Nucleotide depletion promotes cell fate transitions by inducing DNA replication stress.

Control of cellular identity requires coordination of developmental programs with environmental factors such as nutrient availability, suggesting that perturbing metabolism can alter cell state. Here, we find that nucleotide depletion and DNA replication stress drive differentiation in human and murine normal and transformed hematopoietic systems, including patient-derived acute myeloid leukemia (AML) xenografts. These cell state transitions begin during S phase and are independent of ATR/ATM checkpoint signaling, double-stranded DNA break formation, and changes in cell cycle length. In systems where differentiation is blocked by oncogenic transcription factor expression, replication stress activates primed regulatory loci and induces lineage-appropriate maturation genes despite the persistence of progenitor programs. Altering the baseline cell state by manipulating transcription factor expression causes replication stress to induce genes specific for alternative lineages. The ability of replication stress to selectively activate primed maturation programs across different contexts suggests a general mechanism by which changes in metabolism can promote lineage-appropriate cell state transitions.

mRNA trans-splicing dual AAV vectors for (epi)genome editing and gene therapy.

Large genes including several CRISPR-Cas modules like gene activators (CRISPRa) require dual adeno-associated viral (AAV) vectors for an efficient in vivo delivery and expression. Current dual AAV vector approaches have important limitations, e.g., low reconstitution efficiency, production of alien proteins, or low flexibility in split site selection. Here, we present a dual AAV vector technology based on reconstitution via mRNA trans-splicing (REVeRT). REVeRT is flexible in split site selection and can efficiently reconstitute different split genes in numerous in vitro models, in human organoids, and in vivo. Furthermore, REVeRT can functionally reconstitute a CRISPRa module targeting genes in various mouse tissues and organs in single or multiplexed approaches upon different routes of administration. Finally, REVeRT enabled the reconstitution of full-length ABCA4 after intravitreal injection in a mouse model of Stargardt disease. Due to its flexibility and efficiency REVeRT harbors great potential for basic research and clinical applications.

Screening in serum-derived medium reveals differential response to compounds targeting metabolism.

A challenge for screening new anticancer drugs is that efficacy in cell culture models is not always predictive of efficacy in patients. One limitation of standard cell culture is a reliance on non-physiological nutrient levels, which can influence cell metabolism and drug sensitivity. A general assessment of how physiological nutrients affect cancer cell response to small molecule therapies is lacking. To address this, we developed a serum-derived culture medium that supports the proliferation of diverse cancer cell lines and is amenable to high-throughput screening. We screened several small molecule libraries and found that compounds targeting metabolic enzymes were differentially effective in standard compared to serum-derived medium. We exploited the differences in nutrient levels between each medium to understand why medium conditions affected the response of cells to some compounds, illustrating how this approach can be used to screen potential therapeutics and understand how their efficacy is modified by available nutrients.

Physical fitness, hormonal profile, nutritional and psychological aspects assessment of transgender women volleyball players submitted to physical tests: protocol paper of a prospective cohort.

To evaluate aerobic capacity, strength and other physiological, nutritional, and psychological variables which may influence the performance of transgender women (TW) athletes and compare them to cisgender women (CW) and cisgender men (CM) athletes, as well as changes in TW performance over the course of a year. Prospective cohort study including three groups: TW, CW and CM volleyball athletes. Subjects will be comprehensively assessed at two different moments: baseline and after 6-12 months of adequate hormonal therapy. Evaluation will comprise clinical, medical, nutritional and psychological interviews, incremental treadmill cardiopulmonary exercise testing, hand grip strength test, vertical jump test, analysis of sleep quality (Pittsburgh Sleep Quality Index), hormonal profile, echocardiogram, analysis of resting energy expenditure, assessment of bone mass and body composition through dual-energy X-ray absorptiometry scans, and untargeted metabolomic analysis. CW and CM matched by age, body mass index and level of physical activity will undergo a similar evaluation. The assessment of the strength, aerobic capacity, haematological, nutritional and psychological status of TW using gold-standard tests will contribute to understanding the impact of oestrogen therapy on the exercise performance of these athletes and how they compare with CW and CM.

