Negative impacts of social isolation on behavior and neuronal functions are recovered after short-term social reintroduction in zebrafish.

Recently, social isolation measures were crucial to prevent the spread of the coronavirus pandemic. However, the lack of social interactions affected the population mental health and may have long-term consequences on behavior and brain functions. Here, we evaluated the behavioral, physiological, and molecular effects of a social isolation (SI) in adult zebrafish, and whether the animals recover such changes after their reintroduction to the social environment. Fish were submitted to 12 days of SI, and then reintroduced to social context (SR). Behavioral analyses to evaluate locomotion, anxiety-like and social-related behaviors were performed after SI protocol, and 3 and 6 days after SR. Cortisol and transcript levels from genes involved in neuronal homeostasis (c-fos, egr, bdnf), and serotonergic (5-HT) and dopaminergic (DA) neurotransmission (thp, th) were also measured. SI altered social behaviors in zebrafish such as aggression, social preference, and shoaling. Fish submitted to SI also presented changes in the transcript levels of genes related to neural activity, and 5-HT/DA signaling. Interestingly, most of the behavioral and molecular changes induced by SI were not found again 6 days after SR. Thus, we highlight that SR of zebrafish to their conspecifics played a positive role in social behaviors and in the expression of genes involved in different neuronal signaling pathways that were altered after 12 days of SI. This study brings unprecedented data on the effects of SR in the recovery from SI neurobehavioral alterations, and reinforces the role of zebrafish as a translational model for understanding the neurobiological mechanisms adjacent to SI and resocialization.

Kinetics and molecular modeling studies on the inhibition mechanism of GH13 α-glycosidases by small molecule ligands.

Alpha-glucosidase inhibitors play an important role in Diabetes Mellitus (DM) treatment since they prevent postprandial hyperglycemia. The Glycoside Hydrolase family 13 (GH13) is the major family of enzymes acting on substrates containing α-glucoside linkages, such as maltose and amylose/amylopectin chains in starch. Previously, our group identified glycoconjugate 1H-1,2,3-triazoles (GCTs) inhibiting two GH13 α-glycosidases: yeast maltase (MAL12) and porcine pancreatic amylase (PPA). Here, we combined kinetic studies and computational methods on nine GCTs to characterize their inhibitory mechanism. They all behaved as reversible inhibitors, and kinetic models encompassed noncompetitive and various mechanisms of mixed-type inhibition for both enzymes. Most potent inhibitors displayed Ki values of 30 µM for MAL12 (GPESB16) and 37 µM for PPA (GPESB15). Molecular dynamics and docking simulations indicated that on MAL12, GPESB15 and GPESB16 bind in a cavity adjacent to the active site, while on the PPA, GPESB15 was predicted to bind at the entrance of the catalytic site. Notably, despite its putative location within the active site, the binding of GPESB15 does not obstruct the substrate's access to the cleavage site. Our study contributes to paving the way for developing novel therapeutic strategies for managing DM-2 through GH13 α-glycosidases inhibition.

Recent Advances on Targeting Proteases for Antiviral Development.

Viral proteases are an important target for drug development, since they can modulate vital pathways in viral replication, maturation, assembly and cell entry. With the (re)appearance of several new viruses responsible for causing diseases in humans, like the West Nile virus (WNV) and the recent severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), understanding the mechanisms behind blocking viral protease's function is pivotal for the development of new antiviral drugs and therapeutical strategies. Apart from directly inhibiting the target protease, usually by targeting its active site, several new pathways have been explored to impair its activity, such as inducing protein aggregation, targeting allosteric sites or by inducing protein degradation by cellular proteasomes, which can be extremely valuable when considering the emerging drug-resistant strains. In this review, we aim to discuss the recent advances on a broad range of viral proteases inhibitors, therapies and molecular approaches for protein inactivation or degradation, giving an insight on different possible strategies against this important class of antiviral target.

JM-20 potently prevents the onset of caffeine-induced anxiogenic phenotypes in zebrafish (Danio rerio).

