Genetic variability of the endangered fish lake minnow (Eupallasella percnurus) in populations newly established by translocation and those existing long term in Poland.

The lake minnow Eupallasella percnurus is a small leuciscid fish. In Poland, this species has been in a continuous decline since the mid-20th century and is presently considered as a extremely endangered. According to Polish law, E. percnurus is a strictly protected species that requires active conservation measures. In Poland, one the most common and effective measure of active protection E. percnurus is initiation of new populations. For this purpose, in 2004-2012, juvenile individuals originating from aquaculture conditions were translocated to group of isolated water bodies not inhabited by this species. The juveniles were offspring of parental fish belonging to the same local population, which is extinct at present. Five of those attempts were successful. The aim of the present study was to assess the genetic variation in a group new populations and compare genetic variation indicators with 13 old populations that had existed for decades. The polymorphism of 13 microsatellite markers was investigated, significance of differences in the genetic variation indicators between the groups were tested using a one-way analysis of variance (ANOVA). The mean values of all summary statistics under study, i.e. observed heterozygosity, expected heterozygosity and the total number of alleles, were higher in the group of new populations compared to almost all old ones. A similar dependence was found for Garza-Williamson M values, where the mean for the group of new populations was higher than in almost all old populations. Our results indicate that all recently established E. percnurus populations have not yet experienced any extensive founder effects or bottlenecks. They have preserved a large part of the genetic variability typical of their maternal population, which might also have been relatively high. This feature of new populations, may give them a relatively high ability to adapt to changing environments in the future.

TRPV1 channel in the pathophysiology of epilepsy and its potential as a molecular target for the development of new antiseizure drug candidates.

Identification of transient receptor potential cation channel, subfamily V member 1 (TRPV1), also known as capsaicin receptor, in 1997 was a milestone achievement in the research on temperature sensation and pain signalling. Very soon after it became evident that TRPV1 is implicated in a wide array of physiological processes in different peripheral tissues, as well as in the central nervous system, and thereby could be involved in the pathophysiology of numerous diseases. Increasing evidence suggests that modulation of TRPV1 may also affect seizure susceptibility and epilepsy. This channel is localized in brain regions associated with seizures and epilepsy, and its overexpression was found both in animal models of seizures and in brain samples from epileptic patients. Moreover, modulation of TRPV1 on non-neuronal cells (microglia, astrocytes, and/or peripheral immune cells) may have an impact on the neuroinflammatory processes that play a role in epilepsy and epileptogenesis. In this paper, we provide a comprehensive and critical overview of currently available data on TRPV1 as a possible molecular target for epilepsy management, trying to identify research gaps and future directions. Overall, several converging lines of evidence implicate TRPV1 channel as a potentially attractive target in epilepsy research but more studies are needed to exploit the possible role of TRPV1 in seizures/epilepsy and to evaluate the value of TRPV1 ligands as candidates for new antiseizure drugs.

Novel Alaninamide Derivatives with Drug-like Potential for Development as Antiseizure and Antinociceptive Therapies─In Vitro and In Vivo Characterization.

In the present study, a series of original alaninamide derivatives have been designed applying a combinatorial chemistry approach, synthesized, and characterized in the in vivo and in vitro assays. The obtained molecules showed potent and broad-spectrum activity in basic seizure models, namely, the maximal electroshock (MES) test, the 6 Hz (32 mA) seizure model, and notably, the 6 Hz (44 mA) model of pharmacoresistant seizures. Most potent compounds 26 and 28 displayed the following pharmacological values: ED50 = 64.3 mg/kg (MES), ED50 = 15.6 mg/kg (6 Hz, 32 mA), ED50 = 29.9 mg/kg (6 Hz, 44 mA), and ED50 = 34.9 mg/kg (MES), ED50 = 12.1 mg/kg (6 Hz, 32 mA), ED50 = 29.5 mg/kg (6 Hz, 44 mA), respectively. Additionally, 26 and 28 were effective in the ivPTZ seizure threshold test and had no influence on the grip strength. Moreover, lead compound 28 was tested in the PTZ-induced kindling model, and then, its influence on glutamate and GABA levels in the hippocampus and cortex was evaluated by the high-performance liquid chromatography (HPLC) method. In addition, 28 revealed potent efficacy in formalin-induced tonic pain, capsaicin-induced pain, and oxaliplatin- and streptozotocin-induced peripheral neuropathy. Pharmacokinetic studies and in vitro ADME-Tox data proved favorable drug-like properties of 28. The patch-clamp recordings in rat cortical neurons showed that 28 at a concentration of 10 µM significantly inhibited fast sodium currents. Therefore, 28 seems to be an interesting candidate for future preclinical development in epilepsy and pain indications.

