Comprehensive transcription factor perturbations recapitulate fibroblast transcriptional states.

Cell atlas projects have nominated recurrent transcriptional states as drivers of biological processes and disease, but their origins, regulation, and properties remain unclear. To enable complementary functional studies, we developed a scalable approach for recapitulating cell states in vitro using CRISPR activation (CRISPRa) Perturb-seq. Aided by a novel multiplexing method, we activated 1,836 transcription factors in two cell types. Measuring 21,958 perturbations showed that CRISPRa activated targets within physiological ranges, that epigenetic features predicted activatable genes, and that the protospacer seed region drove an off-target effect. Perturbations recapitulated in vivo fibroblast states, including universal and inflammatory states, and identified KLF4 and KLF5 as key regulators of the universal state. Inducing the universal state suppressed disease-associated states, highlighting its therapeutic potential. Our findings cement CRISPRa as a tool for perturbing differentiated cells and indicate that in vivo states can be elicited via perturbation, enabling studies of clinically relevant states ex vivo.

Progression of subclinical cardiovascular disease in patients with HIV.

OBJECTIVE: Human immunodeficiency virus (HIV) infected patients are at increased risk of cardiovascular disease (CVD). Multidetector computed tomography (MDCT) stratifies cardiovascular risk in asymptomatic patients with subclinical atherosclerosis. The aim of this study was to determine the ability of MCTD and clinical and laboratory parameters to assess subclinical CVD progression in HIV patients. METHODS: Prospective longitudinal cohort study of patients with at least 10 years of HIV infection and 5 years of antiretroviral therapy history, low cardiovascular risk and monitored for 6 years (2015-2021). All patients underwent clinical assessment, blood analysis, carotid ultrasound, and gated MDCT in 2015 and 2021. RESULTS: Sixty-three patients (63.5% male) with a mean age of 49.9 years (standard deviation [SD], 10.5) were included in 2015; 63 of them were followed until 2021. Comparing the results from 2015 with those from 2021, Systematic Coronary Risk Estimation-2 (SCORE2) was 2.9% (SD, 2.1) vs. 4.4% (SD,3.1); Multi-Ethnic Study of Atherosclerosis score (MESA risk) was 3.4 (SD 5.8) vs. 6.0 (SD 8.6); coronary artery calcification CAC) score >100 was 11.1% vs. 25.4% (P < 0.05); and 11% vs. 27% had carotid plaques (P = 0.03). CONCLUSIONS: After six years of follow-up, an increase in SCORE2, carotid plaques, CAC scoring and MESA risk was observed. MDCT findings, along with other clinical and laboratory parameters, could play an important role as a marker of CVD progression in the evaluation of patients with HIV and low cardiovascular risk.

Identification of Surfactant Impact on a Monoclonal Antibody Characterization via HPLC-Separation Based and Biophysical Methods.

PURPOSE OR OBJECTIVE: Surfactants, including polysorbates and poloxamers, play a crucial role in the formulation of therapeutic proteins by acting as solubilizing and stabilizing agents. They help prevent protein aggregation and adsorption, thereby enhancing the stability of drug substance and products., However, it is important to note that utilizing high concentrations of surfactants in protein formulations can present significant analytical challenges, which can ultimately affect the product characterization. METHODS: In our study, we specifically investigated the impact of elevated surfactant concentrations on the characterization of monoclonal antibodies. We employed various analytical techniques including size-exclusion chromatography (SEC), capillary electrophoresis (CE-SDS), a cell based functional assay, and biophysical characterization. RESULTS: The findings of our study indicate that higher levels of Polysorbate 80 (PS-80) have adverse effects on the measured purity, biological activity, and biophysical characterization of biologic samples. Specifically, the elevated levels of PS-80 cause analytical interferences, which can significantly impact the accuracy and reliability of analytical studies. CONCLUSIONS: Our study results highlight a significant risk in analytical investigations, especially in studies involving the isolation and characterization of impurities. It is important to be cautious of surfactant concentrations, as they can become more concentrated during common sample manipulations like buffer exchange. Indeed, the research presented in this work emphasizes the necessity to evaluate the impact on analytical assays when there are substantial alternations in the matrix composition. By doing so, valuable insights can be gained regarding potential challenges associated with assay development and characterization of biologics with complex formulations.

Drug-resistant EGFR mutations promote lung cancer by stabilizing interfaces in ligand-free kinase-active EGFR oligomers.