Anxiety is among the most prevalent mental disorders present in the general population. Benzodiazepines are the most commonly prescribed drugs for the treatment of anxiety. Using zebrafish as a model organism, we investigated the anxiolytic activity of JM-20, a novel hybrid molecule with a 1,5-benzodiazepine ring fused to a dihydropyridine moiety. Firstly, we carried out some assays to analyze the possible toxicity mediated by JM-20. For this, zebrafish were exposed to different JM-20 concentrations (0-5 µM) for 96 h. Then, using the novel tank test, we evaluated both locomotor and anxiety-like behavior of the animals. Furthermore, brain, liver and plasma were removed to assess toxicity parameters. JM-20 exposure did not cause changes on novel tank, and also did not alter brain viability, hepatic LDH and plasma ALT levels. Afterward, we investigated whether a pre-exposure to JM-20 would prevent the anxiogenic effect evoked by caffeine. In the novel tank test, caffeine significantly decreased the time spent at the top, as well as the number of transitions to the top area. Moreover, caffeine decreased both the total and average time spent in the lit area, as well as increased the number of risk episodes evaluated by the light-dark test. Whole-body cortisol levels were also increased by caffeine exposure. Interestingly, pre-treatment with JM-20 abolished all alterations induced by caffeine. The anxiolytic effect profile of JM-20 was similar to those found for diazepam (positive control). Our findings show, for the first time, the anxiolytic effect of JM-20 in zebrafish, and its relationship with cortisol regulation.

Screening for Differentially Expressed Memory Genes on a Diabetes Model Induced by High-Sugar Diet in Drosophila melanogaster: Potential Markers for Memory Deficits.

Type 2 diabetes mellitus (T2DM) has been shown to affect a series of cognitive processes including memory, increasing the risk for dementia, particularly Alzheimer's disease (AD). Although increasing evidence has supported that both diseases share common features, the pathophysiological mechanisms connecting these two disorders remain to be fully elucidated. Herein, we used Drosophila melanogaster fed on a high-sugar diet (HSD) to mimic T2DM, and investigate its effects on memory as well as identify potential molecular players associated with the memory deficits induced by HSD. Flies hatched from and reared on HSD for 7 days had a substantial decrease in short-term memory (STM). The screening for memory-related genes using transcriptome data revealed that HSD altered the expression of 33% of memory genes in relation to the control. Among the differentially expressed genes (DEGs) with a fold change (FC) higher than two, we found five genes, related to synapse and memory trace formation, that could be considered strong candidates to underlie the STM deficits in HSD flies: Abl tyrosine kinase (Abl), bruchpilot (Brp), minibrain (Mnb), shaker (Sh), and gilgamesh (Gish). We also analyzed genes from the dopamine system, one of the most relevant signaling pathways for olfactory memory. Interestingly, the flies fed on HSD presented a decreased expression of the Tyrosine hydroxylase (Ple) and Dopa decarboxylase (Ddc) genes, signals of a possible dopamine deficiency. In this work, we present promising biomarkers to investigate molecular networks shared between T2DM and AD.

VEGF-C-expressing TAMs rewire the metastatic fate of breast cancer cells.

The expression of pro-lymphangiogenic VEGF-C in primary tumors is associated with sentinel lymph node metastasis in most solid cancer types. However, the impact of VEGF-C on distant organ metastasis remains unclear. Perivascular tumor-associated macrophages (TAMs) play a crucial role in guiding hematogenous spread of cancer cells by establishing metastatic pathways within the tumor microenvironment. This process supports breast cancer cell intravasation and metastatic dissemination. We show here that VEGF-C-expressing TAMs reduce the dissemination of mammary cancer cells to the lungs while concurrently increasing lymph node metastasis. These TAMs express podoplanin and interact with normalized tumor blood vessels expressing VEGFR3. Moreover, clinical data suggest inverse association between VEGF-C-expressing TAMs and breast cancer malignancy. Thus, our study elucidates the paradoxical role of VEGF-C-expressing TAMs in redirecting cancer cells to preferentially disseminate to lymph nodes rather than to lungs, partially achieved by normalizing tumor blood vessels and promoting lymphangiogenesis.

Photodegradable antimicrobial agents: towards structure optimization.

