Effect of PI3K/AKT/HIF-1α signaling pathway on oxidative stress and apoptosis in nucleus pulposus cells.

OBJECTIVE: The aim of the study was to explore the expression of the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (PKB/AKT)/hypoxia-induced factor-1 alpha (HIF-1α) signaling pathway and the apoptosis of nucleus pulposus cells (NPCs) under different oxygen concentrations to clarify the biological characteristics of NPCs and the molecular mechanism of intervertebral disc degeneration (IVDD). MATERIALS AND METHODS: Normal and degenerated human NPCs were collected. Leibovitz's medium with 100 µmol/L CoCl2 was used to establish a hypoxic culture environment, and 100 µmol/L H2O2 was used to establish an oxidative stress culture environment. Third-generation NPCs were divided into 6 groups: normal NPCs + hypoxia, normal NPCs + normoxia, normal NPCs + oxidative stress, degenerated NPCs + hypoxia, degenerated NPCs + normoxia, and degenerated NPCs + oxidative stress. Normal NPCs + hypoxia was used as the experimental control group. Cell viability and proliferation were detected by using the Cell Counting Kit-8 (CCK-8) method. Cell apoptosis rate was assessed by flow cytometry, and expression levels of PI3K, AKT, and HIF-1α were determined by Real-Time-Polymerase Chain Reaction (RT-PCR) and Western blotting. RESULTS: The cell proliferation rate of both normal and degenerated NPCs decreased with increasing oxygen concentration. Conversely, the apoptosis rate increased as the oxygen concentration increased (p<0.05). Compared with the control group, whether the cells degenerated had a very significant impact on the apoptosis rate (p<0.001), and oxygen concentration also had a highly significant impact on both the cell proliferation rate and apoptosis rate (both p<0.001). The interaction between cell degeneration and oxygen concentration significantly affected both cell proliferation and apoptosis rates (p<0.05). Considering the expression levels of PI3K, AKT, and HIF-1α, normal NPCs had the highest levels under low oxygen concentrations, followed by oxidative stress and normoxia. In degenerated NPCs, the expression levels also decrease as the oxygen concentration increases. CONCLUSIONS: The PI3K/Akt/HIF-1α signaling pathway plays a significant role in inhibiting oxidative stress, antagonizing NPC apoptosis, and consequently delaying IVDD.

[Utilization of basic medical insurance data in cancer-related researches in China].

Objective: To explore the utilization of cancer-related data from basic medical insurance databases in China, and promote the application of medical insurance data in cancer prevention and treatment. Methods: Database PubMed, Web of Science, Wanfang, and CNKI were used to select related research papers using data from basic medical insurance system in China published by December 2021. Descriptive analysis was conducted in terms of the number of publications, types of cancer, primary research contents and author affiliations. Results: A total of 65 papers were included in the study. The number of publications increased rapidly after 2016. The most studied cancer type was lung cancer, and healthcare costs were the most common research contents. Fujian, Beijing, and Anhui have made a better use of cancer-related medical insurance databases compared to other provincial regions. The accessibility of the New Rural Cooperative Medical Scheme data was limited due to the high regional barriers, while the accessibility of the urban basic medical insurance data was relatively high. The researchers from Peking University and Fudan University had higher utilization of basic medical insurance data compared with those from other institutions. Conclusions: The utilization of cancer-related data from basic medical insurance databases in China is limited because of poor accessibility, insufficient data sharing, and regional restrictions. Thus, it is urgent to improve data accessibility and promote the integration and utilization of regional medical insurance data.

[Analysis of time for diagnosis of nontuberculous mycobacterial lung disease and its associated factors in a tuberculosis-designated hospital in Shanghai].

