Modulation of Apoptosis by Bovine Gammaherpesvirus 4 Infection in Bovine Endometrial Cells and the Possible Role of LPS in This Process.

The prevalent pathogens associated with bovine uterine infections are bacteria that appear to increase the host's susceptibility to secondary infections with other bacteria or viruses, among which BoGHV4 is the most frequently found. In this work, the study of the pathways of apoptosis induction was carried out on an experimental model of primary culture of endometrial cells, in order to know the implication of BoGHV4 and the presence of bacterial LPS in the pathogenesis of the bovine reproductive tract. For this, different staining techniques and molecular analysis by RT-PCR were used. The results obtained allowed us to conclude that the level of cell death observed in the proposed primary culture is directly related to the time of viral infection and the presence of LPS in BoGHV4 infection. The apoptosis indices in cells infected with BoGHV4 and BoGHV4 + LPS revealed a maximum that correlated with the appearance of cytopathic effects and the maximum viral titers in the model studied. However, morphological, biochemical, and molecular changes were evident during both early and late stages of apoptosis. These findings provide information on the factors that may influence the pathogenesis of BoGHV4 and help to better understand the mechanisms involved in virus infection.

LC-MS analysis of polar and highly polar organic pollutants in Barcelona urban groundwater using orthogonal LC separation modes.

Groundwater samples may contain thousands of organic pollutants from infiltration of surface water, sewer leakages, and to a minor extent from public water supply network losses. Polar (0 < log D < - 2.0) and very polar substances (log D < - 2.0) have been largely beyond the scope of applied analytical methodologies in environmental monitoring because of challenges related to their extraction from the sample and subsequent chromatographic separation. In this study, we developed an analytical workflow for 96 pollutants covering a broad polarity range, including pharmaceuticals, industrial chemicals, and artificial sweeteners, potentially seeping through the soil in urban areas. The Besos aquifer located at the Northern-eastern edge of the city of Barcelona was chosen as a study area due to the deterioration of the quality of the aquifers over the past years and the proven presence of numerous pollutants. The methodology consisted of vacuum-assisted evaporation (VAE) followed by chromatographic separation of the sample on two columns with orthogonal retention mechanisms, namely, an HSS T3 column (modified C18) and a BEH amide column (HILIC). The analytes were detected by high-resolution mass spectrometry on a Q Exactive Orbitrap system in data-independent acquisition mode. Taking into consideration the retention as well as the peak shape, a Quality Score (QS) was assigned for each analyte to evaluate the quality of each chromatographic peak of each compound. While 67 compounds, including 19 polar and 48 moderately polar, were satisfactorily retained on an HSS T3, 29 compounds, including 14 highly polar, 14 polar, and one moderately polar, were analyzed in the BEH amide column. The optimized methodology was applied for the analysis of 89 out of 96 validated contaminants with satisfactory recoveries in samples collected from seven wells, providing low LODs (0.02 to 0.45 ng L-1) and LOQs (0.06 to 1.34 ng L-1). A number of highly polar and polar compounds not previously reported to occur in GW, including artificial sweeteners, pharmaceuticals, and industrial chemicals, were detected at concentrations as high as few µ g L-1.

Abnormal cell sorting and altered early neurogenesis in a human cortical organoid model of Protocadherin-19 clustering epilepsy.

