Non-Invasive Biomarkers for Duchenne Muscular Dystrophy and Carrier Detection.

Non-invasive biological indicators of the absence/presence or progress of the disease that could be used to support diagnosis and to evaluate the effectiveness of treatment are of utmost importance in Duchenne Muscular Dystrophy (DMD). This neuromuscular disorder affects male children, causing weakness and disability, whereas female relatives are at risk of being carriers of the disease. A biomarker with both high sensitivity and specificity for accurate prediction is preferred. Until now creatine kinase (CK) levels have been used for DMD diagnosis but these fail to assess disease progression. Herein we examined the potential applicability of serum levels of matrix metalloproteinase 9 and matrix metalloproteinase 2, tissue inhibitor of metalloproteinases 1, myostatin (GDF-8) and follistatin (FSTN) as non-invasive biomarkers to distinguish between DMD steroid naïve patients and healthy controls of similar age and also for carrier detection. Our data suggest that serum levels of MMP-9, GDF-8 and FSTN are useful to discriminate DMD from controls (p < 0.05), to correlate with some neuromuscular assessments for DMD, and also to differentiate between Becker muscular dystrophy (BMD) and Limb-girdle muscular dystrophy (LGMD) patients. In DMD individuals under steroid treatment, GDF-8 levels increased as FSTN levels decreased, resembling the proportions of these proteins in healthy controls and also the baseline ratio of patients without steroids. GDF-8 and FSTN serum levels were also useful for carrier detection (p < 0.05). Longitudinal studies with larger cohorts are necessary to confirm that these molecules correlate with disease progression. The biomarkers presented herein could potentially outperform CK levels for carrier detection and also harbor potential for monitoring disease progression.

Were metabolic and other chronic diseases the driven onset epidemic forces of COVID-19 in Mexico?

The underline hypothesis of this study was that SARS-CoV-2 can infect individuals regardless of health condition, sex, and age in opposition to the classical epidemiological assumption of an identifiable susceptible subpopulation for epidemic development. To address this issue, a population cohort with 24.4 million metadata associated with 226,089 official RT-qPCR positive and 283,450 negative cases, including 27,769 deceased, linked putatively to B.1. and B.1.1. SARS-CoV-2 lineages were analyzed. The analysis baseline was to determine the infection and mortality structure of the diseased cohort at the onset-exponential phase of the first epidemic wave in Mexico under the assumption of limited herd immunity. Individuals with nonchronic diseases (NOCDs) were compared with those exhibiting at least one of 10 chronic diseases (CDs) adjusted by age and sex. Risk factors for infection and mortality were estimated with classification and regression tree (CART) and cluster analysis based on Spearman's matrix of rho-values in RStudio®, complemented with two proposed mortality indices. SARS-CoV-2 infection was independent of health condition (52.8% NOCD vs. 47.2% CDs; p = 0.001-0.009) but influenced by age >46 in one risk analysis scenario (p < 0.001). Sex contributed 9.7% to the overall risk. The independent effect was supported by the health structure of negative cases with a similar tendency but a higher proportion of NOCDs (61.4%, p = 0.007). The infection probability in individuals with one CD was determined by the disease type and age, which was higher in those older individuals (≥56 years) exhibiting diabetes (12.3%, cp = 0.0006), hypertension (10.1%, cp < 0.0001), and obesity (7.8%, cp = 0.001). In contrast, the mortality risk was heavily influenced by CD conditioned by sex and age, accounting for 72.3% of total deaths (p = 0.001-0.008). Significant mortality risk (48%) was comprised of women and men (w, m) aged ≥56 years with diabetes (19% w and 27.9% m, cp < 0.0004), hypertension (11.5% w, cp = 0.0001), and CKD (3.5% w and 5.3% m, cp = 0.0009). Older people with diabetes and hypertension comorbidity increased the risk to 60.5% (p = 0.001). Based on a mortality-weighted index, women were more vulnerable to preexisting metabolic or cardiovascular diseases. These findings support our hypothesis and justify the need for surveillance systems at a communitarian level. This is the first study addressing this fundamental epidemiological question.

Transcriptome analysis identifies genes involved in ethanol response of Saccharomyces cerevisiae in Agave tequilana juice.

