Cas9 interaction with the tracrRNA nexus modulates the repression of type II-A CRISPR-cas genes.

Immune responses need to be regulated to prevent autoimmunity. CRISPR-Cas systems provide adaptive immunity in prokaryotes through the acquisition of short DNA sequences from invading viruses (bacteriophages), known as spacers. Spacers are inserted into the CRISPR locus and serve as templates for the transcription of guides used by RNA-guided nucleases to recognize complementary nucleic acids of the invaders and start the CRISPR immune response. In type II-A CRISPR systems, Cas9 uses the guide RNA to cleave target DNA sequences in the genome of infecting phages, and the tracrRNA to bind the promoter of cas genes and repress their transcription. We previously isolated a Cas9 mutant carrying the I473F substitution that increased the frequency of spacer acquisition by 2-3 orders of magnitude, leading to a fitness cost due to higher levels of autoimmunity. Here, we investigated the molecular basis underlying these findings. We found that the I473F mutation decreases the association of Cas9 to tracrRNA, limiting its repressor function, leading to high levels of expression of cas genes, which in turn increase the strength of the type II-A CRISPR-Cas immune response. We obtained similar results for a related type II-A system, and therefore our findings highlight the importance of the interaction between Cas9 and its tracrRNA cofactor in tuning the immune response to balanced levels that enable phage defense but avoid autoimmunity.

Context-aware geometric deep learning for protein sequence design.

Protein design and engineering are evolving at an unprecedented pace leveraging the advances in deep learning. Current models nonetheless cannot natively consider non-protein entities within the design process. Here, we introduce a deep learning approach based solely on a geometric transformer of atomic coordinates and element names that predicts protein sequences from backbone scaffolds aware of the restraints imposed by diverse molecular environments. To validate the method, we show that it can produce highly thermostable, catalytically active enzymes with high success rates. This concept is anticipated to improve the versatility of protein design pipelines for crafting desired functions.

Extended Computed Tomography Angiography for the Successful Diagnosis of Cardioaortic Thrombus in Acute Ischemic Stroke and TIA: Study Protocol for a Randomized Controlled Trial.

INTRODUCTION: Cardiac imaging is one of the main components of the etiological investigation of ischemic strokes. However, basic and advanced cardiac imaging remain underused in most stroke centers globally. Computed tomography angiography (CTA) of the supra-aortic and intracranial arteries is the most frequent imaging modality applied during the evaluation of patients with acute ischemic stroke to identify the presence of a large vessel occlusion. Recent evidence from retrospective observational studies has shown a high detection of cardiac thrombi, ranging from 6.6 to 17.4%, by extending a CTA a few cm below the carina to capture cardiac images. However, this approach has never been prospectively compared against usual care in a randomized controlled trial. The Extended Computed Tomography Angiography for the Successful Screening of Cardioaortic Thrombus in Acute Ischemic Stroke and TIA (DAYLIGHT) prospective, randomized, controlled trial evaluates whether an extended CTA (eCTA) + standard-of-care stroke workup results in higher detection rates of cardiac and aortic source of embolism compared to standard-of-care CTA (sCTA) + standard-of-care stroke workup. METHODS: DAYLIGHT is a single-center, prospective, randomized, open-blinded endpoint trial, aiming to recruit 830 patients with suspected acute ischemic stroke or transient ischemic attack (TIA) being assessed under acute code stroke at the emergency department or at a dedicated urgent stroke prevention clinic. Patients are randomized 1:1 to eCTA versus sCTA. The eCTA expands image acquisition caudally, 6 cm below the carina. All patients receive standard-of-care cardiac imaging and diagnostic stroke workup. The primary efficacy endpoint is the diagnosis of a cardioaortic thrombus after at least 30 days of follow-up. The primary safety endpoint is door-to-CTA completion time. The diagnosis of a qualifying ischemic stroke or TIA is independently adjudicated by a stroke neurologist, blinded to the study arm allocation. Patients without an adjudicated ischemic stroke or TIA are excluded from the analysis. The primary outcome events are adjudicated by a board-certified radiologist with subspecialty training in cardiothoracic radiology and a cardiologist with formal training in cardiac imaging. The primary analysis is performed according to the modified intention-to-diagnose principle and without adjustment by logistic regression models. Results are presented with odds ratios and 95% confidence intervals. CONCLUSION: The DAYLIGHT trial will provide evidence on whether extending a CTA to include the heart results in an increased detection of cardioaortic thrombi compared to standard-of-care stroke workup.

Neurologists Preferences on Basic and Advanced Cardiac Imaging Utilization in Ischemic Stroke Patients.

