Novel Reassortant Highly Pathogenic H5N2 Avian Influenza Viruses in Poultry in China

There has been multiple evidence that domestic poultry may act as a vessel for the generation of novel influenza A viruses. In this study, we have analyzed the evolution and pathogenicity of 4 H5N2 avian influenza viruses isolated from apparently healthy poultry from H5N1 virus endemic areas in China. Phylogenetic analysis revealed that two of these viruses, A/duck/Eastern China/1111/2011 (DK/EC/1111/11) and A/goose/Eastern China/1112/2011 (GS/EC/1112/11) were derived from reassortment events in which clade 2.3.4 highly pathogenic avian influenza (HPAI) H5N1 viruses acquired novel neuraminidase and nonstructural protein genes. Another two isolates, A/chicken/Hebei/1102/2010 (CK/HB/1102/10) and A/duck/Hebei/0908/2009 (DK/HB/0908/09), possess hemagglutinin (HA) gene belong to clade 7 H5 viruses and other genes from endemic H9N2 viruses, or from viruses of various subtypes of the natural gene pool. All of these H5N2 isolates bear characteristic sequences of HPAI virus at the cleavage site of HA, and animal experiments indicated that all of these viruses but DK/HB/0908/09 is highly pathogenic to chickens. In particular, DK/EC/1111/11 and GS/EC/1112/11 are also highly pathogenic to ducks and moderately pathogenic to mice. All of these 4 viruses were able to replicate in domestic ducks and mice without prior adaptation. The emergence of these novel H5N2 viruses adds more evidence for the active evolution of H5 viruses in Asia. The maintenance of the highly pathogenic phenotype of some of these viruses even after reassortment with a new NA subtypes, their ability to replicate and transmit in domestic poultry, and the pathogenicity in the mammalian mouse model, highlight the potential threat posed by these viruses to both veterinary and public health.     

Dianyuea gen. nov. (Salicaceae: Scyphostegioideae) from southwestern China

Dianyuea C. Shang, S. Liao & Z. X. Zhang, a new monotypic genus of Salicaceae based on Flacourtia turbinata H. J. Dong & H. Peng, is described and illustrated. Morphologically, Dianyuea differs from Flacourtia Comm. ex L'Hér. by having six connate stamens, basal placentation and lobed seed appendages. All those features indicate that Dianyuea is allied with Scyphostegia Stapf. A molecular phylogenetic analysis using plastid trnL‐F, matK, and rbcL sequence data for representatives of 16 genera in Salicaceae s.l. shows that Dianyuea is sister to Scyphostegia. The new combination Dianyuea turbinata (H. J. Dong & H. Peng) C. Shang, S. Liao & Z. X. Zhang is proposed.     

RECK Gene Polymorphisms Influence NSCLC Susceptibility,but not the Chemotherapy Response Status in Chinese Cohort

To test the possible association between reversion-inducing cysteine-rich protein with Kazal motifs (RECK) genetic variants and susceptibility as well as the chemotherapy response status to in patients with advanced non-small cell lung cancer (NSCLC). We recruited 304 patients who were histologically diagnosed as advanced NSCLC (IIIa, IIIb, and IV stage) in our hospital from September 2003 to January 2008. We also enrolled 409 sex- and age-matched healthy volunteers as controls. RECK Gene Polymorphisms were determined. Only the genotype distributions and allele frequencies of rs10814325 T>C were significantly different between NSCLC and controls (both P < 0.001). By multivariate analyses, markedly higher risk for NSCLC was observed in rs10814325 CC genotype (adjusted OR = 2.302, P = 0.012, with TT as reference) after adjustment with age, sex, smoking status, histology, differentiation, and stage. Haplotypes analyses showed that the A_rs11788747-G_rs16932912-C_rs10814325 and A_rs11788747-A_rs16932912A-C_rs10814325 were associated with higher risk for NSCLC; however, G_rs11788747-G_rs16932912-T_rs10814325 and G_rs11788747-A_rs16932912-T_rs10814325 haplotypes showed significantly protective roles in the NSCLC risk. The genotype and the allele frequencies of RECK gene were not significantly different between chemotherapy responder and non-responders. Multivariate logistic regression analysis showed no association between the RECK polymorphism and chemotherapy response status in this study. To the best of our knowledge, this is the first study documenting the etiological role of RECK genetic polymorphisms in NSCLC.     