Objective: To investigate the pathogenic characteristics, bacteriological diagnosis time and its associated factors among patients with nontuberculous mycobacterial (NTM) lung disease in a large tuberculosis-designated hospital in Shanghai from 2020 to 2021, in order to improve diagnosis efficiency and formulate precision treatment. Methods: On the basis of the Tuberculosis Database in Shanghai Pulmonary Hospital, NTM patients diagnosed by the Department of Tuberculosis between January 2020 and December 2021 were screened. Demographic, clinical and bacterial information were retrospectively collected. Chi-square test, paired-sample nonparametric test and logistic regression model were used to analyze the factors associated with the diagnosis time of NTM lung disease. Results: A total of 294 patients with bacteriologically confirmed NTM lung disease were included in this study, 147 males and 147 females with a median age of 61(46, 69) years. Of them, 227 (77.2%) patients had comorbidity of bronchiectasis. Species identification results showed that Mycobacterium Avium-Intracellulare Complex was the main pathogen of NTM lung disease (56.1%), followed by Mycobacterium kansasii (19.0%) and Mycobacterium abscessus (15.3%). Species such as Mycobacterium xenopi and Mycobacterium malmoense were rarely identified, accounting for a total proportion of only 3.1%. Positive culture rates for sputum, bronchoalveolar lavage fluid and puncture fluid were 87.4%, 80.3% and 61.5%, respectively. Paired-sample analysis showed that the positive rate of sputum culture was significantly higher than that of smear microscopy (87.1% vs. 48.4%, P<0.01), while no statistical difference was observed between sputum and bronchoalveolar lavage fluid on positive culture rate (78.7% vs. 77.3%, P>0.05). Patients with cough or expectoration were observed with 4.04-fold (95%CI 1.80-9.05) or 2.95-fold (95%CI 1.34-6.52) higher probability of positive sputum culture, compared to those without. Regarding bronchoalveolar lavage fluid, female or patients with bronchiectasis had a 2.82-fold (95%CI 1.16-6.88) or 2.38-fold (95%CI 1.01-5.63) higher probability to achieve a positive culture. The median time to diagnosis of NTM lung disease was 32 (interquartile range: 26-42) days. The results of multivariable analysis showed that patients with symptom of expectoration (aOR=0.48, 95%CI 0.29-0.80) needed a shorter diagnosis time in comparison with patients without expectoration. With Mycobacterium Avium-Intracellulare Complex as a reference, lung disease caused by Mycobacterium abscessus needed shorter diagnosis time (aOR=0.43, 95%CI 0.21-0.88), whereas those caused by rare NTM species were observed to require a longer diagnosis time (aOR=8.31, 95%CI 1.01-68.6). Conclusion: The main pathogen causing NTM lung disease in Shanghai was Mycobacterium Avium-Intracellulare Complex. Sex, clinical symptoms and bronchiectasis had an impact on the positive rate of mycobacterial culture. The majority of patients in study hospital were timely diagnosed. Clinical symptoms and NTM species were associated with the bacteriological diagnosis time of NTM lung disease.

[Evaluation of screening strategies of multidrug-resistant tuberculosis among pulmonary tuberculosis patients of the different risk levels].

Objective: To evaluate the detection of MDR-TB and XDR-TB patients and to provide evidence for further improvement of MDR-TB and XDR-TB screening strategy. Methods: Patients who were under drug resistance surveillance, registered and reported by the TB Management Information System of the Chinese Disease Prevention and Control Information System from 2012 to 2019 and resided in Guizhou province were retrospectively analyzed. The contribution of five high-risk subgroups to detection of MDR/XDR-TB were evaluated using population attributable risk proportion (PARP). Results: Of the 18 506 cases under drug resistance surveillance, patients who were male, aged between 25 and 54 years, with drug-resistant TB or with MDR/XDR-TB accounted for 68.65% (12 705/18 506), 47.69% (8 826/18 506), 15.90% (2 943/18 506) or 5.42% (1 003/18 506), respectively. Five high-risk subgroups made significant contributions to the detection of MDR/XDR-TB with a PARP of 57.00%. Specifically, the PARP were 21.70%, 19.49%, 11.90% and 2.30% for patients that were relapse and return, failed initial treatment, chronic/retreatment failure and smear-positive at the end of the second or third month, respectively. The detection rate of MDR/XDR-TB in high-risk groups was 15.89% (578/3 637) while in low-risk groups was 2.86% (425/14 869). Conclusions: Number of patients under drug resistance surveillance and the detection of MDR/XDR-TB trended to increase in Guizhou province from 2012 to 2019. The detection rate of MDR/XDR-TB in high-risk groups was higher than low-risk groups.

Genomic origin and EGFR-TKI treatments of pulmonary adenosquamous carcinoma.

