Functional conservatism in mitochondrial evolution: insight from hybridization of arctic and brook charrs

To assess the potential adaptive value of mtDNA, we evaluated functional properties and thermal sensitivity of key mitochondrial enzymes in two species that have originally evolved in different thermal environments (arctic charr, Salvelinus alpinus, and brook charr, S. fontinalis), as well as in their hybrids. We measured the activity of two enzymes of the electron transport system (cytochrome c oxidase and NADH-ubiquinone oxidoreductase), one enzyme of the mitochondrial matrix (citrate synthase), and one enzyme of the anaerobic glycolysis (lactate dehydrogenase) in the red muscle at three temperatures (6 degrees C, 12 degrees C and 18 degrees C). Surprisingly, the species presented no significant differences in enzyme activity, thermal sensitivity or thermostability of key metabolic enzymes even though they evolved in different thermal environments and present important differences in amino acid sequences. It seems that amino acid substitutions between those species have minor impact on the functional properties of mitochondrial enzymes studied. The thermal sensitivity results (Q(10)) obtained for inner-membrane mitochondrial enzymes differed somewhat from those of mitochondrial matrix or cytosolic enzymes. This result indicates the modulation of thermal sensitivity of all mitochondrial inner-membrane processes by a common parameter, which could be the structural and functional properties of membrane phospholipids.     

A tandem repeats database for bacterial genomes: application to the genotyping of Yersinia pestis and Bacillus anthracis

Background  

Some pathogenic bacteria are genetically very homogeneous, making strain discrimination difficult. In the last few years, tandem repeats have been increasingly recognized as markers of choice for genotyping a number of pathogens. The rapid evolution of these structures appears to contribute to the phenotypic flexibility of pathogens. The availability of whole-genome sequences has opened the way to the systematic evaluation of tandem repeats diversity and application to epidemiological studies.     

Two new miR‐16 targets: caprin‐1 and HMGA1, proteins implicated in cell proliferation

Background information. miRNAs (microRNAs) are a class of non‐coding RNAs that inhibit gene expression by binding to recognition elements, mainly in the 3′ UTR (untranslated region) of mRNA. A single miRNA can target several hundred mRNAs, leading to a complex metabolic network. miR‐16 (miRNA‐16), located on chromosome 13q14, is involved in cell proliferation and apoptosis regulation; it may interfere with either oncogenic or tumour suppressor pathways, and is implicated in leukaemogenesis. These data prompted us to search for and validate novel targets of miR‐16. Results. In the present study, by using a combined bioinformatics and molecular approach, we identified two novel putative targets of miR‐16, caprin‐1 (cytoplasmic activation/proliferation‐associated protein‐1) and HMGA1 (high‐mobility group A1), and we also studied cyclin E which had been previously recognized as an miR‐16 target by bioinformatics database. Using luciferase activity assays, we demonstrated that miR‐16 interacts with the 3′ UTR of the three target mRNAs. We showed that miR‐16, in MCF‐7 and HeLa cell lines, down‐regulates the expression of caprin‐1, HMGA1a, HMGA1b and cyclin E at the protein level, and of cyclin E, HMGA1a and HMGA1b at the mRNA levels. Conclusions. Taken together, our data demonstrated that miR‐16 can negatively regulate two new targets, HMGA1 and caprin‐1, which are involved in cell proliferation. In addition, we also showed that the inhibition of cyclin E expression was due, at least in part, to a decrease in its mRNA stability.     

Aminobisphosphonate-pretreated dendritic cells trigger successful Vγ9Vδ2 T cell amplification for immunotherapy in advanced cancer patients

Hepatocellular carcinoma (HCC) and colorectal carcinoma with hepatic metastases (mCRC) are cancers with poor prognosis and limited therapeutic options. New approaches are needed and adoptive immunotherapy with Vγ9Vδ2 T lymphocytes represents an attractive strategy. Indeed, Vγ9Vδ2 T cells were shown to exhibit efficient lytic activity against various human tumor cell lines, and in vitro Vγ9Vδ2 T expansion protocol based on single phosphoantigen stimulation could be easily performed for healthy donors. However, a low proliferative response of Vγ9Vδ2 T cells was observed in about half of the cancer patients, leading to an important limitation in the development of Vγ9Vδ2 T cell-based immunotherapy. Here, for the first time in the context of cancer patients, Vγ9Vδ2 T cell expansions were performed by co-culturing peripheral blood mononuclear cell (PBMCs) with autologous dendritic cells (DCs) pretreated with aminobisphosphonate zoledronate. For patients not responding to the conventional culture protocol, co-culture of PBMC with zoledronate-pretreated DCs induced strong cell expansion and allowed reaching a minimal rate of purity of 70% of Vγ9Vδ2 T cells. The potent immunostimulatory activity of zoledronate-treated DCs was associated with higher amount of isopentenyl pyrophosphate (IPP) in the culture and was correlated with better ability to activate Vγ9Vδ2 T cells as measured by IFN-γ production. Moreover, we demonstrated that the cytotoxic level of Vγ9Vδ2 T cells against freshly autologous tumor cells isolated from patients could be significantly increased by pretreating the tumor cells with zoledronate. Thus, this method of generating Vγ9Vδ2 T cells leads eligible for Vγ9Vδ2 T cell adoptive immunotherapy the HCC and mCRC patients.     

