OFF-times before, during, and after nighttime sleep periods in Parkinson's disease patients with motor fluctuations and the effects of opicapone: A post hoc analysis of diary data from BIPARK-1 and -2.

INTRODUCTION: In BIPARK-1 and BIPARK-2, addition of once-daily opicapone to levodopa/carbidopa significantly reduced daily "OFF"-time relative to placebo in adults with Parkinson's disease (PD) and motor fluctuations. Diary data from these studies were pooled and analyzed post hoc to characterize "OFF"-times around nighttime sleep and to explore the effects of opicapone 50 mg. METHODS: "OFF" before sleep (OBS), "OFF during the nighttime sleep period" (ODNSP), early morning "OFF" (EMO), and duration of nighttime sleep and awake periods were analyzed descriptively at baseline. Mean changes from baseline to Week 14/15 (end of double-blind treatment) were analyzed using two-sided t-tests in participants with data for both visits. RESULTS: At baseline, 88.3 % (454/514) of participants reported having OBS (34.0 %), ODNSP (17.1 %), or EMO (79.6 %). Those with ODNSP had substantially shorter mean duration of uninterrupted sleep (4.4 h) than the overall pooled population (7.1 h). At Week 14/15, mean decrease from baseline in ODNSP duration was significantly greater with opicapone than with placebo (-0.9 vs. -0.4 h, P < 0.05). In participants with ODNSP at baseline, the decrease in total time spent awake during the night-time sleep period was significantly greater with opicapone than with placebo (-1.0 vs. -0.4 h, P < 0.05), as was the reduction in percent time spent awake during the night-time sleep period (-12.8 % vs. -4.5 %, P < 0.05). CONCLUSION: "OFF"-times around nighttime sleep were common in BIPARK-1 and BIPARK-2. Opicapone may improve sleep by decreasing the amount of time spent awake during the night in patients with PD who have night-time sleep period "OFF" episodes.

The Rectal Gland of the Shark: The Road to Understanding the Mechanism and Regulation of Transepithelial Chloride Transport.

Pictured, described, and speculated on, for close to 400 years, the function of the rectal gland of elasmobranchs remained unknown. In the late 1950s, Burger discovered that the rectal gland of Squalus acanthias secreted an almost pure solution of sodium chloride, isosmotic with blood, which could be stimulated by volume expansion of the fish. Twenty five years later, Stoff discovered that the secretion of the gland was mediated by adenyl cyclase. Studies since then have shown that vasoactive intestinal peptide (VIP) is the neurotransmitter responsible for activating adenyl cyclase; however, the amount of circulating VIP does not change in response to volume expansion. The humoral factor involved in activating the secretion of the gland is C-type natriuretic peptide, secreted from the heart in response to volume expansion. C-type natriuretic peptide circulates to the gland where it stimulates the release of VIP from nerves within the gland, but it also has a direct effect, independent of VIP. Sodium, potassium, and chloride are required for the gland to secrete, and the secretion of the gland is inhibited by ouabain or furosemide. The current model for the secretion of chloride was developed from this information. Basolateral NaKATPase maintains a low intracellular concentration of sodium, which establishes the large electrochemical gradient for sodium directed into the cell. Sodium moves from the blood into the cell (together with potassium and chloride) down this electrochemical gradient, through a coupled sodium, potassium, and two chloride cotransporter (NKCC1). On activation, chloride moves from the cell into the gland lumen, down its electrical gradient through apical cystic fibrosis transmembrane regulator. The fall in intracellular chloride leads to the phosphorylation and activation of NKCC1 that allows more chloride into the cell. Transepithelial sodium secretion into the lumen is driven by an electrical gradient through a paracellular pathway. The aim of this review was to examine the history of the origin of this model for the transport of chloride and suggest that it is applicable to many epithelia that transport chloride, both in resorptive and secretory directions.

SARS-CoV-2 infection in brown-headed spider monkeys (Ateles fusciceps) at a wildlife rescue center on the coast of Ecuador-South America.

