Yanrui Li

miR-499 regulates mitochondrial dynamics by targeting calcineurin and dynamin-related protein-1.

Myocardial infarction is a leading cause of mortality worldwide. Here we report that modulation of microRNA-499 (miR-499) levels affects apoptosis and the severity of myocardial infarction and cardiac dysfunction induced by ischemia-reperfusion. We found that both the α- and β-isoforms of the calcineurin catalytic subunit are direct targets of miR-499 and that miR-499 inhibits cardiomyocyte apoptosis through its suppression of calcineurin-mediated dephosphorylation of dynamin-related protein-1 (Drp1), thereby decreasing Drp1 accumulation in mitochondria and Drp1-mediated activation of the mitochondrial fission program. We also found that p53 transcriptionally downregulates miR-499 expression. Our data reveal a role for miR-499 in regulating the mitochondrial fission machinery and we suggest that modulation of miR-499 levels may provide a therapeutic approach for treating myocardial infarction.

miR-30 regulates mitochondrial fission through targeting p53 and the dynamin-related protein-1 pathway.

miRNAs participate in the regulation of apoptosis. However, it remains largely unknown as to how miRNAs are integrated into the apoptotic program. Mitochondrial fission is involved in the initiation of apoptosis. It is not yet clear whether miRNAs are able to regulate mitochondrial fission. Here we report that miR-30 family members are able to regulate apoptosis by targeting the mitochondrial fission machinery. Our data show that miR-30 family members can inhibit mitochondrial fission and the consequent apoptosis. In exploring the underlying molecular mechanism, we identified that miR-30 family members can suppress p53 expression. In response to the apoptotic stimulation, the expression levels of miR-30 family members were reduced, whereas p53 was upregulated. p53 transcriptionally activated the mitochondrial fission protein, dynamin-related protein-1 (Drp1). The latter conveyed the apoptotic signal of p53 by initiating the mitochondrial fission program. miR-30 family members inhibited mitochondrial fission through suppressing the expression of p53 and its downstream target Drp1. Our data reveal a novel model in which a miRNA can regulate apoptosis through targeting the mitochondrial fission machinery.

A Novel Lectin with Antiproliferative and HIV-1 Reverse Transcriptase Inhibitory Activities from Dried Fruiting Bodies of the Monkey Head Mushroom Hericium erinaceum

A lectin designated as Hericium erinaceum agglutinin (HEA) was isolated from dried fruiting bodies of the mushroom Hericium erinaceum with a chromatographic procedure which entailed DEAE-cellulose, CM-cellulose, Q-Sepharose, and FPLC Superdex 75. Its molecular mass was estimated to be 51 kDa and its N-terminal amino acid sequences was distinctly different from those of other isolated mushroom lectins. The hemagglutinating activity of HEA was inhibited at the minimum concentration of 12.5 mM by inulin. The lectin was stable at pH 1.9–12.1 and at temperatures up to 70°C, but was inhibited by Hg2+, Cu2+, and Fe3+ ions. The lectin exhibited potent mitogenic activity toward mouse splenocytes, and demonstrated antiproliferative activity toward hepatoma (HepG2) and breast cancer (MCF7) cells with an IC50 of 56.1 μM and 76.5 μM, respectively. It manifested HIV-1 reverse transcriptase inhibitory activity with an IC50 of 31.7 μM. The lectin exhibited potent mitogenic activity toward murine splenocytes but was devoid of antifungal activity.

Novel cardiac apoptotic pathway: the dephosphorylation of apoptosis repressor with caspase recruitment domain by calcineurin.

Background— Apoptosis repressor with caspase recruitment domain (ARC) is abundantly expressed in cardiomyocytes. Protein kinase CK2 can phosphorylate ARC at threonine-149, thereby enabling ARC to antagonize apoptosis. ARC phosphorylation occurs in a constitutive manner. Nevertheless, cardiomyocytes still undergo apoptosis that is related to cardiac diseases such as myocardial infarction and heart failure. Whether the occurrence of apoptosis is related to the loss of protection by ARC under pathological conditions remains unknown. Methods and Results— ARC phosphorylation levels are decreased in cardiomyocytes treated with isoproterenol or aldosterone. We explored the molecular mechanism by which ARC phosphorylation levels are decreased. Our results reveal that either direct incubation or coexpression with calcineurin leads to a decrease in ARC phosphorylation levels. Inhibition of calcineurin can attenuate the reduction in ARC phosphorylation levels on treatment with isoproterenol or aldosterone. These data indicate that the reduction in ARC phosphorylation levels is related to its dephosphorylation by calcineurin. Our results further reveal that ARC can prevent isoproterenol- and aldosterone-induced apoptosis, but this function depends on its phosphorylation status. Isoproterenol and aldosterone upregulate Fas ligand expression, and Fas ligand and caspase-8 are required for isoproterenol and aldosterone to induce apoptosis. However, phosphorylated but not dephosphorylated ARC is able to inhibit caspase-8–mediated apoptosis. Phosphorylated ARC exerts its effects against caspase-8 by directly associating with procaspase-8 and inhibiting its interaction with Fas-associated protein with death domain. Conclusions— Our study identifies a novel cardiac apoptotic pathway in which ARC is dephosphorylated by calcineurin. This pathway could be a component in the cardiac apoptotic machinery.