INTRODUCTION: It is unknown how cardiac imaging studies are used by neurologists to investigate cardioembolic sources in ischemic stroke patients. METHODS: Between August 12, 2023, and December 8, 2023, we conducted an international survey among neurologists from Europe, North America, South America, and Asia, to investigate the frequency of utilization of cardiac imaging studies for the detection of cardioembolic sources of ischemic stroke. Questions were structured into deciles of percentage utilization of transthoracic echocardiography (TTE), transesophageal echocardiography (TEE), ECG-gated cardiac computed tomography (G-CCT), and cardiac magnetic resonance imaging (CMRI). We estimated the weighted proportion (x¯) of utilization of each cardiac imaging modality, both globally and by continent. We also investigated the use of head and neck computed tomography angiography (CTA) as an emerging approach to the screening of cardioembolic sources. RESULTS: A total of 402 neurologists from 64 countries completed the survey. Globally, TTE was the most frequently used cardiac imaging technology (x¯ = 71.2%), followed by TEE (x¯ = 15.8%), G-CCT (x¯ = 10.9%), and CMRI (x¯ = 7.7%). Findings were consistent across all continents. A total of 288 respondents routinely used a CTA in the acute ischemic stroke phase (71.6%), but the CTA included a non-gated CCT in only 15 cases (5.2%). CONCLUSIONS: This survey suggests that basic cardiac imaging is not done in all ischemic stroke patients evaluated in 4 continents. We also found a substantially low utilization of advanced cardiac imaging studies. Easier to adopt screening methods for cardioembolic sources of embolism are needed.

Embolic Stroke of Undetermined Source: New Data and New Controversies on Cardiac Monitoring and Anticoagulation.

Embolic strokes of undetermined source (ESUS) represent 9%-25% of all ischemic strokes. Based on the suspicion that a large proportion of cardioembolic sources remain undetected among embolic stroke of undetermined source patients, it has been hypothesized that a universal approach of anticoagulation would be better than aspirin for preventing recurrent strokes. However, 4 randomized controlled trials (RCTs), with different degrees of patient selection, failed to confirm this hypothesis. In parallel, several RCTs consistently demonstrated that prolonged cardiac monitoring increased atrial fibrillation detection and anticoagulation initiation compared with usual care in patients with ESUS, and later in individuals with ischemic stroke of known cause (e.g., large or small vessel disease). However, none of these trials or subsequent meta-analyses of all available RCTs have shown a reduction in stroke recurrence associated with the use of prolonged cardiac monitoring. In this article, we review the clinical and research implications of recent RCTs of antithrombotic therapy in patients with ESUS and in high-risk populations with and without stroke, with device-detected asymptomatic atrial fibrillation.

Cardiolipin clustering promotes mitochondrial membrane dynamics.

Cardiolipin (CL) is a mitochondria-specific phospholipid that forms heterotypic interactions with membrane-shaping proteins and regulates the dynamic remodeling and function of mitochondria. However, the precise mechanisms through which CL influences mitochondrial morphology are not well understood. In this study, employing molecular dynamics (MD) simulations, we observed CL localize near the membrane-binding sites of the mitochondrial fusion protein Optic Atrophy 1 (OPA1). To validate these findings experimentally, we developed a bromine-labeled CL probe to enhance cryoEM contrast and characterize the structure of OPA1 assemblies bound to the CL-brominated lipid bilayers. Our images provide direct evidence of interactions between CL and two conserved motifs within the paddle domain (PD) of OPA1, which control membrane-shaping mechanisms. We further observed a decrease in membrane remodeling activity for OPA1 in lipid compositions with increasing concentrations of monolyso-cardiolipin (MLCL). Suggesting that the partial replacement of CL by MLCL accumulation, as observed in Barth syndrome-associated mutations of the tafazzin phospholipid transacylase, compromises the stability of protein-membrane interactions. Our analyses provide insights into how biological membranes regulate the mechanisms governing mitochondrial homeostasis.

Extracellular volume measured by whole body CT scans predicts chronic cardiotoxicity in breast cancer patients treated with neoadjuvant therapies based on anthracyclines: A retrospective study.