During ethanol fermentation, yeast cells are exposed to stress due to the accumulation of ethanol, cell growth is altered and the output of the target product is reduced. For Agave beverages, like tequila, no reports have been published on the global gene expression under ethanol stress. In this work, we used microarray analysis to identify Saccharomyces cerevisiae genes involved in the ethanol response. Gene expression of a tequila yeast strain of S. cerevisiae (AR5) was explored by comparing global gene expression with that of laboratory strain S288C, both after ethanol exposure. Additionally, we used two different culture conditions, cells grown in Agave tequilana juice as a natural fermentation media or grown in yeast-extract peptone dextrose as artificial media. Of the 6368 S. cerevisiae genes in the microarray, 657 genes were identified that had different expression responses to ethanol stress due to strain and/or media. A cluster of 28 genes was found over-expressed specifically in the AR5 tequila strain that could be involved in the adaptation to tequila yeast fermentation, 14 of which are unknown such as yor343c, ylr162w, ygr182c, ymr265c, yer053c-a or ydr415c. These could be the most suitable genes for transforming tequila yeast to increase ethanol tolerance in the tequila fermentation process. Other genes involved in response to stress (RFC4, TSA1, MLH1, PAU3, RAD53) or transport (CYB2, TIP20, QCR9) were expressed in the same cluster. Unknown genes could be good candidates for the development of recombinant yeasts with ethanol tolerance for use in industrial tequila fermentation.

Surveillance Web System and Mouthwash-Saliva qPCR for Labor Ambulatory SARS-CoV-2 Detection and Prevention.

This study provides a safe and low-cost in-house protocol for RT-qPCR-based detection of SARS-CoV-2 using mouthwash-saliva self-collected specimens to achieve clinical and epidemiological surveillance in a real-time web environment applied to ambulatory populations. The in-house protocol comprises a mouthwash-saliva self-collected specimen, heat virus inactivation, and primers to target virus N-gene region and the human RPP30-gene. Aligning with 209 SARS-CoV-2 sequences confirmed specificity including the Alpha variant from the UK. Development, validation, and statistical comparison with official nasopharyngeal swabbing RT-qPCR test were conducted with 115 specimens of ambulatory volunteers. A web-mobile application platform was developed to integrate a real-time epidemiological and clinical core baseline database with mouthwash-saliva RT-qPCR testing. Nine built-in algorithms were generated for decision-making on testing, confining, monitoring, and self-reports to family, social, and work environments. Epidemiological and clinical follow-up and SARS-CoV-2 testing generated a database of 37,351 entries allowing individual decision-making for prevention. Mouthwash-saliva had higher sensitivity than nasopharyngeal swabbing in detecting asymptomatic and mild symptomatic cases with 720 viral copy number (VCN)/mL as the detection limit (Ct = 37.6). Cycling threshold and viral loading were marginally different (p = 0.057) between asymptomatic (35 Ct ± 2.8; 21,767.7 VCN/mL, range 720-77,278) and symptomatic (31.3 Ct ± 4.5; 747,294.3 VCN/mL, range 1433.6-3.08 × 106). We provided proof-of-concept evidence of effective surveillance to target asymptomatic and moderate symptomatic ambulatory individuals based on integrating a bio-safety level II laboratory, self-collected, low-risk, low-cost detection protocol, and a real-time digital monitoring system. Mouthwash-saliva was effective for SARS-CoV-2 sampling for the first time at the community level.

Characterization of genetic diversity and clonal complexes by whole genome sequencing of Mycobacterium tuberculosis isolates from Jalisco, Mexico.

Whole genome sequencing (WGS) analysis in tuberculosis allows the prediction of drug-resistant phenotypes, identification of lineages, and to better understanding of the epidemiology and transmission chains. Nevertheless the procedure has been scarcely assessed in Mexico, in this work we analyze by WGS isolates of Mycobacterium tuberculosis circulating in Jalisco, Mexico. Lineage and phylogenetic characterization, drug resistant prediction, "in silico" spoligotyping determination, were provided by WGS in 32 M. tuberculosis clinical isolates. Lineage 4 (L4), with 28 isolates (87%) and eleven sublineages was dominant. Forty SNPs and INDELs were found in genes related to first-, and second-line drugs. Eleven isolates were sensitive, seven (22%) were predicted to be resistant to isoniazid, two resistant to rifampicin (6%) and two (6%) were multidrug-resistant tuberuclosis. Spoligotyping shows that SIT 53 (19%) and SIT 119 (16%) were dominant. Four clonal transmission complexes were found. This is the first molecular epidemiological description of TB isolates circulating in western Mexico, achieved through WGS. L4 was dominant and included a high diversity of sublineages. It was possible to track the transmission route of two clonal complexes. The WGS demonstrated to be of great utility and with further implications for clinical and epidemiological study of TB in the region.

The First exploratory spatial distribution analysis of tuberculosis and associated factors in Tonala, Mexico.

