Shigella promotes major alteration of gut epithelial physiology and tissue invasion by shutting off host intracellular transport.

Intracellular trafficking pathways in eukaryotic cells are essential to maintain organelle identity and structure, and to regulate cell communication with its environment. Shigella flexneri invades and subverts the human colonic epithelium by the injection of virulence factors through a type 3 secretion system (T3SS). In this work, we report the multiple effects of two S. flexneri effectors, IpaJ and VirA, which target small GTPases of the Arf and Rab families, consequently inhibiting several intracellular trafficking pathways. IpaJ and VirA induce large-scale impairment of host protein secretion and block the recycling of surface receptors. Moreover, these two effectors decrease clathrin-dependent and -independent endocytosis. Therefore, S. flexneri infection induces a global blockage of host cell intracellular transport, affecting the exchange between cells and their external environment. The combined action of these effectors disorganizes the epithelial cell polarity, disturbs epithelial barrier integrity, promotes multiple invasion events, and enhances the pathogen capacity to penetrate into the colonic tissue in vivo.

OTX2 Homeoprotein Functions in Adult Choroid Plexus.

The choroid plexus is an important blood barrier that secretes cerebrospinal fluid, which essential for embryonic brain development and adult brain homeostasis. The OTX2 homeoprotein is a transcription factor that is critical for choroid plexus development and remains highly expressed in adult choroid plexus. Through RNA sequencing analyses of constitutive and conditional knockdown adult mouse models, we reveal putative functional roles for OTX2 in adult choroid plexus function, including cell signaling and adhesion, and show that OTX2 regulates the expression of factors that are secreted into the cerebrospinal fluid, notably transthyretin. We also show that Otx2 expression impacts choroid plexus immune and stress responses, and affects splicing, leading to changes in the mRNA isoforms of proteins that are implicated in the oxidative stress response and DNA repair. Through mass spectrometry analysis of OTX2 protein partners in the choroid plexus, and in known non-cell-autonomous target regions, such as the visual cortex and subventricular zone, we identify putative targets that are involved in cell adhesion, chromatin structure, and RNA processing. Thus, OTX2 retains important roles for regulating choroid plexus function and brain homeostasis throughout life.

Nucleolin Discriminates Drastically between Long-Loop and Short-Loop Quadruplexes.

We investigate herein the interaction between nucleolin (NCL) and a set of G4 sequences derived from the CEB25 human minisatellite that adopt a parallel topology while differing in the length of the central loop (from nine nucleotides to one nucleotide). It is revealed that NCL strongly binds to long-loop (five to nine nucleotides) G4 while interacting weakly with the shorter variants (loop with fewer than three nucleotides). Photo-cross-linking experiments using 5-bromo-2'-deoxyuridine (BrU)-modified sequences further confirmed the loop-length dependency, thereby indicating that the WT-CEB25-L191 (nine-nucleotide loop) is the best G4 substrate. Quantitative proteomic analysis (LC-MS/MS) of the product(s) obtained by photo-cross-linking NCL to this sequence enabled the identification of one contact site corresponding to a 15-amino acid fragment located in helix α2 of RNA binding domain 2 (RBD2), which sheds light on the role of this structural element in G4-loop recognition. Then, the ability of a panel of benchmark G4 ligands to prevent the NCL-G4 interaction was explored. It was found that only the most potent ligand PhenDC3 can inhibit NCL binding, thereby suggesting that the terminal guanine quartet is also a strong determinant of G4 recognition, putatively through interaction with the RGG domain. This study describes the molecular mechanism by which NCL recognizes G4-containing long loops and leads to the proposal of a model implying a concerted action of RBD2 and RGG domains to achieve specific G4 recognition via a dual loop-quartet interaction.

An epigenetic silencing pathway controlling T helper 2 cell lineage commitment.

