Gabapentin Prevents Progressive Increases in Excitatory Connectivity and Epileptogenesis Following Neocortical Trauma.

Neocortical injury initiates a cascade of events, some of which result in maladaptive epileptogenic reorganization of surviving neural circuits. Research focused on molecular and organizational changes that occur following trauma may reveal processes that underlie human post-traumatic epilepsy (PTE), a common and unfortunate consequence of traumatic brain injury. The latency between injury and development of PTE provides an opportunity for prophylactic intervention, once the key underlying mechanisms are understood. In rodent neocortex, injury to pyramidal neurons promotes axonal sprouting, resulting in increased excitatory circuitry that is one important factor promoting epileptogenesis. We used laser-scanning photostimulation of caged glutamate and whole-cell recordings in in vitro slices from injured neocortex to assess formation of new excitatory synapses, a process known to rely on astrocyte-secreted thrombospondins (TSPs), and to map the distribution of maladaptive circuit reorganization. We show that this reorganization is centered principally in layer V and associated with development of epileptiform activity. Short-term blockade of the synaptogenic effects of astrocyte-secreted TSPs with gabapentin (GBP) after injury suppresses the new excitatory connectivity and epileptogenesis for at least 2 weeks. Results reveal that aberrant circuit rewiring is progressive in vivo and provide further rationale for prophylactic anti-epileptogenic use of gabapentinoids following cortical trauma.

Resistance mutations generate divergent antibiotic susceptibility profiles against translation inhibitors.

Mutations conferring resistance to translation inhibitors often alter the structure of rRNA. Reduced susceptibility to distinct structural antibiotic classes may, therefore, emerge when a common ribosomal binding site is perturbed, which significantly reduces the clinical utility of these agents. The translation inhibitors negamycin and tetracycline interfere with tRNA binding to the aminoacyl-tRNA site on the small 30S ribosomal subunit. However, two negamycin resistance mutations display unexpected differential antibiotic susceptibility profiles. Mutant U1060A in 16S Escherichia coli rRNA is resistant to both antibiotics, whereas mutant U1052G is simultaneously resistant to negamycin and hypersusceptible to tetracycline. Using a combination of microbiological, biochemical, single-molecule fluorescence transfer experiments, and X-ray crystallography, we define the specific structural defects in the U1052G mutant 70S E. coli ribosome that explain its divergent negamycin and tetracycline susceptibility profiles. Unexpectedly, the U1052G mutant ribosome possesses a second tetracycline binding site that correlates with its hypersusceptibility. The creation of a previously unidentified antibiotic binding site raises the prospect of identifying similar phenomena in antibiotic-resistant pathogens in the future.

Negamycin induces translational stalling and miscoding by binding to the small subunit head domain of the Escherichia coli ribosome.

Negamycin is a natural product with broad-spectrum antibacterial activity and efficacy in animal models of infection. Although its precise mechanism of action has yet to be delineated, negamycin inhibits cellular protein synthesis and causes cell death. Here, we show that single point mutations within 16S rRNA that confer resistance to negamycin are in close proximity of the tetracycline binding site within helix 34 of the small subunit head domain. As expected from its direct interaction with this region of the ribosome, negamycin was shown to displace tetracycline. However, in contrast to tetracycline-class antibiotics, which serve to prevent cognate tRNA from entering the translating ribosome, single-molecule fluorescence resonance energy transfer investigations revealed that negamycin specifically stabilizes near-cognate ternary complexes within the A site during the normally transient initial selection process to promote miscoding. The crystal structure of the 70S ribosome in complex with negamycin, determined at 3.1 Å resolution, sheds light on this finding by showing that negamycin occupies a site that partially overlaps that of tetracycline-class antibiotics. Collectively, these data suggest that the small subunit head domain contributes to the decoding mechanism and that small-molecule binding to this domain may either prevent or promote tRNA entry by altering the initial selection mechanism after codon recognition and before GTPase activation.

