Genomotyping of Coxiella burnetii using microarrays reveals a conserved genomotype for hard tick isolates

C. burnetii is a Gram-negative intracellular Y-proteobacteria that causes the zoonotic disease Q fever. Q fever can manifest as an acute or chronic illness. Different typing methods have been previously developed to classify C. burnetii isolates to explore its pathogenicity. Here, we report a comprehensive genomotyping method based on the presence or absence of genes using microarrays. The genomotyping method was then tested in 52 isolates obtained from different geographic areas, different hosts and patients with different clinical manifestations. The analysis revealed the presence of 10 genomotypes organized into 3 groups, with a topology congruent with that obtained through multi-spacer typing. We also found that only 4 genomotypes were specifically associated with acute Q fever, whereas all of the genomotypes could be associated to chronic human infection. Serendipitously, the genomotyping results revealed that all hard tick isolates, including the Nine Mile strain, belong to the same genomotype.     

Process length variation in cysts of a dinoflagellate,Lingulodinium machaerophorum,in surface sediments: Investigating its potential as salinity proxy

A biometrical analysis of the dinoflagellate cyst Lingulodinium machaerophorum [Deflandre, G., Cookson, I.C., 1955. Fossil microplankton from Australia late Mesozoic and Tertiary sediments. Australian journal of Marine and Freshwater Research 6: 242–313.] Wall, 1967 in 144 globally distributed surface sediment samples revealed that the average process length is related to summer salinity and temperature at a water depth of 30 m by the equation (salinity/temperature) = (0.078?average process length + 0.534) with R² = 0.69. This relationship can be used to reconstruct palaeosalinities, albeit with caution. The particular ecological window can be associated with known distributions of the corresponding motile stage Lingulodinium polyedrum (Stein) Dodge, 1989. Confocal laser microscopy showed that the average process length is positively related to the average distance between process bases (R² = 0.78), and negatively related to the number of processes (R² = 0.65). These results document the existence of two end members in cyst formation: one with many short, densely distributed processes and one with a few, long, widely spaced processes, which can be respectively related to low and high salinity/temperature ratios. Obstruction during formation of the cysts causes anomalous distributions of the processes. From a biological perspective, processes function to facilitate sinking of the cysts through clustering.     

Structure, internal motions and association-dissociation kinetics of the i-motif dimer of d(5mCCTCACTCC)

At slightly acidic pH, the association of two d(5mCCTCACTCC) strands results in the formation of an i-motif dimer. Using NMR methods, we investigated the structure of [d(5mCCTCACTCC)]₂, the internal motion of the base pairs stacked in the i-motif core, the dimer formation and dissociation kinetics versus pH. The excellent resolution of the ¹H and ³¹P spectra provided the determination of dihedral angles, which together with a large set of distance restraints, improve substantially the definition of the sugar-phosphate backbone by comparison with previous NMR studies of i-motif structures. [d(5mCCTCACTCC)]₂ is built by intercalation of two symmetrical hairpins held together by six symmetrical C•C⁺ pairs and by pair T7•T7. The hairpin loops that are formed by a single residue, A5, cross the narrow grooves on the same side of the i-motif core. The base pair intercalation order is C9•C9⁺/5mC1•5mC1⁺/C8•C8⁺/C2•C2⁺/T7.T7/C6•C6⁺/C4•C4⁺. The T3 bases are flipped out in the wide grooves. The core of the structure includes four long-lived pairs whose lifetimes at 15°C range from 100 s (C8•C8⁺) to 0.18 s (T7•T7). The formation rate and the lifetime of [d(5mCCTCACTCC)]₂ were measured between pH 6.8 and 4.8. The dimer formation rate is three to four magnitude orders slower than that of a B-DNA duplex. It depends on pH, as it must occur for a bimolecular process involving non cooperative association of neutral and protonated residues. In the range of pH investigated, the dimer lifetime, 500 s at 0°C, pH 6.8, varies approximately as 10^−pH.     