During immune responses, naive CD4+ T cells differentiate into several T helper (TH) cell subsets under the control of lineage-specifying genes. These subsets (TH1, TH2 and TH17 cells and regulatory T cells) secrete distinct cytokines and are involved in protection against different types of infection. Epigenetic mechanisms are involved in the regulation of these developmental programs, and correlations have been drawn between the levels of particular epigenetic marks and the activity or silencing of specifying genes during differentiation. Nevertheless, the functional relevance of the epigenetic pathways involved in TH cell subset differentiation and commitment is still unclear. Here we explore the role of the SUV39H1­H3K9me3­HP1α silencing pathway in the control of TH2 lineage stability. This pathway involves the histone methylase SUV39H1, which participates in the trimethylation of histone H3 on lysine 9 (H3K9me3), a modification that provides binding sites for heterochromatin protein 1α (HP1α) and promotes transcriptional silencing. This pathway was initially associated with heterochromatin formation and maintenance but can also contribute to the regulation of euchromatic genes. We now propose that the SUV39H1­H3K9me3­HP1α pathway participates in maintaining the silencing of TH1 loci, ensuring TH2 lineage stability. In TH2 cells that are deficient in SUV39H1, the ratio between trimethylated and acetylated H3K9 is impaired, and the binding of HP1α at the promoters of silenced TH1 genes is reduced. Despite showing normal differentiation, both SUV39H1-deficient TH2 cells and HP1α-deficient TH2 cells, in contrast to wild-type cells, expressed TH1 genes when recultured under conditions that drive differentiation into TH1 cells. In a mouse model of TH2-driven allergic asthma, the chemical inhibition or loss of SUV39H1 skewed T-cell responses towards TH1 responses and decreased the lung pathology. These results establish a link between the SUV39H1­H3K9me3­HP1α pathway and the stability of TH2 cells, and they identify potential targets for therapeutic intervention in TH2-cell-mediated inflammatory diseases.

Beneficial effects of omega-3 fatty acids on the consequences of a fructose diet are not mediated by PPAR delta or PGC1 alpha.

PURPOSE: To study, in high-fructose-fed rats, the effect of a dietary enrichment in omega-3 polyunsaturated fatty acids (n-3 PUFA) on the expression of genes involved in lipid metabolism and cardiovascular function. METHODS: Twenty-eight male "Wistar Han" rats received for 8 weeks, either a standard chow food or an isocaloric 67% fructose diet enriched or not in alpha-linolenic acid (ALA) or in docosahexaenoic (DHA) and eicosapentaenoic acids (EPA) mix (DHA/EPA). After sacrifice, blood was withdrawn for biochemical analyses; heart, periepididymal adipose tissue and liver were collected and analyzed for the expression of 22 genes by real-time PCR. RESULTS: Fructose intake resulted in an increase in liver weight and triglyceride content, plasma triglyceride and cholesterol concentrations, although no difference in glucose and insulin. In the liver, lipogenesis was promoted as illustrated by an increase in stearoyl-CoA desaturase and fatty acid synthase (Fasn) together with a decrease in PPAR gamma, delta and PPAR gamma coactivator 1 alpha (PGC1 alpha) expression. In the heart, Fasn and PPAR delta expression were increased. The addition of ALA or DHA/EPA into the diet resulted in a protection against fructose effects except for the decreased expression of PPARs in the liver that was not counterbalanced by n-3 PUFA suggesting that n-3 PUFA and fructose act independently on the expression of PPARs and PGC1 alpha. CONCLUSIONS: In liver, but not in heart, the fructose-enriched diet induces an early tissue-specific reduction in PPAR gamma and delta expression, which is insensitive to n-3 PUFA intake and dissociated from lipogenesis.

Vitamin D Toxicity from an Unusual and Unexpected Source: A Report of 2 Cases.

