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The gene expression platform assay allows for robust and highly reproducible quantification of the expression of up to 800 transcripts (mRNA or miRNAs) in a single reaction. The miRNA assay counts transcripts by directly imaging and digitally counting miRNA molecules that are labeled with color-coded fluorescent barcoded probe sets (a reporter probe and a capture probe). Barcodes are hybridized directly to mature miRNAs that have been elongated by ligating a unique oligonucleotide tag (miRtag) to the 3' end. Reverse transcription and amplification of the transcripts are not required. Reporter probes contain a sequence of six color positions populated using a combination of four fluorescent colors. The four colors over six positions are used to construct a gene-specific color barcode sequence. Post-hybridization processing is automated on a robotic prep station. After hybridization, the excess probes are washed away, and the tripartite structures (capture probe-miRNA-reporter probe) are fixed to a streptavidin-coated slide via the biotin-labeled capture probe. Imaging and barcode counting is done using a digital analyzer. The immobilized barcoded miRNAs are visualized and imaged using a microscope and camera, and the unique barcodes are decoded and counted. Data quality control (QC), normalization, and analysis are facilitated by a custom-designed data processing and analysis software that accompanies the assay software. The assay demonstrates high linearity over a broad range of expression, as well as high sensitivity. Sample and assay preparation does not involve enzymatic reactions, reverse transcription, or amplification; has few steps; and is largely automated, reducing investigator effects and resulting in high consistency and technical reproducibility. Here, we describe the application of this technology to identifying circulating miRNA perturbations in irritable bowel syndrome.

Hypertension and cardiovascular disease rates vary by race/ethnicity in nonpregnant adults. We aimed to examine racial/ethnic differences in prevalence and severity of hypertensive diseases during pregnancy in nulliparous women.Nulliparous women with singleton deliveries and electronic medical record data on demographics and pregnancy outcomes (n = 56,617) were selected from the Consortium on Safe Labor (2002-2008). Multivariable logistic regression was performed to calculate the adjusted odds of gestational hypertension, mild preeclampsia, severe preeclampsia, eclampsia, chronic hypertension, superimposed preeclampsia, and unspecified hypertension for women who were non-Hispanic Black, Hispanic, Asian/Pacific Islander, and multiracial/other race/ethnicity, compared with non-Hispanic White women.Non-Hispanic Black women had higher odds of entering pregnancy with chronic hypertension (adjusted odds ratio (AOR) = 1.43, 95% confidence interval (CI) 1.11-1.84) and had higher odds of developing mild (AOR = 1.26, 95% Cl 1.10-1.45), severe (AOR = 1.31, 95% Cl 1.10-1.57) or superimposed preeclampsia (AOR = 1.98, 95% ClI 1.40-2.80) compared to non-Hispanic White women. Hispanic women and Asian/Pacific Islanders had higher odds of remaining normotensive (AOR = 1.22, 95% CI 1.12-1.33 and AOR=1.55, 95% CI 1.31-1.84, respectively). Conclusions: Odds for specific gestational hypertensive diseases varied by race/ethnicity among women during their first pregnancy. Non-Hispanic Black women experienced more severe disease, while Hispanic women and Asian/Pacific Islanders had an overall decreased risk compared to non-Hispanic Whites. Patterns of racial/ethnic variation associated with hypertensive diseases during pregnancy were similar to racial/ethnic associations reported for adult-onset cardiovascular disease, suggesting that there may be common pathways and shared risk factors.

Ethn Dis. 2021;31(Suppl 1):289-292;doi:10.18865/ed.31.S1.289

AbstractThere is now widespread acceptance of the universal coverage approach, presented in the 2010 World Health Report. There are more and more voices for the benefit of creating a single national risk pool. Now, a body of literature is emerging on institutional design and organizational practice for universal coverage, related to management of the three health-financing functions: collection, pooling and purchasing. While all countries can move towards universal coverage, lower-income countries face particular challenges, including scarce resources and limited capacity. Recently, the Lao PDR has been preparing options for moving to a single national health insurance scheme. The aim is to combine four different social health protection schemes into a national health insurance authority (NHIA) with a single national fund- and risk-pool. This paper investigates the main institutional and organizational challenges related to the creation of the NHIA. The paper uses a qualitative approach, drawing on the World Health Organization's institutional and Organizational Assessment for Improving and Strengthening health financing (OASIS) conceptual framework for data analysis. Data were collected from a review of key health financing policy documents and from 17 semi-structured key informant interviews. Policy makers and advisors are confronting issues related to institutional arrangements, funding sources for the authority and government support for subsidies to the demand-side health financing schemes. Compulsory membership is proposed, but the means for covering the informal sector have not been resolved. While unification of existing schemes may be the basis for creating a single risk pool, challenges related to administrative capacity and cross-subsidies remain. The example of Lao PDR illustrates the need to include consideration of national context, the sequencing of reforms and the time-scale appropriate for achieving universal coverage.

