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.

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.

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.

Discovery and structural characterization of chicoric acid as a SARS-CoV-2 nucleocapsid protein ligand and RNA binding disruptor.

The nucleocapsid (N) protein plays critical roles in coronavirus genome transcription and packaging, representing a key target for the development of novel antivirals, and for which structural information on ligand binding is scarce. We used a novel fluorescence polarization assay to identify small molecules that disrupt the binding of the N protein to a target RNA derived from the SARS-CoV-2 genome packaging signal. Several phenolic compounds, including L-chicoric acid (CA), were identified as high-affinity N-protein ligands. The binding of CA to the N protein was confirmed by isothermal titration calorimetry, 1H-STD and 15N-HSQC NMR, and by the crystal structure of CA bound to the N protein C-terminal domain (CTD), further revealing a new modulatory site in the SARS-CoV-2 N protein. Moreover, CA reduced SARS-CoV-2 replication in cell cultures. These data thus open venues for the development of new antivirals targeting the N protein, an essential and yet underexplored coronavirus target.

Isolation, culture, and immunostaining of neonatal rat ventricular myocytes.

Isolation and culture of ventricular cardiomyocytes from neonatal rats (NRVMs) is a powerful model to study neonatal cardiac development, cell cycle regulation, and cardiac physiology and pathology in vitro. Here, we present our modified enzymatic digestion protocol followed by two-step discontinuous Percoll gradient centrifugation to isolate a high yield of viable ventricular cardiomyocytes from neonatal rats. Finally, here we describe an immunostaining protocol for cytosolic and nuclear staining of NRVMs. For complete details on the use and execution of this protocol, please refer to Pereira et al. (2020).

Molecular and cellular basis of hyperassembly and protein aggregation driven by a rare pathogenic mutation in DDX3X.

Current studies estimate that 1-3% of females with unexplained intellectual disability (ID) present de novo splice site, nonsense, frameshift, or missense mutations in the DDX3X protein (DEAD-Box Helicase 3 X-Linked). However, the cellular and molecular mechanisms by which DDX3X mutations impair brain development are not fully comprehended. Here, we show that the ID-linked missense mutation L556S renders DDX3X prone to aggregation. By using a combination of biophysical assays and imaging approaches, we demonstrate that this mutant assembles solid-like condensates and amyloid-like fibrils. Although we observed greatly reduced expression of the mutant allele in a patient who exhibits skewed X inactivation, this appears to be enough to sequestrate healthy proteins into solid-like ectopic granules, compromising cell function. Therefore, our data suggest ID-linked DDX3X L556S mutation as a disorder arising from protein misfolding and aggregation.

High-resolution synchrotron-based X-ray microtomography as a tool to unveil the three-dimensional neuronal architecture of the brain.

The assessment of neuronal number, spatial organization and connectivity is fundamental for a complete understanding of brain function. However, the evaluation of the three-dimensional (3D) brain cytoarchitecture at cellular resolution persists as a great challenge in the field of neuroscience. In this context, X-ray microtomography has shown to be a valuable non-destructive tool for imaging a broad range of samples, from dense materials to soft biological specimens, arisen as a new method for deciphering the cytoarchitecture and connectivity of the brain. In this work we present a method for imaging whole neurons in the brain, combining synchrotron-based X-ray microtomography with the Golgi-Cox mercury-based impregnation protocol. In contrast to optical 3D techniques, the approach shown here does neither require tissue slicing or clearing, and allows the investigation of several cells within a 3D region of the brain.

Adenosine Kinase couples sensing of cellular potassium depletion to purine metabolism.

