Dynamics of transcription is affected by oxygen tension and developmental speed during in vitro production of bovine embryos.

This study examines the impact of oxygen tension and embryo kinetics on gene transcription dynamics in pathways crucial for embryonic preimplantation development, including lipid metabolism, carbohydrate transport and metabolism, mitochondrial function, stress response, apoptosis and transcription regulation. Bovine embryos were generated in vitro and allocated into two groups based on oxygen tension (20% or 5%) at 18 h post insemination (hpi). At 40 hpi, embryos were categorized into Fast (≥4 cells) or Slow (2 cells) groups, resulting in four experimental groups: FCL20, FCL5, SCL20 and SCL5. Embryo collection also occurred at 72 hpi (16-cell stage; groups FMO20, FMO5, SMO20 and SMO5) and at 168 hpi (expanded blastocyst (BL) stage; groups FBL20, FBL5, SBL20 and SBL5). Pools of three embryos per group were analysed in four replicates using inventoried TaqMan assays specific for Bos taurus, targeting 93 genes. Gene expression patterns were analysed using the K-means algorithm, revealing three main clusters: genes with low relative abundance at the cleavage (CL) and 16-cell morula (MO) stages but increased at the BL stage (cluster 1); genes with higher abundances at CL but decreasing at MO and BL (cluster 2); and genes with low levels at CL, higher levels at MO and decreased levels at BL (cluster 3). Within each cluster, genes related to epigenetic mechanisms, cell differentiation events and glucose metabolism were particularly influenced by differences in developmental kinetics and oxygen tension. Fast-developing embryos, particularly those cultured under low oxygen tension, exhibited transcript dynamics more closely resembling that reported in vivo-produced embryos.

Biochar-nanoparticle combinations enhance the biogeochemical recovery of a post-mining soil.

Here we addressed the capacity of distinct amendments to reduce arsenic (As), copper (Cu), selenium (Se) and zinc (Zn) associated risks and improve the biogeochemical functions of post-mining soil. To this, we examined nanoparticles (NPs) and/or biochar effects, combined with phytostabilization using Lolium perenne L. Soil samples were taken in a former metal mine surroundings. Ryegrass seeds were sown in pots containing different combinations of NPs (zero-valent iron (nZVI) or hydroxyapatite (nH)) (0 and 2 %), and biochar (0, 3 and 5 %). Plants were grown for 45 days and the plant yield and element accumulation were evaluated, also soil properties (element distribution within the soil fractions, fertility, and enzymatic activities associated with microbiota functionality and nutrient cycling) were determined. Results showed biochar-treated soil had a higher pH, and much higher organic carbon (C) content than control soil and NP-treated soils, and it revealed increased labile C, total N, and available P concentrations. Soil treatment with NP-biochar combinations increased exchangeable non-acid cation concentrations and reduced exchangeable Na%, improved soil fertility, reduced sodicity risk, and increased ryegrass biomass. Enzymatic activities, particularly dehydrogenase and glucosidase, increased upon the addition of biochar, and this effect was fostered by NPs. Most treatments led to a significant reduction of metal(loid)s contents in biomass, mitigating contamination risks. The two different NPs had similar effects in many parameters, nH outperformed nZVI in terms of increased nutrients, C content, and enzymatic activities. On the basis of our results, combined biochar-NP amendments use, specially nH, emerges as a potential post-mining soil restoration strategy.

Association of the IFNG +874T/A Polymorphism with Symptomatic COVID-19 Susceptibility.