CICIoT2023: A Real-Time Dataset and Benchmark for Large-Scale Attacks in IoT Environment.

Nowadays, the Internet of Things (IoT) concept plays a pivotal role in society and brings new capabilities to different industries. The number of IoT solutions in areas such as transportation and healthcare is increasing and new services are under development. In the last decade, society has experienced a drastic increase in IoT connections. In fact, IoT connections will increase in the next few years across different areas. Conversely, several challenges still need to be faced to enable efficient and secure operations (e.g., interoperability, security, and standards). Furthermore, although efforts have been made to produce datasets composed of attacks against IoT devices, several possible attacks are not considered. Most existing efforts do not consider an extensive network topology with real IoT devices. The main goal of this research is to propose a novel and extensive IoT attack dataset to foster the development of security analytics applications in real IoT operations. To accomplish this, 33 attacks are executed in an IoT topology composed of 105 devices. These attacks are classified into seven categories, namely DDoS, DoS, Recon, Web-based, brute force, spoofing, and Mirai. Finally, all attacks are executed by malicious IoT devices targeting other IoT devices. The dataset is available on the CIC Dataset website.

Rumpel-Leede phenomenon as a rare complication after transulnar percutaneous coronary angiography and intervention.

The Rumpel-Leede sign, characterized by a non-blanching petechial rash distal to venous occlusion, has historically been associated with thrombocytopenia and capillary fragility. This phenomenon has been observed in various situations involving pressure application, such as tourniquet tests and continuous non-invasive pressure monitoring. Here, we present a case of Rumpel-Leede sign occurring after transulnar percutaneous coronary angiography in a 55-year-old female patient with a history of myocardial infarction. The patient had an uneventful recovery, highlighting the benign nature of the rash and the lack of intervention required. This underscores the importance of recognizing this sign and its association with specific procedures.

Screening in serum-derived medium reveals differential response to compounds targeting metabolism.

A challenge for screening new candidate drugs to treat cancer is that efficacy in cell culture models is not always predictive of efficacy in patients. One limitation of standard cell culture is a reliance on non-physiological nutrient levels to propagate cells. Which nutrients are available can influence how cancer cells use metabolism to proliferate and impact sensitivity to some drugs, but a general assessment of how physiological nutrients affect cancer cell response to small molecule therapies is lacking. To enable screening of compounds to determine how the nutrient environment impacts drug efficacy, we developed a serum-derived culture medium that supports the proliferation of diverse cancer cell lines and is amenable to high-throughput screening. We used this system to screen several small molecule libraries and found that compounds targeting metabolic enzymes were enriched as having differential efficacy in standard compared to serum-derived medium. We exploited the differences in nutrient levels between each medium to understand why medium conditions affected the response of cells to some compounds, illustrating how this approach can be used to screen potential therapeutics and understand how their efficacy is modified by available nutrients.

Plasmin and plasminogen prevent sepsis severity by reducing neutrophil extracellular traps and systemic inflammation.

Sepsis is a lethal syndrome characterized by systemic inflammation and abnormal coagulation. Despite therapeutic advances, sepsis mortality remains substantially high. Herein, we investigated the role of the plasminogen/plasmin (Plg/Pla) system during sepsis. Plasma levels of Plg were significantly lower in mice subjected to severe compared with nonsevere sepsis, whereas systemic levels of IL-6, a marker of sepsis severity, were higher in severe sepsis. Plg levels correlated negatively with IL-6 in both septic mice and patients, whereas plasminogen activator inhibitor-1 levels correlated positively with IL-6. Plg deficiency render mice susceptible to nonsevere sepsis induced by cecal ligation and puncture (CLP), resulting in greater numbers of neutrophils and M1 macrophages, liver fibrin(ogen) deposition, lower efferocytosis, and increased IL-6 and neutrophil extracellular trap (NET) release associated with organ damage. Conversely, inflammatory features, fibrin(ogen), and organ damage were substantially reduced, and efferocytosis was increased by exogenous Pla given during CLP- and LPS-induced endotoxemia. Plg or Pla protected mice from sepsis-induced lethality and enhanced the protective effect of antibiotics. Mechanistically, Plg/Pla-afforded protection was associated with regulation of NET release, requiring Pla-protease activity and lysine binding sites. Plg/Pla are important host-protective players during sepsis, controlling local and systemic inflammation and collateral organ damage.

