Informing investment in health workforce in Bangladesh: a health labour market analysis.

BACKGROUND: As the 2016 Global Strategy on Human Resources for Health: Workforce 2030 (GSHRH) outlines, health systems can only function with health workforce (HWF). Bangladesh is committed to achieving universal health coverage (UHC) hence a comprehensive understanding of the existing HWF was deemed necessary informing policy and funding decisions to the health system. METHODS: The health labour market analysis (HLMA) framework for UHC cited in the GSHRH was adopted to analyse the supply, need and demand of all health workers in Bangladesh. Government's information systems provided data to document the public sector HWF. A national-level assessment (2019) based on a country representative sample of 133 geographical units, served to estimate the composition and distribution of the private sector HWF. Descriptive statistics served to characterize the formal and informal HWF. RESULTS: The density of doctors, nurses and midwives in Bangladesh was only 9.9 per 10 000 population, well below the indicative sustainable development goals index threshold of 44.5 outlined in the GSHRH. Considering all HWFs in Bangladesh, the estimated total density was 49 per 10 000 population. However, one-third of all HWFs did not hold recognized roles and their competencies were unknown, taking only qualified and recognized HWFs into account results in an estimated density 33.2. With an estimate 75 nurses per 100 doctors in Bangladesh, the second area, where policy attention appears to be warranted is on the competencies and skill-mix. Thirdly, an estimated 82% of all HWFs work in the private sector necessitates adequate oversight for patient safety. Finally, a high proportion of unfilled positions in the public sector, especially in rural areas where 67% of the population lives, account only 11% of doctors and nurses. CONCLUSION: Bangladesh is making progress on many of the milestones of the GSHRH, notably, the establishment of the HWF unit and reporting through the national health workforce accounts. However, particular investment on strengthening the intersectoral HWF coordination across sectors; regulation for assurance of patient safety and adequate oversight of the private sector; establishing accreditation mechanisms for training institutions; and halving inequalities in access to a qualified HWF are important towards advancing UHC in Bangladesh.

Hypermethylation of antisense long noncoding RNAs in acute lymphoblastic leukemia.

AIM: Long noncoding RNAs serve critical regulatory functions highly specific for a tissue and its developmental stage. Antisense long ncRNA (AS-lncRNA) methylation changes in acute lymphoblastic leukemia (ALL) versus normal pre-B-cell lymphoblasts were evaluated to identify potential differential methylation in this group of genes. MATERIALS & METHODS: The methylome of ALL and normal lymphoblasts was examined by the methylated CpG island recovery assay followed by NGS. CONCLUSION: The potential effect of trans regulation by AS-lncRNA through DNA/RNA binding is significant as sequence alignment analysis of the 25 most differentially methylated AS-lncRNAs revealed 368 genes containing highly similar sequences with a median nucleotide identity of 90.8% and binding span of 122 base pairs. Regulation of biological processes and anatomical structure development were over represented. ALL classification schemes based on AS-lncRNA methylation can provide new insights into its pathogenesis and treatment.

Inferring a role for methylation of intergenic DNA in the regulation of genes aberrantly expressed in precursor B-cell acute lymphoblastic leukemia.

A complete understanding of the mechanisms involved in the development of pre-B ALL is lacking. In this study, we integrated DNA methylation data and gene expression data to elucidate the impact of aberrant intergenic DNA methylation on gene expression in pre-B ALL. We found a subset of differentially methylated intergenic loci that were associated with altered gene expression in pre-B ALL patients. Notably, 84% of these regions were also bound by transcription factors (TF) known to play roles in differentiation and B-cell development in a lymphoblastoid cell line. Further, an overall downregulation of eRNA transcripts was observed in pre-B ALL patients and these transcripts were associated with the downregulation of putative target genes involved in B-cell migration, proliferation, and apoptosis. The identification of novel putative regulatory regions highlights the significance of intergenic DNA sequences and may contribute to the identification of new therapeutic targets for the treatment of pre-B ALL.

Integrated methylome and transcriptome analysis reveals novel regulatory elements in pediatric acute lymphoblastic leukemia.

