Both TRIF and IPS-1 Adaptor Proteins Contribute to the Cerebral Innate Immune Response against Herpes Simplex Virus 1 Infection

Toll-like receptors (TLRs) and RNA helicases (RLHs) are important cell sensors involved in the immunological control of viral infections through production of type I interferon (IFN). The impact of a deficiency in the TRIF and IPS-1 adaptor proteins, respectively, implicated in TLR3 and RLH signaling pathways, was investigated during herpes simplex virus 1 (HSV-1) encephalitis. TRIF^−/−, IPS-1^−/−, and C57BL/6 wild-type (WT) mice were infected intranasally with 7.5 × 10⁵ PFU of HSV-1. Mice were monitored for neurological signs and survival over 20 days. Groups of mice were sacrificed on days 3, 5, 7, 9, and 11 postinfection for determination of brain viral replication by quantitative PCR (qPCR), plaque assay, and immunohistochemistry and for alpha/beta interferon (IFN-α/β) levels and phosphorylation of interferon regulatory factors 3 and 7 (IRF-3 and -7) in brain homogenates by enzyme-linked immunosorbent assay (ELISA) and Western blotting, respectively. TRIF^−/− and IPS-1^−/− mice had higher mortality rates than WT mice (P = 0.02 and P = 0.09, respectively). Viral antigens were more disseminated throughout the brain, correlating with a significant increase in brain viral load for TRIF^−/− (days 5 to 9) and IPS-1^−/− (days 7 and 9) mice compared to results for the WT. IFN-β production was reduced in brain homogenates of TRIF^−/− and IPS-1^−/− mice on day 5 compared to results for the WT, whereas IFN-α levels were increased on day 7 in TRIF^−/− mice. Phosphorylation levels of IRF-3 and IRF-7 were decreased in TRIF^−/− and IPS-1^−/− mice, respectively. These data suggest that both the TRIF and IPS-1 signaling pathways are important for the control of HSV replication in the brain and survival through IFN-β production.     

Anoctamin 1 dysregulation alters bronchial epithelial repair in cystic fibrosis

Cystic fibrosis (CF) airway epithelium is constantly subjected to injury events due to chronic infection and inflammation. Moreover, abnormalities in CF airway epithelium repair have been described and contribute to the lung function decline seen in CF patients. In the last past years, it has been proposed that anoctamin 1 (ANO1), a Ca^2 +-activated Cl^? channel, might offset the CFTR deficiency but this protein has not been characterized in CF airways. Interestingly, recent evidence indicates a role for ANO1 in cell proliferation and tumor growth. Our aims were to study non-CF and CF bronchial epithelial repair and to determine whether ANO1 is involved in airway epithelial repair. Here, we showed, with human bronchial epithelial cell lines and primary cells, that both cell proliferation and migration during epithelial repair are delayed in CF compared to non-CF cells. We then demonstrated that ANO1 Cl^? channel activity was significantly decreased in CF versus non-CF cells. To explain this decreased Cl^? channel activity in CF context, we compared ANO1 expression in non-CF vs. CF bronchial epithelial cell lines and primary cells, in lung explants from wild-type vs. F508del mice and non-CF vs. CF patients. In all these models, ANO1 expression was markedly lower in CF compared to non-CF. Finally, we established that ANO1 inhibition or overexpression was associated respectively with decreases and increases in cell proliferation and migration. In summary, our study demonstrates involvement of ANO1 decreased activity and expression in abnormal CF airway epithelial repair and suggests that ANO1 correction may improve this process.     

Variation in Wolbachia cidB gene,but not cidA,is associated with cytoplasmic incompatibility mod phenotype diversity in Culex pipiens

Endosymbiotic Wolbachia bacteria are, to date, considered the most widespread symbionts in arthropods and are the cornerstone of major biological control strategies. Such a high prevalence is based on the ability of Wolbachia to manipulate their hosts' reproduction. One manipulation called cytoplasmic incompatibility (CI) is based on the death of the embryos generated by crosses between infected males and uninfected females or between individuals infected with incompatible Wolbachia strains. CI can be seen as a modification‐rescue system (or mod‐resc) in which paternal Wolbachia produce mod factors, inducing embryonic defects, unless the maternal Wolbachia produce compatible resc factors. Transgenic experiments in Drosophila melanogaster and Saccharomyces cerevisiae converged towards a model where the cidB Wolbachia gene is involved in the mod function while cidA is involved in the resc function. However, as cidA expression in Drosophila males was required to observe CI, it has been proposed that cidA could be involved in both resc and mod functions. A recent correlative study in natural Culex pipiens mosquito populations has revealed an association between specific cidA and cidB variations and changes in mod phenotype, also suggesting a role for both these genes in mod diversity. Here, by studying cidA and cidB genomic repertoires of individuals from newly sampled natural C. pipiens populations harbouring wPipIV strains from North Italy, we reinforce the link between cidB variation and mod phenotype variation fostering the involvement of cidB in the mod phenotype diversity. However, no association between any cidA variants or combination of cidA variants and mod phenotype variation was observed. Taken together our results in natural C. pipiens populations do not support the involvement of cidA in mod phenotype variation.     

