Massive expansions of Dscam splicing diversity via staggered homologous recombination during arthropod evolution

The arthropod Down syndrome cell adhesion molecule (Dscam) gene can generate tens of thousands of protein isoforms via combinatorial splicing of numerous alternative exons encoding immunoglobulin variable domains organized into three clusters referred to as the exon 4, 6, and 9 clusters. Dscam protein diversity is important for nervous system development and immune functions. We have performed extensive phylogenetic analyses of Dscam from 20 arthropods (each containing between 46 and 96 alternative exons) to reconstruct the detailed history of exon duplication and loss events that built this remarkable system over 450 million years of evolution. Whereas the structure of the exon 4 cluster is ancient, the exon 6 and 9 clusters have undergone massive, independent expansions in each insect lineage. An analysis of nearly 2000 duplicated exons enabled detailed reconstruction of the timing, location, and boundaries of these duplication events. These data clearly show that new Dscam exons have arisen continuously throughout arthropod evolution and that this process is still occurring in the exon 6 and 9 clusters. Recently duplicated regions display boundaries corresponding to a single exon and the adjacent intron. The boundaries, homology, location, clustering, and relative frequencies of these duplication events strongly suggest that staggered homologous recombination is the major mechanism by which new Dscam exons evolve. These data provide a remarkably detailed picture of how complex gene structure evolves and reveal the molecular mechanism behind this process.     

Functionally diverse rhizobacteria of Saccharum munja (a native wild grass) colonizing abandoned morrum mine in Aravalli hills (Delhi)

Characterization of the rhizobacteria of native grasses naturally colonizing abandoned mine sites may help in identification of microbial inoculants for ecological-restoration programmes. Eighty one strains of Saccharum munja rhizobacteria isolated from an abandoned mine located on Aravalli mountain and 50 from bulk-region were identified using 16S rRNA sequence analyses. Based on chemical- and biological-assays they were categorized into ecologically diverse functional groups (siderophore-, IAA-, ACC-deaminase-, HCN-, polyphosphate-producers; phosphate-solubilizer; antagonistic). Eight genera, 25 species from rhizosphere and 2 genera, 5 species from bulk-region were dominated by Bacillus spp. (B. barbaricus, B. cereus, B. firmus, B. flexus, B. foraminis, B. licheniformis, B. megaterium, B. pumilus, B. subtilis, B. thuringiensis) and Paenibacillus spp. (P. alvei, P. apiarius, P. lautus, P. lentimorbus, P. polymyxa, P. popillae). Siderophore-producers were common in rhizosphere and bulk soil, whereas IAA-producers, N₂-fixers and FePO₄-solubilizers dominated rhizosphere samples. During the reproductive phase (winter) of S. munja, siderophore-, ACC-deaminase- and polyP-producers were predominant; however dominance of HCN-producers in summer might be associated with termite-infestation. In vivo ability of selected rhizobacteria (B. megaterium BOSm201, B. subtilis BGSm253, B. pumilus BGSm157, P. alvei BGSm255, P. putida BOSm217, P. aeruginosa BGSm 306) to enhance seed-germination and seedling-growth of S. munja in mine-spoil suggest their significance in natural colonization and potential for ecological-restoration of Bhatti mine.     

Metabolic connections during apoptotic cell engulfment

Billions of cells die via apoptosis every day and are swiftly removed. When a phagocyte engulfs an apoptotic cell, it essentially doubles its cellular contents, raising the question of how a phagocyte may manage the excess metabolic load. This Minireview discusses phagocyte cellular metabolism, the digestion of the ingested apoptotic cell, and the impact of these processes on engulfment.     

Hypoxia-inducible factor-1α (HIF-1α) and autophagy in polycystic kidney disease (PKD)