Tumor necrosis factor (TNF) and interferon-gamma (IFNγ) are important inflammatory mediators in the development of cytokine storm syndrome (CSS). Single nucleotide polymorphisms (SNPs) regulate the expression of these cytokines, making host genetics a key factor in the prognosis of COVID-19. In this study, we investigated the associations of the TNF -308G/A and IFNG +874T/A polymorphisms with COVID-19. We analyzed the frequencies of the two polymorphisms in the control groups (CG: TNF -308G/A, n = 497; IFNG +874T/A, n = 397), a group of patients with COVID-19 (CoV, n = 222) and among the subgroups of patients with nonsevere (n = 150) and severe (n = 72) COVID-19. We found no significant difference between the genotypic and allelic frequencies of TNF -308G/A in the groups analyzed; however, both the frequencies of the high expression genotype (TT) (CoV: 13.51% vs. CG: 6.30%; p = 0.003) and the *T allele (CoV: 33.56% vs. CG: 24. 81%; p = 0.001) of the IFNG +874T/A polymorphism were higher in the COVID-19 group than in the control group, with no differences between the subgroups of patients with nonsevere and severe COVID-19. The *T allele of IFNG +874T/A (rs2430561) is associated with susceptibility to symptomatic COVID-19. These SNPs provided valuables clues about the potential mechanism involved in the susceptibility to developing symptomatic COVID-19.

High-risk clones of carbapenem resistant Klebsiella pneumoniae recovered from pediatric patients in Southern Brazil.

Carbapenem-resistant Klebsiella pneumoniae (CRKP) exhibit high mortality rates in pediatric patients and usually belong to international high-risk clones. This study aimed to investigate the molecular epidemiology and carbapenem resistance mechanisms of K. pneumoniae isolates recovered from pediatric patients, and correlate them with phenotypical data. Twenty-five CRKP isolates were identified, and antimicrobial susceptibility was assessed using broth microdilution. Carbapenemase production and ß-lactamase genes were detected by phenotypic and genotypic tests. Multilocus sequence typing was performed to differentiate the strains and whole-genome sequencing was assessed to characterize a new sequence type. Admission to the intensive care unit and the use of catheters were significantly positive correlates of CRKP infection, and the mortality rate was 36%. Almost all isolates showed multidrug-resistant phenotype, and most frequent resistant gene was blaKPC. We observed the dissemination of ST307 and clones belonging to CG258, which are considered high risk. In pediatric patients, these clones present with high genomic plasticity, favoring adaptation of the KPC and NDM enzymes to healthcare environments.

Severe COVID-19 and long COVID are associated with high expression of STING, cGAS and IFN-α.

The cGAS-STING pathway appears to contribute to dysregulated inflammation during coronavirus disease 2019 (COVID-19); however, inflammatory factors related to long COVID are still being investigated. In the present study, we evaluated the association of cGAS and STING gene expression levels and plasma IFN-α, TNF-α and IL-6 levels with COVID-19 severity in acute infection and long COVID, based on analysis of blood samples from 148 individuals, 87 with acute COVID-19 and 61 in the post-COVID-19 period. Quantification of gene expression was performed by real-time PCR, and cytokine levels were quantified by ELISA and flow cytometry. In acute COVID-19, cGAS, STING, IFN-α, TNF-α, and IL-6 levels were higher in patients with severe disease than in those with nonsevere manifestations (p < 0.05). Long COVID was associated with elevated cGAS, STING and IFN-α levels (p < 0.05). Activation of the cGAS-STING pathway may contribute to an intense systemic inflammatory state in severe COVID-19 and, after infection resolution, induce an autoinflammatory disease in some tissues, resulting in long COVID.

What have adolescents in Rio de Janeiro city, Brazil, done to reduce their community violence exposure? / O que os adolescentes do município do Rio de Janeiro, Brasil, têm feito para reduzir sua exposição à violência comunitária?

This study aims to identify the individual community strategies to avoid violence exposure most used by adolescents from public and private schools in the IX Administrative Region of Rio de Janeiro and investigate the profile of co-occurrence and its prevalence in specific population subgroups. This is a cross-sectional study with 693 individuals. A multidimensional questionnaire collected information regarding strategies to avoid community violence exposure and was self-completed in the classroom. The most used strategies were avoiding walking close to armed people (55.5%), avoiding walking alone (30.5%), and avoiding returning home at dawn (24.7%). Girls adopt more of all (concurrently) the four limiting behaviors to reduce their community violence exposure (53% vs. 32%). Notably, the adoption of such strategies differed by socioeconomic indicators and was higher among adolescents from lower-income households. These findings point to the high frequency of use of such strategies by adolescents, which may hinder and limit the full development of their social and cultural skills.