The role of a novel auxiliary pocket in bacterial phenylalanyl-tRNA synthetase druggability.

The antimicrobial activity of phenyl-thiazolylurea-sulfonamides against Staphylococcus aureus PheRS are dependent upon phenylalanine levels in the extracellular fluids. Inhibitor efficacy in animal models of infection is substantially diminished by dietary phenylalanine intake, thereby reducing the perceived clinical utility of this inhibitor class. The search for novel antibacterial compounds against Gram-negative pathogens led to a re-evaluation of this phenomenon, which is shown here to be unique to S. aureus. Inhibition of macromolecular syntheses and characterization of novel resistance mutations in Escherichia coli demonstrate that antimicrobial activity of phenyl-thiazolylurea-sulfonamides is mediated by PheRS inhibition, validating this enzyme as a viable drug discovery target for Gram-negative pathogens. A search for novel inhibitors of PheRS yielded three novel chemical starting points. NMR studies were used to confirm direct target engagement for phenylalanine-competitive hits. The crystallographic structure of Pseudomonas aeruginosa PheRS defined the binding modes of these hits and revealed an auxiliary hydrophobic pocket that is positioned adjacent to the phenylalanine binding site. Three viable inhibitor-resistant mutants were mapped to this pocket, suggesting that this region is a potential liability for drug discovery.

Overexpression of Pseudomonas aeruginosa LpxC with its inhibitors in an acrB-deficient Escherichia coli strain.

In Gram-negative bacteria, the cell wall is surrounded by an outer membrane, the outer leaflet of which is comprised of charged lipopolysaccharide (LPS) molecules. Lipid A, a component of LPS, anchors this molecule to the outer membrane. UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine deacetylase (LpxC) is a zinc-dependent metalloamidase that catalyzes the first committed step of biosynthesis of Lipid A, making it a promising target for antibiotic therapy. Formation of soluble aggregates of Pseudomonas aeruginosa LpxC protein when overexpressed in Escherichia coli has limited the availability of high quality protein for X-ray crystallography. Expression of LpxC in the presence of an inhibitor dramatically increased protein solubility, shortened crystallization time and led to a high-resolution crystal structure of LpxC bound to the inhibitor. However, this approach required large amounts of compound, restricting its use. To reduce the amount of compound needed, an overexpression strain of E. coli was created lacking acrB, a critical component of the major efflux pump. By overexpressing LpxC in the efflux deficient strain in the presence of LpxC inhibitors, several structures of P. aeruginosa LpxC in complex with different compounds were solved to accelerate structure-based drug design.

Identification through structure-based methods of a bacterial NAD(+)-dependent DNA ligase inhibitor that avoids known resistance mutations.

In an attempt to identify novel inhibitors of NAD(+)-dependent DNA ligase (LigA) that are not affected by a known resistance mutation in the adenosine binding pocket, a detailed analysis of the binding sites of a variety of bacterial ligases was performed. This analysis revealed several similarities to the adenine binding region of kinases, which enabled a virtual screen of known kinase inhibitors. From this screen, a thienopyridine scaffold was identified that was shown to inhibit bacterial ligase. Further characterization through structure and enzymology revealed the compound was not affected by a previously disclosed resistance mutation in Streptococcus pneumoniae LigA, Leu75Phe. A subsequent medicinal chemistry program identified substitutions that resulted in an inhibitor with moderate activity across various Gram-positive bacterial LigA enzymes.

Factors associated with the rabies vaccination status of dogs in households in Beni City, D.R. Congo.