Antibiotic resistance continues to be an ominous threat facing human health globally and urgent action is required to limit the loss of human life. The pollution of antibiotics into the environment is one of the drivers behind the crisis. With this in mind, we have developed novel photodecomposable antimicrobial agents based on an ethanolamine scaffold, which upon photoirradiation decomposes into two major inactive fragments. Herein we describe our further work on the synthesis of novel ethanolamines with a particular focus on structure activity relationship, resulting in four new active compounds which photodecomposed into inactive fragments.

Visible Light and Triselenium Dicyanide (TSD): New Horizons in the Synthesis of Organic Selenocyanates.

Herein, we describe a new method for the synthesis of α-carbonyl selenocyanates by reacting triselenium dicyanide (TSD) and styrenes under blue light irradiation and O2 atmosphere. The reactions are triggered by the formation of Se-centered radical species, followed by the addition/oxidation of the styrene π-bond. α-Carbonyl selenocyanates and α-hydroxy selenocyanates were obtained in moderate to excellent yields from aryl- and alkyl-substituted alkenes, respectively. It was demonstrated that α-carbonyl selenocyanates could be used as a synthetic platform in a multicomponent reaction strategy to prepare 2-phenylimidazo[1,2-a]pyridine derivatives, which were evaluated for their photophysical properties. Overall, this new method provides a useful tool for synthesizing α-carbonyl selenocyanates, and demonstrates their potential for use in the synthesis of other compounds, thus giving new synthetic opportunities to construct organic selenocyanate compounds.

AI-Driven Discovery of SARS-CoV-2 Main Protease Fragment-like Inhibitors with Antiviral Activity In Vitro.

SARS-CoV-2 is the causative agent of COVID-19 and is responsible for the current global pandemic. The viral genome contains 5 major open reading frames of which the largest ORF1ab codes for two polyproteins, pp1ab and pp1a, which are subsequently cleaved into 16 nonstructural proteins (nsp) by two viral cysteine proteases encoded within the polyproteins. The main protease (Mpro, nsp5) cleaves the majority of the nsp's, making it essential for viral replication and has been successfully targeted for the development of antivirals. The first oral Mpro inhibitor, nirmatrelvir, was approved for treatment of COVID-19 in late December 2021 in combination with ritonavir as Paxlovid. Increasing the arsenal of antivirals and development of protease inhibitors and other antivirals with a varied mode of action remains a priority to reduce the likelihood for resistance emerging. Here, we report results from an artificial intelligence-driven approach followed by in vitro validation, allowing the identification of five fragment-like Mpro inhibitors with IC50 values ranging from 1.5 to 241 µM. The three most potent molecules (compounds 818, 737, and 183) were tested against SARS-CoV-2 by in vitro replication in Vero E6 and Calu-3 cells. Compound 818 was active in both cell models with an EC50 value comparable to its measured IC50 value. On the other hand, compounds 737 and 183 were only active in Calu-3, a preclinical model of respiratory cells, showing selective indexes twice as high as those for compound 818. We also show that our in silico methodology was successful in identifying both reversible and covalent inhibitors. For instance, compound 818 is a reversible chloromethylamide analogue of 8-methyl-γ-carboline, while compound 737 is an N-pyridyl-isatin that covalently inhibits Mpro. Given the small molecular weights of these fragments, their high binding efficiency in vitro and efficacy in blocking viral replication, these compounds represent good starting points for the development of potent lead molecules targeting the Mpro of SARS-CoV-2.

Dataset of feed bunk score images of cattle feedlot.

Bunk management is an important technique to minimize the variations in consumption in feedlot cattle and can be performed according to the South Dakota State University classification system. The use of information and communication technology (ICT) can help, in an objective way, in the interpretation of these measurements. We created a dataset with the objective to develop an automatic classification method of feed bunk score. In May, September and October on the 2021 and September on the 2022 we captured 1511 images in the morning on the farms, in natural lighting conditions with different angles and backgrounds and at a height of about 1.5 m from the bunk. After acquisition data, each image was classified according to its score classification. Additionally, we resized the images to 500 × 500 pixels, generated annotations files, and organized the dataset in folders. The images in this dataset can be used to train and validate a machine learning model to classify feed bunk images. This model can be used to develop an application to support bunk management.