Molecular Biology of Meniscal Healing: A Narrative Review.

This review provides insights at the molecular level into the current and old methods for treating meniscal injuries. Meniscal injuries have been found to have a substantial impact on the progression of osteoarthritis. In line with the "save the meniscus" approach, meniscectomy is considered a last-resort treatment. Nevertheless, it is important to note that mechanical repair alone may not achieve the complete restoration of the meniscus. A deep understanding of the healing pathways could lead to future improvements in meniscal healing. The inclusion of cytokines and chemokines has the potential to facilitate the process of tear repair or impede the inflammatory catabolic cascade. MicroRNA (miRNA) could serve as a potential biomarker for meniscal degeneration, and RNA injections might promote collagen and growth factor production. The critical aspect of the healing process is angiogenesis within the inner zone of the meniscus. The use of collagen scaffolds and the implantation of autologous meniscus fragments have been successfully integrated into clinical settings. These findings are encouraging and underscore the need for well-designed clinical trials to explore the most effective factors that can enhance the process of meniscal repair.

Effects of long-term administration of various dietary prebiotic supplements on the growth, immune cell activity and digestive tract histology of juvenile vimba (Vimba vimba).

Introduction: The experiment was set to determine the effects of long-term (55-day) use of three commercial prebiotics including Saccharomyces cerevisiae-derived ß-glucans and one including inulin on juvenile vimba (Vimba vimba) reared intensively under controlled conditions. Material and Methods: Six-month-old fish were fed commercial feed (Control group, n = 90), or the same feed supplemented with 0.02% Leiber Beta-S (BS group, n = 90), 0.20% Biolex MB40 (MB group, n = 90), 0.30% CeFi (CE group, n = 90) or 1.00% inulin Orafti GR (IN group, n = 90) for 55 days. Results: In the BS group, the final growth parameters were significantly lower than in the Control group, while the feed conversion ratio was significantly higher. No significant differences were found between any other group and the Control group in the respective parameters. The respiratory burst activity of the head-kidney phagocytes was significantly lower in all fish groups fed the prebiotic-supplemented diets compared to the Control group. The proliferative response of the head-kidney lymphocytes stimulated by concanavalin A was lower in the BS group than in the Control group, while in other groups this response was not affected. No significant differences were found in histopathological analyses of the digestive tract, liver or pancreas. Conclusion: The long-term supplementation of fish diets with prebiotics can negatively influence the growth, feed conversion, nonspecific cellular resistance and proliferative activity of the T lymphocytes of vimba juveniles.

Lipid nanocarrier targeting activated macrophages for antiretroviral therapy of HIV reservoir.

Aim: To develop lipid nano-antiretrovirals (LNAs) for the treatment of HIV-infected macrophages. Materials & methods: LNAs were prepared with docosahexaenoic acid to facilitate brain penetration and surface-decorated with folate considering that infected macrophages often overexpress folate receptors. Results: Folate-decorated LNAs loading rilpivirine (RPV) were efficiently taken up by folate receptor-expressing cell types including activated macrophages. The intracellular Cmax of the RPV-LNAs in activated macrophages was 2.54-fold and the area under the curve was 3.4-fold versus free RPV, translating to comparable or higher (p < 0.01; RPV ≤6.5 ng/ml) activities against HIV infectivity and superior protection (p < 0.05) against HIV cytotoxicity. LNAs were also effective in monocyte-derived macrophages. Conclusion: These findings demonstrate the potential of LNAs for the treatment of infected macrophages, which are key players in HIV reservoirs.