The HLA phenotypes of 41 patients (22 males and 19 females) with classical nephropathic cystinosis were analyzed and compared with those reported from a control group of 1,465 Americans of Caucasian origin. There was a significantly increased association of cystinosis with HLA B7, and a reduced association with A9. The calculated A3B7 and A1B7 haplotypes were also significantly increased in cystinosis. Lod score analysis of five families indicated that linkage between the gene(s) for cystinosis and the HLA loci is unlikely. While associations of other genetic diseases with specific HLA types should be sought, such relationships, as documented here, do not imply true genetic linkage.

Interference reflection microscopy (IRM) is an optical technique used to study cell adhesion or cell mobility on a glass coverslip. The interference of reflected light waves generates images with high contrast and definition. IRM can be used to examine almost any cell that will rest upon a glass surface, although it is most useful in examining sites of close contact between a cell and substratum. This unit presents methods for obtaining IRM images of cells with particular emphasis on IRM imaging with a laser scanning confocal microscope (LSCM), as most LSCM are already capable of recording these images without any modification of the instrument. Techniques are presented for imaging fixed and live cells, as well as simultaneous multi-channel capture of fluorescence and reflection images.

Background and purpose Although the genetic contribution to stroke risk is well known, it remains unclear if young-onset stroke has a stronger genetic contribution than old-onset stroke. This study aims to compare the heritability of ischaemic stroke risk between young and old, using common genetic variants from whole-genome array data in population-based samples. Methods This analysis included 4050 ischaemic stroke cases and 5765 controls from six study populations of European ancestry; 47% of cases were young-onset stroke (age < 55 years). To quantify the heritability for stroke risk in these unrelated individuals, the pairwise genetic relatedness was estimated between individuals based on their whole-genome array data using a mixed linear model. Heritability was estimated separately for young-onset stroke and old-onset stroke (age ≥ 55 years). Results Heritabilities for young-onset stroke and old-onset stroke were estimated at 42% (±8%, P < 0.001) and 34% (±10%, P < 0.001), respectively. Conclusions Our data suggest that the genetic contribution to the risk of stroke may be higher in young-onset ischaemic stroke, although the difference was not statistically significant.

Ground glass hepatocytes (GGHs), a histological hallmark of chronic hepatitis B virus (HBV) infection, contain excessive hepatitis surface antigen (HBsAg) in the endoplasmic reticulum (ER), which is linked to unfolded protein response (UPR). The mechanism by which HBV activates UPR has not been fully defined. To investigate this, HepG2-NTCP cells and primary human hepatocytes (PHHs) were either infected with HBV or transduced with adenoviral vectors expressing replication-competent HBV genome or individual HBV genes. UPR markers were evaluated by qPCR, Western blotting, and immunofluorescence. Apoptosis and cell viability were measured by Caspase-3/7 and ATPlite assay respectively. We found that UPR markers were induced by the overexpression of HBsAg in HepG2-NTCP cells and PHHs. Elevation of UPR-induced genes showed a dose-dependent correlation with HBsAg levels. In HBV-infected livers, GGHs also demonstrated excessive accumulation of HBsAg associated with increased BIP/GRP78 staining, a marker of UPR. Prolonged activation of UPR by HBsAg overexpression induced signs of apoptosis. Overexpression of HBsAg can induce ER stress through protein kinase RNA-like endoplasmic reticulum kinase (PERK) pathway in vitro, and may be linked to the appearance of GGHs. The activation of UPR by HBsAg may sensitize hepatocytes to cell death and result in possible subsequent cellular changes leading to a premalignant phenotype.

AbstractThe Genea050 human embryonic stem cell line was derived from a donated, fully commercially consented ART blastocyst, carrying a deletion in 4q35 with only 5 D4Z4 repeats by PGD linkage analysis, indicative of FSHD1. Following ICM outgrowth on inactivated human feeders, karyotype was confirmed as 46, XY and STR analysis demonstrated a male Allele pattern. The hESC line had pluripotent cell morphology, 92% of cells expressed Nanog, 97% Oct4, 79% Tra1-60 and 99% SSEA4, gave a Pluritest Pluripotency score of 25.45, Novelty of 1.45 demonstrated Alkaline Phosphatase activity and tri-lineage teratoma formation. The cell line was negative for Mycoplasma and visible contamination.