Metamicrobiomics in herbivore beetles of the genus Cryptocephalus (Chrysomelidae): toward the understanding of ecological determinants in insect symbiosis

The Cryptocephalus marginellus (Coleoptera: Chrysomelidae) complex is composed by six species that are supposed to have originated by events of allo‐ or parapatric speciation. In the present study we investigated the alternative hypotheses that the bacterial communities associated with six populations of this species complex are shaped by environmental factors, or reflect the proposed pattern of speciation. The microbiota associated with the six populations, from five species of the complex, have been characterized through 16S rRNA pyrotag sequencing. Based on a 97% sequence similarity threshold, data were clustered into 381 OTUs, which were analyzed using a variety of diversity indices. The microbiota of C. acquitanus and C. marginellus (Calanques) were the most diverse (over 100 OTUs), while that from C. zoiai yielded less bacterial diversity (45 OTUs). Taxonomic assignment revealed Proteobacteria, Tenericutes and Firmicutes as the dominant components of these beetles’ microbiota. The most abundant genera were Ralstonia, Sphingomonas, Rickettsia, and Pseudomonas. Different strains of Rickettsia were detected in C. eridani and C. renatae. The analysis of β‐diversity revealed high OTU turnover among the populations of C. marginellus complex, with only few shared species. Hierarchical clustering taking into account relative abundances of OTUs does not match the phylogeny of the beetles, therefore we hypothesize that factors other than phylogenetic constraints play a role in shaping the insects’ microbiota. Environmental factors that could potentially affect the composition of bacterial communities were tested by fitting them on the results of a multi‐dimensional scaling analysis. No significant correlations were observed towards the geographic distances or the host plants, while the composition of the microbiota appeared associated with altitude. The metabolic profiles of the microbiotas associated with each population were inferred from bacterial taxonomy, and interestingly, the obtained clustering pattern was consistent with the host phylogeny.     

Establishment and characterization of new mammary adenocarcinoma cell lines derived from double transgenic mice expressing GFP and neu oncogene

A new murine cell line, named GFPneu, was established from a mammary adenocarcinoma arising in double transgenic MMTVneu x CMV-GFP mice. Breast tumours develop in 100% of females after 2 months latency, as a result of the over-expression of the activated rat neu oncogene in the mammary glands. All tissues, and in particular the breast tumours, express the GFP protein. This cell line was tumorigenic when inoculated into nude mice and the derived tumours showed the same histological features as the primaries from which they were isolated. Their histopathology reproduces many characteristics of human breast adenocarcinomas, in particular their ability to metastasize. The GFP marker allows us to visualize the presence of lung metastases in fresh tissues immediately, to confirm the histopathology. From a lung metastatic fluorescent nodule, we derived a further cell line, named MTP-GFP, which we also characterized. These two cell lines could be useful to study the role played by the neu oncogene in the maintenance of the transformed phenotype, in the metastatic process, to test novel therapeutic strategies to inhibit primary tumour growth and to observe the generation of distant metastases.     

Vitamin D deficiency in rheumatoid arthritis: prevalence,determinants and associations with disease activity and disability

Introduction  

The aim of this study was to estimate the prevalence and determinants of vitamin D deficiency in patients with rheumatoid arthritis (RA) as compared to healthy controls and to analyze the association between 25-hydroxyvitamin D (25(OH)D) with disease activity and disability.     

Of mice and MEN1: Insulinomas in a conditional mouse knockout

Patients with multiple endocrine neoplasia type 1 (MEN1) develop multiple endocrine tumors, primarily affecting the parathyroid, pituitary, and endocrine pancreas, due to the inactivation of the MEN1 gene. A conditional mouse model was developed to evaluate the loss of the mouse homolog, Men1, in the pancreatic beta cell. Men1 in these mice contains exons 3 to 8 flanked by loxP sites, such that, when the mice are crossed to transgenic mice expressing cre from the rat insulin promoter (RIP-cre), exons 3 to 8 are deleted in beta cells. By 60 weeks of age, >80% of mice homozygous for the floxed Men1 gene and expressing RIP-cre develop multiple pancreatic islet adenomas. The formation of adenomas results in elevated serum insulin levels and decreased blood glucose levels. The delay in tumor appearance, even with early loss of both copies of Men1, implies that additional somatic events are required for adenoma formation in beta cells. Comparative genomic hybridization of beta cell tumor DNA from these mice reveals duplication of chromosome 11, potentially revealing regions of interest with respect to tumorigenesis.     

A novel calcium-stimulated adenylyl cyclase from Trypanosoma cruzi,which interacts with the structural flagellar protein paraflagellar rod

Trypanosoma cruzi adenylyl cyclases are encoded by a large polymorphic gene family. Although several genes have been identified in this parasite, little is known about the properties and regulation of these enzymes. Here we report the cloning and characterization of TczAC, a novel member of T. cruzi adenylyl cyclase family. The TczAC gene is expressed in all of the parasite life forms and encodes a 1,313-amino acid protein that can complement a Saccharomyces cerevisiae mutant deficient in adenylyl cyclase activity. The recombinant enzyme expressed in yeasts is constitutively active, has a low affinity for ATP (K(m) = 406 microm), and requires a divalent cation for catalysis. TczAC is inhibited by Zn(2+) and the P-site inhibitor 2'-deoxyadenosine 3'-monophosphate, suggesting some level of conservation in the catalytic mechanism with mammalian adenylyl cyclases. It shows a dose-dependent stimulation by Ca(2+) which can be reversed by high concentrations of phenothiazinic calmodulin inhibitors. However, bovine calmodulin fails to stimulate the enzyme. Using a yeast two-hybrid screen it was found that TczAC interacts through its catalytic domain with the paraflagellar rod protein, a component of the flagellar structure. Furthermore, we demonstrate that TczAC can dimerize through the same domain. These results provide novel evidence of the possible localization and regulation of this protein.     

