Assessment of tropane alkaloid levels in Brazilian buckwheat flour products: A novel LC-UHPLC-MS/MS approach using solid-liquid extraction at low temperature.

In recent years, the monitoring of tropane alkaloids, specifically hyoscyamine and scopolamine, in food has become a pressing concern. This is due to increasing reports of food contamination with these compounds worldwide, raising awareness about the potential risks associated with their consumption. A novel method is proposed here for the determination of the sum of (+)-hyoscyamine, (-)-hyoscyamine, and (-)-scopolamine in buckwheat-based matrices, using solid-liquid extraction at low temperature and quantification by bidimensional chromatography coupled to tandem mass spectrometry. The validated method presented a linear response in the concentration range of 2.5-15 µg kg-1 (r > 0.99). The precision and accuracy were in the ranges from 0.8 to 11.0 % and from 96 to 103 %, respectively. The limit of quantification (LOQ) was 2.5 µg kg-1. No contamination was found at levels above the LOQ in any of the 18 samples analyzed (buckwheat flour, grains, and gluten-free mix).

In vitro Assessment of Efferocytic Capacity of Human Macrophages Using Flow Cytometry.

Clearance of dying cells, named efferocytosis, is a pivotal function of professional phagocytes that impedes the accumulation of cell debris. Efferocytosis can be experimentally assessed by differentially tagging the target cells and professional phagocytes and analyzing by cell imaging or flow cytometry. Here, we describe an assay to evaluate the uptake of apoptotic cells (ACs) by human macrophages in vitro by labeling the different cells with commercially available dyes and analysis by flow cytometry. We detail the methods to prepare and label human macrophages and apoptotic lymphocytes and the in vitro approach to determine AC uptake. This protocol is based on previously published literature and allows for in vitro modeling of the efficiency of AC engulfment during continual efferocytosis process. Also, it can be modified to evaluate the clearance of different cell types by diverse professional phagocytes.

Myelin Disruption, Neuroinflammation, and Oxidative Stress Induced by Sulfite in the Striatum of Rats Are Mitigated by the pan-PPAR agonist Bezafibrate.

Sulfite predominantly accumulates in the brain of patients with isolated sulfite oxidase (ISOD) and molybdenum cofactor (MoCD) deficiencies. Patients present with severe neurological symptoms and basal ganglia alterations, the pathophysiology of which is not fully established. Therapies are ineffective. To elucidate the pathomechanisms of ISOD and MoCD, we investigated the effects of intrastriatal administration of sulfite on myelin structure, neuroinflammation, and oxidative stress in rat striatum. Sulfite administration decreased FluoromyelinTM and myelin basic protein staining, suggesting myelin abnormalities. Sulfite also increased the staining of NG2, a protein marker of oligodendrocyte progenitor cells. In line with this, sulfite also reduced the viability of MO3.13 cells, which express oligodendroglial markers. Furthermore, sulfite altered the expression of interleukin-1ß (IL-1ß), interleukin-6 (IL-6), interleukin-10 (IL-10), cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS) and heme oxygenase-1 (HO-1), indicating neuroinflammation and redox homeostasis disturbances. Iba1 staining, another marker of neuroinflammation, was also increased by sulfite. These data suggest that myelin changes and neuroinflammation induced by sulfite contribute to the pathophysiology of ISOD and MoCD. Notably, post-treatment with bezafibrate (BEZ), a pan-PPAR agonist, mitigated alterations in myelin markers and Iba1 staining, and IL-1ß, IL-6, iNOS and HO-1 expression in the striatum. MO3.13 cell viability decrease was further prevented. Moreover, pre-treatment with BEZ also attenuated some effects. These findings show the modulation of PPAR as a potential opportunity for therapeutic intervention in these disorders.

C5aR1 signaling triggers lung immunopathology in COVID-19 through neutrophil extracellular traps.

Patients with severe COVID-19 develop acute respiratory distress syndrome (ARDS) that may progress to cytokine storm syndrome, organ dysfunction, and death. Considering that complement component 5a (C5a), through its cellular receptor C5aR1, has potent proinflammatory actions and plays immunopathological roles in inflammatory diseases, we investigated whether the C5a/C5aR1 pathway could be involved in COVID-19 pathophysiology. C5a/C5aR1 signaling increased locally in the lung, especially in neutrophils of critically ill patients with COVID-19 compared with patients with influenza infection, as well as in the lung tissue of K18-hACE2 Tg mice (Tg mice) infected with SARS-CoV-2. Genetic and pharmacological inhibition of C5aR1 signaling ameliorated lung immunopathology in Tg-infected mice. Mechanistically, we found that C5aR1 signaling drives neutrophil extracellular traps-dependent (NETs-dependent) immunopathology. These data confirm the immunopathological role of C5a/C5aR1 signaling in COVID-19 and indicate that antagonists of C5aR1 could be useful for COVID-19 treatment.

