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Significance We provide a comprehensive catalog of novel genetic variants influencing gene expression and metabolic phenotypes in human pancreatic islets. The data also show that the path from genetic variation (SNP) to gene expression is more complex than hitherto often assumed, and that we need to consider that genetic variation can also influence function of a gene by influencing exon usage or splice isoforms (sQTL), allelic imbalance, RNA editing, and expression of noncoding RNAs, which in turn can influence expression of target genes. Genetic variation can modulate gene expression, and thereby phenotypic variation and susceptibility to complex diseases such as type 2 diabetes (T2D). Here we harnessed the potential of DNA and RNA sequencing in human pancreatic islets from 89 deceased donors to identify genes of potential importance in the pathogenesis of T2D. We present a catalog of genetic variants regulating gene expression (eQTL) and exon use (sQTL), including many long noncoding RNAs, which are enriched in known T2D-associated loci. Of 35 eQTL genes, whose expression differed between normoglycemic and hyperglycemic individuals, siRNA of tetraspanin 33 (TSPAN33), 5′-nucleotidase, ecto (NT5E), transmembrane emp24 protein transport domain containing 6 (TMED6), and p21 protein activated kinase 7 (PAK7) in INS1 cells resulted in reduced glucose-stimulated insulin secretion. In addition, we provide a genome-wide catalog of allelic expression imbalance, which is also enriched in known T2D-associated loci. Notably, allelic imbalance in paternally expressed gene 3 (PEG3) was associated with its promoter methylation and T2D status. Finally, RNA editing events were less common in islets than previously suggested in other tissues. Taken together, this study provides new insights into the complexity of gene regulation in human pancreatic islets and better understanding of how genetic variation can influence glucose metabolism.

Epigenetic dysregulation may influence disease progression. Here we explore whether epigenetic alterations in human pancreatic islets impact insulin secretion and type 2 diabetes (T2D). In islets, 5,584 DNA methylation sites exhibit alterations in T2D cases versus controls and are associated with HbA1c in individuals not diagnosed with T2D. T2D-associated methylation changes are found in enhancers and regions bound by β-cell-specific transcription factors and associated with reduced expression of e.g. CABLES1 , FOXP1 , GABRA2 , GLR1A , RHOT1 , and TBC1D4 . We find RHOT1 (MIRO1) to be a key regulator of insulin secretion in human islets. Rhot1 -deficiency in β-cells leads to reduced insulin secretion, ATP/ADP ratio, mitochondrial mass, Ca 2+ , and respiration. Regulators of mitochondrial dynamics and metabolites, including L-proline, glycine, GABA, and carnitines, are altered in Rhot1 -deficient β-cells. Islets from diabetic GK rats present Rhot1-deficiency. Finally, RHOT1 methylation in blood is associated with future T2D. Together, individuals with T2D exhibit epigenetic alterations linked to mitochondrial dysfunction in pancreatic islets. Type 2 diabetes (T2D) is characterized by hyperglycemia caused by insufficient insulin release from pancreatic islets, often in combination with insulin resistance. Here the authors present an epigenetic case-control study in human pancreatic islets revealing changes that contribute to type 2 diabetes development, e.g., epigenetic downregulation of RHOT1.

