Differential CpG methylation at Nnat in the early establishment of beta cell heterogeneity.

AIMS/HYPOTHESIS: Beta cells within the pancreatic islet represent a heterogenous population wherein individual sub-groups of cells make distinct contributions to the overall control of insulin secretion. These include a subpopulation of highly connected 'hub' cells, important for the propagation of intercellular Ca2+ waves. Functional subpopulations have also been demonstrated in human beta cells, with an altered subtype distribution apparent in type 2 diabetes. At present, the molecular mechanisms through which beta cell hierarchy is established are poorly understood. Changes at the level of the epigenome provide one such possibility, which we explore here by focusing on the imprinted gene Nnat (encoding neuronatin [NNAT]), which is required for normal insulin synthesis and secretion. METHODS: Single-cell RNA-seq datasets were examined using Seurat 4.0 and ClusterProfiler running under R. Transgenic mice expressing enhanced GFP under the control of the Nnat enhancer/promoter regions were generated for FACS of beta cells and downstream analysis of CpG methylation by bisulphite sequencing and RNA-seq, respectively. Animals deleted for the de novo methyltransferase DNA methyltransferase 3 alpha (DNMT3A) from the pancreatic progenitor stage were used to explore control of promoter methylation. Proteomics was performed using affinity purification mass spectrometry and Ca2+ dynamics explored by rapid confocal imaging of Cal-520 AM and Cal-590 AM. Insulin secretion was measured using homogeneous time-resolved fluorescence imaging. RESULTS: Nnat mRNA was differentially expressed in a discrete beta cell population in a developmental stage- and DNA methylation (DNMT3A)-dependent manner. Thus, pseudo-time analysis of embryonic datasets demonstrated the early establishment of Nnat-positive and -negative subpopulations during embryogenesis. NNAT expression is also restricted to a subset of beta cells across the human islet that is maintained throughout adult life. NNAT+ beta cells also displayed a discrete transcriptome at adult stages, representing a subpopulation specialised for insulin production, and were diminished in db/db mice. 'Hub' cells were less abundant in the NNAT+ population, consistent with epigenetic control of this functional specialisation. CONCLUSIONS/INTERPRETATION: These findings demonstrate that differential DNA methylation at Nnat represents a novel means through which beta cell heterogeneity is established during development. We therefore hypothesise that changes in methylation at this locus may contribute to a loss of beta cell hierarchy and connectivity, potentially contributing to defective insulin secretion in some forms of diabetes. DATA AVAILABILITY: The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD048465.

Safeguarding Accuracy: The Impact of Interventions on Reducing Blood Culture Contamination.

Introduction: Blood culture is the gold standard for diagnosing bacteremia and direct the physicians to select appropriate antimicrobials. In hospitals, blood culture contamination (BCC) is a common problem that has a detrimental effect on patient outcomes. Hence, we implemented strategies in our tertiary care setup, for training phlebotomists and nurses in proper blood sampling techniques, and assessed their effectiveness in reducing BCC rates. Methods: This interventional study was conducted at the Indus Hospital, Karachi, Pakistan from 1st January 2021 to 30th June 2023. All blood cultures received from different departments of the hospital were included. The 2.5-year study period was divided into pre-intervention and intervention periods, with monthly monitoring of BCC. The BCC data between 1st January 2021 and 31st December 2021 was taken as the baseline pre-intervention period and the next 1.5 years comprised the intervention period (1st January 2022-30th June 2023). To improve compliance, various strategies were implemented, such as regular training sessions, didactic sessions, and re-competencies. Results: A total of 86 774 Blood cultures were received from all departments of the hospital, out of which n = 30 672 were received in the pre-intervention period whereas, n = 56 102 were received in the intervention period. Mean BCC rate in the pre-intervention period was found to be 4.6%. However, after the implementation of different measures to reduce BCC, the contamination rate decreased to a mean of 3.1% by the end of the intervention period. Emergency department accounted for the highest proportion of BCC in the pre-intervention and intervention periods. Conclusion: We decreased BCC in our tertiary care setup by implementing a simple and inexpensive collaborative intervention, and came to the conclusion that the higher incidence of BCC was probably caused by factors unique to the emergency department and provided measures to successfully address them.

CO2-Free Ethylene Oxide Production via Liquid-Phase Epoxidation with Fe2O3/MSM Catalyst.

In this study, we present an approach for ethylene oxide (EO) production that addresses environmental concerns by eliminating greenhouse gas emissions. Our catalyst, Fe2O3/MSM, was synthesized using a hydrothermal method, incorporating Fe2O3 nanoparticles into a well-structured mesoporous silica matrix (MSM). We selected peracetic acid as the oxidant, enabling CO2-free EO production while yielding valuable by-products such as acetic acid, monoethylene glycol, and diethylene glycol. X-ray diffraction (XRD), X- ray photoelectron spectroscopy (XPS), and Brunauer-Emmett-Teller (BET) analyses confirmed the heteroatom structure of the catalysts and porosity, while Transmission electron microscopy (TEM) analysis provided insights into its morphology. Then, the synthesized catalyst was used in the liquid-phase epoxidation of ethylene for EO production. Our systematic experiments involved varying critical parameters such as temperature, ethylene to oxidant ratio, catalyst dosage, and solvent to optimize EO selectivity and ethylene conversion. The results of this study demonstrated an 80.2 % ethylene conversion to EO with an EO selectivity of 87.6 %. The production process yielded valuable by-products without CO2 emissions, highlighting its environmental friendliness.