Protein profiling in the gut of Penaeus monodon gavaged with oral WSSV‐vaccines and live white spot syndrome virus

White spot syndrome virus (WSSV) is a pathogen that causes considerable mortality of the farmed shrimp, Penaeus monodon. Candidate ‘vaccines’, WSSV envelope protein VP28 and formalin‐inactivated WSSV, can provide short‐lived protection against the virus. In this study, P. monodon was orally intubated with the aforementioned vaccine candidates, and protein expression in the gut of immunised shrimps was profiled. The alterations in protein profiles in shrimps infected orally with live‐WSSV were also examined. Seventeen of the identified proteins in the vaccine and WSSV‐intubated shrimps varied significantly compared to those in the control shrimps. These proteins, classified under exoskeletal, cytoskeletal, immune‐related, intracellular organelle part, intracellular calcium‐binding or energy metabolism, are thought to directly or indirectly affect shrimp's immunity. The changes in the expression levels of crustacyanin, serine proteases, myosin light chain, and ER protein 57 observed in orally vaccinated shrimp may probably be linked to immunoprotective responses. On the other hand, altered expression of proteins linked to exoskeleton, calcium regulation and energy metabolism in WSSV‐intubated shrimps is likely to symbolise disturbances in calcium homeostasis and energy metabolism.     

Architecture of the eIF2B regulatory subcomplex and its implications for the regulation of guanine nucleotide exchange on eIF2

Eukaryal translation initiation factor 2B (eIF2B) acts as guanine nucleotide exchange factor (GEF) for eIF2 and forms a central target for pathways regulating global protein synthesis. eIF2B consists of five non-identical subunits (α–ϵ), which assemble into a catalytic subcomplex (γ, ϵ) responsible for the GEF activity, and a regulatory subcomplex (α, β, δ) which regulates the GEF activity under stress conditions. Here, we provide new structural and functional insight into the regulatory subcomplex of eIF2B (eIF2B^RSC). We report the crystal structures of eIF2Bβ and eIF2Bδ from Chaetomium thermophilum as well as the crystal structure of their tetrameric eIF2B(βδ)₂ complex. Combined with mutational and biochemical data, we show that eIF2B^RSC exists as a hexamer in solution, consisting of two eIF2Bβδ heterodimers and one eIF2Bα₂ homodimer, which is homologous to homohexameric ribose 1,5-bisphosphate isomerases. This homology is further substantiated by the finding that eIF2Bα specifically binds AMP and GMP as ligands. Based on our data, we propose a model for eIF2B^RSC and its interactions with eIF2 that is consistent with previous biochemical and genetic data and provides a framework to better understand eIF2B function, the molecular basis for Gcn⁻, Gcd⁻ and VWM/CACH mutations and the evolutionary history of the eIF2B complex.     

The ability of land plants to synthesize glucuronoxylans predates the evolution of tracheophytes

Glucuronoxylans with a backbone of 1,4-linked β-D-xylosyl residues are ubiquitous in the secondary walls of gymnosperms and angiosperms. Xylans have been reported to be present in hornwort cell walls, but their structures have not been determined. In contrast, the presence of xylans in the cell walls of mosses and liverworts remains a subject of debate. Here we present data that unequivocally establishes that the cell walls of leafy tissue and axillary hair cells of the moss Physcomitrella patens contain a glucuronoxylan that is structurally similar to glucuronoxylans in the secondary cell walls of vascular plants. Some of the 1,4-linked β-D-xylopyranosyl residues in the backbone of this glucuronoxylan bear an α-D-glucosyluronic acid (GlcpA) sidechain at O-2. In contrast, the lycopodiophyte Selaginella kraussiana synthesizes a glucuronoxylan substituted with 4-O-Me-α-D-GlcpA sidechains, as do many hardwood species. The monilophyte Equisetum hyemale produces a glucuronoxylan with both 4-O-Me-α-D-GlcpA and α-D-GlcpA sidechains, as does Arabidopsis. The seedless plant glucuronoxylans contain no discernible amounts of the reducing-end sequence that is characteristic of gymnosperm and eudicot xylans. Phylogenetic studies showed that the P. patens genome contains genes with high sequence similarity to Arabidopsis CAZy family GT8, GT43 and GT47 glycosyltransferases that are likely involved in xylan synthesis. We conclude that mosses synthesize glucuronoxylan that is structurally similar to the glucuronoxylans present in the secondary cell walls of lycopodiophytes, monilophytes, and many seed-bearing plants, and that several of the glycosyltransferases required for glucuronoxylan synthesis evolved before the evolution of tracheophytes.     

