Preclinical studies of COVID-19 mRNA vaccine BNT162b2, developed by Pfizer and BioNTech, showed reversible hepatic effects in animals that received the BNT162b2 injection. Furthermore, a recent study showed that SARS-CoV-2 RNA can be reverse-transcribed and integrated into the genome of human cells. In this study, we investigated the effect of BNT162b2 on the human liver cell line Huh7 in vitro. Huh7 cells were exposed to BNT162b2, and quantitative PCR was performed on RNA extracted from the cells. We detected high levels of BNT162b2 in Huh7 cells and changes in gene expression of long interspersed nuclear element-1 (LINE-1), which is an endogenous reverse transcriptase. Immunohistochemistry using antibody binding to LINE-1 open reading frame-1 RNA-binding protein (ORFp1) on Huh7 cells treated with BNT162b2 indicated increased nucleus distribution of LINE-1. PCR on genomic DNA of Huh7 cells exposed to BNT162b2 amplified the DNA sequence unique to BNT162b2. Our results indicate a fast up-take of BNT162b2 into human liver cell line Huh7, leading to changes in LINE-1 expression and distribution. We also show that BNT162b2 mRNA is reverse transcribed intracellularly into DNA in as fast as 6 h upon BNT162b2 exposure.

Fine-tuning of insulin release from pancreatic β-cells is essential to maintain blood glucose homeostasis. Here, we report that insulin secretion is regulated by a circular RNA containing the lariat sequence of the second intron of the insulin gene. Silencing of this intronic circular RNA in pancreatic islets leads to a decrease in the expression of key components of the secretory machinery of β-cells, resulting in impaired glucose- or KCl-induced insulin release and calcium signaling. The effect of the circular RNA is exerted at the transcriptional level and involves an interaction with the RNA-binding protein TAR DNA-binding protein 43 kDa (TDP-43). The level of this circularized intron is reduced in the islets of rodent diabetes models and of type 2 diabetic patients, possibly explaining their impaired secretory capacity. The study of this and other circular RNAs helps understanding β-cell dysfunction under diabetes conditions, and the etiology of this common metabolic disorder.

Type 2 diabetes (T2D) is caused by insufficient insulin secretion from pancreatic β-cells. To identify candidates contributing to T2D pathophysiology, we studied human pancreatic islets from ~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 islet expression changes may predispose to diabetes, as they associated with HbA1c in individuals not previously diagnosed with T2D. Most DEGs were expressed in human β-cells based on single-cell RNA-sequencing data. Additionally, DEGs displayed alterations in open chromatin and associated with T2D-SNPs. Mouse knock-out strains demonstrated that T2D-associated candidates 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 T2Dassociated DEGs in human islets. Overall, we identified molecular alterations in human pancreatic islets contributing to β-cell dysfunction in T2D pathophysiology.

Cystic fibrosis-related diabetes (CFRD) is a common complication for patients with cystic fibrosis (CF), a disease caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR). The cause of CFRD is unclear, but a commonly observed reduction in first-phase insulin secretion suggests defects at the beta cell level. Here we aimed to examine alpha and beta cell function in the Cftr tm1 EUR/F508del mouse model (C57BL/6J), which carries the most common human mutation in CFTR, the F508del mutation. CFTR expression, beta cell mass, insulin granule distribution, hormone secretion and single cell capacitance changes were evaluated using islets (or beta cells) from F508del mice and age-matched wild type (WT) mice aged 7–10 weeks. Granular pH was measured with DND-189 fluorescence. Serum glucose, insulin and glucagon levels were measured in vivo, and glucose tolerance was assessed using IPGTT. We show increased secretion of proinsulin and concomitant reduced secretion of C-peptide in islets from F508del mice compared to WT mice. Exocytosis and number of docked granules was reduced. We confirmed reduced granular pH by CFTR stimulation. We detected decreased pancreatic beta cell area, but unchanged beta cell number. Moreover, the F508del mutation caused failure to suppress glucagon secretion leading to hyperglucagonemia. In conclusion, F508del mice have beta cell defects resulting in (1) reduced number of docked insulin granules and reduced exocytosis and (2) potential defective proinsulin cleavage and secretion of immature insulin. These observations provide insight into the functional role of CFTR in pancreatic islets and contribute to increased understanding of the pathogenesis of CFRD.