Valor prognóstico do escore SYNTAX residual nos desfechos clínicos intra- hospitalares e de acompanhamento em pacientes com infarto do miocárdio com supradesnivelamento do segmento ST submetidos à estratégia farmacoinvasiva / Prognostic value of residual SYNTAX score on in-hospital and follow-up clinical outcomes in patients with ST-segment elevation myocardial infarction undergoing pharmacoinvasive strategy

Introdução: A doença arterial coronariana multiarterial é um desafio na prática clínica. Uma abordagem individualizada deve considerar não apenas as características do paciente, mas também um enfoque multidisciplinar, com o Heart Team. Diversos escores angiográficos foram propostos com o objetivo de quantificar o risco associado à doença arterial coronariana multiarterial. O escore SYNTAX residual foi proposto como um método para caracterizar e quantificar a doença coronariana residual, de forma sistemática, após intervenção coronária percutânea. Existem poucos dados na literatura que correlacionam o escore SYNTAX residual em pacientes com infarto do miocárdio com supradesnivelamento do segmento ST submetidos a uma estratégia farmacoinvasiva. O objetivo deste estudo foi avaliar o escore SYNTAX e o escore SYNTAX residual como preditores de desfechos intra-hospitalares e de médio prazo (180 a 380 dias), em pacientes com doença coronária multiarterial no contexto de infarto do miocárdio com supradesnivelamento do segmento ST, após terapia fibrinolítica bem-sucedida. Métodos: Em um estudo transversal, analítico e prospectivo, avaliamos o escore SYNTAX residual como preditor de desfechos intra-hospitalares e de médio prazo (6 meses a 1 ano), em pacientes com doença arterial coronariana multiarterial, no contexto de infarto do miocárdio com supradesnivelamento do segmento ST após estratégia farmacoinvasiva. Resultados: Entre agosto de 2019 e dezembro de 2020, foram analisados 108 pacientes com infarto do miocárdio com supradesnivelamento do segmento ST após fibrinólise, com critérios de reperfusão. O escore SYNTAX médio foi 13,98 (±4,87) e o escore SYNTAX residual médio foi 7,56 (±4,47). O escore SYNTAX residual elevado foi associado à nefropatia induzida por contraste e evento cardíaco adverso maior. Também foi um preditor independente de evento cardíaco adverso maior, com risco aumentado 9,69 vezes (p=0,0274). Conclusão: O escore SYNTAX residual elevado confere pior prognóstico em pacientes com infarto do miocárdio com elevação do segmento ST após estratégia farmacoinvasiva.

