GPR81 (HCAR1) is a lactate-sensing G protein-coupled receptor (GPCR) involved in tumor progression, immune evasion, and therapeutic resistance across various cancers. Despite their clinical relevance and druggable nature, selective HCAR1 antagonists have yet to be identified. This study aimed to construct a statistically significant Support Vector Machine (SVM) model for binary classification (agonists versus antagonists) of HCAR1's potential ligands and the prioritization of molecular substructures driving antagonism and receptor selectivity. An SVM model was trained on 144 ligands (66 agonists, 78 antagonists), listed in the IUPHAR/BPS Guide to Pharmacology, from 12 structurally related Class A GPCRs (HCAR1, HCAR2, HCAR3, OXER1, GPR35, SUCNR1, P2Y2, MCHR1, OPRD1, AGTR1, ADORA2A, and ADRA1A). Their ligands were encoded using physicochemical descriptors, 2048-bit ECFP4 fingerprints, and ΔAffinity scores from molecular docking to active and inactive receptor conformations. The data set was split 80/20 for training and testing, respectively, with hyperparameters (C, γ) being optimized via 5-fold cross-validation. SHAP analysis was performed for feature interpretation. The final SVM model achieved a test set accuracy of 79.3%, with a sensitivity of 69.2% and specificity of 87.5%. The ROC analysis yielded an AUC of 0.94, while bootstrapping confirmed robust performance with a mean AUC of 0.874 and a 95% confidence interval [0.711, 1.000]. SHAP analysis highlighted polar, rigid, and aromatic substructures as selectivity-driving features. We applied the model to screen 3,377 compounds from natural products, synthetic libraries, and FDA-approved drugs, prioritizing potential HCAR1 ligands with antagonist-like features. Based on ΔAffinity, off-target scores, and prediction confidence, Ketanserin, Cryptopyranmoscatone A1 diacetate, and Cefuroxime emerged as reference ligands with promising antagonistic potential, two of which are FDA-approved drugs. Rather than representing final hits, these molecules illustrate how structural and electronic features can favor the stabilization of inactive states in HCAR1. Overall, this work presents a proof-of-concept framework that integrates conformational docking, machine learning, and substructure interpretation to elucidate the chemical and structural determinants of HCAR1 antagonism. The findings provide fragment-level insights that may guide future bioisosteric and fragment-based design of selective antagonists for lactate-driven tumors.

Toxoplasmosis is a relevant parasitic infection in sheep, with ovine meat an important source of human exposure. Accurate detection of the early immune response to Toxoplasma gondii is essential for preventing reproductive losses and improving diagnostic strategies. This study evaluated the kinetics of the acute immune response in eighteen sheep experimentally exposed to live tachyzoites or immunized with parasite-derived glycoconjugates (GlyC). Animals were divided into three groups and injected with saline solution, tachyzoites, or glycoconjugates combined with an adjuvant. Infected sheep developed specific IgM antibodies against both lysate and glycoconjugate antigens from day 4, and IgG against glycoconjugates from day 12 post-infection. Glycoconjugate-immunized sheep produced IgM against lysate antigens from day 4, and IgG against both antigens from day 12. Flow cytometry revealed a significant increase in circulating CD8+ T cells and a reduction in MHC class II+ cells on day 60 in the infected group. These findings demonstrate the early humoral and cellular immune response profiles following infection or GlyC immunization. This supports their future application in diagnostic tests or as vaccine candidates against toxoplasmosis in sheep.

