Enhancing genomic prediction with Stacking Ensemble Learning in Arabica Coffee.

Coffee Breeding programs have traditionally relied on observing plant characteristics over years, a slow and costly process. Genomic selection (GS) offers a DNA-based alternative for faster selection of superior cultivars. Stacking Ensemble Learning (SEL) combines multiple models for potentially even more accurate selection. This study explores SEL potential in coffee breeding, aiming to improve prediction accuracy for important traits [yield (YL), total number of the fruits (NF), leaf miner infestation (LM), and cercosporiosis incidence (Cer)] in Coffea Arabica. We analyzed data from 195 individuals genotyped for 21,211 single-nucleotide polymorphism (SNP) markers. To comprehensively assess model performance, we employed a cross-validation (CV) scheme. Genomic Best Linear Unbiased Prediction (GBLUP), multivariate adaptive regression splines (MARS), Quantile Random Forest (QRF), and Random Forest (RF) served as base learners. For the meta-learner within the SEL framework, various options were explored, including Ridge Regression, RF, GBLUP, and Single Average. The SEL method was able to predict the predictive ability (PA) of important traits in Coffea Arabica. SEL presented higher PA compared with those obtained for all base learner methods. The gains in PA in relation to GBLUP were 87.44% (the ratio between the PA obtained from best Stacking model and the GBLUP), 37.83%, 199.82%, and 14.59% for YL, NF, LM and Cer, respectively. Overall, SEL presents a promising approach for GS. By combining predictions from multiple models, SEL can potentially enhance the PA of GS for complex traits.

CRISPR/Cas genome editing in soybean: challenges and new insights to overcome existing bottlenecks.

BACKGROUND: Soybean is a worldwide-cultivated crop due to its applications in the food, feed, and biodiesel industries. Genome editing in soybean began with ZFN and TALEN technologies; however, CRISPR/Cas has emerged and shortly became the preferable approach for soybean genome manipulation since it is more precise, easy to handle, and cost-effective. Recent reports have focused on the conventional Cas9 nuclease, Cas9 nickase (nCas9) derived base editors, and Cas12a (formally Cpf1) as the most commonly used genome editors in soybean. Nonetheless, several challenges in the complex plant genetic engineering pipeline need to be overcome to effectively edit the genome of an elite soybean cultivar. These challenges include (1) optimizing CRISPR cassette design (i.e., gRNA and Cas promoters, gRNA design and testing, number of gRNAs, and binary vector), (2) improving transformation frequency, (3) increasing the editing efficiency ratio of targeted plant cells, and (4) improving soybean crop production. AIM OF REVIEW: This review provides an overview of soybean genome editing using CRISPR/Cas technology, discusses current challenges, and highlights theoretical (insights) and practical suggestions to overcome the existing bottlenecks. KEY SCIENTIFIC CONCEPTS OF REVIEW: The CRISPR/Cas system was discovered as part of the bacterial innate immune system. It has been used as a biotechnological tool for genome editing and efficiently applied in soybean to unveil gene function, improve agronomic traits such as yield and nutritional grain quality, and enhance biotic and abiotic stress tolerance. To date, the efficiency of gRNAs has been validated using protoplasts and hairy root assays, while stable plant transformation relies on Agrobacterium-mediated and particle bombardment methods. Nevertheless, most steps of the CRISPR/Cas workflow require optimizations to achieve a more effective genome editing in soybean plants.

Ballast water transport of alien phytoplankton species to the brazilian amazon coast.

The aim of this study was to investigate the presence of alien phytoplankton species transported through ballast water of ships that docked on the Amazon coast. Phytoplankton samples were collected from 25 ships between 2012 and 2014, revealing 215 identified species, mostly comprising oceanic planktonic marine species. However, several coastal and freshwater species not yet documented on the Maranhão coast were also observed. The identification of several coastal and freshwater species not yet recorded for Amazonian environments in the ballast water of the Ponta da Madeira Maritime Terminal (TMPM), as well as toxic microalgae genera such as the dinoflagellates Alexandrium and Gymnodinium and of some diatom species from the genus Pseudo-nitzchia, raises concerns regarding the possibility of introducing species. This indicates that ballast water can be responsible for the introduction of alien species in Amazonian aquatic environments, thereby highlighting the TMPM as a critical hotspot in the Amazonian region.