Background: Multivessel coronary artery disease is a challenge in clinical practice. An individualized approach should consider not only the patient characteristics, but also a multidisciplinary approach, together with the Heart Team. Multiple angiographic scores have been proposed with the aim of quantifying the risk associated with multivessel coronary artery disease. Residual SYNTAX score has been proposed as a method to systematically characterize and quantify residual coronary disease after percutaneous coronary intervention. There are few data in the literature correlating the residual SYNTAX score in patients with ST-segment elevation myocardial infarction undergoing pharmacoinvasive strategy. The objective of this study was to evaluate the SYNTAX score and residual SYNTAX score as predictors of in-hospital and medium-term outcomes (180 to 380 days) in patients with multivessel coronary artery disease in the setting of ST-segment elevation myocardial infarction, after successful fibrinolytic therapy. Methods: In a cross-sectional, analytical, and prospective study, we evaluated residual SYNTAX score as predictor of in-hospital and medium-term outcomes (6 months to 1 year), in patients with multivessel coronary artery disease, in the setting of ST-segment elevation myocardial infarction after pharmacoinvasive strategy. Results: Between August 2019 and December 2020, 108 patients with ST-segment elevation myocardial infarction after fibrinolysis, with reperfusion criteria, were analyzed. The mean SYNTAX score was 13.98 (±4.87) and the mean residual SYNTAX score was 7.56 (±4.47). High residual SYNTAX score was associated with contrast-induced nephropathy and major adverse cardiac event. It was also an independent predictor of major adverse cardiac event with a 9.69-fold increased risk (p=0.0274). Conclusion: High residual SYNTAX score confers worse prognosis in patients with ST-segment elevation myocardial infarction after pharmacoinvasive strategy.

Fluorescence-Activated Cell Sorting as a Tool for Recombinant Strain Screening.

Metabolic engineering of microbial cells is the discipline of optimizing microbial metabolism to enable and improve the production of target molecules ranging from biofuels and chemical building blocks to high-value pharmaceuticals. The advances in genetic engineering have eased the construction of highly engineered microbial strains and the generation of genetic libraries. Intracellular metabolite-responsive biosensors facilitate high-throughput screening of these libraries by connecting the levels of a metabolite of interest to a fluorescence output. Fluorescent-activated cell sorting (FACS) enables the isolation of highly fluorescent single cells and thus genotypes that produce higher levels of the metabolite of interest. Here, we describe a high-throughput screening method for recombinant yeast strain screening based on intracellular biosensors and FACS.

Rational gRNA design based on transcription factor binding data.

The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system has become a standard tool in many genome engineering endeavors. The endonuclease-deficient version of Cas9 (dCas9) is also a powerful programmable tool for gene regulation. In this study, we made use of Saccharomyces cerevisiae transcription factor (TF) binding data to obtain a better understanding of the interplay between TF binding and binding of dCas9 fused to an activator domain, VPR. More specifically, we targeted dCas9-VPR toward binding sites of Gcr1-Gcr2 and Tye7 present in several promoters of genes encoding enzymes engaged in the central carbon metabolism. From our data, we observed an upregulation of gene expression when dCas9-VPR was targeted next to a TF binding motif, whereas a downregulation or no change was observed when dCas9 was bound on a TF motif. This suggests a steric competition between dCas9 and the specific TF. Integrating TF binding data, therefore, proved to be useful for designing guide RNAs for CRISPR interference or CRISPR activation applications.

Laboratory-Generated DNA Can Cause Anomalous Pathogen Diagnostic Test Results.

The coronavirus disease 2019 (COVID-19) pandemic has brought about the unprecedented expansion of highly sensitive molecular diagnostics as a primary infection control strategy. At the same time, many laboratories have shifted focus to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) research and diagnostic development, leading to large-scale production of SARS-CoV-2 nucleic acids that can interfere with these tests. We have identified multiple instances, in independent laboratories, in which nucleic acids generated in research settings are suspected to have caused researchers to test positive for SARS-CoV-2 in surveillance testing. In some cases, the affected individuals did not work directly with these nucleic acids but were exposed via a contaminated surface or object. Though researchers have long been vigilant of DNA contaminants, the transfer of these contaminants to SARS-CoV-2 testing samples can result in anomalous test results. The impact of these incidents stretches into the public sphere, placing additional burdens on public health resources, placing affected researchers and their contacts in isolation and quarantine, removing them from the testing pool for 3 months, and carrying the potential to trigger shutdowns of classrooms and workplaces. We report our observations as a call for increased stewardship over nucleic acids with the potential to impact both the use and development of diagnostics. IMPORTANCE To meet the challenges imposed by the COVID-19 pandemic, research laboratories shifted their focus and clinical diagnostic laboratories developed and utilized new assays. Nucleic acid-based testing became widespread and, for the first time, was used as a prophylactic measure. We report 15 cases of researchers at two institutes testing positive for SARS-CoV-2 on routine surveillance tests, in the absence of any symptoms or transmission. These researchers were likely contaminated with nonhazardous nucleic acids generated in the laboratory in the course of developing new SARS-CoV-2 diagnostics. These contaminating nucleic acids were persistent and widespread throughout the laboratory. We report these findings as a cautionary tale to those working with nucleic acids used in diagnostic testing and as a call for careful stewardship of diagnostically relevant molecules. Our conclusions are especially relevant as at-home COVID-19 testing gains traction in the marketplace and these amplicons may impact on the general public.