INTRODUCTION: Hypervitaminosis D is a relatively uncommon etiology of hypercalcemia. Toxicity is usually caused by very high doses, mostly secondary to erroneous prescription or administration of vitamin D, and less commonly, contaminated foods or manufacturing errors of vitamin D-containing supplements. CASE PRESENTATION: A 16-year-old male, previously healthy, presented with 2-week history of nonspecific symptoms (fatigue, gastrointestinal complaints). Investigations showed acute kidney injury and hypercalcemia (total calcium 3.81 mmol/L). Further diagnostic workup revealed markedly elevated 25-hydroxyvitamin D levels (1,910 nmol/L). He denied taking any vitamin D supplements; however, he reported consumption of creatine and protein supplements. Mass spectrometry analysis of the creatine supplement estimated a vitamin D content of 425,000 IU per serving (100 times the upper tolerable daily dose). A few months later, another previously healthy adolescent presented with severe hypercalcemia and acute kidney injury secondary to hypervitaminosis D. He was also using a creatine supplement, from the same manufacturer brand and lot. Both patients were treated with intravenous hydration, calcitonin, and pamidronate. They maintained normocalcemia after their initial presentation but required low-calcium diets and laboratory testing for months after this exposure. DISCUSSION/CONCLUSION: We present 2 cases of hypervitaminosis D caused by a manufacturing error of a natural health product which did not claim to contain vitamin D. The use of dietary supplements is highly prevalent; this should be incorporated while taking medical history, and considered a potential source of toxicity when an alternative source cannot be found, regardless of the product label.

Splicing Patterns in SF3B1-Mutated Uveal Melanoma Generate Shared Immunogenic Tumor-Specific Neoepitopes.

Disruption of splicing patterns due to mutations of genes coding splicing factors in tumors represents a potential source of tumor neoantigens, which would be both public (shared between patients) and tumor-specific (not expressed in normal tissues). In this study, we show that mutations of the splicing factor SF3B1 in uveal melanoma generate such immunogenic neoantigens. Memory CD8+ T cells specific for these neoantigens are preferentially found in 20% of patients with uveal melanoma bearing SF3B1-mutated tumors. Single-cell analyses of neoepitope-specific T cells from the blood identified large clonal T-cell expansions, with distinct effector transcription patterns. Some of these expanded T-cell receptors are also present in the corresponding tumors. CD8+ T-cell clones specific for the neoepitopes specifically recognize and kill SF3B1-mutated tumor cells, supporting the use of this new family of neoantigens as therapeutic targets. SIGNIFICANCE: Mutations of the splicing factor SF3B1 in uveal melanoma generate shared neoantigens that are uniquely expressed by tumor cells, leading to recognition and killing by specific CD8 T cells. Mutations in splicing factors can be sources of new therapeutic strategies applicable to diverse tumors.This article is highlighted in the In This Issue feature, p. 1861.

Exploring the evolving concept of 'patient ownership' in the era of resident duty hour regulations-experience of residents and faculty in an internal medicine night float system.

BACKGROUND: Despite the use of 'patient ownership' as an embodiment of professionalism and increasing concerns over its loss among trainees, how its development in residents has been affected by duty hour regulations has not been well described. In this qualitative study, we aim to outline the key features of patient ownership in internal medicine, factors enabling its development, and how these have been affected by the adoption of a night float system to comply with duty hour regulations. METHODS: In this qualitative descriptive study, we interviewed 18 residents and 12 faculty internists at one university centre and conducted a thematic analysis of the data focused on the concept of patient ownership. RESULTS: We identified three key features of patient ownership: personal concern for patients, professional capacity for autonomous decision-making, and knowledge of patients' issues. Within the context of a night float system, factors that facilitate development of patient ownership include improved fitness for duty and more consistent interactions with patients/families resulting from working the same shift over consecutive days (or nights). Conversely, the increase in patient handovers, if done poorly, is a potential threat to patient ownership development. Trainees often struggle to develop ownership when autonomy is not supported with supervision and when role-modelling by faculty is lacking. DISCUSSION: These features of patient ownership can be used to frame discussions when coaching trainees. Residency programs should be mindful of the downstream effects of shift-based scheduling. We propose strategies to optimize factors that enable trainee development of patient ownership.

Auranofin/Vitamin C: A Novel Drug Combination Targeting Triple-Negative Breast Cancer.