Mitochondrial fission controls DNA fragmentation by regulating endonuclease G.

Mitochondria constantly undergo fusion and fission that are necessary for the maintenance of organelle fidelity. However, growing evidence has shown that abnormal mitochondrial fusion and fission participate in the regulation of apoptosis. Mitochondrial fusion is able to inhibit apoptosis, whereas mitochondrial fission is involved in the initiation of apoptosis. It remains elusive as to whether mitochondrial fission can regulate DNA fragmentation during apoptosis. Mitochondrial fission is triggered by dynamin-related protein-1 (Drp1), whereas mitofusin 1 (Mfn 1) is able to induce mitochondrial fusion. Here, we report that Drp1 is required for the release of endonuclease G from mitochondria. Knockdown of Drp1 can attenuate DNA fragmentation. Our data further show that Mfn 1 prevents endonuclease G release from mitochondria and the consequent DNA fragmentation. Intriguingly, Mfn 1 could inhibit the activation of caspase-3 and caspase-9, which are necessary for endonuclease G translocation to the nucleus. Our results provide novel evidence that DNA fragmentation is regulated by the mitochondrial fission machinery.

DNA vaccines encoding membrane‐bound or secreted forms of heat shock protein 70 exhibit improved potency

Traditional vaccine strategies are inefficient against challenge with complex pathogens including HIV; therefore, novel vaccine technologies are required. DNA vaccines are attractive as they are relatively cheap and easy to manufacture, but a major limitation has been their lack of immunogenicity in humans, which may be overcome with the incorporation of an adjuvant. HSP70 is a recognised damage‐associated molecular pattern, which is a potential adjuvant. We investigated the immunogenicity of a DNA vaccine encoding HIV gag and HSP70; the latter was genetically modified to produce cytoplasmic, secreted or membrane‐bound HSP70, the expression of which was controlled by an independent promoter. The DNA was administered to C57BL/6 mice to evaluate gag‐specific T‐cell responses. Our results demonstrated the ability of membrane‐bound and secreted HSP70 to significantly enhance gag‐specific T‐cell responses and increase the breadth of T‐cell responses to include subdominant epitopes. Membrane‐bound or secreted HSP70 also significantly improved the multifunctionality of HIV‐specific T cells and T‐cell proliferation, which is important for maintaining T‐cell integrity. Most importantly, the inclusion of membrane‐bound HSP70, secreted HSP70 or a combination significantly increased protection in mice challenged with EcoHIV, a chimeric virus that replicates in mouse leukocytes in vivo.

A Multiantigenic DNA Vaccine That Induces Broad Hepatitis C Virus-Specific T-Cell Responses in Mice

ABSTRACT There are 3 to 4 million new hepatitis C virus (HCV) infections annually around the world, but no vaccine is available. Robust T-cell mediated responses are necessary for effective clearance of the virus, and DNA vaccines result in a cell-mediated bias. Adjuvants are often required for effective vaccination, but during natural lytic viral infections damage-associated molecular patterns (DAMPs) are released, which act as natural adjuvants. Hence, a vaccine that induces cell necrosis and releases DAMPs will result in cell-mediated immunity (CMI), similar to that resulting from natural lytic viral infection. We have generated a DNA vaccine with the ability to elicit strong CMI against the HCV nonstructural (NS) proteins (3, 4A, 4B, and 5B) by encoding a cytolytic protein, perforin (PRF), and the antigens on a single plasmid. We examined the efficacy of the vaccines in C57BL/6 mice, as determined by gamma interferon enzyme-linked immunosorbent spot assay, cell proliferation studies, and intracellular cytokine production. Initially, we showed that encoding the NS4A protein in a vaccine which encoded only NS3 reduced the immunogenicity of NS3, whereas including PRF increased NS3 immunogenicity. In contrast, the inclusion of NS4A increased the immunogenicity of the NS3, NS4B, andNS5B proteins, when encoded in a DNA vaccine that also encoded PRF. Finally, vaccines that also encoded PRF elicited similar levels of CMI against each protein after vaccination with DNA encoding NS3, NS4A, NS4B, and NS5B compared to mice vaccinated with DNA encoding only NS3 or NS4B/5B. Thus, we have developed a promising “multiantigen” vaccine that elicits robust CMI. IMPORTANCE Since their development, vaccines have reduced the global burden of disease. One strategy for vaccine development is to use commercially viable DNA technology, which has the potential to generate robust immune responses. Hepatitis C virus causes chronic liver infection and is a leading cause of liver cancer. To date, no vaccine is currently available, and treatment is costly and often results in side effects, limiting the number of patients who are treated. Despite recent advances in treatment, prevention remains the key to efficient control and elimination of this virus. Here, we describe a novel DNA vaccine against hepatitis C virus that is capable of inducing robust cell-mediated immune responses in mice and is a promising vaccine candidate for humans.

