[Quantitative analysis of lipid metabolism-related differential proteins in patients with alcoholic liver disease].

Objective: To screen and identify differential proteins, analyze lipid metabolism-related proteins and pathways, and explore their functions and biological processes in liver tissue of patients with alcoholic liver disease using tandem mass tag (TMT) labeling technology. Methods: Liver tissues that met the inclusion criteria were collected. Eight samples from patients with alcoholic cirrhosis and three samples from the normal control group were screened out. The TMT technique was used to screen differential proteins, perform signaling pathway enrichment analysis, and analyze protein interaction networks to explore the biological processes involved in them. Results: Proteomic analysis identified 2 741 kinds of differentially expressed proteins in the two groups of data with statistical significance (P < 0.05). The standard criteria of P < 0.05 and |log2(foldchange)| > 1 had screened out 106 kinds of differentially expressed proteins. Compared with the control group, the alcoholic liver disease group had 12 kinds of up-regulated proteins and 94 kinds of down-regulated proteins. Among them, there were 2 kinds of up-regulated differential proteins related to lipid metabolism and 14 kinds of down-regulated differential proteins. The results of bioinformatics analysis showed that these proteins were primarily involved in biological processes such as lipid transport, regulation of lipase activity, fatty acid binding, and cholesterol metabolism in lipid metabolism and also had a close link to signal pathways related to lipid metabolism such as peroxisome proliferator-activated receptor signaling pathways, cholesterol metabolism, triglyceride metabolism, and regulation of lipolysis in adipocytes. Conclusion: The 16 kinds of lipid metabolism-related differential proteins may be the key proteins in the pathogenesis of alcoholic liver disease.

[Sorafenib regulates vascular endothelial growth factor by runt-related transcription factor-3 to inhibit angiogenesis in hepatocellular carcinoma].

Objective: To investigate the molecular mechanism of sorafenib against hepatocellular carcinoma. Methods: Sorafenib efficacy was screened and verified by the hepatocellular carcinoma patient-derived tumor xenograft (PDX) model. Veterinary B-mode ultrasonography and in vivo confocal laser scanning microscopy were used to observe PDX angiogenesis. Immunohistochemistry was used to observe the expression of proliferation and angiogenesis-related proteins in PDX tissue. Real-time quantitative PCR technology was used to observe the RUNX3 gene in PDX tissues. SPSS 17.0 statistical software was used for statistical analysis. Results: Four cases of PDX were used to screen the efficacy of sorafenib. PDX1 had a significant response to sorafenib, with an inhibition rate of 68.07%. Compared with the control group, sorafenib had significantly inhibited PDX1 relative tumor volume (5.76±2.14 vs. 11.71±2.87, P<0.05). Cell division index (39.50±7.72 vs. 67.10±9.14, P<0.05) and Ki67 expression (288.6±43.40 vs. 531.70±55.60, P<0.05) were significantly decreased. Veterinary B-mode ultrasonography showed evident blood flow signals in PDX1 tumors. In vivo confocal laser scanning microscopy results showed that sorafenib had significantly reduced the total vessel length (1573.00±236.21 vs. 2675.03±162.00, P<0.05) and area (11 145.33±1931.97 vs. 20 105.37±885.93, P<0.05)) of PDX1 tumors. Immunohistochemical results showed that sorafenib had significantly down-regulated the protein expressions of CD34 (27.55±3.76 vs. 45.47±5.57, P<0.05), VEGF (16.33±2.86 vs. 22.77±3.20, P<0.05) and MVD (38.75±6.01 vs. 55.50±8.61, P<0.05). Real-time PCR results showed that sorafenib had significantly up-regulated RUNX3 gene expression (2.14±0.71 vs. 1.00±0.36, P<0.05). However, there was a negative correlation between the expression of RUNX3 gene and the ratio of VEGF-positive cells in sorafenib group (R2=0.509 7). Conclusion: Sorafenib may inhibit the PDX angiogenesis and the growth of hepatocellular carcinoma by regulating the RUNX3-VEGF pathway.

