A novel frameshift mutation in DNAH6 associated with male infertility and asthenoteratozoospermia.

Introduction: Asthenoteratozoospermia is one of the most common causes of male infertility. Several genes have been identified as genetic causative factors, but there is a considerable genetic heterogeneity underlying asthenoteratozoospermia. In this study, we performed a genetic analysis of two brothers from a consanguineous Uighur family in China to identify gene mutations causative for asthenoteratozoospermia-related male infertility. Methods: Two related patients with asthenoteratozoospermia from a large consanguineous family were sequenced by whole-exome sequencing and Sanger sequencing to identify disease-causing genes. Scanning and transmission electron microscopy analysis revealed ultrastructural abnormalities of spermatozoa. Quantitative real-time PCR (qRT-PCR) analysis and immunofluorescence (IF) analysis were used to assess the expression of the mutant messenger RNA (mRNA) and protein. Results: A novel homozygous frameshift mutation (c.2823dupT, p.Val942Cysfs*21) in DNAH6 was identified in both affected individuals and was predicted to be pathogenic. Papanicolaou staining and electron microscopy revealed multiple morphological and ultrastructural abnormalities of affected spermatozoa. qRT-PCR and IF analysis showed abnormal expression of DNAH6 in affected sperm, probably due to premature termination code and decay of abnormal 3' untranslated region (UTR) region of mRNA. Furthermore, intracytoplasmic sperm injection could achieve successful fertilization in infertile men with DNAH6 mutations. Discussion: The novel frameshift mutation identified in DNAH6 may contribute to asthenoteratozoospermia. These findings expand the spectrum of genetic mutations and phenotypes associated with asthenoteratozoospermia and may be useful for genetic and reproductive counseling in male infertility.

Autophagy is involved in the replication of H9N2 influenza virus via the regulation of oxidative stress in alveolar epithelial cells.

BACKGROUND: Oxidative stress is an important pathogenic factor in influenza A virus infection. It has been found that reactive oxygen species induced by the H9N2 influenza virus is associated with viral replication. However, the mechanisms involved remain to be elucidated. METHODS: In this study, the role of autophagy was investigated in H9N2 influenza virus-induced oxidative stress and viral replication in A549 cells. Autophagy induced by H9N2 was inhibited by an autophagy inhibitor or RNA interference, the autophagy level, viral replication and the presence of oxidative stress were detected by western blot, TCID50 assay, and Real-time PCR. Then autophagy and oxidative stress were regulated, and viral replication was determined. At last, the Akt/TSC2/mTOR signaling pathways was detected by western blot. RESULTS: Autophagy was induced by the H9N2 influenza virus and the inhibition of autophagy reduced the viral titer and the expression of nucleoprotein and matrix protein. The blockage of autophagy suppressed the H9N2 virus-induced increase in the presence of oxidative stress, as evidenced by decreased reactive oxygen species production and malonaldehyde generation, and increased superoxide dismutase 1 levels. The changes in the viral titer and NP mRNA level caused by the antioxidant, N-acetyl-cysteine (NAC), and the oxidizing agent, H2O2, confirmed the involvement of oxidative stress in the control of viral replication. NAC plus transfection with Atg5 siRNA significantly reduced the viral titer and oxidative stress compared with NAC treatment alone, which confirmed that autophagy was involved in the replication of H9N2 influenza virus by regulating oxidative stress. Our data also revealed that autophagy was induced by the H9N2 influenza virus through the Akt/TSC2/mTOR pathway. The activation of Akt or the inhibition of TSC2 suppressed the H9N2 virus-induced increase in the level of LC3-II, restored the decrease in the expression of phospho-pAkt, phospho-mTOR and phospho-pS6 caused by H9N2 infection, suppressed the H9N2-induced increase in the presence of oxidative stress, and resulted in a decrease in the viral titer. CONCLUSION: Autophagy is involved in H9N2 virus replication by regulating oxidative stress via the Akt/TSC2/mTOR signaling pathway. Thus, autophagy maybe a target which may be used to improve antiviral therapeutics.

A Novel Cleavage Pattern of Complement C5 Induced by Chlamydia trachomatis Infection via the Chlamydial Protease CPAF.

Urogenital Chlamydia trachomatis infection is one of the most common bacterial sexually transmitted diseases globally. Untreated C. trachomatis infections can ascend to the upper genital tract and establish a series of severe complications. Previous studies using C3-/- and C5-/- mice models demonstrated that C3-independent activation of C5 occurred during C. trachomatis infection. However, the mechanism of how chlamydial infection activates C5 in the absence of C3 has yet to be elucidated. To delineate interactions between C5 and chlamydial infection, cleavage products in a co-incubation system containing purified human C5 and C. trachomatis-HeLa229 cell lysates were analyzed, and a novel cleavage pattern of C5 activation induced by C. trachomatis infection was identified. C5 was cleaved efficiently at the previously unidentified site K970, but was cleaved poorly at site R751. C5b was modified to C5bCt, which later formed C5bCt-9, which had enhanced lytic ability compared with C5b-9. The chlamydial serine protease CPAF contributed to C3-independent C5 activation during C. trachomatis infection. Nafamostat mesylate, a serine protease inhibitor with a good safety profile, had a strong inhibitory effect on C5 activation induced by chlamydial infection. These discoveries reveal the mechanism of C3-independent C5 activation induced by chlamydial infection, and furthermore provide a potential therapeutic target and drug for preventing tubal fibrosis caused by chlamydial infection.

