Human T-cell receptor triggering requires inactivation of Lim kinase-1 by Slingshot-1 phosphatase.

Actin dynamics control early T-cell receptor (TCR) signalling during T-cell activation. However, the precise regulation of initial actin rearrangements is not completely understood. Here, we have investigated the regulatory role of the phosphatase Slingshot-1 (SSH1) in this process. Our data show that SSH1 rapidly polarises to nascent cognate synaptic contacts and later relocalises to peripheral F-actin networks organised at the mature immunological synapse. Knockdown of SSH1 expression by CRISPR/Cas9-mediated genome editing or small interfering RNA reveal a regulatory role for SSH1 in CD3ε conformational change, allowing Nck binding and proper downstream signalling and immunological synapse organisation. TCR triggering induces SSH1-mediated activation of actin dynamics through a mechanism mediated by Limk-1 inactivation. These data suggest that during early TCR activation, SSH1 is required for rapid F-actin rearrangements that mediate initial conformational changes of the TCR, integrin organisation and proximal signalling events for proper synapse organisation. Therefore, the SSH1 and Limk-1 axis is a key regulatory element for full T cell activation.

[Recommendations based on the management of patients with Crohn disease in the Peruvian context. Position paper of the Association for the Study of the Intestine (ASPEIN)]. / Recomendaciones basadas en el manejo de pacientes con diagnóstico de enfermedad de Crohn en el contexto peruano. Documento de posición de la Asociación Peruana para el Estudio del Intestino (ASPEIN).

Crohn's disease is a chronic inflammatory disease of the gastrointestinal tract whose etiology is unknown, which can transmurally affect any segment of the intestine and/or the perineal region. Worldwide, the incidence of inflammatory bowel disease has increased in recent decades, and the same upward trend can be seen in South America. At national level, there are no official data, however, the increase in the number of publications in the last 20 years confirms this upward trend. Crohn's disease is a forgotten disease and does not have implemented clinical guidelines based on evidence that contribute to clinicians in decision making. In this sense, the Peruvian Association for the Study of the Intestine considers the preparation of this document relevant and timely. clinical contextualized for Peru.

Combining Loop and Thiazide Diuretics Across the Left Ventricular Ejection Fraction Spectrum: The CLOROTIC Trial.

BACKGROUND: The addition of hydrochlorothiazide (HCTZ) to furosemide in the CLOROTIC (Combining Loop with Thiazide Diuretics for Decompensated Heart Failure) trial improved the diuretic response in patients with acute heart failure (AHF). OBJECTIVES: This work aimed to evaluate if these results differ across the spectrum of left ventricular ejection fraction (LVEF). METHODS: This post hoc analysis of the randomized, double-blind, placebo-controlled CLOROTIC trial enrolled 230 patients with AHF to receive either HCTZ or a placebo in addition to an intravenous furosemide regimen. The influence of LVEF on primary and secondary outcomes was evaluated. RESULTS: The median LVEF was 55%: 166 (72%) patients had LVEF >40%, and 64 (28%) had LVEF ≤40%. Patients with a lower LVEF were younger, more likely to be male, had a higher prevalence of ischemic heart disease, and had higher natriuretic peptide levels. The addition of HCTZ to furosemide was associated with the greatest weight loss at 72 of 96 hours, better metrics of diuretic response, and greater 24-hour diuresis compared with placebo, with no significant differences according to the LVEF category (using 2 LVEF cutoff points: 40% and 50%) or LVEF as a continuous variable (all P values were insignificant). There were no significant differences observed with the addition of HCTZ in terms of mortality, rehospitalizations, or safety endpoints (impaired renal function, hyponatremia, and hypokalemia) among the 2 LVEF groups (all P values were insignificant). CONCLUSIONS: Adding HCTZ to intravenous furosemide seems to be effective strategy for improving diuretic response in AHF without treatment effect modification according to baseline LVEF. (Combining Loop with Thiazide Diuretics for Decompensated Heart Failure [CLOROTIC], NCT01647932; Randomized, double blinded, multicenter study, to asses Safety and Efficacy of the Combination of Loop With Thiazide-type Diuretics vs Loop diuretics with placebo in Patients With Decompensated, EudraCT Number 2013-001852-36).