INTRODUCTION: Neoadjuvant chemotherapies for breast cancer (BC) are effective but potentially cardiotoxic, and expose long survivors at risk of chemotherapy-related cardiac dysfunction (CTRCD). Unfortunately, early screening for CTRCD has actual diagnostic limits. Myocardial extracellular volume (mECV) is a radiological marker used in cardiac CT scans and cardiac magnetic resonance for diagnosis and follow-up of CTRCD. It can be measured in whole-body CT (WB-CT) scan, routinely performed in patients at high risk of relapse, to evaluate CTRCD occurrence during oncological follow-up. METHODS: 82 WB-CT scans were examined at baseline (T0) and during oncological follow-up at first year (T1) and fifth year (T5) after the end of neoadjuvant treatment. mECV was measured at 1 min (PP) and 5 min (DP) after contrast injection. 31 echocardiograms were retrieved in T1 to perform a linear correlation between mECV and left ventricular ejection fraction (LVEF). RESULTS: mECV values in T0 were similar between the two groups both in PP and in DP. Significant results were found for PP values in T1 (37.0 % vs 32 %, p = 0.0005) and in T5 (27.2 % vs 31.2 %, p = 0.025). A cut-off value of 35 % in PP proved significant in T1 (OR = 12.4, p = 0.004), while mECV was inversely correlated with LVEF both in PP (adj-S = -3.54, adj-p = 0.002) and in DP (adj-S = -2.51, adj-p = 0.0002), suggesting a synergistic action with the age at diagnosis (p < 0.0001, respectively). CONCLUSIONS: WB-CT scans performed during oncological reassessment in patients at high-risk of recurrence could be used for CTRCD screening in cardiovascular low-risk patients, especially in aging patients with mECV values above 35 %.

Sodium-glucose cotransporter 2 inhibitor dapagliflozin prevents ejection fraction reduction, reduces myocardial and renal NF-κB expression and systemic pro-inflammatory biomarkers in models of short-term doxorubicin cardiotoxicity.

Background: Anthracycline-mediated adverse cardiovascular events are among the leading causes of morbidity and mortality in patients with cancer. Sodium-glucose cotransporter 2 inhibitors (SGLT2i) exert multiple cardiometabolic benefits in patients with/without type 2 diabetes, chronic kidney disease, and heart failure with reduced and preserved ejection fraction. We hypothesized that the SGLT2i dapagliflozin administered before and during doxorubicin (DOXO) therapy could prevent cardiac dysfunction and reduce pro-inflammatory pathways in preclinical models. Methods: Cardiomyocytes were exposed to DOXO alone or combined with dapagliflozin (DAPA) at 10 and 100 nM for 24 h; cell viability, iATP, and Ca++ were quantified; lipid peroxidation products (malondialdehyde and 4-hydroxy 2-hexenal), NLRP3, MyD88, and cytokines were also analyzed through selective colorimetric and enzyme-linked immunosorbent assay (ELISA) methods. Female C57Bl/6 mice were treated for 10 days with a saline solution or DOXO (2.17 mg/kg), DAPA (10 mg/kg), or DOXO combined with DAPA. Systemic levels of ferroptosis-related biomarkers, galectin-3, high-sensitivity C-reactive protein (hs-CRP), and pro-inflammatory chemokines (IL-1α, IL-1ß, IL-2, IL-4, IL-6, IL-10, IL-12, IL17-α, IL-18, IFN-γ, TNF-α, G-CSF, and GM-CSF) were quantified. After treatments, immunohistochemical staining of myocardial and renal p65/NF-kB was performed. Results: DAPA exerts cytoprotective, antioxidant, and anti-inflammatory properties in human cardiomyocytes exposed to DOXO by reducing iATP and iCa++ levels, lipid peroxidation, NLRP-3, and MyD88 expression. Pro-inflammatory intracellular cytokines were also reduced. In preclinical models, DAPA prevented the reduction of radial and longitudinal strain and ejection fraction after 10 days of treatment with DOXO. A reduced myocardial expression of NLRP-3 and MyD-88 was seen in the DOXO-DAPA group compared to DOXO mice. Systemic levels of IL-1ß, IL-6, TNF-α, G-CSF, and GM-CSF were significantly reduced after treatment with DAPA. Serum levels of galectine-3 and hs-CRP were strongly enhanced in the DOXO group; on the other hand, their expression was reduced in the DAPA-DOXO group. Troponin-T, B-type natriuretic peptide (BNP), and N-Terminal Pro-BNP (NT-pro-BNP) were strongly reduced in the DOXO-DAPA group, revealing cardioprotective properties of SGLT2i. Mice treated with DOXO and DAPA exhibited reduced myocardial and renal NF-kB expression. Conclusion: The overall picture of the study encourages the use of DAPA in the primary prevention of cardiomyopathies induced by anthracyclines in patients with cancer.

Differential Effects of Post-translational Modifications on the Membrane Interaction of Huntingtin Protein.