Effect of non-esterified fatty acids on bovine granulosa cell steroidogenesis and proliferation in vitro

In high-yielding dairy cows, the negative energy balance (NEB) during the first weeks post partum may influence dominant follicle growth and steroidogenesis. Since non-esterified fatty acid (NEFA) concentrations are elevated during NEB and are shown to be toxic for several cell types, we investigated the individual and combined effects of the three main NEFA's on granulosa cell proliferation and steroidogenesis in vitro. Granulosa cells from large follicles were cultured for two days in serum free medium in the presence of palmitic (C16:0) (PA), stearic (C18:0) (SA) and/or oleic acid (C18:1) (OA). Addition of 150, 300 or 500 microM of PA and SA inhibited cell proliferation (P<0.05) while OA only elicited such an effect at 500 microM (P<0.01). In the combination treatment (150 microM of each fatty acid), cell numbers were also reduced (P<0.01). These inhibitory effects on cell number are partly due to the induction of apoptosis by these NEFA's, as was demonstrated by annexin V-FITC/propidium iodide staining of the granulosa cells. Oestradiol-17beta production was stimulated by all doses of PA, by 300 and 500 microM of SA and by 500 microM of OA (P<0.05). Combined treatment with 150 microM of each fatty acid also stimulated oestradiol-17beta production per 10(4) cells (P<0.05). We can conclude that PA, SA and to a lesser degree OA modulate granulosa cell proliferation and steroidogenesis in vitro. These effects may be involved in the occurrence of ovarian dysfunction during the postpartum period in high-yielding dairy cows.     

Blockade of PI3Kgamma suppresses joint inflammation and damage in mouse models of rheumatoid arthritis

Phosphoinositide 3-kinases (PI3K) have long been considered promising drug targets for the treatment of inflammatory and autoimmune disorders as well as cancer and cardiovascular diseases. But the lack of specificity, isoform selectivity and poor biopharmaceutical profile of PI3K inhibitors have so far hampered rigorous disease-relevant target validation. Here we describe the identification and development of specific, selective and orally active small-molecule inhibitors of PI3Kgamma (encoded by Pik3cg). We show that Pik3cg(-/-) mice are largely protected in mouse models of rheumatoid arthritis; this protection correlates with defective neutrophil migration, further validating PI3Kgamma as a therapeutic target. We also describe that oral treatment with a PI3Kgamma inhibitor suppresses the progression of joint inflammation and damage in two distinct mouse models of rheumatoid arthritis, reproducing the protective effects shown by Pik3cg(-/-) mice. Our results identify selective PI3Kgamma inhibitors as potential therapeutic molecules for the treatment of chronic inflammatory disorders such as rheumatoid arthritis.     

Dimannosyldiacylglycerol serves as a lipid anchor precursor in the assembly of the membrane-associated lipomannan in Micrococcus luteus

Based on recent analytical and enzymological studies, a topological model for the role of alpha-D-mannosyl-(1-->3)-alpha-D-mannosyl-(1-->3)-diacylglycerol (Man(2)-DAG) as a lipid anchor precursor and mannosylphosphorylundecaprenol (Man-P-Und) as a mannosyl donor in the assembly of a membrane-associated lipomannan (LM) in Micrococcus luteus has been proposed. In this study, a [(3)H]mannose-suicide selection procedure has been used to identify temperature-sensitive (ts) mutants defective in LM assembly. Two micrococcal mutants with abnormal levels of Man(2)-DAG and LM at the nonpermissive temperature (37 degrees C), mms1 and mms2, have been isolated and characterized. In vivo and in vitro biochemical assays indicate that mms1 cells have a defect in the mannosyltransferase catalyzing the conversion of Man-DAG to Man(2)-DAG, and mms2 has a temperature-sensitive defect in the synthesis of Man-P-Und. Because mms1 cells are depleted of endogenous Man(2)-DAG, membranes from this mutant efficiently converted purified, exogenous [(3)H]Man(2)-DAG to [(3)H]LM by a Man-P-Und-dependent process. An obligatory role for Man-P-Und as a mannosyl donor in the elongation process was also demonstrated by showing that the conversion of exogenous [(3)H]Man(2)-DAG to [(3)H]LM by membranes from mms1 cells in the presence of GDP-Man was inhibited by amphomycin. In addition, consistent with Man(2)-DAG serving as a lipid anchor precursor for LM assembly, endogenous, prelabeled [(3)H]Man(2)-DAG was converted to [(3)H]LM when membranes from mms2 cells were incubated with purified, exogenous Man-P-Und. These studies provide the first direct proof for the role of Man(2)-DAG as the lipid anchor precursor for LM, and suggest that Man(2)-DAG may be essential for the normal growth of M. luteus cells.     

