Exposure to titanium dioxide nanoparticles induces autophagy in primary human keratinocytes.

Understanding the mechanisms of cell-nanomaterial interactions is vital in harnessing the potential of using nanomaterials in biomedical applications. By immuno-labeling of LC3 and TEM analysis, it is found that titanium dioxide nanoparticles are internalized by human keratinocytes and induce autophagy. Autophagy appears to play a cytoprotective role in response to toxicity influence exerted by the nanoparticles.

In vivo and in vitro studies on the antiviral activities of viperin against influenza H1N1 virus infection.

Influenza A virus has caused a number of pandemics in past decades, including the recent H1N1-2009 pandemic. Viperin is an interferon (IFN)-inducible protein of innate immunity, and acts as a broad-spectrum antiviral protein. We explored the antiviral activities and mechanisms of viperin during influenza virus (IFV) infection in vitro and in vivo. Wild-type (WT) HeLa and viperin-expressing HeLa cells were infected with influenza A/WSN/33/H1N1 (WSN33) virus, and subjected to virological, light and electron microscopic analyses. Viperin expression reduced virus replication and titres, and restricted viral budding. Young and old viperin-knockout (KO) mice and WT control animals were challenged with influenza WSN33 at lethal doses of 10(3) and 10(4) p.f.u. via the intratracheal route. Lungs were subjected to histopathological, virological and molecular studies. Upon lethal IFV challenge, both WT and KO mice revealed similar trends of infection and recovery with similar mortality rates. Viral quantification assay and histopathological evaluation of lungs from different time points showed no significant difference in viral loads and lung damage scores between the two groups of mice. Although the in vitro studies demonstrated the ability of viperin to restrict influenza H1N1 virus replication, the viperin-deficient mouse model indicated that absence of viperin enhanced neither the viral load nor pulmonary damage in the lungs of infected mice. This may be due to the compensation of IFN-stimulated genes in the lungs and/or the influenza non-structural protein 1-mediated IFN antagonism dampening the IFN response, thereby rendering the loss of viperin insignificant. Nevertheless, further investigations that exploit the antiviral mechanisms of viperin as prophylaxis are still warranted.

Staurosporine-induced programmed cell death in Blastocystis occurs independently of caspases and cathepsins and is augmented by calpain inhibition.

Previous studies have shown that the protozoan parasite Blastocystis exhibits apoptotic features with caspase-like activity upon exposure to a cytotoxic monoclonal antibody or the anti-parasitic drug metronidazole. The present study reports that staurosporine (STS), a common apoptosis inducer in mammalian cells, also induces cytoplasmic and nuclear features of apoptosis in Blastocystis, including cell shrinkage, phosphatidylserine (PS) externalization, maintenance of plasma membrane integrity, extensive cytoplasmic vacuolation, nuclear condensation and DNA fragmentation. STS-induced PS exposure and DNA fragmentation were abolished by the mitochondrial transition pore blocker cyclosporine A and significantly inhibited by the broad-range cysteine protease inhibitor iodoacetamide. Interestingly, the apoptosis phenotype was insensitive to inhibitors of caspases and cathepsins B and L, while calpain-specific inhibitors augmented the STS-induced apoptosis response. While the identities of the proteases responsible for STS-induced apoptosis warrant further investigation, these findings demonstrate that programmed cell death in Blastocystis is complex and regulated by multiple mediators.

Enterovirus 71 infection of motor neuron-like NSC-34 cells undergoes a non-lytic exit pathway.