Factors associated with relapse and hospital death in patients coinfected with visceral leishmaniasis and HIV: a longitudinal study.

OBJECTIVE: Visceral leishmaniasis (VL) is an endemic parasitic disease in Latin America, and its clinical picture is aggravated in coinfections with the human immunodeficiency virus (HIV). The objective of this study was to investigate clinical factors and laboratory variables associated with VL relapse and death in VL/HIV coinfected patients. METHODS: A prospective longitudinal study was conducted from January 2013 to July 2020 among 169 patients coinfected with VL and HIV. The outcomes investigated were the occurrence of VL relapse and death. Chi-square test, Mann-Whitney test and logistic regression models were used for statistical analysis. RESULTS: The occurrence rates were 41.4% for VL relapse and 11.2% for death. Splenomegaly and adenomegaly were associated with the increased risk of VL relapse. Patients with VL relapse had higher levels of urea (p = .005) and creatinine (p < .001). Patients who died had lower red blood cell counts (p = .012), hemoglobin (p = .017) and platelets (p < .001). The adjusted model showed that antiretroviral therapy for more than 6 months was associated with a decrease in VL relapse, and adenomegaly was associated with an increase in VL relapse. In addition, edema, dehydration, poor general health status, and paleness were associated with an increase in hospital death. CONCLUSION: The findings suggest that adenomegaly, antiretroviral therapy, and renal abnormalities can be associated with VL relapse, while hematological abnormalities, and clinical manifestations like paleness, and edema can be associated with an increased odds of hospital death. TRIAL REGISTRATION NUMBER: The study was submitted to the Ethics and Research Committee of the Federal University of Maranhão (Protocol: 409.351).

Emetic Tartar-Loaded Liposomes as a New Strategy for Leishmaniasis Treatment.

Emetic tartar (ET), was used in the treatment of leishmaniasis but its use was discontinued due to its low therapeutic index. Liposomes have been shown to be a promising strategy for delivery of bioactive substances in the region of interest, in order to reduce and/or eliminate undesirable effects. In the present study, liposomes containing ET were prepared and characterized to evaluate acute toxicity as well as their leishmanicidal action using BALB/c mice with an inoculum of Leishmania (Leishmania) infantum. Liposomes were composed of egg phosphatidylcholine and 3ß-[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol, with an average diameter of 200 nm, zeta potential of +18 mV, and ET encapsulated into liposomes at a concentration near 2 g/L. Healthy mice were treated with ET or liposome containing ET (Lip-ET) in a single dose of 16 mg/kg of Sb3+ intravenously and observed for 14 days. The death of two animals in the ET-treated group and no deaths in the Lip-ET-treated group was observed. Higher hepatic and cardiac toxicity were observed in animals treated with ET when compared to animals treated with Lip-ET, blank liposomes (Blank-Lip) and PBS. The study of antileishmanial efficacy was conducted by intraperitoneal administration of Lip-ET, for ten consecutive days. It was observed by limiting dilution that treatments with liposomal formulations containing ET, as well as Glucantime®, led to a significant reduction in parasitic load in spleen and liver (p < 0.05) when compared to the untreated control group.

Incidental Findings Protocol Implementation at a Level-I Trauma Center: A Review of Patient Follow-Up.

OBJECTIVES: Pan scanning in trauma patients has become routine, resulting in increased identification of incidental findings (IF), findings unrelated to the reason for the scan. This has posed a conundrum of ensuring patients have appropriate follow-up for these findings. We sought to evaluate our compliance and follow-up for patients after implementation of an IF protocol at our level-I trauma center. METHODS: We performed a retrospective review from 9/2020 to 4/2021, to encompass before and after protocol implementation. Patients were separated into PRE and POST groups. Charts were reviewed evaluating several factors including three- and six-month follow-ups on IF. Data were analyzed comparing PRE and POST groups. RESULTS: A total of 1989 patients were identified, 31.22% (n = 621) with an IF. 612 patients were included in our study. Compared to PRE, POST showed a significant increase in PCP notification (35% vs 22%, P < .001) and patient notification (82% vs 65%, P < .001). As a result, patient follow-up regarding IF at six months was significantly higher in POST (44%) v. PRE (29%), (P < .001). There was no difference in follow-up based on insurance carrier. There was no difference in patient age for PRE (63 y) and POST (66 y) overall, (P = .089); nor in age of patients who followed up; 68.8 PRE vs 68.2 years POST (P = .819). CONCLUSION: Implementation of an IF protocol with patient and PCP notification was significantly improved in overall patient follow-up for category one and two IF. Utilizing the results of this study, the protocol will be further revised to improve patient follow-up.