Human populations can be affected in unpredictable ways by the emergence and spread of zoonotic diseases. The COVID-19 (coronavirus disease of 2019) pandemic was a reminder of how devastating these events can be if left unchecked. However, once they have spread globally, the impact of these diseases when entering non-exposed wildlife populations is unknown. The current study reports the infection of brown-headed spider monkeys (Ateles fusciceps) at a wildlife rescue center in Ecuador. Four monkeys were hospitalized, and all tested positive for SARS-CoV-2 (Severe acute respiratory syndrome coronavirus 2) by RT-qPCR (Quantitative Reverse Transcription PCR). Fecal samples (n = 12) from monkeys at the rescue center also tested positive; three zookeepers responsible for feeding and deworming the monkeys also tested positive, suggesting human-animal transmission. Whole genome sequencing identified most samples' omicron clade 22B BA.5 lineage. These findings highlight the threat posed by an emerging zoonotic disease in wildlife species and the importance of preventing spillover and spillback events during epidemic or pandemic events.IMPORTANCEAlthough COVID-19 (coronavirus disease of 2019) has been primarily contained in humans through widespread vaccination, the impact and incidence of SARS-CoV-2 (Severe acute respiratory syndrome coronavirus) and its transmission and epidemiology in wildlife may need to be addressed. In some natural environments, the proximity of animals to humans is difficult to control, creating perfect scenarios where susceptible wildlife can acquire the virus from humans. In these places, it is essential to understand how transmission can occur and to develop protocols to prevent infection. This study reports the infection of brown-headed spider monkeys with SARS-CoV-2, a red-listed monkey species, at a wildlife recovery center in Ecuador. This study reports the infection of brown-headed spider monkeys with SARS-CoV-2, indicating the potential for transmission between humans and wildlife primates and the importance of preventing such events in the future.

Exploring Plants with Flowers: From Therapeutic Nutritional Benefits to Innovative Sustainable Uses.

Flowers have played a significant role in society, focusing on their aesthetic value rather than their food potential. This study's goal was to look into flowering plants for everything from health benefits to other possible applications. This review presents detailed information on 119 species of flowers with agri-food and health relevance. Data were collected on their family, species, common name, commonly used plant part, bioremediation applications, main chemical compounds, medicinal and gastronomic uses, and concentration of bioactive compounds such as carotenoids and phenolic compounds. In this respect, 87% of the floral species studied contain some toxic compounds, sometimes making them inedible, but specific molecules from these species have been used in medicine. Seventy-six percent can be consumed in low doses by infusion. In addition, 97% of the species studied are reported to have medicinal uses (32% immune system), and 63% could be used in the bioremediation of contaminated environments. Significantly, more than 50% of the species were only analysed for total concentrations of carotenoids and phenolic compounds, indicating a significant gap in identifying specific molecules of these bioactive compounds. These potential sources of bioactive compounds could transform the health and nutraceutical industries, offering innovative approaches to combat oxidative stress and promote optimal well-being.

The salivary peptide histatin-1 enhances bone repair in vivo.

The salivary peptide histatin-1 was recently described as a novel osteogenic factor that stimulates cell adhesion, migration, and differentiation in bone-lineage cells. Since these cell responses collectively contribute to bone regeneration, we hypothesized that histatin-1 harbors the capacity to enhance bone tissue repair at the preclinical level. By using a model of monocortical bone defect, we explored the effects of histatin-1 in tibial mineralization and organic matrix formation in vivo. To this end, different amounts of histatin-1 were embedded in one-mm3 collagen sponges and then applied to tibial monocortical defects in C57bl/6 mice. After seven days, mice were euthanized, and samples were processed for subsequent analysis. Micro-computed tomography screening showed that histatin-1 increased intraosseous mineralization, and this phenomenon was accompanied by augmented collagen matrix deposition and closure of cortical defect edges, as determined by Hematoxylin-Eosin and Masson's Trichrome staining. Moreover, immunohistochemical analyses showed that histatin-1 increased the expression of the osteogenic marker alkaline phosphatase, which was accompanied by augmented blood vessel formation. Collectively, our findings show that histatin-1 itself promotes bone regeneration in an orthotopic model, proposing this molecule as a therapeutic candidate for use in bone regenerative medicine.