Introduction: Protocadherin-19 (PCDH19)-Clustering Epilepsy (PCE) is a developmental and epileptic encephalopathy caused by loss-of-function variants of the PCDH19 gene on the X-chromosome. PCE affects females and mosaic males while male carriers are largely spared. Mosaic expression of the cell adhesion molecule PCDH19 due to random X-chromosome inactivation is thought to impair cell-cell interactions between mutant and wild type PCDH19-expressing cells to produce the disease. Progress has been made in understanding PCE using rodent models or patient induced pluripotent stem cells (iPSCs). However, rodents do not faithfully model key aspects of human brain development, and patient iPSC models are limited by issues with random X-chromosome inactivation. Methods: To overcome these challenges and model mosaic PCDH19 expression in vitro, we generated isogenic female human embryonic stem cells with either HA-FLAG-tagged PCDH19 (WT) or homozygous PCDH19 knockout (KO) using genome editing. We then mixed GFP-labeled WT and RFP-labeled KO cells and generated human cortical organoids (hCOs). Results: We found that PCDH19 is highly expressed in early (days 20-35) WT neural rosettes where it co-localizes with N-Cadherin in ventricular zone (VZ)-like regions. Mosaic PCE hCOs displayed abnormal cell sorting in the VZ with KO and WT cells completely segregated. This segregation remained robust when WT:KO cells were mixed at 2:1 or 1:2 ratios. PCE hCOs also exhibited altered expression of PCDH19 (in WT cells) and N-Cadherin, and abnormal deep layer neurogenesis. None of these abnormalities were observed in hCOs generated by mixing only WT or only KO (modeling male carrier) cells. Discussion: Our results using the mosaic PCE hCO model suggest that PCDH19 plays a critical role in human VZ radial glial organization and early cortical development. This model should offer a key platform for exploring mechanisms underlying PCE-related cortical hyperexcitability and testing of potential precision therapies.

Evaluation of different QuEChERS-based methods for the extraction of 48 wastewater-derived organic contaminants from soil and lettuce root using high-resolution LC-QTOF with MRMHR and SWATH acquisition modes.

The reuse of treated wastewater in agriculture is an important route of introducing a large number of organic contaminants into the agroecosystem. In this study, a modified QuEChERS-based approach was developed for rapid, simple, and simultaneous extraction of 48 organic wastewater-derived contaminants from soil and lettuce root. Twenty-two different (modification) scenarios of the known (or original) QuEChERS method have been tested, in order to obtain best and well-compromised recoveries for all target compounds for soil and roots. Finally, a common method was chosen for both matrices consisting of a single extraction step using EDTA-Mcllvaine buffer and the unbuffered Original QuEChERS salts. Method performance was accomplished by liquid chromatography coupled with high-resolution mass spectrometry on a QToF-MS system using two different acquisition modes, the ultra-fast high-resolution multiple reaction monitoring (MRMHR) mode and the innovative Sequential Window Acquisition of All Theoretical Fragment-Ion (SWATH) mode. Performance characterization was evaluated in terms of recovery, linearity, intra-day precision, method detection limits (MDLs), method quantification limits (MQLs), and matrix effect (ME). Recoveries in MRMHR mode ranged from 63 to 111% and 54 to 104% for lettuce root and soil, respectively, for most of compounds in MRMHR mode and from 56 to 121% and 54 to 104% for lettuce root and soil, respectively, for most of compounds in SWATH. Whereas, MQLs ranged from 0.03 to 0.92 ng g-1 in MRMHR and from 0.03 to 82 ng g-1 in SWATH for lettuce root, and from 0.02 to 0.44 ng g-1 in MRMHR and 0.02 to 0.14 ng g-1 in SWATH for soil. The method was then applied to follow the target compounds in soil and lettuce root, where the system lettuce-soil was irrigated with treated wastewater under real greenhouse conditions. Five and 17 compounds were detected in lettuce root and soil, respectively.

Ivermectin antiviral activity against Varicellovirus bovinealpha 1: assessment of intracellular drug accumulation in virus-infected cells.

Varicellovirus bovinealpha 1 (formerly bovine alphaherpesvirus type 1, BoAHV-1) is associated with several syndromes in cattle, including respiratory disease and is one of the main agents involved in the bovine respiratory disease complex (BRDC). Its infectious cycle is characterized by latent infections with sporadic virus reactivation and transmission. Although the acute disease can be prevented by the use of vaccines, specific therapeutic measures are not available. Ivermectin (IVM) is a semi-synthetic avermectin with a broad-spectrum antiparasitic activity, which has previously shown to have potential as an antiviral drug. In this study, IVM antiviral activity against BoAHV-1 was characterized in two cell lines (MDBK [Madin Darby bovine kidney] and BT [bovine turbinate]), including the measurement of intracellular drug accumulation within virus-infected cells. IVM antiviral activity was assessed at three different drug concentrations (1.25, 2.5 and 5 µM) after incubation for 24, 48 and 72 h. Slight cytotoxicity was only observed with 5 µM IVM. Even the lowest IVM dose was able to induce a significant reduction in virus titers in both cell lines. These findings indicate that the antiviral effects of IVM were evident in our experimental model within the range of concentrations achievable through therapeutic in vivo administration. Consequently, additional in vivo trials are necessary to validate the potential utility of these results in effectively managing BoAHV-1 in infected cattle.