Molecular characterization and pathogenesis of H9N2 avian influenza virus isolated from a racing pigeon.

H9N2 avian influenza viruses (AIVs) can cross species barriers and expand from birds tomammals and humans. It usually leads to economic loss for breeding farms and poses a serious threat to human health.This study investigated the molecular characteristics of H9N2 AIV isolated from a racing pigeon and its pathogenesis in BALB/c mice and pigeons. Phylogenetic analysis indicated that the H9N2 virus belonged to the Ck/BJ/94-like lineage, and acquired multiple specific amino acid substitutions that might contribute to viral transmission from birds to mammals and humans. A pathogenesis study showed that both mice and pigeons infected with H9N2 virus showed clinical signs and mortality. The H9N2 viruses efficiently replicated in mice and pigeons. In our study, high levels of viral shedding were detected in pigeons, but the infection was not transmitted to co-housed pigeons. Histopathological examination revealed the presence of inflammatory responses in the infected mice and pigeons. Immunohistochemical analysis showed the presence of H9N2 virus in multiple organs of the infected mice and pigeons. Moreover, the infected mice and pigeons demonstrated significant cytokine/chemokine production. Our results showed that the H9N2 virus can infect mice and pigeons, and can not be transmitted between pigeons through direct contact.

Autophagy is involved in the acute lung injury induced by H9N2 influenza virus.

Influenza A virus usually leads to economic loss to breeding farms and pose a serious threat to human health. Virus infecting tissues directly and influenza virus-induced excessive production of inflammatory factors play the key role in pathogenesis of the disease, but the mechanism is not well clarified. Here, the role of autophagy was investigated in H9N2 influenza virus-triggered inflammation. The results showed that autophagy was induced by H9N2 virus in A549 cells and in mice. Inhibiting autophagy by an autophagy inhibitor (3-methyladenine, 3-MA) or knockdown of Atg5(autophagy-related gene) by Atg5 siRNA significantly suppressed H9N2 virus replication, H9N2 virus-triggered inflammatory cytokines and chemokines, including IL-1ß, TNF-α, IL-8, and CCL5 in vitro and in vivo, and suppressed H9N2 virus-triggered acute lung injury as indicated as accumulative mortality of mice, inflammatory cellular infiltrate and interstitial edema, thickening of the alveolar walls in mice lung tissues, increased inflammatory cytokines and chemokines, increased W/D ratio in mice. Moreover, autophagy mediated inflammatory responses through Akt-mTOR, NF-κB and MAPKs signaling pathways. Our data showed that autophagy was essential in H9N2 influenza virus-triggered inflammatory responses, and autophagy could be target to treat influenza virus-caused lung inflammation.

Four genetic variants interact to confer susceptibility to atopic dermatitis in Chinese Han population.

Atopic dermatitis is a chronic inflammatory skin disease and is affected by environmental and genetic factors. Gene-gene/environment interactions are strongly believed to contribute to the genetic risk of common diseases. A number of gene-environment interactions of atopic dermatitis were performed. However, there are few comprehensive investigations on the gene-gene (or genetic variants) interactions for atopic dermatitis. We explored the association model of 6 single nucleotide polymorphisms (SNPs) which were most significant (P < 10E-05) in our previous genome wide association study (GWAS) for atopic dermatitis, and search for the possible genetic variant interactions based on the previous GWAS data using Generalized Multifactor Dimensionality Reduction and Plink 1.07 in the combined sample of 4,636 cases and 13,559 controls. The most significant associated evidence was observed under dominant model for SNPs rs3126085, rs12085366, and rs7701890, recessive model for SNP rs17173197, and additive model for SNPs rs2393903 and rs6010620. Three significant pair-way interactions were observed, including PRKAG2 and FLG SNPs (rs17173197 × rs3126085, P combined = 1.11E-15), PRKAG2 and TMEM232-SLC25A46 SNPs (rs17173197 × rs7701890, P combined = 2.22E-15), PRKAG2 and TNFRSF6B-ZGPAT SNPs (rs17173197 × rs6010620, P combined = 6.66E-16). Besides, a three-way significant interaction among PRKAG2, TMEM232-SLC25A46 and TNFRSF6B-ZGPAT SNPs (rs17173197 × rs7701890 × rs6010620, P combined = 5.99E-15) was observed in this study. These four genetic variant interactions confer susceptibility to atopic dermatitis, and highlight the genetic variant interactions in the etiology of atopic dermatitis in Chinese Han population.