Metformin acts in the gut and induces gut-liver crosstalk.

Metformin is the most prescribed drug for DM2, but its site and mechanism of action are still not well established. Here, we investigated the effects of metformin on basolateral intestinal glucose uptake (BIGU), and its consequences on hepatic glucose production (HGP). In diabetic patients and mice, the primary site of metformin action was the gut, increasing BIGU, evaluated through PET-CT. In mice and CaCo2 cells, this increase in BIGU resulted from an increase in GLUT1 and GLUT2, secondary to ATF4 and AMPK. In hyperglycemia, metformin increased the lactate (reducing pH and bicarbonate in portal vein) and acetate production in the gut, modulating liver pyruvate carboxylase, MPC1/2, and FBP1, establishing a gut-liver crosstalk that reduces HGP. In normoglycemia, metformin-induced increases in BIGU is accompanied by hypoglycemia in the portal vein, generating a counter-regulatory mechanism that avoids reductions or even increases HGP. In summary, metformin increases BIGU and through gut-liver crosstalk influences HGP.

Structural dynamics of SARS-CoV-2 nucleocapsid protein induced by RNA binding.

The nucleocapsid (N) protein of the SARS-CoV-2 virus, the causal agent of COVID-19, is a multifunction phosphoprotein that plays critical roles in the virus life cycle, including transcription and packaging of the viral RNA. To play such diverse roles, the N protein has two globular RNA-binding modules, the N- (NTD) and C-terminal (CTD) domains, which are connected by an intrinsically disordered region. Despite the wealth of structural data available for the isolated NTD and CTD, how these domains are arranged in the full-length protein and how the oligomerization of N influences its RNA-binding activity remains largely unclear. Herein, using experimental data from electron microscopy and biochemical/biophysical techniques combined with molecular modeling and molecular dynamics simulations, we show that, in the absence of RNA, the N protein formed structurally dynamic dimers, with the NTD and CTD arranged in extended conformations. However, in the presence of RNA, the N protein assumed a more compact conformation where the NTD and CTD are packed together. We also provided an octameric model for the full-length N bound to RNA that is consistent with electron microscopy images of the N protein in the presence of RNA. Together, our results shed new light on the dynamics and higher-order oligomeric structure of this versatile protein.

Developmental differences in myocardial transmembrane Na+ transport: implications for excitability and Na+ handling.

Little is currently known about possible developmental changes in myocardial Na+ handling, which may have impact on cell excitability and Ca2+ content. Resting intracellular Na+ concentration ([Na+ ]i ), measured in freshly isolated rat ventricular myocytes with CoroNa green, was not significantly different in neonates (3-5 days old) and adults, but electrical stimulation caused marked [Na+ ]i rise only in neonates. Inhibition of L-type Ca2+ current by CdCl2 abolished not only systolic Ca2+ transients, but also activity-dependent intracellular Na+ accumulation in immature cells. This indicates that the main Na+ influx pathway during activity is the Na+ /Ca2+ exchanger, rather than voltage-dependent Na+ current (INa ), which was not affected by CdCl2 . In immature myocytes, INa density was two-fold greater, inactivation was faster, and the current peak occurred at less negative transmembrane potential (Em ) than in adults. Na+ channel steady-state activation and inactivation curves in neonates showed a rightward shift, which should increase channel availability at diastolic Em , but also require greater depolarization for excitation, which was observed experimentally and reproduced in computer simulations. Ventricular mRNA levels of Nav 1.1, Nav 1.4 and Nav 1.5 pore-forming isoforms were greater in neonate ventricles, while a decrease was seen for the ß1 subunit. Both molecular and biophysical changes in the channel profile may contribute to the differences in INa density and voltage-dependence, and also to the less negative threshold Em , in neonates compared to adults. The apparently lower excitability in immature ventricle may confer protection against the development of spontaneous activity in this tissue. KEY POINTS: Previous studies showed that myocardial preparations from immature rats are less sensitive to electrical field stimulation than adult preparations. Freshly isolated ventricular myocytes from neonatal rats showed lower excitability than adult cells, e.g. less negative threshold membrane potential and greater membrane depolarization required for action potential triggering. In addition to differences in mRNA levels for Na+ channel isoforms and greater Na+ current (INa ) density, Na+ channel voltage-dependence was shifted to the right in immature myocytes, which seems to be sufficient to decrease excitability, according to computer simulations. Only in neonatal myocytes did cyclic activity promote marked cytosolic Na+ accumulation, which was prevented by abolition of systolic Ca2+ transients by blockade of Ca2+ currents. Developmental changes in INa may account for the difference in action potential initiation parameters, but not for cytosolic Na+ accumulation, which seems to be due mainly to Na+ /Ca2+ exchanger-mediated Na+ influx.