Single-cell and single-nuclei RNA-sequencing (scRNA-seq and snRNA-seq) analyze cell-specific transcriptomes. However, only snRNA-seq applies to frozen biobanked samples. For human pancreatic islets, marker genes and reference-based cell type annotation methods are mainly from scRNA-seq datasets and may not be suitable for snRNA-seq. We compared human islet scRNA-seq and snRNA-seq data from the same donors (N = 4) and evaluated annotation methods by studying cell type composition and gene detection, and identified novel marker genes. We compared cell type annotations: (1) manual annotation based on identified marker genes, (2) reference-based annotation using Azimuth’s scRNA-seq pancreasref dataset, or (3) Seurat’s label transfer from the Human Pancreas Analysis Program (HPAP) scRNA-seq dataset. ScRNA-seq and snRNA-seq identified the same cell types, but predicted cell type proportions differed. Cell type proportion-differences between annotation methods were larger for snRNA-seq. Reference-based annotations generated higher cell type prediction and mapping scores for scRNA-seq than snRNA-seq. Manual annotation identified the novel snRNA-seq markers DOCK10, KIRREL3 (beta cells), STK32B (alpha cells), MECOM , AC007368.1 (acinar cells), LAMC2 and SLC28A3 (ductal cells), which improve snRNA-seq-based annotation. We confirmed ZNF385D as a snRNA-seq beta cell marker and ZNF385D silencing reduced insulin secretion. In conclusion, this study discovered novel snRNA-seq cell type marker genes in human pancreatic islets, and highlights the need for tailored snRNA-seq annotation strategies.

Type 2 diabetes (T2D) is caused by insufficient insulin secretion from pancreatic β cells. To identify candidate genes contributing to T2D pathophysiology, we studied human pancreatic islets from approximately 300 individuals. We found 395 differentially expressed genes (DEGs) in islets from individuals with T2D, including, to our knowledge, novel (OPRD1, PAX5, TET1) and previously identified (CHL1, GLRA1, IAPP) candidates. A third of the identified expression changes in islets may predispose to diabetes, as expression of these genes associated with HbA1c in individuals not previously diagnosed with T2D. Most DEGs were expressed in human β cells, based on single-cell RNA-Seq data. Additionally, DEGs displayed alterations in open chromatin and associated with T2D SNPs. Mouse KO strains demonstrated that the identified T2D-associated candidate genes regulate glucose homeostasis and body composition in vivo. Functional validation showed that mimicking T2D-associated changes for OPRD1, PAX5, and SLC2A2 impaired insulin secretion. Impairments in Pax5-overexpressing β cells were due to severe mitochondrial dysfunction. Finally, we discovered PAX5 as a potential transcriptional regulator of many T2D-associated DEGs in human islets. Overall, we have identified molecular alterations in human pancreatic islets that contribute to β cell dysfunction in T2D pathophysiology.

MicroRNAs have emerged as important players of gene regulation with significant impact in diverse disease processes. In type-2 diabetes, in which impaired insulin secretion is a major factor in disease progression, dysregulated microRNA expression in the insulin-secreting pancreatic beta cell has been widely-implicated. Here, we show that miR-130a-3p, miR-130b-3p, and miR-152-3p levels are elevated in the pancreatic islets of hyperglycaemic donors, corroborating previous findings about their upregulation in the islets of type-2 diabetes model Goto-Kakizaki rats. We demonstrated negative regulatory effects of the three microRNAs on pyruvate dehydrogenase E1 alpha (PDHA1) and on glucokinase (GCK) proteins, which are both involved in ATP production. Consequently, we found both proteins to be downregulated in the Goto-Kakizaki rat islets, while GCK mRNA expression showed reduced trend in the islets of type-2 diabetes donors. Overexpression of any of the three microRNAs in the insulin-secreting INS-1 832/13 cell line resulted in altered dynamics of intracellular ATP/ADP ratio ultimately perturbing fundamental ATP-requiring beta cell processes such as glucose-stimulated insulin secretion, insulin biosynthesis and processing. The data further strengthen the wide-ranging influence of microRNAs in pancreatic beta cell function, and hence their potential as therapeutic targets in type-2 diabetes.