BACKGROUND: Cancer cells from different origins exhibit various basal redox statuses and thus respond differently to intrinsic or extrinsic oxidative stress. These intricate characteristics condition the success of redox-based anticancer therapies that capitalize on the ability of reactive oxygen species to achieve selective and efficient cancer cell killing. METHODS: Redox biology methods, stable isotope labeling by amino acids in cell culture (SILAC)-based proteomics, and bioinformatics pattern comparisons were used to decipher the underlying mechanisms for differential response of lung and breast cancer cell models to redox-modulating molecule auranofin (AUF) and to combinations of AUF and vitamin C (VC). The in vivo effect of AUF, VC, and two AUF/VC combinations on mice bearing MDA-MB-231 xenografts (n = 5 mice per group) was also evaluated. All statistical tests were two-sided. RESULTS: AUF targeted simultaneously the thioredoxin and glutathione antioxidant systems. AUF/VC combinations exerted a synergistic and hydrogen peroxide (H2O2)-mediated cytotoxicity toward MDA-MB-231 cells and other breast cancer cell lines. The anticancer potential of AUF/VC combinations was validated in vivo on MDA-MB-231 xenografts in mice without notable side effects. On day 14 of treatments, mean (SD) tumor volumes for the vehicle-treated control group and the two AUF/VC combination-treated groups (A/V1 and A/V2) were 197.67 (24.28) mm3, 15.66 (10.90) mm3, and 10.23 (7.30)mm3, respectively; adjusted P values of the differences between mean tumor volumes of vehicle vs A/V1 groups and vehicle vs A/V2 groups were both less than .001. SILAC proteomics, bioinformatics analysis, and functional experiments linked prostaglandin reductase 1 (PTGR1) expression levels with breast cancer cell sensitivity to AUF/VC combinations. CONCLUSION: The combination of AUF and VC, two commonly available drugs, could be efficient against triple-negative breast cancer and potentially other cancers with similar redox properties and PTGR1 expression levels. The redox-based anticancer activity of this combination and the discriminatory potential of PTGR1 expression are worth further assessment in preclinical and clinical studies.

Long-chain (n-3) polyunsaturated fatty acids prevent metabolic and vascular disorders in fructose-fed rats.

The crossover relationship between cardiometabolic risk, in terms of insulin resistance and vascular dysfunction, and the fatty acid (FA) profile of insulin-sensitive tissues as well as the dietary FA impact has almost never been explored in the same experiment. In this study, the intake of alpha-linolenic acid (ALA) alone and/or with its higher metabolites, eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA) were evaluated in a nonobese, hypertriglyceridemic and insulin-resistant rat model, that exhibits the 2 main characteristics of metabolic syndrome. Wistar rats were fed either a cornstarch and (n-6) PUFA-based diet (C-N6) or a 66% fructose diet over a 10-wk period. Fructose-fed rats received a diet containing ALA alone (F-ALA group) or ALA plus EPA and DHA (F-LC3 group) or no (n-3) PUFA (F-N6 group). The 10-wk high-fructose diet (F-N6) induced an insulin-resistant state, as assessed by glucose and insulin tolerance tests. Insulin resistance was linked to a specific FA pattern in insulin-sensitive tissues, which probably involved modifications of Delta9, Delta6, and Delta5-desaturases. This pathological status was related to high cardiovascular risk as assessed by increases in systolic and diastolic blood pressures and particularly by the increase of pulse pressure, an index of vascular stiffness obtained from telemetry investigations. The (n-3) experimental diets prevented changes in the FA patterns in insulin-sensitive tissues, insulin resistance, and vascular dysfunction. This beneficial effect was large with an intake of long chain (n-3) PUFA (ALA+EPA+DHA) and to a lesser extent with dietary ALA alone.

A single intravenous sTNFR-Fc administration at the time of reperfusion limits infarct size--implications in reperfusion strategies in man.