Comparison of transcription stimulating, phenol-soluble non-histone chromosomal proteins in normal rat liver and transplantable hepatocellular carcinomas.

The non-histone chromosomal proteins (NHCP) of a rapidly and slowly proliferating transplantable hepatocellular carcinoma (THC) were compared to those of normal and regenerating rat liver. The total quantity of NHCP is approximately threefold higher in the THCs than in either normal rat liver at 4 h and 44 h regenerating rat liver. Only those NHCP that can be extracted from chromatin by 0.35 M NaCl were further examined and it was observed that the proteins of this highly complex fraction could be further fractionated by their differential phenol-solubility. The phenol-soluble 0.35 NHCP contained protein(s) capable of stimulating the level of DNA-directed RNA synthesis in vitro. The total amount of this stimulatory activity was 5 times higher in the rapidly growing THC and 1.6 times higher in the slowly growing THC than in normal rat liver. In order to assess the contribution of cell-cycle dependent alterations on the increase in the amount of stimulatory activity in the THCs, 44 h regenerating rat livers were examined. This tissue represents a mix of cells in various stages of the cell cycle which is similar to that found in the THCs. It was found that the total quantity of NHCP in the 44 h regenerating rat liver was the same as in normal rat liver. The total amount of the stimulatory activity also was similar in both the normal and 44 h regenerating rat liver. The amount of the stimulatory activity was found to double in 4 h regenerating rat liver, however. These data suggest that the alterations observed in the NHCP of the THCs are not due solely to cell cycle dependent changes, but may represent malignancy dependent alterations.     

Legionella Contamination in Hot Water of Italian Hotels

A cross-sectional multicenter survey of Italian hotels was conducted to investigate Legionella spp. contamination of hot water. Chemical parameters (hardness, free chlorine concentration, and trace element concentrations), water systems, and building characteristics were evaluated to study risk factors for colonization. The hot water systems of Italian hotels were strongly colonized by Legionella; 75% of the buildings examined and 60% of the water samples were contaminated, mainly at levels of ≥10³ CFU liter⁻¹, and Legionella pneumophila was the most frequently isolated species (87%). L. pneumophila serogroup 1 was isolated from 45.8% of the contaminated sites and from 32.5% of the hotels examined. When a multivariate logistic model was used, only hotel age was associated with contamination, but the risk factors differed depending on the contaminating species and serogroup. Soft water with higher chlorine levels and higher temperatures were associated with L.pneumophila serogroup 1 colonization, whereas the opposite was observed for serogroups 2 to 14. In conclusion, Italian hotels, particularly those located in old buildings, represent a major source of risk for Legionnaires' disease due to the high frequency of Legionella contamination, high germ concentration, and major L. pneumophila serogroup 1 colonization. The possible role of chlorine in favoring the survival of Legionella species is discussed.     

Development of an immunomagnetic bead-immunoliposome fluorescence assay for rapid detection of Escherichia coli O157:H7 in aqueous samples and comparison of the assay with a standard microbiological method

The objective of this study was to develop and optimize a protocol for the rapid detection of Escherichia coli O157:H7 in aqueous samples by a combined immunomagnetic bead-immunoliposome (IMB/IL) fluorescence assay. The protocol consisted of the filtration or centrifugation of 30- to 100-ml samples followed by incubation of the filter membranes or pellet with anti-E. coli O157:H7 immunomagnetic beads in growth medium specific for E. coli O157:H7. The resulting E. coli O157:H7-immunomagnetic bead complexes were isolated by magnetic separation, washed, and incubated with sulforhodamine B-containing immunoliposomes specific for E. coli O157:H7; the final immunomagnetic bead-E. coli O157:H7-immunoliposome complexes were again isolated by magnetic separation, washed, and lysed with a n-octyl-beta-d-glucopyranoside to release sulforhodamine B. The final protocol took less than 8 h to complete and had a detection limit of less than 1 CFU of E. coli O157:H7 per ml in various aqueous matrices, including apple juice and cider. To validate the protocol at an independent facility, 100-ml samples of groundwater with and without E. coli O157:H7 (15 CFU) were analyzed by a public health laboratory using the optimized protocol and a standard microbiological method. While the IMB/IL fluorescence assay was able to identify E. coli O157:H7-containing samples with 100% accuracy, the standard microbiological method was unable to distinguish E. coli O157:H7-spiked samples from negative controls without further extensive workup. These results demonstrate the feasibility of using immunomagnetic beads in combination with sulforhodamine B-encapsulating immunoliposomes for the rapid detection of E. coli O157:H7 in aqueous samples.