The CMNR group comprises bacteria of the genera Corynebacterium, Mycobacterium, Nocardia, and Rhodococcus and share cell wall and DNA content characteristics. Many pathogenic CMNR bacteria cause diseases such as mastitis, lymphadenitis, and pneumonia in farmed animals, which cause economic losses for breeders and represent a threat to public health. Traditional diagnosis in CMNR involves isolating target bacteria on general or selective media and conducting metabolic analyses with the assistance of laboratory biochemical identification systems. Advanced mass spectrometry may also support diagnosing these bacteria in the clinic's daily routine despite some challenges, such as the need for isolated bacteria. In difficult identification among some CMNR members, molecular methods using polymerase chain reaction (PCR) emerge as reliable options for correct specification that is sometimes achieved directly from clinical samples such as tracheobronchial aspirates and feces. On the other hand, immunological diagnostics such as the skin test or Enzyme-Linked Immunosorbent Assay (ELISA) for Mycobacterium tuberculosis yield promising results in subclinical infections with no bacterial growth involved. In this review, we present the methods most commonly used to diagnose pathogenic CMNR bacteria and discuss their advantages and limitations, as well as challenges and perspectives on adopting new technologies in diagnostics.

AbstractIntroduction: Corynebacterium pseudotuberculosis is an intracellular facultative bacterium that affects macrophages, causing caseous lymphadenitis in goats and sheep. This disease is characterised by the formation of granulomas and results in significant economic losses for farmers of goats and sheep around the world. Phagosome maturation is essential for the death of intracellular pathogens, involving the fusion of the phagosome with early and late endocytic organelles, a process partially regulated by GTPase Rab5 and Rab7 proteins, respectively. Methodology: we evaluated the ability of an attenuated T1 strain and a wild-type C57 strain of C. pseudotuberculosis to alter Rab5 and Rab7 protein expression in peritoneal CBA mouse macrophages following in vitro infection. Results: Rab5 and Rab7 expression decreased in macrophages infected with the C57 strain compared to uninfected macrophages, while a significant reduction only in Rab7 expression was observed in cells infected with T1. Nonetheless, Rab5 and Rab7 expression was higher in macrophages infected with T1 than in cells infected with the wild-type C57 strain. Conclusions: our studies indicate a greater degree of modulation of the endosomal proteins directly involved in phagosome maturation by Corynebacterium pseudotuberculosis wild type.Keywords: Corynebacterium pseudotuberculosis; macrophages; phagolysosome; Rab proteins.

A caprinocultura é um importante segmento no agronegócio. Porém, existem alguns entraves na produção que causam graves prejuízos, dentre eles, patógenos responsáveis por doenças persistentes, o C. pseudotuberculosis - agente etiológico da linfadenite caseosa. Para analisar a infecção da doença, 18 (dezoito) caprinos da raça Canindé foram divididos em três grupos, sendo um grupo controle (sem infecção) e dois grupos infectados, sendo um grupo com uma cepa de C. pseudotuberculosis produtora e outro grupo com uma cepa não produtora de biofilme. Antes da infecção foram coletados 4 mL de sangue para realização do hemograma. Após a infecção dos animais foram realizadas coletas de sangue e exames clínicos nos tempos 7, 14, 28, 45, 60, 90, 120, 150 e 180 dias pós-infecção. Após esse período os animais foram abatidos tecnicamente para análise post mortem. Foi possível observar a presença de lesões em todos os animais infectados, podendo-se depreender que a cepa produtora de biofilme causou maiores lesões nos animais. Para as condições de infecção experimental foram comprovados picos de haptoglobina na infecção aguda, bem como os valores dos hemogramas apresentando-se dentro da normalidade, no entanto foi observada a elevação do número de leucócitos com aproximação do valor máximo de referência na infecção aguda e na fase crônica da doença, podendo sugerir uma reinfecção da doença.