HIV can infect and hide inside certain types of white blood cells that make up the immune system and help defend our body, such as macrophages. Because these infected cells tend to carry the virus to certain organs where antiviral drugs have a hard time reaching, the virus is able to avoid treatment from the drug. In this study, the authors developed very small devices known as nanocarriers to carry antiviral drugs. These nanocarriers were designed to seek out infected macrophages. The nanocarriers were successfully built with oils and lipids that are safe for patients and could easily deliver antiviral drugs to macrophages infected by HIV. Excellent anti-HIV effects were observed using these nanocarriers. In summary, the authors developed a promising device with the potential to fight HIV in a smart and safe manner.

Preclinical safety and biodistribution of CRISPR targeting SIV in non-human primates.

In this study, we demonstrate the safety and utility of CRISPR-Cas9 gene editing technology for in vivo editing of proviral DNA in ART-treated, virally controlled simian immunodeficiency virus (SIV) infected rhesus macaques, an established model for HIV infection. EBT-001 is an AAV9-based vector delivering SaCas9 and dual guide RNAs designed to target multiple regions of the SIV genome: the viral LTRs, and the Gag gene. The results presented here demonstrate that a single IV inoculation of EBT-001 at each of 3 dose levels (1.4 × 1012, 1.4 × 1013 and 1.4 × 1014 genome copies/kg) resulted in broad and functional biodistribution of AAV9-EBT-001 to known tissue reservoirs of SIV. No off-target effects or abnormal pathology were observed, and animals returned to their normal body weight after receiving EBT-001. Importantly, the macaques that received the 2 highest doses of EBT-001 showed improved absolute lymphocyte counts as compared to antiretroviral-treated controls. Taken together, these results demonstrate safety, biodistribution, and in vivo proviral DNA editing following IV administration of EBT-001, supporting the further development of CRISPR-based gene editing as a potential therapeutic approach for HIV in humans.

Translocator protein PET imaging in temporal lobe epilepsy: A reliable test-retest study using asymmetry index.

Objective: Translocator protein (TSPO) targeting positron emission tomography (PET) imaging radioligands have potential utility in epilepsy to assess the efficacy of novel therapeutics for targeting neuroinflammation. However, previous studies in healthy volunteers have indicated limited test-retest reliability of TSPO ligands. Here, we examine test-retest measures using TSPO PET imaging in subjects with epilepsy and healthy controls, to explore whether this biomarker can be used as an endpoint in clinical trials for epilepsy. Methods: Five subjects with epilepsy and confirmed mesial temporal lobe sclerosis (mean age 36 years, 3 men) were scanned twice-on average 8 weeks apart-using a second generation TSPO targeting radioligand, [11C]PBR28. We evaluated the test-retest reliability of the volume of distribution and derived hemispheric asymmetry index of [11C]PBR28 binding in these subjects and compared the results with 8 (mean age 45, 6 men) previously studied healthy volunteers. Results: The mean (± SD) of the volume of distribution (VT), of all subjects, in patients living with epilepsy for both test and retest scans on all regions of interest (ROI) is 4.49 ± 1.54 vs. 5.89 ± 1.23 in healthy volunteers. The bias between test and retest in an asymmetry index as a percentage was small (-1.5%), and reliability is demonstrated here with Bland-Altman Plots (test mean 1.062, retest mean 2.56). In subjects with epilepsy, VT of [11C]PBR28 is higher in the (ipsilateral) hippocampal region where sclerosis is present than in the contralateral region. Conclusion: When using TSPO PET in patients with epilepsy with hippocampal sclerosis (HS), an inter-hemispheric asymmetry index in the hippocampus is a measure with good test-retest reliability. We provide estimates of test-retest variability that may be useful for estimating power where group change in VT represents the clinical outcome.