In-Flow Heterogeneous Triplet-Triplet Annihilation Upconversion.

Photon upconversion based on triplet-triplet annihilation (TTA-UC) is an attractive wavelength conversion with increasing use in organic synthesis in the homogeneous phase; however, this technology has not performed with canonical solid catalysts yet. Herein, a BOPHY dye covalently anchored on silica is successfully used as a sensitizer in a TTA system that efficiently catalyzes Mizoroki-Heck coupling reactions. This procedure has enabled the implementation of in-flow reaction conditions for the synthesis of a variety of aromatic compounds, and mechanistic proof has been obtained by means of transient absorption spectroscopy.

Unlocking Photocatalytic Activity of Acridinium Salts by Anion-Binding Co-Catalysis.

The in situ generation of active photoredox organic catalysts upon anion-binding co-catalysis by making use of the ionic nature of common photosensitizers is reported. Hence, the merge of anion-binding and photocatalysis permitted the modulation of the photocatalytic activity of simple acridinium halide salts, building an effective anion-binding - photoredox ion pair complex able to promote a variety of visible light driven transformations, such as anti-Markovnikov addition to olefins, Diels-Alder and the desilylative C-C bond forming reactions. Anion-binding studies, together with steady-state and time-resolved spectroscopy analysis, supported the postulated ion pair formation between the thiourea hydrogen-bond donor organocatalyst and the acridinium salt, which proved essential for unlocking the photocatalytic activity of the photosensitizer.

Rapid and Accurate Detection of the SARS-CoV-2 Omicron Variant with a CRISPR-Cas12a Reaction in the RT-qPCR Pot.

Gene sequencing in back of reverse transcription-quantitative polymerase chain reaction (RT-qPCR) is the current approach for discriminating infections produced by different severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants in the clinic. However, sequencing is often a time-consuming step, which hinders the deployment of a very fast response during a pandemic. Here, we propose to run a CRISPR-Cas12a reaction after completing the RT-qPCR and in the very same pot to detect with high specificity genetic marks characterizing variants of concern. A crRNA was appropriately designed to detect the S gene of the SARS-CoV-2 Omicron BA.1 variant. A significant response with >20-fold dynamic range was obtained for the Omicron BA.1 S gene, while the Delta S gene did not produce any detectable signal. The sensitivity of the method was analyzed with a series of diluted samples and different Cas12a nucleases. A correlation between the RT-qPCR CT values and the CRISPR-Cas12a reaction signals was observed. Variant discrimination with the CRISPR-Cas12a reaction was possible in some minutes with high accuracy from patient samples. In conclusion, CRISPR-Cas systems seem ready to be exploited in the clinic to boost personalized diagnoses and accelerate epidemiological surveillance in a cost-effective way.

Disparities in HIV Testing and PrEP Awareness for Black Women who Inject Drugs in San Francisco, California.

HIV disproportionately affects Black/African Americans (AA), while PrEP is under-utilized by Black/AA, women, and people who inject drugs (PWID). In San Francisco, California's National HIV Behavioral Surveillance among PWID in 2022, Black/AA women were the least likely to be tested for HIV among all groups by sex and race/ethnicity and the least likely to be aware of PrEP among women. Yet, Black/AA women were no less likely to see a healthcare provider in the last year. Data suggest that providers' failure to discuss and address HIV risk with Black/AA female PWID is a major barrier to accessing effective care and prevention. El VIH afecta de manera desproporcionada a Black/afroamericanos (AA), mientras que la PrEP está infrautilizada por los Black/AA, las mujeres y las personas que se inyectan drogas (PWID). En la National HIV Behavioral Surveillance de PWID de San Francisco, California en 2022, las mujeres Black/AA eran las que menos probabilidades tenían de someterse a la prueba del VIH entre todos los grupos por sexo y raza/etnia y las que menos probabilidades tenían de conocer la PrEP entre las mujeres. Sin embargo, las mujeres Black/AA no tenían menos probabilidades de acudir a un profesional sanitario en el último año. Los datos sugieren que el hecho de que los proveedores no hablen ni aborden el riesgo de VIH con las PWID de raza Black/AA es un obstáculo importante para acceder a una atención y prevención eficaces.