The pandemic caused by the SARS-CoV-2 virus has represented a global challenge with a significant impact on public health since its emergence in 2019. Understanding the interactions between this virus and the human immune system is essential for the development of new strategies more effective therapies and diagnoses. This study aimed to predict epitopes for SARS-CoV-2 T and B cells, as well as evaluate the interaction of the spike protein with other viral proteins, using bioinformatics methods. SARS-CoV-2 protein sequences were collected from UniProt. Epitopes for T cells were predicted in silico using specific HLA alleles from the Bahia population. Epitopes for B cells were predicted using the IEDB server with multiple methods based on amino acid features. Protein-protein interaction was analyzed using the STRING database. The result is 10,671 peptides related to various SARS-CoV-2 viral proteins, including the spike, essential for the infection and pathogenesis of COVID-19. In addition to spike, proteins such as ORF3a, ORF7a, and ORF8 showed significant immunogenic potential. Protein-protein interaction analysis revealed that proteases such as TMPRSS2 and TMPRSS11D are crucial for viral entry and are potential therapeutic targets. This study expands the understanding of the molecular interactions of SARS-CoV-2, highlighting new therapeutic targets and clinical complications associated with COVID-19. The results provide valuable insights for the development of targeted therapeutic strategies and improved diagnostics, contributing to the mitigation of the global pandemic.

Multiple Myeloma (MM) is a hematological malignancy characterized by the clonal proliferation of plasma cells within the bone marrow. Diagnosing MM presents considerable challenges, involving the identification of plasma cells in cytology examinations on hematological slides. At present, this is still a time-consuming manual task and has high labor costs. These challenges have adverse implications, which rely heavily on medical professionals’ expertise and experience. To tackle these challenges, we present an investigation using Artificial Intelligence, specifically a Machine Learning analysis of hematological slides with a Deep Neural Network (DNN), to support specialists during the process of diagnosing MM. In this sense, the contribution of this study is twofold: in addition to the trained model to diagnose MM, we also make available to the community a fully-curated hematological slide dataset with thousands of images of plasma cells. Taken together, the setup we established here is a framework that researchers and hospitals with limited resources can promptly use. Our contributions provide practical results that have been directly applied in the public health system in Brazil. Given the open-source nature of the project, we anticipate it will be used and extended to diagnose other malignancies.

This work evaluated aspects of the immune response of BALB/c mice infected with Corynebacterium pseudotuberculosis (T1 and C57). The fifteen BALB/c mice were euthanized after 70 days of infection and morphologically evaluated, also analyzing the innate and adaptive immune responses. The C57 strain induced more pronounced morphological changes than the T1 strain. There was an increase in CD4+ and CD8+ T cells identified during infection with the C57 strain. Cytokines of the inflammatory profile IL-1α and IL-6 and regulatory IL-13 and IL-10 presented significant differences. Cytokines IL-2, IL-4, INF-γ, IL-22, IL-21, and IL-27 did not differ significantly between groups. The obtained results contribute to a better understanding of the type of response and the immunological mechanisms involved during infection with different strains of C. pseudotuberculosis.

Equine strangles is a prevalent disease that affects the upper respiratory in horses and is caused by the Gram-positive bacterium Streptococcus equi. In addition to strangles, other clinical conditions are caused by the two S. equi subspecies, equi and zooepidemicus, which present relevant zoonotic potential. Treatment of infections caused by S. equi has become challenging due to the worldwide spreading of infected horses and the unavailability of effective therapeutics and vaccines. Penicillin treatment is often recommended, but multidrug resistance issues arised. We explored the whole genome sequence of 18 S. equi isolates to identify candidate proteins to be targeted by natural drug-like compounds or explored as immunogens. We considered only proteins shared among the sequenced strains of subspecies equi and zooepidemicus, absent in the equine host and predicted to be essential and involved in virulence. Of these, 4 proteins with cytoplasmic subcellular location were selected for molecular docking with a library of 5008 compounds, while 6 proteins were proposed as prominent immunogens against S. equi due to their probabilities of behaving as adhesins. The molecular docking analyses revealed the best ten ligands for each of the 4 drug target candidates, and they were ranked according to their binding affinities and the number of hydrogen bonds for complex stability. Finally, the natural 5-ring compound C25H20F3N5O3 excelled in molecular dynamics simulations for the increased stability in the interaction with UDP-N-acetylenolpyruvoylglucosamine reductase (MurB). This research paves the way to developing new therapeutics to minimize the impacts caused by S. equi infections.

