Genome-edited zebrafish model of ABCC8 loss-of-function disease

KATP channel gain- (GOF) and loss-of-function (LOF) mutations underlie human neonatal diabetes mellitus (NDM) and hyperinsulinism (HI), respectively. Genetically modified mice with transgenic overexpression of GOF mutations recapitulate many features of human NDM but, importantly, there are currently no gene knock-in mouse models of GOF mutations. Moreover, while transgenic mice expressing incomplete KATP LOF do reiterate mild hyperinsulinism, KATP knockout animals do not exhibit persistent hyperinsulinism. We have shown that islet excitability and glucose homeostasis are regulated by identical KATP channels in zebrafish. SUR1 truncation mutation (K499X) was introduced into the abcc8 gene to explore the possibility of using zebrafish for modeling human NDM and HI. Patch-clamp analysis confirmed complete absence of channel activity in β-cells from K499X (SUR1-/-) fish. No difference in random blood glucose was detected in heterozygous SUR1+/- fish, nor in homozygous SUR1-/- fish, mimicking findings in SUR1 knockout mice. Mutant fish did however, demonstrate impaired glucose tolerance, similar to partial LOF mouse models. In paralleling features of mammalian diabetes and hyperinsulinism resulting from equivalent gain- or loss-of-function mutations, these gene-edited animals provide valid zebrafish models of KATP LOF driven-dependent pancreatic disease.

A truncation mutation in the L1CAM gene in a child with hydrocephalus

<abstract> <p>Hydrocephalus is a neurodevelopmental, X-linked recessive disorder caused by mutations in the <italic>L1CAM</italic> gene. The <italic>L1CAM</italic> gene encodes for L1CAM protein which is essential for the nervous system development including adhesion between neurons, Myelination, Synaptogenesis etc. Herein, the present study has reported mutations in L1 syndrome patient with Hydrocephalus and Adducted thumb. Genomic DNA was extracted from patients whole blood (n = 18). The 11 exons of the <italic>L1CAM</italic> gene were amplified using specific PCR primers. The sequenced data was analysed and the pathogenicity of the mutation was predicted using the various bioinformatics programs: PROVEAN, PolyPhen2, and MUpro. The results revealed that the proband described here had nonsense mutation G1120→T at position 1120 in exon 9 which is in extracellular immunoglobulin domain (Ig4) of the <italic>L1CAM</italic> gene. This nonsense mutation is found to be truncated with a deleterious effect on developing brain of the child, and this is the first report of this novel mutation in patient with X-linked Hydrocephalus in India.</p> </abstract>

SARS-CoV-2 Spike Alterations Enhance Pseudoparticle Titers and Replication-Competent VSV-SARS-CoV-2 Virus

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the most recent global pandemic that has caused more than a million deaths around the world. The spike glycoprotein (S) drives the entry and fusion of this virus and is the main determinant of cell tropism. To explore S requirements for entry under BSL2 conditions, S has been pseudotyped onto vesicular stomatitis virus (VSV) or retroviral particles with varied success. Several alterations to S were demonstrated to improve pseudoparticle titers, but they have not been systematically compared. In this study, we produced pseudotyped VSV particles with multiple modifications to S, including truncation, mutation, and tagging strategies. The main objective of this study was to determine which modifications of the S protein optimize cell surface expression, incorporation into pseudotyped particles, and pseudoparticle entry. Removal of the last 19 residues of the cytoplasmic tail produced a hyper-fusogenic S, while removal of 21 residues increased S surface production and VSV incorporation. Additionally, we engineered a replication-competent VSV (rVSV) virus to produce the S-D614G variant with a truncated cytoplasmic tail. While the particles can be used to assess S entry requirements, the rVSV∆G/SMet1D614G∆21 virus has a poor specific infectivity (particle to infectious titer ratio).