Identification of Smith-Magenis syndrome cases through an experimental evaluation of machine learning methods.

This research work introduces a novel, nonintrusive method for the automatic identification of Smith-Magenis syndrome, traditionally studied through genetic markers. The method utilizes cepstral peak prominence and various machine learning techniques, relying on a single metric computed by the research group. The performance of these techniques is evaluated across two case studies, each employing a unique data preprocessing approach. A proprietary data "windowing" technique is also developed to derive a more representative dataset. To address class imbalance in the dataset, the synthetic minority oversampling technique (SMOTE) is applied for data augmentation. The application of these preprocessing techniques has yielded promising results from a limited initial dataset. The study concludes that the k-nearest neighbors and linear discriminant analysis perform best, and that cepstral peak prominence is a promising measure for identifying Smith-Magenis syndrome.

Continuing Low Awareness and Use of Pre-exposure Prophylaxis (PrEP) for HIV among People Who Inject Drugs (PWID), San Francisco, 2022.

Clinical trials provide evidence that pre-exposure prophylaxis (PrEP) prevents HIV acquisition including through sharing of injection equipment among people who inject drugs (PWID). However, uptake among many populations at risk for HIV has been slow, particularly among PWID. We examined data from the National HIV Behavioral Surveillance (NHBS) from San Francisco in 2022 to measure PrEP uptake and identify factors associated with PrEP awareness among PWID. Of 479 PWID with HIV-negative or unknown HIV status, 54.9% were aware of PrEP, 5.9% had discussed PrEP with a healthcare provider, and 1.5% had used PrEP in the past year. Lack of PrEP awareness was associated with being age 50 years and older (adjusted odds ratio [aOR] 0.40, 95% CI 0.27-0.60), being men who have sex with women (vs. men who have sex with men, aOR 0.47, 95% CI 0.24-0.92), having a disability (aOR 0.58, 95% CI 0.35-0.95), using heroin as their most frequently injected drug (aOR 0.51, 95% CI, 0.34-0.78), not having tested for HIV, HCV, or an STD in the past year (aOR 0.43, 95% CI 0.28-0.64), and not having access to new sterile needles in the past year (aOR 0.28, 95%CI 0.08-1.00). We found negligible change in the awareness and uptake of PrEP among PWID since previously measured in NHBS in 2018. Low PrEP use among PWID may be addressed by increasing provider discussion of PrEP with their PWID patients and clients during routine care, expanding testing for injection-related infections among PWID, and integrating PrEP access into harm reduction programs.

Loop diuretic down-titration at discharge in patients hospitalized for acute heart failure.

