Association of Urinary Phthalates with Self-Reported Eye Affliction/Retinopathy in Individuals with Diabetes: National Health and Nutrition Examination Survey, 2001-2010

Background. An epidemiological association between exposure to phthalates and type 2 diabetes (T2D) is known. However, the potential role of environmental phthalates in the complications of T2D is unknown. Methods. Using data from the National Health and Nutrition Examination Survey (NHANES) 2001-2010, we studied the association of 12 urinary phthalate metabolites with self-reported eye affliction/retinopathy in 1,004 participants with diabetes. Data from retinal imaging was used to validate this outcome. Independence of the phthalates→T2D association was studied by adjusting for age, sex, race, marital status, educational attainment, poverty income ratio, physical activity, glycated hemoglobin levels, total serum cholesterol, serum high-density lipoprotein cholesterol, serum triglycerides, blood pressure, duration of diabetes, total calorie intake, and obesity. Results. Self-reported eye affliction/retinopathy had 82% accuracy with Cohen\u27s kappa of 0.31 (p \u3c 0.001). Urinary mono-n-octyl phthalate (MOP) was independently associated with the likelihood of self-reported eye affliction/retinopathy in subjects with T2D after accounting for all the confounders. This significance of this association was robust to the potential misclassification in cases and controls of retinopathy. Further, a significant dose-response relationship between MOP and self-reported eye affliction/retinopathy was demonstrable. Conclusions. We show a novel epidemiological link between the environment and diabetic complications in NHANES 2001-2010 participants

Association of Urinary Phthalates with Self-Reported Eye Affliction/Retinopathy in Individuals with Diabetes: National Health and Nutrition Examination Survey, 2001-2010

Background. An epidemiological association between exposure to phthalates and type 2 diabetes (T2D) is known. However, the potential role of environmental phthalates in the complications of T2D is unknown. Methods. Using data from the National Health and Nutrition Examination Survey (NHANES) 2001-2010, we studied the association of 12 urinary phthalate metabolites with self-reported eye affliction/retinopathy in 1,004 participants with diabetes. Data from retinal imaging was used to validate this outcome. Independence of the phthalates→T2D association was studied by adjusting for age, sex, race, marital status, educational attainment, poverty income ratio, physical activity, glycated hemoglobin levels, total serum cholesterol, serum high-density lipoprotein cholesterol, serum triglycerides, blood pressure, duration of diabetes, total calorie intake, and obesity. Results. Self-reported eye affliction/retinopathy had 82% accuracy with Cohen\u27s kappa of 0.31 (p \u3c 0.001). Urinary mono-n-octyl phthalate (MOP) was independently associated with the likelihood of self-reported eye affliction/retinopathy in subjects with T2D after accounting for all the confounders. This significance of this association was robust to the potential misclassification in cases and controls of retinopathy. Further, a significant dose-response relationship between MOP and self-reported eye affliction/retinopathy was demonstrable. Conclusions. We show a novel epidemiological link between the environment and diabetic complications in NHANES 2001-2010 participants

Utility of Lymphoblastoid Cell Lines for Induced Pluripotent Stem Cell Generation

A large number of EBV immortalized LCLs have been generated and maintained in genetic/epidemiological studies as a perpetual source of DNA and as a surrogate in vitro cell model. Recent successes in reprograming LCLs into iPSCs have paved the way for generating more relevant in vitro disease models using this existing bioresource. However, the overall reprogramming efficiency and success rate remain poor and very little is known about the mechanistic changes that take place at the transcriptome and cellular functional level during LCL-to-iPSC reprogramming. Here, we report a new optimized LCL-to-iPSC reprogramming protocol using episomal plasmids encoding pluripotency transcription factors and mouse p53DD (p53 carboxy-terminal dominant-negative fragment) and commercially available reprogramming media. We achieved a consistently high reprogramming efficiency and 100% success rate using this optimized protocol. Further, we investigated the transcriptional changes in mRNA and miRNA levels, using FC-abs ≥ 2.0 and FDR ≤ 0.05 cutoffs; 5,228 mRNAs and 77 miRNAs were differentially expressed during LCL-to-iPSC reprogramming. The functional enrichment analysis of the upregulated genes and activation of human pluripotency pathways in the reprogrammed iPSCs showed that the generated iPSCs possess transcriptional and functional profiles very similar to those of human ESCs