Comparison of Metarhizium isolates for biocontrol of Helicoverpa armigera (Lepidoptera: Noctuidae) in chickpea

Metarhizium isolates from soil (53) and insect hosts (10) were evaluated for extracellular production of cuticle degrading enzyme (CDE) activities such as chitinase, chitin deacetylase (CDA), chitosanase, protease and lipase. Regression analysis demonstrated the relation of CDE activities with Helicoverpa armigera mortality. On basis of this relation, ten isolates were selected for further evaluation. Subsequently, based on LT₅₀ of the 10 isolates towards H. armigera, five isolates were selected. Out of these five isolates, three were selected on the basis of higher conidia production (60–75 g/kg rice), faster sedimentation time (ST₅₀) (2.3–2.65 h in 0.1% (w/v) Tween 80) and lower LC₅₀ (1.4–5.7×10³ conidia/mL) against H. armigera. Finally, three Metarhizium isolates were selected for the molecular fingerprinting using ITS sequencing and RAPD patterning. All three isolates, M34412, M34311 and M81123, showed comparable RAPD patterns with a 935G primer. These were further evaluated for their field performance against H. armigera in a chickpea crop. The percent efficacies with the three Metarhizium isolates were from 65 to 72%, which was comparable to the chemical insecticide, endosulfan (74%).     

Cellular nucleic acid binding protein binds G-rich single-stranded nucleic acids and may function as a nucleic acid chaperone

Cellular nucleic acid binding protein (CNBP) is a small single-stranded nucleic acid binding protein made of seven Zn knuckles and an Arg-Gly rich box. CNBP is strikingly conserved among vertebrates and was reported to play broad-spectrum functions in eukaryotic cells biology. Neither its biological function nor its mechanisms of action were elucidated yet. The main goal of this work was to gain further insights into the CNBP biochemical and molecular features. We studied Bufo arenarum CNBP (bCNBP) binding to single-stranded nucleic acid probes representing the main reported CNBP putative targets. We report that, although bCNBP is able to bind RNA and single-stranded DNA (ssDNA) probes in vitro, it binds RNA as a preformed dimer whereas both monomer and dimer are able to bind to ssDNA. A systematic analysis of variant probes shows that the preferred bCNBP targets contain unpaired guanosine-rich stretches. These data expand the knowledge about CNBP binding stoichiometry and begins to dissect the main features of CNBP nucleic acid targets. Besides, we show that bCNBP presents a highly disordered predicted structure and promotes the annealing and melting of nucleic acids in vitro. These features are typical of proteins that function as nucleic acid chaperones. Based on these data, we propose that CNBP may function as a nucleic acid chaperone through binding, remodeling, and stabilizing nucleic acids secondary structures. This novel CNBP biochemical activity broadens the field of study about its biological function and may be the basis to understand the diverse ways in which CNBP controls gene expression.     

UNC-16 alters DLK-1 localization and negatively regulates actin and microtubule dynamics in Caenorhabditis elegans regenerating neurons

Neuronal regeneration after injury depends on the intrinsic growth potential of neurons. Our study shows that UNC-16, a Caenorhabditis elegans JIP3 homolog, inhibits axonal regeneration by regulating initiation and rate of regrowth. This occurs through the inhibition of the regeneration-promoting activity of the long isoform of DLK-1 and independently of the inhibitory short isoform of DLK-1. We show that UNC-16 promotes DLK-1 punctate localization in a concentration-dependent manner limiting the availability of the long isoform of DLK-1 at the cut site, minutes after injury. UNC-16 negatively regulates actin dynamics through DLK-1 and microtubule dynamics partially via DLK-1. We show that post-injury cytoskeletal dynamics in unc-16 mutants are also partially dependent on CEBP-1. The faster regeneration seen in unc-16 mutants does not lead to functional recovery. Our data suggest that the inhibitory control by UNC-16 and the short isoform of DLK-1 balances the intrinsic growth-promoting function of the long isoform of DLK-1 in vivo. We propose a model where UNC-16’s inhibitory role in regeneration occurs through both a tight temporal and spatial control of DLK-1 and cytoskeletal dynamics.