Strategic self-limiting production of infectious HIV particles by CRISPR in permissive cells.

Post-translational glycosylation of the HIV-1 envelope protein involving precursor glycan trimming by mannosyl oligosaccharide glucosidase (MOGS) is critically important for morphogenesis of virions and viral entry. Strategic editing of the MOGS gene in T lymphocytes and myeloid origin cells harboring latent proviral DNA results in the production of non-infectious particles upon treatment of cells with latency reversal agents. Controlled activation of CRISPR-MOGS by rebound HIV-1 mitigates production of infectious particles that exhibit poor ability of the virus to penetrate uninfected cells. Moreover, exclusive activation of CRISPR in cells infected with HIV-1 alleviates concern for broad off-target impact of MOGS gene ablation in uninfected cells. Combination CRISPR treatment of peripheral blood lymphocytes prepared from blood of people with HIV-1 (PWH) tailored for editing the MOGS gene (CRISPR-MOGS) and proviral HIV-1 DNA (CRISPR-HIV) revealed a cooperative impact of CRISPR treatment in inhibiting the production of infectious HIV-1 particles. Our design for genetic inactivation of MOGS by CRISPR exhibits no detectable off-target effects on host cells or any deleterious impact on cell survival and proliferation. Our findings offer the development of a new combined gene editing-based cure strategy for the diminution of HIV-1 spread after cessation of antiretroviral therapy (ART) and its elimination.

CRISPR editing of CCR5 and HIV-1 facilitates viral elimination in antiretroviral drug-suppressed virus-infected humanized mice.

Treatment of HIV-1ADA-infected CD34+ NSG-humanized mice with long-acting ester prodrugs of cabotegravir, lamivudine, and abacavir in combination with native rilpivirine was followed by dual CRISPR-Cas9 C-C chemokine receptor type five (CCR5) and HIV-1 proviral DNA gene editing. This led to sequential viral suppression, restoration of absolute human CD4+ T cell numbers, then elimination of replication-competent virus in 58% of infected mice. Dual CRISPR therapies enabled the excision of integrated proviral DNA in infected human cells contained within live infected animals. Highly sensitive nucleic acid nested and droplet digital PCR, RNAscope, and viral outgrowth assays affirmed viral elimination. HIV-1 was not detected in the blood, spleen, lung, kidney, liver, gut, bone marrow, and brain of virus-free animals. Progeny virus from adoptively transferred and CRISPR-treated virus-free mice was neither detected nor recovered. Residual HIV-1 DNA fragments were easily seen in untreated and viral-rebounded animals. No evidence of off-target toxicities was recorded in any of the treated animals. Importantly, the dual CRISPR therapy demonstrated statistically significant improvements in HIV-1 cure percentages compared to single treatments. Taken together, these observations underscore a pivotal role of combinatorial CRISPR gene editing in achieving the elimination of HIV-1 infection.

Anterior Cruciate Ligament Reconstruction Using a 4-Strand Semitendinosus Tendon Graft or a Doubled Semitendinosus and Gracilis Tendon Graft: A 4.5-Year Prospective, Randomized, Double-Blind, Parallel-Group Study.

