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Virions produced from cell culture is the primary source for production of formalin-inactivated whole virus vaccines for enteroviruses. EV-A71 particles produced from culture system comprise two major types, the immature/empty (E)-particle and the mature/full (F)-particle, which both exhibit low isoelectric point (pI) values but have distinct differences in infectivity and immunogenicity. Although EV-A71 particles can conventionally be separated into E-particle and F-particle using sucrose gradient ultracentrifugation, this procedure is cumbersome and difficult to put into practice for vaccine production. Methods based on ion-exchange chromatography have been exploited to improve the purification efficacy; however, none of them are capable of separating the E- and F-particles efficiently. In this study, we aimed to develop an approach to isolate and purify the highly immunogenic mature EV-A71 particles. By applying a step gradient elution procedure, we successfully isolated the viral structure protein VP0-cleaved particles of EV-A71 from a mixture of cultured viral solution using the Q-membrane anion-exchange chromatography. The elution started with 0.1x phosphate buffered saline (PBS) solution while increasing the percentage of 1x PBS containing 1M NaCl in sequential steps. By this procedure, the VP0-cleaved mature particles and VP0-uncleaved immature particles of EV-A71 could be separated into different fractions in Q-membrane with gradually increased NaCl concentration in elution buffer. The purified VP0-cleaved particles were shown to have characteristics equivalent to those of the highly infectious F-particles of EV-A71. The overall recovery rate for the mature EV-A71 particles by Q-membrane is 56% and its purity was shown to be equivalent to those isolated by the sucrose gradient ultracentrifugation. Our approach provides a simple and efficient purification method for recovering mature, highly infectious virus particles from the EV-A71 culture bulk.

Childhood exanthema caused by different serotypes of coxsackievirus (CV-A) and enterovirus A71 (EV-A71) has become a serious global health problem; it is commonly known as hand, foot, and mouth disease (HFMD). Current EV-A71 vaccine clinical trials have demonstrated that human antibody responses generated by EV-A71 vaccinations do not cross-neutralize coxsackievirus A16 (CV-A16). An effective multivalent HFMD vaccine is urgently needed. From molecular epidemiological studies in Southeast Asia, CV-A6 and CV-A10 are commonly found in HFMD outbreaks. In this study, CV-A6 and CV-A10 were individually cultured in rhabdomyosarcoma (RD) cells grown in medium containing serum, harvested and concentrated. In viral downstream purification, two viral fractions were separated by sucrose gradient zonal ultracentrifugation and detected using a SDS-PAGE analysis and a virus infectivity assay. These two viral fractions were formalin-inactivated, and only the infectious particle fraction was found to be capable of inducing CV-A serotype-specific neutralizing antibody responses in animal immunogenicity studies. These mouse and rabbit antisera also failed to cross-neutralize EV-A71 and CV-A16 infections. Only a combination of formalin-inactivated EV-A71, CV-A6, CV-A10 and CV-A16 multivalent vaccine candidates elicited cross-neutralizing antibody responses in both mouse and rabbit immunogenicity studies. The current results certainly provide important information for multivalent HFMD vaccine development.

In alphaviruses, here represented by Semliki Forest virus, infection requires an acid-responsive spike configuration to facilitate membrane fusion. The creation of this relies on the chaperon function of glycoprotein E2 precursor (p62) and its maturation cleavage into the small external E3 and the membrane-anchored E2 glycoproteins. To reveal how the E3 domain of p62 exerts its control of spike functions, we determine the structure of a p62 cleavage-impaired mutant virus particle (SQL) by electron cryomicroscopy. A comparison with the earlier solved wild type virus structure reveals that the E3 domain of p62SQL forms a bulky side protrusion in the spike head region. This establishes a gripper over part of domain II of the fusion protein, with a cotter-like connection downward to a hydrophobic cluster in its central β-sheet. This finding reevaluates the role of the precursor from being only a provider of a shield over the fusion loop to a structural playmate in formation of the fusogenic architecture.

Nanomaterials were already identified to exhibit anti-cancer property without carrying drugs. We previously revealed the selective anti-cancer activity of Fe@Au nanoparticles (NP) by impairing mitochondria. The zero-valent iron (ZVI) core contributes the major cytotoxicity. Here, we screen oral cancer cells for resistance profile to ZVI NP and identify OECM1, OC3 and SCC9 to be sensitive, while HSC3, SAS and OC2 tolerate ZVI NP. Resistance clones of OECM1 also can be derived by pulsed NPs treatment. We identify that ZVI NP initiated Fenton reaction and induced free radicals with distinct difference in the mitochondria or cytosol between the refractory and sensitive cancer cells. Lipid peroxidation plays critical roles in the ZVI NP derived ROS induction and subsequent declined mitochondrial respiration. We further discovered the decrease of GPx upon ZVI treatment. GPx inhibitors and glutathione deprivation are able to sensitize ZVI refractory cancer cells accompanied by enhancing mitochondrial depolarization. These results reveal a potential anti-cancer mechanism of ZVI. We also demonstrate how to manipulate the ZVI resistance by combination therapy. This study provides biomarkers that predicts ZVI NP efficacy and new strategies to overcome ZVI resistance through integration of small molecular inhibitors in such new generational anti-cancer nanomedicine.

Single-point mutation of mitochondrial DNA (mtDNA) has been confirmed to be involved in some inheritance of mitochondrial myopathy such as Mitochondrial Encephalopathy, Lactic acidosis, And Stroke-like syndrome (MELAS) and Myoclonic Epilepsy and Ragged-Red Fibers (MERRF). The A-to-G mutation at nucleotide 8344 accounts for 80 to 90% of MERRF syndrome. More than 80% of MELAS patients carry the A3243G mutation. The location of these two point mutations were identified in the transfer RNA (tRNA). It remains unknown why these particular tRNA point mutations caused the abnormal phenotypes. Some studies have demonstrated that Reactive Oxygen Species (ROS) production, alteration in antioxidant defenses and detoxification enzymes is involved in the pathogenesis of MERRF and MELAS syndrome. More and more reports revealed a potential role of oxidative stress within dysregulated mitochondrial network in lesion tissue. However, structural integrity and appropriate distribution and dynamics of mitochondria have to be maintained to execute normal physiological functions. Therefore, we integrated light and electron microscopy to illuminate mosaic mechanisms of the altered mitochondrial distribution and dynamics in cultured skin fibroblasts from patients with mitochondrial tRNA mutations.