Pharmaceutical and pesticide mixtures in a Mediterranean coastal wetland: comparison of sampling methods, ecological risks, and removal by a constructed wetland.

Pharmaceuticals and pesticides can be considered hazardous compounds for Mediterranean coastal wetland ecosystems. Although many of these compounds co-occur in environmental samples, only a few studies have been dedicated to assessing the ecotoxicological risks of complex contaminant mixtures. We evaluated the occurrence of 133 pharmaceuticals and pesticides in 12 sites in a protected Mediterranean wetland, the Albufera Natural Park (ANP), based on conventional grab sampling and polar organic chemical integrative samplers (POCIS). We assessed acute and chronic ecological risks posed by these contaminant mixtures using the multi-substance Potentially Affected Fraction (msPAF) approach and investigated the capacity of a constructed wetland to reduce chemical exposure and risks. This study shows that pharmaceuticals and pesticides are widespread contaminants in the ANP, with samples containing up to 75 different compounds. POCIS samplers were found to be useful for the determination of less predictable exposure profiles of pesticides occurring at the end of the rice cultivation cycle, while POCIS and grab samples provide an accurate method to determine (semi-)continuous pharmaceutical exposure. Acute risks were identified in one sample, while chronic risks were determined in most of the collected samples, with 5-25% of aquatic species being potentially affected. The compounds that contributed to the chronic risks were azoxystrobin, ibuprofen, furosemide, caffeine, and some insecticides (diazinon, imidacloprid, and acetamiprid). The evaluated constructed wetland reduced contaminant loads by 45-73% and reduced the faction of species affected from 25 to 6%. Our study highlights the need of addressing contaminant mixture effects in Mediterranean wetlands and supports the use of constructed wetlands to reduce contaminant loads and risks in areas with high anthropogenic pressure.

Combination of multivariate data analysis and mixing modelling to assess tracer potential of contaminants of emerging concern in aquifers.

Collected evidence has shown that contaminants of emerging concern (CECs) in conjunction with more conventional tracers (major ions, nutrients, isotopes etc.) can be used to trace pollution origin in aquatic systems. However, in highly mixed aquifer systems signals obtained from conventional tracers overlap diminishing their potential to be used as tracers. In this study, we present an approach that incorporates multivariate statistical analysis (principal component analysis (PCA) and Kohonen's Self-Organizing Map method (SOM)) and mixing modelling to identify the most suitable CECs to be employed as anthropogenic tracers. The study area is located in the Besòs River Delta (Barcelona, NE Spain) and represents the highly mixed aquifer system. A one-year monthly based monitoring campaign was performed to collect the information about the concentrations of 105 CECs as well as major and minor ions in the river and along the groundwater flow. The dimensionality of the obtained dataset was reduced to 25 CECs, based on their estimated health risk effects, for multivariate data analysis. The obtained results showed the overlap of conventional tracers' signals obtained from PCA. In case of CECs, PCA revealed differences in their distributions allowing the differentiation of the roles of natural attenuation processes, local and regional flows on their occurrence in different parts of the aquifer. This was not possible to do using solely CECs' distribution profiles. SOMs provided the lacking information about the modality of the distribution of each CECs, revealing their ability to represent factors controlling the groundwater hydrochemistry, which assist in defining their tracer potential. Based on the obtained results four identified persistent CECs, two with unimodal (lamotrigine and 5-Desamino-5-oxo-lamotrigine) and two with bimodal (carbamazepine and diazepam (higher modality was not revealed)) distributions, were selected to run a mixing model to compare their tracer performance.

Perceived burdensomeness, thwarted belongingness, and hopelessness as predictors of future suicidal ideation in Spanish university students.