CASP4/11 Contributes to NLRP3 Activation and COVID-19 Exacerbation.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection triggers activation of the NLRP3 inflammasome, which promotes inflammation and aggravates severe COVID-19. Here, we report that SARS-CoV-2 induces upregulation and activation of human caspase-4/CASP4 (mouse caspase-11/CASP11), and this process contributes to NLRP3 activation. In vivo infections performed in transgenic hACE2 humanized mice, deficient or sufficient for Casp11, indicate that hACE2 Casp11-/- mice were protected from disease development, with the increased pulmonary parenchymal area, reduced clinical score of the disease, and reduced mortality. Assessing human samples from fatal cases of COVID-19, we found that CASP4 was expressed in patient lungs and correlated with the expression of inflammasome components and inflammatory mediators, including CASP1, IL1B, IL18, and IL6. Collectively, our data establish that CASP4/11 promotes NLRP3 activation and disease pathology, revealing a possible target for therapeutic interventions for COVID-19.

First molecular detection of Mycoplasma ovis in horses from Brazil.

This study aimed to determine the occurrence of hemoplasmas and tick-borne pathogens (TBP) (Theileria equi, Babesia caballi, and Ehrlichia sp.) in horses and ticks' salivary glands, and determine the factors associated with exposure/infection in a rural settlement in southern Brazil. Blood samples from 22 horses were screened for anti-T. equi and anti-Ehrlichia sp. antibodies by an indirect fluorescent antibody test (IFAT) assays. Samples were also tested by PCR assays for T. equi and B. caballi (18S rRNA and rap-1 genes, respectively), hemoplasmas (16S rRNA gene), and Ehrlichia sp. (dsb gene). Ticks were removed from the animals (inspection) and the environment (flannel trawling and dry ice traps), and morphologically identified. Additionally, salivary glands DNA was extracted from 28 adult ticks infesting the animals and four nymphs from the environment, and further screened for Ehrlichia sp. and hemotropic Mycoplasma sp. Anti-T. equi and anti-Ehrlichia sp. antibodies were detected in 40.91% (nine/22; 95% CI: 23.26-61.27) and 31.81% (seven/22; 95% CI: 16.36-52.68) horses, respectively. Theileria equi, B. caballi, and hemotropic Mycoplasma sp. DNA was detected in 59.09% (13/22), 4.55% (one/22), and 50% (11/22) horses, respectively. All horses tested negative in the PCR for Ehrlichia sp. All sequences showed ≥99% identity with multiple T. equi, B. caballi, and Mycoplasma ovis sequences deposited in GenBank database. Adult ticks were identified as Dermacentor nitens (44/47; 93.62%) and Rhipicephalus microplus (three/47; 6.38%). Ticks' salivary glands were negative for Ehrlichia sp., while 39.29% from adults (11/28) and 50% from nymphs (two/four) from the environment were positive for hemotropic Mycoplasma sp. This is the first report of M. ovis infection in horses from Brazil and the first detection of hemoplasma DNA in salivary glands of D. nitens and R. microplus ticks. Further studies are needed to elucidate the vector competence of ticks to transmit hemoplasmas.

One-year clinical evaluation of class II bulk-fill restorations in primary molars: a randomized clinical trial.

This double-blind, randomized clinical trial aimed to compare the clinical performance and clinical time to restore occluso-proximal cavities in primary molars withbulk-fillresin and conventional resin. A total of 140 class II restorations in primary molars of 65 participants (mean age of 6.7 + 1.5) were placed in two random groups:bulk-filland conventional resin. The restorations were evaluated using FDI criteria at the baseline, 6-month, and one year by a single calibrated examiner, and the clinical restorative time was measured with a digital timer. The success and survival of the restorations were evaluated with Kaplan-Meier graphs. The log-rank test compared the curves. Differences in restorative clinical time were compared using the Mann-Whitney U test. The level of significance was 5%. After one year, 115 restorations were evaluated. The success probability was 88.7% for Filtek Z350 XT and 85.9% for FiltekTM Bulk-fill, and for the survival probability, Filtek Z350 XT presented 90%, and FiltekTM Bulk-fill presented 93.7%. No significant difference was found between the success and survival curves (p=0.62), (p=0.51). The main reason for failure was marginal adaptation.Bulk-fillresin required 30% less time than the conventional resin (p<0.001).Bulk-fillresin presented similar clinical performance to the conventional resin and required less restorative clinical time. It is an option to restore class II lesions of primary molars.

