Rotavirus activates a noncanonical ATM‐Chk2 branch of DNA damage response during infection to positively regulate viroplasm dynamics

Surveillance for maintaining genomic pristineness, a protective safeguard of great onco‐preventive significance, has been dedicated in eukaryotic cells to a highly conserved and synchronised signalling cascade called DNA damage response (DDR). Not surprisingly, foreign genetic elements like those of viruses are often potential targets of DDR. Viruses have evolved novel ways to subvert this genome vigilance by twisting canonical DDR to a skewed, noncanonical response through selective hijacking of some DDR components while antagonising the others. Though reported for many DNA and a few RNA viruses, potential implications of DDR have not been addressed yet in case of infection with rotavirus (RV), a double‐stranded RNA virus. In the present study, we aimed at the modulation of ataxia telangiectasia mutated (ATM)‐checkpoint kinase 2 (Chk2) branch of DDR in response to RV infection in vitro. We found activation of the transducer kinase ATM and its downstream effector Chk2 in RV‐SA11‐infected cells, the activation response being maximal at 6‐hr post infection. Moreover, ATM activation was found to be dependent on induction of the upstream sensor Mre11‐Rad50‐Nbs1 (MRN) complex. Interestingly, RV‐SA11‐mediated maximal induction of ATM‐Chk2 pathway was revealed to be neither preceded by occurrence of nuclear DNA damage nor transduced to formation of damage‐induced canonical nuclear foci. Subsequent investigations affirmed sequestration of MRN components as well as ATM‐Chk2 proteins away from nucleus into cytosolic RV replication factories (viroplasms). Chemical intervention targeting ATM and Chk2 significantly inhibited fusion and maturation of viroplasms leading to attenuated viral propagation. Cumulatively, the current study describes RV‐mediated activation of a noncanonical ATM‐Chk2 branch of DDR skewed in favour of facilitated viroplasm fusion and productive viral perpetuation.     

Noninvasive Method of DNA Isolation From Fecal Epithelial Tissue of Dairy Animals

A novel noninvasive genomic DNA isolation protocol from fecal tissue, by the proteinase K digestion and guanidine hydrochloride extraction method, was assessed for the genotyping of cattle and buffalo. The epithelial tissues present on the surface of the feces were used as source for isolation of genomic DNA. The DNA isolated from fecal tissue was found to be similar as those obtained from other body tissues such as skin, brain, liver, kidney, and muscle. The quality of DNA was checked by agarose gel electrophoresis and polymerase chain reaction (PCR). We successfully amplified a 320 bp MHC class II DRB gene and a 125 bp mt-DNA D-loop region from isolated genomic DNA of cattle. Thus, the DNA isolated using this method was suitable for common molecular biology methods, such as restriction enzyme digestion and genotyping of dairy animals through PCR.     

Proteomics analysis to compare the venom composition between Naja naja and Naja kaouthia from the same geographical location of eastern India: Correlation with pathophysiology of envenomation and immunological cross-reactivity towards commercial polyantivenom

ABSTRACT

Background : Cobra bite is frequently reported across the Indian subcontinent and is associated with a high rate of death and morbidity. In eastern India (EI) Naja naja and Naja kaouthia are reported to be the two most abundant species of cobra.

Research design and methods : The venom proteome composition of N. naja (NnV) and N. kaouthia (NkV) from Burdwan districts of EI were compared by separation of venom proteins by 1D-SDS-PAGE followed by LC-MS/MS analysis of protein bands. The potency of commercial polyantivenom (PAV) was assessed by neutralization, ELISA, immuno-blot and venom-PAV immunoaffinity chromatography studies.

Results : Proteomic analysis identified 52 and 55 proteins for NnV and NkV, respectively, when searched against the Elapidae database. A small quantitative difference in venom composition between these two species of cobra was observed. PAVs exhibited poor cross-reactivity against low molecular mass toxins (<20 kDa) of both cobra venoms, which was substantiated by a meager neutralization of their phospholipase A₂ activity. Phospholipase A₂ and 3FTx, the two major classes of nonenzymatic and enzymatic proteins, respectively, were partially recognized by PAVs.

Conclusions : Efforts must be made to improve immunization protocols and supplement existing antivenoms with antibodies raised against the major toxins of these venoms.     

Structural and Functional Characterization of the Redβ Recombinase from Bacteriophage λ

The Red system of bacteriophage λ is responsible for the genetic rearrangements that contribute to its rapid evolution and has been successfully harnessed as a research tool for genome manipulation. The key recombination component is Redβ, a ring-shaped protein that facilitates annealing of complementary DNA strands. Redβ shares functional similarities with the human Rad52 single-stranded DNA (ssDNA) annealing protein although their evolutionary relatedness is not well established. Alignment of Rad52 and Redβ sequences shows an overall low level of homology, with 15% identity in the N-terminal core domains as well as important similarities with the Rad52 homolog Sak from phage ul36. Key conserved residues were chosen for mutagenesis and their impact on oligomer formation, ssDNA binding and annealing was probed. Two conserved regions were identified as sites important for binding ssDNA; a surface basic cluster and an intersubunit hydrophobic patch, consistent with findings for Rad52. Surprisingly, mutation of Redβ residues in the basic cluster that in Rad52 are involved in ssDNA binding disrupted both oligomer formation and ssDNA binding. Mutations in the equivalent of the intersubunit hydrophobic patch in Rad52 did not affect Redβ oligomerization but did impair DNA binding and annealing. We also identified a single amino acid substitution which had little effect on oligomerization and DNA binding but which inhibited DNA annealing, indicating that these two functions of Redβ can be separated. Taken together, the results provide fresh insights into the structural basis for Redβ function and the important role of quaternary structure.     

Linkage Maps of Lowland and Upland Tetraploid Switchgrass Ecotypes

Switchgrass (Panicum virgatum L.) is a native perennial warm season (C₄) grass that has been identified as a promising species for bioenergy research and production. Consequently, biomass yield and feedstock quality improvements are high priorities for switchgrass research. The objective of this study was to develop a switchgrass genetic linkage map using a full-sib pseudo-testcross mapping population derived from a cross between two heterozygous genotypes selected from the lowland cultivar ‘Alamo’ (AP13) and the upland cultivar ‘Summer’ (VS16). The female parent (AP13) map consists of 515 loci in 18 linkage groups (LGs) and spans 1,733 cM. The male parent (VS16) map arranges 363 loci in 17 LGs and spans 1,508 cM. No obvious cause for the lack of one LG in VS16 could be identified. Comparative analyses between the AP13 and VS16 maps showed that the two major ecotypic classes of switchgrass have highly colinear maps with similar recombination rates, suggesting that chromosomal exchange between the two ecotypes should be able to occur freely. The AP13 and VS16 maps are also highly similar with respect to marker orders and recombination levels to previously published switchgrass maps. The genetic maps will be used to identify quantitative trait loci associated with biomass and quality traits. The AP13 genotype was used for the whole genome-sequencing project and the map will thus also provide a tool for the anchoring of the switchgrass genome assembly.