BACKGROUND: Adenosquamous carcinoma (ASC) of the lung is a heterogeneous disease that is composed of both adenocarcinoma components (ACC) and squamous cell carcinoma components (SCCC). Their genomic profile, genetic origin, and clinical management remain controversial. PATIENTS AND METHODS: Resected ASC and metastatic tumor in regional lymph nodes (LNs) were collected. The ACC and SCCC were separated by microdissection of primary tumor. The 1021 cancer-related genes were evaluated by next-generation sequencing independently in ACC and SCCC and LNs. Shared and private alterations in the two components were investigated. In addition, genomic profiles of independent cohorts of adenocarcinomas and squamous cell carcinomas were examined for comparison. We have also carried out a retrospective study of ASCs with known EGFR mutation status from 11 hospitals in China for their clinical outcomes. RESULTS: The most frequent alterations in 28 surgically resected ASCs include EGFR (79%), TP53 (68%), MAP3K1 (14%) mutations, EGFR amplifications (32%), and MDM2 amplifications (18%). Twenty-seven patients (96%) had shared variations between ACC and SCCC, and pure SCCC metastases were not found in metastatic LNs among these patients. Only one patient with geographically separated ACC and SCCC had no shared mutations. Inter-component heterogeneity was a common genetic event of ACC and SCCC. The genomic profile of ASC was similar to that of 170 adenocarcinomas, but different from that of 62 squamous cell carcinomas. The incidence of EGFR mutations in the retrospective analysis of 517 ASCs was 51.8%. Among the 129 EGFR-positive patients who received EGFR-TKIs, the objective response rate was 56.6% and the median progression-free survival was 10.1 months (95% confidence interval: 9.0-11.2). CONCLUSIONS: The ACC and SCCC share a monoclonal origin, a majority with genetically inter-component heterogeneity. ASC may represent a subtype of adenocarcinoma with EGFR mutation being the most common genomic anomaly and sharing similar efficacy to EGFR TKI.

[The study of exposure levels of dioxin-like compounds in cord blood of newborns in an e-waste dismantling area in Guangdong Province].

Objective: To study the pollution status of polybrominated diphenyl ethers (PBDEs), polychlorinated biphenyls (PCB), polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDD/Fs) in cord blood of newborns in an e-waste dismantling area of Guangdong Province. Methods: We recruited 20 eligible mothers and newborns who could meet the inclusion criteria in local hospitals of Guiyu in 2007. The inclusion criteria included directly engaged in dismantling e-waste during pregnancy and within 1 year before pregnancy; living in the e-waste dismantling workshops or the distance between living place and the e-waste dismantling areas was ≤200 m; the father of newborn was directly engaged in electronic waste dismantling for more than 1 year; the frequency of visiting the e-waste dismantling workshop during pregnancy was ≥3 times in a week. Questionnaires and physical examinations were performed on maternal and neonatal, and cord blood was collected from newborns to detect PCDD/Fs, PCB and PBDE. The concentration level of organic pollutants was corrected by the blood lipid content, and the total toxicity equivalent was calculated. The correlation between three compounds was analyzed by Spearman correlation. Results: The mothers of the 20 newborns were (23.45±3.27) years old and lived for more than 5 years. The number of one parent engaged in e-waste dismantling, the mother or father smoking, and parent engaged in e-waste dismantling work were 3, 13, 15 and 19, respectively. The weight of newborns ranged from 2.5 to 3.6 kilogram and the Apgar score was 10 points. No adverse birth outcomes such as preterm birth, malformation or stillbirth were found. The median (maximum, minimum) concentration of PCBs, PCDD/Fs and PBDEs in cord blood were 263.22 (328.29, 244.19), 38.42 (147.49, 12.68), 39.33 (265.11, 14.81) pg/g lipid, respectively. The median (maximum, minimum) of toxic equivalence concentrations of PCDD/Fs and PCB were 3.94 (9.24, 2.69) and 15.95 (26.64, 9.28) pg TEQ/g lipid. PBDE, the proportion of PBDE, PCB and PCDD/Fs in cord blood was 50.41%, 49.25% and 0.34%, respectively. PCBs and PBDEs were positively correlated (r=0.733, P=0.039). Conclusion: The high concentrations of PCDD/Fs, PCB, and PBDE were detected in the e-waste dismantling area. It is recommended that the risk of such substances on the health of local people should be assessed in a timely manner.

[Impact of informal e-waste recycling on human health].