Hemoplasmas and ticks in cattle from Somalia.

Hemotropic mycoplasmas (hemoplasmas) are small Gram-negative bacteria that parasitize red blood cells and can cause mild to severe anemia in a wide range of vertebrates, including ruminants. Cattle population in Somalia is around 3.9 million heads, with animals more concentrated around the river areas, mainly in the Juba River and Shabelle River Valleys. Information on hemoplasmas in Sub-Saharan Africa are scarce, mainly in Somalia, where no studies have been performed to date. Accordingly, this study aimed to assess the molecular occurrence of hemoplasmas in 131 cattle blood samples from Somalia. Thirty out of 131 (22.90%; 95% CI: 16.54-30.81%) cattle were infested by ticks: Rhipicephalus pulchellus (68.18%), Amblyomma gemma (18.18%), Amblyomma lepidum (9.09%), Hyalomma marginatum (1.51%), Hyalmomma rufipes (1.51%), and Rhipicephalus pravus (1.51%). A total of 74/131 (56.48%; 95% CI: 47.93-64.67%) cattle were positive for hemotropic Mycoplasma spp. by real-time PCR (qPCR) based on the 16S rRNA gene. Hemoplasma-positive samples were later subjected to species-specific PCR assays for Mycoplasma wenyonii and 'Candidatus Mycoplasma haematobovis' based on the 16S rRNA gene. A total of 34/74 (45.94%; 95% CI: 35.07-57.22%) animals were coinfected by both species; 31/74 (41.89%; 95% CI: 31.32-53.26%) and 3/74 (4.05%; 95% CI: 01.39-11.25%) cattle were solely positive to M. wenyonii and 'Ca. M. haematobovis', respectively. Six out of 74 (8.1%; 95% CI: 03.77-16.58%) cattle were negative on species-specific conventional PCR assays but tested positive by a semi-nested PCR assay based on the 16S rRNA gene of hemoplasmas. Sequencing of the detected hemotropic Mycoplasma sp. 16S rRNA gene confirmed that animals were infected by M. wenyonii and 'Ca. M. haematobovis'. To the best of our knowledge, this is the first study on the detection of hemoplasmas in cattle from Somalia.

Identification of immunomodulatory drugs that inhibit multiple inflammasomes and impair SARS-CoV-2 infection.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) induces mild or asymptomatic COVID-19 in most cases, but some patients develop an excessive inflammatory process that can be fatal. As the NLRP3 inflammasome and additional inflammasomes are implicated in disease aggravation, drug repositioning to target inflammasomes emerges as a strategy to treat COVID-19. Here, we performed a high-throughput screening using a 2560 small-molecule compound library and identified FDA-approved drugs that function as pan-inflammasome inhibitors. Our best hit, niclosamide (NIC), effectively inhibits both inflammasome activation and SARS-CoV-2 replication. Mechanistically, induction of autophagy by NIC partially accounts for inhibition of NLRP3 and AIM2 inflammasomes, but NIC-mediated inhibition of NAIP/NLRC4 inflammasome are autophagy independent. NIC potently inhibited inflammasome activation in human monocytes infected in vitro, in PBMCs from patients with COVID-19, and in vivo in a mouse model of SARS-CoV-2 infection. This study provides relevant information regarding the immunomodulatory functions of this promising drug for COVID-19 treatment.

Targeting C5aR1 signaling reduced neutrophil extracellular traps and ameliorates COVID-19 pathology

Patients with severe COVID-19 develop acute respiratory distress syndrome (ARDS) that may progress to cytokine storm syndrome, organ dysfunction, and death. Considering that complement component 5a (C5a), through its cellular receptor C5aR1, has potent proinflammatory actions, and plays immunopathological roles in inflammatory diseases, we investigated whether C5a/C5aR1 pathway could be involved in COVID-19 pathophysiology. C5a/C5aR1 signaling increased locally in the lung, especially in neutrophils of critically ill COVID-19 patients compared to patients with influenza infection, as well as in the lung tissue of K18-hACE2 Tg mice (Tg mice) infected with SARS-CoV-2. Genetic and pharmacological inhibition of C5aR1 signaling ameliorated lung immunopathology in Tg-infected mice. Mechanistically, we found that C5aR1 signaling drives neutrophil extracellular trap (NET)s-dependent immunopathology. These data confirm the immunopathological role of C5a/C5aR1 signaling in COVID-19 and indicate that antagonist of C5aR1 could be useful for COVID-19 treatment.