BACKGROUND: Reperfusion of the ischemic myocardium is associated with increased inflammatory processes that can exert deleterious effects and therefore contribute to cardiac dysfunction. The aim of the present study was to verify whether the administration of sTNFR-Fc, a scavenger of the pro-inflammatory cytokine TNF-alpha, at the time of reperfusion would protect against myocardial infarction and reduce the severity of early mechanical dysfunction. METHODS: Male Wistar rats were subjected to 60 min coronary occlusion followed by reperfusion. A bolus of sTNFR-Fc (10 microg/kg, i.v.) (MI + sTNFR-Fc group) or a placebo (MI group) was injected prior to reperfusion. Cardiac geometry was assessed by echocardiography 1, 3 and 7 days after reperfusion. Eight days after reperfusion, left ventricular (LV) function was evaluated under basal conditions and during an experimental challenge of volume overload. Finally, infarct size was measured after euthanasia. RESULTS: sTNFR-Fc administration markedly reduced infarct size (P < 0.01) and decreased LV dilation as assessed by the echocardiographic measurement of the LV end diastolic area, 7 days post-MI (P < 0.01). Moreover, LV end-diastolic pressure was significantly preserved by sTNFR-Fc 1 week after myocardial infarction, under basal conditions (P < 0.05) as well as during cardiac overload (P < 0.05). CONCLUSION: A single administration of sTNFR-Fc at the time of reperfusion after myocardial infarction is able to limit infarct size and to reduce early LV diastolic dysfunction in rats. These findings suggest that intravenous neutralization of TNF-alpha during surgical cardiac reperfusion might improve the outcome of myocardial infarction in humans.

Auranofin/Vitamin C: A Novel Drug Combination Targeting Triple-Negative Breast Cancer.

BACKGROUND: Cancer cells from different origins exhibit various basal redox statuses and thus respond differently to intrinsic or extrinsic oxidative stress. These intricate characteristics condition the success of redox-based anticancer therapies that capitalize on the ability of reactive oxygen species to achieve selective and efficient cancer cell killing. METHODS: Redox biology methods, stable isotope labeling by amino acids in cell culture (SILAC)-based proteomics, and bioinformatics pattern comparisons were used to decipher the underlying mechanisms for differential response of lung and breast cancer cell models to redox-modulating molecule auranofin (AUF) and to combinations of AUF and vitamin C (VC). The in vivo effect of AUF, VC, and two AUF/VC combinations on mice bearing MDA-MB-231 xenografts (n = 5 mice per group) was also evaluated. All statistical tests were two-sided. RESULTS: AUF targeted simultaneously the thioredoxin and glutathione antioxidant systems. AUF/VC combinations exerted a synergistic and hydrogen peroxide (H2O2)-mediated cytotoxicity toward MDA-MB-231 cells and other breast cancer cell lines. The anticancer potential of AUF/VC combinations was validated in vivo on MDA-MB-231 xenografts in mice without notable side effects. On day 14 of treatments, mean (SD) tumor volumes for the vehicle-treated control group and the two AUF/VC combination-treated groups (A/V1 and A/V2) were 197.67 (24.28) mm3, 15.66 (10.90) mm3, and 10.23 (7.30)mm3, respectively; adjusted P values of the differences between mean tumor volumes of vehicle vs A/V1 groups and vehicle vs A/V2 groups were both less than .001. SILAC proteomics, bioinformatics analysis, and functional experiments linked prostaglandin reductase 1 (PTGR1) expression levels with breast cancer cell sensitivity to AUF/VC combinations. CONCLUSION: The combination of AUF and VC, two commonly available drugs, could be efficient against triple-negative breast cancer and potentially other cancers with similar redox properties and PTGR1 expression levels. The redox-based anticancer activity of this combination and the discriminatory potential of PTGR1 expression are worth further assessment in preclinical and clinical studies.

DET1-mediated degradation of a SAGA-like deubiquitination module controls H2Bub homeostasis.

DE-ETIOLATED 1 (DET1) is an evolutionarily conserved component of the ubiquitination machinery that mediates the destabilization of key regulators of cell differentiation and proliferation in multicellular organisms. In this study, we provide evidence from Arabidopsis that DET1 is essential for the regulation of histone H2B monoubiquitination (H2Bub) over most genes by controlling the stability of a deubiquitination module (DUBm). In contrast with yeast and metazoan DUB modules that are associated with the large SAGA complex, the Arabidopsis DUBm only comprises three proteins (hereafter named SGF11, ENY2 and UBP22) and appears to act independently as a major H2Bub deubiquitinase activity. Our study further unveils that DET1-DDB1-Associated-1 (DDA1) protein interacts with SGF11 in vivo, linking the DET1 complex to light-dependent ubiquitin-mediated proteolytic degradation of the DUBm. Collectively, these findings uncover a signaling path controlling DUBm availability, potentially adjusting H2Bub turnover capacity to the cell transcriptional status.