Microstructured Polymer System Containing Proanthocyanidin-Enriched Extract from Limonium brasiliense as a Prophylaxis Strategy to Prevent Recurrence of Porphyromonas gingivalis.

Periodontal diseases are a global oral health problem affecting almost 10% of the global population. Porphyromonas gingivalis is one of the main bacteria involved in the initiation and progression of inflammatory processes as a result of the action of the cysteine proteases lysin- and arginine-gingipain. Surelease/polycarbophil microparticles containing a lyophilized proanthocyanidin-enriched fraction from the rhizomes of Limonium brasiliense, traditionally named "baicuru" (ethyl acetate fraction), were manufactured. The ethyl acetate fraction was characterized by UHPLC by the presence of samarangenins A and B (12.10 ± 0.07 and 21.05 ± 0.44%, respectively) and epigallocatechin-3-O-gallate (13.44 ± 0.27%). Physiochemical aspects of Surelease/polycarbophil microparticles were characterized concerning particle size, zeta potential, entrapment efficiency, ethyl acetate fraction release, and mucoadhesion. Additionally, the presence of the ethyl acetate fraction-loaded microparticles was performed concerning potential influence on viability of human buccal KB cells, P. gingivalis adhesion to KB cells, gingipain activity, and P. gingivalis biofilm formation. In general, all Surelease/polycarbophil microparticles tested showed strong adhesion to porcine cheek mucosa (93.1 ± 4.2% in a 30-min test), associated with a prolonged release of the ethyl acetate fraction (up to 16.5 ± 0.8% in 24 h). Preincubation of KB cells with Surelease/polycarbophil microparticles (25 µg/mL) resulted in an up to 93 ± 2% reduced infection rate by P. gingivalis. Decreased activity of the P. gingivalis-specific virulence factors lysin- and arginine-gingipain proteases by Surelease/polycarbophil microparticles was confirmed. Surelease/polycarbophil microparticles decreased biofilm formation of P. gingivalis (97 ± 2% at 60 µg/mL). Results from this study prove the promising activity of Surelease/polycarbophil microparticles containing ethyl acetate fraction microparticles as a prophylaxis strategy to prevent the recurrence of P. gingivalis.

Design as strategy for evaluation of the mechanical properties of binary mixtures composed of poly(methyl vinyl ether-alt-maleic anhydride) and Pluronic F127 for biomedical applications.

The synergism between thermoresponsive and bioadhesive polymers can lead to the optimization of materials with enhanced mechanical and bioadhesive properties. Quality by Design can assure the understanding and control of formulation variables. In this approach, Design of Experiment has been widely utilized as an important strategy. Poly(methyl vinyl ether-alt-maleic anhydride) (PVMMA) is a bioadhesive polymer and Pluronic F127 (PF127) shows thermoresponsiveness. The association of these two polymers has been poorly investigated. The aim of this work was to study the mechanical, bioadhesive and rheological properties of polymer mixtures composed of PVMMA and PF127, in order to select the best conditions and formulations for biomedical applications. Textural properties (hardness, compressibility, adhesiveness, cohesiveness and elasticity), softness index, bioadhesion and rheological characteristics (flow and viscoelasticity) showed that 17.5-20% (w/w) PF127-polymer mixtures displayed improved values of the parameters. However, the rheological interaction parameter showed low synergism, due to the polymers' characteristics and system organization. The formulations displayed gelation temperatures suitable for administration, with improved bioadhesive properties mainly at 34 °C and suggests the formulations can be used for biomedical applications. DoE constituted an important tool to investigate these systems showing the main effects that significantly influence the binary mixtures.

Assessment of Pharmaceutical Services for Smoking Cessation: An Effectiveness-Implementation Hybrid Study.