BACKGROUND: Hamstring tendon grafts are the most common choice for anterior cruciate ligament (ACL) reconstruction (ACLR). Previous studies have provided evidence that offers conflicting opinions concerning the most favorable graft choice. PURPOSE: To identify whether the use of a quadrupled semitendinosus tendon (ST) or doubled semitendinosus tendon and gracilis tendon (ST/G) graft provides comparable anterior tibial translation (ATT) with similar functional results and similar donor site morbidity. STUDY DESIGN: Randomized controlled trial; Level of evidence, 1. METHODS: This was a prospective, patient- and surgeon-blinded, randomized trial set in a busy orthopaedic and traumatology department at a university hospital. Between 2015 and 2017, a total of 162 patients with ACL lesions were randomized to undergo ACLR with either a quadrupled ST or doubled ST/G graft. The primary endpoint was ATT assessed with the KT-1000 arthrometer. Clinical outcomes were assessed using the patient-reported outcome measures (PROMs) of the visual analog scale (VAS), International Knee Documentation Committee (IKDC) subjective evaluation form, Knee injury and Osteoarthritis Outcome Score (KOOS), Tegner activity scale, and Lysholm knee scoring scale. RESULTS: Preoperative demographic data, ATT, and PROM scores showed no significant differences. At 4.5 years, no significant differences were found between the ST and ST/G groups with respect to PROM scores and KT-1000 arthrometer, Lachman test, and pivot-shift test findings. Differences in functional results between groups were significant (muscle strength). Subgroup analysis revealed significantly increased ATT in female patients undergoing ACLR with a quadrupled ST graft during the 4.5-year observation period, as assessed by the KT-1000 arthrometer, as well as inferior KOOS, IKDC, Lysholm, and VAS scores. CONCLUSION: This study showed a significant increase in ATT as well as inferior results on PROMs during a 4.5-year observation period in female patients undergoing ACLR with a quadrupled ST graft. In male patients, the study provided evidence of the noninferiority of ACLR with an ST graft, with no influence on donor site morbidity. TRIAL REGISTRATION: clinicaltrials.gov: NCT03626883.

PK/PD analysis of trazodone and gabapentin in neuropathic pain rodent models: Translational PK-PD modeling from nonclinical to clinical development.

A pharmacokinetic/pharmacodynamic (PK/PD) model was developed to describe the time course of writhings after intraperitoneal injection of acetic acid in mice. The model was applied to investigate the antinociceptive effect of trazodone and gabapentin alone and in combination. Writhings time course was described by a transit compartment model with the delay due to the transit of the acetic acid being represented by a chain of intermediate compartments. In the drug-treated animals, the number of writhings decreases according to a k2 factor linking drug concentration and antinociceptive effect. Compounds' potency parameters were 10.9 and 0.0459 L/µmoles/min for trazodone and gabapentin, respectively, indicating a much higher in vivo potency of trazodone in the PD writhing test. The PK/PD parameters were used to simulate the expected writhing counts in mice at combined doses without efficacy alone, assuming pharmacological additivity. Simulation results indicated that, at low dose combinations, experimental data were mostly below the simulated writhings median, suggesting possible synergic effect. Such hypothesis was tested by adding the γ parameter in the PK/PD model to represent the deviation from the assumption of no-interaction, leading to a reduction of the objective function compared to the additive model. On this basis, several simulations were performed to identify possible starting dose combinations of trazodone and gabapentin in humans, by selecting doses yielding systemic exposures close to those being synergic in the mouse. Simulations indicated that doses of 50-100 mg trazodone could enhance gabapentin antinociceptive effect in humans, supporting the development of a low dose combination for optimal analgesia treatment.

Coronavirus infection in chemosensory cells.

Clinical manifestations of human coronavirus (HCoV)-related diseases are mostly related to the respiratory system, although secondary complications such as headache, anosmia, ageusia, and myalgia have been reported. HCoV infection and replication in chemosensory cells associated with ageusia and anosmia is poorly understood. Here, we characterized HCoV-OC43 and SARS-CoV-2 infection in two types of chemosensory cells, olfactory and taste cells, with their unique molecular and histological characteristics. We first assessed HCoV-OC43 infection in in vitro cultured human olfactory epithelial cells (hOECs) and fungiform taste papilla (HBO) cells. Interestingly, while both cell types were susceptible to HCoV-OC43 infection, viral replication rates were significantly reduced in HBO cells compared to hOECs. More interestingly, while culture media from hOECs was able to produce secondary infection in Vero cells, there was very limited secondary infection from HBO cells, suggesting that HBO cells may not be able to release infectious virus. On the other hand, unlike HCoV-OC43, SARS-CoV-2 showed comparable levels of viral infection rates in both hOECs and HBO cells. Furthermore, our RT-qPCR-based gene array studies revealed that several key genes involved in taste and olfactory functions were significantly altered by SARS-CoV-2 infection. These results may suggest a possible mechanism associated with chemosensory symptoms, such as anosmia and ageusia in patients infected with SARS-CoV-2.

