GAA Deficiency in Pompe Disease Is Alleviated by Exon Inclusion in iPSC-Derived Skeletal Muscle Cells

Pompe disease is a metabolic myopathy caused by deficiency of the acid α-glucosidase (GAA) enzyme and results in progressive wasting of skeletal muscle cells. The c.-32-13T>G (IVS1) GAA variant promotes exon 2 skipping during pre-mRNA splicing and is the most common variant for the childhood/adult disease form. We previously identified antisense oligonucleotides (AONs) that promoted GAA exon 2 inclusion in patient-derived fibroblasts. It was unknown how these AONs would affect GAA splicing in skeletal muscle cells. To test this, we expanded induced pluripotent stem cell (iPSC)-derived myogenic progenitors and differentiated these to multinucleated myotubes. AONs restored splicing in myotubes to a similar extent as in fibroblasts, suggesting that they act by modulating the action of shared splicing regulators. AONs targeted the putative polypyrimidine tract of a cryptic splice acceptor site that was part of a pseudo exon in GAA intron 1. Blocking of the cryptic splice donor of the pseudo exon with AONs likewise promoted GAA exon 2 inclusion. The simultaneous blocking of the cryptic acceptor and cryptic donor sites restored the majority of canonical splicing and alleviated GAA enzyme deficiency. These results highlight the relevance of cryptic splicing in human disease and its potential as therapeutic target for splicing modulation using AONs

Early Life Stress Delays Sexual Maturation in Female Mice

In humans, some forms of early life stress (ELS) have been linked with precocious puberty, altered brain maturation, and increased risk for a variety of forms of pathology. Interestingly, not all forms of ELS have been found to equally impact these metrics of maturation. In recent work, we have found that ELS in the form of limited bedding (LB) from P4 to P11, was associated with precocious hippocampus maturation in males and increased risk for depressive-like pathology and attentional disturbance in female mice. Here, we sought to test whether ELS in the form of LB also impacted the timing of sexual maturation in female mice. To establish rate of somatic and sexual development, distinct cohorts of mice were tested for weight gain, timing of vaginal opening, and development of estrous cycling. ELS animals weighed significantly less than controls at every timepoint measured. Onset of vaginal opening was tracked from P21 to 40, and ELS was found to significantly delay the onset of vaginal opening. To test the impact of ELS on estrous cycle duration and regularity, vaginal cytology was assessed in independent groups of animals using either a continuous sampling (daily from P40 to P57) or random sampling approach (single swab at P35, P50, or P75). ELS did impact measures of estrous cycling, but these effects were dependent upon the sampling method used. We also tested the impact of ELS on anxiety-like behaviors over development and across the estrous cycle. We observed a developmental increase in anxiety-like behavior in control but not ELS mice. No effect of estrous cycle stage was found on anxiety-like behavior for either group of mice. Together these results provide evidence that ELS in the form of LB delays somatic and sexual development. Additional work will be required to determine the mechanism by which ELS impacts these measures, and if these effects are common to other models of ELS in rodents

Promoting advance planning for health care and research among older adults: A randomized controlled trial

<p>Abstract</p> <p>Background</p> <p>Family members are often required to act as substitute decision-makers when health care or research participation decisions must be made for an incapacitated relative. Yet most families are unable to accurately predict older adult preferences regarding future health care and willingness to engage in research studies. Discussion and documentation of preferences could improve proxies' abilities to decide for their loved ones. This trial assesses the efficacy of an advance planning intervention in improving the accuracy of substitute decision-making and increasing the frequency of documented preferences for health care and research. It also investigates the financial impact on the healthcare system of improving substitute decision-making.</p> <p>Methods/Design</p> <p>Dyads (<it>n </it>= 240) comprising an older adult and his/her self-selected proxy are randomly allocated to the experimental or control group, after stratification for type of designated proxy and self-report of prior documentation of healthcare preferences. At baseline, clinical and research vignettes are used to elicit older adult preferences and assess the ability of their proxy to predict those preferences. Responses are elicited under four health states, ranging from the subject's current health state to severe dementia. For each state, we estimated the public costs of the healthcare services that would typically be provided to a patient under these scenarios. Experimental dyads are visited at home, twice, by a specially trained facilitator who communicates the dyad-specific results of the concordance assessment, helps older adults convey their wishes to their proxies, and offers assistance in completing a guide entitled <it>My Preferences </it>that we designed specifically for that purpose. In between these meetings, experimental dyads attend a group information session about <it>My Preferences</it>. Control dyads attend three monthly workshops aimed at promoting healthy behaviors. Concordance assessments are repeated at the end of the intervention and 6 months later to assess improvement in predictive accuracy and cost savings, if any. Copies of completed guides are made at the time of these assessments.</p> <p>Discussion</p> <p>This study will determine whether the tested intervention guides proxies in making decisions that concur with those of older adults, motivates the latter to record their wishes in writing, and yields savings for the healthcare system.</p> <p>Trial Registration</p> <p><a href="http://www.controlled-trials.com/ISRCTN89993391">ISRCTN89993391</a></p

The pediatric NAFLD fibrosis index: a predictor of liver fibrosis in children with non-alcoholic fatty liver disease