Randomized clinical trial of BCG vaccine in patients with convalescent COVID-19: Clinical evolution, adverse events, and humoral immune response.

BACKGROUND: The Bacillus Calmette-Guérin (BCG) vaccine may confer cross-protection against viral diseases in adults. This study evaluated BCG vaccine cross-protection in adults with convalescent coronavirus disease 2019 (COVID-19). METHOD: This was a multicenter, prospective, randomized, placebo-controlled, double-blind phase III study (ClinicalTrials.gov: NCT04369794). SETTING: University Community Health Center and Municipal Outpatient Center in South America. PATIENTS: a total of 378 adult patients with convalescent COVID-19 were included. INTERVENTION: single intradermal BCG vaccine (n = 183) and placebo (n = 195). MEASUREMENTS: the primary outcome was clinical evolution. Other outcomes included adverse events and humoral immune responses for up to 6 months. RESULTS: A significantly higher proportion of BCG patients with anosmia and ageusia recovered at the 6-week follow-up visit than placebo (anosmia: 83.1% vs. 68.7% healed, p = 0.043, number needed to treat [NNT] = 6.9; ageusia: 81.2% vs. 63.4% healed, p = 0.032, NNT = 5.6). BCG also prevented the appearance of ageusia in the following weeks: seven in 113 (6.2%) BCG recipients versus 19 in 126 (15.1%) placebos, p = 0.036, NNT = 11.2. BCG did not induce any severe or systemic adverse effects. The most common and expected adverse effects were local vaccine lesions, erythema (n = 152; 86.4%), and papules (n = 111; 63.1%). Anti-severe acute respiratory syndrome coronavirus 2 humoral response measured by N protein immunoglobulin G titer and seroneutralization by interacting with the angiotensin-converting enzyme 2 receptor suggest that the serum of BCG-injected patients may neutralize the virus at lower specificity; however, the results were not statistically significant. CONCLUSION: BCG vaccine is safe and offers cross-protection against COVID-19 with potential humoral response modulation. LIMITATIONS: No severely ill patients were included.

Sequential IgG antibody monitoring for virus-inactivated and adenovirus-vectored COVID-19 vaccine in Brazilian healthcare workers.

Vaccination certainly is the best way to fight against the COVID-19 pandemic. In this study, the seroconversion effectiveness of two vaccines against severe acute respiratory syndrome coronavirus 2 was assessed in healthcare workers: virus-inactivated CoronaVac (CV, n = 303), and adenovirus-vectored Oxford-AstraZeneca (AZ, n = 447). The immunoglobulin G (IgG) antibodies anti-spike glycoprotein and anti-nucleocapsid protein were assessed by enzyme-linked immunosorbent assay at the time before vaccination (T1), before the second dose (T2), and 30 days after the second dose (T3). Of all individuals vaccinated with AZ, 100% (n = 447) exhibited seroconversion, compared to 91% (n = 276) that were given CV vaccine. Among individuals who did not respond to the CV, only three individuals showed a significant increase in the antibody level 4 months later the booster dose. A lower seroconversion rate was observed in elders immunized with the CV vaccine probably due to the natural immune senescence, or peculiarity of this vaccine. The AZ vaccine induced a higher humoral response; however, more common side effects were also observed. Nonvaccinated convalescent individuals revealed a similar rate of anti-spike IgG to individuals that were given two doses of CV vaccine, which suggests that only a one-shot COVID-19 vaccine could produce an effective immune response in convalescents.