Smoking is the main preventable cause of illness and early death worldwide. Thus, it is better to promote smoking cessation than to treat tobacco-related diseases. The objective of this study was to assess the implementation and effectiveness of smoking cessation pharmaceutical services offered in primary health care (PHC) in a large Brazilian city through a type 1 effectiveness-implementation hybrid study. The services were offered through individual or group approaches (Jan/2018-Dec/2019). The service indicators were described and the incidence of cessation in the services was evaluated. Factors associated with cessation were assessed by Poisson regression analysis. The services were offered in most PHC centers (61.2%) and by most pharmacists (81.3%). In total, 170 individual (9.7%) and 1591 group (90.3%) approaches occurred, leading to cessation in 39.4% (n = 67) and 44.8% (n = 712) of these, respectively. The use of nicotine plus antidepressants (RR = 1.30; 95%CI = 1.08-1.57; p = 0.006) and the number of sessions with pharmacists (RR = 1.21; 95%CI = 1.19-1.23; p < 0.001) were positively associated with cessation; a very high level of dependence was negatively associated (RR = 0.77; 95%CI = 0.67-0.89; p = 0.001). The smoking cessation services were effective and should be encouraged.

Tumor MHC class I expression alters cancer-associated myelopoiesis driven by host NK cells.

Downregulation of MHC class I (MHCI) molecules on tumor cells is recognized as a resistance mechanism of cancer immunotherapy. Given that MHCI molecules are potent regulators of immune responses, we postulated that the expression of MHCI by tumor cells influences systemic immune responses. Accordingly, mice-bearing MHCI-deficient tumor cells showed reduced tumor-associated extramedullary myelopoiesis in the spleen. Depletion of natural killer (NK) cells abrogated these differences, suggesting an integral role of immune-regulatory NK cells during tumor progression. Cytokine-profiling revealed an upregulation of TNF-α by NK cells in tumors and spleen in mice-bearing MHCI expressing tumors, and inhibition of TNF-α enhanced host myelopoiesis in mice receiving adoptive transfer of tumor-experienced NK cells. Our study highlights a critical role of NK cells beyond its identity as a killer lymphocyte and more importantly, the potential host responses to a localized tumor as determined by its MHCI expression.

A hybrid of 1-deoxynojirimycin and benzotriazole induces preferential inhibition of butyrylcholinesterase (BuChE) over acetylcholinesterase (AChE).

The synthesis of four heterodimers in which the copper(I)-catalysed azide-alkyne cycloaddition was employed to connect a 1-deoxynojirimycin moiety with a benzotriazole scaffold is reported. The heterodimers were investigated as inhibitors against acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE). The heterodimers displayed preferential inhibition (> 9) of BuChE over AChE in the micromolar concentration range (IC50 = 7-50 µM). For the most potent inhibitor of BuChE, Cornish-Bowden plots were used, which demonstrated that it behaves as a mixed inhibitor. Modelling studies of the same inhibitor demonstrated that the benzotriazole and 1-deoxynojirimycin moiety is accommodated in the peripheral anionic site and catalytic anionic site, respectively, of AChE. The binding mode to BuChE was different as the benzotriazole moiety is accommodated in the catalytic anionic site.

Study on the synthesis and structure-activity relationship of 1,2,3-triazoles against toxic activities of Bothrops jararaca venom.

Snakebite envenoming is a health concern and has been a neglected tropical disease since 2017, according to the World Health Organization. In this study, we evaluated the ability of ten 1,2,3-triazole derivatives AM001 to AM010 to inhibit pertinent in vitro (coagulant, hemolytic, and proteolytic) and in vivo (hemorrhagic, edematogenic, and lethal) activities of Bothrops jararaca venom. The derivatives were synthesized, and had their molecular structures fully characterized by CHN element analysis, Fourier-transform infrared spectroscopy and Nuclear magnetic resonance. The derivatives were incubated with the B. jararaca venom (incubation protocol) or administered before (prevention protocol) or after (treatment protocol) the injection of B. jararaca venom into the animals. Briefly, the derivatives were able to inhibit the main toxic effects triggered by B. jararaca venom, though with varying efficacies, and they were devoid of toxicity through in vivo, in silico or in vitro analyses. However, it seemed that the derivatives AM006 or AM010 inhibited more efficiently hemorrhage or lethality, respectively. The derivatives were nontoxic. Therefore, the 1,2,3-triazole derivatives may be useful as an adjuvant to more efficiently treat the local toxic effects caused by B. jararaca envenoming.