Influence of sleep on motor skill acquisition in children: a systematic review.

Effects of sleep on procedural (implicit) memory consolidation in children remain controversial. The aim of this systematic review was to synthesise the evidence on the influence of sleep on motor skills acquisition in children. Four electronic databases were searched: PubMed, Cochrane Central Register of Controlled Trials (CENTRAL), Excerpta Medica Database (Embase), and Biblioteca Virtual em Saúde (BVS). Original studies, published until October 17, 2023, on motor skill acquisition in children aged ≤12 years, in which the intervention group slept after motor skill training, while the control group remained awake, were considered for inclusion. Risk of bias was evaluated using the Cochrane's Risk of Bias 2 tool. The review protocol was pre-registered at the International Prospective Register of Systematic Reviews (PROSPERO protocol number: CRD42022363868) and all reported items followed the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. Of the 7241 articles initially retrieved, nine met the primary criteria and were included in this review. Of these, six studies reported that daytime or night-time sleep intervention improved motor skill acquisition, as compared to wakefulness. All studies presented a high risk of bias. In conclusion, the evidence summarised suggests that sleep may enhance motor skills acquisition and could be important for motor development in childhood. However, due to the high risk of bias in the included studies, future randomised controlled trials with high methodological quality are necessary to better clarify this topic.

Quantitative Assessment of the Inadequate Intake of Macronutrients, Minerals, and Vitamins Associated with Ultra-Processed Food Consumption.

Studies indicate that ultra-processed food (UP) consumption correlates negatively with essential vitamin and mineral intake and positively with sodium and lipid intake. The objective of this study was to explore the relationship between UP consumption and deviations from nutritional guidelines. An observational, cross-sectional analytical study was conducted on a probability sample of manufacturing workers in the state of Rio Grande do Norte, Brazil. Food consumption was assessed with a 24 h recall survey, and nutrient intake inadequacies were calculated as the difference between individuals' intake of energy, macronutrients, minerals and vitamins, and the dietary reference intakes for individuals of the same sex and age group, and then analyzed for trends across the percentage contribution of UP to total energy intake with nonparametric multiple regression adjusted for covariates. The study included 921 workers from 33 industries, 55.9% male, with a mean age of 32 years. Overall, the study population exhibited deficits in energy, all macronutrients, and in some micronutrients. With increasing UP contribution to total energy intake, there is a trend towards a greater intake of energy (p < 0.001), total, saturated, monounsaturated, and trans fats (p < 0.001), n6-polyunsaturated fatty acids (p = 0.03), carbohydrates (p < 0.001), calcium (p = 0.008), and manganese (p < 0.001), thiamin (p < 0.001), and vitamin B6 (p = 0.01); however, this comes with a negative consequence in terms of reducing the protein consumption (p = 0.037), fiber (p = 0.035), copper (p = 0.033), and vitamin E (p = 0.002) intake. The results show that correcting energy and micronutrient deficiencies by increasing UP consumption can also lead to a decrease in diet quality.

Chemical composition, antibacterial potential, and toxicity of the extracts from the stem bark of Hancornia speciosa Gomes (Apocynaceae).