Adenosine Kinase (ADK) regulates the cellular levels of adenosine (ADO) by fine-tuning its metabolic clearance. The transfer of γ-phosphate from ATP to ADO by ADK involves regulation by the substrates and products, as well as by Mg2+ and inorganic phosphate. Here we present new crystal structures of mouse ADK (mADK) binary (mADK:ADO; 1.2 Å) and ternary (mADK:ADO:ADP; 1.8 Å) complexes. In accordance with the structural demonstration of ADO occupancy of the ATP binding site, kinetic studies confirmed a competitive model of auto-inhibition of ADK by ADO. In the ternary complex, a K+ ion is hexacoordinated between loops adjacent to the ATP binding site, where Asp310 connects the K+ coordination sphere to the ATP binding site through an anion hole structure. Nuclear Magnetic Resonance 2D 15N-1H HSQC experiments revealed that the binding of K+ perturbs Asp310 and residues of adjacent helices 14 and 15, engaging a transition to a catalytically productive structure. Consistent with the structural data, the mutants D310A and D310P are catalytically deficient and loose responsiveness to K+. Saturation Transfer Difference spectra of ATPγS provided evidence for an unfavorable interaction of the mADK D310P mutant for ATP. Reductions in K+ concentration diminish, whereas increases enhance the in vitro activity of mADK (maximum of 2.5-fold; apparent Kd = 10.4 mM). Mechanistically, K+ increases the catalytic turnover (Kcat) but does not affect the affinity of mADK for ADO or ATP. Depletion of intracellular K+ inhibited, while its restoration was accompanied by a full recovery of cellular ADK activity. Together, this novel dataset reveals the molecular basis of the allosteric activation of ADK by K+ and highlights the role of ADK in connecting depletion of intracellular K+ to the regulation of purine metabolism.

Human mitochondrial pyruvate carrier 2 as an autonomous membrane transporter.

The active transport of glycolytic pyruvate across the inner mitochondrial membrane is thought to involve two mitochondrial pyruvate carrier subunits, MPC1 and MPC2, assembled as a 150 kDa heterotypic oligomer. Here, the recombinant production of human MPC through a co-expression strategy is first described; however, substantial complex formation was not observed, and predominantly individual subunits were purified. In contrast to MPC1, which co-purifies with a host chaperone, we demonstrated that MPC2 homo-oligomers promote efficient pyruvate transport into proteoliposomes. The derived functional requirements and kinetic features of MPC2 resemble those previously demonstrated for MPC in the literature. Distinctly, chemical inhibition of transport is observed only for a thiazolidinedione derivative. The autonomous transport role for MPC2 is validated in cells when the ectopic expression of human MPC2 in yeast lacking endogenous MPC stimulated growth and increased oxygen consumption. Multiple oligomeric species of MPC2 across mitochondrial isolates, purified protein and artificial lipid bilayers suggest functional high-order complexes. Significant changes in the secondary structure content of MPC2, as probed by synchrotron radiation circular dichroism, further supports the interaction between the protein and ligands. Our results provide the initial framework for the independent role of MPC2 in homeostasis and diseases related to dysregulated pyruvate metabolism.

FAK Forms a Complex with MEF2 to Couple Biomechanical Signaling to Transcription in Cardiomyocytes.

Focal adhesion kinase (FAK) has emerged as a mediator of mechanotransduction in cardiomyocytes, regulating gene expression during hypertrophic remodeling. However, how FAK signaling is relayed onward to the nucleus is unclear. Here, we show that FAK interacts with and regulates myocyte enhancer factor 2 (MEF2), a master cardiac transcriptional regulator. In cardiomyocytes exposed to biomechanical stimulation, FAK accumulates in the nucleus, binds to and upregulates the transcriptional activity of MEF2 through an interaction with the FAK focal adhesion targeting (FAT) domain. In the crystal structure (2.9 Å resolution), FAT binds to a stably folded groove in the MEF2 dimer, known to interact with regulatory cofactors. FAK cooperates with MEF2 to enhance the expression of Jun in cardiomyocytes, an important component of hypertrophic response to mechanical stress. These findings underscore a connection between the mechanotransduction involving FAK and transcriptional regulation by MEF2, with potential relevance to the pathogenesis of cardiac disease.

MEF2C DNA-binding activity is inhibited through its interaction with the regulatory protein Ki-1/57.