Increasing e-waste has become a major problem for global environment and public health. In the process of dismantling and recycling of disordered electronic waste, heavy metals such as lead and brominated flame retardants and organic substances are released into environmental media such as air, soil, dust and water, which is harmful to the health of local residents. Taking an e-waste dismantling area in Guangdong Province as an example, this paper reviews exposure levels of heavy metals and organic matters in e-waste recycling areas in China, as well as the health effects of local residents. Previous studies have found that e-waste recycling activities led to serious environmental pollution and high exposure levels of heavy metals and organic matters in local residents, which has a certain impact on the physiological functions of various human systems. The establishment of a centralized dismantling zone can effectively reduce the load level of various pollutants.

[Neuronavigation-assisted percutaneous radiofrequency thermocoagulation of trigeminal gasserian ganglion for refractory craniofacial pain].

Objective: Percutaneous radiofrequency thermocoagulation (RFT) of the Gasserian ganglion is an effective treatment for refractory trigeminal craniofacial pain.In the present study, we assessed the feasibility of accessing the Gasserian ganglion through the foramen ovale with neuronavigation guidance in the patients of trigeminal craniofacial pain. Method: We retrospectively analyzed forty-four patients with type Ⅰ trigeminal neuralgia who had undergone percutaneous RFT treatment in our hospital from June 2014 to December 2016.The patients were divided into fluoroscopy group and navigation group according to the intraoperative guiding manners for foramen ovale cannulation.We compared groups in terms of the duration of the whole RFT procedure and times of intraoperative fluoroscopy.We also analyzed the immediate and late outcome accessing by Barrow Neurological Institute (BNI) pain scale, as well as the complication rates in groups. Result: There were 32 patients in the fluoroscopy group and 12 in the navigation group.The duration of the surgical procedure in navigation group was less than that in fluoroscopy group (46±12 min versus 67±16 min, P=0.00), and times of intraoperative fluoroscopy was reduced (6.3±2.2 versus 1.3±1.6, P=0.00). The learning curve of navigation-aid RFT was not steep in the present study overall.There was no significant difference between groups regarding pain reduction at the immediate (P=0.07) or late follow-up (P=0.400) time points.However, the rate of pain reduction to BNI-Ⅰ grade was greater in navigation group (P=0.026). No significant difference in the complication rate between both groups, and no serious complications were observed in the both groups. Conclusion: Neuronavigation may be encouraged in trigeminal Gasserian ganglion RFT with better operating efficiency and less radiation exposure.The immediate and late therapeutic effects for craniofacial pain control were positive, whereas further studies are necessary.

Detection and characterization of ciRS-7: a potential promoter of the development of cancer.

Circular RNAs (circRNAs) are a class of newly-identified non-coding RNA molecules. CircRNAs are conserved across different species and display specific organization, sequence, and expression in disease. Moreover, circRNAs' closed ring structure, insensitivity to RNase, and stability are advantages over linear RNAs in terms of development and application as a new kind of clinical marker. In addition, according to recent studies, circular RNA-7 (ciRS-7) acts as a sponge of miR-7 and thus inhibits its activity. Numerous evidences have confirmed expression of miR-7 is dysregulated in cancer tissues, however, whether ciRS-7 invovled in oncogenesis by acting as sponge of miR-7 remains unclear. Most recently, a study reported ciRS-7 acted as an oncogene in hepatocellular carcinoma through targeting miR-7 expression. This suggest ciRS-7/ miR-7 axis affects oncogenesis, and it provides a new perspective on the mechanisms of decreased miR-7 expression in cancer tissues. Discovery of sponge role of circRNAs caused researchers to more closely explore the underlying mechanism of carcinogenesis and has significant clinical implications, and may open a new chapter in research on the pathology and treatment of cancers. This review summarizes the structure and function of circRNAs and provides evidence for the impact of ciRS-7 in promoting the development of cancer by acting as sponge of miR-7.

[Effect of exendin-4 on lipid deposition in skeletal muscle of diet-induced obese mice and its underlying mechanism].