Environmentally relevant manganese concentrations evoke anxiety phenotypes in adult zebrafish.

Manganese (Mn) is an essential metal for living organisms. However, the excess of Mn can be toxic, especially for the central nervous system. Herein, we used adult zebrafish as model organism to investigate the relationship of an environmentally relevant Mn exposure with the onset of neurobehavioral disturbances and brain biochemical alterations. Fish were exposed to MnCl2 at 0.5, 2.0, 7.5 and 15.0 mg/L for 96 h, and after submitted to trials for examining exploratory, locomotor and anxiety-related behaviors. The neurobehavioral parameters were followed by the analyses of cell viability, Mn accumulation and acetylcholinesterase activity in the brain, and whole-body cortisol levels. By Novel tank, Light dark and Social preference test, we found that the exposure to Mn, along with locomotor deficits induced anxiety-like phenotypes in zebrafish. Most of these behavioral changes were evoked by the highest concentrations, which also caused cell viability loss, higher accumulation of Mn and increased AChE activity in the brain, and an increase in the whole-body cortisol content. Our findings demonstrated that zebrafish are quite sensitive to levels of Mn found in the environment, and that the magnitude of the neurotoxic effects may be associated with the levels of manganese accumulated in the brain. Interestingly, we showed that Mn exposure in addition to motor deficits may also cause psychiatric abnormalities, namely anxiety.

Factors associated with the identification of drug therapy problems among older patients in Primary Health Care.

OBJECTIVE: To determine the frequency of drug therapy problems among older adults in Primary Health Care, and to analyze the factors associated with their identification in the initial patient assessment, carried out by pharmacists offering medication therapy management services. METHODS: A cross-sectional study conducted with data from 758 older adults followed up in medication therapy management services in Primary Health Care in the cities of Belo Horizonte, Betim, and Lagoa Santa (MG, Brazil). Univariate and multivariate analyses were performed to evaluate the factors associated with identification of four or more drug therapy problems in the initial clinical assessment. RESULTS: A total of 1,683 drug therapy problems were identified, 73.6% of older patients had at least one problem. The most frequent problems were nonadherence (23.0%) and the need for additional drug therapy (18.0%). Polypharmacy, chronic obstructive pulmonary disease, hypertension, diabetes mellitus, heart failure, and aged 75 years or older remained positively and statistically associated with identification of four or more drug therapy problems (p<0.05). CONCLUSION: There is a high frequency of problems related to medication use among older users of Primary Health Care, and the medication therapy management services should be prioritized to the older patients, who present with polypharmacy, chronic obstructive pulmonary disease, hypertension, diabetes mellitus, heart failure, and age ≥ 75 years, since they are more likely to have more drug therapy problems.

Aging-related episodic-like memory decline in dogs.

Episodic memory, in humans, is the memory most affected by age-related deterioration or the constitution of neurodegenerative pathologies, such as Alzheimer's disease. However, it is unknown whether this relationship is also present in nonhuman animals. Since studies in birds, rats, primates, and dogs have been shown to have episodic-like memory, more studies aiming to improve the present understanding of this relationship in nonhuman animals are important to aid the development of new translational models for neurodegenerative disorders. Knowing that dogs (Canis familiaris) represent a promising experimental model for neurodegenerative disorders, a memory retrieval test was conducted with 90 clinically healthy domestic dogs of different ages, both sexes, and distinct breeds, for the purpose of evaluating episodic-like memory. The present study adapted a test that corroborates episodic memory requirements through incidental codification of experienced events. We performed a test with two exposure phases, with different characteristics between them, so that in the third phase it was necessary to integrate previous experiences in order to achieve success in the test. In our study, it was possible to verify the decline of episodic memory in elderly dogs, even clinically healthy, regardless of the dogs' sex and size. This episodic-like memory decline observed in elderly dogs may be related to the physiological process of aging or preclinical pathological manifestation of cognitive impairment, similar as reported in humans. More studies should be carried out evaluating episodic-like memory in dogs with suspected of canine cognitive dysfunction syndrome in order to better understand the physiological and pathological behavior of this type of memory in canine species.