ETHNOPHARMACOLOGICAL RELEVANCE: Hancornia speciosa is a medicinal plant popularly used to treat different medical issues, including infectious diseases. Exploring the therapeutic potentialities of the extracts from medicinal plants combined with conventional antibiotic drugs is a promising horizon, especially considering the rising microbial resistance. AIM OF THE STUDY: This study aimed to characterize the chemical composition of the ethereal (EEHS) and methanolic (MEHS) extracts of the stem bark of H. speciosa, and also evaluate their antibacterial and drug-modifying activity, and toxicity. MATERIALS AND METHODS: The extracts were characterized by gas chromatography coupled to mass spectrometry (GC-MS). Additionally, total phenol and flavonoid contents were determined. The antibacterial and antibiotic-modifying activity was evaluated against strains of Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa using the serial microdilution method, obtaining the minimum inhibitory concentration (MIC). The toxicity assay was carried out using the Drosophila melanogaster model. RESULTS: Thirty compounds were identified in the extracts of the stem bark of H. speciosa, with triterpenoids being predominant in both extracts. Additionally, fatty alcohols, carbohydrates, fatty acids, phenolic acids, and phytosterols were identified in both extracts. EEHS and MEHS extracts had considerable phenol contents (346.4 and 340.0 mg GAE/g, respectively). Flavonoids were detected in a lower proportion (7.6 and 6.9 mg QE/g, respectively). H. speciosa extracts did not display intrinsic antibacterial activity against the bacterial strains evaluated, however, they were capable of modifying the activity of gentamicin, erythromycin, and norfloxacin. EEHS increased the efficacy of norfloxacin against E. coli and S. aureus, reducing MIC values by 50%. MEHS potentiated the action of gentamicin against all bacterial strains, especially against E. coli. The extracts did not display toxicity at clinically relevant concentrations against D. melanogaster. CONCLUSION: The stem bark of H. speciosa was considered a rich source of bioactive compounds. Our findings evidenced the therapeutic potential of H. speciosa extracts for the development of new pharmaceutical therapeutics against bacteria. Although the extracts did not exhibit intrinsic antibacterial activity, they enhanced the efficacy of commercial antibiotic drugs and were non-toxic at clinically relevant concentrations. Future studies are needed to elucidate the mechanisms of action of these extracts, ensuring their safety and efficacy.

Genetic ablation of the isoform γ of PI3K decreases antidepressant efficacy of ketamine in male mice.

About one-third of major depressive disorder (MDD) patients demonstrate unresponsiveness to classic antidepressants, and even the clinical efficacy of fast-acting drugs such as ketamine varies significantly among patients with treatment-resistant depression. Nevertheless, the lack of suitable animal models that mimic a possible ketamine-resistant phenotype challenges the understanding of resistance to drug treatment. In this study, we showed that PI3Kγ knock-out (KO) mice do not respond to classical doses of ketamine and classical antidepressants. PI3Kγ KO mice were unresponsive to both the rapid and sustained antidepressant-like effects of a single dose of ketamine in the forced swimming test. Additionally, they were unresponsive to the antidepressant-like effects induced by the tricyclic antidepressant imipramine and the selective serotonin reuptake inhibitor fluoxetine. However, acute pharmacological inhibition of PI3Kγ did not block the antidepressant-like effect of ketamine, showing that a chronic deficiency of the PI3Kγ-mediated pathway is necessary for the effects of classic doses of ketamine and antidepressants. Therefore, we propose that PI3Kγ participates in the antidepressant activity and is likely implicated in the neurobiology and phenotype observed in patients with MDD who demonstrate treatment resistance.

Cannabidiol and epilepsy.

Cannabidiol (CBD) has been investigated as a pharmacological approach for treating a myriad of neurological and psychiatric disorders, the most successful of them being its use as an antiseizure drug (ASD). Indeed, CBD has reached the clinics for the treatment of certain epileptic syndromes. This chapter aims to overview the pharmacology of CBD and its potential mechanisms of action as an ASD. First, we give an outline of the concepts, mechanisms and pharmacology pertaining to the field of study of epilepsy and epileptic seizures. In the second section, we will summarize the effects of CBD as an ASD. Next, we will discuss its potential mechanisms of action to alleviate epileptic seizures, which seem to entail multiple neurotransmitters, receptors and intracellular pathways. Finally, we will conclude and present some limitations and perspectives for future studies.

MODCEL-MHUS: a comprehensive multilayer hydrodynamic unified simulation for stormwater, sanitary sewer systems, and urban surface.