Human rabies transmitted by dogs still kills thousands of people each year worldwide. Dog bites are common in the city of Beni (Democratic Republic of Congo), which shows low rabies vaccination coverage. This study aimed to determine the factors associated with the rabies vaccination status of dogs. A cross-sectional analytical study was conducted in the town of Beni among dog owners, during a household survey selected using a multistage sampling. The information sought concerned the knowledge and characteristics of the dog owners as well as the vaccination status of these dogs. Logistic regression was used to investigate associations between the vaccination status of the dogs and the main independent factors. Rabies vaccination coverage in Beni was 26% (95% confidence interval [CI]: 22% - 30%). The main factors associated with the rabies vaccination status of the dog were primary education level of household head (adjusted odds ratio [aOR]:4.8; 95% CI: 1.2- 19.8); university education level of household head (aOR: 5.9; 95% CI: 1.6-22); perceived rabies severity (aOR: 44. 4; 95% CI: 10.4-188), having more than one dog in the household (aOR: 2.6; 95% CI: 1.6-4.3); age range 7-12 months (aOR: 0.2; 95% CI: 0.1-0.6) and confined dog breeding (aOR: 3.9; 95% CI: 1.1-14.9). The low vaccination coverage in Beni requires mass vaccination campaigns against canine rabies targeting the dog owners with low education levels, those raising more than one dog, with stray dogs or dogs less than 12 months old.

Ghanaian clients' perception of fitness instructors' adherence to exercise delivery services codes of conduct: An exploratory study.

Background and Aims: Patronage for fitness and wellness activities has increased in Ghana, but the perception of primary consumers regarding exercise delivery service codes of conduct (EDCC) remains undocumented. This study reported the perception of clients about fitness instructors' adherence to EDCC. Methods: Three hundred and seventy-nine (mean age = 26.12 ± 8.83 years) clients registered with National Sports for ALL Association, Ghana (NASFAAG) were recruited. The participants were not living with any diagnosed disability, using psychiatric medication, and had consistently participated in fitness training, at least three times a week for a year, and not below the age of 18 years. The participants were recruited from fitness and wellness centers, gyms, and fitness clubs in three regions (Greater Accra-GA, Upper East-UE, and Ashanti-A) of Ghana. A closed-ended, self-structured and validated awareness and adherence exercise delivery codes of conduct questionnaires was administered. The questionnaire focused on data protection and responsibility, informed consent, competence, and professional and personal conduct adapted from the British Association of Sports and Exercise Science codes of conduct was administered. Statistical Package for Social Sciences (SPSS) version 23.0 was used to run factor analysis which determined factorial distribution of clients' perception of instructors on codes of conduct. Results: In total, 50.99% (UE), 47.68% (A), and 46.02% (GA) clients indicated that identities were unprotected when trainers displayed information. In all, 31.05% (UE), 40.34% (A), and 36.48% (GA) showed they were introduced to substances without consent. In total, 38.89% (UE), 32.70% (A), and 53.55% (GA) clients participated in training to realize that the expertise expected was not provided. In all, 38.10% (UE) and 36.23% (A) agreed that instructors put safety at risk, while 23.02% (GA) exploited clients for personal gain. Conclusion: Fitness instructors need enlightenment to adhere ethically to EDCC activities in Ghana. Activities related to wellness and fitness in Ghana require regulations.

Acetic acid-induced colitis modulating potential of total crude alkaloidal extract of Picralima nitida seeds in rats.