Transmitochondrial Cybrid Generation Using Cancer Cell Lines.

In recent years, the number of studies dedicated to ascertaining the connection between mitochondria and cancer has significantly risen. However, more efforts are still needed to fully understand the link involving alterations in mitochondria and tumorigenesis, as well as to identify tumor-associated mitochondrial phenotypes. For instance, to evaluate the contribution of mitochondria in tumorigenesis and metastasis processes, it is essential to understand the influence of mitochondria from tumor cells in different nuclear environments. For this purpose, one possible approach consists of transferring mitochondria into a different nuclear background to obtain the so-called cybrid cells. In the traditional cybridization techniques, a cell line lacking mtDNA (ρ0, nuclear donor cell) is repopulated with mitochondria derived from either enucleated cells or platelets. However, the enucleation process requires good cell adhesion to the culture plate, a feature that is partially or completely lost in many cases in invasive cells. In addition, another difficulty found in the traditional methods is achieving complete removal of the endogenous mtDNA from the mitochondrial-recipient cell line to obtain pure nuclear and mitochondrial DNA backgrounds, avoiding the presence of two different mtDNA species in the generated cybrid. In this work, we present a mitochondrial exchange protocol applied to suspension-growing cancer cells based on the repopulation of rhodamine 6G-pretreated cells with isolated mitochondria. This methodology allows us to overcome the limitations of the traditional approaches, and thus can be used as a tool to expand the comprehension of the mitochondrial role in cancer progression and metastasis.

Antiepileptogenesis after stroke-trials and tribulations: Methodological challenges and recruitment results of a Phase II study with eslicarbazepine acetate.

There is currently no evidence to support the use of antiseizure medications to prevent unprovoked seizures following stroke. Experimental animal models suggested a potential antiepileptogenic effect for eslicarbazepine acetate (ESL), and a Phase II, multicenter, randomized, double-blind, placebo-controlled study was designed to test this hypothesis and assess whether ESL treatment for 1 month can prevent unprovoked seizures following stroke. We outline the design and status of this antiepileptogenesis study, and discuss the challenges encountered in its execution to date. Patients at high risk of developing unprovoked seizures after acute intracerebral hemorrhage or acute ischemic stroke were randomized to receive ESL 800 mg/d or placebo, initiated within 120 hours after primary stroke occurrence. Treatment continued until Day 30, then tapered off. Patients could receive all necessary therapies for stroke treatment according to clinical practice guidelines and standard of care, and are being followed up for 18 months. The primary efficacy endpoint is the occurrence of a first unprovoked seizure within 6 months after randomization ("failure rate"). Secondary efficacy assessments include the occurrence of a first unprovoked seizure during 12 months after randomization and during the entire study; functional outcomes (Barthel Index original 10-item version; National Institutes of Health Stroke Scale); post-stroke depression (Patient Health Questionnaire-9; PHQ-9); and overall survival. Safety assessments include the evaluation of treatment-emergent adverse events; laboratory parameters; vital signs; electrocardiogram; suicidal ideation and behavior (PHQ-9 question 9). The protocol aimed to randomize approximately 200 patients (1:1), recruited from 21 sites in seven European countries and Israel. Despite the challenges encountered, particularly during the COVID-19 pandemic, the study progressed and included a remarkable number of patients, with 129 screened and 125 randomized. Recruitment was stopped after 30 months, the first patient entered in May 2019, and the study is ongoing and following up on patients according to the Clinical Trial Protocol.

Local Temperature Increments and Induced Cell Death in Intracellular Magnetic Hyperthermia.