Background Women exposed to Plasmodium infection develop antibodies and become semi-immune. This immunity is suppressed during pregnancy making both the pregnant woman and the foetus vulnerable to the adverse effects of malaria, particularly by Plasmodium falciparum. Intermittent preventive treatment of malaria in pregnancy (IPTp) with Sulfadoxine–pyrimethamine (SP) tablets is one of the current interventions to mitigate the effects of malaria on both the pregnant woman and the unborn child. The extent to which IPTp may interfere with the acquisition of protective immunity against pregnancy-associated malaria (PAM) is undefined in Ghana. Methods Three-hundred-and-twenty pregnant women were randomly enrolled at the antenatal clinic (ANC) in Madina, Accra. Venous blood samples were obtained at first ANC registration and at 4-week intervals (post-IPTp administration). Placental and cord blood samples were obtained at delivery and the infants were followed monthly for 6 months after birth. Anti-IgG and IgM antibodies against a crude antigen preparation and the glutamate-rich protein (GLURP) of P. falciparum were quantified by the enzyme-linked immunosorbent assay (ELISA). Results There was a general decline in the trend of mean concentrations of all the antibodies from enrolment to delivery. The levels of antibodies in cord blood and placenta were well correlated. Children did not show clinical signs of malaria at 6 months after birth. Conclusions IgG against both crude antigen and GLURP were present in placenta and cord blood and it is therefore concluded that there is a trend of declining antibody from enrolment to delivery and IPTp-SP may have reduced malaria exposure, however, this does not impact on the transfer of antibodies to the foetus in utero. The levels of maternal and cord blood antibodies at delivery showed no adverse implications on malaria among the children at 6 months. However, the quantum and quality of the antibody transferred needs further investigation to ensure that the infants are protected from severe episodes of malaria.

MicroRNAs contribute to the maintenance of optimal cellular functions by fine‐tuning protein expression levels. In the pancreatic β‐cells, imbalances in the exocytotic machinery components lead to impaired insulin secretion and type 2 diabetes (T2D). We hypothesize that dysregulated miRNA expression exacerbates β‐cell dysfunction, and have earlier shown that islets from the diabetic GK‐rat model have increased expression of miRNAs, including miR‐335‐5p (miR‐335). Here, we aim to determine the specific role of miR‐335 during development of T2D, and the influence of this miRNA on glucose‐stimulated insulin secretion and Ca2+‐dependent exocytosis. We found that the expression of miR‐335 negatively correlated with secretion index in human islets of individuals with prediabetes. Overexpression of miR‐335 in human EndoC‐βH1 and in rat INS‐1 832/13 cells (OE335) resulted in decreased glucose‐stimulated insulin secretion, and OE335 cells showed concomitant reduction in three exocytotic proteins: SNAP25, Syntaxin‐binding protein 1 (STXBP1), and synaptotagmin 11 (SYT11). Single‐cell capacitance measurements, complemented with TIRF microscopy of the granule marker NPY‐mEGFP demonstrated a significant reduction in exocytosis in OE335 cells. The reduction was not associated with defective docking or decreased Ca2+ current. More likely, it is a direct consequence of impaired priming of already docked granules. Earlier reports have proposed reduced granular priming as the cause of reduced first‐phase insulin secretion during prediabetes. Here, we show a specific role of miR‐335 in regulating insulin secretion during this transition period. Moreover, we can conclude that miR‐335 has the capacity to modulate insulin secretion and Ca2+‐dependent exocytosis through effects on granular priming. Earlier reports have proposed reduced granular priming as the cause of reduced first‐phase insulin secretion during prediabetes. Here, we show a negative correlation between miR‐335 expression and insulin secretion index during this transition period. Moreover, overexpression of miR‐335 in β‐cells resulted in decreased glucose‐stimulated insulin secretion, reduced Ca2+‐dependent exocytosis through effects on granular priming, and concomitant reduction in three exocytotic proteins: SNAP25, Syntaxin‐binding protein 1 (STXBP1), and synaptotagmin 11 (SYT11). PM, plasma membrane; VDCC, voltage‐dependent calcium channel.