PURPOSE: The total crude alkaloidal extract of Picralima nitida seeds (PNE) is known to possess anti-inflammatory activity among other therapeutic benefits although its benefits in colitis has not been investigated. The current study therefore seeks to investigate the anti-colitis potential of PNE using acetic acid-induced colitis model in rats. METHODS: Sprague Dawley rats were treated with oral 500 mg/kg sulphasalazine or 30, 100, and 300 mg/kg of PNE daily for 8 days with induction of colitis on the fourth day with acetic acid. Rats were killed 24 h after the last treatment and whole blood was obtained from the jugular vein for hematological analysis and biochemical assays. Colons were extirpated for assessment of macroscopic and histological damage to the colon. RESULTS: Treatment with PNE protected against colonic injury induced with acetic acid by decreasing mucosal ulceration, epithelial erosion, inflammatory cell infiltration, and colonic edema. Thus, PNE preserved mucosal architecture and suppressed goblet cells depletion. Moreover, treatment with PNE was associated with improved hematological parameters and reductions in the expression of serum tumor necrosis factor-alpha, interleukin-1ß, and p38 mitogen-activated protein kinase. Also, PNE treatment exerted antioxidant effects by reducing nitric oxide production and increasing glutathione levels. In addition, PNE inhibited colonic lipid peroxidation by decreasing myeloperoxidase activity and malondialdehyde production. CONCLUSION: It can be concluded that PNE attenuates intestinal oxidative and inflammatory damages following intrarectal acetic acid challenge. Thus, demonstrates potential for use in chronic intestinal inflammatory diseases such as ulcerative colitis.

Shared community effects and the non-genetic maternal environment shape cortisol levels in wild chimpanzees.

Mechanisms of inheritance remain poorly defined for many fitness-mediating traits, especially in long-lived animals with protracted development. Using 6,123 urinary samples from 170 wild chimpanzees, we examined the contributions of genetics, non-genetic maternal effects, and shared community effects on variation in cortisol levels, an established predictor of survival in long-lived primates. Despite evidence for consistent individual variation in cortisol levels across years, between-group effects were more influential and made an overwhelming contribution to variation in this trait. Focusing on within-group variation, non-genetic maternal effects accounted for 8% of the individual differences in average cortisol levels, significantly more than that attributable to genetic factors, which was indistinguishable from zero. These maternal effects are consistent with a primary role of a shared environment in shaping physiology. For chimpanzees, and perhaps other species with long life histories, community and maternal effects appear more relevant than genetic inheritance in shaping key physiological traits.

Clinical experience of tocilizumab treatment among a cohort of patients with COVID-19 infection from Western India.

Background: Initiation of tocilizumab (TCZ) treatment in patients with coronavirus disease 2019 (COVID-19) during the early phases of cytokine storm is crucial. This study evaluated the clinical experience of TCZ use in the treatment of patients with COVID-19. Methods: This retrospective observational study included patients (>18 years) with confirmed COVID19 treated with TCZ alone/in combination with other drugs. Data related to demographics, clinical characteristics, radiological parameters, oxygen/ventilator/vasopressor support, treatment parameters, laboratory investigations pre- and post-TCZ treatment, and clinical outcomes were retrieved from medical records. Results: Out of 95 patients (mean age, 55 years), 68.4% and 31.6% of patients had moderate and severe COVID-19 disease, respectively. The mean time to TCZ administration from symptom onset was 8.7 days. At the time of admission, the mean oxygen saturation (SpO2) was 90.4% and mean concentration of fraction of inspired oxygen (FiO2) was 80.6%. The most commonly received dose of TCZ was 400 mg (84.2%) intravenously. The mean concentration of FiO2 and SpO2 improved significantly during the treatment (P < 0.001) compared to before TCZ initiation. The change in median levels of C-reactive protein (CRP) from baseline to post-treatment (63.0 vs. 4.5 mg/dL; P < 0.001) was significant. Post TCZ treatment, 73.6% of patients improved; whereas 26.4% of patients died. Acute respiratory distress syndrome (23.2%) and elevated transaminases (12.6%) were the most commonly reported adverse events. Conclusion: Tocilizumab administration during earlier phase of cytokine storm syndrome leads to reversal of abnormal SpO2 and FiO2 concentrations to normal levels and rapid decline of elevated CRP levels in patients with COVID-19.

Determinants of COVID-19 vaccine acceptance and hesitancy among healthcare professionals in the Kintampo North Municipality, Bono East Region, Ghana.