Transition from pre-diabetes to diabetes and predictors of risk in Mexican-Americans

Background: No studies have examined risk factors for the transition from pre-diabetes to diabetes in populations with widespread obesity and diabetes. We determined proximal changes and factors affecting the transition among Mexican-Americans with pre-diabetes. Methods: Participants with pre-diabetes (n=285) were recruited from our randomly sampled population-based Cameron County Hispanic Cohort. These participants were followed for an average of 27 months with repeat examination every 3 to 4 months. Metabolic health was defined as having less than 2 metabolic abnormalities (e.g., hypertension, elevated low-density lipoprotein, etc). Diabetes was identified as fasting blood glucose ≥126 mg/dL, glycated hemoglobin ≥6.5% and/or on hypoglycemic medication. Results: Ninety-six of 285 (33.7%) participants transitioned to overt diabetes. The increased risk of diabetes in the metabolically unhealthy varying with follow-up time was 81% (adjusted odds ratio [OR]: 1.81; 95% CI: 1.09–3.02). The risk of diabetes increased 8% for each kg/m2 of increase in body mass index (BMI, OR: 1.08; 95% CI: 1.05–1.11) independent of covariates. Transition to diabetes was accompanied by a mean increase in BMI of 0.28 kg/m2, and deterioration in metabolic health of 9% (OR: 1.09; 95% CI: 1.003–1.18) compared with those who did not transition. Conclusions: Deteriorating metabolic health and/or increasing BMI significantly raises the risk of transitioning from pre-diabetes to diabetes. Transition itself was accompanied by further increase in BMI and deterioration in metabolic health. These data underline the importance of improving metabolic health and avoiding weight gain in pre-diabetes as simple but clear diabetes prevention targets, and emphasize the importance of lifestyle management

Large scale mitochondrial sequencing in Mexican Americans suggests a reappraisal of Native American origins

<p>Abstract</p> <p>Background</p> <p>The Asian origin of Native Americans is largely accepted. However uncertainties persist regarding the source population(s) within Asia, the divergence and arrival time(s) of the founder groups, the number of expansion events, and migration routes into the New World. mtDNA data, presented over the past two decades, have been used to suggest a single-migration model for which the Beringian land mass plays an important role.</p> <p>Results</p> <p>In our analysis of 568 mitochondrial genomes, the coalescent age estimates of shared roots between Native American and Siberian-Asian lineages, calculated using two different mutation rates, are A4 (27.5 ± 6.8 kya/22.7 ± 7.4 kya), C1 (21.4 ± 2.7 kya/16.4 ± 1.5 kya), C4 (21.0 ± 4.6 kya/20.0 ± 6.4 kya), and D4e1 (24.1 ± 9.0 kya/17.9 ± 10.0 kya). The coalescent age estimates of pan-American haplogroups calculated using the same two mutation rates (A2:19.5 ± 1.3 kya/16.1 ± 1.5 kya, B2:20.8 ± 2.0 kya/18.1 ± 2.4 kya, C1:21.4 ± 2.7 kya/16.4 ± 1.5 kya and D1:17.2 ± 2.0 kya/14.9 ± 2.2 kya) and estimates of population expansions within America (~21-16 kya), support the pre-Clovis occupation of the New World. The phylogeography of sublineages within American haplogroups A2, B2, D1 and the C1b, C1c andC1d subhaplogroups of C1 are complex and largely specific to geographical North, Central and South America. However some sub-branches (B2b, C1b, C1c, C1d and D1f) already existed in American founder haplogroups before expansion into the America.</p> <p>Conclusions</p> <p>Our results suggest that Native American founders diverged from their Siberian-Asian progenitors sometime during the last glacial maximum (LGM) and expanded into America soon after the LGM peak (~20-16 kya). The phylogeography of haplogroup C1 suggest that this American founder haplogroup differentiated in Siberia-Asia. The situation is less clear for haplogroup B2, however haplogroups A2 and D1 may have differentiated soon after the Native American founders divergence. A moderate population bottle neck in American founder populations just before the expansion most plausibly resulted in few founder types in America. The similar estimates of the diversity indices and Bayesian skyline analysis in North America, Central America and South America suggest almost simultaneous (~ 2.0 ky from South to North America) colonization of these geographical regions with rapid population expansion differentiating into more or less regional branches across the pan-American haplogroups.</p