Numerous countries and regions have embraced implementing a separate sewer system, segregating sanitary and storm sewers into distinct systems. However, the functionality of these systems often needs to improve due to irregular interconnections, resulting in a mixed and malfunctioning system. Sewage collection is crucial for residential sanitation, but untreated collection significantly contributes to environmental degradation. Analyzing the simultaneous operation of both systems becomes vital for effective management. Using mathematical tools for precise and unified diagnosis and prognosis becomes imperative. However, municipal professionals and companies need more tools specifically designed to evaluate these systems in a unified way, mapping all the hydraulic connections observed in practice. This study proposes a unified simulation method for stormwater and sanitary sewer urban systems, addressing real-world scenarios and potential interferences. The primary goal is to develop a simulation method for both systems, considering system interconnections and urban layouts, involving hydrodynamic and water quality simulations. The practical application of this method, the Multilayer Hydrodynamic Simulation Method (MODCEL-MHUS), successfully identifies issues in urban water networks and suggests solutions, making it a valuable tool for urban water management and environmental engineering professionals.

Identification of SNP Markers and Candidate Genes Associated with Major Agronomic Traits in Coffea arabica.

Genome-wide association studies (GWASs) allow for inferences about the relationships between genomic variants and phenotypic traits in natural or breeding populations. However, few have used this methodology in Coffea arabica. We aimed to identify chromosomal regions with significant associations between SNP markers and agronomic traits in C. arabica. We used a coffee panel consisting of 195 plants derived from 13 families in F2 generations and backcrosses of crosses between leaf rust-susceptible and -resistant genotypes. The plants were phenotyped for 18 agronomic markers and genotyped for 21,211 SNP markers. A GWAS enabled the identification of 110 SNPs with significant associations (p < 0.05) for several agronomic traits in C. arabica: plant height, plagiotropic branch length, number of vegetative nodes, canopy diameter, fruit size, cercosporiosis incidence, and rust incidence. The effects of each SNP marker associated with the traits were analyzed, such that they can be used for molecular marker-assisted selection. For the first time, a GWAS was used for these important agronomic traits in C. arabica, enabling applications in accelerated coffee breeding through marker-assisted selection and ensuring greater efficiency and time reduction. Furthermore, our findings provide preliminary knowledge to further confirm the genomic loci and potential candidate genes contributing to various structural and disease-related traits of C. arabica.

Developing a consensus-based motivational care pathway for individuals with lower limb fractures: a Delphi protocol.

Older adults with lower limb fractures often harbor concerns about losing their mobility, fearing a loss of independence. It is vital to develop strategies that foster their active engagement in the rehabilitation process. The present protocol aims to create a care pathway tailored to motivate individuals with lower limb fractures to adhere to rehabilitation. We will develop an observational, cross-sectional, and descriptive study using the Delphi data-gathering approach. Purposive sampling will recruit a panel of healthcare professionals and experts who care for patients with lower limb fractures. Aligned with the Delphi method, a series of iterative rounds will be developed to gather consensus around the motivational strategies used by health professionals in the rehabilitation of people with lower limb fractures. We will employ the Qualtrics platform for data collection and analysis, and a consensus target of 75% has been predetermined. For quantitative data analysis, we will use descriptive statistics encompassing a range of measures, including count, mean, standard deviation, median, minimum, maximum, and range. An inductive thematic analysis procedure will be employed to extract meaningful themes and patterns from qualitative data. The study results are expected to significantly impact clinical practice by creating a specialized care pathway to motivate individuals with lower limb fractures to adhere to rehabilitation. Adopting these explicit standards by professionals will ensure uniform and high-quality care.

Study protocol for the development and validation of a questionnaire evaluating predisposition to immunosuppressant medication non-adherence of kidney pre-transplant patients. The KATITA project.

