Towards revealing the global diversity and community assembly of soil eukaryotes

Soil fungi, protists, and animals (i.e., the eukaryome) play a critical role in key ecosystem functions in terrestrial ecosystems. Yet, we lack a holistic understanding of the processes shaping the global distribution of the eukaryome. We conducted a molecular analysis of 193 composite soil samples spanning the world's major biomes. Our analysis showed that the importance of selection processes was higher in the community assemblage of smaller-bodied and wider niche breadth organisms. Soil pH and mean annual precipitation were the primary determinants of the community structure of eukaryotic microbes and animals, respectively. We further found contrasting latitudinal diversity patterns and strengths for soil eukaryotic microbes and animals. Our results point to a potential link between body size and niche breadth of soil eukaryotes and the relative effect of ecological processes and environmental factors in driving their biogeographic patterns.     

Neospora caninum protein disulfide isomerase is involved in tachyzoite-host cell interaction

We have previously shown that treatment of Neospora caninum tachyzoites with the aspartyl protease inhibitor pepstatin A reduces host cell invasion [Naguleswaran, A., Muller, N., Hemphill, A., 2003. Neospora caninum and Toxoplasma gondii: a novel adhesion/invasion assay reveals distinct differences in tachyzoite-host cell interactions. Exp. Parasitol. 104, 149-158]. Pepstatin A-affinity-chromatography led to the isolation of a major band of approximately 52 kDa which was identified as a homologue of a previously described Toxoplasma gondii putative protein disulfide isomerase (TgPDI) through tandem mass spectrometry. A BLAST search against N. caninum expressed sequence tags (ESTs) on the ApiDots server using TgPDI cDNA as query sequence revealed a 2251 bp PDI-like consensus (NcPDI), which shows 94% identity to the T. gondii homologue. In N. caninum tachyzoites, NcPDI was found mainly in the soluble hydrophilic fraction. Immunofluorescence showed that expression of NcPDI was dramatically down-regulated in the bradyzoite stage, and immunogold-EM on tachyzoites localised the protein to the cytoplasm, mostly in close vicinity to the nuclear membrane, to the micronemes, and to the parasite cell surface. However, NcPDI was absent in rhoptries and dense granules. Preincubation of tachyzoites with the sulfhydryl blocker 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), p-chloromercuribenzoic acid (pCMBA), and with the PDI inhibitor bacitracin reduced adhesion of parasites to host cells. In addition, incubation of N. caninum tachyzoites with affinity-purified anti-NcPDI antibodies reduced host cell adhesion. PDIs catalyse the formation, reduction or isomerisation of disulfide bonds. Many major components of the adhesion and invasion machinery of apicomplexan parasites are cysteine-rich and dependent on correct folding via disulfide bond formation. Thus, our data points towards an important role for surface-associated NcPDI in Neospora-host cell interaction.     

Isolation of Sertoli Cells and Peritubular Cells from Rat Testes

The testis, and in particular the male gamete, challenges the immune system in a unique way because differentiated sperm first appear at the time of puberty - more than ten years after the establishment of systemic immune tolerance. Spermatogenic cells express a number of proteins that may be seen as non-self by the immune system. The testis must then be able to establish tolerance to these neo-antigens on the one hand but still be able to protect itself from infections and tumor development on the other hand. Therefore the testis is one of a few immune privileged sites in the body that tolerate foreign antigens without evoking a detrimental inflammatory immune response. Sertoli cells play a key role for the maintenance of this immune privileged environment of the testis and also prolong survival of cotransplanted cells in a foreign environment. Therefore primary Sertoli cells are an important tool for studying the immune privilege of the testis that cannot be easily replaced by established cell lines or other cellular models. Here we present a detailed and comprehensive protocol for the isolation of Sertoli cells - and peritubular cells if desired - from rat testes within a single day.     

Human ENS regulates the intestinal epithelial barrier permeability and a tight junction-associated protein ZO-1 via VIPergic pathways

Although the enteric nervous system (ENS) has been shown to regulate various mucosal functions, its role in the physiological control of the human intestinal epithelial barrier is unknown. The aim of this study was to investigate whether the ENS is able to modulate epithelial barrier permeability and a key tight junction-associated protein, zonula occludens-1 (ZO-1). Therefore, we developed a co-culture model, consisting of human submucosa containing the submucosal neuronal network and human polarized colonic epithelial monolayers (HT29-Cl.16E or Caco-2). Submucosal neurons were activated by electrical field stimulation (EFS). Permeability was assessed by measuring the flux of paracellular permeability markers (FITC-dextran or FITC-inulin) across epithelial monolayers. Expression of ZO-1 was determined by immunofluorescence, quantitative immunoblot analysis, and real time RT-PCR. Using the coculture model, we showed that EFS of submucosal neurons resulted in a reduction in FITC-dextran or FITC-inulin fluxes, which was blocked by TTX. In HT29-Cl.16E, the effect of submucosal neuron activation was blocked by a VIP receptor antagonist (VIPra) and reproduced by VIP. Furthermore, ZO-1 expression (mRNA, protein) assessed in HT29-Cl.16E, was significantly increased after submucosal neuron activation by EFS. These effects on ZO-1 expression were blocked by TTX and VIPra and reproduced by VIP. In conclusion, our results strongly suggest a modulatory role of VIPergic submucosal neuronal pathways on intestinal epithelial barrier permeability and ZO-1 expression.     