Corynebacterium pseudotuberculosis is a gram-positive bacterium and is the etiologic agent of caseous lymphadenitis (CL) in small ruminants. This disease is characterized by the development of encapsulated granulomas in visceral and superficial lymph nodes, and its clinical treatment is refractory to antibiotic therapy. An important virulence factor of the Corynebacterium genus is the ability to produce biofilm; however, little is known about the characteristics of the biofilm produced by C. pseudotuberculosis and its resistance to antimicrobials. Silver nanoparticles (AgNPs) are considered as promising antimicrobial agents, and are known to have several advantages, such as a broad-spectrum activity, low resistance induction potential, and antibiofilm activity. Therefore, we evaluate herein the activity of AgNPs in C. pseudotuberculosis, through the determination of minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), antibiofilm activity, and visualization of AgNP-treated and AgNP-untreated biofilm through scanning electron microscopy. The AgNPs were able to completely inhibit bacterial growth and inactivate C. pseudotuberculosis at concentrations ranging from 0.08 to 0.312 mg/mL. The AgNPs reduced the formation of biofilm in reference strains and clinical isolates of C. pseudotuberculosis, with interference values greater than 80% at a concentration of 4 mg/mL, controlling the change between the planktonic and biofilm-associated forms, and preventing fixation and colonization. Scanning electron microscopy images showed a significant disruptive activity of AgNP on the consolidated biofilms. The results of this study demonstrate the potential of AgNPs as an effective therapeutic agent against CL.

There are few reports of Trypanosoma in snakes, as well as little information about its pathogenicity in these animals. Thus, the present study aimed to characterize Trypanosoma found in Boa constrictor snakes, to verify the influence of the parasitism on hematological and clinical biochemistry parameters, and to perform a phylogenetic study of the isolates. Blood samples from sixty-one boas were analyzed for the presence of trypanosomatids and by hematological and clinical biochemistry assays. The flagellates that were found in this analysis were used for cell culture, morphometry, and molecular analysis. Later, molecular typing phylogenetic studies were performed. Nine positive animals (14.75%) were identified by microscopy analysis. The hematological results showed that parasitized animals presented significantly lower levels of packed cell volume, hemoglobin, mean corpuscular volume, and mean corpuscular hemoglobin. In the leukogram, eosinophils and heterophils counts were higher in parasitized animals. Considering the molecular analyses, the isolates presented a higher identity of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and the 18S small subunit ribosomal RNA (SSU rRNA) gene fragments with Trypanosoma serpentis . The phylogenetic tree, using the GAPDH, clustered all isolates with T. serpentis and Trypanosoma cascavelli . This is the first description of T. serpentis parasitizing boas and of the clinical changes caused by trypanosomatid infection in snakes.

Caseous lymphadenitis can be diagnosed using serological tests, mainly the enzyme-linked immunosorbent assay (ELISA). In this work, we evaluated interferon-gamma production in goats infected and not infected with Corynebacterium pseudotuberculosis, correlating it with the ELISA diagnostic test and analyzing the cellular and humoral responses, respectively. Eighteen Canindé goats experimentally infected with biofilm-producing and non-biofilm-producing strains were used. The sensitivity of the antigens was above 80%, with seroconversion observed from 14 days post-infection. A significant band between 64 and 70 kDa was shown in Western blotting. The production of IFN-y shows a tendency towards greater production in goats infected with the biofilm-producing strain stimulated by the TPP antigen of the same strain. The results help better understand the cellular and humoral response and antigenicity of the strains under study, correlated with the production of IFN-y, which plays a vital role in infection.

This work evaluated aspects of the immune response of BALB/c mice infected with Corynebacterium pseudotuberculosis (T1 and C57). The fifteen BALB/c mice were euthanized after 70 days of infection and morphologically evaluated, also analyzing the innate and adaptive immune responses. The C57 strain induced more pronounced morphological changes than the T1 strain. There was an increase in CD4+ and CD8+ T cells identified during infection with the C57 strain. Cytokines of the inflammatory profile IL-1 alpha and IL-6 and regulatory IL-13 and IL-10 presented significant differences. Cytokines IL-2, IL-4, INF-gamma, IL-22, IL-21, and IL-27 did not differ significantly between groups. The obtained results contribute to a better understanding of the type of response and the immunological mechanisms involved during infection with different strains of C. pseudotuberculosis.