<p>Abstract</p> <p>Background</p> <p>Liver fibrosis is a stage of non-alcoholic fatty liver disease (NAFLD) which is responsible for liver-related morbidity and mortality in adults. Accordingly, the search for non-invasive markers of liver fibrosis has been the subject of intensive efforts in adults with NAFLD. Here, we developed a simple algorithm for the prediction of liver fibrosis in children with NAFLD followed at a tertiary care center.</p> <p>Methods</p> <p>The study included 136 male and 67 female children with NAFLD aged 3.3 to 18.0 years; 141 (69%) of them had fibrosis at liver biopsy. On the basis of biological plausibility, readily availability and evidence from adult studies, we evaluated the following potential predictors of liver fibrosis at bootstrapped stepwise logistic regression: gender, age, body mass index, waist circumference, alanine transaminase, aspartate transaminase, gamma-glutamyl-transferase, albumin, prothrombin time, glucose, insulin, triglycerides and cholesterol. A final model was developed using bootstrapped logistic regression with bias-correction. We used this model to develop the 'pediatric NAFLD fibrosis index' (PNFI), which varies between 0 and 10.</p> <p>Results</p> <p>The final model was based on age, waist circumference and triglycerides and had a area under the receiver operating characteristic curve of 0.85 (95% bootstrapped confidence interval (CI) with bias correction 0.80 to 0.90) for the prediction of liver fibrosis. A PNFI ≥ 9 (positive likelihood ratio = 28.6, 95% CI 4.0 to 201.0; positive predictive value = 98.5, 95% CI 91.8 to 100.0) could be used to rule in liver fibrosis without performing liver biopsy.</p> <p>Conclusion</p> <p>PNFI may help clinicians to predict liver fibrosis in children with NAFLD, but external validation is needed before it can be employed for this purpose.</p

Confirmed adult dengue deaths in Singapore: 5-year multi-center retrospective study

10.1186/1471-2334-11-123BMC Infectious Diseases11-BIDM

A

Abstract:&nbsp; SARS-CoV-2 is causing COVID-19, a new respiratory virus from Wuhan, China, since December 2019 and devolved into the current pandemic. Viral circulation and infection would become endemic, making direct immunofluorescence (IF) a cost-effective diagnostic methodology for sustained sentinel surveillance. We proposed to develop an IF for SARS-CoV-2 in slides&nbsp;with respiratory samples from patients challenged with antibodies from the serum of convalescent patients as an intermediate reagent for subsequent labeling with fluorochrome conjugate. With Ethics Committee approvals, nasopharyngeal swabs and sera were obtained from patients with PCR-confirmed COVID-19. The slides were prepared following the standard method in a biological biosafety cabinet. The conditions for performing the IF technique were: 1-Sera were assayed with CovidAR, CMIA Arquitect-ABBOTT and Neutralization-titrated challenge sera. 2-Anti-FC IgG of human IgG obtained in mouse and goat conjugated with fluorescein. 3-Blocking with albumin and tween 20 at different stages of the technique. The results obtained so far indicate that the best combination of conditions involves the use of blocking with 1% albumin + 0.05% tween 20 on the slides&nbsp;for 20 minutes and a layer of human serum with specific antibody titer of 1/80 NT. In addition, the goat conjugate performed better. Validation and standardization with monoclonal antibodies are still pending. Descriptive analyses will be performed by means of tables and graphs, establishing absolute and relative frequencies (%). &nbsp;Chi-square analysis will be applied estimating sensitivity, specificity, positive and negative predictive values. Interspecific and intraspecific validation assays and statistical analysis of DIF with respect to molecular biology will be performed. The R-Medic software will be used and in all cases the significance level will be 5%.&nbsp; Although IF has lower sensitivity than molecular methods, it offers practicality in the initial screening task. The relevance of this work lies in being able to implement in the future, once the technique is standardized and validated, the detection of SARS-Cov-2 as differential diagnosis by IF, to distinguish it among the other viral agents of the respiratory panel, contributing to the diagnosis in clinical practice and epidemiological surveillance in Public Health.Resumen:&nbsp; El SARS-CoV-2 es causante de COVID-19, un nuevo virus respiratorio de&nbsp;Wuhan, China, desde&nbsp;diciembre de 2019 y devino en la pandemia actual. La circulación e infección viral se volverían endémicas, por lo que la Inmunofluorescencia (IF) directa sería una metodología diagnóstica económica para la vigilancia centinela sostenida. Se propuso desarrollar una IF para SARS-CoV-2 en improntas con muestras respiratorias de pacientes&nbsp;enfrentadas&nbsp;con anticuerpos del suero de pacientes convalecientes como reactivo intermediario para el posterior marcaje con conjugado con fluorocromo. Con aprobaciones del Comités de Ética, se obtuvieron hisopados nasofaríngeos y sueros de pacientes con COVID-19 confirmado por PCR. Las improntas se elaboraron siguiendo el método estándar en cabina de bioseguridad biológica. Las condiciones&nbsp;para la realización de la técnica de IF fueron: 1-Se ensayó con sueros problemas titulados por CovidAR, CMIA Arquitect-ABBOTT y Neutralización. 2-IgG anti-FC de IgG humana obtenidas en ratón y cabra conjugada con fluoresceína. 3-Bloqueo con albúmina y tween 20 en diferentes etapas de la técnica. Los resultados obtenidos hasta ahora indican que la mejor combinación de condiciones implica el uso de bloqueo con albúmina al 1% + tween 20 al 0,05% sobre la impronta durante 20 minutos y una capa&nbsp;del suero humano con título de anticuerpos específicos de 1/80 NT.&nbsp; Además, funcionó mejor el conjugado de cabra. Permanece a la espera la validación y estandarización con anticuerpos monoclonales. Se realizarán análisis descriptivos mediante tablas y gráficos, estableciendo frecuencias absolutas y relativas (%). Se aplicarán análisis Chi cuadrado estimando&nbsp;sensibilidad, especificidad, valores predictivos positivos y negativos. Se harán ensayos de validación interespecíficos e intraespecíficos y análisis estadísticos de IFD con respecto a biología molecular.&nbsp;Se utilizará el soft R-Medic&nbsp;y en todos los casos el nivel de significación será del 5%.&nbsp; Si bien la IF posee menor sensibilidad que los métodos moleculares, ofrece practicidad en la tarea inicial de tamizaje. La relevancia del trabajo radica en poder implementar a futuro, una vez estandarizada y validada la técnica, la detección de SARS-Cov-2 como diagnóstico diferencial por IF, para distinguirlo entre los otros agentes virales del panel respiratorio, aportando al diagnóstico en la práctica clínica y a la vigilancia epidemiológica&nbsp;en Salud Pública.