Myocyte enhancer factor (MEF2) are MADS box transcription factors that play important roles in the regulation of myogenesis and morphogenesis of muscle cells. MEF2 proteins are activated by mechanical overload in the heart. In this study, we found the interaction of MEF2C with the regulatory protein Ki-1/57 using yeast two-hybrid system. This interaction was confirmed by GST-pull down assay in vitro and by co-immunoprecipitation in vivo. This interaction is also dependent on pressure overload in the heart. Co-imunoprecipitation assay with anti-MEF2 and anti-Ki-1/57 antibodies demonstrated a basal association between these proteins in the left ventricles of control rats. Pressure overload caused a reduction in this association. Ki-1/57 co-localizes with MEF2 in the nucleus of myocytes of control rats. However, after submitting the animals to pressure overload Ki-1/57 leaves the nucleus thereby decreasing this co-localization. Ki-1/57 also exerts an inhibitory effect upon MEF2C DNA binding activity. These results suggest that Ki-1/57 is a new interacting partner of MEF2 protein and may be involved in the regulation of MEF2 at the onset of hypertrophy.

High performance liquid chromatography analysis of a 4-anilinoquinazoline derivative (PD153035), a specific inhibitor of the epidermal growth factor receptor tyrosine kinase, in rat plasma.

The quinazoline derivative, 4-N-(3'-bromo-phenyl)amino-6,7-dimethoxyquinazoline (PD153035), has recently been identified as a potential drug for the treatment of proliferative disease. Here, we report a sensitive high performance liquid chromatography (HPLC)-based quantitative detection method for measurement of PD153035 levels in rat plasma. Sample pretreatment involved a two-step extraction with chloroform. The analytes were separated on a column packed with OmniSpher C18 material and eluted with acetonitrile-0.1 M ammonium acetate, pH 7.2 (70:30, v/v). The column effluent was monitored by UV detection at 330 nm. A linear response was achieved over the concentration range 0.50-100.00 microM using multilevel calibration with an internal standard. The analytical method inter- and intra-run accuracy and precision were better than +/-15%. The lower limit of quantification was 0.50 microM. The method has been applied to study the preclinical pharmacokinetics of this compound in rats.

Immediate response of myocardium to pressure overload includes transient regulation of genes associated with mitochondrial bioenergetics and calcium availability.

Ventricular hypertrophy is one of the major myocardial responses to pressure overload (PO). Most studies on early myocardial response focus on the days or even weeks after induction of hypertrophic stimuli. Since mechanotransduction pathways are immediately activated in hearts undergoing increased work load, it is reasonable to infer that the myocardial gene program may be regulated in the first few hours. In the present study, we monitored the expression of some genes previously described in the context of myocardial hypertrophic growth by using the Northern blot technique, to estimate the mRNA content of selected genes in rat myocardium for the periods 1, 3, 6, 12 and 48 h after PO stimuli. Results revealed an immediate switch in the expression of genes encoding alpha and beta isoforms of myosin heavy chain, and up-regulation of the cardiac isoform of alpha actin. We also detected transitory gene regulation as the increase in mitochondrial cytochrome c oxidase 1 gene expression, parallel to down-regulation of genes encoding sarco(endo)plasmic reticulum Ca(+2) ATPase and sodium-calcium exchanger. Taken together, these results indicate that initial myocardial responses to increased work load include alterations in the contractile properties of sarcomeres and transitory adjustment of mitochondrial bioenergetics and calcium availability.

Analysis of adenosine by RP-HPLC method and its application to the study of adenosine kinase kinetics.

An RP-HPLC method for the analysis of adenosine (ADO) has been developed and validated. In the present study, we report an RP-HPLC-based method with modifications of mobile phase and shorter retention time that substantially improved the efficiency of ADO analysis. The HPLC separation of the ADO was achieved on a C18 column, using a mobile phase consisting of water, containing 7% v/v ACN, at a flow rate of 0.8 mL/min. The column effluent was monitored by UV detection at 260 nm. A linear response was achieved over the concentration range of 0.25-100.00 micromol/L. The analytical method inter- and intra-run accuracy and precision were better than +/- 15%. The LOQ was 0.25 micromol/L, with ADO detection in the range of 6.25 pmol per sample. The method has been applied to the study of adenosine kinase (AK) kinetics.