AIMS: The current literature provides limited guidance on the best diuretic strategy post-hospitalization for acute heart failure (AHF). It is postulated that the efficacy and safety of the outpatient diuretic regimen may be significantly influenced by the degree of fluid overload (FO) encountered during hospitalization. We hypothesize that in patients with more pronounced FO, reducing their regular oral diuretic dosage might be associated with an elevated risk of unfavourable clinical outcomes. METHODS AND RESULTS: It was a retrospective observational study of 410 patients hospitalized for AHF in which the dose of furosemide at admission and discharge was collected. Patients were categorized across diuretic dose status into two groups: (i) the down-titration group and (ii) the stable/up-titration group. FO status was evaluated by a clinical congestion score and circulating biomarkers. The endpoint of interest was the composite of time to all-cause death and/or heart failure readmission. A multivariable Cox proportional hazard regression model was constructed to analyse the endpoints. The median age was 86 (78-92) years, 256 (62%) were women, and 80% had heart failure with preserved ejection fraction. After multivariate adjustment, the down-titration furosemide equivalent dose remained not associated with the risk of the combined endpoint in the whole sample (hazard ratio 1.34, 95% confidence interval 0.86-2.06, P = 0.184). The risk of the combination of death and/or worsening heart failure associated with the diuretic strategy at discharge was significantly influenced by FO status, including clinical congestion scores and circulating proxies of FO like BNP and cancer antigen 125. CONCLUSIONS: In patients hospitalized for AHF, furosemide down-titration does not imply an increased risk of mortality and/or heart failure readmission. However, FO status modifies the effect of down-titration on the outcome. In patients with severe congestion or residual congestion at discharge, down-titration was associated with an increased risk of mortality and/or heart failure readmission.

Repurposing the mammalian RNA-binding protein Musashi-1 as an allosteric translation repressor in bacteria.

The RNA recognition motif (RRM) is the most common RNA-binding protein domain identified in nature. However, RRM-containing proteins are only prevalent in eukaryotic phyla, in which they play central regulatory roles. Here, we engineered an orthogonal post-transcriptional control system of gene expression in the bacterium Escherichia coli with the mammalian RNA-binding protein Musashi-1, which is a stem cell marker with neurodevelopmental role that contains two canonical RRMs. In the circuit, Musashi-1 is regulated transcriptionally and works as an allosteric translation repressor thanks to a specific interaction with the N-terminal coding region of a messenger RNA and its structural plasticity to respond to fatty acids. We fully characterized the genetic system at the population and single-cell levels showing a significant fold change in reporter expression, and the underlying molecular mechanism by assessing the in vitro binding kinetics and in vivo functionality of a series of RNA mutants. The dynamic response of the system was well recapitulated by a bottom-up mathematical model. Moreover, we applied the post-transcriptional mechanism engineered with Musashi-1 to specifically regulate a gene within an operon, implement combinatorial regulation, and reduce protein expression noise. This work illustrates how RRM-based regulation can be adapted to simple organisms, thereby adding a new regulatory layer in prokaryotes for translation control.

Trajectory of Urine Albumin-Creatinine Ratio in Patients with Acute Heart Failure.

INTRODUCTION: Albuminuria is prevalent in patients with chronic heart failure and is a risk factor for disease progression. However, its clinical meaning in acute heart failure remains elusive. This study analyzed the trajectory of urine albumin to creatinine ratio (UACR) between admission and discharge and its association with decongestion. METHODS: In this prospective observational study, 63 patients were enrolled. UACR, B-type natriuretic peptide (BNP), and clinical congestion score (CCS) were obtained at admission and discharge. We used linear mixed regression analysis to compare changes in the natural logarithm of UACR (logUACR) and its association with changes in markers of decongestion. Estimates were reported as least squares mean with their respective 95% CIs. RESULTS: The median age of the study population was 87 years, 68.5% were women, and 69.8% had a left ventricular ejection fraction >50%. LogUACR at discharge significantly decreased in the overall population compared to admission (Δ -0.47, 95% CI: -0.78 to -0.15, p value = 0.003). The magnitude of UACR drop at discharge was associated with changes in surrogate markers of decongestion. Patients who showed a greater reduction in BNP at discharge exhibited a greater reduction in UACR (p = 0.016). The same trend was also found with clinical decongestion, as assessed by changes in CCS, however, without achieving statistical significance (p = 0.171). UACR change at discharge was not associated with changes in serum creatinine (p value = 0.923). CONCLUSION: In elderly patients with AHF and volume overload, the level of UACR significantly decreased upon discharge compared to admission. This reduction in UACR was closely linked to decreases in BNP.

A new age of precision gene therapy.

