Mycobiota and molecular detection of Aspergillus flavus and A. parasiticus aflatoxin contamination of Strawberry (Fragaria ananassa Duch.) fruits

Strawberry fungi were isolated from fresh fruits and juice on the two types of media (Sabouraud dextrose agar, SDA and potato-dextrose agar, PDA) at 28 °C. Nineteen fungal species belong to 12 genera were isolated from fruits and juice on both isolation media. The most common fungal genera and species were Aspergillus flavus, A. niger, Mucor racemosus, Neurospora crassa, Penicillium chrysogenum, Rhizopus stolonifer and Trichoderma harzianum. Twenty A. flavus and A. parasitics isolates were assayed for their abilities to produce aflatoxins. The concentration of aflatoxins ranged between 25.8–75.2 and 23.6–71.1 ng/ml at 350 and 365 nm, respectively. Among A. flavus and A. parasiticus strains tested, aflatoxin B contributed 30–60% of total isolates. However, G type contributed 85–90%. The R_f values of B₁, B₂, G₁ and G₂ were 0.79, 0.61, 0.44 and 0.32, respectively. High-performance liquid chromatography analysis of extracts revealed the presence of aflatoxins with variable levels.     

SG2NA is a regulator of endoplasmic reticulum (ER) homeostasis as its depletion leads to ER stress

SG2NA belongs to a three-member striatin subfamily of WD40 repeat superfamily of proteins. It has multiple protein-protein interaction domains involved in assembling supramolecular signaling complexes. Earlier, we had demonstrated that there are at least five variants of SG2NA generated by alternative splicing, intron retention, and RNA editing. Such versatile and dynamic mode of regulation implicates it in tissue development. In order to shed light on its role in cell physiology, total proteome analysis was performed in NIH3T3 cells depleted of 78 kDa SG2NA, the only isoform expressing therein. A number of ER stress markers were among those modulated after knockdown of SG2NA. In cells treated with the ER stressors thapsigargin and tunicamycin, expression of SG2NA was increased at both mRNA and protein levels. The increased level of SG2NA was primarily in the mitochondria and the microsomes. A mouse injected with thapsigargin also had an increase in SG2NA in the liver but not in the brain. Cell cycle analysis suggested that while loss of SG2NA reduces the level of cyclin D1 and retains a population of cells in the G1 phase, concurrent ER stress facilitates their exit from G1 and traverse through subsequent phases with concomitant cell death. Thus, SG2NA is a component of intrinsic regulatory pathways that maintains ER homeostasis.     

Highly efficient CRISPR-mediated large DNA docking and multiplexed prime editing using a single baculovirus

CRISPR-based precise gene-editing requires simultaneous delivery of multiple components into living cells, rapidly exceeding the cargo capacity of traditional viral vector systems. This challenge represents a major roadblock to genome engineering applications. Here we exploit the unmatched heterologous DNA cargo capacity of baculovirus to resolve this bottleneck in human cells. By encoding Cas9, sgRNA and Donor DNAs on a single, rapidly assembled baculoviral vector, we achieve with up to 30% efficacy whole-exon replacement in the intronic β-actin (ACTB) locus, including site-specific docking of very large DNA payloads. We use our approach to rescue wild-type podocin expression in steroid-resistant nephrotic syndrome (SRNS) patient derived podocytes. We demonstrate single baculovirus vectored delivery of single and multiplexed prime-editing toolkits, achieving up to 100% cleavage-free DNA search-and-replace interventions without detectable indels. Taken together, we provide a versatile delivery platform for single base to multi-gene level genome interventions, addressing the currently unmet need for a powerful delivery system accommodating current and future CRISPR technologies without the burden of limited cargo capacity.     

Mutations of MYO6 are associated with recessive deafness,DFNB37

Cosegregation of profound, congenital deafness with markers on chromosome 6q13 in three Pakistani families defines a new recessive deafness locus, DFNB37. Haplotype analyses reveal a 6-cM linkage region, flanked by markers D6S1282 and D6S1031, that includes the gene encoding unconventional myosin VI. In families with recessively inherited deafness, DFNB37, our sequence analyses of MYO6 reveal a frameshift mutation (36-37insT), a nonsense mutation (R1166X), and a missense mutation (E216V). These mutations, along with a previously published missense allele linked to autosomal dominant progressive hearing loss (DFNA22), provide an allelic spectrum that probes the relationship between myosin VI dysfunction and the resulting phenotype.     

A novel FtsZ-like protein is involved in replication of the anthrax toxin-encoding pXO1 plasmid in Bacillus anthracis

Plasmid pXO1 encodes the tripartite anthrax toxin, which is the major virulence factor of Bacillus anthracis. In spite of the important role of pXO1 in anthrax pathogenesis, very little is known about its replication and maintenance in B. anthracis. We cloned a 5-kb region of the pXO1 plasmid into an Escherichia coli vector and showed that this plasmid can replicate when introduced into B. anthracis. Mutational analysis showed that open reading frame 45 (repX) of pXO1 was required for the replication of the miniplasmid in B. anthracis. Interestingly, repX showed limited homology to bacterial FtsZ proteins that are involved in cell division. A mutation in the predicted GTP binding domain of RepX abolished its replication activity. Genes almost identical to repX are contained on several megaplasmids in members of the Bacillus cereus group, including a B. cereus strain that causes an anthrax-like disease. Our results identify a novel group of FtsZ-related initiator proteins that are required for the replication of virulence plasmids in B. anthracis and possibly in related organisms. Such replication proteins may provide novel drug targets for the elimination of plasmids encoding the anthrax toxin and other virulence factors.     

Sulphur application improves the growth, seed yield and oil quality of canola

Sulphur (S) nutrition is very important for harvesting potential seed and oil yield of rapeseed. This study evaluated response of foliage applied thiourea on the performance of two canola cultivars Shiralee and Dunkeld. Sulphur was applied to soil (40 kg ha^?1) or foliage (500 and 1,000 mg L^?1) at rosette, bud initiation and flowering stages using elemental S or thiourea as source, respectively; no S application was taken as control. Among all the treatments, soil application of S improved the crop growth, yield and oil quality in both cultivars and was followed by foliar application of thiourea at 1,000 mg L^?1 compared with no application. Soil applied S and foliar thiourea (1,000 mg L^?1) delayed the flowering and maturity. Soil and foliar applied S significantly improved leaf area index, crop growth rate, net assimilation rate and chlorophyll contents. Plant height, number of branches, siliqua per plant, seed number per siliqua, 1,000-seed weight, biological and seed yield were also increased by soil applied S and foliage applied thiourea (1,000 mg L^?1). Nonetheless, improvement in harvest index, seed oil, protein and glucosinolate contents was only observed from foliage applied thiourea (1,000 mg L^?1). Response of cv. Shiralee to sulphur application was better than cv. Dunkeld. In conclusion, foliar applied thiourea (1,000 mg L^?1) can have potential to improve growth, yield and oil quality in canola and can be economically viable and attractive alternative source.