Discovery of (R)-N-Benzyl-2-(2,5-dioxopyrrolidin-1-yl)propanamide [(R)-AS-1], a Novel Orally Bioavailable EAAT2 Modulator with Drug-like Properties and Potent Antiseizure Activity In Vivo.

(R)-7 [(R)-AS-1] showed broad-spectrum antiseizure activity across in vivo mouse seizure models: maximal electroshock (MES), 6 Hz (32/44 mA), acute pentylenetetrazol (PTZ), and PTZ-kindling. A remarkable separation between antiseizure activity and CNS-related adverse effects was also observed. In vitro studies with primary glia cultures and COS-7 cells expressing the glutamate transporter EAAT2 showed enhancement of glutamate uptake, revealing a stereoselective positive allosteric modulator (PAM) effect, further supported by molecular docking simulations. (R)-7 [(R)-AS-1] was not active in EAAT1 and EAAT3 assays and did not show significant off-target activity, including interactions with targets reported for marketed antiseizure drugs, indicative of a novel and unprecedented mechanism of action. Both in vivo pharmacokinetic and in vitro absorption, distribution, metabolism, excretion, toxicity (ADME-Tox) profiles confirmed the favorable drug-like potential of the compound. Thus, (R)-7 [(R)-AS-1] may be considered as the first-in-class small-molecule PAM of EAAT2 with potential for further preclinical and clinical development in epilepsy and possibly other CNS disorders.

Osteotomies and Total Knee Arthroplasty: Systematic Review and Meta-Analysis.

Total knee replacement (TKA) is a frequent modality performed in patients with osteoarthritis. Specific circumstances can make it much more difficult to execute successfully, and additional procedures such as osteotomy may be required. The aim of this study was to perform a meta-analysis and systematic review of osteotomies combined with TKA. METHODS: In June 2022, a search PubMed, Embase, Cochrane, and Clinicaltrials was undertaken, adhering to PRISMA guidelines. The search included the terms "osteotomy" and "total knee arthroplasty". RESULTS: Two subgroups (tibial tubercle osteotomy and medial femoral condyle osteotomy) were included in the meta-analysis. Further subgroups were described as a narrative review. The primary outcome showed no significant difference in favor to TTO. Secondary outcomes showed improved results in all presented subgroups compared to preoperative status. CONCLUSION: This study showed a significant deficit of randomized control trials treated with osteotomies, in addition to TKA, and a lack of evidence-based surgical guidelines for the treatment of patients with OA in special conditions: posttraumatic deformities, stiff knee, severe varus, and valgus axis or patella disorders.

Connectivity Mapping Using a Novel sv2a Loss-of-Function Zebrafish Epilepsy Model as a Powerful Strategy for Anti-epileptic Drug Discovery.