O objetivo do estudo é conhecer as estratégias individuais mais utilizadas por adolescentes de escolas públicas e privadas da IX Região Administrativa do município do Rio de Janeiro para evitar a exposição à violência comunitária, bem como investigar o perfil de coocorrência e sua prevalência em subgrupos populacionais específicos. Trata-se de um estudo seccional com 693 indivíduos. As informações referentes às estratégias para evitar a exposição à violência comunitária foram coletadas por meio de questionário multidimensional autopreenchido em sala de aula. As estratégias mais utilizadas foram: evitar passar onde há pessoas armadas (55,5%), evitar andar sozinho (30,5%) e evitar voltar para casa de madrugada (24,7%). Observou-se que as meninas adotam mais todos (concomitantemente) os quatro tipos de comportamento limitantes para reduzir sua exposição à violência comunitária (53% vs. 32%). Ressalta-se que a adoção de tais estratégias diferiu segundo os indicadores socioeconômicos, sendo maior entre os adolescentes oriundos de família de estratos de renda mais baixos. Tais achados chamam a atenção para a alta frequência de utilização de tais estratégias por adolescentes, o que pode cercear e limitar o pleno desenvolvimento de suas habilidades sociais e culturais.

The central role of pyruvate metabolism on the epigenetic maturation and transcriptional profile of bovine oocytes.

In brief: Pyruvate metabolism is one of the main metabolic pathways during oocyte maturation. This study demonstrates that pyruvate metabolism also regulates the epigenetic and molecular maturation in bovine oocytes. Abstract: Pyruvate, the final product of glycolysis, undergoes conversion into acetyl-CoA within the mitochondria of oocytes, serving as a primary fuel source for the tricarboxylic acid (TCA) cycle. The citrate generated in the TCA cycle can be transported to the cytoplasm and converted back into acetyl-CoA. This acetyl-CoA can either fuel lipid synthesis or act as a substrate for histone acetylation. This study aimed to investigate how pyruvate metabolism influences lysine 9 histone 3 acetylation (H3K9ac) dynamics and RNA transcription in bovine oocytes during in vitro maturation (IVM). Bovine cumulus-oocyte complexes were cultured in vitro for 24 h, considering three experimental groups: Control (IVM medium only), DCA (IVM supplemented with sodium dichloroacetate, a stimulant of pyruvate oxidation into acetyl-CoA), or IA (IVM supplemented with sodium iodoacetate, a glycolysis inhibitor). The results revealed significant alterations in oocyte metabolism in both treatments, promoting the utilization of lipids as an energy source. These changes during IVM affected the dynamics of H3K9ac, subsequently influencing the oocyte's transcriptional activity. In the DCA and IA groups, a total of 148 and 356 differentially expressed genes were identified, respectively, compared to the control group. These findings suggest that modifications in pyruvate metabolism trigger the activation of metabolic pathways, particularly lipid metabolism, changing acetyl-CoA availability and H3K9ac levels, ultimately impacting the mRNA content of in vitro matured bovine oocytes.

Metabolic stressful environment drives epigenetic modifications in oviduct epithelial cells.