The generation of temperature gradients on nanoparticles heated externally by a magnetic field is crucially important in magnetic hyperthermia therapy. But the intrinsic low heating power of magnetic nanoparticles, at the conditions allowed for human use, is a limitation that restricts the general implementation of the technique. A promising alternative is local intracellular hyperthermia, whereby cell death (by apoptosis, necroptosis, or other mechanisms) is attained by small amounts of heat generated at thermosensitive intracellular sites. However, the few experiments conducted on the temperature determination of magnetic nanoparticles have found temperature increments that are much higher than the theoretical predictions, thus supporting the local hyperthermia hypothesis. Reliable intracellular temperature measurements are needed to get an accurate picture and resolve the discrepancy. In this paper, we report the real-time variation of the local temperature on γ-Fe2O3 magnetic nanoheaters using a Sm3+/Eu3+ ratiometric luminescent thermometer located on its surface during exposure to an external alternating magnetic field. We measure maximum temperature increments of 8 °C on the surface of the nanoheaters without any appreciable temperature increase on the cell membrane. Even with magnetic fields whose frequency and intensity are still well within health safety limits, these local temperature increments are sufficient to produce a small but noticeable cell death, which is enhanced considerably as the magnetic field intensity is increased to the maximum level tolerated for human use, consequently demonstrating the feasibility of local hyperthermia.

How hot can mitochondria be? Incubation at temperatures above 43 °C induces the degradation of respiratory complexes and supercomplexes in intact cells and isolated mitochondria.

Mitochondrial function generates an important fraction of the heat that contributes to cellular and organismal temperature maintenance, but the actual values of this parameter reached in the organelles is a matter of debate. The studies addressing this issue have reported divergent results: from detecting in the organelles the same temperature as the cell average or the incubation temperature, to increasing differences of up to 10 degrees above the incubation value. Theoretical calculations based on physical laws exclude the possibility of relevant temperature gradients between mitochondria and their surroundings. These facts have given rise to a conundrum or paradox about hot mitochondria. We have examined by Blue-Native electrophoresis, both in intact cells and in isolated organelles, the stability of respiratory complexes and supercomplexes at different temperatures to obtain information about their tolerance to heat stress. We observe that, upon incubation at values above 43 °C and after relatively short periods, respiratory complexes, and especially complex I and its supercomplexes, are unstable even when the respiratory activity is inhibited. These results support the conclusion that high temperatures (>43 °C) cause damage to mitochondrial structure and function and question the proposal that these organelles can physiologically work at close to 50 °C.

Long-term effect of Toll-like receptor-2, -4, -5, -7 and NOD2 stimulation on Na+,K+-ATPase activity and expression in intestinal epithelial cells.

Inappropriate activation of Toll-like receptors (TLRs) and nucleotide-binding oligomerization domain receptors (NOD) is involved in many chronic disorders, including inflammatory bowel disease (IBD). Altered function and/or expression of Na+,K+-ATPase (NKA) and epithelial ion channels are the main cause of electrolyte absorption imbalance in patients with IBD, leading to diarrhea. We aimed to evaluate the effect of TLRs and NOD2 stimulation upon NKA activity and expression in human intestinal epithelial cells (IECs) using RT-qPCR, Western blot, and electrophysiology techniques. TLR2, TLR4, and TLR7 activation inhibited NKA activity [(means ± SE) -20.0 ± 1.2%, -34.0 ± 1.5%, and -24.5 ± 2.0% in T84 cells; and -21.6 ± 7.4%, -37.7 ± 3.5%, and -11.0 ± 2.3% in Caco-2 cells]. On the other hand, activation of TLR5 increased NKA activity (16.2 ± 2.9% in T84 and 36.8 ± 5.2% in Caco-2 cells) and ß1-NKA mRNA levels (21.8 ± 7.8% in T84 cells). The TLR4 agonist synthetic monophosphoryl lipid A (MPLAs) reduced α1-NKA mRNA levels in both T84 and Caco-2 cells (-28.5 ± 3.6% and -18.7 ± 2.8%), and this was accompanied by a decrease in α1-NKA protein expression (-33.4 ± 11.8% and -39.4 ± 11.2%). NOD2 activation upregulated NKA activity (12.2 ± 5.1%) and α1-NKA mRNA levels (6.8 ± 1.6%) in Caco-2 cells. In summary, TLR2, TLR4, and TLR7 activation induce downregulation of NKA in IECs, whereas TLR5 and NOD2 activation has the opposite effect. A comprehensive understanding of the cross talk between TLRs, NOD2, and NKA is of utmost relevance for developing better IBD treatments.

