Non-random organization of the Biomphalaria glabrata genome in interphase Bge cells and the spatial repositioning of activated genes in cells co-cultured with Schistosomamansoni

Biomphalaria glabrata is a major intermediate host for the parasitic trematode Schistosoma mansoni, a causative agent of human schistosomiasis. To decipher the molecular basis of this host-parasite interaction, the Bge embryonic cell line provides a unique in vitro model system to assess whether interactions between the snail and parasite affect the cell and genome biology in either organism. The organization of the B. glabrata genome in Bge cells was studied using image analysis through positioning territories of differently sized chromosomes within cell nuclei. The snail chromosome territories are similar in morphology as well as in non-random radial positioning as those found in other derived protostome and deuterostome organisms. Specific monitoring of four gene loci, piwi, BgPrx, actin and ferritin, revealed non-random radial positioning of the genome. This indicates that specific parts of the snail genome reside in reproducible nuclear addresses. To determine whether exposure to parasite is reflected in genome organization, the interphase spatial positioning of genes was assessed after co-culturing Bge cells with either normal or irradiation attenuated miracidia for 30 min to 24 h. The loci of actin and ferritin, genes that are up-regulated in the snail when subjected to infection, were visualized by fluorescence in situ hybridisation (FISH) and their radial nuclear positions i.e. their position in the interphase nucleus with respect to the nuclear edge/envelope, mapped. Interestingly, large scale gene repositioning correlated to temporal kinetics of gene expression levels in Bge cells co-cultured with normal miracidia while irradiated parasites failed to elicit similar gene expression or gene loci repositioning as demonstrated using the ferritin gene. This indicates that normal but not attenuated schistosomes provide stimuli that evoke host responses that are reflected in the host’s nuclear architecture. We believe that this is not only the first time that gene-repositioning studies have been attempted in a mollusc but also demonstrates a parasite influencing the interphase genome organization of its host.     

Phylogenetic diversity of rhizobia associated with horsegram [Macrotyloma uniflorum (Lam.) Verdc.] grown in South India based on glnII, recA and 16S-23S intergenic sequence analyses

Horsegram [Macrotyloma uniflorum (Lam.) Verdc.) is an important grain legume and fodder crop in India. Information on root nodule endosymbionts of this legume in India is limited. In the present study, 69 isolates from naturally occurring root nodules of horsegram collected from two agro-eco-climatic regions of South India was analyzed by generation rate, acid/alkali reaction on YMA medium, restriction fragment length polymorphism analysis of 16S-23S rDNA intergenic spacer region (IGS), and sequence analyses of IGS and housekeeping genes glnII and recA. Based on the rDNA IGS RFLP by means of three restriction enzymes rhizobia were grouped in five clusters (I–V). By sequence analysis of 16S-23S rDNA IGS identified genotypes of horsegram rhizobia were distributed into five divergent lineages of Bradyrhizobium genus which comprised (I) the IGS type IV rhizobia and valid species B. yuanmingense, (II) the strains of IGS type I and Bradyrhizobium sp. ORS 3257 isolated from Vigna sp., (III) the strains of the IGS type II and Bradyrhizobium sp. CIRADAc12 from Acacia sp., (IV) the IGS type V strains and Bradyrhizobium sp. genospecies IV, and (V) comprising genetically distinct IGS type III strains which probably represent an uncharacterized new genomic species. Nearly, 87% of indigenous horsegram isolates (IGS types I, II, III, and V) could not be related to any other species within the genus Bradyrhizobium. Phylogeny based on housekeeping glnII and recA genes confirmed those results found by the analysis of the IGS sequence. All the isolated rhizobia nodulated Macrotyloma sp. and Vigna spp., and only some of them formed nodules on Arachis hypogeae. The isolates within each IGS type varied in their ability to fix nitrogen. Selection for high symbiotic effective strains could reward horsegram production in poor soils of South India where this legume is largely cultivated.     

Nitrogen status dependent oxidative stress tolerance conferred by overexpression of MnSOD and FeSOD proteins in Anabaena sp. strain PCC7120

The heterocystous nitrogen-fixing cyanobacterium, Anabaena sp. strain PCC7120 displayed two superoxide dismutase (SOD) activities, namely FeSOD and MnSOD. Prolonged exposure of Anabaena PCC7120 cells to methyl viologen mediated oxidative stress resulted in loss of both SOD activities and induced cell lysis. The two SOD proteins were individually overexpressed constitutively in Anabaena PCC7120, by genetic manipulation. Under nitrogen-fixing conditions, overexpression of MnSOD (sodA) enhanced oxidative stress tolerance, while FeSOD (sodB) overexpression was detrimental. Under nitrogen supplemented conditions, overexpression of either SOD protein, especially FeSOD, conferred significant tolerance against oxidative stress. The results demonstrate a nitrogen status-dependent protective role of individual superoxide dismutases in Anabaena PCC7120 during oxidative stress.     

