Deletion of RFX6 impairs iPSC-derived islet organoid development and survival, with no impact on PDX1+/NKX6.1+ progenitors.

AIMS/HYPOTHESIS: Homozygous mutations in RFX6 lead to neonatal diabetes accompanied by a hypoplastic pancreas, whereas heterozygous mutations cause MODY. Recent studies have also shown RFX6 variants to be linked with type 2 diabetes. Despite RFX6's known function in islet development, its specific role in diabetes pathogenesis remains unclear. Here, we aimed to understand the mechanisms underlying the impairment of pancreatic islet development and subsequent hypoplasia due to loss-of-function mutations in RFX6. METHODS: We examined regulatory factor X6 (RFX6) expression during human embryonic stem cell (hESC) differentiation into pancreatic islets and re-analysed a single-cell RNA-seq dataset to identify RFX6-specific cell populations during islet development. Furthermore, induced pluripotent stem cell (iPSC) lines lacking RFX6 were generated using CRISPR/Cas9. Various approaches were then employed to explore the consequences of RFX6 loss across different developmental stages. Subsequently, we evaluated transcriptional changes resulting from RFX6 loss through RNA-seq of pancreatic progenitors (PPs) and endocrine progenitors (EPs). RESULTS: RFX6 expression was detected in PDX1+ cells in the hESC-derived posterior foregut (PF). However, in the PPs, RFX6 did not co-localise with pancreatic and duodenal homeobox 1 (PDX1) or NK homeobox 1 (NKX6.1) but instead co-localised with neurogenin 3, NK2 homeobox 2 and islet hormones in the EPs and islets. Single-cell analysis revealed high RFX6 expression levels in endocrine clusters across various hESC-derived pancreatic differentiation stages. Upon differentiating iPSCs lacking RFX6 into pancreatic islets, a significant decrease in PDX1 expression at the PF stage was observed, although this did not affect PPs co-expressing PDX1 and NKX6.1. RNA-seq analysis showed the downregulation of essential genes involved in pancreatic endocrine differentiation, insulin secretion and ion transport due to RFX6 deficiency. Furthermore, RFX6 deficiency resulted in the formation of smaller islet organoids due to increased cellular apoptosis, linked to reduced catalase expression, implying a protective role for RFX6. Overexpression of RFX6 reversed defective phenotypes in RFX6-knockout PPs, EPs and islets. CONCLUSIONS/INTERPRETATION: These findings suggest that pancreatic hypoplasia and reduced islet cell formation associated with RFX6 mutations are not due to alterations in PDX1+/NKX6.1+ PPs but instead result from cellular apoptosis and downregulation of pancreatic endocrine genes. DATA AVAILABILITY: RNA-seq datasets have been deposited in the Zenodo repository with accession link (DOI: https://doi.org/10.5281/zenodo.10656891 ).

A descriptive study of frequency of short term complications in late preterm neonates at a tertiary care hospital Rawalpindi.

OBJECTIVE: To determine the frequency of short-term complications in late preterm neonates in a tertiary care setting. METHODS: The observational study was conducted at Benazir Bhutto Hospital, Rawalpindi, Pakistan, from December 1, 2020, to May 31, 2021, and comprised late preterm neonates. Frequency of complications were noted in the subjects. Data was analysed using SPSS 22. RESULTS: Of the 200 subjects, 108(54%) were males, 84(42%) were aged 34-35 weeks. Among the complications, sepsis was the most frequent 88(44%), followed by respiratory distress syndrome RDS 58(29%). Mean weight was 2±0.42 kg and mean day of life at presentation was 2±1.9. Most common maternal risk factor was premature rupture of membrane 48(24%). CONCLUSIONS: Late preterm neonates had critical complications. The health authorities should formulate policies in this regard.

Transcriptome Analysis of Host Inflammatory Responses to the Ectoparasitic Mite Sarcoptes scabiei var. hominis.

Scabies, a human skin infestation caused by the ectoparasitic mite Sarcoptes scabiei var. hominis, affects more than 200 million people globally. The prevailing knowledge of the disease process and host immune response mechanisms is limited. A better understanding of the host-parasite relationship is essential for the identification of novel vaccine and drug targets. Here we aimed to interrogate the transcriptomic profiles of mite-infested human skin biopsies with clinical manifestations of ordinary scabies subjects ("OS"; n = 05) and subjects naive to scabies ("control"; n = 03) using RNASeq data analysis. A combined clustering, network, and pathway mapping approach enabled us to identify key signaling events in the host immune and pro-inflammatory responses to S. scabiei infestation. The clustering patterns showed various differentially expressed genes including inflammatory responses and innate immunity genes (DEFB4A, IL-19, CXCL8, CSF3, SERPINB4, S100A7A, HRNR) and notably upregulation of the JAK-STAT pathway in scabies-infested samples. Mite-infested human skin biopsies (GSE178563) were compared with an ex-vivo porcine infested model (E-MTAB-6433) and human skin equivalents (GSE48459). Marked enrichment of immune response pathways (JAK-STAT signaling, IL-4 and IL-13 pathway, and Toll receptor cascade), chemokine ligands and receptors (CCL17, CCL18, CCL3L1, CCL3L3, CCR7), and cytokines (IL-13 and IL-20) were observed. Additionally, genes known for their role in psoriasis and atopic dermatitis were upregulated, e.g., IL-19. The detailed transcriptomic profile has provided an insight into molecular functions, biological processes, and immunological responses and increased our understanding about transcriptomic regulation of scabies in human.

A Computational Systems Analyses to Identify Biomarkers and Mechanistic Link in Psoriasis and Cutaneous Squamous Cell Carcinoma.

Psoriasis is the most common and chronic skin disease that affects individuals from every age group. The rate of psoriasis is increasing over the time in both developed and developing countries. Studies have revealed the possibility of association of psoriasis with skin cancers, particularly non-melanoma skin cancers (NMSC), which, include basal cell carcinoma and cutaneous squamous cell carcinoma (cSCC). There is a need to analyze the disease at molecular level to propose potential biomarkers and therapeutic targets in comparison to cSCC. Therefore, the second analyzed disease of this study is cSCC. It is the second most common prevalent skin cancer all over the world with the potential to metastasize and recur. There is an urge to validate the proposed biomarkers and discover new potential biomarkers as well. In order to achieve the goals and objectives of the study, microarray and RNA-sequencing data analyses were performed followed by network analysis. Afterwards, quantitative systems biology was implemented to analyze the results at a holistic level. The aim was to predict the molecular patterns that can lead psoriasis to cancer. The current study proposed potential biomarkers and therapeutic targets for psoriasis and cSCC. IL-17 signaling pathway is also identified as significant pathway in both diseases. Moreover, the current study proposed that autoimmune pathology, neutrophil recruitment, and immunity to extracellular pathogens are sensitive towards MAPKs (MAPK13 and MAPK14) and genes for AP-1 (FOSL1 and FOS). Therefore, these genes should be further studied in gene knock down based studies as they may play significant role in leading psoriasis towards cancer.