Acute lymphoblastic leukemia (ALL) is the most common cancer diagnosed in children under the age of 15. In addition to genetic aberrations, epigenetic modifications such as DNA methylation are altered in cancer and impact gene expression. To identify epigenetic alterations in ALL, genome-wide methylation profiles were generated using the methylated CpG island recovery assay followed by next-generation sequencing. More than 25,000 differentially methylated regions (DMR) were observed in ALL patients with ∼ 90% present within intronic or intergenic regions. To determine the regulatory potential of the DMR, whole-transcriptome analysis was performed and integrated with methylation data. Aberrant promoter methylation was associated with the altered expression of genes involved in transcriptional regulation, apoptosis, and proliferation. Novel enhancer-like sequences were identified within intronic and intergenic DMR. Aberrant methylation in these regions was associated with the altered expression of neighboring genes involved in cell cycle processes, lymphocyte activation and apoptosis. These genes include potential epi-driver genes, such as SYNE1, PTPRS, PAWR, HDAC9, RGCC, MCOLN2, LYN, TRAF3, FLT1, and MELK, which may provide a selective advantage to leukemic cells. In addition, the differential expression of epigenetic modifier genes, pseudogenes, and non-coding RNAs was also observed accentuating the role of erroneous epigenetic gene regulation in ALL.

Genome-wide DNA methylation analysis in precursor B-cells.

DNA methylation is responsible for regulating gene expression and cellular differentiation and for maintaining genomic stability during normal human development. Furthermore, it plays a significant role in the regulation of hematopoiesis. In order to elucidate the influence of DNA methylation during B-cell development, genome-wide DNA methylation status of pro-B, pre-BI, pre-BII, and naïve-B-cells isolated from human umbilical cord blood was determined using the methylated CpG island recovery assay followed by next generation sequencing. On average, 182-200 million sequences were generated for each precursor B-cell subset in 10 biological replicates. An overall decrease in methylation was observed during the transition from pro-B to pre-BI, whereas no differential methylation was observed in the pre-BI to pre-BII transition or in the pre-BII to naïve B-cell transition. Most of the methylated regions were located within intergenic and intronic regions not present in a CpG island context. Putative novel enhancers were identified in these regions that were differentially methylated between pro-B and pre-BI cells. The genome-wide methylation profiles are publically available and may be used to gain a better understanding of the involvement of atypical DNA methylation in the pathogenesis of malignancies associated with precursor B-cells.

Isolation of precursor B-cell subsets from umbilical cord blood.

Umbilical cord blood is highly enriched for hematopoietic progenitor cells at different lineage commitment stages. We have developed a protocol for isolating precursor B-cells at four different stages of differentiation. Because genes are expressed and epigenetic modifications occur in a tissue specific manner, it is vital to discriminate between tissues and cell types in order to be able to identify alterations in the genome and the epigenome that may lead to the development of disease. This method can be adapted to any type of cell present in umbilical cord blood at any stage of differentiation. This method comprises 4 main steps. First, mononuclear cells are separated by density centrifugation. Second, B-cells are enriched using biotin conjugated antibodies that recognize and remove non B-cells from the mononuclear cells. Third the B-cells are fluorescently labeled with cell surface protein antibodies specific to individual stages of B-cell development. Finally, the fluorescently labeled cells are sorted and individual populations are recovered. The recovered cells are of sufficient quantity and quality to be utilized in downstream nucleic acid assays.

Isolation of RNA and DNA from single preimplantation embryos and a small number of Mammalian oocytes for imprinting studies.

Researchers whose experimental models are mammalian oocytes and preimplantation embryos are often limited by the yield of nucleic acids that can be isolated from such a small sample size. In addition, the limited number of cells from these types of samples makes the simultaneous recovery of RNA and DNA very difficult and often sample pooling is necessary to increase nucleic acid yield. Here we report a simple set of procedures using commercially available kits that results in consistent yield and quality of nucleic acids. After sample lysis, RNA is isolated and converted to a reusable cDNA library. Following RNA isolation, DNA is precipitated, isolated, and bisulfite converted for DNA methylation studies. Our results demonstrate the feasibility of isolating RNA and DNA from a small number of cells with repeatability of results.