Habilidades de pensamento crítico por estudantes e professores universitários da área da saúde / Critical thinking skills by university health students and professors / Habilidades de pensamiento crítico en estudiantes y profesores universitarios de salud

Objetivo:analisar o uso do pensamento crítico por estudantes e professores da área da saúde. Método: estudo transversal e quantitativo, realizado entre fevereiro e outubro de 2021, com participação de estudantes regularmente matriculados em cursos de graduação ou pós-graduação, de uma Universidade de Santa Catarina, e professoresda mesma instituição, vinculados a cursos da área da saúde. Para a análise do pensamento crítico foi utilizado o instrumento Questionário de Pensamento Crítico para Estudantes e Profissionais de Saúde. Para a análise, utilizou-se o teste de qui-quadrado, teste t de Studente ANOVA. Resultados: todos os participantes tiveram grau de competência de pensamento crítico médio ou elevado, com destaque para o domínio "interpretação", que foi mais elevado nos professores. Na competência "análise" foram observadas médias mais baixas para todos participantes. Conclusão: os participantes demonstraram boas habilidades de pensamento crítico, entretanto algumas habilidades se mostraram mais frágeis em alunos, devendo ser incentivadas.

Objective: to analyze the use of critical thinking by students and professors in the health area. Method: cross-sectional and quantitative study, carried out between February and October 2021, with the participation of students regularly enrolled in undergraduate or graduate courses at a University of Santa Catarina, and professors from the same institution, linked to courses at the University of Santa Catarina. Health area. For the analysis of critical thinking, the Critical Thinking Questionnaire for Students and Health Professionals was used. For the analysis, the chi-square test, Student's t test and ANOVA were used. Results:53 individuals participated, 43 (81.1%) undergraduate students, six (11.3%) graduate students and four (7.5%) professors. It was observed that all participants had a medium or high level of competence in critical thinking, with emphasison the Interpretation domain, which was higher among teachers. In the Analysis competency, lower averages were observed for all participants. Conclusion:the participants showed good critical thinking skills, however some skills were more fragile in students and should be encouraged

Objetivo: analizar el uso del pensamiento crítico por parte de estudiantes y profesores del área de la salud. Método: estudio transversal y cuantitativo, realizado entre febrero y octubre de 2021, con la participación de estudiantes matriculados regularmente en cursos de pregrado o posgrado, de una Universidad de Santa Catarina, y profesores de la misma institución, vinculados a cursos en la area de salud. Para el análisis del pensamiento crítico se utilizó el Cuestionario de Pensamiento Crítico para Estudiantes y Profesionales de la Salud. Para el análisis se utilizaron las pruebas de chi-cuadrado, t de Student y ANOVA. Resultados: participaron 53 personas, 43 (81,1%) estudiantes de pregrado, seis (11,3%) estudiantes de posgrado y cuatro (7,5%) profesores. Se observó que todos los participantes tenían un nivel medio o alto de competencia en pensamiento crítico, con énfasis en el dominio Interpretación, que fue mayor entre los docentes. En la competencia Análisis, se observaron promedios más bajos para todos los participantes. Conclusión: los participantes mostraron buenas habilidades de pensamiento crítico, sin embargo, algunas habilidades eran más frágiles enlos estudiantes y deberían fomentarse.

Exploring the mechanism of inhibition of human telomerase by cysteine-reactive compounds.

Telomerase is an almost universal cancer target that consists minimally of a core protein human telomerase reverse transcriptase (hTERT) and a RNA component human telomerase RNA (hTR). Some inhibitors of this enzyme are thought to function by the covalent binding to one or several cysteine residues; however, this inhibition mechanism has never been investigated because of the difficulty in producing telomerase. In this study, we use a recent method to produce recombinant hTERT to analyze the effect of cysteine-reactive inhibitors on telomerase. Using mass spectrometry and mutagenesis analysis, we identify several targeted residues in separated domains of the hTERT protein and show that cysteine-reactive reagents abolish the interaction with the CR4/5 region of hTR.