The Interpersonal Theory of Suicide (IPTS) has received support for its role in understanding suicidal thoughts and behaviors. However, few longitudinal studies have focused on testing this theory in university students. The present study aimed to confirm the theoretical model of the IPTS in a sample of 225 Spanish university students, using path analysis in a longitudinal study. We assessed thwarted belongingness and perceived burdensomeness at T1 and hopelessness and suicidal ideation at T2, 12-14 weeks later. Moreover, we assessed suicidal ideation weekly for 14 weeks. Path analyses confirmed the Interpersonal Theory of Suicide model, with thwarted belongingness and perceived burdensomeness as direct and indirect predictors of suicidal ideation through hopelessness. Providers of guidance and clinical services in university settings should be trained to identify perceived burdensomeness, social belongingness, hopelessness, and suicidal ideation when screening for suicide prevention.

Antioxidant and Anti-Inflammatory Effects of Garlic in Ischemic Stroke: Proposal of a New Mechanism of Protection through Regulation of Neuroplasticity.

Stroke represents one of the main causes of death and disability in the world; despite this, pharmacological therapies against stroke remain insufficient. Ischemic stroke is the leading etiology of stroke. Different molecular mechanisms, such as excitotoxicity, oxidative stress, and inflammation, participate in cell death and tissue damage. At a preclinical level, different garlic compounds have been evaluated against these mechanisms. Additionally, there is evidence supporting the participation of garlic compounds in other mechanisms that contribute to brain tissue recovery, such as neuroplasticity. After ischemia, neuroplasticity is activated to recover cognitive and motor function. Some garlic-derived compounds and preparations have shown the ability to promote neuroplasticity under physiological conditions and, more importantly, in cerebral damage models. This work describes damage/repair mechanisms and the importance of garlic as a source of antioxidant and anti-inflammatory agents against damage. Moreover, we examine the less-explored neurotrophic properties of garlic, culminating in proposals and observations based on our review of the available information. The aim of the present study is to propose that garlic compounds and preparations could contribute to the treatment of ischemic stroke through their neurotrophic effects.

Transplacental infection by bovine alphaherpesvirus type 1 induces protein expression of COX-2, iNOS and inflammatory cytokines in fetal lungs and placentas.

Bovine alphaherpesvirus type 1 (BoAHV-1) is associated with respiratory and reproductive syndromes. Until present the immunologic mechanisms involved in BoAHV-1 abortion are partially known. We studied key elements of the innate immune response in the placentas and fetal lungs from cattle experimentally-inoculated with BoAHV-1. These tissues were analyzed by histopathology. Furthermore, virus identification was performed by qPCR and the expression of the inflammatory cytokines such as tumor necrosis factor-alpha, interleukin 1-alpha and inflammatory mediators like inducible nitric oxide synthase and cyclooxeganse-2 was evaluated by immunohistochemistry. The viral transplacental infection was confirmed by the detection of BoAHV-1 by qPCR in the placenta and fetal organs, which revealed mild inflammatory lesions. Inducible nitric oxide synthase immunolabelling was high in the lungs of infected fetuses and placentas, as well as for tumor necrosis factor-alpha in the pulmonary parenchyma and cyclooxeganse-2 in fetal annexes. However, the expression of interleukin 1-alpha was weak in these organs. To our knowledge, this is the first study that provides strong evidence of an early immune response to BoAHV-1 infection in the conceptus. Advances in the knowledge of the complex immunological interactions at the feto-maternal unit during BoAHV-1 infection are needed to clarify the pathogenesis of abortion.

Developing Evidence for Equitable Assessment Characteristics Based on Clinical Learner Preferences Using Discrete Choice Experiments.