Voltage-gated Ca2+ (CaV) channels trigger glucose-induced insulin secretion in pancreatic beta-cell and their dysfunction increases diabetes risk. These heteromeric complexes include the main subunit alpha1, and the accessory ones, including subunit gamma that remains unexplored. Here, we demonstrate that CaV gamma subunit 4 (CaVγ4) is downregulated in islets from human donors with diabetes, diabetic Goto-Kakizaki (GK) rats, as well as under conditions of gluco-/lipotoxic stress. Reduction of CaVγ4 expression results in decreased expression of L-type CaV1.2 and CaV1.3, thereby suppressing voltage-gated Ca2+ entry and glucose stimulated insulin exocytosis. The most important finding is that CaVγ4 expression is controlled by the transcription factor responsible for beta-cell specification, MafA, as verified by chromatin immunoprecipitation and experiments in beta-cell specific MafA knockout mice (MafAΔβcell). Taken together, these findings suggest that CaVγ4 is necessary for maintaining a functional differentiated beta-cell phenotype. Treatment aiming at restoring CaVγ4 may help to restore beta-cell function in diabetes.

Alzheimer’s disease is a neurodegenerative condition which involves heavy neuronal cell death linked to oligomers formed during the aggregation process of the amyloid β peptide 42 (A β 42). The aggregation of A β 42 involves both primary and secondary nucleation. Secondary nucleation dominates the generation of oligomers and involves the formation of new aggregates from monomers on catalytic fibril surfaces. Understanding the molecular mechanism of secondary nucleation may be crucial in developing a targeted cure. Here, the self-seeded aggregation of WT A β 42 is studied using direct stochastic optical reconstruction microscopy (dSTORM) with separate fluorophores in seed fibrils and monomers. Seeded aggregation proceeds faster than nonseeded reactions because the fibrils act as catalysts. The dSTORM experiments show that monomers grow into relatively large aggregates on fibril surfaces along the length of fibrils before detaching, thus providing a direct observation of secondary nucleation and growth along the sides of fibrils. The experiments were repeated for cross-seeded reactions of the WT A β 42 monomer with mutant A β 42 fibrils that do not catalyze the nucleation of WT monomers. While the monomers are observed by dSTORM to interact with noncognate fibril surfaces, we fail to notice any growth along such fibril surfaces. This implies that the failure to nucleate on the cognate seeds is not a lack of monomer association but more likely a lack of structural conversion. Our findings support a templating role for secondary nucleation, which can only take place if the monomers can copy the underlying parent structure without steric clashes or other repulsive interactions between nucleating monomers.

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Fluorescence-based single molecule techniques provide important tools towards understanding the molecular mechanism of complex neurodegenerative diseases. This requires efficient covalent attachment of fluorophores. Here we create a series of cysteine mutants (S8C, Y10C, S26C, V40C, and A42C) of Aβ42, involved in Alzheimer’s disease, based on exposed positions in the fibril structure and label them with the Alexa-fluorophores using maleimide chemistry. Direct stochastic optical reconstruction microscopy imaging shows that all the labelled mutants form fibrils that can be detected by virtue of Alexa fluorescence. Aggregation assays and cryo-electron micrographs establish that the careful choice of labelling position minimizes the perturbation of the aggregation process and fibril structure. Peptides labelled at the N-terminal region, S8C and Y10C, form fibrils independently and with wild-type. Peptides labelled at the fibril core surface, S26C, V40C and A42C, form fibrils only in mixture with wild-type peptide. This can be understood on the basis of a recent fibril model, in which S26, V40 and A42 are surface exposed in two out of four monomers per fibril plane. We provide a palette of fluorescently labelled Aβ42 peptides that can be used to gain understanding of the complex mechanisms of Aβ42 self-assembly and help to develop a more targeted approach to cure the disease.