Objectives: To assess the determinants of COVID-19 vaccine acceptance and hesitation among Health Care Professionals (HCPs) in the Kintampo North Municipality of Ghana. Design: An analytical cross-sectional study. Setting: The study was carried out in the Kintampo North Municipality. Participants: All health care professionals within the Kintampo North Municipality of Ghana. Main outcome measure: Acceptance of COVID-19 vaccine. Results: In all, 215 HCPs were included in this study. The overall vaccine acceptance was 78.6% among HCPs, while 21.4% were hesitant to receive the COVID-19 vaccine. Majority (57.7%) of HCPs believed that COVID-19 vaccines were safe. The following factors were found to influence vaccine acceptance significantly; those who knew someone who has taken the vaccine (adjusted Odds Ratio [aOR]; 14.9, 95% Confidence Interval [95% CI];5.0-45.0, p<0.001), those who think COVID -19 vaccine in Ghana was safe (AOR;9.2, 95%CI;3.3-25.8, P<0.001), those who said vaccines are effective in controlling COVID-19 transmission (aOR=5.0, 95%CI;2.1-12.4, p<0.001), and those who have never refused vaccines in the past (aOR=7.8, 95CI;1.6-37.8, p=0.01). Conclusion: The study indicated high COVID-19 vaccination acceptability among HCPs. However, some HCPs are hesitant to take COVID-19 vaccinations immediately. Increased adoption of COVID-19 vaccinations among HCPs and the broader Ghanaian population requires concerted efforts, including strengthening public health education on the perceived risks and safety of COVID-19 vaccines. Funding: None declared.

Dendritic mechanisms underlying penicillin-induced epileptiform activity.

The action of penicillin on synaptically evoked dendritic activity was examined with the use of hippocampal slice preparations. Orthodormic activation of CA1 pyramidal neurons produced an excitatory-inhibitory postsynaptic potential sequence recorded intracellularly in the dendrites. Treatment with penicillin resulted in the appearance of spontaneous and synaptically evoked multipeaked field potentials and associated depolarization shifts and spike burst generation in CA1 cells. Intracellular recordings revealed that penicillin produced no detectable change in passive membrane properties of the postsynaptic dendrites. However, the inhibitory postsynaptic potential was suppressed by penicillin, resulting in the release of intrinsic dendritic burst firing during synaptic activation. These findings emphasize the role of normal patterns of dendritic burst generation in the production of intense neuronal discharge during penicillin-induced epileptiform activities.

Dye coupling and possible electrotonic coupling in the guinea pig neocortical slice.

Iontophoretic injection of the fluorescent dye Lucifer Yellow CH into single neurons of guinea pig neocortical slices resulted in the staining of more than one cell. Dye-coupled neuronal aggregates were found only in the superficial cortical layers and were often organized in vertical columns. Antidromic stimuli evoked all-or-none, subthreshold depolarizations in some superficial cells. These potentials were not eliminated by manganese and did not collide with spikes originating in the soma, suggesting that they arose from electrotonic interaction between superficial cortical neurons.

Cholinergic switching within neocortical inhibitory networks.

Differential actions of acetylcholine on the excitability of two subtypes of interneurons in layer V of the rat visual cortex were examined. Acetylcholine excited low-threshold spike (LTS) cells through nicotinic receptors, whereas it elicited hyperpolarization in fast spiking (FS) cells through muscarinic receptors. Axons of LTS cells were mainly distributed vertically to upper layers, and those of FS cells were primarily confined to layer V. Thus, cortical cholinergic activation may reduce some forms of intralaminar inhibition, promote intracolumnar inhibition, and change the direction of information flow within cortical circuits.

Thalamocortical relay neurons: antidromic invasion of spikes from a cortical epileptogenic focus.

Thalamocortical relay neurons whose axons project into a penicillininduced cortical epileptogenic focus generate bursts of action potentials during spontaneous interictal epileptiform discharges. These bursts originate in intracortical axons and propagate antidromically into thalamic neurons. Repetitive spike generation in cortical axons and presynaptic terminals could produce a potent excitatory drive and contribute to the generation of the large depolarization shifts which are seen in cortical elements during focal epileptogenesis.