PURPOSE: Medical education is only beginning to explore the factors that contribute to equitable assessment in clinical settings. Increasing knowledge about equitable assessment ensures a quality medical education experience that produces an excellent, diverse physician workforce equipped to address the health care disparities facing patients and communities. Through the lens of the Anti-Deficit Achievement framework, the authors aimed to obtain evidence for a model for equitable assessment in clinical training. METHOD: A discrete choice experiment approach was used which included an instrument with 6 attributes each at 2 levels to reveal learner preferences for the inclusion of each attribute in equitable assessment. Self-identified underrepresented in medicine (UIM) and not underrepresented in medicine (non-UIM) (N = 306) fourth-year medical students and senior residents in medicine, pediatrics, and surgery at 9 institutions across the United States completed the instrument. A mixed-effects logit model was used to determine attributes learners valued most. RESULTS: Participants valued the inclusion of all assessment attributes provided except for peer comparison. The most valued attribute of an equitable assessment was how learner identity, background, and trajectory were appreciated by clinical supervisors. The next most valued attributes were assessment of growth, supervisor bias training, narrative assessments, and assessment of learner's patient care, with participants willing to trade off any of the attributes to get several others. There were no significant differences in value placed on assessment attributes between UIM and non-UIM learners. Residents valued clinical supervisors valuing learner identity, background, and trajectory and clinical supervisor bias training more so than medical students. CONCLUSIONS: This study offers support for the components of an antideficit-focused model for equity in assessment and informs efforts to promote UIM learner success and guide equity, diversity, and inclusion initiatives in medical education.

Deriving early single-rosette brain organoids from human pluripotent stem cells.

Brain organoid methods are complicated by multiple rosette structures and morphological variability. We have developed a human brain organoid technique that generates self-organizing, single-rosette cortical organoids (SOSR-COs) with reproducible size and structure at early timepoints. Rather than patterning a 3-dimensional embryoid body, we initiate brain organoid formation from a 2-dimensional monolayer of human pluripotent stem cells patterned with small molecules into neuroepithelium and differentiated to cells of the developing dorsal cerebral cortex. This approach recapitulates the 2D to 3D developmental transition from neural plate to neural tube. Most monolayer fragments form spheres with a single central lumen. Over time, the SOSR-COs develop appropriate progenitor and cortical laminar cell types as shown by immunocytochemistry and single-cell RNA sequencing. At early time points, this method demonstrates robust structural phenotypes after chemical teratogen exposure or when modeling a genetic neurodevelopmental disorder, and should prove useful for studies of human brain development and disease modeling.

A systematic review of the effectiveness of meaning-centred psychotherapies in depressed participants.

Depression is a mental disorder that is associated with low quality of life, increased risk of suicide and a high economic cost for society. Meta-analyses indicate that Meaning Centred Psychotherapies (MCP) are an efficacious psychotherapy to reduce depression in participants with chronic illness and cancer. However, to date, no systematic review has analysed the effectiveness of MCP in depressed participants who do not have a physical illness or cancer. The objective of this study was to carry out a systematic review to analyse the effectiveness of MCP in participants with depression and no physical illness. The search was conducted following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines (PRISMA). Six studies (two randomised controlled trials and four quasi-experimental studies) were included in the systematic review. The results indicate that MCP is more effective than a control group as waitlist (four studies), psychoeducation group (one study), and cognitive behavioural therapy (CBT, one study), in reducing depressive symptoms, improving meaning in life and reducing anxiety in participants without physical illness. MCP would be considered a probably efficacious treatment for depression. However, further research with better experimental designs will be necessary to confirm the effectiveness of MCP for depression.

Analysis of vaccine responses after anti-CD20 maintenance in B-cell lymphoma in the Balearic Islands. A single reference center experience.

Introduction: The use of maintenance approaches with anti-CD20 monoclonal antibodies has improved the outcomes of B-cell indolent lymphomas but may lead to significant peripheral B-cell depletion. This depletion can potentially hinder the serological response to neoantigens. Methods: Our objective was to analyze the effect of anti-CD20 maintenance therapy in a reliable model of response to neoantigens: SARS-CoV-2 vaccine responses and the incidence/severity ofCOVID-19 in a reference hospital. Results: In our series (n=118), the rate of vaccination failures was 31%. Through ROC curve analysis, we determined a cutoff for SARS-CoV-2 vaccine serologic response at 24 months from the last anti-CD20 dose. The risk of severe COVID-19 was notably higher within the first 24months following the last anti-CD20 dose (52%) compared to after this period (just 18%) (p=0.007). In our survival analysis, neither vaccine response nor hypogammaglobulinemia significantly affected OS. While COVID-19 led to a modest mortality rate of 2.5%, this figure was comparable to the OS reported in the general immunocompetent population. However, most patients with hypogammaglobulinemia received intravenous immunoglobulin therapy and all were vaccinated. In conclusion, anti-CD20 maintenance therapy impairs serological responses to SARS-CoV-2 vaccines. Discussion: We report for the first time that patients during maintenance therapy and up to 24 months after the last anti-CD20 dose are at a higher risk of vaccine failure and more severe cases of COVID-19. Nevertheless, with close monitoring, intravenous immunoglobulin supplementation or proper vaccination, the impact on survival due to the lack of serological response in this high-risk population can be mitigated, allowing for the benefits of anti-CD20 maintenance therapy, even in the presence of hypogammaglobulinemia.