The screening and preliminary construction of quality mutant population for cultivar “Nipponbare” in japonica rice (Oryza sativa)

Progenies of Oryza sativa cv. Nipponbare induced with 0.4% ethyl methane sulphonate (EMS) were screened for quality mutants and the preliminary quality mutant population was constructed in present experiment. A total of 2210 materials were first screened using near infrared reflectance spectroscopy (NIRS) from which 208 quality mutants were obtained for a second screening and then yielded 73 quality mutants including amylase content (AC), gel consistency (GC), gelatinization temperature (GT), protein content (PC), rapid viscosity analysis (RVA) parameters and amino acid contents. The screening yielded 11 PC mutants with a mutation frequency of 4.98??, followed by 7 rice floury viscosity mutants (3.17??), 5 AC mutants (2.26??), 4 chalky mutants, GT and GC mutants (1.81??), and 2 ASV mutants (0.9??). The relative contents of 17 kinds of amino acid mutations, including 7 kinds for essential amino acids and 10 kinds for nonessential amino acids were identified. With the variation of 10% as the screening standard, mutants were obtained for lysine and leucine at 0.45?? and for valine at 4.98??, but no mutants were found for isoleucine, phenylalanine, threonine. For nonessential amino acids, mutants of glutamic (0.45??), arginine (3.62??), alanine (3.17??), serine (0.45??), glycine (0.45??), tyrosine (1.81??), proline (2.71??), and histidine (0.45??) were obtained, but none was found for aspartic, phenylalanine nor threonine. At 100% as the screening standard for methionine and cysteines, the mutation frequency of these two amino acid mutants were 0.9?? and 4.98?? respectively. Quality mutants in this preliminary library of rice could play important role in gene function and breeding of rice quality.     

Matrix metalloproteinase-9 induces cardiac fibroblast migration, collagen and cytokine secretion: inhibition by salvianolic acid B from Salvia miltiorrhiza

Cardiac fibroblasts play the key role in cardiac function and matrix metalloproteinases-9 (MMP-9) is a well known contributor to the development of myocardial remodeling. However, the direct regulation of MMP-9 on the function of cardiac fibroblasts and the underlying mechanism are far from elucidation. In the present research, recombinant protein encoding catalytic domain of MMP-9 (MMP-9 CD) was constructed and the function of neonatal cardiac fibroblasts was investigated by cell proliferation assay, migration assay, picrosirius red assay, multiplex cytokine assay and fibroblast phenotype detection. 200 nM MMP-9 CD stimulated cardiac fibroblasts migration (169.4±22.5% versus 100±0%, p<0.01), increased collagen synthesis (1.5±0.2 fold, p<0.05), up-regulated the secretion of ICAM (574.0±40.1 versus 268.5±8.6pg/ml, p<0.01), TNF-α (192.6±11.0 versus 14.4±1.8pg/ml, p<0.001), IL-6 (1500.9±70.2 versus 323.4±40.6pg/ml, p<0.001) and sVCAM-1 (30.3±4.3 versus 7.0±0.1 pg/ml, p<0.05) and down-regulated VEGF (436.5±148.9 versus 1034.3±28.1 pg/ml, p<0.05) significantly with modest effects on proliferation. Accompanying with these regulations, transition of fibroblasts to myofibroblast was confirmed by immunofluorescent stain of α-smooth muscle actin (α-SMA) with MMP-9 CD treatment. Furthermore, salvianolic acid B (SalB) inhibited the effects of MMP-9 CD significantly. In conclusion, our results provide evidence for a direct influence of MMP-9 on cardiac fibroblast migration, collagen and cytokine secretion, which can be attenuated by SalB.     

Synergistic inhibitory effects by the combination of gefitinib and genistein on NSCLC with acquired drug-resistance in vitro and in vivo

In clinical practice, most patients with non small cell lung cancer (NSCLC) who respond to tyrosine kinase inhibitors eventually progress because of an acquired resistance mutation, T790M, in epidermal growth factor receptor (EGFR). Thus, it is important to identify a new drug to reduce resistance. The aim of this study was to test whether genistein combined with gefitinib is effective against NSCLC in a cell line carrying T790M, and to clarify the underlying mechanisms. The human lung cancer cell line H1975 was used as an in vitro and in vivo model. Cells were treated with gefitinib, genistein, or a combination at a range of concentrations. Cell proliferation was calculated to assess the anticancer effects of the compounds in vitro. Flow cytometry and Western blotting were employed to determine the inhibitory effects on proliferation and the induction of apoptosis. The in vivo effects of the compounds were examined using a xenografted nude mouse model for validation. Gefitinib together with genistein enhanced both growth inhibition and apoptosis; however, the greatest synergistic effect was observed at low concentrations. p-EGFR, p-Akt, and p-mTOR expressions in vitro were reduced more by the combined use of the drugs, whereas caspase-3 and PARP activities were increased. Significantly more tumor growth inhibition was detected following combination treatment in the in vivo model. These findings suggest that genistein enhanced the antitumor effects of gefitinib in a NSCLC cell line carrying the T790M mutation. This synergistic activity may be due to increased inhibition of the downstream molecular and pro-apoptotic effects of EGFR.     