Non-adherence to immunosuppressive medication after kidney transplant is an important cause of graft rejection and loss. Approaches to minimization of non-adherence have focused on the identification of episodes of medication non-adherence, but by then irreparable harm to the graft may already have occurred, and a more effective approach would be to adopt preventive measures in patients who may have difficulty in adhering to medication. The aim of this study protocol is to develop and validate a clinical questionnaire for assessing, in kidney transplant candidate patients in the pre-transplant setting, the predisposition to non-adherence to immunosuppressive medication. In this multicenter, prospective study, a pilot questionnaire in Brazilian Portuguese language, composed of Likert-scaled statements expressing patients' beliefs, behaviors and barriers regarding medication taking will be assembled from a literature review, from focus groups, and an expert panel. The pilot questionnaire will be administered to a minimum of 300 patients in kidney transplant waiting lists and exploratory factor analysis will be used for development of the definitive questionnaire. A random subsample of a minimum of 60 patients will have the scale re-administered after one month for evaluation of test-retest reliability. A multicenter, external validation study will include 364 kidney transplant candidates who will be evaluated immediately before surgery and at months 3, 6 and 12 post-transplant for assessment of concurrent validity, by comparison with two scales that assess medication non-adherence, and for determination of predictive validity using a triangulation method for assessment of medication non-adherence. Structural validity will be assessed with confirmatory factor analysis using structural equation modeling. Cross-cultural generalizability and validity will be assessed by a multicenter study, in which a translation of the scale to another language will be administered to kidney transplant candidate patients from a different culture, with a subsample being selected for test-retest. This study will be conducted in Spain with a Spanish translation of the scale.

Prevalence of consumption of mechanically separated meat, consumer profile, nutrient intake and food choices among manufacturing workers in Northeastern Brazil.

Mechanically separated meat (MSM) is widely used in the food industry, however, there is a lack of studies on its consumption in populations. The objective of this study was to identify the frequency and amount of MSM consumption, factors associated with MSM consumption, nutrient intake and preferential choice of food groups among MSM consumers. This was an observational, cross-sectional prospective study based on a probability sample of manufacturing workers, conducted in Brazil. Logistic and linear multiple regression with robust standard errors were used. 921 workers from 33 manufacturing companies were studied, with an average age of 38.2 ± 10.7 years, 55.9% males. MSM products are consumed by 28.8% and represent in average 10% of total daily caloric intake, and 47.3% of the daily kcal from ultra-processed products. Younger age and greater waist circumference are associated with MSM consumption. Younger age and lesser educational level are associated with increased contribution of MSM to total daily kcal intake. MSM consumers have greater consumption of energy, fats, carbohydrates and sodium. Their dietary patterns are characterized by lower consumption of in natura and minimally processed foods, such as tubers and roots, fruits, white and red meat, and eggs and greater consumption of ultra-processed foods and beverages.

Activated AMP-protein kinase (pAMPK) is overexpressed in human somatotroph pituitary adenomas.

INTRODUCTION: AMPK (AMP-activated protein kinase) is an enzyme that acts as a metabolic sensor and regulates multiple pathways via phosphorylating proteins in metabolic and proliferative pathways. The aim of this work was to study the activated cellular AMPK (phosphorylated-AMPK at Thr172, pAMPK) levels in pituitary tumor samples from patients with sporadic and familial acromegaly, as well as in samples from normal human pituitary gland. METHODS: We studied pituitary adenoma tissue from patients with sporadic somatotroph adenomas, familial acromegaly with heterozygote germline variants in the aryl hydrocarbon receptor interacting protein (AIP) gene (p.Q164*, p.R304* and p.F269_H275dup) and autopsy from normal pituitary glands without structural alterations. RESULTS: Cellular levels of pAMPK were significantly higher in patients with sporadic acromegaly compared to normal pituitary glands (p < 0.0001). Tissues samples from patients with germline AIP mutations also showed higher cellular levels of pAMPK compared to normal pituitary glands. We did not observe a significant difference in cellular levels of pAMPK according to the cytokeratin (CAM5.2) pattern (sparsely or densely granulated) for tumor samples of sporadic acromegaly. CONCLUSION: Our data show, for the first time in human cells, an increase of cellular levels of pAMPK in sporadic somatotropinomas, regardless of cytokeratin pattern, as well as in GH-secreting adenomas from patients with germline AIP mutations.

Mechanical stimulation-induced purinome priming fosters osteogenic differentiation and osteointegration of mesenchymal stem cells from the bone marrow of post-menopausal women.