Enterovirus 71 (EV71) causing Hand, Foot and Mouth Disease, is regarded as the most important neurotropic virus worldwide. EV71 is believed to replicate in muscles and infect motor neurons to reach the central nervous system (CNS). To further investigate the mechanisms involved, we have employed the motor neuron cell line NSC-34. NSC-34 cells were permissive to EV71 and virus production yields were strain-dependent with differential efficacy at the entry, replication and egress steps. Furthermore, unlike all the other cell lines previously reported, EV71-infected NSC-34 cells neither displayed cytopathic effect nor underwent apoptosis. Instead, autophagy was markedly up-regulated and virus-containing autophagic vacuoles were isolated from the culture supernatant, providing the first experimental evidence that EV71 can adopt a non-lytic exit pathway. Finally, the ability of EV71 to infect productively NSC-34 cells correlated with its ability to invade the CNS in vivo, supporting the relevance of NSC-34 cells to study the intrinsic neurovirulence of EV71 strains.

Evidence for an intact polysaccharide capsule in Bordetella pertussis.

Polysaccharide capsules contribute to the pathogenesis of many bacteria species by providing resistance against various defense mechanisms. The production of a capsule in Bordetella pertussis, the etiologic agent of whooping cough, has remained controversial; earlier studies reported this pathogen as a capsulated microorganism whereas the recent B. pertussis genome analysis revealed the presence of a truncated capsule locus. In this work, using transmission electron microscopy and immunostaining approaches, we provide a formal evidence for the presence of an intact microcapsule produced at the surface of both laboratory strain and clinical isolates of B. pertussis. In agreement with previous studies, we found that the capsule is optimally produced in avirulent phase. Unexpectedly, the presence of the capsule was also detected at the surface of virulent B. pertussis bacteria. Consistently, a substantial transcriptional activity of the capsule operon was detected in virulent phase, suggesting that the capsular polysaccharide may play a role during pertussis pathogenesis. In vitro assays indicated that the presence of the capsule does not affect B. pertussis adherence to mammalian cells and does not further protect the bacterium from phagocytosis, complement-mediated killing or antimicrobial peptide attack.

Tyrosine 78 of premembrane protein is essential for assembly of West Nile virus.

Flavivirus premembrane (prM) protein plays an important role in conformational folding of the envelope (E) protein and protects it against premature fusion in acidic vesicles of the Golgi network. Currently, molecular determinants on the prM protein ectodomain which mediate critical steps during the flavivirus assembly process are poorly characterized. In this study, bioinformatics analysis and alanine scanning mutagenesis showed that the amino acid triplet valine 76, tyrosine 78 and glycine 79 is absolutely conserved among flavivirus prM ectodomains. Triple mutations engineered at these residues in prM ectodomain of West Nile virus (WNV) completely abrogated virus infectivity. Site-directed mutagenesis of prM protein revealed that tyrosine 78 of the amino acid triplet was required for virus infectivity and secretion. The mutation did not affect folding, post-translational modifications and trafficking of the prM and E proteins. Ultrastructural studies using transmission electron microscopy confirmed that virus particle formation was blocked by tyrosine 78 mutation. Specificity of assembly defect conferred by tyrosine 78 mutation was demonstrated by positive and negative trans complementation studies. Collectively, these results defined tyrosine 78 as a novel critical determinant present on prM protein ectodomain that is required for flavivirus assembly. Molecular dissection of prM protein function provides the crucial knowledge much needed in the elucidation of flavivirus particle formation.

Ultrastructural and phylogenetic studies on Blastocystis isolates from cockroaches.

Four Blastocystis isolates from cockroaches were established and these isolates were morphologically confirmed as Blastocystis organisms by light and/or electron microscopy. As these isolates were morphologically indistinguishable from Blastocystis isolated from other animals, phylogenetic analyses were conducted using their small subunit ribosomal RNA genes. A analyses of these sequences with previously reported ones that had been classified into nine Blastocystis clades indicated the presence of a new clade that comprised only Blastocystis organisms from cockroaches (clade X). A clade comprised of amphibian and reptilian Blastocystis organisms (clade IX) was located at the basal position of the Blastocystis tree together with the common ancestor of Proteromonas and Protoopalina, clade X emerged after the divergences of these two basal clades and its branching position was clearly supported by bootstrap analysis.