[The safety of atropine for myopia prevention and control].

Atropine is a classical drug with a wide use in clinical practice. In ophthalmology, atropine can be used for cycloplegia before optometry, and the treatment of amblyopia, iridocyclitis, malignant glaucoma, etc. In recent years, the "old drugs with new application " research and application of atropine for myopia prevention and control has become a hotspot and the efficacy of atropine has been preliminarily recognized. However, before the widely used in clinical, the safety of atropine draws attention. Researches concerning side effects of atropine were searched. The most common problem is photophobia due to dilated pupils, followed by poor near visual acuity, allergy and inflammation, local irritation. Other side effects include withdraw rebound, dry eyes, elevation of intraocular pressure, system reactions, photic damage and toxicity. Among them, some side effects are theoretical yet, and the long-term effects of some side reactions are not clear. Further research and exploration is needed to serve clinical evidence. At present, investigational usage for myopia prevention and control in clinical trials of atropine can be beneficial. Safety observation and efficacy evaluation are equally important in the course of application. (Chin J Ophthalmol, 2021, 57: 299-304).

[Construction of apoptosis-stimulating of p53 protein 2 gene knockout mice and its effect on diethylnitrosamine-induced liver cancer].

Objective: To construct apoptosis-stimulating of p53 protein 2 (ASPP2) gene knockout mice using diethylnitrosamine (DEN)-induced liver cancer model to study the biological functions of ASPP2. Methods: The sgRNA oligonucleotides were constructed, and ASPP2 knockout mice were prepared with the CRISPR/Cas9 system. PCR and sequencing methods were used to identify the genotypes of F0 and F1 generations and their progeny. DEN was used to induce ASPP2+/- mice to establish liver cancer model. Results: PCR and sequencing results showed that ASPP2 gene was successfully knocked out in F0 generation mice. The genotype of F1 generation mice was accorded with ASPP2+/- and had obtained stable heredity. The success rate of DEN-induced liver cancer model (7/8 and 3 / 8) of ASPP2 + /-mice obtained by self-hybridization of F1 generation was significantly higher than that of wild-type mice. Conclusion: ASPP2 knockout mice were successfully constructed based on the CRISPR/Cas9 system. The success rate of DEN-induced liver cancer model of ASPP2 knockout mice was significantly higher than that of the wild-type mice.

[Effect of Δ40p53 isoform on enhancing the pro-apoptotic function of p53 in tumor cells].