Immediate response of myocardium to pressure overload includes transient regulation of genes associated with mitochondrial bioenergetics and calcium availability

Ventricular hypertrophy is one of the major myocardial responses to pressure overload (PO). Most studies on early myocardial response focus on the days or even weeks after induction of hypertrophic stimuli. Since mechanotransduction pathways are immediately activated in hearts undergoing increased work load, it is reasonable to infer that the myocardial gene program may be regulated in the first few hours. In the present study, we monitored the expression of some genes previously described in the context of myocardial hypertrophic growth by using the Northern blot technique, to estimate the mRNA content of selected genes in rat myocardium for the periods 1, 3, 6, 12 and 48 h after PO stimuli. Results revealed an immediate switch in the expression of genes encoding alpha and beta isoforms of myosin heavy chain, and up-regulation of the cardiac isoform of alpha actin. We also detected transitory gene regulation as the increase in mitochondrial cytochrome c oxidase 1 gene expression, parallel to down-regulation of genes encoding sarco(endo)plasmic reticulum Ca+2 ATPase and sodium-calcium exchanger. Taken together, these results indicate that initial myocardial responses to increased work load include alterations in the contractile properties of sarcomeres and transitory adjustment of mitochondrial bioenergetics and calcium availability.

Simvastatin prevents load-induced protein tyrosine nitration in overloaded hearts.

Hydroxymethylglutaryl-coenzyme A reductase inhibitors prevent load-induced left ventricular hypertrophy (LVH). Whether this effect is related to antioxidant properties of this class of drugs is poorly understood. The aim of the present report was to evaluate the regulation of nitrotyrosine production during the development of load-induced LVH and the effect of simvastatin treatment in this process. Rats were subjected to aortic constriction up to 15 days. LVH was evaluated by left/right ventricle mass ratio. Myocardial content of nitrotyrosine, nitric oxide synthase (NOS) isoforms, and phagocyte-type NAD(P)H-oxidase subunits (p67-phox and p22-phox) were analyzed by immunoblotting and immunohistochemistry assays. Another group of rats received treatment with either simvastatin or placebo for 15 days after the onset of pressure overload, and their hearts were also studied. Myocardial nitrotyrosine content was increased from 3 to 15 days of pressure overload in regions of cardiac myocytes in close apposition to myocardial stroma during LVH. Neuronal NOS (nNOS), inducible NOS (iNOS), and endothelial NOS (eNOS) isoforms had their expression increased in coronary vessels (nNOS and iNOS) and in myocardial stroma (eNOS) from day 3 to day 7 of aortic constriction. However, p67-phox and p22-phox expression was increased in cells of myocardial stroma in parallel to augmented myocardial nitrotyrosine content. Simvastatin treatment inhibited the increases in myocardial nitrotyrosine content and in p67-phox and p22-phox expression, and significantly reduced LVH. In conclusion, antioxidant properties of simvastatin might play a role in myocardial remodeling induced by pressure overload.

Cor triatriatum dexter: diagnóstico ecocardiográfico; relato de caso / Cor triatriatum dexter: echocardiographic diagnosis; a case report

Os autores apresentam um caso de cor triatriatum dexter, diagnosticado por ecodoplercardiografia bidimensional, associado a comuniçäo interatrial e estenose pulmonar valvar. A anomalia näo foi detectada no estudo hemodinâmico. O ecodopplercardiograma mostrou uma ampla membrana dividindo o átrio em duas cavidades, estendendo-se de sua porçäo inferior, imediatamente acima do anel tricúspide, até os orifícios da veias cavas. Esses achados foram confirmados pela necrópsia

Implicações cardiovasculares da síndrome metabólica: importância da hiperinsulinemia na hipertrofia cardíaca e do vaso / Cardiovascular implications of metabolic syndrome: role of hyperinsulinemia on cardiac and vascular hypertrophy