A Mutation in Mouse MT-ATP6 Gene Induces Respiration Defects and Opposed Effects on the Cell Tumorigenic Phenotype.

As the last step of the OXPHOS system, mitochondrial ATP synthase (or complex V) is responsible for ATP production by using the generated proton gradient, but also has an impact on other important functions linked to this system. Mutations either in complex V structural subunits, especially in mtDNA-encoded ATP6 gene, or in its assembly factors, are the molecular cause of a wide variety of human diseases, most of them classified as neurodegenerative disorders. The role of ATP synthase alterations in cancer development or metastasis has also been postulated. In this work, we reported the generation and characterization of the first mt-Atp6 pathological mutation in mouse cells, an m.8414A>G transition that promotes an amino acid change from Asn to Ser at a highly conserved residue of the protein (p.N163S), located near the path followed by protons from the intermembrane space to the mitochondrial matrix. The phenotypic consequences of the p.N163S change reproduce the effects of MT-ATP6 mutations in human diseases, such as dependence on glycolysis, defective OXPHOS activity, ATP synthesis impairment, increased ROS generation or mitochondrial membrane potential alteration. These observations demonstrate that this mutant cell line could be of great interest for the generation of mouse models with the aim of studying human diseases caused by alterations in ATP synthase. On the other hand, mutant cells showed lower migration capacity, higher expression of MHC-I and slightly lower levels of HIF-1α, indicating a possible reduction of their tumorigenic potential. These results could suggest a protective role of ATP synthase inhibition against tumor transformation that could open the door to new therapeutic strategies in those cancer types relying on OXPHOS metabolism.

Boosting caffeic acid performance as antioxidant and monoamine oxidase B/catechol-O-methyltransferase inhibitor.

Increased oxidative stress (OS) and depletion of nigrostriatal dopamine (DA) are closely linked to the neurodegeneration observed in Parkinson's Disease (PD). Caffeic acid (CA)-based antioxidants were developed, and their inhibitory activities towards monoamine oxidases (MAOs) and catechol O-methyltransferases (COMT) were screened. The results showed that the incorporation of an extra double bond maintained or even boosted the antioxidant properties of CA. α-CN derivatives displayed redox potentials (Ep) similar to CA (1) and inhibited hMAO-B with low µM IC50 values. Moreover, catechol amides acted as MB-COMT inhibitors, showing IC50 values within the low µM range. In general, CA derivatives presented safe cytotoxicity profiles at concentrations up to 10 µM. The formation of reactive oxygen species (ROS) induced by CA derivatives may be underlying the cytotoxic effects observed at higher concentrations. Catechol amides 3-6, 8-11 at 10 µM protected cells against oxidative damage. Compounds 3 and 8 were predicted to cross the blood-brain barrier (BBB) by passive diffusion. In summary, we report for the first time BBB-permeant CA-based multitarget lead compounds that may restore DAergic neurotransmission (dual hMAO-B/MB-COMT inhibition) and prevent oxidative damage. The data represents a groundbreaking advancement towards the discovery of the next generation of new drugs for PD.

Voltage-clamp evidence of GABAA receptor subunit-specific effects: pharmacodynamic fingerprint of chlornordiazepam, the major active metabolite of mexazolam, as compared to alprazolam, bromazepam, and zolpidem.