The pathogenesis of acute myeloid leukemia (AML) involves mutations in genes such as FLT3 and NPM1, which are also associated with the prognosis of the disease. The immune system influences disease progression, but the mechanisms underlying the interaction between the immune system and AML are not clear. In this study, the profiles of lymphocytes and cytokines were described in individuals with AML stratified by molecular changes associated with prognosis. The participants included in this study were newly diagnosed AML patients (n = 43) who were about to undergo chemotherapy. Subtypes of lymphocytes in peripheral blood, including B cells, T cells, and natural killer cells, and serum concentrations of cytokines, including Th1, Th2, and Th17, were studied by flow cytometry assays (BD FACSCanto II). The correlations between lymphocyte subsets, cytokines, and genetic/prognostic risk stratification (based on the FLT3 and NPM1 genes) were analyzed. The differences in B lymphocytes (%), T lymphocytes (%), plasmablasts (%), leukocytes (cells/µl), and tumor necrosis factor (pg/ml) were determined between groups with FLT3-ITD+ and FLT3-ITD− mutations. The presence of mutations in NPM1 and FLT3-ITD and age suggested changes in the lymphocyte and cytokine profile in individuals with AML.

Identifying individuals and factors associated with severe cases of COVID-19 is crucial as the pandemic continues to spread globally. Effective biomarkers for predicting severe cases are essential for optimizing clinical man-agement, therapy, and preventing unfavorable outcomes. This exploratory observational study aimed to inves-tigate the expression of dysregulated immune response genes (ARG1, NOS2, ITGA4, and SELPLG) in total leukocytes, plasmatic levels of P-selectin and PSGL-1, and their clinical associations in patients with mild and severe COVID-19. Data from 117 confirmed COVID-19 patients (severe = 58, mild = 59) were collected upon admission. Gene expression was measured using RT-qPCR, and plasma protein levels assessed with ELISA assay. The severe COVID-19 patient group had a higher median age of 62.0 (p = 0.0001), a higher proportion of black individuals (86.2%, p < 0.0001), and more males (65.5%, p = 0.007). The neutrophil-lymphocyte ratio (NLR) and platelet-lymphocyte ratio (PLR) were significantly higher in the severe COVID-19 patient group (p < 0.0001), indicating ongoing systemic inflammation. Severe COVID-19 patients also exhibited increased expression of ARG1 (p < 0.05) and SELPLG (p < 0.0001) genes, as well as higher concentrations of soluble P-selectin (p < 0.005) and PSGL-1 (p < 0.05) proteins. Multivariate analysis revealed that NLR, PLR, the expression of SELPLG and sPSGL-1 were independent predictors of COVID-19 severity. In conclusion, this study suggests that biomarkers of endothelial dysfunction and dysregulated leukocyte responses are associated with COVID-19 severity, serving as promising predictive tools for optimizing clinical management and patient monitoring.