The Epidermal Growth Factor Receptor (EGFR) is frequently found to be mutated in non-small cell lung cancer. Oncogenic EGFR has been successfully targeted by tyrosine kinase inhibitors, but acquired drug resistance eventually overcomes the efficacy of these treatments. Attempts to surmount this therapeutic challenge are hindered by a poor understanding of how and why cancer mutations specifically amplify ligand-independent EGFR auto-phosphorylation signals to enhance cell survival and how this amplification is related to ligand-dependent cell proliferation. Here we show that drug-resistant EGFR mutations manipulate the assembly of ligand-free, kinase-active oligomers to promote and stabilize the assembly of oligomer-obligate active dimer sub-units and circumvent the need for ligand binding. We reveal the structure and assembly mechanisms of these ligand-free, kinase-active oligomers, uncovering oncogenic functions for hitherto orphan transmembrane and kinase interfaces, and for the ectodomain tethered conformation of EGFR. Importantly, we find that the active dimer sub-units within ligand-free oligomers are the high affinity binding sites competent to bind physiological ligand concentrations and thus drive tumor growth, revealing a link with tumor proliferation. Our findings provide a framework for future drug discovery directed at tackling oncogenic EGFR mutations by disabling oligomer-assembling interactions.

Co-delivery of PD-L1- and EGFR-targeting siRNAs by synthetic PEG12-KL4 peptide to the lungs as potential strategy against non-small cell lung cancer.

BACKGROUND: Small interfering RNA (siRNA) holds great promise for treating various lung diseases, but the lack of safe and efficient pulmonary siRNA delivery systems has hindered its advance into the clinics. The epidermal growth factor receptor (EGFR) which promotes cell proliferation, and the programmed cell death ligand 1 (PD-L1) which plays a crucial role in suppressing cytotoxic T cells activity, are two important targets for treating non-small cell lung cancer (NSCLC). Here, we explored the potential of PEG12-KL4, a synthetic peptide, to deliver siRNA to various NSCLC cells and to lung tissues in mice. METHODS: PEG12-KL4 was used to transfect siRNAs targeted at both EGFR and PD-L1 into NSCLC cells. Immunoblotting was used to evaluate the siRNA silencing effects in HCC827 and NCI-H1975 NSCLC cells. CD8+ T cell-mediated NSCLC cell killing was employed to demonstrate the functional effects of PD-L1 siRNA knock-down. Fluorescent siRNAs were used to visualise siRNA uptake in cells as well as to enable biodistribution studies in BALB/c mice. RESULTS: Our results showed that PEG12-KL4 was efficient in mediating siRNA knock-down of EGFR and PD-L1 in various NSCLC cells. Importantly, the PEG12-KL4 peptide enabled significantly better siRNA delivery than the commercial Lipofectamine 2000 reagent. We hypothesised that PEG12-KL4 peptide enabled siRNA to either escape from or bypass endosomal degradation as indicated by confocal fluorescence imaging. Notably, combined knock-down of EGFR and PD-L1 in NCI-H1975 cells resulted in better effector T cell-mediated cancer cell killing than knock-down of PD-L1 alone. Moreover, biodistribution of PEG12-KL4/siRNA complexes following intravenous administration revealed poor lung delivery with the fluorescent siRNA accumulating in the liver. In contrast, intratracheal delivery of PEG12-KL4/siRNA complexes resulted in the fluorescent siRNA to be detected in the lung with retarded renal excretion. CONCLUSION: In conclusion, we demonstrated that the co-delivery of siRNAs targeting EGFR and PD-L1 using PEG12-KL4 is feasible and represents a promising future strategy to treat NSCLC, whereby pulmonary siRNA delivery is favourable to intravenous administration.

Infecciones del tracto urinario bajo en adultos y embarazadas: consenso para el manejo empírico / Lower Urinary Tract Infections in Adults and Pregnant Women: A Consensus for Empirical Treatment

Introducción: La infección no complicada del tracto urinario bajo y la bacteriuria asintomática son causas frecuentes de consulta médica ambulatoria y en el servicio de urgencias en Colombia y el mundo. La falta de pautas y consenso para el manejo, así como la emergencia de resistencia a las múltiples opciones terapéuticas disponibles en los uropatógenos provenientes de la comunidad, hacen necesario elaborar unas recomendaciones que orienten al clínico sobre el abordaje óptimo de estas entidades. Objetivo: Definir un consenso sobre el manejo empírico de la bacteriuria asintomática y la infección del tracto urinario (ITU) bajo en adultos y mujeres embarazadas en Colombia. Metodología: Se lleva a cabo una metodología de consenso con expertos en urología, infectología, medicina interna, ginecología y microbiología basada en la revisión de las referencias bibliográficas disponibles en los términos de búsqueda relacionados, haciendo énfasis en estudios locales. Resultados: Se elaboró un algoritmo de manejo para el tratamiento empírico de la ITU baja no complicada en adultos con antisépticos urinarios como nitrofurantoína y fosfomicina trometamol como primera línea, con recomendaciones terapéuticas específicas para el tratamiento de la bacteriuria asintomática en mujeres embarazadas. Adicionalmente, se formuló un algoritmo de decisión para el procesamiento de cultivos de orina. La recurrencia o recaída frecuente justifica remisión a urología e infectología. Conclusiones: Se generan recomendaciones prácticas de fácil implementación en el diagnóstico y manejo de la ITU bajo en adultos y embarazadas, y de los casos donde es necesario tratar la bacteriuria asintomática, con opciones terapéuticas efectivas y de espectro reducido.