The oviduct provides a suitable microenvironment from the gametes' final maturation until initial embryo development. Dynamic functional changes are observed in the oviduct cells, mainly controlled by steroid hormones and well-orchestrated during the estrous cycle. However, based on the roles played by the oviduct, additional layers of complexity might be present in its regulatory process. There is a cellular process that includes metabolic adaptation that can guide molecular modifications. This process is known as metaboloepigenetics. Therefore, we aimed to better understand how this crosstalk occurs in oviductal epithelial cells (OEC). Due to limited in situ access to the oviduct, we used the primary in vitro cell culture as a culture model and glucose as a metabolic disturbed factor. For that, cells derived from the oviductal epithelial layer were collected from cows at either follicular or luteal stages (n = 4 animals per group). They were cultured on a monolayer culture system under normoglycemic (2.7 mM glucose) or hyperglycemic conditions (27 mM glucose). On day five of culture, attached cells were submitted to analysis of mitochondrial metabolism (mitochondrial membrane potential - MMP) and epigenetics markers (5- methylcytosine - 5 mC and histone 3 lysine 9 acetylation - H3K9ac). Moreover, the culture media were submitted to the metabolites analysis profile by Raman spectrometry. Data were analyzed considering the effect of glucose level (normoglycemic vs. hyperglycemic), stages when OEC were harvested (follicular vs. luteal), and their interaction (glucose level * cycle stage) by two-way ANOVA. As a result, the high glucose level decreased the H3K9ac and MMP levels but did not affect the 5 mC. Regardless of the metabolic profile of the culture media, the glucose level was the only factor that changed the Raman shifts abundance. Although this present study evaluated oviductal epithelial cells after being submitted to an in vitro monolayer culture system, which is known to lead to cell dedifferentiation, yet, these results provide evidence of a relationship between epigenetic reprogramming and energy metabolism under these cell culture conditions. In conclusion, the levels of metabolites in culture media may be crucial for cellular function and differentiation, meaning that it should be considered in studies culturing oviductal cells.

Outbreak of vancomycin-resistant Enterococcus faecium ST1133 in paediatric patients with acute lymphoblastic leukaemia from southern Brazil.

OBJECTIVES: We aimed to investigate an outbreak of vancomycin-resistant Enterococcus faecium (VREfm) in paediatric patients from Hospital Pequeno Príncipe. The susceptibility profile was determined, and whole-genome sequencing (WGS) was used to analyse the genetic context of the strains. METHODS: Five VREfm isolates were recovered from sterile sites and surveillance cultures of two paediatric patients with acute lymphoblastic leukaemia. Species identification was performed using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS), and the minimum inhibitory concentration (MIC) was assessed according to the European Committee for Antimicrobial Susceptibility Testing (EUCAST). WGS was performed to analyse the genetic context of virulence and resistance genes, and in silico multilocus sequence typing was performed to identify the sequence typing of the strains. RESULTS: High-level vancomycin resistance was observed in all isolates (≥256 mg/L). WGS revealed the presence of mobile genetic elements, such as plasmids (rep2, rep11a, repUS15, rep17, and rep18a), insertion sequences, and phages. Multiple resistance genes (aac(6')-aph(2"), dfrG, ermB, and vanA) and virulence genes (acm and efaAfm) were identified. All the isolates were assigned to ST117 (ST1133 - via a novel MLST), an important epidemic lineage associated with nosocomial infections and outbreaks. CONCLUSION: Our results show that the ST117 (ST1133) VREfm isolates are circulating in paediatric patients, which raises a great concern. The development of new drugs as well as the implementation of an antimicrobial stewardship program are necessary for their correct management, limiting the spread of resistance in oncohematological patients.

O que os adolescentes do município do Rio de Janeiro, Brasil, têm feito para reduzir sua exposição à violência comunitária? / What have adolescents in Rio de Janeiro city, Brazil, done to reduce their community violence exposure?

Resumo O objetivo do estudo é conhecer as estratégias individuais mais utilizadas por adolescentes de escolas públicas e privadas da IX Região Administrativa do município do Rio de Janeiro para evitar a exposição à violência comunitária, bem como investigar o perfil de coocorrência e sua prevalência em subgrupos populacionais específicos. Trata-se de um estudo seccional com 693 indivíduos. As informações referentes às estratégias para evitar a exposição à violência comunitária foram coletadas por meio de questionário multidimensional autopreenchido em sala de aula. As estratégias mais utilizadas foram: evitar passar onde há pessoas armadas (55,5%), evitar andar sozinho (30,5%) e evitar voltar para casa de madrugada (24,7%). Observou-se que as meninas adotam mais todos (concomitantemente) os quatro tipos de comportamento limitantes para reduzir sua exposição à violência comunitária (53% vs. 32%). Ressalta-se que a adoção de tais estratégias diferiu segundo os indicadores socioeconômicos, sendo maior entre os adolescentes oriundos de família de estratos de renda mais baixos. Tais achados chamam a atenção para a alta frequência de utilização de tais estratégias por adolescentes, o que pode cercear e limitar o pleno desenvolvimento de suas habilidades sociais e culturais.