Fibrogenic cytokine levels in bronchoalveolar lavage aspirates 15 years after exposure to sulfur mustard

Over 100,000 Iranian war veterans suffer from chronic effects of mustard gas exposure. Sulfur mustard was used by Iraq during the Iraqi-imposed war on Iran (between 1980 and 1988). The major complaints of these patients are mild interstitial fibrosis and bronchiolitis. We aimed to determine the state of fibrosis progression and assessed transforming growth factor (TGF)-beta1 levels in pulmonary samples and in bronchoalveolar lavage (BAL) aspirates. A total of 126 war veterans confirmed for lung disease were assessed and compared with three control groups: 1) 64 veterans not exposed to chemical agents, 2) 12 idiopathic pulmonary fibrosis civilian patients, and 3) 33 normal persons. BAL was performed via a flexible fiber-optic bronchoscope and the standard manual method. Total protein was measured by Bradford assay, and samples were corrected with regard to coefficients. Samples were concentrated 15-fold by lyophilization and resolubilization. Samples were double-checked using an ELISA test kit. The Mann-Whitney test was used for the data analysis using commercial software. We detected that significant differences between TGF-beta1 levels between the case group and control group 1 (P = 0.001) and control group 3 (P = 0.003). No significant differences were found between the case group and control group 2 (P = 0.57). Inflammation and fibrotic processes in lung tissue of patients exposed to sulfur mustard may be progressive so IFN-gamma may be a useful drug to these patients' treatment.     

Reduced infection and protection from clinical signs of cerebral neosporosis in C57BL/6 mice vaccinated with recombinant microneme antigen NcMIC1

NcMIC1 is a 460 amino acid Neospora caninum microneme protein implicated in host cell adhesion and invasion processes. In this study, we assessed the potential protectivity of NcMIC1-based vaccination against experimental N. caninum infection in mice, employing both recombinant antigen vaccines and DNA vaccines. Recombinant NcMIC1 (recNcMIC1) was expressed in Escherichia coli as gluthatione-S-transferase-fusion protein. The corresponding NcMIC1 cDNA was cloned into the pcDNA3.1 expression plasmid (pcDNA-MIC1), and expression was checked in transfected Vero cells. Mice (10 animals/group) were vaccinated either with recNcMIC1 antigen suspended in Ribi-adjuvant (3 intraperitoneal injections), pcDNA-NcMIC1 (3 intramuscular injections), or pcDNA-NcMIC1 (twice intramuscularly), followed by 1 intraperitoneal recNcMIC1 antigen boost. Control groups included corresponding treatments with adjuvant, pcDNA3.1 without insert, and PBS (= infection control). All vaccinated and control groups were then challenged intraperitoneally with 2 x 10(6) N. caninum tachyzoites. Animals were inspected daily for a period of 3 wk postinfection (PI). At day 21, all animals were killed and assessed for infection. Before day 21 PI, clinical signs such as walking disorders, rounded back, apathy, and paralysis occurred in infection controls (50% of the mice), pcDNA and adjuvant controls (20% each), and the combined pcDNA-NcMIC1/recNcMIC1-treated group (30%). No clinical symptoms were observed in the recNcMIC1 and pcDNA-NcMIC1 vaccinated groups. All mice were positive for cerebral N. caninum infection as assessed by PCR of brain tissue. However, quantitative real-time PCR revealed that the infection intensity was significantly reduced in the group vaccinated with recNcMIC1 antigen. Immunohistochemistry confirmed these findings. In contrast, the infection intensity was highest in the group vaccinated with the pcDNA-NcMIC1/recNcMIC1 combination, indicating that the sequential application of the DNA vaccine and recombinant antigen had a deleterious effect. Serological analysis showed that only recNcMIC1-immunized animals generated detectable antibody levels recognizing native NcMIC1. Thus, of all protocols applied here, only recNcMIC1 vaccination appears to be suited to reduce cerebral infection in mice challenged with N. caninum tachyzoites.     

Application of an Effective Statistical Technique for an Accurate and Powerful Mining of Quantitative Trait Loci for Rice Aroma Trait

When a phenotype of interest is associated with an external/internal covariate, covariate inclusion in quantitative trait loci (QTL) analyses can diminish residual variation and subsequently enhance the ability of QTL detection. In the in vitro synthesis of 2-acetyl-1-pyrroline (2AP), the main fragrance compound in rice, the thermal processing during the Maillard-type reaction between proline and carbohydrate reduction produces a roasted, popcorn-like aroma. Hence, for the first time, we included the proline amino acid, an important precursor of 2AP, as a covariate in our QTL mapping analyses to precisely explore the genetic factors affecting natural variation for rice scent. Consequently, two QTLs were traced on chromosomes 4 and 8. They explained from 20% to 49% of the total aroma phenotypic variance. Additionally, by saturating the interval harboring the major QTL using gene-based primers, a putative allele of fgr (major genetic determinant of fragrance) was mapped in the QTL on the 8th chromosome in the interval RM223-SCU015RM (1.63 cM). These loci supported previous studies of different accessions. Such QTLs can be widely used by breeders in crop improvement programs and for further fine mapping. Moreover, no previous studies and findings were found on simultaneous assessment of the relationship among 2AP, proline and fragrance QTLs. Therefore, our findings can help further our understanding of the metabolomic and genetic basis of 2AP biosynthesis in aromatic rice.