Deciphering the "m6A Code" via Antibody-Independent Quantitative Profiling.

N6-methyladenosine (m6A) is the most abundant modification on mRNA and is implicated in critical roles in development, physiology, and disease. A major limitation has been the inability to quantify m6A stoichiometry and the lack of antibody-independent methodologies for interrogating m6A. Here, we develop MAZTER-seq for systematic quantitative profiling of m6A at single-nucleotide resolution at 16%-25% of expressed sites, building on differential cleavage by an RNase. MAZTER-seq permits validation and de novo discovery of m6A sites, calibration of the performance of antibody-based approaches, and quantitative tracking of m6A dynamics in yeast gametogenesis and mammalian differentiation. We discover that m6A stoichiometry is "hard coded" in cis via a simple and predictable code, accounting for 33%-46% of the variability in methylation levels and allowing accurate prediction of m6A loss and acquisition events across evolution. MAZTER-seq allows quantitative investigation of m6A regulation in subcellular fractions, diverse cell types, and disease states.

txtools: an R package facilitating analysis of RNA modifications, structures, and interactions.

We present txtools, an R package that enables the processing, analysis, and visualization of RNA-seq data at the nucleotide-level resolution, seamlessly integrating alignments to the genome with transcriptomic representation. txtools' main inputs are BAM files and a transcriptome annotation, and the main output is a table, capturing mismatches, deletions, and the number of reads beginning and ending at each nucleotide in the transcriptomic space. txtools further facilitates downstream visualization and analyses. We showcase, using examples from the epitranscriptomic field, how a few calls to txtools functions can yield insightful and ready-to-publish results. txtools is of broad utility also in the context of structural mapping and RNA:protein interaction mapping. By providing a simple and intuitive framework, we believe that txtools will be a useful and convenient tool and pave the path for future discovery. txtools is available for installation from its GitHub repository at https://github.com/AngelCampos/txtools.

Multiplexed profiling facilitates robust m6A quantification at site, gene and sample resolution.

N6-methyladenosine (m6A) is the most prevalent modification of messenger RNA in mammals. To interrogate its functions and dynamics, there is a critical need to quantify m6A at three levels: site, gene and sample. Current approaches address these needs in a limited manner. Here we develop m6A-seq2, relying on multiplexed m6A-immunoprecipitation of barcoded and pooled samples. m6A-seq2 allows a big increase in throughput while reducing technical variability, requirements of input material and cost. m6A-seq2 is furthermore uniquely capable of providing sample-level relative quantitations of m6A, serving as an orthogonal alternative to mass spectrometry-based approaches. Finally, we develop a computational approach for gene-level quantitation of m6A. We demonstrate that using this metric, roughly 30% of the variability in RNA half life in mouse embryonic stem cells can be explained, establishing m6A as a main driver of RNA stability. m6A-seq2 thus provides an experimental and analytic framework for dissecting m6A-mediated regulation at three different levels.

Visual-functional impact of people affected by severe ocular trauma during social protests in Chile in 2019.

PURPOSE: To determine the impact on the functionality associated with visual loss (VFIP) in people with severe ocular trauma (SOT) caused by kinetic impact projectiles used in police crowd control through a prioritization tool in people admitted to a rehabilitation program in Santiago de Chile from December 02, 2019, to November 13, 2020. METHODS: A cross-sectional descriptive study of SOT victims (N = 85), average age 31.4 ± 11.9. The data were recorded through a new 9-item screening instrument for assessment and prioritization of rehabilitation created for this emergency scenario. RESULTS: The impact of the use of kinetic weapons resulted in monocular blindness in the majority of those affected (n = 68; 80.0%). The highest VFIP observed was among young men from lower social strata. There were extreme difficulties in the performance of productive tasks (occupational and/or educational) (n = 42; 49.4%) and the pursuit of hobbies and pastimes (n = 23; 27.1%), as well as a high difficulty in adapting to changes in brightness (n = 29; 34.1%) and handling objects accurately (n = 22; 25.9%). CONCLUSION: The use of kinetic weapons for crowd control resulted in high and extreme VFIP and, in most cases, monocular blindness, causing major difficulties in work, study, and development of hobbies and pastimes in the affected population, highlighting the urgent need for effective rehabilitative care, which requires special attention in order to generate an adequate rehabilitation program. The use of kinetic weapons for crowd control contravenes international goals, policies, and plans set by the WHO and the International Agency for the Prevention of Blindness on strategies to prevent avoidable blindness worldwide until 2020. It is essential to ban the use of these weapons in Chile and worldwide, as well as to revise police protocols for crowd control.