Highly efficient induced pluripotent stem cell reprogramming of cryopreserved lymphoblastoid cell lines

Tissue culture based in-vitro experimental modeling of human inherited disorders provides insight into the cellular and molecular mechanisms involved and the underlying genetic component influencing the disease phenotype. The breakthrough development of induced pluripotent stem cell (iPSC) technology represents a quantum leap in experimental modeling of human diseases, providing investigators with a self-renewing and thus unlimited source of pluripotent cells for targeted differentiation into functionally relevant disease specific tissue/cell types. The existing rich bio-resource of Epstein-Barr virus (EBV) immortalized lymphoblastoid cell line (LCL) repositories generated from a wide array of patients in genetic and epidemiological studies worldwide, many of them with extensive genotypic, genomic and phenotypic data already existing, provides a great opportunity to reprogram iPSCs from any of these LCL donors in the context of their own genetic identity for disease modeling and disease gene identification. However, due to the low reprogramming efficiency and poor success rate of LCL to iPSC reprogramming, these LCL resources remain severely underused for this purpose. Here, we detailed step-by-step instructions to perform our highly efficient LCL-to-iPSC reprogramming protocol using EBNA1/OriP episomal plasmids encoding pluripotency transcription factors (i.e., OCT3/4, SOX2, KLF4, L-MYC, and LIN28), mouse p53DD (p53 carboxy-terminal dominant-negative fragment) and commercially available reprogramming media. We achieved a consistently high reprogramming efficiency and 100% success rate (\u3e 200 reprogrammed iPSC lines) using this protocol

microRNA and mRNA interactions in induced pluripotent stem cell reprogramming of lymphoblastoid cell lines

A large number of Epstein Barr virus (EBV) immortalized lymphoblastoid cell lines (LCLs) have been generated and maintained in genetic/epidemiological studies as a perpetual source of DNA and as a surrogate in vitro cell model. Recent successes in reprograming LCLs into induced pluripotent stem cells (iPSCs) has paved the way to generate more relevant in vitro disease models using this existing bioresource. However, the latent EBV infection in the LCLs make them a unique cell type by altering expression of many cellular genes and miRNAs. These EBV induced changes in the LCL miRNome and transcriptome are reversed upon reprogramming into iPSCs, which allows a unique opportunity to better understand the miRNA and mRNA interactions that are EBV induced in LCLs and the changes that takes place during iPSC reprogramming. To identify the potential miRNA-mRNA interactions and better understand their role in regulating the cellular transitions in LCLs and their reprogrammed iPSCs, we performed a parallel genome-wide miRNA and mRNA expression analysis in six LCLs and their reprogrammed iPSCs. A total of 85 miRNAs and 5,228 mRNAs were significantly differentially expressed (DE). The target prediction of the DE miRNAs using TargetScan-Human, TarBase and miRecords databases identified 1,842 mRNA targets that were DE between LCLs and their reprogrammed iPSCs. The functional annotation, upstream regulator and gene expression analysis of the predicted DE mRNA targets suggest the role of DE miRNAs in regulating EBV induced changes in LCLs and self-renewal, pluripotency and differentiation in iPSCs

Human iPSC derived cardiomyocyte model reveals the transcriptomic bases of COVID-19 associated myocardial injury