Efferocytosis of SARS-CoV-2-infected dying cells impairs macrophage anti-inflammatory functions and clearance of apoptotic cells.

COVID-19 is a disease of dysfunctional immune responses, but the mechanisms triggering immunopathogenesis are not established. The functional plasticity of macrophages allows this cell type to promote pathogen elimination and inflammation or suppress inflammation and promote tissue remodeling and injury repair. During an infection, the clearance of dead and dying cells, a process named efferocytosis, can modulate the interplay between these contrasting functions. Here, we show that engulfment of SARS-CoV-2-infected apoptotic cells exacerbates inflammatory cytokine production, inhibits the expression of efferocytic receptors, and impairs continual efferocytosis by macrophages. We also provide evidence supporting that lung monocytes and macrophages from severe COVID-19 patients have compromised efferocytic capacity. Our findings reveal that dysfunctional efferocytosis of SARS-CoV-2-infected cell corpses suppresses macrophage anti-inflammation and efficient tissue repair programs and provides mechanistic insights for the excessive production of pro-inflammatory cytokines and accumulation of tissue damage associated with COVID-19 immunopathogenesis.

Mediating factors of statistics anxiety in university students: a systematic review and meta-analysis.

Statistics plays a key role in many areas of modern society, including technology, social and behavior studies, economics, and the sciences. Statistics anxiety (SA) has a detrimental impact on academic experiences in university populations, although the mediating factors remain underexplored. We conducted the first systematic review and meta-analysis focused on SA in university students in the context of statistical performance, individual differences in statistical learning, self-perceptions regarding the statistics course and instructor, and sociodemographic factors. Searches were carried out in the PsycINFO, PubMed, Scielo, and Web of Science databases according to our preregistration. Forty studies were selected for systematic review. Seventeen were included in a series of six meta-analyses concerning academic achievement, attitudes, self-perception, procrastination, and gender. The findings reveal learning strategies, procrastination, self-efficacy, and self-awareness as predictors of SA. However, the impact of sociodemographic data in these moderators is still uncharted. We conclude with a critical appraisal of the selected studies and present future directions for research in SA.

Cortical bone density by quantitative computed tomography mirrors disorders of bone structure in bone biopsy of non-dialysis CKD patients.

Bone biopsy is still the gold standard tool to evaluate either trabecular or cortical bone, though the quantitative computed tomography of the vertebrae (QCT), a non-invasive technique, could be useful to evaluate bone structure in patients with chronic kidney disease (CKD). Cortical bone microstructure derangements have been associated with poor outcomes in the general population. An association between trabecular bone density, assessed by QCT, and bone volume and microarchitecture by histomorphometry, has been previously documented. This relationship has not yet been fully evaluated in cortical bone in the CKD scenario. The aim of this study was to evaluate the relationship among vertebrae density measured by QCT, structural histomorphometric parameters of cortical bone and biochemical and hormonal data in 50 CKD stage 2-5ND patients. This was a post hoc analysis of a cross-sectional study where cortical porosity and cortical thickness were analyzed in undecalcified bone samples from the iliac crest. The cortical bone density was obtained by QCT from the thoracic vertebrae. The patients were 52 ± 10 years, 68% men, 30% diabetes and the estimated glomerular filtration rate 34 ± 16 mL/min/1.73 m2. Cortical porosity was 4.6% (3.6; 6.6) and cortical thickness was 578.4 ± 151.8 µm, while cortical bone density was 149.2 ± 58.3 HU. Cortical density correlated with cortical thickness (p = 0.001) but not with cortical porosity (p = 0.30). Higher porosity was associated with older age (p = 0.02), higher levels of PTH (p = 0.04) and lower renal function (p = 0.03), while smaller thickness was associated with higher levels of PTH (p = 0.02). Lower density was associated with older age (p = 0.02) and higher levels of PTH (p = 0.01). In conclusion, cortical bone density measured by QCT was able to mirror the cortical thickness of bone biopsy in pre-dialysis CKD patients. In addition, PTH action on cortical bone can be already seen in this population.

Novel Anaplasmataceae agents Candidatus Ehrlichia hydrochoerus and Anaplasma spp. Infecting Capybaras, Brazil.

We amplified Ehrlichia and Anaplasma DNA from Amblyomma dubitatum tick-infested capybaras (Hydrochoerus hydrochaeris) in southern Brazil. Sequencing of 16S rRNA, sodB, and groEL indicated a novel Ehrlichia species, and sequencing of 16S rRNA from 2 capybaras indicated a novel Anaplasma species. The tick vectors remain unknown.