Objective: To investigate the effect of exendin-4, a glucagon-like peptide-1 (GLP-1) receptor agonist, on reducing lipid deposition and improving insulin resistance in skeletal muscle and the underlying mechanisms in high-fat diet (HFD)-induced obese mice. Methods: Twelve male C57BL/6J mice were challenged with HFD for 12 weeks to induce obesity and then randomly divided into two groups: exendin-4 group (intraperitoneal injection of 24 nmol·kg-1·d-1 exendin-4 for 4 weeks) and HFD group (intraperitoneal injection of normal saline for 4 weeks), with 6 mice in each group. Additional 6 mice were also selected as control group. Body weight, fasting blood glucose were recorded. Serum triglyceride (TG), total cholesterol (TC), insulin and skeletal muscle triglyceride levels were measured by enzyme-linked immunosobent assay (ELISA). Oil red O staining was used for morphologic changes of frozen sections from skeletal muscle. The protein levels of lipid metabolic pathway mediated by AMP-activated protein kinase (AMPK) and insulin signailing pathway were determined by Western blot. Results: Compared with mice in HFD group, exendin-4 significantly decreased body weight[(37.68±1.80) vs (46.03±5.00) g, P<0.025], fasting blood glucose[(5.40±0.33) vs (7.65±1.92) mmol/L, P<0.025], serum TG[(37.78±7.14) vs (80.76±34.22) mg/dl, P<0.025], TC[(180.13±18.75) vs (217.57±22.52) mg/dl, P<0.025], insulin[(0.58±0.01) vs (1.67±1.23) ng/ml, P<0.025]and skeletal muscle TG levels[(9.84±1.08) vs (19.35±7.44) mg/g, P<0.025]of obese mice. Oil red O staining revealed that exendin-4 alleviated the accumulation of larger lipid droplets in skeletal muscle. The protein expressions of lipolysis and lipid oxidation mediated by AMPK and insulin signailing pathway were up-regulated, and the protein expressions of lipogenesis mediated by AMPK were down-regulated after intervention of exendin-4. Conclusion: Exendin-4 reduces lipid deposition and insulin resistance in skeletal muscle of HFD-induced obese mice via activating AMPK and up-regulating insulin signailing pathway.

[The relationship of A Proliferation-Inducing Ligand expression with the clinical pathology and prognosis in colorectal carcinoma].

Objective: To investigate the protein expression of A Proliferation-Inducing Ligand (APRIL) in colorectal cancer (CRC) tissue, and its correlation with clinical pathology and prognosis. Methods: One hundred and twenty cases of CRC tissue and related clinical data were collected from the Department of Pathology of Affiliated Hospital of Nantong University. The specimens were made into tissue microarray. The protein expression of APRIL was detected by Envision immunohistochemistry. The relationship of the protein expression of APRIL with clinicopathological parameters and follow-up data were statistically analyzed. Results: APRIL protein expression positive rate of CRC tissue and cancer adjacent normal mucosal tissue were 83.33% (100/120) and 12.5% (15/120), with a statistical significance (P<0.001). The expression of APRIL was related to TNM staging (χ2=10.222, P=0.006) and the depth of tumor invasion (χ2=5.987, P=0.014). Kaplan-Meier survival curves showed that five years survival rate of high APRIL expression group was lower than that of low APRIL expression group. Univariate survival analysis showed that the expression of APRIL (P<0.001), the degree of tumor differentiation (P=0.033), TNM staging (P<0.001), depth of tumor invasion (P=0.011), lymph node metastasis (P<0.001) and serum CEA (P=0.003) time were associated with postoperative survival. Multivariate COX proportional hazard regression model analysis further revealed the expression of APRIL (P=0.001), the degree of tumor differentiation (P=0.005), TNM staging (P=0.001) and serum CEA (P=0.039) were the independent risk factors for prognosis. Conclusion: The expression of APRIL in CRC tumor tissue is closely related to TNM stage and depth of tumor invasion. The high expression of APRIL prompts bad prognosis. APRIL is one of the independent prognostic factors in patients with CRC, and could be a potential target for the therapy of CRC.

[Effect of SIRT1 deficiency on function of brown adipose tissue in obese mice].