Diet Is Critical for Prolonged Glycemic Control after Short-Term Insulin Treatment in High-Fat Diet-Induced Type 2 Diabetic Male Mice

Background

Clinical studies suggest that short-term insulin treatment in new-onset type 2 diabetes (T2DM) can promote prolonged glycemic control. The purpose of this study was to establish an animal model to examine such a “legacy” effect of early insulin therapy (EIT) in long-term glycemic control in new-onset T2DM. The objective of the study was to investigate the role of diet following onset of diabetes in the favorable outcomes of EIT.

Methodology

As such, C57BL6/J male mice were fed a high-fat diet (HFD) for 21 weeks to induce diabetes and then received 4 weeks of daily insulin glargine or sham subcutaneous injections. Subsequently, mice were either kept on the HFD or switched to a low-fat diet (LFD) for 4 additional weeks.

Principal Findings

Mice fed a HFD gained significant fat mass and displayed increased leptin levels, increasing insulin resistance (poor HOMA-IR) and worse glucose tolerance test (GTT) performance in comparison to mice fed a LFD, as expected. Insulin-treated diabetic mice but maintained on the HFD demonstrated even greater weight gain and insulin resistance compared to sham-treated mice. However, insulin-treated mice switched to the LFD exhibited a better HOMA-IR compared to those mice left on a HFD. Further, between the insulin-treated and sham control mice, in spite of similar HOMA-IR values, the insulin-treated mice switched to a LFD following insulin therapy did demonstrate significantly better HOMA-B% values than sham control and insulin-treated HFD mice.

Conclusion/Interpretation

Early insulin treatment in HFD-induced T2DM in C57BL6/J mice was only beneficial in animals that were switched to a LFD after insulin treatment which may explain why a similar legacy effect in humans is achieved clinically in only a portion of cases studied, emphasizing a vital role for diet adherence in diabetes control.     

The subtype-specific molecular function of SPDEF in breast cancer and insights into prognostic significance

Breast cancer (BC) is a molecular diverse disease which becomes the most common malignancy among women worldwide. There are four BC subtypes (Luminal A, Luminal B, HER2-enriched and Basal-like) robustly established following gene expression pattern-based characterization, behave significant differences in terms of their incidence, risk factors, prognosis and therapeutic sensitivity. Thus, there is an urgent need to provide mechanism research, treatment strategies and/or prognosis evaluation based on the patient stratification of BC subtypes. The prostate-derived ETS factor SPDEF was first identified as an activator of prostate specific antigen, and then, the involvements in many aspects of BC have been proposed. However, the subtype-specific molecular function of SPDEF in BC and insights into prognostic significance have not been clearly elucidated. This study demonstrated for the first time that SPDEF may play a diversity role in the expression levels, clinicopathologic importance, biological function and prognostic evaluation in BC via bioinformatics and experimental evidence, which mainly depends on different BC subtyping. In summary, our findings would help to better understand the possible mechanisms of various BC subtypes and to find possible candidate genes for prognostic and therapeutic usage.     

Metformin promotes survivin degradation through AMPK/PKA/GSK-3β-axis in non–small cell lung cancer

Metformin, a first-line antidiabetic drug, has been reported with anticancer activities in many types of cancer. However, its molecular mechanisms remain largely unknown. As a member of inhibitor of apoptosis proteins, survivin plays an important role in the regulation of cell death. In the present study, we investigated the role of survivin in metformin-induced anticancer activity in non–small cell lung cancer in vitro. Metformin mainly induced apoptotic cell death in A549 and H460 cell lines. It remarkably suppressed the expression of survivin, decreased the stability of this protein, then promoted its proteasomal degradation. Moreover, metformin greatly suppressed protein kinase A (PKA) activity and induced its downstream glycogen synthase kinase 3β (GSK-3β) activation. PKA activators, both 8-Br-cAMP and forskolin, significantly increased the expression of survivin. Consistently both GSK-3β inhibitor LiCl and siRNA restored the expression of survivin in lung cancer cells. Furthermore, metformin induced adenosine 5′-monophosphate-activated protein kinase (AMPK) activation. Suppression of the activity of AMPK with Compound C reversed the degradation of survivin induced by metformin, and meanwhile, restored the activity of PKA and GSK-3β. These results suggest that metformin kills lung cancer cells through AMPK/PKA/GSK-3β-axis–mediated survivin degradation, providing novel insights into the anticancer effects of metformin.