BACKGROUND: Anxiolytic benzodiazepines, due to their clinical effectiveness, are one of the most prescribed drugs worldwide, despite being associated with sedative effects and impaired psychomotor and cognitive performance. Not every GABAA receptor functions in the same manner. Those containing α1 subunits are associated with sleep regulation and have a greater effect on the sedative-hypnotic benzodiazepines, whereas those containing α2 and/or α3 subunits are associated with anxiety phenomena and have a greater effect on the anxiolytic benzodiazepines. Therefore, characterization of the selectivity profile of anxiolytic drugs could translate into a significant clinical impact. METHODS: The present study pharmacodynamically evaluated chlornordiazepam, the main active metabolite of mexazolam, upon GABAA receptors containing α2 and/or α3, anxiety-related, and those containing an α1 subunit, associated with sleep modulation. RESULTS: As shown by whole-cell patch-clamp data, chlornordiazepam potentiated GABA-evoked current amplitude in α2 and α3 containing receptors without changing the current amplitude in α1 containing receptors. However, current decay time increased, particularly in GABAA receptors containing α1 subunits. In contrast, other anxiolytic benzodiazepines such as alprazolam, bromazepam, and zolpidem, all increased currents associated with GABAA receptors containing the α1 subunit. CONCLUSIONS: This novel evidence demonstrates that mexazolam (through its main metabolite chlornordiazepam) has a "pharmacodynamic fingerprint" that correlates better with an anxiolytic profile and fewer sedative effects, when compared to alprazolam, bromazepam and zolpidem, explaining clinical trial outcomes with these drugs. This also highlights the relevance of the pharmacological selectivity over GABAA receptor subtypes in the selection of benzodiazepines, in addition to their clinical performance and pharmacokinetic characteristics.

The safety/tolerability of opicapone when used early in Parkinson's disease patients with levodopa-induced motor fluctuations: A post-hoc analysis of BIPARK-I and II.

Introduction: Post-hoc analyses of the BIPARK-I and II trials previously demonstrated that opicapone (OPC) 50 mg was efficacious over the whole trajectory of motor fluctuation evolution in patients with Parkinson's disease (PD) and end-of-dose motor fluctuations, with enhanced efficacy in patients who were earlier vs. later in their disease course and levodopa treatment pathway. Complementary post-hoc analyses were performed to evaluate the safety/tolerability of OPC following the same pre-defined segmentation of the wide spectrum of duration of both PD and levodopa therapy, as well as of motor fluctuation history, in this patient population. Materials and methods: Data from matching treatment arms in BIPARK-I and II were combined for the placebo (PLC) and OPC 50 mg groups and exploratory post-hoc analyses were performed to investigate the safety/tolerability of OPC 50 mg and PLC in 22 subgroups of patients who were in "earlier" vs. "later" stages of both their disease course (e.g., duration of PD <6 years vs. ≥6 years) and levodopa treatment pathway (e.g., levodopa treatment duration <4 vs. ≥4 years). Safety/tolerability assessments included evaluation of treatment-emergent adverse events (TEAEs). Results: The Safety Set included 522 patients (PLC, n = 257; OPC 50 mg, n = 265). For OPC 50 mg, incidences of TEAEs, related TEAEs, related serious TEAEs, and related TEAEs leading to discontinuation were lower for patients in earlier vs. later stages of their disease course and levodopa treatment pathway in 86.4, 86.4, 63.6, and 68.2% of the 22 pairwise comparisons conducted, respectively (compared with 63.6, 77.3, 18.2, and 45.5%, respectively, in the 22 corresponding PLC comparisons). Conclusion: OPC 50 mg was generally well-tolerated when used to treat patients with PD with end-of-dose fluctuations, with an even more favorable tolerability profile in patients who were earlier, as opposed to later, in their disease course and levodopa treatment pathway, further supporting its use as an early adjunct to levodopa in PD.

Effect of Opicapone on Levodopa Pharmacokinetics in Patients with Fluctuating Parkinson's Disease.