Cyst expansion in polycystic kidney disease (PKD) results in localized hypoxia in the kidney that may activate hypoxia-inducible factor-1α (HIF-1α). HIF-1α and autophagy, a form of programmed cell repair, are induced by hypoxia. The purposes were to determine HIF-1α expression and autophagy in rat and mouse models of PKD. HIF-1α was detected by electrochemiluminescence. Autophagy was visualized by electron microscopy (EM). LC3 and beclin-1, markers of autophagy, were detected by immunoblotting. Eight-week-old male heterozygous (Cy/+) and 4-wk-old homozygous (Cy/Cy) Han:SPRD rats, 4-wk-old cpk mice, and 112-day-old Pkd2WS25/- mice with a mutation in the Pkd2 gene were studied. HIF-1α was significantly increased in massive Cy/Cy and cpk kidneys and not smaller Cy/+ and Pkd2WS25/- kidneys. On EM, features of autophagy were seen in wild-type (+/+), Cy/+, and cpk kidneys: autophagosomes, mitophagy, and autolysosomes. Specifically, autophagosomes were found on EM in the tubular cells lining the cysts in cpk mice. The increase in LC3-II, a marker of autophagosome production and beclin, a regulator of autophagy, in Cy/Cy and cpk kidneys, followed the same pattern of increase as HIF-1α. To determine the role of HIF-1α in cyst formation and/or growth, Cy/+ rats, Cy/Cy rats, and cpk mice were treated with the HIF-1α inhibitor 2-methoxyestradiol (2ME2). 2ME2 had no significant effect on kidney volume or cyst volume density. In summary, HIF-1α is highly expressed in the late stages of PKD and is associated with an increase in LC3-II and beclin-1. The first demonstration of autophagosomes in PKD kidneys is reported. Inhibition of HIF-1α did not have a therapeutic effect.     

Autoimmune disease risk variant of IFIH1 is associated with increased sensitivity to IFN-α and serologic autoimmunity in lupus patients

Increased IFN-α signaling is a heritable risk factor for systemic lupus erythematosus (SLE). IFN induced with helicase C domain 1 (IFIH1) is a cytoplasmic dsRNA sensor that activates IFN-α pathway signaling. We studied the impact of the autoimmune-disease-associated IFIH1 rs1990760 (A946T) single nucleotide polymorphism upon IFN-α signaling in SLE patients in vivo. We studied 563 SLE patients (278 African-American, 179 European-American, and 106 Hispanic-American). Logistic regression models were used to detect genetic associations with autoantibody traits, and multiple linear regression was used to analyze IFN-α-induced gene expression in PBMCs in the context of serum IFN-α in the same blood sample. We found that the rs1990760 T allele was associated with anti-dsDNA Abs across all of the studied ancestral backgrounds (meta-analysis odds ratio = 1.34, p = 0.026). This allele also was associated with lower serum IFN-α levels in subjects who had anti-dsDNA Abs (p = 0.0026). When we studied simultaneous serum and PBMC samples from SLE patients, we found that the IFIH1 rs1990760 T allele was associated with increased IFN-induced gene expression in PBMCs in response to a given amount of serum IFN-α in anti-dsDNA-positive patients. This effect was independent of the STAT4 genotype, which modulates sensitivity to IFN-α in a similar way. Thus, the IFIH1 rs1990760 T allele was associated with dsDNA Abs, and in patients with anti-dsDNA Abs this risk allele increased sensitivity to IFN-α signaling. These studies suggest a role for the IFIH1 risk allele in SLE in vivo.     

EmrA1 membrane fusion protein of Francisella tularensis LVS is required for resistance to oxidative stress,intramacrophage survival and virulence in mice

Francisella tularensis is a category A biodefence agent that causes a fatal human disease known as tularaemia. The pathogenicity of F. tularensis depends on its ability to persist inside host immune cells primarily by resisting an attack from host‐generated reactive oxygen and nitrogen species (ROS/RNS). Based on the ability of F. tularensis to resist high ROS/RNS levels, we have hypothesized that additional unknown factors act in conjunction with known antioxidant defences to render ROS resistance. By screening a transposon insertion library of F. tularensis LVS in the presence of hydrogen peroxide, we have identified an oxidant‐sensitive mutant in putative EmrA1 (FTL_0687) secretion protein. The results demonstrate that the emrA1 mutant is highly sensitive to oxidants and several antimicrobial agents, and exhibits diminished intramacrophage growth that can be restored to wild‐type F. tularensis LVS levels by either transcomplementation, inhibition of ROS generation or infection in NADPH oxidase deficient (gp91Phox^?/?) macrophages. The emrA1 mutant is attenuated for virulence, which is restored by infection in gp91Phox^?/? mice. Further, EmrA1 contributes to oxidative stress resistance by affecting secretion of Francisella antioxidant enzymes SodB and KatG. This study exposes unique links between transporter activity and the antioxidant defence mechanisms of F. tularensis.