Chemical modifications of imidazole-containing alkoxyamines increase C–ON bond homolysis rate: Effects on their cytotoxic properties in glioblastoma cells

Previously, we described alkoxyamines bearing a pyridine ring as new pro-drugs with low molecular weights and theranostic activity. Upon chemical stimulus, alkoxyamines undergo homolysis and release free radicals, which can, reportedly, enhance magnetic resonance imaging and trigger cancer cell death. In the present study, we describe the synthesis and the anti-cancer activity of sixteen novel alkoxyamines that contain an imidazole ring. Activation of the homolysis was conducted by protonation and/or methylation. These new molecules displayed cytotoxic activities towards human glioblastoma cell lines, including the U251-MG cells that are highly resistant to the conventional chemotherapeutic agent Temozolomide. We further showed that the biological activities of the alkoxyamines were not only related to their half-life times of homolysis. We lastly identified the alkoxyamine (RS/SR)-4a, with both a high antitumour activity and favourable logD_7.4 and pK_a values, which make it a robust candidate for blood-brain barrier penetrating therapeutics against brain neoplasia.     

Molecular characterization of four Haemophilus influenzae serotype a strains isolated from patients in Quebec, Canada

Four epidemiologically unrelated Haemophilus influenzae serotype a (Hia) strains from patients in Quebec, Canada, were characterized and found to represent 3 distinct groups. One isolate, found to be biotype I and sequence type (ST)-62 by multilocus sequence typing, was shown to possess the copper- and zinc-containing superoxide dismutase gene, sodC, and was suspected to belong to clonal division II. The other 3 isolates were classified as clonal division I based on the absence of the sodC gene. Among the 3 sodC-negative Hia strains, 2 were biotype II and had related STs (ST-23 and ST-403) and highly similar DNA fingerprints, similar to a group of previously described Hia isolates causing invasive disease in Manitoba, Canada. The remaining sodC-negative strain belonged to biotype I and ST-4 and shared no common allele with ST-23, ST-403, or ST-62. This isolate also possessed the IS1016-bexA partial deletion, which is often associated with increased virulence. Despite the small number of isolates used in this study, our finding of 3 distinct groups shows the existence of a potential genetic diversity not previously described for Hia. Whether this genetic diversity is related to the severity and epidemiology of Hia disease requires further studies.     

Capsular arabinomannans from Mycobacterium avium with morphotype-specific structural differences but identical biological activity

The capsules of two colony morphotypes of Mycobacterium avium strain 2151 were investigated, i.e. the virulent smooth-transparent (SmT1) and the nonvirulent smooth-opaque (SmO) types. From both morphotypes we separated a nonacylated arabinomannan (AM) from an acylated polysaccharide fraction by affinity chromatography, of which the AMs were structurally characterized. The AMs from the virulent morphotype, in contrast to that from the nonvirulent form, possessed a larger mannan chain and a shorter arabinan chain. Incubation of murine bone marrow-derived macrophages and human dendritic cells showed that the acylated polysaccharide fractions were potent inducers of tumor necrosis factor-alpha, interleukin-12, and interleukin-10 compared with nonacylated AMs, which led to only a marginal cytokine release. Further in vitro experiments showed that both the acylated polysaccharide fractions and the nonacylated AMs were able to induce in vitro anti-tumor cytotoxicity of human peripheral blood mononuclear cells. Thus, morphotype-specific structural differences in the capsular AMs of M. avium do not correlate with biological activity; however, their acylation is a prerequisite for effective stimulation of murine macrophages and human dendritic cells.     

Substrate-specific interactions with the heme-bound oxygen molecule of nitric-oxide synthase

We report the characterization by resonance Raman spectroscopy of the oxygenated complex (Fe(II)O(2)) of nitric-oxide synthases of Staphylococcus aureus (saNOS) and Bacillus subtilis (bsNOS) saturated with N(omega)-hydroxy-l-arginine. The frequencies of the nu(Fe-O) and nu(O-O) modes were 530 and 1135 cm(-), respectively, in both the presence and absence of tetrahydrobiopterin. On the basis of a comparison of these frequencies with those of saNOS and bsNOS saturated with l-arginine (nu(Fe-O) at 517 cm(-1) and nu(O-O) at 1123 cm(-1)) and those of substrate-free saNOS (nu(Fe-O) at 517 and nu(O-O) at 1135 cm(-1)) (Chartier, F. J. M., Blais, S. P., and Couture, M. (2006) J. Biol. Chem. 281, 9953-9962), we propose two models that account for the frequency shift of nu(Fe-O) (but not nu(O-O)) upon N(omega)-hydroxy-l-arginine binding as well as the frequency shift of nu(O-O) (but not nu(Fe-O)) upon l-arginine binding. The implications of these substrate-specific interactions with respect to catalysis by NOSs are discussed.     

BMI1 nuclear location is critical for RAD51-dependent response to replication stress and drives chemoresistance in breast cancer stem cells

Replication stress (RS) has a pivotal role in tumor initiation, progression, or therapeutic resistance. In this study, we depicted the mechanism of breast cancer stem cells’ (bCSCs) response to RS and its clinical implication. We demonstrated that bCSCs present a limited level of RS compared with non-bCSCs in patient samples. We described for the first time that the spatial nuclear location of BMI1 protein triggers RS response in breast cancers. Hence, in bCSCs, BMI1 is rapidly located to stalled replication forks to recruit RAD51 and activate homologous-recombination machinery, whereas in non-bCSCs BMI1 is trapped on demethylated 1q12 megasatellites precluding effective RS response. We further demonstrated that BMI1/RAD51 axis activation is necessary to prevent cisplatin-induced DNA damage and that treatment of patient-derived xenografts with a RAD51 inhibitor sensitizes tumor-initiating cells to cisplatin. The comprehensive view of replicative-stress response in bCSC has profound implications for understanding and improving therapeutic resistance.Subject terms: Breast cancer, Cancer stem cells