INTRODUCTION: The US-Mexico region is at high risk of elevated tuberculosis (TB) incidence due to mobility and migration. Knowledge of how socio-demographic factors varies geographically, provides clues to understanding the determinants of tuberculosis and may provide guidance for regional prevention and control strategies to improve public health in Mexico. The aim of the present study was to describe the epidemiologic characteristics and spatial patterns of the incidence of tuberculosis in Tonala, Jalisco (Mexico) from 2013-2015. METHODOLOGY: The Surveillance System Database from the Health Department, complemented by information from the National Institute of Statistics and Geography, was used to obtain data for a spatial-temporal analysis of TB cases. For the geographical analysis map creation and geoinformation storing, ArcGIS software was used. RESULTS: This study sought to characterize problem areas and jurisdictional locations of TB via a spatial approach based on analyses of case distributions and individual patient variables. The study found that tuberculosis cases were dispersed throughout Tonala County and were mainly concentrated on the Guadalajara city border. The TB cases were mainly individuals between 31 and 45 years old. Most of the cases reported during the observation period were male patients, and most cases primarily had lung involvement; however, there were quite a few cases with lymph node and intestinal disease. CONCLUSION: Our findings show that TB cases are essentially located in areas close to the city of Guadalajara and that most TB cases were pulmonary cases spread throughout the whole jurisdiction.

Genetic diversity of Mycobacterium tuberculosis and transmission associated with first-line drug resistance: a first analysis in Jalisco, Mexico.

OBJECTIVES: The objectives of this study were to analyse the frequency of gene mutations associated with antitubercular drug resistance in clinical samples from the population of Jalisco State (Mexico) and to evaluate the genetic variability of Mycobacterium tuberculosis and multidrug-resistant (MDR) M. tuberculosis strains to describe the frequency of various families. METHODS: Clinical isolates of M. tuberculosis obtained from Jalisco State were analysed. Isolates were subjected to drug susceptibility testing, and mutations were characterised by sequencing, followed by genotyping using spoligotyping and mycobacterial interspersed repetitive units-variable-number of tandem repeats (MIRU-VNTR). Moreover, the prevalence of mutations was analysed by phylogenetic lineages. RESULTS: Resistant strains were analysed by sequencing of katG, inhA and rpoB genes to determine the presence of mutations associated with isoniazid and rifampicin resistance. In MDR, monoresistant and polyresistant isolates, mutations were found in 17 (54.84%) of 31 strains. Spoligotyping identified six different strain lineages [T1 (25.40%), H3 (7.94%), MANU (4.76%), X1 (3.17%), EAI5 (1.59%) and LAM1 (1.59%)], with the remaining strains identified as orphans. In additional tree-based identification, a dendrogram of spoligotype patterns generated five different similarity clusters. When combining 24-loci MIRU-VNTR and spoligotyping approaches, the results shows that there is no cluster formation, indicating low transmission of the samples. CONCLUSIONS: This study using spoligotyping and MIRU-VNTR showed that the analysed strains were not related to each other since no two identical strains were found. Families with the highest prevalence in the study were orphans followed by T family.

Characterization of liver histopathology in a transgenic mouse model expressing genotype 1a hepatitis C virus core and envelope proteins 1 and 2.

Hepatitis C virus (HCV) is a major cause of chronic hepatitis and hepatocellular carcinoma worldwide. The purpose of this study was to determine how the HCV structural proteins affect the dynamic structural and functional properties of hepatocytes and measure the extra-hepatic manifestations induced by these viral proteins. A transgenic mouse model was established by expressing core, E1 and E2 proteins downstream of a CMV promoter. HCV RNA was detected using RT-PCR in transgenic mouse model tissues, such as liver, kidney, spleen and heart. Expression of the transgene was analysed by real-time PCR to quantify viral RNA in different tissues at different ages. Immunofluorescence analysis revealed the expression of core, E1 and E2 proteins predominantly in hepatocytes. Lower levels of protein expression were detected in spleen and kidneys. HCV RNA and viral protein expression increased in the liver with age. Histological analysis of liver cells demonstrated steatosis in transgenic mice older than 3 months, which was more progressed with age. Electron microscopy analysis revealed alterations in nuclei, mitochondria and endoplasmic reticulum. HCV structural proteins induce a severe hepatopathy in the transgenic mouse model. These mice became more prone to liver and lymphoid tumour development and hepatocellular carcinoma. In this model, the extra-hepatic effects of HCV, which included swelling of renal tubular cells, were mild. It is likely that the HCV structural proteins mediate some of the histological alterations in hepatocytes by interfering with lipid transport and liver metabolism.