OBJECTIVE: To investigate the effect of silent mating type information regulation 2 homolog 1 (SIRT1) deficiency on function of brown adipose tissue (BAT) in high-fat diet (HFD)-induced obese mice. METHODS: Male SIRT1 deficient heterozygous (SIRT1(+ /-)) mice and their wild-type (WT) littermates were challenged with a HFD diet for 16 weeks to induce obesity model.Energy metabolic cages were used to measure oxygen consumption and heat production, and cold tolerance test was to evaluate the adaptive thermogenic function.With brown fat collected after the diet intervention, determination techniques were adopted included HE staining for morphologic changes, immunohistochemical staining and Western blotting for uncoupling protein 1 (UCP1) expression, quantitative real-time PCR for relative content of mitochondria DNA (mtDNA). RESULTS: Compared to WT controls, SIRT1(+ /-) mice displayed significant decreases in both oxygen consumption and heat production[(2 681±297) vs (3 017±313) ml·kg(-1)·h(-1,) (19.05±2.40) vs (21.15±2.49) kcal·kg(-1)·h(-1,) both P<0.05)], as well as an impairment in maintaining their body temperature during the cold challenge.HE staining revealed the accumulation of larger lipid droplets in BAT of SIRT1(+ /-) mice, and both immunohistochemical staining and Western blotting indicated an obvious reduction in expression of UCP1 (P<0.05). Quantitative real-time PCR showed a significant decrease in the relative mtDNA content in BAT of SIRT1(+ /-) mice (0.38±0.10 vs 1.00±0.40, P<0.05). CONCLUSION: SIRT1 deficiency promotes BAT dysfunction, meaning that whitening in obese mice.

[The influence of patatin-like phospholipase domain-containing protein 3 on palmitic acid-induced hepatocyte apoptosis].

OBJECTIVE: To explore the influence of patatin-like phospholipase domain-containing protein 3 (PNPLA3) on palmitic acid (PA)-induced hepatocyte apoptosis and its mechanism. METHODS: Human hepatocarcinoma cell line HepG2 cells were transfected with PNPLA3(WT)-pcDNA3.1 (PNPLA3(WT) group) and PNPLA3(I148M)-pcDNA3.1 (PNPLA3(I148M) group) plasmids respectively to overexpress wild type or mutant type PNPLA3, and cells transfected with empty vector pcDNA3.1 (NC group) were set as control group.After 24 h PA incubation, Oil red staining was used to determine lipid deposition, and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling assay (TUNEL) was used to measure apoptosis.Western blot was used to detect the protein level of endoplasmic reticulum (ER) stress and associated apoptosis.Enzyme-linked immunosorbent assay (ELISA) was used to test lysolecithin (LPC) levels in the cellular supernatant. RESULTS: After 24 h PA incubation, there was no significant difference in lipid deposition among three groups.Compared to NC group, the cell apoptosis rates of PNPLA3(WT) and PNPLA3(I148M) groups were increased by 2 times and 3 times respectively.The levels of ER stress PRKR-like endoplasmic reticulum kinase (PERK) pathway associated proteins, immunoglobulin-binding protein (BIP), p-PERK, p-eIF2α, and ER stress associated apoptosis pathway proteins, CCAAT/enhancer binding homologous protein (CHOP), p53 upregulated modulator of apoptosis (PUMA), Bax, caspase-3 were higher, and were more significant in PNPLA3(I148M) group.The LPC level in the supernatant of PNPLA3(WT) and PNPLA3(I148M) groups were about 5 times and 1.5 times of NC group respectively after PA incubation. CONCLUSION: PNPLA3 may be involved in palmitic acid-induced apoptosis mediated by hepatocyte ER stress through regulating LPC metabolism.

shRNA inhibits the expression of chicken telomerase reverse transcriptase in MDCC-MSB1 cells.

Here, we investigated the effects of blocking chicken telomerase reverse transcriptase (chTERT) in MDCC-MSB1 cells, using small-hairpin RNAs (shRNAs) to interfere with gene expression. shRNAs specific to chTERT mRNA were designed, cloned into DNA plasmid vectors, and transfected into MDCC-MSB1 cells. The transfected chTERT RNAs were expressed by the RNA polymerase machinery of the MDCC-MSB1 cells. mRNA expression in transfected MDCC-MSB1 cells was detected using real-time PCR. After transfection, telomerase activity was monitored via a modified telomeric repeat amplification protocol assay, and cell cycle analysis was performed using a flow cytometer. At 72 h after transfection, chTERT expression was considerably reduced in cells transfected with shRNA; the highest inhibition rate was 89%. Compared with the control group, telomerase activity was significantly reduced and the cells failed to progress to S phase. shRNA effectively reduced telomerase activity and prohibited the transition of MDCC-MSB1 cells from G2/M to S phase.