Synaptic vesicle glycoprotein 2A (SV2A) regulates action potential-dependent neurotransmitter release and is commonly known as the primary binding site of an approved anti-epileptic drug, levetiracetam. Although several rodent knockout models have demonstrated the importance of SV2A for functional neurotransmission, its precise physiological function and role in epilepsy pathophysiology remains to be elucidated. Here, we present a novel sv2a knockout model in zebrafish, a vertebrate with complementary advantages to rodents. We demonstrated that 6 days post fertilization homozygous sv2a-/- mutant zebrafish larvae, but not sv2a +/- and sv2a+/+ larvae, displayed locomotor hyperactivity and spontaneous epileptiform discharges, however, no major brain malformations could be observed. A partial rescue of this epileptiform brain activity could be observed after treatment with two commonly used anti-epileptic drugs, valproic acid and, surprisingly, levetiracetam. This observation indicated that additional targets, besides Sv2a, maybe are involved in the protective effects of levetiracetam against epileptic seizures. Furthermore, a transcriptome analysis provided insights into the neuropathological processes underlying the observed epileptic phenotype. While gene expression profiling revealed only one differentially expressed gene (DEG) between wildtype and sv2a +/- larvae, there were 4386 and 3535 DEGs between wildtype and sv2a-/- , and sv2a +/- and sv2a-/- larvae, respectively. Pathway and gene ontology (GO) enrichment analysis between wildtype and sv2a-/- larvae revealed several pathways and GO terms enriched amongst up- and down-regulated genes, including MAPK signaling, synaptic vesicle cycle, and extracellular matrix organization, all known to be involved in epileptogenesis and epilepsy. Importantly, we used the Connectivity map database to identify compounds with opposing gene signatures compared to the one observed in sv2a-/- larvae, to finally rescue the epileptic phenotype. Two out of three selected compounds rescued electrographic discharges in sv2a-/- larvae, while negative controls did not. Taken together, our results demonstrate that sv2a deficiency leads to increased seizure vulnerability and provide valuable insight into the functional importance of sv2a in the brain in general. Furthermore, we provided evidence that the concept of connectivity mapping represents an attractive and powerful approach in the discovery of novel compounds against epilepsy.

New Phenylglycinamide Derivatives with Hybrid Structure as Candidates for New Broad-Spectrum Anticonvulsants.

In the present study, a focused combinatorial chemistry approach was applied to merge structural fragments of well-known TRPV1 antagonists with a potent anticonvulsant lead compound, KA-104, that was previously discovered by our group. Consequently, a series of 22 original compounds has been designed, synthesized, and characterized in the in vivo and in vitro assays. The obtained compounds showed robust in vivo antiseizure activity in the maximal electroshock (MES) test and in the 6 Hz seizure model (using both 32 and 44 mA current intensities). The most potent compounds 53 and 60 displayed the following pharmacological profile: ED50 = 89.7 mg/kg (MES), ED50 = 29.9 mg/kg (6 Hz, 32 mA), ED50 = 68.0 mg/kg (6 Hz, 44 mA), and ED50 = 73.6 mg/kg (MES), ED50 = 24.6 mg/kg (6 Hz, 32 mA), and ED50 = 56.3 mg/kg (6 Hz, 44 mA), respectively. Additionally, 53 and 60 were effective in the ivPTZ seizure threshold and had no influence on the grip strength and body temperature in mice. The in vitro binding and functional assays indicated a multimodal mechanism of action for 53 and 60. These molecules, beyond TRPV1 antagonism, inhibited calcium currents and fast sodium currents in patch-clamp assays. Further studies proved beneficial in vitro ADME-Tox properties for 53 and 60 (i.e., high metabolic stability, weak influence on CYPs, no neurotoxicity, etc.). Overall, 53 and 60 seem to be interesting candidates for future preclinical development in epilepsy and pain indications due to their interaction with the TRPV1 channel.

13C Natural Isotope Abundance in Urothelium as a New Marker in the Follow-Up of Patients with Bladder Cancer.

Bladder cancer (BC) is the most common urological malignancy and has a high incidence of recurrence. BC cells alter their nutrient uptake and metabolic pathways in order to continue the production of sufficient levels of ATP and metabolic intermediates for proliferation and survival. Changes in metabolic pathways regarding the rate of the enzymatic reaction and transport lead to differences in the content of natural isotopes (13C, 15N, 34S) between normal and cancerous tissues. The assessment of the stable isotopes of carbon, nitrogen, and sulfur in normal urothelium and bladder cancer samples was performed using Isotope Ratio Mass Spectrometry (IRMS). The natural abundance of 15N and 13C was decreased in bladder cancer samples when compared to normal urothelium. No significant correlation was observed in BC specimens depending on the tumor grade and stage. Samples derived from bladder tumors and normal urothelium had a different pattern of 15N and 13C isotope abundance. Decreased 13C natural isotopes in the normal urothelium of BC patients were significantly associated with a shorter DFS. Our results suggest that isotopic analysis of normal urothelium of BC patients can be used to predict bladder cancer recurrence.