Sepsis expands a CD39+ plasmablast population that promotes immunosuppression via adenosine-mediated inhibition of macrophage antimicrobial activity.

Sepsis results in elevated adenosine in circulation. Extracellular adenosine triggers immunosuppressive signaling via the A2a receptor (A2aR). Sepsis survivors develop persistent immunosuppression with increased risk of recurrent infections. We utilized the cecal ligation and puncture (CLP) model of sepsis and subsequent infection to assess the role of adenosine in post-sepsis immune suppression. A2aR-deficient mice showed improved resistance to post-sepsis infections. Sepsis expanded a subset of CD39hi B cells and elevated extracellular adenosine, which was absent in mice lacking CD39-expressing B cells. Sepsis-surviving B cell-deficient mice were more resistant to secondary infections. Mechanistically, metabolic reprogramming of septic B cells increased production of ATP, which was converted into adenosine by CD39 on plasmablasts. Adenosine signaling via A2aR impaired macrophage bactericidal activity and enhanced interleukin-10 production. Septic individuals exhibited expanded CD39hi plasmablasts and adenosine accumulation. Our study reveals CD39hi plasmablasts and adenosine as important drivers of sepsis-induced immunosuppression with relevance in human disease.

Systematic reviews on interventions for COVID-19 have rarely graded the certainty of the evidence.

BACKGROUND: Numerous systematic reviews on coronavirus disease-19 (COVID-19) treatment have been developed to provide syntheses of the large volume of primary studies. However, the methodological quality of most of these reviews is questionable and the results provided may therefore present bias. OBJECTIVE: To investigate how many systematic reviews on the therapeutic or preventive options for COVID-19 assessed the certainty of the evidence through the Grading of Recommendations Assessment, Development and Evaluation (GRADE) approach. METHODS: We conducted a sensitive search in MEDLINE (via PubMed) and included all systematic reviews that assessed any intervention for COVID-19. The systematic reviews included were examined to identify any planned and/or actual assessment using the GRADE approach (or absence thereof) regarding the certainty of the evidence. RESULTS: We included 177 systematic reviews and found that only 37 (21%; 37/177) assessed and reported the certainty of the evidence using the GRADE approach. This number reduced to 27 (16.2%; 27/167) when Cochrane reviews (n = 10), in which an evaluation using GRADE is mandatory, were excluded. CONCLUSION: Most of the systematic reviews on interventions relating to COVID-19 omitted assessment of the certainty of the evidence. This is a critical methodological omission that must not be overlooked in further research, so as to improve the impact and usefulness of syntheses relating to COVID-19.

FATTY ACID SYNTHESIS IS REQUIRED FOR BREAST CANCER BRAIN METASTASIS.