Personalized medicine using omics approaches in acute respiratory distress syndrome to identify biological phenotypes.

In the last decade, research on acute respiratory distress syndrome (ARDS) has made considerable progress. However, ARDS remains a leading cause of mortality in the intensive care unit. ARDS presents distinct subphenotypes with different clinical and biological features. The pathophysiologic mechanisms of ARDS may contribute to the biological variability and partially explain why some pharmacologic therapies for ARDS have failed to improve patient outcomes. Therefore, identifying ARDS variability and heterogeneity might be a key strategy for finding effective treatments. Research involving studies on biomarkers and genomic, metabolomic, and proteomic technologies is increasing. These new approaches, which are dedicated to the identification and quantitative analysis of components from biological matrixes, may help differentiate between different types of damage and predict clinical outcome and risk. Omics technologies offer a new opportunity for the development of diagnostic tools and personalized therapy in ARDS. This narrative review assesses recent evidence regarding genomics, proteomics, and metabolomics in ARDS research.

Early use of nitazoxanide in mild COVID-19 disease: randomised, placebo-controlled trial.

BACKGROUND: Nitazoxanide is widely available and exerts broad-spectrum antiviral activity in vitro. However, there is no evidence of its impact on severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. METHODS: In a multicentre, randomised, double-blind, placebo-controlled trial, adult patients presenting up to 3 days after onset of coronavirus disease 2019 (COVID-19) symptoms (dry cough, fever and/or fatigue) were enrolled. After confirmation of SARS-CoV-2 infection using reverse transcriptase PCR on a nasopharyngeal swab, patients were randomised 1:1 to receive either nitazoxanide (500 mg) or placebo, three times daily, for 5 days. The primary outcome was complete resolution of symptoms. Secondary outcomes were viral load, laboratory tests, serum biomarkers of inflammation and hospitalisation rate. Adverse events were also assessed. RESULTS: From June 8 to August 20, 2020, 1575 patients were screened. Of these, 392 (198 placebo, 194 nitazoxanide) were analysed. Median (interquartile range) time from symptom onset to first dose of study drug was 5 (4-5) days. At the 5-day study visit, symptom resolution did not differ between the nitazoxanide and placebo arms. Swabs collected were negative for SARS-CoV-2 in 29.9% of patients in the nitazoxanide arm versus 18.2% in the placebo arm (p=0.009). Viral load was reduced after nitazoxanide compared to placebo (p=0.006). The percentage viral load reduction from onset to end of therapy was higher with nitazoxanide (55%) than placebo (45%) (p=0.013). Other secondary outcomes were not significantly different. No serious adverse events were observed. CONCLUSIONS: In patients with mild COVID-19, symptom resolution did not differ between nitazoxanide and placebo groups after 5 days of therapy. However, early nitazoxanide therapy was safe and reduced viral load significantly.

Mechanistic insights revealed by a UBE2A mutation linked to intellectual disability.

Ubiquitin-conjugating enzymes (E2) enable protein ubiquitination by conjugating ubiquitin to their catalytic cysteine for subsequent transfer to a target lysine side chain. Deprotonation of the incoming lysine enables its nucleophilicity, but determinants of lysine activation remain poorly understood. We report a novel pathogenic mutation in the E2 UBE2A, identified in two brothers with mild intellectual disability. The pathogenic Q93E mutation yields UBE2A with impaired aminolysis activity but no loss of the ability to be conjugated with ubiquitin. Importantly, the low intrinsic reactivity of UBE2A Q93E was not overcome by a cognate ubiquitin E3 ligase, RAD18, with the UBE2A target PCNA. However, UBE2A Q93E was reactive at high pH or with a low-pKa amine as the nucleophile, thus providing the first evidence of reversion of a defective UBE2A mutation. We propose that Q93E substitution perturbs the UBE2A catalytic microenvironment essential for lysine deprotonation during ubiquitin transfer, thus generating an enzyme that is disabled but not dead.