Gene therapy has become a clinical reality as market-approved advanced therapy medicinal products for the treatment of distinct monogenetic diseases and B-cell malignancies. This Therapeutic Review aims to explain how progress in genome editing technologies offers the possibility to expand both therapeutic options and the types of diseases that will become treatable. To frame these impressive advances in the context of modern medicine, we incorporate examples from human clinical trials into our discussion on how genome editing will complement currently available strategies in gene therapy, which still mainly rely on gene addition strategies. Furthermore, safety considerations and ethical implications, including the issue of accessibility, are addressed as these crucial parameters will define the impact that gene therapy in general and genome editing in particular will have on how we treat patients in the near future.

An Arabidopsis mutant line lacking the mitochondrial calcium transport regulator MICU shows an altered metabolite profile.

Plant metabolism is constantly changing and requires input signals for efficient regulation. The mitochondrial calcium uniporter (MCU) couples organellar and cytoplasmic calcium oscillations leading to oxidative metabolism regulation in a vast array of species. In Arabidopsis thaliana, genetic deletion of AtMICU leads to altered mitochondrial calcium handling and ultrastructure. Here we aimed to further assess the consequences upon genetic deletion of AtMICU. Our results confirm that AtMICU safeguards intracellular calcium transport associated with carbohydrate, amino acid, and phytol metabolism modifications. The implications of such alterations are discussed.

A human genome editing-based MLL::AF4 ALL model recapitulates key cellular and molecular leukemogenic features.

Cellular ontogeny and MLL breakpoint site influence the capacity of MLL-edited CD34+ hematopoietic cells to initiate and recapitulate infant patients' features in pro-B-cell acute lymphoblastic leukemia (B-ALL). We provide key insights into the leukemogenic determinants of MLL-AF4+ infant B-ALL.

Neuroblastoma RAS viral oncogene homolog (N-RAS) deficiency aggravates liver injury and fibrosis.

Progressive hepatic damage and fibrosis are major features of chronic liver diseases of different etiology, yet the underlying molecular mechanisms remain to be fully defined. N-RAS, a member of the RAS family of small guanine nucleotide-binding proteins also encompassing the highly homologous H-RAS and K-RAS isoforms, was previously reported to modulate cell death and renal fibrosis; however, its role in liver damage and fibrogenesis remains unknown. Here, we approached this question by using N-RAS deficient (N-RAS-/-) mice and two experimental models of liver injury and fibrosis, namely carbon tetrachloride (CCl4) intoxication and bile duct ligation (BDL). In wild-type (N-RAS+/+) mice both hepatotoxic procedures augmented N-RAS expression in the liver. Compared to N-RAS+/+ counterparts, N-RAS-/- mice subjected to either CCl4 or BDL showed exacerbated liver injury and fibrosis, which was associated with enhanced hepatic stellate cell (HSC) activation and leukocyte infiltration in the damaged liver. At the molecular level, after CCl4 or BDL, N-RAS-/- livers exhibited augmented expression of necroptotic death markers along with JNK1/2 hyperactivation. In line with this, N-RAS ablation in a human hepatocytic cell line resulted in enhanced activation of JNK and necroptosis mediators in response to cell death stimuli. Of note, loss of hepatic N-RAS expression was characteristic of chronic liver disease patients with fibrosis. Collectively, our study unveils a novel role for N-RAS as a negative controller of the progression of liver injury and fibrogenesis, by critically downregulating signaling pathways leading to hepatocyte necroptosis. Furthermore, it suggests that N-RAS may be of potential clinical value as prognostic biomarker of progressive fibrotic liver damage, or as a novel therapeutic target for the treatment of chronic liver disease.

Expression of HMGCS2 in intestinal epithelial cells is downregulated in inflammatory bowel disease associated with endoplasmic reticulum stress.