Abstract This study aims to identify the individual community strategies to avoid violence exposure most used by adolescents from public and private schools in the IX Administrative Region of Rio de Janeiro and investigate the profile of co-occurrence and its prevalence in specific population subgroups. This is a cross-sectional study with 693 individuals. A multidimensional questionnaire collected information regarding strategies to avoid community violence exposure and was self-completed in the classroom. The most used strategies were avoiding walking close to armed people (55.5%), avoiding walking alone (30.5%), and avoiding returning home at dawn (24.7%). Girls adopt more of all (concurrently) the four limiting behaviors to reduce their community violence exposure (53% vs. 32%). Notably, the adoption of such strategies differed by socioeconomic indicators and was higher among adolescents from lower-income households. These findings point to the high frequency of use of such strategies by adolescents, which may hinder and limit the full development of their social and cultural skills.

Adaptative response to changes in pyruvate metabolism on the epigenetic landscapes and transcriptomics of bovine embryos.

The epigenetic reprogramming that occurs during the earliest stages of embryonic development has been described as crucial for the initial events of cell specification and differentiation. Recently, the metabolic status of the embryo has gained attention as one of the main factors coordinating epigenetic events. In this work, we investigate the link between pyruvate metabolism and epigenetic regulation by culturing bovine embryos from day 5 in the presence of dichloroacetate (DCA), a pyruvate analog that increases the pyruvate to acetyl-CoA conversion, and iodoacetate (IA), which inhibits the glyceraldehyde-3-phosphate dehydrogenase (GAPDH), leading to glycolysis inhibition. After 8 h of incubation, both DCA and IA-derived embryos presented higher mitochondrial membrane potential. Nevertheless, in both cases, lower levels of acetyl-CoA, ATP-citrate lyase and mitochondrial membrane potential were found in blastocysts, suggesting an adaptative metabolic response, especially in the DCA group. The metabolic alteration found in blastocysts led to changes in the global pattern of H3K9 and H3K27 acetylation and H3K27 trimethylation. Transcriptome analysis revealed that such alterations resulted in molecular differences mainly associated to metabolic processes, establishment of epigenetic marks, control of gene expression and cell cycle. The latter was further confirmed by the alteration of total cell number and cell differentiation in both groups when compared to the control. These results corroborate previous evidence of the relationship between the energy metabolism and the epigenetic reprogramming in preimplantation bovine embryos, reinforcing that the culture system is decisive for precise epigenetic reprogramming, with consequences for the molecular control and differentiation of cells.

Association of Polymorphisms of IL-6 Pathway Genes (IL6, IL6R and IL6ST) with COVID-19 Severity in an Amazonian Population.

Interleukin-6 has been recognized as a major role player in COVID-19 severity, being an important regulator of the cytokine storm. Hence, the evaluation of the influence of polymorphisms in key genes of the IL-6 pathway, namely IL6, IL6R, and IL6ST, may provide valuable prognostic/predictive markers for COVID-19. The present cross-sectional study genotyped three SNPs (rs1800795, rs2228145, and rs7730934) at IL6. IL6R and IL6ST genes, respectively, in 227 COVID-19 patients (132 hospitalized and 95 non-hospitalized). Genotype frequencies were compared between these groups. As a control group, published data on gene and genotype frequencies were gathered from published studies before the pandemic started. Our major results point to an association of the IL6 C allele with COVID-19 severity. Moreover, IL-6 plasmatic levels were higher among IL6 CC genotype carriers. Additionally, the frequency of symptoms was higher at IL6 CC and IL6R CC genotypes. In conclusion, the data suggest an important role of IL6 C allele and IL6R CC genotype on COVID-19 severity, in agreement with indirect evidence from the literature about the association of these genotypes with mortality rates, pneumonia, and heightening of protein plasmatic levels pro-inflammatory driven effects.

