The MAPK Kinase Kinase GmMEKK1 Regulates Cell Death and Defense Responses.

MAPK signaling pathways play critical roles in plant immunity. Here, we silenced multiple genes encoding MAPKs using virus-induced gene silencing mediated by Bean pod mottle virus to identify MAPK genes involved in soybean (Glycine max) immunity. Surprisingly, a strong hypersensitive response (HR) cell death was observed when soybean MAPK KINASE KINASE1 (GmMEKK1), a homolog of Arabidopsis (Arabidopsis thaliana) MEKK1, was silenced. The HR was accompanied by the overaccumulation of defense signaling molecules, salicylic acid (SA) and hydrogen peroxide. Genes involved in primary metabolism, translation/transcription, photosynthesis, and growth/development were down-regulated in GmMEKK1-silenced plants, while the expression of defense-related genes was activated. Accordingly, GmMEKK1-silenced plants were more resistant to downy mildew (Peronospora manshurica) and Soybean mosaic virus compared with control plants. Silencing GmMEKK1 reduced the activation of GmMPK6 but enhanced the activation of GmMPK3 in response to flg22 peptide. Unlike Arabidopsis MPK4, GmMPK4 was not activated by either flg22 or SA. Interestingly, transient overexpression of GmMEKK1 in Nicotiana benthamiana also induced HR. Our results indicate that GmMEKK1 plays both positive and negative roles in immunity and appears to differentially activate downstream MPKs by promoting GmMPK6 activation but suppressing GmMPK3 activation in response to flg22. The involvement of GmMPK4 kinase activity in cell death and in flg22- or SA-triggered defense responses in soybean requires further investigation.

Dual Roles of GSNOR1 in Cell Death and Immunity in Tetraploid Nicotiana tabacum.

S-nitrosoglutathione reductase 1 (GSNOR1) is the key enzyme that regulates cellular homeostasis of S-nitrosylation. Although extensively studied in Arabidopsis, the roles of GSNOR1 in tetraploid Nicotiana species have not been investigated previously. To study the function of NtGSNOR1, we knocked out two NtGSNOR1 genes simultaneously in Nicotiana tabacum using clustered regularly interspaced short palindromic repeats (CRISPR)/caspase 9 (Cas9) technology. To our surprise, spontaneous cell death occurred on the leaves of the CRISPR/Cas9 lines but not on those of the wild-type (WT) plants, suggesting that NtGSNOR1 negatively regulates cell death. The natural cell death on the CRISPR/Cas9 lines could be a result from interactions between overaccumulated nitric oxide (NO) and hydrogen peroxide (H2O2). This spontaneous cell death phenotype was not affected by knocking out two Enhanced disease susceptibility 1 genes (NtEDS11a/1b) and thus was independent of the salicylic acid (SA) pathway. Unexpectedly, we found that the NtGSNOR1a/1b knockout plants displayed a significantly (p < 0.001) enhanced resistance to paraquat-induced cell death compared to WT plants, suggesting that NtGSNOR1 functions as a positive regulator of the paraquat-induced cell death. The increased resistance to the paraquat-induced cell death of the NtGSNOR1a/1b knockout plants was correlated with the reduced level of H2O2 accumulation. Interestingly, whereas the N gene-mediated resistance to Tobacco mosaic virus (TMV) was significantly enhanced (p < 0.001), the resistance to Pseudomonas syringae pv. tomato DC3000 was significantly reduced (p < 0.01) in the NtGSNOR1a/1b knockout lines. In summary, our results indicate that NtGSNOR1 functions as both positive and negative regulator of cell death under different conditions and displays distinct effects on resistance against viral and bacterial pathogens.

Silencing GmFLS2 enhances the susceptibility of soybean to bacterial pathogen through attenuating the activation of GmMAPK signaling pathway.

The plasma membrane (PM)-localized receptor-like kinases (RLKs) play important roles in pathogen defense. One of the first cloned RLKs is the Arabidopsis receptor kinase FLAGELLIN SENSING 2 (FLS2), which specifically recognizes a conserved 22 amino acid N-terminal sequence of Pseudomonas syringae pv．tomato DC3000 (Pst) flagellin protein (flg22). Although extensively studied in Arabidopsis, the functions of RLKs in crop plants remain largely uninvestigated. To understand the roles of RLKs in soybean (Glycine max), GmFLS2 was silenced via virus induced gene silencing (VIGS) mediated by Bean pod mottle virus (BPMV). No significant morphological differences were observed between GmFLS2-silenced plants and the vector control plants. However, silencing GmFLS2 significantly enhanced the susceptibility of the soybean plants to Pseudomonas syringae pv．glycinea (Psg). Kinase activity assay showed that silencing GmFLS2 significantly reduced the phosphorylation level of GmMPK6 in response to flg22 treatment. However, reduced phosphorylation level of both GmMPK3 and GmMPK6 in response to Psg infection was observed in GmFLS2-silenced plants, implying that defense response is likely transduced through activation of the downstream GmMAPK signaling pathway upon recognition of bacterial pathogen by GmFLS2. The core peptides of flg22 from Pst and Psg were highly conserved and only 4 amino acid differences were seen at their N-termini. Interestingly, it appeared that the Psg-flg22 was more effective in activating soybean MAPKs than activating Arabidopsis MAPKs, and conversely, Pst-flg22 was more effective in activating Arabidopsis MAPKs than activating soybean MAPKs, suggesting that the cognate recognition is more potent than heterologous recognition in activating downstream signaling. Taken together, our results suggest that the function of FLS2 is conserved in immunity against bacteria pathogens across different plant species.