Large-scale purification of human BACE expressed in mammalian cells and removal of the prosegment with HIV-1 protease to improve crystal diffraction.

BACE, or beta-secretase, is an attractive target in the treatment of Alzheimer's Disease because of its involvement in the generation of amyloid beta peptides. BACE is a type I transmembrane aspartyl protease composed of pre-, pro-, catalytic, transmembrane and cytoplasmic domains. For the present study, the coding sequence was truncated just before the transmembrane domain and the resulting construct was extended with the C-terminal addition of a (His)(6) and expressed in several mammalian host cells. The enzyme expressed in CHO cells had the best crystallographic behavior and was purified in large quantities in a three step procedure. The purified BACE was comprised of two forms, namely the full length proBACE construct beginning with Thr(1), and a derivative missing the first 24 amino acids beginning with E(25). These BACE precursors co-crystallized in the presence of inhibitors yielding structures to 3.2 A resolution. HIV-1 protease treatment of this mixture resulted in complete cleavage of the F(39)-V(40) bond, leaving the V(40)EM...ES(432) (His)(6) derivative that was purified yielding an enzyme that was no more active than untreated BACE but co-crystallized with inhibitors producing well shaped, bipyramidal co-crystals diffracting to 2.6 A resolution.

High yield expression of human BACE constructs in Eschericia coli for refolding, purification, and high resolution diffracting crystal forms.

BACE (beta-site APP cleaving enzyme) or beta-secretase, the enzyme responsible for processing APP to give the N-terminal portion of the Abeta peptide, is a membrane bound aspartyl protease consisting of an ectodomain catalytic unit, a C-terminal transmembrane segment and a cytoplasmic domain. Three BACE constructs, pET11a-BACE, pQE80L-BACE, and pQE70-BACE were designed to terminate at a position just before the transmembrane domain (Ser(432)) and are described schematically below. (1) pET11a-T7.Tag-G-S-M-(A-8GV......QTDES(432)), (2) pQE80L-Met-R-G-S-(His)(6)-G-S-I-E-T-D-(T(1)QH...QTDES(432)), and (3) pQE70-Met-BACE (R(36)GSFVEMG....PQTDES(432) (His) (6)) Each construct was over-expressed in Escherichia coli as inclusion bodies. The inclusion body proteins were solubilized in urea and refolded by dilution in water to yield active enzyme. Maximal activity for pET11a-BACE and pQE80L-BACE was usually reached at day 3 to 4, while construct pQE70-BACE required about 21 days. Active BACE was purified to homogeneity by anion-exchange chromatography and affinity chromatography over a column of immobilized peptide inhibitor. The process, easily scalable to 60 liters of cell culture, yielded in excess of 400 mg of active enzyme for crystallographic analysis. Highly purified pET11a-BACE and pQE70-BACE formed complexes with various inhibitors, the latter protein giving crystals diffracting up to 1.45 A resolution. In addition, a crystal form that does not require the presence of an inhibitor has been obtained for pQE70-BACE. This ligand-free crystal form has proven useful for the preparation of BACE-inhibitor complexes in soaking experiments.

TRAFIC: Fiber Tract Classification Using Deep Learning.

We present TRAFIC, a fully automated tool for the labeling and classification of brain fiber tracts. TRAFIC classifies new fibers using a neural network trained using shape features computed from previously traced and manually corrected fiber tracts. It is independent from a DTI Atlas as it is applied to already traced fibers. This work is motivated by medical applications where the process of extracting fibers from a DTI atlas, or classifying fibers manually is time consuming and requires knowledge about brain anatomy. With this new approach we were able to classify traced fiber tracts obtaining encouraging results. In this report we will present in detail the methods used and the results achieved with our approach.