First Report of Root Rot Caused by Phytophthora sansomeana on Soybean in China.

Phytophthora sansomeana E.M. Hansen & Reeser is a newly described species and infects Douglas-fir, alfalfa, and soybean (1). Soybean production is an important part of the local economy in Yili State in Xinjiang Uygur Autonomous Region, northwest China. Unfortunately since 2005, root and stem rot disease has emerged on a number of farms. To identify the causal agent, plant samples with symptoms, including whole plant wilting or yellowing and stunting, were collected from fields during 2005 and 2008. Tissue from the edges of stem lesions was placed on selective lima bean agar (LBA) at 20°C for 3 to 4 days (2,3). Four single zoospore isolates of Phytophthora were obtained and maintained on LBA or 10% V8 juice liquid medium for examination of morphological and physiological characteristics. The colonies on LBA were aerial and rosaceous. The isolates were homothallic, and oogonia and oospores were readily produced in culture after 7 days on LBA plates. Oogonia averaged 38 µm and oospore width ranged from 23 to 48 µm and averaged 31 µm. Antheridia were approximately 15 × 12 µm and predominantly amphigynous in V8 juice. Sporangia were terminal or paragynous on persistent sporangiophores, nonpapillate, ovoid to obpyriform, and measured 52 × 35 µm with an average length/breadth ratio of 1.5. Hyphal swellings were produced in V8 juice 2 days after inoculation. The optimum temperature for growth was approximately 25°C and none occurred at 0 or 35°C. The internal transcribed spacer (ITS) sequence of this Phytophthora species (GenBank FJ966880) agreed 100% with sequences of P. sansomeana isolates deposited in GenBank (GQ853880 and EU925375). Pathogenicity tests were performed by hypocotyl inoculation method (2) using isolate Yili71 and potted soybean cv. Williams. Plants were grown in a growth chamber for 10 days before inoculation in 16-cm-diameter pots (2). Plants were inoculated with 2- × 2-mm plugs of mycelium grown for 4 days on LBA at 25°C, the plugs were adhered to the sides of wounded lower hypocotyls. As controls, plants were inoculated with LBA agar plugs without mycelium (2). Inoculated plants were maintained in a growth chamber at approximately 25°C with a 10-h dark/14-h light cycle and 50% relative humidity and symptom development was monitored daily for 1 week. Wounded stems inoculated with mycelium developed water-soaked lesions, which were similar to those seen on naturally infected plants. A Phytophthora sp. was reisolated from the margins of expanding lesions on wounded stems. To our knowledge, this is the first report of P. sansomeana infection of soybean in China and the threat it may pose to soybean production is unclear. References: (1) E. M. Hansen et al. Mycologia 101:129. 2009. (2) Z. Y. Wang et al. Fungal Genet. Biol. 43:826, 2006. (3) X. B. Zheng. Methods in Phytophthora. Chinese Agriculture Press. Beijing, China, 1995.

Molecular mapping of two cultivar-specific avirulence genes in the rice blast fungus Magnaporthe grisea.

Rice blast, caused by the fungus Magnaporthe grisea, is a globally important disease of rice that causes annual yield losses. The segregation of genes controlling the virulence of M. grisea on rice was studied to establish the genetic basis of cultivar specificity in the interaction of rice and M. grisea. The segregation of avirulence and virulence was studied in 87 M. grisea F(1) progeny isolates from a cross of two isolates, Guy11 and JS153, using resistance-gene-differential rice cultivars. The segregation ratio indicated that avirulence and virulence in the rice cultivars Aichi-asahi and K59, respectively, are controlled by single major genes. Genetic analyses of backcrosses and full-sib crosses in these populations were also performed. The chi(2 )test of goodness-of-fitness for a 1:1 ratio indicated that one dominant gene controls avirulence in Aichi-asahi and K59 in this population. Based on the resistance reactions of rice differential lines harboring known resistance genes to the parental isolates, two genetically independent avirulence genes, AVR-Pit and AVR-Pia, were identified. Genetic linkage analysis showed that the SSR marker m355-356 is closely linked to AVR-Pit, on the telomere of chromosome 1 at a distance of approximately 2.3 cM. The RAPD marker S487, which was converted to a sequence-characterized amplified region (SCAR) marker, was found to be closely linked to AVR-Pia, on the chromosome 7 telomere at a distance of 3.5 cM. These molecular markers will facilitate the positional cloning of the two AVR genes, and can be applied to molecular-marker-assisted studies of M. grisea populations.

