated method was used to characterize mouse kidney plasma membrane proteins, mouse brain cortical vessels and rat brain cortical microvessels. Most of the results agree with previously reported values, although some differences are seen due to different sample treatment, heterogeneity of the sample or peptide used. Importantly, the use of three peptides allowed the quantification of P-gp in mouse kidney plasma membrane proteins which was below the limit of quantification of the previously NTTGALTTR peptide. The different levels obtained for each peptide highlight the importance and difficulty of choosing surrogate peptides for protein quantification. In addition, using isoform-specific peptides for the quantification of the Na+/K + ATPase pump, we evaluated the presence of neuronal and glial cells on rat and mouse brain cortical vessels in addition to endothelial cells. In mouse liver and kidney, only the alpha-1 isoform was detected.

Litigation and political transformation: the case of Russia.

This article analyzes some recent developments in the system of public law in the Russian Federation, focusing in particular on changing patterns of litigation and increases in use of administrative law, linked to new acts of legislation. It argues that discussion of the Russian case provides a sociological perspective in which we can understand the importance of legal actions in hybrid polities. It explains that litigation in Russia, even where it may have counter-systemic outcomes, is partly incentivized by the government, as promotion of access to law is seen as a means to formalize interactions between citizens and government and so to extend the societal penetration of the political system more generally. Litigation thus forms a mode of practice that, dialectically, possesses both inner- and counter-systemic status. In addition, the article argues that the case of Russia allows us to comprehend litigation as an element in processes of nation building and social integration more widely, and Russia illuminates the systemic significance of litigation in other societies.