A genome-wide meta-analysis of six type 1 diabetes cohorts identifies multiple associated loci

Diabetes impacts approximately 200 million people worldwide, of whom approximately 10% are affected by type 1 diabetes (T1D). The application of genome-wide association studies (GWAS) has robustly revealed dozens of genetic contributors to the pathogenesis of T1D, with the most recent meta-analysis identifying in excess of 40 loci. To identify additional genetic loci for T1D susceptibility, we examined associations in the largest meta-analysis to date between the disease and ∼2.54 million SNPs in a combined cohort of 9,934 cases and 16,956 controls. Targeted follow-up of 53 SNPs in 1,120 affected trios uncovered three new loci associated with T1D that reached genome-wide significance. The most significantly associated SNP (rs539514, P = 5.66×10⁻¹¹) resides in an intronic region of the LMO7 (LIM domain only 7) gene on 13q22. The second most significantly associated SNP (rs478222, P = 3.50×10⁻⁹) resides in an intronic region of the EFR3B (protein EFR3 homolog B) gene on 2p23; however, the region of linkage disequilibrium is approximately 800 kb and harbors additional multiple genes, including NCOA1, C2orf79, CENPO, ADCY3, DNAJC27, POMC, and DNMT3A. The third most significantly associated SNP (rs924043, P = 8.06×10⁻⁹) lies in an intergenic region on 6q27, where the region of association is approximately 900 kb and harbors multiple genes including WDR27, C6orf120, PHF10, TCTE3, C6orf208, LOC154449, DLL1, FAM120B, PSMB1, TBP, and PCD2. These latest associated regions add to the growing repertoire of gene networks predisposing to T1D.     

Food restriction affects circadian and photoperiodic response in black-headed munia (Lonchura malacca malacca)

We determined the effect of food restriction on locomotor activity pattern and gonadal growth on black-headed munia (Lonchura malacca malacca) exposed to12L:12D light–dark cycle. Two groups of male and female munia (n = 5 pairs each) were exposed to 12L:12D (L = 265 lux; D = 0.3 lux) for day 1–7. On day 8, group one received 4 h food (ZT 0–4; ZT 0 = Zeitgeber time, light onset) and group two received food ad libitum (ZT 0–24 h). Locomotor activity pattern and gonadal growth were recorded over the experiment period of one year. Birds were highly active in group one, with a high daily and total activity. However, activity behavior was fully synchronized with light onset in the group two, when food was available ad libitum. Gonadal maturation was initiated in both the groups but the maturation of testis and ovarian follicle was noticed only in group two (food ad libitum). It appears that food restriction affects the locomotor activity and gonadal growth in black-headed munia.     

Functional interaction between RAFT1/FRAP/mTOR and protein kinase cdelta in the regulation of cap-dependent initiation of translation

Hormones and growth factors induce protein translation in part by phosphorylation of the eukaryotic initiation factor 4E (eIF4E) binding protein 1 (4E-BP1). The rapamycin and FK506-binding protein (FKBP)-target 1 (RAFT1, also known as FRAP) is a mammalian homolog of the Saccharomyces cerevisiae target of rapamycin proteins (mTOR) that regulates 4E-BP1. However, the molecular mechanisms involved in growth factor-initiated phosphorylation of 4E-BP1 are not well understood. Here we demonstrate that protein kinase Cdelta (PKCdelta) associates with RAFT1 and that PKCdelta is required for the phosphorylation and inactivation of 4E-BP1. PKCdelta-mediated phosphorylation of 4E-BP1 is wortmannin resistant but rapamycin sensitive. As shown for serum, phosphorylation of 4E-BP1 by PKCdelta inhibits the interaction between 4E-BP1 and eIF4E and stimulates cap-dependent translation. Moreover, a dominant-negative mutant of PKCdelta inhibits serum-induced phosphorylation of 4E-BP1. These findings demonstrate that PKCdelta associates with RAFT1 and thereby regulates phosphorylation of 4E-BP1 and cap-dependent initiation of protein translation.     