Background: Uncomplicated infection of the lower urinary tract and asymptomatic bacteriuria are frequent causes of visits to outpatient clinics and emergency departments in Colombia and worldwide. The lack of guidelines and a consensus for their management, and the emergence of resistance of community-based uropathogens to the available therapeutic options, make it necessary to develop recommendations to guide clinicians on the optimal approach to these entities. Objective: Define a consensus for the empiric management of asymptomatic bacteriuria and Urinary Tract Infection (UTI) in adults and pregnant women in Colombia. Methods: We carried out a consensus methodology with experts in urology, infectious diseases, internal medicine, gynecology and clinical microbiology; based on reviewing the available literature on the related terms, and emphasizing local studies. Results: We developed a management algorithm for the empirical treatment of uncomplicated lower UTI in adults with urinary antiseptics such as nitrofurantoin and fosfomycin trometamol as first line, with specific therapeutic recommendations for the treatment of asymptomatic bacteriuria in pregnant women. Additionally, we created a decision algorithm for processing urine cultures. Frequent recurrence or relapse justifies referral to urology and infectious disease professionals. Conclusions: We generated straightforward and easy-to-implement recommendations for the diagnosis and management of UTI in adults and pregnant women, and in cases where it is necessary to treat asymptomatic bacteriuria, with effective therapeutic and narrow spectrum options.

Diagnosis and therapeutic considerations of uterine infections in dairy cattle: [review] / Consideraciones diagnósticas y terapéuticas para infecciones uterinas en vacas lecheras: [revisión]

From an animal health-well being, welfare, and performance perspective, the postpartum period is comprised of an early window where acute health and cow survival is an issue with the development of septic metritis. Subsequently, sustained presence of endometritis is likely to be contributing to sub-fertility in lactating dairy cows with an overall herd pregnancy rate of approximately 16%. The challenge is to integrate on commercial dairies preventive medicine programs with reproductive management to improve herd fertility. Cows affected by periparturient disorders such as hypocalcemia, dystocia, and retained fetal membranes are more likely to contract uterine infections than cows that calve normally are. Thus, appropriate management of the transition period is critical in the prevention of uterine infections at the herd level. In addition, a postpartum program to monitor health to provide treatment to cows in the early stages of disease should be implemented.

Desde el punto de vista del bienestar animal y de la perspectiva de productividad, el periodo postparto puede estar comprometido por una pequeña ventana durante el postparto temprano, en la cual la salud y supervivencia de las vacas es un problemática, debido a la presentación de metritis séptica. Esta se perpetuán subsecuentemente con la presentación de endometritis, la cual muy posiblemente contribuye con los patrones de sub-fertilidad en vacas lactantes, generando en general bajas tasas de preñez del 16 %. El reto es entonces integrar en las explotaciones comerciales un programa de medicina veterinaria preventiva con el manejo reproductivo, con el fin de mejorar la fertilidad del hato. Se ha descrito que las vacas que sufren problemas durante el periparto tales como hipocalcemia y retención de las membranas fetales son más susceptibles de contraer infecciones uterinas, comparadas con aquellas vacas que tienen un parto normal. Por ello un manejo apropiado del periodo de transición es crítico en la prevención de infecciones uterinas a nivel del hato. Así mismo, es necesario implementar un programa adecuado de monitoreo postparto con el fin de proveer tratamiento oportuno a las vacas durante los estados iniciales de enfermedad.

Indice de compromiso cumplido ICC Bolivia: un instrumento de control ciudadano / Index of fulfilled commitment ICC Bolivia: a citizen control instrument

El Indice de Compromiso Cumplido (ICC) es un instrumento técnico-político que esta orientado a medir el cumplimiento de los compromisos nacionales e internacionales que ha suscrito el gobierno boliviano en materia de equidad de género y adelanto de las mujeres

Indice de compromiso cumplido ICC Bolivia: un instrumento de control ciudadano ( resumen) / Index of fulfilled commitment ICC Bolivia: a citizen control instrument (summary)

El ICC es un sistema de indicadores relacionados entre si: contiene tres áreas temáticas estratégicas: participación ciudadana y acceso al poder; autonomia y empleo; salud de la mujer y derechos sexuales y reproductivos. asignando valores a cada una de las áreas y a su vez da un valor final a los distintos indicadores que los componen: indicadores de voluntad política, de proceso y de resultado