Time interval from the end of sperm processing to artificial intrauterine in semination with husband's sperm correlates to the rate of clinical pregnancy / 中华男科学杂志

<p><b>OBJECTIVE</b>To investigate the influence of the time interval from the end of semen processing to artificial intrauterine in semination with husband's sperm (AIH-IUI) on the rate of clinical pregnancy.</p><p><b>METHODS</b>This study involved 191 AIH-IUI cycles with the same ovulation induction protocol. After Percoll density gradient centrifugation, we divided the sperm into four groups based on the incubation time: 0-19, 20-39, 40-59, and 60-80 min, and again into another four groups according to the total progressively motile sperm count (TPMC): (0-9), (10-20), (21-30), and > 30 x 10(6). We analyzed the correlation of the clinical pregnancy rate with the time interval from the end of sperm processing to AIH-IUI and with other influencing factors, such as maternal age, infertility duration, and semen quality.</p><p><b>RESULTS</b>The rate of clinical pregnancy was significantly higher in the 20-39 min group (18.3%) than in the 0-19, 40-59, and 60-80 min groups (12.7, 11.4 and 9.1%) (all P < 0.05). The (10-20) x 10(6) group achieved a remarkably higher pregnancy rate (16.7%) than the (0-9), (21-30), and > 30 x 10(6) groups (0, 11.4, and 8.3%) (all P < 0.05). Logistic multivariate analysis showed that the rate of clinical pregnancy was decreased with the increased age of the women (OR 0.89, 95% CI 0.83-0.94) but significantly elevated in the 20-39 min group (OR 2.11, 95% CI 1.34-3.13) and of (10-20) x 10(6) group (OR 2.06, 95% CI 1.32-3.46).</p><p><b>CONCLUSION</b>The time interval from the end of sperm processing to AIH-IUI is a most significant factor influencing the rate of clinical pregnancy of AIH-IUI.</p>

A Study on Relationship between Anti -diarrhea Medicine Sales Volume and the Incidence of Diarrhea in Hangzhou / 预防医学

Objective To examine the association between anti -diarrhea medicine sales volume and the reported cases of intestinal infectious diseases in Hangzhou.Methods Sales data of three over -the -counter drugs including G dysentery Sha,berberine and Smecta from 2006 to 2010 were collected and standardize.Correlations between out -patient cases of infectious and non -infectious diarrheal from 9 hospitals and medicine sales volume were analyzed respectively.Results From 2006 to 2010,berberine had the highest standardized daily drug dose (DDD)with median 1 600.32.The peak of DDD occurred on July in year 2007,2008 and 2012;while on August in year 2006,2009 and 2010.There was positive correlation between medicine sales volume and infectious diarrhea in year 2006,2007,2008 and 2010,the correlation coefficients were 0.635,0.448,0.298 and 0.217 respectively (P <0.01).The same correlation was occurred in non -infectious diarrhea in year 2006 to 2008 as well and the total correlation coefficient of the three years was 0.419 (P <0.01).Conclusion A positive correlation has been found between the anti -diarrheal medicine sales volume and incidence of infectious and non -infectious intestinal diseases,demonstrating that pharmacy monitoring could have a positive effect on early detection of outbreaks of intestinal diseases.

A survey on eperythrozoon infection in the population from Hangzhou city, Zhejiang province / 中华流行病学杂志