Brain metastases are refractory to therapies that control systemic disease in patients with human epidermal growth factor receptor 2 (HER2+) breast cancer, and the brain microenvironment contributes to this therapy resistance. Nutrient availability can vary across tissues, therefore metabolic adaptations required for brain metastatic breast cancer growth may introduce liabilities that can be exploited for therapy. Here, we assessed how metabolism differs between breast tumors in brain versus extracranial sites and found that fatty acid synthesis is elevated in breast tumors growing in brain. We determine that this phenotype is an adaptation to decreased lipid availability in brain relative to other tissues, resulting in a site-specific dependency on fatty acid synthesis for breast tumors growing at this site. Genetic or pharmacological inhibition of fatty acid synthase (FASN) reduces HER2+ breast tumor growth in the brain, demonstrating that differences in nutrient availability across metastatic sites can result in targetable metabolic dependencies.

Systematic reviews on interventions for COVID-19 have rarely graded the certainty of the evidence

ABSTRACT BACKGROUND: Numerous systematic reviews on coronavirus disease-19 (COVID-19) treatment have been developed to provide syntheses of the large volume of primary studies. However, the methodological quality of most of these reviews is questionable and the results provided may therefore present bias. OBJECTIVE: To investigate how many systematic reviews on the therapeutic or preventive options for COVID-19 assessed the certainty of the evidence through the Grading of Recommendations Assessment, Development and Evaluation (GRADE) approach. METHODS: We conducted a sensitive search in MEDLINE (via PubMed) and included all systematic reviews that assessed any intervention for COVID-19. The systematic reviews included were examined to identify any planned and/or actual assessment using the GRADE approach (or absence thereof) regarding the certainty of the evidence. RESULTS: We included 177 systematic reviews and found that only 37 (21%; 37/177) assessed and reported the certainty of the evidence using the GRADE approach. This number reduced to 27 (16.2%; 27/167) when Cochrane reviews (n = 10), in which an evaluation using GRADE is mandatory, were excluded. CONCLUSION: Most of the systematic reviews on interventions relating to COVID-19 omitted assessment of the certainty of the evidence. This is a critical methodological omission that must not be overlooked in further research, so as to improve the impact and usefulness of syntheses relating to COVID-19.

Effects of Caffeine Supplementation on Physical Performance of Soccer Players: Systematic Review and Meta-Analysis.

CONTEXT: Caffeine is 1 of the most popular supplements consumed by athletes, and the evidence for improving soccer performance remains limited. OBJECTIVE: To investigate and update the effects (benefits and harms) of caffeine to improve performance on soccer players. DATA SOURCES: Electronic search in Medline (via PubMed), CENTRAL, Embase, SPORTDiscus, and LILACS, from inception to March 28, 2020. STUDY SELECTION: Randomized clinical trials (RCTs) assessing the effects of caffeine on the performance of soccer players. STUDY DESIGN: Systematic review with meta-analysis. LEVEL OF EVIDENCE: Level 1. DATA EXTRACTION: Data extraction was conducted independently by 2 authors using a piloted form. We assessed methodological quality (Cochrane risk-of-bias [RoB] table) and the certainty of the evidence (GRADE [Grading of Recommendations Assessment, Development and Evaluation] approach). RESULTS: Sixteen RCTs were included. Overall methodological quality was classified as unclear to low risk of bias. When assessing aerobic endurance, meta-analyses did not demonstrate the differences between caffeine and placebo (mean difference [MD], 44.9 m; 95% confidence interval [CI], -77.7 to 167.6). Similarly, no difference was observed during time to fatigue test (MD, 169.8 seconds; 95% CI, -71.8 to 411.6). Considering anaerobic power, meta-analyses also did not find differences for vertical jump (MD, 1.01 cm; 95% CI, -0.68 to 2.69) and repeated sprint tests (MD, -0.02 seconds; 95% CI, -0.09 to 0.04), as well as reaction time agility test (MD, 0.02 seconds; 95% CI, -0.01 to 0.04) and rating of perceived exertion (MD, 0.16 points; 95% CI, -0.55 to 0.87). Regarding safety, a few minor adverse events were reported. Based on the GRADE approach, the certainty of this evidence was classified as very low to low. CONCLUSION: We found no significant improvement in soccer-related performance with caffeine compared with placebo or no intervention. However, caffeine appears to be safe.