Identification of bis-quindolines as new antiinfective agents

Several N-substituted quindolines were made to further evaluate the role of N-alkylation on the activity of indoloquinolines as antifungal agents. While N-5 substitution is required for these activities, N-10 alkylation alone leads to inactive products but is tolerated in the presence of N-5 alkyl groups. It was also discovered that bis-quindolines appear to have a more expanded antimicrobial spectrum and lower cytotoxicity than their monomeric counterparts.     

Potent antitumor 9-anilinoacridines bearing an alkylating N-mustard residue on the anilino ring: synthesis and biological activity

A series of N-mustard derivatives of 9-anilinoacridine was synthesized for antitumor and structure-activity relationship studies. The alkylating N-mustard residue was linked to the C-3' or C-4' position of the anilino ring with an O-ethylene (O-C(2)), O-butylene (O-C(4)), and methylene (C(1)) spacer. All of the new N-mustard derivatives exhibited significant cytotoxicity in inhibiting human lymphoblastic leukemic cells (CCRF-CEM) in culture. Of these agents, (3-(acridin-9-ylamino)-5-{2-[bis (2-chloroethyl)amino]ethoxy}phenyl)methanol (10) was subjected to antitumor studies, resulting in an approximately 100-fold more potent effect than its parent analogue 3-(9-acridinylamino)-5-hydroxymethylaniline (AHMA) in inhibiting the growth of human lymphoblastic leukemic cells (CCRF-CEM) in vitro. This agent did not exhibit cross-resistance against vinblastine-resistant (CCRF-CEM/VBL) or Taxol-resistant (CCRF-CEM/Taxol) cells. Remarkably, the therapeutic effect of 10 at a dose as low as one tenth of the Taxol therapeutic dose [i.e., 1-2mg/kg (Q3Dx7) or 3mg/kg (Q4Dx5); intravenous injection] on nude mice bearing human breast carcinoma MX-1 xenografts resulted in complete tumor remission in two out of three mice. Furthermore, 10 yielded xenograft tumor suppression of 81-96% using human T-cell acute lymphoblastic leukemia CCRF-CEM, colon carcinoma HCT-116, and ovarian adenocarcinoma SK-OV-3 tumor models.     

Spectrum of CFTR mutations on Réunion Island: impact on neonatal screening

The large heterogeneity in the cystic fibrosis (CF) gene is the main difficulty for genotype characterization. Numerous studies have reported considerable variations in frequencies of CF transmembrane conductance regulator (CFTR) mutations in different populations, such as African, Asian, or European populations. To completely characterize the spectrum of mutations in the CFTR gene in the Réunion Island population, we screened 228 CF chromosomes using denaturing high-pressure liquid chromatography and denaturing gradient gel electrophoresis following by direct sequencing. We identified 27 mutations, accounting for 93% of CF chromosomes. They included three novel mutations (M1T, 3121-3C-->G, and L1324P), which are described in this paper. The detection of such a high proportion of Réunion Island CFTR mutations is important for improving neonatal screening of CF on Réunion Island.