The Trw Type IV Secretion System of Bartonella Mediates Host-Specific Adhesion to Erythrocytes

Bacterial pathogens typically infect only a limited range of hosts; however, the genetic mechanisms governing host-specificity are poorly understood. The α-proteobacterial genus Bartonella comprises 21 species that cause host-specific intraerythrocytic bacteremia as hallmark of infection in their respective mammalian reservoirs, including the human-specific pathogens Bartonella quintana and Bartonella bacilliformis that cause trench fever and Oroya fever, respectively. Here, we have identified bacterial factors that mediate host-specific erythrocyte colonization in the mammalian reservoirs. Using mouse-specific Bartonella birtlesii, human-specific Bartonella quintana, cat-specific Bartonella henselae and rat-specific Bartonella tribocorum, we established in vitro adhesion and invasion assays with isolated erythrocytes that fully reproduce the host-specificity of erythrocyte infection as observed in vivo. By signature-tagged mutagenesis of B. birtlesii and mutant selection in a mouse infection model we identified mutants impaired in establishing intraerythrocytic bacteremia. Among 45 abacteremic mutants, five failed to adhere to and invade mouse erythrocytes in vitro. The corresponding genes encode components of the type IV secretion system (T4SS) Trw, demonstrating that this virulence factor laterally acquired by the Bartonella lineage is directly involved in adherence to erythrocytes. Strikingly, ectopic expression of Trw of rat-specific B. tribocorum in cat-specific B. henselae or human-specific B. quintana expanded their host range for erythrocyte infection to rat, demonstrating that Trw mediates host-specific erythrocyte infection. A molecular evolutionary analysis of the trw locus further indicated that the variable, surface-located TrwL and TrwJ might represent the T4SS components that determine host-specificity of erythrocyte parasitism. In conclusion, we show that the laterally acquired Trw T4SS diversified in the Bartonella lineage to facilitate host-restricted adhesion to erythrocytes in a wide range of mammals.     

Further Insights into the Roles of GTP and the C Terminus of the Hepatitis C Virus Polymerase in the Initiation of RNA Synthesis

The hepatitis C virus (HCV) NS5b protein is an RNA-dependent RNA polymerase essential for replication of the viral RNA genome. In vitro and presumably in vivo, NS5b initiates RNA synthesis by a de novo mechanism. Different structural elements of NS5b have been reported to participate in RNA synthesis, especially a so-called “β-flap” and a C-terminal segment (designated “linker”) that connects the catalytic core of NS5b to a transmembrane anchor. High concentrations of GTP have also been shown to stimulate de novo RNA synthesis by HCV NS5b. Here we describe a combined structural and functional analysis of genotype 1 HCV-NS5b of strains H77 (subtype 1a), for which no structure has been previously reported, and J4 (subtype 1b). Our results highlight the linker as directly involved in lifting the first boundary to processive RNA synthesis, the formation of the first dinucleotide primer. The transition from this first dinucleotide primer state to processive RNA synthesis requires removal of the linker and of the β-flap with which it is shown to strongly interact in crystal structures of HCV NS5b. We find that GTP specifically stimulates this transition irrespective of its incorporation in neosynthesized RNA.     

Raw cow milk bacterial population shifts attributable to refrigeration

We monitored the dynamic changes in the bacterial population in milk associated with refrigeration. Direct analyses of DNA by using temporal temperature gel electrophoresis (TTGE) and denaturing gradient gel electrophoresis (DGGE) allowed us to make accurate species assignments for bacteria with low-GC-content (low-GC%) (<55%) and medium- or high-GC% (>55%) genomes, respectively. We examined raw milk samples before and after 24-h conservation at 4 degrees C. Bacterial identification was facilitated by comparison with an extensive bacterial reference database ( approximately 150 species) that we established with DNA fragments of pure bacterial strains. Cloning and sequencing of fragments missing from the database were used to achieve complete species identification. Considerable evolution of bacterial populations occurred during conservation at 4 degrees C. TTGE and DGGE are shown to be a powerful tool for identifying the main bacterial species of the raw milk samples and for monitoring changes in bacterial populations during conservation at 4 degrees C. The emergence of psychrotrophic bacteria such as Listeria spp. or Aeromonas hydrophila is demonstrated.     

Diversity in the ability of Agaricus bisporus wild isolates to fruit at high temperature (25°C)

The button mushroom Agaricus bisporus commercially cultivated requires 16-19 °C during the fruiting period. Wild strains are also present in natural habitat, and in light of their wide range of geographic distribution reported, from boreal region to tropical region, questions on the development adaptation to temperature arose. Isolates from various geographic areas were screened for their ability to fruit at higher temperature (FHT ability) than commercial cultivars. The FHT trait discriminated at the varietal rank. Agaricus bisporus var. eurotetrasporus was unable to develop any sporophores whilst A. bisporus var. burnettii adapted perfectly to 25 °C for fruiting, suggesting that the FHT ability is a fixed trait in these varieties. In contrast, FHT ability of A. bisporus var. bisporus appeared variable and correlated neither with climate/microclimate nor with habitat. However, FHT ability taken as a whole appeared higher in North American populations than in European ones. Some A. bisporus var. bisporus isolates revealed a good potential for cultivation at 25 °C.