BACKGROUND: Mechanical stimulation (MS) significantly increases the release of adenine and uracil nucleotides from bone marrow-derived mesenchymal stem cells (BM-MSCs) undergoing osteogenic differentiation. Released nucleotides acting via ionotropic P2X7 and metabotropic P2Y6 purinoceptors sensitive to ATP and UDP, respectively, control the osteogenic commitment of BM-MSCs and, thus, bone growth and remodelling. Yet, this mechanism is impaired in post-menopausal (Pm)-derived BM-MSCs, mostly because NTPDase3 overexpression decreases the extracellular accumulation of nucleotides below the levels required to activate plasma membrane-bound P2 purinoceptors. This prompted us to investigate whether in vitro MS of BM-MSCs from Pm women could rehabilitate their osteogenic commitment and whether xenotransplantation of MS purinome-primed Pm cells promote repair of critical bone defects in an in vivo animal model. METHODS: BM-MSCs were harvested from the neck of femora of Pm women (70 ± 3 years old) undergoing total hip replacement. The cells grew, for 35 days, in an osteogenic-inducing medium either submitted (SS) or not (CTR) to MS (90 r.p.m. for 30 min) twice a week. Increases in alkaline phosphatase activity and in the amount of osteogenic transcription factors, osterix and osteopontin, denoted osteogenic cells differentiation, while bone nodules formation was ascertain by the alizarin red-staining assay. The luciferin-luciferase bioluminescence assay was used to quantify extracellular ATP. The kinetics of the extracellular ATP (100 µM) and UDP (100 µM) catabolism was assessed by HPLC. The density of P2Y6 and P2X7 purinoceptors in the cells was assessed by immunofluorescence confocal microscopy. MS-stimulated BM-MSCs from Pm women were xenotransplanted into critical bone defects drilled in the great trochanter of femora of one-year female Wistar rats; bone repair was assessed by histological analysis 10 days after xenotransplantation. RESULTS: MS-stimulated Pm BM-MSCs in culture (i) release 1.6-fold higher ATP amounts, (ii) overexpress P2X7 and P2Y6 purinoceptors, (iii) exhibit higher alkaline phosphatase activity and overexpress the osteogenic transcription factors, osterix and osteopontin, and (iv) form larger bone nodules, than CTR cells. Selective blockage of P2X7 and P2Y6 purinoceptors with A438079 (3 µM) and MRS 2578 (0.1 µM), respectively, prevented the osteogenic commitment of cultured Pm BM-MSCs. Xenotransplanted MS purinome-primed Pm BM-MSCs accelerated the repair of critical bone defects in the in vivo rat model. CONCLUSIONS: Data suggest that in vitro MS restores the purinergic cell-to-cell communication fostering the osteogenic differentiation and osteointegration of BM-MSCs from Pm women, a strategy that may be used in bone regeneration and repair tactics.

GlyT1 Inhibition by NFPS Promotes Neuroprotection in Amyloid-ß-Induced Alzheimer's Disease Animal Model.

Alzheimer's disease (AD) is a neurodegenerative disorder characterized by the accumulation of amyloid-ß, leading to N-methyl-D-aspartate (NMDA) receptor-dependent synaptic depression, spine elimination, and memory deficits. Glycine transporter type 1 (GlyT1) modulates glutamatergic neurotransmission via NMDA receptors (NMDAR), presenting a potential alternative therapeutic approach for AD. This study investigates the neuroprotective potential of GlyT1 inhibition in an amyloid-ß-induced AD mouse model. C57BL/6 mice were treated with N-[3-([1,1-Biphenyl]-4-yloxy)-3-(4-fluorophenyl)propyl]-N-methylglycine (NFPS), a GlyT1 inhibitor, 24 h prior to intrahippocampal injection of amyloid-ß. NFPS pretreatment prevented amyloid-ß-induced cognitive deficits in short-term and long-term memory, evidenced by novel object recognition and spatial memory tasks. Moreover, NFPS pretreatment curbed microglial activation, astrocytic reactivity, and subsequent neuronal damage from amyloid-ß injection. An extensive label-free quantitative UPLC-MSE proteomic analysis was performed on the hippocampi of mice treated with NFPS. In proteomics, KEGG enrichment analysis revealed increased in dopaminergic synapse, purine-containing compound biosynthetic process and long-term potentiation, and a reduction in Glucose catabolic process and glycolytic process pathways. The western blot analysis confirmed that NFPS treatment elevated BDNF levels, correlating with enhanced TRKB phosphorylation and mTOR activation. Moreover, NFPS treatment reduced the GluN2B expression after 6 h, which was associated with an increase on CaMKIV and CREB phosphorylation. Collectively, these findings demonstrate that GlyT1 inhibition by NFPS activates diverse neuroprotective pathways, enhancing long-term potentiation signaling and countering amyloid-ß-induced hippocampal damage.