Background: Multi-organ complications have been the hallmark of severe COVID-19; cardiac injuries were reported in 20% to 30% of hospitalized COVID-19 patients, although the disease etiology remains poorly understood. This study leveraged genome-wide RNA-sequence data generated using induced pluripotent stem cell (iPSC) differentiated cardiomyocytes (CMs) and in vitro modeling of SARS-CoV-2 infection in CMs, to understand the molecular mechanisms of COVID-19 myocardial injuries for novel diagnostic and therapeutic development. Methods: Raw RNA-sequence data sets, GSE165242 and GSE150392 were aligned to human genome assembly GRCh38 and gene expressions were quantified. Differentially expressed (DE) genes between experimental groups were identified using moderated t-statistics (FDR-corrected p-value ≤ 0.05) and Fold-Change analysis (FC absolute ≥ 2.0). Results: A total of 2,148 genes were significantly DE between SARS-CoV-2 infected and vehicle treated CMs and showed significant enrichment in cytokine signaling pathways (p-value=4.89E-25) and regulation of heart contraction (p-value=2.51E-19) gene-ontology biological processes. 606 of these DE genes were significantly upregulated during iPSC to CM differentiation. Disease and function annotation analysis of these 606 genes showed significant enrichment and activation of angiogenesis (p-value=4.04E-23; activation Z-score=3.7) and downregulation of heart contraction and related functions (p-value=4.24E-29; activation Z-score=-2.2) in SARS-CoV-2 infected CMs. The upstream regulator analysis identified upregulation of AGT associated proinflammatory genes and significant downregulation of TBX5 and MYOCD transcription factors and their gene networks, suggesting remodeling of CM contractility architecture. Conclusions: This study identified several AGT associated proinflammatory genes and TBX5 and MYOCD gene networks as potential targets for drug development to address COVID-19 associated cardiac injury

Expression of glucocorticoid and progesterone nuclear receptor genes in archival breast cancer tissue

BACKGROUND: Previous studies in our laboratory have shown associations of specific nuclear receptor gene variants with sporadic breast cancer. In order to investigate these findings further, we conducted the present study to determine whether expression levels of the progesterone and glucocorticoid nuclear receptor genes vary in different breast cancer grades. METHODS: RNA was extracted from paraffin-embedded archival breast tumour tissue and converted into cDNA. Sample cDNA underwent PCR using labelled primers to enable quantitation of mRNA expression. Expression data were normalized against the 18S ribosomal gene multiplex and analyzed using analysis of variance. RESULTS: Analysis of variance indicated a variable level of expression of both genes with regard to breast cancer grade (P = 0.00033 for glucocorticoid receptor and P = 0.023 for progesterone receptor). CONCLUSION: Statistical analysis indicated that expression of the progesterone nuclear receptor is elevated in late grade breast cancer tissue

Functional characterization of the iPSC generated hepatocytes using genome-wide transcriptomic analysis

Advances in iPSC technologies now allow us to consider non-invasive large-scale in-vitro disease modeling experiments on disease appropriate cell types in human subjects to better understand human disease pathophysiology, disease genetics and to develop better diagnostic and therapeutic technologies. We performed differential gene expression and functional annotation analysis using genome wide mRNA sequencing data to evaluate the functional and disease modeling potential of iPSC generated hepatocytes. Following the criteria moderated t statistics FDR corrected p-value ≤ 0.05 and fold change-absolute ≥ 2.0, 7,246 genes/transcripts were significantly differentially expressed iPSCs and hepatocytes. The 3,791 of these DE genes/transcripts were significantly upregulated in the hepatocytes and accounted for about 55% of the hepatocytes total expressed transcriptome. The heatmap and principal component analysis suggests a discrete and uniform resetting of cellular transcriptome during iPSC to hepatocyte differentiation. The functional annotation analysis of the 3,791 significantly upregulated hepatocytes genes/transcripts showed significant enrichment hepatocytes associated cellular functions and canonical pathways. The gene known to be associated with various common human liver disorders and toxicities were also significantly enriched in hepatocytes upregulated 3,791 genes/transcripts. These results suggest that iPSC generated hepatocytes have a functional profile very similar to human primary hepatocytes and are suitable for in-vitro modeling of human liver disorders and toxicities