Mitochondrial fission leads to Smac/DIABLO release quenched by ARC

Apoptosis plays a critical role for the development of a variety of cardiac diseases. Cardiomyocytes are enriched in mitochondria, while mitochondrial fission can regulate apoptosis. The molecular mechanism governing cardiomyocyte apoptosis remain to be fully elucidated. Our results showed that Smac/DIABLO is necessary for apoptosis in cardiomyocytes, and it is released from mitochondria into cytosol in response to apoptotic stimulation. Smac/DIABLO release is a consequence of mitochondrial fission mediated by dynamin-related protein-1 (Drp1). Upon release Smac/DIABLO binds to X-linked inhibitor of apoptosis protein (XIAP), resulting in the activation of caspase-9 and caspase-3. Their activation is a prerequisite for the initiation of apoptosis because the administration of z-LEHD-fmk and z-DQMD-fmk, two relatively specific inhibitors for caspase-9, and caspase-3, respectively, could significantly attenuate apoptosis. Smac/DIABLO release could not be blocked by these caspase inhibitors, indicating that it is an event upstream of caspase activation. ARC (apoptosis repressor with caspase recruitment domain), an abundantly expressed apoptotic repressor in cardiomyocytes, could inhibit mitochondrial fission and Smac/DIABLO release. Our data reveal that Smac/DIABLO is a target of ARC in counteracting apoptosis.

Current understanding of cancer stem cells: Review of their radiobiology and role in head and neck cancers

Evidence of cancer cells that bear attributes analogous to those of normal stem cells has developed a hierarchical model of cancers architecture and progression. This subset of cancer stem cells (CSCs) drives the progression and therapy resistance of cancers. Research to identify the phenotypes of these CSCs presents evidence of a subpopulation that is more resistant to therapy and may proliferate in response. Literature shows that CSCs typically represent around 1%‐10% of cell populations in head and neck cancer but this proportion may increase in response to a therapeutic radiation dose. This is shown to be not just as a result of preferential killing, but also their capacity to alter divisional dynamics and enlist the support of a complicit microenvironment in therapy resistance and proliferation. The CSCs represent the apex of a hierarchy in the heterogeneity of cancer cells and may be seen as the agents of treatment failure, metastasis, and tumor recurrence, the principal cause of mortality in head and neck cancers. Greater than 90% of head and neck cancers are squamous cell carcinomas (HNSCCs), and among these an increasing incidence of the involvement of the human papillomavirus (HPV) is reported. Chemoradiotherapy along with surgical resection are the interventions of choice for control and cure of HNSCC, but given CSCs therapy resistance and proliferative responses to radiation, the identification and understanding of the radiobiology of this subpopulation is critical to their targeted elimination. This article reviews the current evidence on CSC generally and in HNSCC specifically to identify their phenotype, evaluate their responses to radiotherapy, and evaluate the defensive mechanisms used to resist therapeutic control.

A HIV-Tat/C4-binding protein chimera encoded by a DNA vaccine is highly immunogenic and contains acute EcoHIV infection in mice

DNA vaccines are cost-effective to manufacture on a global scale and Tat-based DNA vaccines have yielded protective outcomes in preclinical and clinical models of human immunodeficiency virus (HIV), highlighting the potential of such vaccines. However, Tat-based DNA vaccines have been poorly immunogenic, and despite the administration of multiple doses and/or the addition of adjuvants, these vaccines are not in general use. In this study, we improved Tat immunogenicity by fusing it with the oligomerisation domain of a chimeric C4-binding protein (C4b-p), termed IMX313, resulting in Tat heptamerisation and linked Tat to the leader sequence of tissue plasminogen activator (TPA) to ensure that the bulk of heptamerised Tat is secreted. Mice vaccinated with secreted Tat fused to IMX313 (pVAX-sTat-IMX313) developed higher titres of Tat-specific serum IgG, mucosal sIgA and cell-mediated immune (CMI) responses, and showed superior control of EcoHIV infection, a surrogate murine HIV challenge model, compared with animals vaccinated with other test vaccines. Given the crucial contribution of Tat to HIV-1 pathogenesis and the precedent of Tat-based DNA vaccines in conferring some level of protection in animal models, we believe that the virologic control demonstrated with this novel multimerised Tat vaccine highlights the promise of this vaccine candidate for humans.