Polymorphisms in the MBL2 gene are associated with the plasma levels of MBL and the cytokines IL-6 and TNF-α in severe COVID-19.

Introduction: Mannose-binding lectin (MBL) promotes opsonization, favoring phagocytosis and activation of the complement system in response to different microorganisms, and may influence the synthesis of inflammatory cytokines. This study investigated the association of MBL2 gene polymorphisms with the plasma levels of MBL and inflammatory cytokines in COVID-19. Methods: Blood samples from 385 individuals (208 with acute COVID-19 and 117 post-COVID-19) were subjected to real-time PCR genotyping. Plasma measurements of MBL and cytokines were performed by enzyme-linked immunosorbent assay and flow cytometry, respectively. Results: The frequencies of the polymorphic MBL2 genotype (OO) and allele (O) were higher in patients with severe COVID-19 (p< 0.05). The polymorphic genotypes (AO and OO) were associated with lower MBL levels (p< 0.05). IL-6 and TNF-α were higher in patients with low MBL and severe COVID-19 (p< 0.05). No association of polymorphisms, MBL levels, or cytokine levels with long COVID was observed. Discussion: The results suggest that, besides MBL2 polymorphisms promoting a reduction in MBL levels and therefore in its function, they may also contribute to the development of a more intense inflammatory process responsible for the severity of COVID-19.

DOHaD: A Menagerie of Adaptations and Perspectives: The interplay between early embryo metabolism and mitoepigenetic programming of development.

In brief: This review discusses advances in the knowledge of epigenetic mechanisms regulating mitochondrial DNA and the relationship with reproductive biology. Abstract: Initially perceived simply as an ATP producer, mitochondria also participate in a wide range of other cellular functions. Mitochondrial communication with the nucleus, as well as signaling to other cellular compartments, is critical to cell homeostasis. Therefore, during early mammalian development, mitochondrial function is reported as a key element for survival. Any mitochondrial dysfunction may reflect in poor oocyte quality and may impair embryo development with possible long-lasting consequences to cell functions and the overall embryo phenotype. Growing evidence suggests that the availability of metabolic modulators can alter the landscape of epigenetic modifications in the nuclear genome providing an important layer for the regulation of nuclear-encoded gene expression. However, whether mitochondria could also be subjected to such similar epigenetic alterations and the mechanisms involved remain largely obscure and controversial. Mitochondrial epigenetics, also known as 'mitoepigenetics' is an intriguing regulatory mechanism in mitochondrial DNA (mtDNA)-encoded gene expression. In this review, we summarized the recent advances in mitoepigenetics, with a special focus on mtDNA methylation in reproductive biology and preimplantation development. A better comprehension of the regulatory role of mitoepigenetics will help the understanding of mitochondrial dysfunction and provide novel strategies for in vitro production systems and assisted reproduction technologies, as well as prevent metabolic related stress and diseases.

New pathogenic variant in DLX5: New clues for a clinical spectrum from split-hand-foot malformation to fibular aplasia, tibial campomelia and oligosyndactyly.

Introduction: FATCO (Fibular Aplasia, Tibial Campomelia and Oligosyndactyly) is a very infrequent skeletal dysplasia classified within the limb hypoplasia-reduction defects group whose genetic cause has not yet been identified. The advent of next-generation sequencing is enabling the diagnosis of diseases with no previously known genetic cause. Methods: We performed a thorough autopsy on a fetus whose pregnancy was legally terminated due to severe malformations detected by ultrasound. A trio exome was run to identify the genetic cause and risk of recurrence. Previous literature of similar cases was systematically searched. Results: Anatomopathological analyses revealed complete fibular aplasia, shortened and campomelic tibia, absent ankle joint, club right foot and a split foot malformation, leading to the diagnosis of FATCO. Exome sequencing showed that the female fetus carried a de novo nonsense variant in DLX5. The literature search permitted the collection of information on 43 patients with FATCO, the majority of whom were males diagnosed postnatally. In most cases, lower limbs were affected exclusively, but in 39.5% of cases the upper limbs were also affected. Conclusion: The pathologies associated with DLX5 variants encompass a wide spectrum of manifestations ranging from abnormalities exclusively in the hands and feet to long bones such as the tibia and fibula.

