Role of post-translational modifications to lens proteins in cataract formation

Cataract is a leading cause of blindness throughout the world, yet the fundamental biochemical causes are unknown. A rodent model of the biochemical processes is selenite cataract. This cataract shows some of the features of human cataracts such as increased lens calcium, proteolysis of proteins, and insolubilization leading to lens opacity. The goals of the current experiments were: (1) To measure changes in transcript levels for calpains and caspase 3 and oxidation of epithelial proteins in selenite cataract. (2) To elucidate changes in calpain 10 and its interaction with other calpains in selenite cataract. (3) To investigate changes in stability of βB1-crystallin caused by deamidation and truncation. These data would provide roles for apoptosis, protein insolubilization, proteolysis and deamidation observed in cataract. To induce cataract, 12-day old rats were injected with an overdose of Na₂SeO₃. Epithelium was analyzed by competitive RT-PCR, zymography, and thiol-blotting. Calpains were detected by western-blotting. For βB1-crystallin stability studies, recombinant βB1-crystallins were denatured by urea or heat. Urea stability was measured by circular dichroism and fluorescence spectrometry, and heat stability was measured by light scattering at 405 nm. During selenite cataract formation, calpains in epithelium were activated resulting in increased proteolysis of crystallins, but mRNA levels for calpains did not show appreciable changes. Oxidation of sulthydryls in epithelial proteins was minimal during cataract formation. These results suggested that calpain-induced proteolysis in the epithelium contribute to selenite cataract. In selenite cataract, calpain 10 proteins disappeared, which appeared to be due to degradation by calpain 2 and Lp82 calpain. Deamidated βB1-crystallin was less stable in urea and heat, compared to wildtype. When the terminal extensions were removed, βB1-crystallin was as stable as wild-type. However, without the extensions, truncated βB1-crystallin caused accelerated precipitation in a complex with αA-crystallin, suggesting that the extensions may contribute to proper association with other crystallins and to stability of the soluble complexes. In summary, proteolysis of proteins by calpains was more pronounced than protein oxidation in lens epithelium of selenite cataract. Deamidation and truncation caused instability of βB1-crystallin and abnormal association with αA-crystallin. Thus, proteolysis and deamidation may increase susceptibility of lenses to cataract

Endothelial FAK is essential for vascular network stability, cell survival, and lamellipodial formation

Morphogenesis of a vascular network requires dynamic vessel growth and regression. To investigate the cellular mechanism underlying this process, we deleted focal adhesion kinase (FAK), a key signaling mediator, in endothelial cells (ECs) using Tie2-Cre mice. Targeted FAK depletion occurred efficiently early in development, where mutants exhibited a distinctive and irregular vasculature, resulting in hemorrhage and lethality between embryonic day (e) 10.5 and 11.5. Capillaries and intercapillary spaces in yolk sacs were dilated before any other detectable abnormalities at e9.5, and explants demonstrate that the defects resulted from the loss of FAK and not from organ failure. Time-lapse microscopy monitoring EC behavior during vascular formation in explants revealed no apparent decrease in proliferation or migration but revealed increases in cell retraction and death leading to reduced vessel growth and increased vessel regression. Consistent with this phenotype, ECs derived from mutant embryos exhibited aberrant lamellipodial extensions, altered actin cytoskeleton, and nonpolarized cell movement. This study reveals that FAK is crucial for vascular morphogenesis and the regulation of EC survival and morphology

Stochastic priming and spatial cues orchestrate heterogeneous clonal contribution to mouse pancreas organogenesis

The pancreas arises from a small population of cells but how individual cells contribute to organ formation is unclear. Here, the authors deconstruct pancreas organogenesis into clonal units, showing that single progenitors give rise to heterogeneous multi-lineage and endocrinogenic single-lineage clones

Short-term calorie restriction ameliorates genomewide, age-related alterations in DNA methylation