Evidências epidemiológicas indicam que a resistência à insulina e a conseqüente hiperinsulinemia são fatores de risco independentes para doenças cardiovasculares. Diversos estudos ecocardiográficos têm demonstrado que a hiperinsulinemia está associada de maneira independente à hipertrofia ventricular esquerda e ao aumento da relação entre a massa ventricular esquerda e o volume diastólico final.Por outro lado, estudos prospectivos e observações epidemiológicas demonstraram associação independente entre hiperinsulinemia e doença isquêmica coronária e vascular cerebral, indicando participação importante da insulina no desenvolvimento da aterosclerose. Embora ainda não estejam estabelecidos os mecanismos pelos quais a hiperinsulinemia induz à hipertrofia tanto cardíaca como vascular em condições de resistência à insulina, observações experimentais recentes têm sugerido que anormalidades na sinalização intracelular ativada pela insulina possam exercer papel fundamental nesse processo. A redução da ativação da via da fosfatidilinositol-3-quinase/Akt, responsável pelos efeitos metabólicos da insulina, em paralelo à ativação preservada da via de crescimento celular mediada pelas quinases ativadas por mitógenos (MAPKs) estão sendo consideradas como eventos fundamentais para o desenvolvimento da hipertrofia tanto miocárdica como do vaso. Além disso, desequilíbrios entre insulina e fatores de crescimento, como a angiotensina 11, parecemtambém contribuir para a hipertrofia em ambos os tecidos. Portanto, a hiperinsulinemia parece ser um importante regulador da hipertrofia cardíaca e vascular, consistindo em um fator de risco independente para a morbidade e a mortalidade cardiovasculares. Ademais, mecanismos fisiopatogênicos similares podem estar envolvidos no crescimento cardíaco e do vaso em condições de resistência à insulina e hiperinsulinemia.

Influência de fatores ambientais e genéticos na hipertrofia e remodelamento cardíacos na hipertensäo arterial / Influence of environmental and genetic factors on cardiac hypertrophy and remodeling in hypertension

A hipertrofia e o remodelamento ventricular representam mudanças estruturais adaptativas do ventrículo esquerdo em resposta à hipertensäo arterial. Apesar de consideradas compensatórias, essas alteraçöes estäo associadas a maior risco de morbimortalidade cardiovascular. Embora a pressäo arterial seja a principal determinante da massa e geometria ventricular esquerda, diversos fatores ambientais e genéticos podem exercer influência no fenótipo ventricular. Fatores ambientais como a ingestäo de sal, o consumo de álcool, o diabetes melito, o estresse psicológico e o exercício físico prolongado podem exercer influências significativas sobre a massa e a geometria do ventrículo esquerdo. Por outro lado, evidências epidemiológicas e análises de ligaçäo de polimorfismos gênicos têm apontado para uma importância significativa do componente genético na determinaçäo da massa ventricular esquerda. Portanto, a massa e a arquitetura do ventrículo esquerdo constituem fenótipos complexos que säo determinados pela interaçäo da carga hemodinâmica imposta dentre outras pela pressäo arterial com fatores genéticos e ambientais.

Atividade colinesterásica no plasma e em eritrócitos de pacientes com as formas cardíaca e indeterminada da doença de Chagas / Cholinesterase activity in plasma and in erythrocytes of patients with cardiac and indeterminate form of Chagas' disease

A atividade colinesterásica foi determinada no plasma e em eritrócitos de 10 indivíduos normais e em 26 pacientes chagásicos crônicos, sendo 6 com insuficiência cardíaca congestiva compensada, 10 com cardiopatia sem insuficiência cardíaca congestiva e 10 com a forma indeterminada. Os valores enzimáticos encontrados foram (média + ou - sd): 2196 + ou - 442 UI/L no plasma e 19,4 + ou - 3,3 UI/g Hb em eritrócitos do grupo controle; 2291 + ou - 317 UI/L no plasma e 19,2 + ou - 3,7 UI/g Hb em eritrócitos dos chagásicos com insuficiência cardíaca congestiva compensada; 2445 + ou - 357 UI/L no plasma e 18,3 + ou - 3,0 UI/g Hb em eritrócitos dos cardiopatas chagásicos sem insuficiência cardíaca congestiva; 2006 + ou - 327 UI/L no plasma e 17,8 + ou - 3,1 UI/g Hb em eritrócitos de chagásicos com a forma indeterminada. Nossos dados mostram que o comprometimento neuronal que ocorre nos cardiopatas chagásicos crônicos näo é suficiente para levar a alteraçöes significativvas (p>0,05) das atividades colinesterásicas no plasma e em eritrócitos