Thrombophilia and Immune-Related Genetic Markers in Long COVID.

Aiming to evaluate the role of ten functional polymorphisms in long COVID, involved in major inflammatory, immune response and thrombophilia pathways, a cross-sectional sample composed of 199 long COVID (LC) patients and a cohort composed of 79 COVID-19 patients whose follow-up by over six months did not reveal any evidence of long COVID (NLC) were investigated to detect genetic susceptibility to long COVID. Ten functional polymorphisms located in thrombophilia-related and immune response genes were genotyped by real time PCR. In terms of clinical outcomes, LC patients presented higher prevalence of heart disease as preexistent comorbidity. In general, the proportions of symptoms in acute phase of the disease were higher among LC patients. The genotype AA of the interferon gamma (IFNG) gene was observed in higher frequency among LC patients (60%; p = 0.033). Moreover, the genotype CC of the methylenetetrahydrofolate reductase (MTHFR) gene was also more frequent among LC patients (49%; p = 0.045). Additionally, the frequencies of LC symptoms were higher among carriers of IFNG genotypes AA than among non-AA genotypes (Z = 5.08; p < 0.0001). Two polymorphisms were associated with LC in both inflammatory and thrombophilia pathways, thus reinforcing their role in LC. The higher frequencies of acute phase symptoms among LC and higher frequency of underlying comorbidities might suggest that acute disease severity and the triggering of preexisting condition may play a role in LC development.

Antibody Response to the SARS-CoV-2 Spike and Nucleocapsid Proteins in Patients with Different COVID-19 Clinical Profiles.

The first case of coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), in Brazil was diagnosed on February 26, 2020. Due to the important epidemiological impact of COVID-19, the present study aimed to analyze the specificity of IgG antibody responses to the S1, S2 and N proteins of SARS-CoV-2 in different COVID-19 clinical profiles. This study enrolled 136 individuals who were diagnosed with or without COVID-19 based on clinical findings and laboratory results and classified as asymptomatic or as having mild, moderate or severe disease. Data collection was performed through a semistructured questionnaire to obtain demographic information and main clinical manifestations. IgG antibody responses to the S1 and S2 subunits of the spike (S) protein and the nucleocapsid (N) protein were evaluated using an enzyme-linked immunosorbent assay (ELISA) according to the manufacturer's instructions. The results showed that among the participants, 87.5% (119/136) exhibited IgG responses to the S1 subunit and 88.25% (120/136) to N. Conversely, only 14.44% of the subjects (21/136) displayed S2 subunit responses. When analyzing the IgG antibody response while considering the different proteins of the virus, patients with severe disease had significantly higher antibody responses to N and S1 than asymptomatic individuals (p ≤ 0.0001), whereas most of the participants had low antibody titers against the S2 subunit. In addition, individuals with long COVID-19 showed a greater IgG response profile than those with symptomatology of a short duration. Based on the results of this study, it is concluded that levels of IgG antibodies may be related to the clinical evolution of COVID-19, with high levels of IgG antibodies against S1 and N in severe cases and in individuals with long COVID-19.

Insulin-like growth factor-1 (IGF-1) selectively modulates the metabolic and lipid profile of bovine embryos according to their kinetics of development.