Tolerance to Oxidative Stress in Budding Yeast by Heterologous Expression of Catalases A and T from Debaryomyces hansenii.

The function of catalases A and T from the budding yeast Saccharomyces cerevisiae (ScCta1 and ScCtt1) is to decompose hydrogen peroxide (H2O2) to mitigate oxidative stress. Catalase orthologs are widely found in yeast, suggesting that scavenging H2O2 is crucial to avoid the oxidative damage caused by reactive oxygen species (ROS). However, the function of catalase orthologs has not yet been experimentally characterized in vivo. Here, we heterologously expressed Debaryomyces hansenii DhCTA1 and DhCTT1 genes, encoding ScCta1 and ScCtt1 orthologs, respectively, in a S. cerevisiae acatalasemic strain (cta1Δ ctt1Δ). We performed a physiological analysis evaluating growth, catalase activity, and H2O2 tolerance of the strains grown with glucose or ethanol as carbon source, as well as under NaCl stress. We found that both genes complement the catalase function in S. cerevisiae. Particularly, the strain harboring DhCTT1 showed improved growth when ethanol was used as carbon source both in the absence or presence of salt stress. This phenotype is attributed to the high catalase activity of DhCtt1 detected at the exponential growth phase, which prevents intracellular ROS accumulation and confers oxidative stress resistance.

Functional impact of multi-omic interactions in lung cancer.

Lung tumors are a leading cause of cancer-related death worldwide. Lung cancers are highly heterogeneous on their phenotypes, both at the cellular and molecular levels. Efforts to better understand the biological origins and outcomes of lung cancer in terms of this enormous variability often require of high-throughput experimental techniques paired with advanced data analytics. Anticipated advancements in multi-omic methodologies hold potential to reveal a broader molecular perspective of these tumors. This study introduces a theoretical and computational framework for generating network models depicting regulatory constraints on biological functions in a semi-automated way. The approach successfully identifies enriched functions in analyzed omics data, focusing on Adenocarcinoma (LUAD) and Squamous cell carcinoma (LUSC, a type of NSCLC) in the lung. Valuable information about novel regulatory characteristics, supported by robust biological reasoning, is illustrated, for instance by considering the role of genes, miRNAs and CpG sites associated with NSCLC, both novel and previously reported. Utilizing multi-omic regulatory networks, we constructed robust models elucidating omics data interconnectedness, enabling systematic generation of mechanistic hypotheses. These findings offer insights into complex regulatory mechanisms underlying these cancer types, paving the way for further exploring their molecular complexity.

Dissecting the sequence and structural determinants guiding m6A deposition and evolution via inter- and intra-species hybrids.

BACKGROUND: N6-methyladenosine (m6A) is the most abundant mRNA modification, and controls mRNA stability. m6A distribution varies considerably between and within species. Yet, it is unclear to what extent this variability is driven by changes in genetic sequences ('cis') or cellular environments ('trans') and via which mechanisms. RESULTS: Here we dissect the determinants governing RNA methylation via interspecies and intraspecies hybrids in yeast and mammalian systems, coupled with massively parallel reporter assays and m6A-QTL reanalysis. We find that m6A evolution and variability is driven primarily in 'cis', via two mechanisms: (1) variations altering m6A consensus motifs, and (2) variation impacting mRNA secondary structure. We establish that mutations impacting RNA structure - even when distant from an m6A consensus motif - causally dictate methylation propensity. Finally, we demonstrate that allele-specific differences in m6A levels lead to allele-specific changes in gene expression. CONCLUSIONS: Our findings define the determinants governing m6A evolution and diversity and characterize the consequences thereof on gene expression regulation.