Metallic copper corrosion rates, moisture content, and growth medium influence survival of copper ion-resistant bacteria

The rapid killing of various bacteria in contact with metallic copper is thought to be influenced by the influx of copper ions into the cells, but the exact mechanism is not fully understood. This study showed that the kinetics of contact killing of copper surfaces depended greatly on the amount of moisture present, copper content of alloys, type of medium used, and type of bacteria. We examined antibiotic- and copper ion-resistant strains of Escherichia coli and Enterococcus faecium isolated from pig farms following the use of copper sulfate as feed supplement. The results showed rapid killing of both copper ion-resistant E. coli and E. faecium strains when samples in rich medium were spread in a thin, moist layer on copper alloys with 85% or greater copper content. E. coli strains were rapidly killed under dry conditions, while E. faecium strains were less affected. Electroplated copper surface corrosion rates were determined from electrochemical polarization tests using the Stern–Geary method and revealed decreased corrosion rates with benzotriazole and thermal oxide coating. Copper ion-resistant E. coli and E. faecium cells suspended in 0.8% NaCl showed prolonged survival rates on electroplated copper surfaces with benzotriazole coating and thermal oxide coating compared to surfaces without anti-corrosion treatment. Control of surface corrosion affected the level of copper ion influx into bacterial cells, which contributed directly to bacterial killing.     

Biomechanical failure properties and microstructural content of ruptured and unruptured abdominal aortic aneurysms

PurposeTo test the hypothesis that ruptured abdominal aortic aneurysms (AAA) are globally weaker than unruptured ones.MethodsFour ruptured and seven unruptured AAA specimens were harvested whole from fresh cadavers during autopsies performed over an 18-month period. Multiple regionally distributed longitudinally oriented rectangular strips were cut from each AAA specimen for a total of 77 specimen strips. Strips were subjected to uniaxial extension until failure. Sections from approximately the strongest and weakest specimen strips were studied histologically and histochemically. From the load-extension data, failure tension, failure stress and failure strain were calculated. Rupture site characteristics such as location, arc length of rupture and orientation of rupture were also documented.ResultsThe failure tension, a measure of the tissue mechanical caliber was remarkably similar between ruptured and unruptured AAA (group mean±standard deviation of within-subject means: 11.2±2.3 versus 11.6±3.6 N/cm; p=0.866 by mixed model ANOVA). In post-hoc analysis, there was little difference between the groups in other measures of tissue mechanical caliber as well such as failure stress (95±28 versus 98±23 N/cm²; p=0.870), failure strain (0.39±0.09 versus 0.36±0.09; p=0.705), wall thickness (1.7±0.4 versus 1.5±0.4 mm; p=0.470) , and % coverage of collagen within tissue cross section (49.6±12.9% versus 60.8±9.6%; p=0.133). In the four ruptured AAA, primary rupture sites were on the lateral quadrants (two on left; one on left-posterior; one on right). Remarkably, all rupture lines had a longitudinal orientation and ranged from 1 to 6 cm in length.ConclusionThe findings are not consistent with the hypothesis that ruptured aortic aneurysms are globally weaker than unruptured ones.     

Endoplasmic reticulum calcium depletion impacts chaperone secretion, innate immunity, and phagocytic uptake of cells

A number of immunological functions are ascribed to cell surface-expressed forms of the endoplasmic reticulum (ER) chaperone calreticulin (CRT). In this study, we examined the impact of ER stress-inducing drugs upon cell surface CRT induction and the resulting immunological consequences. We showed that cell surface expression of CRT and secretion of CRT, BiP, gp96, and PDI were induced by thapsigargin (THP) treatment, which depletes ER calcium, but not by tunicamycin treatment, which inhibits protein glycosylation. Surface expression of CRT in viable, THP-treated fibroblasts correlated with their enhanced phagocytic uptake by bone marrow-derived dendritic cells. Incubation of bone marrow-derived dendritic cells with THP-treated fibroblasts enhanced sterile IL-6 production and LPS-induced generation of IL-1β, IL-12, IL-23, and TNF-α. However, extracellular CRT is not required for enhanced proinflammatory responses. Furthermore, the pattern of proinflammatory cytokine induction by THP-treated cells and cell supernatants resembled that induced by THP itself and indicated that other ER chaperones present in supernatants of THP-treated cells also do not contribute to induction of the innate immune response. Thus, secretion of various ER chaperones, including CRT, is induced by ER calcium depletion. CRT, previously suggested as an eat-me signal in dead and dying cellular contexts, can also promote phagocytic uptake of cells subject to ER calcium depletion. Finally, there is a strong synergy between calcium depletion in the ER and sterile IL-6, as well as LPS-dependent IL-1β, IL-12, IL-23, and TNF-α innate responses, findings that have implications for understanding inflammatory diseases that originate in the ER.     