Objective To understand the situation of eperythrozoon (EPE) infection in the population from Hangzhou. Methods According to the situation in Hangzhou, a questionnaire was designed to investigate the study population. Venous blood specimens from the studied objects were collected before an Improved Wright-Giemsa Fast Dyeing method was used. Microscopic examination was applied to test their condition of infection. SPSS 13.0 software was applied for statistical analysis.Results Totally, 580 persons were under investigation in this study, including 111 with jobs ( 'occupational' ) and another 118 served as internal controls of them. The rest 351 were considered as external controls. Finally, 54 people were detected as having EPE infection with a total positive rate as 9.31%. The infection rates were 20.72%(23/111), 7.12%(25/351) and 5.08%(6/118) among the 'occupational' population, external controls and the internal controls, respectively. The difference among these three rates was statistically significant (χ2=21.60, P<0.05). There was also significant difference found in the infection rate between people who washed their hands promptly after being exposed to animal coat, raw meat or animal excrements and those who did not wash their hands in time. The infection rate of the population who washed hands with soap or other cleaning products was lower than that of those who washed hands with only water (χ2=6.76, P<0.05). We found that residential area, pet feeding, exposure to animal coat/raw meat/animal excrement, trauma were not risk factors of EPE infection. People with higher education degree had lower risk to EPE infection than those with low education. The infection rate was not different between the populations with different eating habits. The Improved Wright-Giemsa Fast Dyeing method used in this study was good in detecting the positive rates and easy to handle. Conclusion The risk factors to EPE infection were livestock contacting, washing their hands not promptly or washing hands without soap or other cleaning products after contacting.

Effect of ginsenoside Rg1 on the microenvironment dependent differentiation of human bone marrow mesenchymal stem cell to vaso-endothelioid formative cells in vitro / 中国中西医结合杂志

<p><b>OBJECTIVE</b>To investigate the effect of ginsenoside Rg1 on the microenvironment dependent differentiation of human mesenchymal stem cells (hMSCs) to vaso-endothelioid cells (VECs) in vitro.</p><p><b>METHODS</b>The in vitro differentiation of hMSCs to VECs were established adopting the in vivo environment simulated semi-permeable membrane separated non-contact co-culturing method. The mRNA expressions of endothelial markers, such as platelet endothelial adhesive factor-1 (CD31), vascular hemophillia factor (vWF) and vascular endothelial cadherin (VE-cadherin) were analyzed by RT-PCR; the protein expressions of CD31 and vascular endothelial adhesive factor-1 (VCAM1) were detected by fluorescence immunohistochemistry; structural identification for the endothelial characteristics of differentiated hMSCs were made under electron microscopy; and the percentage of CD31 expression in differentiated hMSCs was determined by flow cytometry to explore the effect of ginsenoside Rg1 on the differentiation.</p><p><b>RESULTS</b>The bone marrow mesenchymal stem cells co-cultured with mature endothelial membrane showed a microenvironment dependent capacity for differentiating to endothelium, with the morphological changes revealed starting from the 2nd week, showing cell body contraction, polygonal-shaped change; and at the 3rd week, the markedly speedily cell proliferation with elliptic or slabstone-like change of cells. High levels of classic endothelial cell markers, such as mRNA expressions of CD31, vWF, VE-cadherin, and protein expressions of CD31 and VCAM1, were shown; the typical weibel-palade body of endothelial cell was found in the differentiated cells. Moreover, percentage of CD31 expression in the differentiated hMSCs was increased after Rg1 treatment dose-dependently.</p><p><b>CONCLUSION</b>Under the microenvironment of co-culture, hMSCs could differentiate into cells presenting the characteristics of endothelial cell in aspects of the morphology and ultrastructure of cells, as well as the gene and protein expressions of cell markers; ginsenoside Rg1 can promote the microenvironment dependent differentiation of hMSCs to VECs system in vitro.</p>

The study of early application with Dixiong Decoction (地芎汤) for non-small cell lung cancer to decrease the incidence and severity of radiation pneumonitis: A prospective, randomized clinical trial / 中国结合医学杂志

<p><b>OBJECTIVE</b>To evaluate the efficacy of compound Dixiong Decoction (地芎汤, a Chinese herbal decoction) on early prevention of radiation pneumonitis.</p><p><b>METHODS</b>Forty-six patients with non-small cell lung cancer who were planning to receive radiotherapy were randomly assigned to the treatment group treated with the compound Dixiong Decoction and the control group treated with a commonly used herbal decoction which has the effects of supplementing qi and nourishing yin, clearing heat and detoxifying at the time of radiotherapy. Primary measure was the incidence of radiation pneumonitis after radiotherapy. Secondary outcomes included Watters clinical radiographic physiologic (CRP) dyspnea score, the Radiation Therapy Oncology Group (RTOG) grading score, Karnofsky Performance Status (KPS) score, and the application of corticosteroids.</p><p><b>RESULTS</b>The incidence of radiation pneumonitis in the treatment group was 10.0%, while that in the control group was 26.3% (P=0.0032). The Watters CRP dyspnea score and RTOG grading score in the treatment group were significantly =lower than those in the control group (P<0.05). The KPS score in the treatment group was significantly higher than that in the control group (P<0.01). The dosage of corticosteroids was smaller with a shorter duration of therapy in the treatment group than that in the control group.</p><p><b>CONCLUSION</b>The early application of the Chinese herbal decoction compound Dixiong Decoction can decrease the incidence of radiation pneumonitis, reduce the injury of the lung, and improve the life quality of the patients.</p>