BACKGROUND: Inhibiting catechol-O-methyltransferase extends the plasma half-life of levodopa, potentially allowing physicians to optimize the levodopa regimen in patients with Parkinson's disease (PD) experiencing motor fluctuations. OBJECTIVES: To evaluate the effects of once-daily opicapone on levodopa plasma pharmacokinetics and motor response when added to two different levodopa dosing regimens. METHODS: A total of 24 patients with PD and motor fluctuations were enrolled in an exploratory, open-label, modified cross-over trial. Participants first received levodopa/carbidopa 500/125 mg (five intakes) for 2 weeks and were then randomly assigned (1:1) to levodopa/carbidopa 400/100 mg given over either four or five daily intakes plus opicapone 50 mg for an additional 2 weeks. Levodopa 12-hour pharmacokinetics was the primary outcome (ie, excluding the effect of last/evening levodopa/carbidopa intake), with motor complications evaluated as secondary outcomes. RESULTS: Over 12-hour pharmacokinetics and compared with five-intake levodopa/carbidopa 500/125 mg without opicapone, maximal levodopa concentrations were similar or nonsignificantly higher on both levodopa/carbidopa 400/100 mg regimens plus opicapone. Despite a 100 mg lower total levodopa/carbidopa daily dose, adding opicapone 50 mg at least doubled the levodopa plasma half-life and minimal concentrations, with a significant ≈30% increase in total exposure. The levodopa fluctuation index was only significantly lower for the five intakes plus opicapone regimen (difference of -71.8%; P < 0.0001). Modifications to levodopa pharmacokinetics were associated with decreased off time and increased on time. CONCLUSIONS: Combining opicapone 50 mg with a 100 mg lower daily dose of levodopa provides higher levodopa bioavailability with avoidance of trough levels. Despite the lower levodopa dose, modifying the levodopa pharmacokinetic profile with opicapone was associated with decreased off time and increased on time. © 2022 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

Occurrence of SARS-CoV-2 reinfections at regular intervals in Ecuador.

SARS-CoV-2 reinfection is defined as a new infection with a different virus variant in an individual who has already recovered from a previous episode of COVID-19. The first case of reinfection in the world was described in August 2020, since then, reinfections have increased over time and their incidence has fluctuated with specific SARS-CoV-2 variant waves. Initially, reinfections were estimated to represent less than 1% of total COVID-19 infections. With the advent of the Omicron variant, reinfections became more frequent, representing up to 10% of cases (based on data from developed countries). The frequency of reinfections in Latin America has been scarcely reported. The current study shows that in Ecuador, the frequency of reinfections has increased 10-fold following the introduction of Omicron, after 22 months of surveillance in a single center of COVID-19 diagnostics. Suspected reinfections were identified retrospectively from a database of RT-qPCR-positive patients. Cases were confirmed by sequencing viral genomes from the first and second infections using the ONT MinION platform. Monthly surveillance showed that the main incidence peaks of reinfections were reached within four to five months, coinciding with the increase of COVID-19 cases in the country, suggesting that the emergence of reinfections is related to higher exposure to the virus during outbreaks. This study performed the longest monitoring of SARS-CoV-2 reinfections, showing an occurrence at regular intervals of 4-5 months and confirming a greater propensity of Omicron to cause reinfections.

Evaluation of the Anti-Leishmania mexicana and -Trypanosoma brucei Activity and Mode of Action of 4,4'-(Arylmethylene)bis(3-methyl-1-phenyl-1H-pyrazol-5-ol).

Trypanosomiasis and leishmaniasis are neglected infections caused by trypanosomatid parasites. The first-line treatments have many adverse effects, high costs, and are prone to resistance development, hence the necessity for new chemotherapeutic options. In line with this, twenty five 4,4'-(arylmethylene)bis(1H-pyrazol-5-ols) derivatives were synthesized and evaluated in vitro for their anti-trypanosomatid activity. Ten and five compounds from this series showed IC50 ≤ 10 µM against the promastigote and the bloodstream stage of Leishmania mexicana and Trypanosoma brucei brucei, respectively. Overall, derivatives with pyrazole rings substituted with electron-withdrawing groups proved more active than those with electron-donating groups. The hits proved moderately selective towards L. mexicana and T. brucei (selectivity index, SI, compared to murine macrophages = 5−26). The exception was one derivative displaying an SI (>111−189) against T. brucei that surpassed, by >6-fold, the selectivity of the clinical drug nifurtimox (SI = 13−28.5). Despite sharing a common scaffold, the hits differed in their mechanism of action, with halogenated derivatives inducing a rapid and marked intracellular oxidative milieu in infective T. brucei. Notably, most of the hits presented better absorption, distribution, metabolism, and excretion (ADME) properties than the reference drugs. Several of the bioactive molecules herein identified represent a promising starting point for further improvement of their trypanosomatid potency and selectivity.