The polypeptide binding conformation of calreticulin facilitates its cell-surface expression under conditions of endoplasmic reticulum stress

We define two classes of calreticulin mutants that retain glycan binding activity; those that display enhanced or reduced polypeptide-specific chaperone activity, due to conformational effects. Under normal conditions, neither set of mutants significantly impacts the ability of calreticulin to mediate assembly and trafficking of major histocompatibility complex class I molecules, which are calreticulin substrates. However, in cells treated with thapsigargin, which depletes endoplasmic reticulum calcium, major histocompatibility complex class I trafficking rates are accelerated coincident with calreticulin secretion, and detection of cell-surface calreticulin is dependent on its polypeptide binding conformations. Together, these findings identify a site on calreticulin that is an important determinant of the induction of its polypeptide binding conformation and demonstrate the relevance of the polypeptide binding conformations of calreticulin to endoplasmic reticulum stress-induced interactions.     

Association of interleukin 1 receptor antagonist (IL1RN) gene polymorphism with recurrent pregnancy loss risk in the North Indian Population and a meta-analysis

An appropriate ratio of interleukin 1 beta to interleukin 1 receptor antagonist (IL1Ra) is required for successful pregnancy. Our objective was to study the genetic association between IL1RN variable numbers of tandem repeat (VNTR) polymorphism and recurrent pregnancy loss (RPL). To analyze the association between IL1RN VNTR allele and RPL, we investigated the IL1RN VNTR polymorphism in 136 RPL patients and in 200 healthy control women. Meta-analysis on this polymorphism was conducted to support our findings. PCR based approach was used to analyze IL1RN VNTR polymorphism and it was further confirmed by sequencing. Systematic review and meta-analysis was done using electronic database (Pub-Med, Google Scholar and Ovid) up to February 27, 2013. This meta-analysis was assessed by comprehensive meta-analysis software version 2. For meta-analysis 549 cases and 1,450 controls were included. The frequency of IL1RN genotype 2/2 was significantly higher in RPL compared to control group (AORs 3.10, 95 % CI 1.58–6.11, p = 0.001). The presence of rare allele also increased the risk of RPL significantly (ORs 1.63, 95 % CI 1.16–2.29, p = 0.004). The meta-analysis stratified by ethnicity showed that individuals with allele 2 had increased risk of RPL (OR 1.29, 95 % CI 1.04–1.61, p = 0.01), in Asians population by using fixed model. However the data of the present study clearly suggests that IL1RN VNTR polymorphism is a genetic risk factor for pregnancy loss in the study population.     

Altered chemokine production and accumulation of regulatory T cells in intestinal adenomas of APCMin/+ mice

Tumor progression in the colon moves from aberrant crypt foci to adenomatous polyps to invasive carcinomas. The composition of the tumor-infiltrating leukocyte population affects the ability of the immune system to fight the tumor. T cell infiltration into colorectal adenocarcinomas, particularly T helper 1 (Th1) type T cells as well as increased regulatory T cell (Treg) frequencies, is correlated with improved prognosis. However, whether Th1 cells and Tregs are already present at the adenoma stage is not known. In this study, the APC^Min/+ mouse model of intestinal adenomatous polyposis was used to investigate tumor-associated lymphocyte subsets and the mechanisms of their accumulation into gastrointestinal adenomas. Compared to unaffected tissue, adenomas accumulated CD4⁺FoxP3⁺ putative Treg in parallel with lower frequencies of conventional T cells and B cells. The accumulation of Treg was also observed in human adenomatous polyps. Despite high Treg numbers, the function of conventional T cells present in the APC^Min/+ adenomas was not different from those in the unaffected tissue. Adenomas displayed an altered chemokine balance, with higher CCL17 and lower CXCL11 and CCL25 expression than in the unaffected tissue. In parallel, CXCR3⁺ Tregs were largely absent from adenomas. The data indicate that already in early stages of tumor development, the balance of lymphocyte-recruiting chemokines is altered possibly contributing to the observed shift toward higher frequencies of Treg.