Introduction and Objectives: Hepatitis E is an infection caused by the E virus (HEV) with the fecal-oral transmission. The spectrum of the disease varies from acute form to chronic evolution. There are few studies in the rural region of Bahia State. This study aimed to study the seroprevalence of hepatitis E in a city in a rural region in Brazil. Materials and Methods: Held in May 2022 at family health clinics (PSFs) in the city of Serrinha-Bahia State, with 80,000 inhabitants and 184.6 km from Salvador, capital city. Participants who agreed to integrate into the study signed the Free and Informed Consent Term, collected blood samples and answered the questionnaire. The samples were stored at -20°C until the moment of use; those that needed to be retested for IgM were transported on dry ice and frozen at -80°C. IgG and IgM anti-HEV diagnostic kits (mikrogen and Diapro) approved by ANVISA of MS in Brazil were used. Medical-educational activities were carried out in the units on the prevention of viral hepatitis and health Results: Of a sample of 300 volunteers, actually, 150 blood samples were analyzed, the prevalence of anti-HEV IgM and IgG was 2.66%, and anti-HEV IgG alone was 8.6%. Most were women, 75.3%, and the average age was 43 years (18 to 78 years). Median liver enzymes in HEV-positive patients were AST (27.5 IU/L), ALT (21 IU/L), and GGT (58.5 IU/L). Among the samples, 81% of the residences were in rural areas, 90.5% did not have a sewage system, 94.6% had running water, and 39.5% worked or worked with animal husbandry. Only 1.4% experienced flooding. The majority, 95.4%, consume pork and/or derivatives and 19.7% consume hunting meat. Conclusions: A seroprevalence of 8.6% was found, higher than the result in a parallel study of our team in Salvador City (1.8%) and associated with a lack of basic sanitation, especially in rural areas.

Candida haemulonii is an emergent infectious pathogen that affects human presenting comorbidities and/or immunodepression. Little is known about other possible hosts. For the first time, this fungus was found causing a cutaneous infection in a snake, Boa constrictor, characterized by scale opacity and several ulcerative lesions. This C. haemulonii was isolated, identified using molecular techniques and a phylogenetic study, and had its growth totally inhibited by all the drugs tested; however, no fungicide effect was seen for fluconazole and itraconazole. The B. constrictor clinical signals subsided after a treatment using a biogenic silver nanoparticle-based ointment. These findings, along with the B. constrictor presence near human habitats, warn for the necessity of wildlife health monitoring for emergent and opportunistic diseases in peri-urban environments.

Dental cements are widely used in the clinical routine, specifically for root canal sealing. Within this context, it is expected that these materials present antimicrobial activity, since it would help in the prevention of apical and periapical infections. The present study aimed to comparatively verify the antimicrobial activity of four dental cements against microorganisms that are routinely isolated from endodontic infections. Reference strains of Enterococcus faecalis, Candida albicans and Escherichia coli were submitted to the agar diffusion test and to modified direct contact test using four different sealers: an eugenol zinc oxide compound, an epoxy resin associated to calcium hydroxide and bismuth, a mineral trioxide aggregate (MTA) and a bioceramics. Different E. coli, C. albicans and E. faecalis growth inhibition profiles were observed in the agar diffusion assay. In the direct contact test, the bioceramics presented a higher microbicide activity on all microorganisms tested herein. Dental cements have different antimicrobial activities, being that the bioceramics present the most consistent antimicrobial activity, and that the direct contact test presented more uniform results than the agar diffusion test. This study reveals the antimicrobial activities of different cements and allow dentists to decide which material to employ in their daily practice.

Caseous lymphadenitis (CLA), an infectious disease caused by Corynebacterium pseudotuberculosis in small ruminants, is highly prevalent worldwide. Economic losses have already been associated with the disease, and little is known about the host–pathogen relationship associated with the disease. The present study aimed to perform a metabolomic study of the C. pseudotuberculosis infection in goats. Serum samples were collected from a herd of 173 goats. The animals were classified as controls (not infected), asymptomatic (seropositives but without detectable CLA clinical signs), and symptomatic (seropositive animals presenting CLA lesions), according to microbiological isolation and immunodiagnosis. The serum samples were analyzed using nuclear magnetic resonance (1H-NMR), nuclear Overhauser effect spectroscopy (NOESY), and Carr–Purcell–Meiboom–Gill (CPMG) sequences. The NMR data were analyzed using chemometrics, and principal component analysis (PCA) and partial least square discriminant analysis (PLS-DA) were performed to discover specific biomarkers responsible for discrimination between the groups. A high dissemination of the infection by C. pseudotuberculosis was observed, being 74.57 • Sixty-two samples from healthy, CLA asymptomatic, and symptomatic goats were screened • Twenty metabolites of interest were identified by NOESY and 29 by CPMG • 1 H-NMR NOESY and CPMG were complementary and mutually confirming