Body indices and basic vital signs in Helicobacter pylori positive and negative persons

It has been hypothesized that Helicobacter pylori (Hp) infection may contribute to reduced stature, risk of hypertension or obesity. The aim was to evaluate body indices in Hp positive and negative persons. A total of 2436 subjects (4–100 years old) were tested for Hp status by 13Curea breath test. Data on height and weight were collected for 84%, and blood pressure for 80% of the study subjects. The prevalence of Hp infection was 41.6%. The odds ratio for a 10-year increase in age was 1.21 (95% CI 1.17–1.25, p-value <0.001). Statistically significant negative association of Hp positivity with body height was most pronounced in the younger age groups, while a positive association of Hp positivity with body mass index was only seen in those aged 15+ years. There was a negative effect of Hp positivity on systolic and diastolic blood pressure in subjects below 25 and a relatively strong positive effect on blood pressure in subjects over 65 years. Residual confounding by social characteristics as a possible explanation for the associations of Hp positivity with height and blood pressure cannot be excluded. Unmeasured factors related to social and family environment may cause the apparent association between Hp positivity and children’s growth and blood pressure

Insulin resistance and its association with the components of the metabolic syndrome among obese children and adolescents

<p>Abstract</p> <p>Background</p> <p>Insulin resistance is the primary metabolic disorder associated with obesity; yet little is known about its role as a determinant of the metabolic syndrome in obese children. The aim of this study is to assess the association between the degree of insulin resistance and the different components of the metabolic syndrome among obese children and adolescents.</p> <p>Methods</p> <p>An analytical, cross-sectional and population-based study was performed in forty-four public primary schools in Campeche City, Mexico. A total of 466 obese children and adolescents between 11-13 years of age were recruited. Fasting glucose and insulin concentrations, high density lipoprotein cholesterol, triglycerides, waist circumference, systolic and diastolic blood pressures were measured; insulin resistance and metabolic syndrome were also evaluated.</p> <p>Results</p> <p>Out of the total population studied, 69% presented low values of high density lipoprotein cholesterol, 49% suffered from abdominal obesity, 29% had hypertriglyceridemia, 8% presented high systolic and 13% high diastolic blood pressure, 4% showed impaired fasting glucose, 51% presented insulin resistance and 20% metabolic syndrome. In spite of being obese, 13% of the investigated population did not present any metabolic disorder. For each one of the components of the metabolic syndrome, when insulin resistance increased so did odds ratios as cardiometabolic risk factors.</p> <p>Conclusions</p> <p>Regardless of age and gender an increased degree of insulin resistance is associated with a higher prevalence of disorders in each of the components of the metabolic syndrome and with a heightened risk of suffering metabolic syndrome among obese children and adolescents.</p

LRRK2 at the interface of autophagosomes, endosomes and lysosomes

Over the past 20 years, substantial progress has been made in identifying the underlying genetics of Parkinson’s disease (PD). Of the known genes, LRRK2 is a major genetic contributor to PD. However, the exact function of LRRK2 remains to be elucidated. In this review, we discuss how familial forms of PD have led us to hypothesize that alterations in endomembrane trafficking play a role in the pathobiology of PD. We will discuss the major observations that have been made to elucidate the role of LRRK2 in particular, including LRRK2 animal models and high-throughput proteomics approaches. Taken together, these studies strongly support a role of LRRK2 in vesicular dynamics. We also propose that targeting these pathways may not only be beneficial for developing therapeutics for LRRK2-driven PD, but also for other familial and sporadic cases