MicroRNAs are small non-coding RNAs, which negatively regulate the expression of target genes. They have emerged as important modulators in beta cell compensation upon increased metabolic demand, failure of which leads to reduced insulin secretion and type 2 diabetes. To elucidate the function of miRNAs in beta cells, insulin-secreting cell lines, such as the rat insulinoma INS-1 832/13 and the human EndoC-βH1, are widely used. Previous studies in the cancer field have suggested that miRNA expression is influenced by confluency of adherent cells. We therefore aimed to investigate whether one of the most enriched miRNAs in the pancreatic endocrine cells, miR-375, and two of its validated targets in mouse, Cav1 and Aifm1 , were differentially-expressed in cell cultures with different confluences. Additionally, we measured the expression of other miRNAs, such as miR-152, miR-130a, miR-132, miR-212 and miR-200a, with known roles in beta cell function. We did not see any significant expression changes of miR-375 nor any of the two targets, in both the rat and human beta cell lines at different confluences. Interestingly, among the other miRNAs measured, the expression of miR-132 and miR-212 positively correlated with confluence, but only in the INS-1 832/13 cells. Our results show that the expression of miR-375 and other miRNAs with known roles in beta cell function is independent of, or at least minimally influenced by the density of proliferating adherent cells, especially within the confluence range optimal for functional assays to elucidate miRNA-dependent regulatory mechanisms in the beta cell.

Background and AimsA shift from outcrossing to selfing is thought to reduce the long-term survival of populations by decreasing the genetic variation necessary for adaptation to novel ecological conditions. However, theory also predicts an increase in adaptive potential as more of the existing variation becomes expressed as homozygous genotypes. So far, relatively few studies have examined how a transition to selfing simultaneously affects means, variances and covariances for characters that might be under stabilizing selection for a spatially varying optimum, e.g. characters describing leaf morphology.MethodsExperimental crosses within an initially self-sterile population of Crepis tectorum were performed to produce an outbred and inbred progeny population to assess how a shift to selfing affects the adaptive potential for measures of leaf morphology, with special emphasis on the degree of leaf dissection, a major target of diversifying selection within the study species.Key ResultsThree consecutive generations of selfing had a minor impact on survival, the total number of heads produced and the mean leaf phenotype, but caused a proportional increase in the genetic (co)variance matrix for foliar characters. For the degree of leaf dissection, the lowest 50th percentile of the inbred progeny population showed a disproportionate increase in the genetic variance, consistent with the recessive nature of the weakly lobed phenotype observed in interpopulation crosses. Comparison of inbreeding response with large-scale patterns of variation indicates a potential for selection in a (recently) inbred population to drive a large evolutionary reduction in degree of leaf dissection by increasing the frequency of particular sibling lines.ConclusionsThe results point to a positive role for inbreeding in phenotypic evolution, at least during or immediately after a rapid shift in mating system.

Anthropogenic climate change is a serious global environmental issue that threatens food and water security, energy production, and human health and wellbeing, ultimately jeopardizing the attainment of the UN Sustainable Development Goals (SDGs). A good understanding of climate change is essential for societies to adapt to or mitigate it. Yet, studies reveal that most people have limited knowledge, misconceptions and misunderstanding about climate change. Sub-Saharan Africa is projected to experience disproportionately higher adverse effects of climate change, but there is paucity of information about climate change knowledge in the region. Here, we assessed climate change knowledge, attitude and perception of undergraduate students in Ghana and the influential factors using a cross-sectional study and semi-structured questionnaire. The study population was full-time undergraduate students at the University of Ghana, Legon. The data was analyzed using descriptive statistics, logistic regressions, t-test and One-Way ANOVA. The results revealed that a strong majority of the respondents believe that climate change is real and largely human-induced, and they expressed concern about it. Yet, students lack basic knowledge and had some misconceptions about the causes and consequences of climate change. The overall knowledge score of the students on climate change was average (66.9%), although majority (92%) of the respondents claimed they had adequate (75–85%) knowledge of climate change. Our data also showed that respondents’ level of education, programme of study, ethnicity, religion and mother’s occupation had statistically significant association with their knowledge, perception and attitude on aspects of climate change. Our findings highlight knowledge gaps in climate change among undergraduate students in Ghana, underscoring the need to integrate climate change science into the education curricula at all levels of pre-tertiary schools and university for both the science and non-science programme.