Associations between Stigma, Depression, and Adherence to Antiretroviral Therapy in Brazilian Men Who Have Sex with Men Living with HIV.

Adherence to antiretroviral therapy (ART) is a complex and multi-determined process that is influenced by psychosocial variables. Although international studies have pointed to the adverse impact of HIV stigma, sexual stigma, and depression on ART adherence among men who have sex with men (MSM) with HIV, less is known about this association among Brazilians. We aimed to (a) evaluate indicators of depression, stigma related to HIV and homosexuality, and adherence to ART in a sample of Brazilian MSM living with HIV; (b) assess possible correlations between the variables analyzed, and (c) assess the impact of HIV and sexual stigma and depression on ART adherence. This cross-sectional study comprised 138 Brazilian MSM living with HIV as participants. Scales used included: a sociodemographic/clinical questionnaire, the questionnaire for assessment of adherence to antiretroviral therapy (CEAT-HIV), the Beck depression inventory (BDI-II), the internalized homophobia scale, and the HIV stigmatization scale. The mean adherence score was relatively high (78.83, within a range of 17-89 points). However, we observed inadequate ART adherence (CEAT-HIV < 75) in 28 (20.2%) respondents. Participants reported high scores for internalized sexual stigma, perceived sexual stigma in the community, and HIV stigma. Symptoms of depression were identified in 48.47% of participants. We found negative correlations between depression, HIV stigma, and treatment adherence, but not between sexual stigma and ART adherence. HIV-related stigma and sexual stigma were positively correlated with depression. Our regression analysis indicated that each year of age at diagnosis of HIV increased adherence by 0.22 points, on average. Each additional BDI-II score reduced adherence to ART by 0.20 points. The high prevalence of depression, HIV stigma, and sexual stigma, and their adverse effects on ART adherence and mental health, point to the need to implement evidence-based interventions to reduce sexual and serological stigma in the general population, as well as to mitigate the negative impacts of stigma on MSM living in HIV in Brazil. They also highlight the importance of periodically screening for these variables among MSM treated in Brazilian public health services, especially among those with inadequate adherence to ART.

Retrospective application of ICH M7 to anti-hypertensive drugs in Brazil: Risk assessment of potentially mutagenic impurities.

Potentially mutagenic impurities are likely to be formed in any drug substance, since their synthesis requires reactive intermediates which may also react with DNA. The ICH M7 guideline, which defines how to risk assess and control mutagenic impurities, was first published in 2014 and is not to be applied retrospectively; however, some impurities have been found above the permitted limits in drug products which were already on the market. This study assessed the implications of applying ICH M7 retrospectively to anti-hypertensive drugs marketed in Brazil by performing a risk assessment and establishing control strategies. The manufacturing processes of 15 drug substances were evaluated and 262 impurities were identified, from which 21% were classified as potentially mutagenic. Most of the impurities were identified below ICH M7 acceptable limits, except for impurities described in a pharmacopoeial monograph. Compendial specifications are defined based on scientific evidence and play an important role in setting quality and safety standards for pharmaceuticals, however there are opportunities for further alignment with ICH guidelines, aiming for a holistic assessment of the impurities profile to ensure the safety of medicines.

Pharmacological inhibition of receptor-interacting protein kinase 2 (RIPK2) elicits neuroprotective effects following experimental ischemic stroke.