Introduction: The Unfolded Protein Response, a mechanism triggered by the cell in response to Endoplasmic reticulum stress, is linked to inflammatory responses. Our aim was to identify novel Unfolded Protein Response-mechanisms that might be involved in triggering or perpetuating the inflammatory response carried out by the Intestinal Epithelial Cells in the context of Inflammatory Bowel Disease. Methods: We analyzed the transcriptional profile of human Intestinal Epithelial Cell lines treated with an Endoplasmic Reticulum stress inducer (thapsigargin) and/or proinflammatory stimuli. Several genes were further analyzed in colonic biopsies from Ulcerative Colitis patients and healthy controls. Lastly, we generated Caco-2 cells lacking HMGCS2 by CRISPR Cas-9 and analyzed the functional implications of its absence in Intestinal Epithelial Cells. Results: Exposure to a TLR ligand after thapsigargin treatment resulted in a powerful synergistic modulation of gene expression, which led us to identify new genes and pathways that could be involved in inflammatory responses linked to the Unfolded Protein Response. Key differentially expressed genes in the array also exhibited transcriptional alterations in colonic biopsies from active Ulcerative Colitis patients, including NKG2D ligands and the enzyme HMGCS2. Moreover, functional studies showed altered metabolic responses and epithelial barrier integrity in HMGCS2 deficient cell lines. Conclusion: We have identified new genes and pathways that are regulated by the Unfolded Protein Response in the context of Inflammatory Bowel Disease including HMGCS2, a gene involved in the metabolism of Short Chain Fatty Acids that may have an important role in intestinal inflammation linked to Endoplasmic Reticulum stress and the resolution of the epithelial damage.

A variant-dependent molecular clock with anomalous diffusion models SARS-CoV-2 evolution in humans.

The evolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in humans has been monitored at an unprecedented level due to the public health crisis, yet the stochastic dynamics underlying such a process is dubious. Here, considering the number of acquired mutations as the displacement of the viral particle from the origin, we performed biostatistical analyses from numerous whole genome sequences on the basis of a time-dependent probabilistic mathematical model. We showed that a model with a constant variant-dependent evolution rate and nonlinear mutational variance with time (i.e., anomalous diffusion) explained the SARS-CoV-2 evolutionary motion in humans during the first 120 wk of the pandemic in the United Kingdom. In particular, we found subdiffusion patterns for the Primal, Alpha, and Omicron variants but a weak superdiffusion pattern for the Delta variant. Our findings indicate that non-Brownian evolutionary motions occur in nature, thereby providing insight for viral phylodynamics.

Prognostic significance of plasma chloride in elderly patients hospitalized for acute heart failure.

AIMS: Previous studies demonstrated the relationship between hypochloraemia and poor prognosis in patients hospitalized for acute heart failure (AHF). However, the usefulness of chloride in clinical practice remains uncertain, notably in very old patients with predominantly heart failure (HF) with preserved ejection fraction (HFpEF). We aimed to evaluate the prognostic impact of chloride in a cohort of very aged patients with AHF and the possible existence of different phenotypes of hypochloraemia with distinct clinical significance. METHODS AND RESULTS: It was an observational study of 429 patients hospitalized for AHF in which chloraemia was measured. Two different phenotypes of hypochloraemia were identified by their relationship with estimated plasma volume status (ePVS) as a proxy of intravascular congestion. The endpoint of interest was time to all-cause mortality and the composite of death and/or HF readmission. A multivariable Cox proportional hazard regression model was constructed to analyse the endpoints. The median age was 85 (78-92) years, 266 (62%) were women, and 80% had HFpEF. After multivariable analysis, chloraemia, but not natraemia, was associated with the risk of death and HF readmission in a U-shaped pattern. The phenotype characterized by hypochloraemia and low ePVS (depletional) was associated with an increased risk of mortality when compared with patients with normochloraemia [hazard ratio (HR) 1.86, P = 0.008]. In contrast, hypochloraemia with high ePVS (dilutional) had no prognostic significance (HR 0.94, P = 0.855). CONCLUSIONS: In very old patients hospitalized with AHF, plasma chloride was associated with the risk of death and HF readmission in a U-shaped pattern and could potentially be used for congestion phenotyping.