Risk factors for diabetes mellitus and impaired glucose tolerance following allogeneic hematopoietic stem cell transplantation in pediatric patients with hematological malignancies

Long-term surviving recipients of allogeneic hematopoietic stem cell transplantation (HSCT) often suffer from diabetes mellitus (DM). We sought to identify risk factors for the development of post-transplant DM and impaired glucose tolerance (IGT) in pediatric HSCT patients. Glucose tolerance statuses were evaluated in 22 patients aged 6.3–21.8 years who had received allogeneic HSCT between the ages of 0.8–13.5 years. Five patients were diagnosed as having type 2 DM, and treated with insulin or oral hypoglycemic agents. Five patients were included in the IGT group, and the remaining 12 children were in the normal glucose tolerance (NGT) group. The cumulative incidence of DM plus IGT was 11.6 % at 5 years and 69.3 % at 10 years. None of the patients were obese/overweight and none had a family history of DM. There were no significant differences in serum levels of leptin and adiponectin between the DM + IGT and the NGT groups. An average preprandial glucose levels in the DM + IGT group were significantly higher than those in the NGT group from preparative conditioning to 60 days after HSCT. In multivariate analysis, an age of ≥6 years at the time of HSCT was significantly associated with the development of DM + IGT. Additionally, careful follow-up is necessary, even for NGT patients.     

A simple and practical workflow for genotyping of CRISPR–Cas9‐based knockout phenotypes using multiplexed amplicon sequencing

Founder animals carrying high proportions of somatic mutation induced by CRISPR–Cas9 enable a rapid and scalable strategy for the functional screening of numerous target genes in vivo. In this functional screening, genotyping using pooled amplicons with next‐generation sequencing is the most suitable approach for large‐scale management of multiple samples and accurate evaluation of the efficiency of Cas9‐induced somatic mutations at target sites. Here, we present a simple workflow for genotyping of multiple CRISPR–Cas9‐based knockout founders by pooled amplicon sequencing. Using custom barcoded primers, pooled amplicons from multiple individuals can be run in a single‐indexed library on the Illumina MiSeq platform. Additionally, a user‐friendly web tool, CLiCKAR, is available to simultaneously perform demultiplexing of pooled sequence data and evaluation of somatic mutation in each phenotype. CLiCKAR provides users with practical reports regarding the positions of insertions/deletions, as well as the frameshift ratio and tables containing mutation sequences, and read counts of each phenotype, with just a few clicks by the implementation of demultiplexing for pooled sample data and calculation of the frameshift ratio. This genotyping workflow can be harnessed to evaluate genotype–phenotype correlations in CRISPR–Cas9‐based loss‐of‐function screening of numerous target genes in various organisms.     

Protective role of JAK/STAT signaling against renal fibrosis in mice with unilateral ureteral obstruction

Inflammation is involved in renal fibrosis, a final common pathway for kidney diseases. To clarify how JAK/STAT/SOCS system was involved in renal fibrosis, UUO was induced in BALB/c or SOCS3^+/− mice in the presence or absence of JAK inhibitor-incorporated nanoparticle (pyridine6–PGLA). UUO increased pSTAT3 and subsequently elevated SOCS3 levels in the obstructed kidneys. pSTAT3 levels were further increased in SOCS3^+/− mice. UUO-induced renal fibrosis was markedly suppressed in SOCS3^+/− mice, while it was aggravated by pre-treatment with pyridine6–PGLA. Although there were no differences in renal mRNA levels of TGF-β and collagens between wild and SOCS3^+/− mice, MMP-2 activity was enhanced in SOCS3^+/− UUO mice. Activated MMP-2 was completely suppressed by pyridine6–PGLA-pre-treatment. TNF-α one of JAK/STAT activators, increased pSTAT3 levels and subsequently induced MMP-2 activation in proximal tubular cells. These results suggest that JAK/STAT3 signaling may play a role in repair process of renal fibrosis in UUO partly via MMP-2 activation.     

The Body Fat Percentage Rather Than the BMI Is Associated with the CD4 Count among HIV Positive Japanese Individuals

Maintenance of the cluster of differentiation 4 (CD4) positive lymphocyte count (CD4 count) is important for human immunodeficiency virus (HIV) positive individuals. Although a higher body mass index (BMI) is shown to be associated with a higher CD4 count, BMI itself does not reflect body composition. Therefore, we examined the association of body weight, body composition and the CD4 count, and determined the optimal ranges of CD4 count associated factors in Japanese HIV positive individuals. This cross-sectional study included 338 male patients treated with antiretroviral therapy for ≥12 months. Multiple logistic regression analysis was used to identify factors significantly associated with a CD4 count of ≥500 cells (mm³)⁻¹. The cutoff values of factors for a CD4 ≥ 500 cells (mm³)⁻¹ and cardiovascular disease risk were obtained by receiver operating characteristic curves. Age, body fat percentage (BF%), nadir CD4 count, duration of antiretroviral therapy (ART), years since the HIV-positive diagnosis and cholesterol intake showed significant associations with the CD4 count. The cutoff value of BF% for a CD4 ≥ 500 cells (mm³)⁻¹ and lower cardiovascular disease risk were ≥25.1% and ≤25.5%, respectively. The BF%, but not the BMI, was associated with CD4 count. For the management of HIV positive individuals, 25% appears to be the optimal BF% when considering the balance between CD4 count management and cardiovascular disease risk.     

Muscle metabolism during exercise using phosphorus-31 nuclear magnetic resonance spectroscopy in adolescents

Very little has been reported on muscle energetics during exercise in adolescents. This is attributable to the difficulty of subjecting children to muscle biopsy. The purpose of this study was to investigate the characteristics of muscle metabolism during exercisein vivo in adolescents by comparing firstly, with adults and secondly, the differences resulting from physical activity using phosphorus-31 nuclear magnetic resonance (³¹PNMR) spectroscopy. The subjects were boys aged 12 to 15 years, comprising 21 trained boys and 23 control boys, and 6 adults controls. The ratio of phosphocreatine (PCr):(PCr + P_i), where P_i is inorganic phosphate intracellular pH at exhaustion and the time constant of PCr during recovery were measured in all the subjects using³¹PNMR. Both groups of children showed higher values of PCr:(PCr + P_i) and intracellular pH at exhaustion than did the adult control group (P < 0.01 orP < 0.05). However, no significant differences were found between the trained boys and the control boys with respect to PCr:(PCr + P_i) and intracellular pH at exhaustion. On the other hand, we found the same values for PCr time constant in all groups. This result suggested no differences of the muscle oxidative capacity between children and adults. We concluded that the adolescents, aged 12 to 15 years in both the trained and control groups, had less glycolytic ability during exercise than the adults.