DNA methylation plays major roles in many biological processes, including aging, carcinogenesis, and development. Analyses of DNA methylation using next-generation sequencing offer a new way to profile and compare methylomes across the genome in the context of aging. We explored genomewide DNA methylation and the effects of short-term calorie restriction (CR) on the methylome of aged rat kidney. Whole-genome methylation of kidney in young (6 months old), old (25 months old), and OCR (old with 4-week, short-term CR) rats was analyzed by methylated DNA immunoprecipitation and next-generation sequencing (MeDIP-Seq). CpG islands and repetitive regions were hypomethylated, but 5'-UTR, exon, and 3'-UTR hypermethylated in old and OCR rats. The methylation in the promoter and intron regions was decreased in old rats, but increased in OCR rats. Pathway enrichment analysis showed that the hypermethylated promoters in old rats were associated with degenerative phenotypes such as cancer and diabetes. The hypomethylated promoters in old rats related significantly to the chemokine signaling pathway. However, the pathways significantly enriched in old rats were not observed from the differentially methylated promoters in OCR rats. Thus, these findings suggest that short-term CR could partially ameliorate age-related methylation changes in promoters in old rats. From the epigenomic data, we propose that the hypermethylation found in the promoter regions of disease-related genes during aging may indicate increases in susceptibility to age-related diseases. Therefore, the CR-induced epigenetic changes that ameliorate age-dependent aberrant methylation may be important to CR's health-and life-prolonging effects.ope

Long-term feeder-free culture of human pancreatic progenitors on fibronectin or matrix-free polymer potentiates β cell differentiation

With the aim of producing β cells for replacement therapies to treat diabetes, several protocols have been developed to differentiate human pluripotent stem cells to β cells via pancreatic progenitors. While in vivo pancreatic progenitors expand throughout development, the in vitro protocols have been designed to make these cells progress as fast as possible to β cells. Here, we report on a protocol enabling a long-term expansion of human pancreatic progenitors in a defined medium on fibronectin, in the absence of feeder layers. Moreover, through a screening of a polymer library we identify a polymer that can replace fibronectin. Our experiments, comparing expanded progenitors to directly differentiated progenitors, show that the expanded progenitors differentiate more efficiently into glucose-responsive β cells and produce fewer glucagon-expressing cells. The ability to expand progenitors under defined conditions and cryopreserve them will provide flexibility in research and therapeutic production

Intravenous fluid prescription practices among pediatric residents in Korea

PurposeRecent studies have established the association between hypotonic fluids administration and hospital-acquired hyponatremia in children. The present paper investigated the pattern of current practice in intravenous fluid prescription among Korean pediatric residents, to underscore the need for updated education.MethodsA survey-based analysis was carried out. Pediatric residents at six university hospitals in Korea completed a survey consisting of four questions. Each question proposed a unique scenario in which the respondents had to prescribe either a hypotonic or an isotonic fluid for the patient.ResultsNinety-one responses were collected and analyzed. In three of the four scenarios, a significant majority prescribed the hypotonic fluids (98.9%, 85.7%, and 69.2%, respectively). Notably, 69.2% of the respondents selected the hypotonic fluids for postoperative management. Almost all (96.7%) selected the isotonic fluids for hydration therapy.ConclusionIn the given scenarios, the majority of Korean pediatric residents would prescribe a hypotonic fluid, except for initial hydration. The current state of pediatric fluid management, notably, heightens the risk of hospital-acquired hyponatremia. Updated clinical practice education on intravenous fluid prescription, therefore, is urgently required

Occurrence and characterization of oseltamivir-resistant influenza virus in children between 2007-2008 and 2008-2009 seasons

PurposeThere was a global increase in the prevalence of oseltamivir-resistant influenza viruses during the 2007-2008 influenza season. This study was conducted to investigate the occurrence and characteristics of oseltamivir-resistant influenza viruses during the 2007-2008 and 2008-2009 influenza seasons among patients who were treated with oseltamivir (group A) and those that did not receive oseltamivir (group B).MethodsA prospective study was conducted on 321 pediatric patients who were hospitalized because of influenza during the 2007-2008 and 2008-2009 influenza seasons. Drug resistance tests were conducted on influenza viruses isolated from 91 patients.ResultsThere was no significant difference between the clinical characteristics of groups A and B during both seasons. Influenza A/H1N1, isolated from both groups A and B during the 2007-2008 and 2008-2009 periods, was not resistant to zanamivir. However, phenotypic analysis of the virus revealed a high oseltamivir IC50 range and that H275Y substitution of the neuraminidase (NA) gene and partial variation of the hemagglutinin (HA) gene did not affect its antigenicity to the HA vaccine even though group A had a shorter hospitalization duration and fewer lower respiratory tract complications than group B. In addition, there was no significant difference in the clinical manifestations between oseltamivir-susceptible and oseltamivir-resistant strains of influenza A/H1N1.ConclusionEstablishment of guidelines to efficiently treat influenza with oseltamivir, a commonly used drug for treating influenza in Korean pediatric patients, and a treatment strategy with a new therapeutic agent is required