Supplementation of culture media with IGF-1 during in vitro culture of embryos has had controversial results over the years. In the present study, we show that differences previously observed in response to IGF addition might be related to intrinsic heterogeneity of the embryos. In other words, the effects exerted by IGF-1 are dependent on the characteristics of the embryos and their ability to modulate metabolism and overcome stressful conditions, such as the ones found in a non-optimized in vitro culture system. To test this hypothesis, in vitro produced bovine embryos with distinct morphokinetics (fast- and slow-cleavage) were submitted to treatment with IGF-1 and then evaluated for embryo production rates, total cell number, gene expression and lipid profile. Our results show that remarkable differences were found when fast and slow embryos treated with IGF-1 were compared. Fast embryos respond by upregulating genes related to mitochondrial function, stress response, and lipid metabolism, whereas slow embryos presented lower mitochondrial efficiency and lipid accumulation. We conclude that indeed the treatment with IGF-1 selectively affects embryonic metabolism according to early morphokinetics phenotypes, and this information is relevant for decision-making in the design of more appropriate in vitro culture systems.

Lipid availability during in vitro maturation alters oocyte lipid content and blastocyst development and metabolism.

Lipids play a crucial role in various biological functions, including membrane composition, energy storage, cell signalling, and metabolic and epigenetic processes. Abnormal lipid accumulation and metabolism during in vitro maturation (IVM) of oocytes have been linked to the use of fetal bovine serum (FBS), even though it provides several beneficial molecules, contributing to the oocyte competence. Delipidating agents have been used to mitigate these deleterious effects, but they can have adverse effects on embryonic development. In this study, we explored how lipids present in fetal bovine serum (FBS) can impact the composition of oocytes and their resulting blastocysts in vitro. For that, we used organic solvents to separate the polar and nonpolar (lipid enriched) phase of FBS. Oocytes were in vitro matured in the presence of 10% whole FBS (control), 10% FBS plus 10% nonpolar lipids (lipid enriched - OL) or 10% polar lipids only (partially delipidated - ODL). After 24 h, part of the matured oocytes was collected and those remaining in each group underwent in vitro fertilization (IVF) and culture (IVC) under the same conditions and expanded blastocysts were collected at day 7 (control, BL and BDL). Oocytes and embryos were analysed by Multiple Reaction Monitoring mass spectrometry (MRM-MS) to determine their lipid composition. Interestingly, principal component analysis (PCA) revealed a clear distinction in the lipid profile of oocytes and blastocysts from both treatments compared to the control group. Control oocytes and blastocysts had higher triacylglycerol and cholesterol ester enrichment while the OL, ODL, BL and BDL groups had higher amounts of free fatty acids (FFAs). The structural and signalling phospholipids also differed among groups. Our findings suggest that the lipid-enriched fraction of FBS can be manipulated for IVM to ensure proper maturation, resulting in oocytes and blastocysts with less accumulated intracellular lipids and an improved metabolic status.

Laboratory diagnosis of Clostridioides difficile infection in symptomatic patients: what can we do better?

The laboratory diagnosis of Clostridioides difficile infection (CDI) is challenging since this bacteria may be detected in healthy people and toxin production detection is not sensitive enough to be used alone. Thus, there is no single test with adequate sensitivity and specificity to be used in laboratory diagnosis. We evaluated the performance of tests used in the diagnosis of CDI in symptomatic patients with risk factors in hospitals in southern Brazil. Enzyme immunoassays (EIA) for glutamate dehydrogenase antigen (GDH) and toxins A/B, real-time polymerase chain reaction (qPCR), GeneXpert system, and a two-step algorithm comprising GDH/TOXIN EIA performed simultaneously followed by GeneXpert for outliers were evaluated. Toxigenic strain in stool culture was considered CDI positive (gold standard). Among 400 samples tested, 54 (13.5%) were positive for CDI and 346 (86.5%) were negative. The diagnosis of the two-step algorithm and qPCR had an excellent performance with an accuracy of 94.5% and 94.2%, respectively. The Youden index showed that GeneXpert as a single test (83.5%) and the two-step algorithm (82.8%) were the most effective assays. Diagnosing CDI and non-CDI diarrhea could be successfully attained by the combination of clinical data with accuracy of laboratory tests.