Huntington's disease is a neurodegenerative disorder caused by an expanded polyglutamine stretch near the N-terminus of the huntingtin (HTT) protein, rendering the protein more prone to aggregate. The first 17 residues in HTT (Nt17) interact with lipid membranes and harbor multiple post-translational modifications (PTMs) that can modulate HTT conformation and aggregation. In this study, we used a combination of biophysical studies and molecular simulations to investigate the effect of PTMs on the helicity of Nt17 in the presence of various lipid membranes. We demonstrate that anionic lipids such as PI4P, PI(4,5)P2, and GM1 significantly enhance the helical structure of unmodified Nt17. This effect is attenuated by single acetylation events at K6, K9, or K15, whereas tri-acetylation at these sites abolishes Nt17-membrane interaction. Similarly, single phosphorylation at S13 and S16 decreased but did not abolish the POPG and PIP2-induced helicity, while dual phosphorylation at these sites markedly diminished Nt17 helicity, regardless of lipid composition. The helicity of Nt17 with phosphorylation at T3 is insensitive to the membrane environment. Oxidation at M8 variably affects membrane-induced helicity, highlighting a lipid-dependent modulation of the Nt17 structure. Altogether, our findings reveal differential effects of PTMs and crosstalks between PTMs on membrane interaction and conformation of HTT. Intriguingly, the effects of phosphorylation at T3 or single acetylation at K6, K9, and K15 on Nt17 conformation in the presence of certain membranes do not mirror that observed in the absence of membranes. Our studies provide novel insights into the complex relationship between Nt17 structure, PTMs, and membrane binding.

In AF, CHA2DS2-VASc 1 subgroups did not differ for predicting risk for arterial thromboembolism at 1 y.

SOURCE CITATION: Østergaard L, Olesen JB, Petersen JK, et al. Arterial thromboembolism in patients with atrial fibrillation and CHA2DS2-VASc 1: a nationwide study. Circulation. 2024;149:764-773. 38152890.

The landscape of biomedical research funding in Brazil: a current overview

ABSTRACT Objective: The objective of this narrative review is to discuss the current state of research funding in Brazil. Materials and Methods: This study is based on the most recent edition of the course Funding for Research and Innovation in the University of Sao Paulo School of Medicine which was a three-day course with 12 hours of instruction. The course brought together leading experts in the field to comprehensively discuss the current state of research funding in Brazil. Each speaker provided a presentation on a specific topic related to research funding. After the workshop, speakers assembled relevant topics in this manuscript. Results: collaborative research is critical for securing research funding. It optimizes proposal competitiveness, amplifies societal impact, and manages risks effectively. As such, fostering and supporting these collaborations is paramount for both researchers and funding agencies. To maintain the highest integrity in research, investigators involved in these collaborations must disclose any relationships that could potentially influence the outcomes or interpretation of their projects. Conclusions: In Brazil, the mainstay of research funding stems from public entities, with agencies such as CNPq, CAPES, and state bodies like FAPESP, FAPERJ, FAPEMIG and others at the forefront. Concurrently, industry funding offers viable pathways, especially through industry-sponsored studies, investigator-led projects, and collaborative initiatives. The Brazilian funding landscape is further enriched by innovative platforms, including crowdfunding and the contributions of institutions like the Serrapilheira Institute. Internationally, esteemed organizations such as the National Institutes of Health (NIH) and the Bill & Melinda Gates Foundation stand out as potential funders.

Kidney collecting system anatomy applied to endourology - a narrative review

ABSTRACT Objective: To evaluate the surgical anatomy of the kidney collecting system through a narrative review of the literature, highlighting its importance during diagnosis and its approach during surgical procedures for the treatment of renal stones. Material and Methods: We carried out a review about the anatomy of the kidney collecting system. We analyzed papers published in the past 40 years in the databases Pubmed, Embase and Scielo, and we included only papers in English and excluded case reports, editorials and opinions of specialists. Results: Renal collecting system could be divided in four groups: A1 - kidney midzone (KM), drained by minor calyx that are dependent on the superior or the inferior caliceal groups; A2 - KM drained by crossed calyx, one draining into the superior caliceal group and another draining into the inferior caliceal group; B1 - KM drained by a major caliceal group independent of both the superior and inferior groups; and B2 - KM drained by minor calyx entering directly into the renal pelvis. Some details and anatomic variations of the collecting system are related to clinical and radiological aspects, particularly perpendicular calyces, interpyelocalyx space, position of calyces in relation to renal border, classification of the renal collecting system, infundibular diameter and the angle between the lower infundibulum and renal pelvis. Conclusion: The knowledge of intra-renal collecting system divisions and variations as the angle between the renal pelvis and lower infundibula, position of the calices in relationship with renal edge and the diameter and position of the calyces are important for the planning of minimally invasive renal surgeries.

Toward More Personalized Management of Device-Detected Atrial Fibrillation.

This Viewpoint discusses the need to individualize the management of subclinical atrial fibrillation according to burden (among other factors) by modeling stroke risk.