Validated assays for the quantification of C9orf72 human pathology.

A repeat expansion mutation in the C9orf72 gene is the leading known genetic cause of FTD and ALS. The C9orf72-ALS/FTD field has been plagued by a lack of reliable tools to monitor this genomic locus and its RNA and protein products. We have validated assays that quantify C9orf72 pathobiology at the DNA, RNA and protein levels using knock-out human iPSC lines as controls. Here we show that single-molecule sequencing can accurately measure the repeat expansion and faithfully report on changes to the C9orf72 locus in what has been a traditionally hard to sequence genomic region. This is of particular value to sizing and phasing the repeat expansion and determining changes to the gene locus after gene editing. We developed ddPCR assays to quantify two major C9orf72 transcript variants, which we validated by selective excision of their distinct transcriptional start sites. Using validated knock-out human iPSC lines, we validated 4 commercially available antibodies (of 9 tested) that were specific for C9orf72 protein quantification by Western blot, but none were specific for immunocytochemistry. We tested 15 combinations of antibodies against dipeptide repeat proteins (DPRs) across 66 concentrations using MSD immunoassay, and found two (against poly-GA and poly-GP) that yielded a 1.5-fold or greater signal increase in patient iPSC-motor neurons compared to knock-out control, and validated them in human postmortem and transgenic mouse brain tissue. Our validated DNA, RNA and protein assays are applicable to discovery research as well as clinical trials.

The first report of bean common mosaic virus (BCMV) infection of African yam bean (Sphenostylis stenocarpa) in Nigeria.

BACKGROUND: African yam bean (Sphenostylis stenocarpa) is an underutilized crop that has the potential to contribute to sustainable food security. In October 2021, more than 90% African Yam Bean (AYB) plants showed typical virus symptoms of mosaic and necrosis in the grain legumes field of the Institute of Agricultural Research and Training (IAR&T), Nigeria. METHODS AND RESULTS: Subsequently, leaf samples were collected and tested by ELISA and PCR to identify the virus species. Anti-BCMV and anti-potyvirus antibodies both gave positive results when symptomatic leaves were tested, and PCR using primers designed to the coat protein gene of BCMV amplified a band of the expected size (469 bp). The sequence of the PCR product was deposited in GenBank with the accession No. OL763314. The nucleotide sequence of the coat protein gene had 99% identity with BCMV isolate TN2 (KY044818). The identities of the nucleotide and amino acid sequence of the partial CP gene of the isolated virus relative to those of other potyviruses were 82.96-99.12% and 87.33-100%,, respectively. Phylogenetic analyses of the partial CP-nucleotide sequences grouped the isolate from this study (BCMV-IART-AYB) and BCMV-TN2 in the same cluster with other BCMV strains of the peanut stripe (PSt) and the blackeye cowpea (BlC) strains. CONCLUSIONS: In this study, we identified Bean commom mosaic virus (BCMV) infecting AYB for the first time in Nigeria and show that it has high nucleotide and amino acid identity with an Isolate of cowpea-infecting BCMV in India and China respectively than isolate in Nigeria.

First report of Cucumber mosaic virus infecting Ocimum gratissimum (L.) in Calabar, Cross River State, Nigeria.

Leaf samples of Ocimum gratissimum (L.) exhibiting vein banding, mosaic and chlorotic spots were collected randomly from the field. The symptomatic samples reacted positively to specific CMV antibody in antigen coated plate enzyme linked immunosorbent assay and to confirm the presence of CMV, RT-PCR was performed using CMV-specific primers that amplify a 519 bp region from the viral coat protein gene. The expected amplicon shared homology of 97.06% with a Nigerian isolate MH178110. Phylogenetic tree constructed revealed the isolate in close association with CMV strains belonging to subgroup II. This is the first molecular evidence of CMV in O. gratissimum in Nigeria and adds to the list of natural host for the virus.