The impact of national culture in the development of complexity reasoning skills: An international comparison.

There is a clear inequality in gender distribution for the STEM areas (Science, Technology, Engineering, and Mathematics). Furthermore, there is a noticeable lack of diversity and a socio-economic gap that requires actionable solutions. To explore potential factors that affect the participation of women in STEM, this paper reviews two possible groups of determinants: national culture and complexity thinking. A survey with 684 respondents from higher education institutions in Chile, Ecuador, Mexico, and Spain was undertaken. The instrument measured four components of complexity thinking namely critical, scientific, innovative, and systemic). Using analysis of variance between two groups and between multiple groups, differences were observed between the countries' samples and between genders. Once the significance was confirmed, boxplots for each dimension were elaborated to facilitate the visualization of the distributions. The scores were compared with the national culture values to seek possible behavioral patterns in the data. The results reveal two groups between the observed countries. Also, there are clear indications of a relationship between the national culture dimensions and the complex thinking components.

Protocol for selecting single human pluripotent stem cells using a modified micropipetter.

Single-cell clonal selection is a critical procedure for generating a homogeneous population of human pluripotent stem cells. Here, we present a protocol that repurposes the STRIPPER Micropipetter, normally used for in vitro fertilization, to pick single stem cells. We describe steps for tool and reagent preparation, single-cell picking, and colony passaging. We then detail procedures for amplification and analysis. Our protocol does not require cell sorting and produces homogenous clonal cultures with more than 50% survival rate. For complete details on the use and execution of this protocol, please refer to Deng et al.1.

An effective method for the simultaneous extraction of 173 contaminants of emerging concern in freshwater invasive species and its application.

A robust and efficient extraction method was developed to detect a broad range of pollutants of emerging interest in three freshwater invasive species: American red crab (Prokambarus clarkii), Asian clam (Corbicula fluminea), and pumpkinseed fish (Lepomis gibbosus). One native species, "petxinot" clam (Anodonta cygnea), was also evaluated. Invasive species are often more resistant to contamination and could be used in biomonitoring studies to assess the effect of contaminants of emerging concern on aquatic ecosystems while preserving potentially threatened native species. So far, most extraction methods developed for this purpose have focused on analyzing fish and generally focus on a limited number of compounds, especially analyzing compounds from the same family. In this sense, we set out to optimize a method that would allow the simultaneous extraction of 87 PhACs, 11 flame retardants, 21 per- and poly-fluoroalkyl substances, and 54 pesticides. The optimized method is based on ultrasound-assisted solvent extraction. Two tests were performed during method development, one to choose the extraction solvent with the best recovery efficiencies and one to select the best clean-up. The analysis was performed by high-performance liquid chromatography coupled to high-resolution mass spectrometry. The method obtained recoveries between 40 and 120% and relative standard deviations of less than 25% for 85% of the analytes in the four validated matrices. Limits of quantification between 0.01 ng g-1 and 22 ng g-1 were obtained. Application of the method on real samples from the Albufera Natural Park of Valencia (Spain) confirmed the presence of contaminants of emerging concern in all samples, such as acetaminophen, hydrochlorothiazide, tramadol, PFOS, carbendazim, and fenthion. PFAS were the group of compounds with the highest mean concentrations. C. fluminea was the species with the highest detection frequency, and P. clarkii had the highest average concentrations, so its use is prioritized for biomonitoring studies.