Ischemic stroke induces a debilitating neurological insult, where inflammatory processes contribute greatly to the expansion and growth of the injury. Receptor-interacting protein kinase 2 (RIPK2) is most well-known for its role as the obligate kinase for NOD1/2 pattern recognition receptor signaling and is implicated in the pathology of various inflammatory conditions. Compared to a sham-operated control, ischemic stroke resulted in a dramatic increase in the active, phosphorylated form of RIPK2, indicating that RIPK2 may be implicated in the response to stroke injury. Here, we assessed the effects of pharmacological inhibition of RIPK2 to improve post-stroke outcomes in mice subjected to experimental ischemic stroke. We found that treatment at the onset of reperfusion with a RIPK2 inhibitor, which inhibits the phosphorylation and activation of RIPK2, resulted in marked improvements in post-stroke behavioral outcomes compared to the vehicle-administered group assessed 24 h after stroke. RIPK2 inhibitor-treated mice exhibited dramatic reductions in infarct volume, concurrent with reduced damage to the blood-brain barrier, as evidenced by reduced levels of active matrix metalloproteinase-9 (MMP-9) and leakage of blood-borne albumin in the ipsilateral cortex. To explore the protective mechanism of RIPK2 inhibition, we next pretreated mice with RIPK2 inhibitor or vehicle and examined transcriptomic alterations occurring in the ischemic brain 6 h after stroke. We observed a dramatic reduction in neuroinflammatory markers in the ipsilateral cortex of the inhibitor-treated group while also attaining a comprehensive view of the vast transcriptomic alterations occurring in the brain with inhibitor treatment through bulk RNA-sequencing of the injured cortex. Overall, we provide significant novel evidence that RIPK2 may represent a viable target for post-stroke pharmacotherapy and potentially other neuroinflammatory conditions.

Transcriptomics analysis highlights potential ways in human pathogenesis in Leishmania braziliensis infected with the viral endosymbiont LRV1.

The parasite Leishmania (Viannia) braziliensis is widely distributed in Brazil and is one of the main species associated with human cases of different forms of tegumentary leishmaniasis (TL) such as cutaneous leishmaniasis (CL) and mucosal leishmaniasis (ML). The mechanisms underlying the pathogenesis of TL are still not fully understood, but it is known that factors related to the host and the parasite act in a synergistic and relevant way to direct the response to the infection. In the host, macrophages have a central connection with the parasite and play a fundamental role in the defense of the organism due to their ability to destroy intracellular parasites and present antigens. In the parasite, some intrinsic factors related to the species or even the strain analyzed are fundamental for the outcome of the disease. One of them is the presence of Leishmania RNA Virus 1 (LRV1), an endosymbiont virus that parasitizes some species of Leishmania that triggers a cascade of signals leading to a more severe TL phenotype, such as ML. One of the strategies for understanding factors associated with the immune response generated after Leishmania/host interaction is through the analysis of molecular patterns after infection. Thus, the gene expression profile in human monocyte-derived macrophages obtained from healthy donors infected in vitro with L. braziliensis positive (LbLRV1+) and negative (LbLRV1-) for LRV1 was evaluated. For this, the microarray assay was used and 162 differentially expressed genes were identified in the comparison LbLRV1+ vs. LbLRV1-, 126 upregulated genes for the type I and II interferons (IFN) signaling pathway, oligoadenylate synthase OAS/RNAse L, non-genomic actions of vitamin D3 and RIG-I type receptors, and 36 down-regulated. The top 10 downregulated genes along with the top 10 upregulated genes were considered for analysis. Type I interferon (IFNI)- and OAS-related pathways results were validated by RT-qPCR and Th1/Th2/Th17 cytokines were analyzed by Cytometric Bead Array (CBA) and enzyme-linked immunosorbent assay (ELISA). The microarray results validated by RT-qPCR showed differential expression of genes related to IFNI-mediated pathways with overexpression of different genes in cells infected with LbLRV1+ compared to LbLRV1- and to the control. No significant differences were found in cytokine levels between LbLRV1+ vs. LbLRV1- and control. The data suggest the activation of gene signaling pathways associated with the presence of LRV1 has not yet been reported so far. This study demonstrates, for the first time, the activation of the OAS/RNase L signaling pathway and the non-genomic actions of vitamin D3 when comparing infections with LbLRV1+ versus LbLRV1- and the control. This finding emphasizes the role of LRV1 in directing the host's immune response after infection, underlining the importance of identifying LRV1 in patients with TL to assess disease progression.