Use of metabolomics for the identification and validation of clinical biomarkers for preterm birth: Preterm SAMBA.

BACKGROUND: Spontaneous preterm birth is a complex syndrome with multiple pathways interactions determining its occurrence, including genetic, immunological, physiologic, biochemical and environmental factors. Despite great worldwide efforts in preterm birth prevention, there are no recent effective therapeutic strategies able to decrease spontaneous preterm birth rates or their consequent neonatal morbidity/mortality. The Preterm SAMBA study will associate metabolomics technologies to identify clinical and metabolite predictors for preterm birth. These innovative and unbiased techniques might be a strategic key to advance spontaneous preterm birth prediction. METHODS/DESIGN: Preterm SAMBA study consists of a discovery phase to identify biophysical and untargeted metabolomics from blood and hair samples associated with preterm birth, plus a validation phase to evaluate the performance of the predictive modelling. The first phase, a case-control study, will randomly select 100 women who had a spontaneous preterm birth (before 37 weeks) and 100 women who had term birth in the Cork Ireland and Auckland New Zealand cohorts within the SCOPE study, an international consortium aimed to identify potential metabolomic predictors using biophysical data and blood samples collected at 20 weeks of gestation. The validation phase will recruit 1150 Brazilian pregnant women from five participant centres and will collect blood and hair samples at 20 weeks of gestation to evaluate the performance of the algorithm model (sensitivity, specificity, predictive values and likelihood ratios) in predicting spontaneous preterm birth (before 34 weeks, with a secondary analysis of delivery before 37 weeks). DISCUSSION: The Preterm SAMBA study intends to step forward on preterm birth prediction using metabolomics techniques, and accurate protocols for sample collection among multi-ethnic populations. The use of metabolomics in medical science research is innovative and promises to provide solutions for disorders with multiple complex underlying determinants such as spontaneous preterm birth.

Systematic review and meta-analysis of diagnostic test accuracy of ST-segment elevation for acute coronary occlusion.

OBJECTIVE: To evaluate the diagnostic sensitivity and specificity of ST-segment elevation on a 12­lead ECG in detecting ACO across any coronary artery, challenging the current STEMI-NSTEMI paradigm. METHODS: Studies from MEDLINE and Scopus (2012-2023) comparing ECG findings with coronary angiograms were systematically reviewed and analyzed following PRISMA-DTA guidelines. QUADAS-2 assessed the risk of bias. STUDY SELECTION: Studies included focused on AMI patients and provided data enabling the construction of contingency tables for sensitivity and specificity calculation, excluding those with non-ACS conditions, outdated STEMI criteria, or a specific focus on bundle branch blocks or other complex diagnoses. Data were extracted systematically and pooled test accuracy estimates were computed using MetaDTA software, employing bivariate analyses for within- and between-study variation. The primary outcomes measured were the sensitivity and specificity of ST-segment elevation in detecting ACO. RESULTS: Three studies with 23,704 participants were included. The pooled sensitivity of ST-segment elevation for detecting ACO was 43.6% (95% CI: 34.7%-52.9%), indicating that over half of ACO cases may not exhibit ST-segment elevation. The specificity was 96.5% (95% CI: 91.2%-98.7%). Additional analysis using the OMI-NOMI strategy showed improved sensitivity (78.1%, 95% CI: 62.7%-88.3%) while maintaining similar specificity (94.4%, 95% CI: 88.6%-97.3%). CONCLUSION: The findings reveal a significant diagnostic gap in the current STEMI-NSTEMI paradigm, with over half of ACO cases potentially lacking ST-segment elevation. The OMI-NOMI strategy could offer an improved diagnostic approach. The high heterogeneity and limited number of studies necessitate cautious interpretation and further research in diverse settings.