Does PLEX® Elite 9000 OCT Identify and Characterize Most Posterior Pole Lesions in Highly Myopic Patients?

High myopia (HM) is defined as an axial length (AL) ≥ 26 mm that may result in various pathologies that constitute pathologic myopia (PM). The PLEX® Elite 9000 (Carl Zeiss AC, Jena, Germany) is a new swept-source optical coherence tomography (SS-OCT) underdevelopment that allows wider, deeper and more detailed posterior-segment visualization; it can acquire ultra-wide OCT angiography (OCTA) or new ultra-wide high-density scans in one image. We assessed the technology's ability to identify/characterize/quantify staphylomas and posterior pole lesions or image biomarkers in highly myopic Spanish patients and estimate the technology's potential to detect macular pathology. The instrument acquired 6 × 6 OCTA, 12 × 12 or 6 × 6 OCT cubes, and at least two high-definition spotlight single scans. A hundred consecutive patients (179 eyes; age, 51.4 ± 16.8 years; AL, 28.8 ± 2.33 mm) were recruited in one center for this prospective observational study. Six eyes were excluded because images were not acquired. The most common alterations were perforating scleral vessels (88.8%), classifiable staphyloma (68.7%), vascular folds (43%), extrafoveal retinoschisis (24%), dome-shaped macula (15.6%), and more uncommonly, scleral dehiscence (4.46%), intrachoroidal cavitation (3.35%), and macular pit (2.2%). The retinal thickness of these patients decreased, and the foveal avascular zone increased in the superficial plexus compared with normal eyes. SS-OCT is a novel potent tool that can detect most main posterior pole complications in PM and may provide us with a better understanding of the associated pathologies; some pathologies were identifiable only with this new kind of equipment, such as perforating scleral vessels, which seem to be the most common finding and not so frequently related to choroidal neovascularization, as previously reported.

Un procedimiento algebraico para la estimación de la demanda de servicios de salud depués de los sismos / An algebraic method to calculate the future demand of health services after the earthquake

A partir de los sismos de 1985 que afectaron a la ciudad de México, se planteó la necesidad de analizar el futuro mediato del sistema de salud en el área metropolitana. En este trabajo se describe un procedimiento algebraico que se utiliza para estimar la evolución de la demanda futura de algunos servicios de salud por un periodo de cinco años, con base en un crecimiento exponencial de la población

Prospectiva de los servicios de salud a partir de los sismos de septiembre de 1985 / Perspectives health services in the Federal District after the earthquakes of September, 1985

Se presentan algunos resultados del "Grupo de Prospectiva del Sector Salud" formado a raíz de los sismos de septiembre de 1985. Se analizan las características de los daños a la salud en el Distrito Federal y se estudian las consecuencias de diferentes alternativas y estrategias de reconstrucción. Se elabora un modelo prospectivo enfocado en los servicios de salud a la población abierta. En él se analiza alternativa de reforzamiento del nivel primario de atención, como forma de disminuir la hospitalización de segundo nivel, examinándose una posible disminución de un tercio en la relación consulta externa/ingresos hospitalarios. También se analiza una estrategia de redistribución de los servicios hospitalarios en el Distrito Federal, con un reforzamiento de servicios integrales regionalizados. Se eleaboran proyecciones a seis meses y dos años en cuanto a la demanda y oferta de camas hospitalarias de segundo nivel. Por último, se estudia brevemente la posibilidad de la unificación funcional de los servicios de hospitalización para población abierta