Genetic analysis and molecular mapping of the avirulence gene PRE1, a gene for host-species specificity in the blast fungus Magnaporthe grisea.

We analyzed host-species specificity of Magnaporthe grisea on rice using 110 F1 progeny derived from a cross between the Oryza isolate CH87 (pathogenic to rice) and the Digitaria isolate 6023 (pathogenic to crabgrass). To elucidate the genetic mechanisms controlling species specificity in M. grisea, we performed a genetic analysis of species-specific avirulence on this rice population. Avirulent and virulent progeny segregated in a 1:1 ratio on the 2 rice cultivars 'Lijiangxintuanheigu' (LTH) and 'Shin2', suggesting that a single locus, designated PRE1, was involved in the specificity. In a combination between 'Kusabue' and 'Tsuyuake', the segregation of the 4 possible phenotypes of F1 progeny was significantly different from the expected 3:1:3:1 and instead fit a ratio of 2:0:1:1. This indicated that 2 loci, PRE1 and AVR2, were involved in specific parasitism on rice. These results suggest that the species specificity of M. grisea on rice is governed by species-dependent genetic mechanisms that are similar to the gene-for-gene interactions controlling cultivar specificity. Pathogenicity tests with various plant species revealed that the Digitaria isolate 6023 was exclusively parasitic on crabgrass. Genetic linkage analysis showed that PRE1 was mapped on chromosome 3 with respect to RAPD and SSR markers. RAPD marker S361 was linked to the avirulence gene at a distance of ~6.4 cM. Two SSR markers, m677-678 and m77-78, were linked to the PRE1 gene on M. grisea chromosome 3 at distances of 5.9 and 7.1 cM, respectively. Our results will facilitate positional cloning and functional studies of this gene.

Geographical heterogeneity between Far Eastern and Western countries in prevalence of the virulence plasmid, the superantigen Yersinia pseudotuberculosis-derived mitogen, and the high-pathogenicity island among Yersinia pseudotuberculosis strains.

Yersinia pseudotuberculosis produces novel superantigenic toxins designated YPMa (Y. pseudotuberculosis-derived mitogen), YPMb, and YPMc and has a pathogenicity island termed HPI (high-pathogenicity island) and R-HPI (the right-hand part of the HPI with truncation in its left-hand part) on the chromosome. Analysis of the distribution of these virulence factors allowed for differentiation of species Y. pseudotuberculosis into six subgroups, thus reflecting the geographical spread of two main clones: the YPMa(+) HPI(-) Far Eastern systemic pathogenic type belonging to serotypes O1b, -2a, -2b, -2c, -3, -4a, -4b, -5a, -5b, -6, -10, and UT (untypeable) and the YPMs(-) HPI(+) European gastroenteric pathogenic type belonging to serotypes O1a and -1b. The YPMa(+) HPI(+) pathogenic type belonging to serotypes O1b, -3, -5a, -5b, and UT and the YPMb(+) HPI(-) nonpathogenic type belonging to non-melibiose-fermenting serotypes O1b, -5a, -5b, -6, -7, -9, -10, -11, and -12 were prevalent in the Far East. The YPMc(+) R-HPI(+) European low-pathogenicity type belonging to non-melibiose-fermenting serotype O3 and the YPMs(-) HPI(-) pathogenic type belonging to 15 serotypes were found to be prevalent all over the world. This new information is useful for a better understanding of the evolution and spread of Y. pseudotuberculosis.

Putative origin of Yersinia pseudotuberculosis in western and eastern countries. A comparison of restriction endonuclease analysis of virulence plasmids.

Yersinia pseudotuberculosis isolates from Russia east of Moscow, Korea and mainland China were used for restriction endonuclease analysis of virulence plasmid (REAP) and findings were compared with REAP of isolates from Japan and Western countries. An identical REAP pattern of each serogroup 1a, 1b, 3, 4a and 4b strain was observed among isolates from Russia, Korea, mainland China, and Japan but such was absent in West European strains. Therefore, the possibility that the origin of Y. pseudotuberculosis between West Europe and eastern Eurasia east of Moscow may be from a different clone should be considered.