Molecular Interactions between HIV-1 Integrase and the Two Viral DNA Ends within the Synaptic Complex that Mediates Concerted Integration

A macromolecular nucleoprotein complex in retrovirus-infected cells, termed the preintegration complex, is responsible for the concerted integration of linear viral DNA genome into host chromosomes. Isolation of sufficient quantities of the cytoplasmic preintegration complexes for biochemical and biophysical analysis is difficult. We investigated the architecture of HIV-1 nucleoprotein complexes involved in the concerted integration pathway in vitro. HIV-1 integrase (IN) non-covalently juxtaposes two viral DNA termini forming the synaptic complex, a transient intermediate in the integration pathway, and shares properties associated with the preintegration complex. IN slowly processes two nucleotides from the 3′ OH ends and performs the concerted insertion of two viral DNA ends into target DNA. IN remains associated with the concerted integration product, termed the strand transfer complex. The synaptic complex and strand transfer complex can be isolated by native agarose gel electrophoresis. In-gel fluorescence resonance energy transfer measurements demonstrated that the energy transfer efficiencies between the juxtaposed Cy3 and Cy5 5′-end labeled viral DNA ends in the synaptic complex (0.68 ± 0.09) was significantly different from that observed in the strand transfer complex (0.07 ± 0.02). The calculated distances were 46 ± 3 Å and 83 ± 5 Å, respectively. DNaseI footprint analysis of the complexes revealed that IN protects U5 and U3 DNA sequences up to ∼ 32 bp from the end, suggesting two IN dimers were bound per terminus. Enhanced DNaseI cleavages were observed at nucleotide positions 6 and 9 from the terminus on U3 but not on U5, suggesting independent assembly events. Protein-protein cross-linking of IN within these complexes revealed the presence of dimers, tetramers, and a larger multimer (> 120 kDa). Our results suggest a new model where two IN dimers individually assemble on U3 and U5 ends before the non-covalent juxtaposition of two viral DNA ends, producing the synaptic complex.     

Cross-Talk between Malarial Cysteine Proteases and Falstatin: The BC Loop as a Hot-Spot Target

Cysteine proteases play a crucial role in the development of the human malaria parasites Plasmodium falciparum and Plasmodium vivax. Our earlier studies demonstrated that these enzymes are equipped with specific domains for defined functions and further suggested the mechanism of activation of cysteine proteases. The activities of these proteases are regulated by a new class of endogenous inhibitors of cysteine proteases (ICPs). Structural studies of the ICPs of Trypanosoma cruzi (chagasin) and Plasmodium berghei (PbICP) indicated that three loops (termed BC, DE, and FG) are crucial for binding to target proteases. Falstatin, an ICP of P. falciparum, appears to play a crucial role in invasion of erythrocytes and hepatocytes. However, the mechanism of inhibition of cysteine proteases by falstatin has not been established. Our study suggests that falstatin is the first known ICP to function as a multimeric protein. Using site-directed mutagenesis, hemoglobin hydrolysis assays and peptide inhibition studies, we demonstrate that the BC loop, but not the DE or FG loops, inhibits cysteine proteases of P. falciparum and P. vivax via hydrogen bonds. These results suggest that the BC loop of falstatin acts as a hot-spot target for inhibiting malarial cysteine proteases. This finding suggests new strategies for the development of anti-malarial agents based on protease-inhibitor interactions.     

Chloroplasts alter their morphology and accumulate at the pathogen interface during infection by Phytophthora infestans

Upon immune activation, chloroplasts switch off photosynthesis, produce antimicrobial compounds and associate with the nucleus through tubular extensions called stromules. Although it is well established that chloroplasts alter their position in response to light, little is known about the dynamics of chloroplast movement in response to pathogen attack. Here, we report that during infection with the Irish potato famine pathogen Phytophthora infestans, chloroplasts accumulate at the pathogen interface, associating with the specialized membrane that engulfs the pathogen haustorium. The chemical inhibition of actin polymerization reduces the accumulation of chloroplasts at pathogen haustoria, suggesting that this process is partially dependent on the actin cytoskeleton. However, chloroplast accumulation at haustoria does not necessarily rely on movement of the nucleus to this interface and is not affected by light conditions. Stromules are typically induced during infection, embracing haustoria and facilitating chloroplast interactions, to form dynamic organelle clusters. We found that infection-triggered stromule formation relies on BRASSINOSTEROID INSENSITIVE 1-ASSOCIATED KINASE 1 (BAK1)-mediated surface immune signaling, whereas chloroplast repositioning towards haustoria does not. Consistent with the defense-related induction of stromules, effector-mediated suppression of BAK1-mediated immune signaling reduced stromule formation during infection. On the other hand, immune recognition of the same effector stimulated stromules, presumably via a different pathway. These findings implicate chloroplasts in a polarized response upon pathogen attack and point to more complex functions of these organelles in plant–pathogen interactions.     