Effect of Toll-like receptor-2, -4, -5, -7, and NOD2 stimulation on potassium channel conductance in intestinal epithelial cells.

Disproportionate activation of pattern recognition receptors plays a role in inflammatory bowel disease (IBD) pathophysiology. Diarrhea is a hallmark symptom of IBD, resulting at least in part from an electrolyte imbalance that may be caused by changes in potassium channel activity. We evaluated the impact of Toll-like receptors (TLRs) and nucleotide-binding oligomerization domain 2 (NOD2) stimulation on potassium conductance of the basolateral membrane in human intestinal epithelial cells (IECs) and the role of potassium channels through electrophysiological assays under short-circuit current in Ussing chambers. TLRs and NOD2 were stimulated using specific agonists, and potassium channels were selectively blocked using triarylmethane-34 (TRAM-34), adenylyl-imidodiphosphate (AMP-PNP), and BaCl2. Potassium conductance of the basolateral membrane decreased upon activation of TLR2, TLR4, and TLR7 in T84 cells (means ± SE, -11.2 ± 4.5, -40.4 ± 7.2, and -19.4 ± 5.9, respectively) and in Caco-2 cells (-13.1 ± 5.7, -55.7 ± 7.4, and -29.1 ± 7.2, respectively). In contrast, activation of TLR5 and NOD2 increased basolateral potassium conductance, both in T84 cells (18.0 ± 4.1 and 18.4 ± 2.8, respectively) and in Caco-2 cells (21.2 ± 8.4 and 16.0 ± 3.6, respectively). TRAM-34 and AMP-PNP induced a decrease in basolateral potassium conductance upon TLR4 stimulation in both cell lines. Both KCa3.1- and Kir6-channels appear to be important mediators of this effect in IECs and could be potential targets for therapeutic agent development.NEW & NOTEWORTHY This study highlights that PRRs stimulation directly influences K+-channel conductance in IECs. TLR-2, -4, -7 stimulation decreased K+ conductance, whereas TLR5 and NOD2 stimulation had the opposite effect, leading to an increase of it instead. This study reports for the first time that KCa3.1- and Kir6-channels play a role in K+ transport pathways triggered by TLR4 stimulation. These findings suggest that KCa3.1- and Kir6-channels modulation may be a potential target for new therapeutic agents in IBD.

Inactivation Mechanism of the Fatty Acid Amide Hydrolase Inhibitor BIA 10-2474.

BIA 10-2474 is a time-dependent inhibitor of fatty acid amide hydrolase (FAAH) that was under clinical development for the treatment of neurological conditions when the program was terminated after one subject died and four were hospitalized with neurological symptoms during a first-in-human clinical study. The present work describes the mechanism of FAAH inhibition by BIA 10-2474 as a target-specific covalent inhibition, supported by quantum mechanics and molecular modelling studies. The inhibitor incorporates a weakly reactive electrophile which, upon specific binding to the enzyme's active site, is positioned to react readily with the catalytic residues. The reactivity is enhanced on-site by the increased molarity at the reaction site and by specific inductive interactions with FAAH. In the second stage, the inhibitor reacts with the enzyme's catalytic nucleophile to form a covalent enzyme-inhibitor adduct. The hydrolysis of this adduct is shown to be unlikely under physiological conditions, therefore leading to irreversible inactivation of FAAH. The results also reveal the important role played by FAAH Thr236 in the reaction with BIA 10-2474, which is specific to FAAH and is not present in other serine hydrolases. It forms a hydrogen bond with the imidazole nitrogen of the inhibitor and helps lowering the activation free energy of the first step of the reaction, by pre-orienting and stabilizing the inhibitor in a near-reactive configuration. In the second step, Thr236 can also serve as a mechanistic alternative to protonate the leaving group.