Proteomic analysis of the abomasal mucosal response following infection by the nematode, Haemonchus contortus, in genetically resistant and susceptible sheep

Sheep have a variable ability to resist gastrointestinal nematode infection, but the key factors mediating this response are poorly defined. Here we report the first large-scale application of quantitative proteomic technologies to define proteins that are differentially abundant between sheep selectively bred to have an enhanced (resistant) or reduced (susceptible) ability to eliminate nematodes. Samples were collected from the abomasal mucosa three days after experimental challenge with the nematode, Haemonchus contortus. This timing reflects the initial interaction of host and parasite, and the tissue represents the immediate interface. We identified and quantified more than 4400 unique proteins, of which 158 proteins showed >1.5 fold difference between the resistant and susceptible sheep. Trefoil factor 2, a member of RAS oncogene family (RAP1A) and ring finger protein 126 were amongst the proteins found to be highly abundant in the abomasal surface of resistant sheep, whereas adenosine deaminase and the gastrokine-3 like precursor were found at higher levels in susceptible sheep. Construction of gut proteome interaction networks identified mitochondrial function and energetic partitioning as important components of an effective nematode eliminating response. The differentially abundant proteins may be useful targets for phenotypic tests that aim to identify sheep with an enhanced ability to resist nematode infection.     

Hg2+ uptake in a cyanobacterium

The uptake of Hg²⁺ and its regulation in the cyanobacteriumNostoc calcicola Bréb. was studied. Hg²⁺ uptake pattern consisted of two distinct phases: (a) rapid binding of the cation to the negatively charged cell surface (first 10 min) and (b) its subsequent metabolism-dependent intracellular import, at least up to 40 min (saturating concentration 1.5 M Hg²⁺, K_m=1.0M Hg²⁺ and V_max 0.21 nmol Hg²⁺ mg^–1 protein min^–1). Hg²⁺ influx, to a major extent, depended on photosynthetically generated energy, and the supply of exogenous ATP (10 M) or DCMU (5 M) suggested the vital role of PS II-mediated energy to support the process. The significant lowering in Hg²⁺ uptake rate as well as total cellular Hg²⁺ in the presence ofp-chloromercuribenzoate (pCMB), azide (NaN₃), N,N-dicyclohexycarbodiimide (DCCD), and thiol (mercaptoethanol) indicated the role of membrane potential,-SH groups, and ATP hydrolysis in regulating Hg²⁺ transport. While Cu²⁺ antagonized Hg²⁺ intake, Ni²⁺ showed synergism.     

CIPK9 targets VDAC3 and modulates oxidative stress responses in Arabidopsis

Calcium (Ca²⁺) is widely recognized as a key second messenger in mediating various plant adaptive responses. Here we show that calcineurin B-like interacting protein kinase CIPK9 along with its interacting partner VDAC3 identified in the present study are involved in mediating plant responses to methyl viologen (MV). CIPK9 physically interacts with and phosphorylates VDAC3. Co-localization, co-immunoprecipitation, and fluorescence resonance energy transfer experiments proved their physical interaction in planta. Both cipk9 and vdac3 mutants exhibited a tolerant phenotype against MV-induced oxidative stress, which coincided with the lower-level accumulation of reactive oxygen species in their roots. In addition, the analysis of cipk9vdac3 double mutant and VDAC3 overexpressing plants revealed that CIPK9 and VDAC3 were involved in the same pathway for inducing MV-dependent oxidative stress. The response to MV was suppressed by the addition of lanthanum chloride, a non-specific Ca²⁺ channel blocker indicating the role of Ca²⁺ in this pathway. Our study suggest that CIPK9-VDAC3 module may act as a key component in mediating oxidative stress responses in Arabidopsis.