Objective: To investigate the effect of Δ40p53, an alternative spliced isoform of p53 lacking the N-ter minus, on the pro-apoptotic function of p53. Methods: The wild-type p53 was ectopically expressed in HCT116-p53(-/-) (endogenous Δ40p53 expression), HCT116-p53(+ /+) (wild-type p53) and H1299 (p53-null) cells by adenoviral delivery, while Δ40p53 plasmid were transfected into these cells to overexpress Δ40p53. The levels of Δ40p53 and p53 mRNA were detected by reverse transcription-polymerase chain reaction (RT-PCR) and quantitative PCR. The expression of related proteins was deter mined by Western blotting. The interaction of p53 and Δ40p53 was observed by co-immunoprecipitation assay. Calcein-AM/propidium iodide (PI) staining and flow cytometry were used to detect the apoptotic rate of tested cells in each group. Results: HCT116-p53(-/-) cells expressed endogenous Δ40p53 isoform. Neither transcription nor protein expression of wild-type p53 was interfered by the increased expression of Δ40p53. Full length p53 and Δ40p53 could bind to each other. Calcein-AM/PI staining showed that the apoptotic rates of H1299-Control, HCT116-p53(-/-) -Control, H1299+ p53, HCT116-p53(-/-)+ p53, H1299+ oxaliplatin (Oxa), HCT116-p53(-/-)+ Oxa, H1299+ p53+ Oxa and HCT116-p53(-/-)+ p53+ Oxa groups were (2.50±0.47)%, (2.40±0.32)%, (5.20±0.58)%, (4.10±0.18)%, (22.40±1.73)%, (19.30±1.11)%, (29.90±1.15)% and (39.30±2.26)%, respectively. It was statistically significant between H1299+ p53+ Oxa and HCT116-p53(-/-)+ p53+ Oxa groups (t=3.721, P=0.0205). Moreover, the apoptotic rates of H1299-Control, H1299+ Δ40p53, H1299+ p53, H1299+ p53+ Δ40p53, H1299+ Oxa, H1299+ Δ40p53+ Oxa, H1299+ p53+ Oxa and H1299+ p53+ Δ40p53+ Oxa groups were (2.60±0.35)%, (2.20±0.17)%, (4.80±0.49)%, (4.90±1.10)%, (20.30±1.10)%, (19.60±1.45)%, (27.90±1.39)%, (35.20±1.43)%, respectively. Furthermore, flow cytometry assay showed that the apoptotic rates of above cells were (2.70±0.32)%, (2.20±0.24)%, (4.60±0.48)%, (3.90±0.67)%, (19.30±1.11)%, (17.70±0.66)%, (28.30±2.76)% and (37.50±1.51)%, respectively. H1299+ p53+ Δ40p53+ Oxa cells showed higher cell apoptosis than H1299+ p53+ Oxa cells (t=2.930, P=0.042). Conclusion: Δ40p53 isoform can bind to full-length p53, and enhance its pro-apoptotic function in tumor cells.

[Protective effect of glycogen synthase kinase 3ß inhibition via peroxisome proliferator-activated receptor alpha activation in mice with acute liver failure].

Objective: To investigate the role of the glycogen synthase kinase 3ß (GSK3ß) and the peroxisome proliferator-activated receptor alpha (PPARα) signaling pathway in acute liver failure and related mechanisms in a mouse model of acute liver failure induced by D-galactosamine/lipopolysaccharide (D-GalN/LPS). Methods: C57BL/6 mice were given intraperitoneal injection of D-GalN/LPS to establish a mouse model of acute liver failure. SB216763 was used to inhibit the activity of GSK3ß and PPARα siRNA was used to inhibit the expression of PPARα. Western blotting was used to measure the expression of PPARα protein. The changes in liver pathology were observed to evaluate liver injury, and the serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were measured to assess liver function. Quantitative real-time PCR was used to measure the mRNA expression of tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß), interleukin-12p40 (IL-12p40), and PPARα. A one-way analysis of variance was used for comparison of means between multiple groups; the least significant difference test was used for data with homogeneity of variance, and the Games-Howell method was used for data with heterogeneity of variance. Results: In the mice with liver failure induced by D-GalN/LPS, GSK3ß inhibition promoted the mRNA and protein expression of PPARα (F = 13.18 and 301.36, P = 0.00 and 0.00). In the mice with acute liver failure induced by D-GalN/LPS, GSK3ß inhibition alleviated liver bleeding, inflammation, and necrosis and reduced the serum levels of ALT (F = 25.16, P = 0.000) and AST (F = 12.96, P = 0.001), as well as the mRNA expression of TNF-α (F = 32.17, P = 0.00), IL-1ß (F = 11.57, P = 0.005), and IL-12p40 (F = 14.17, P = 0.015) in liver tissue. The inhibition of PPARα expression reversed the liver-protecting effect of GSK3ß inhibition, which manifested as aggravation in liver bleeding, inflammation, and necrosis, increases in the serum levels of ALT (F = 25.16, P = 0.001) and AST (F = 12.96, P = 0.000), and an increase in the mRNA expression of TNF-α (F = 32.17, P = 0.00), IL-1ß (F = 11.57, P = 0.024), and IL-12p40 (F = 14.17, P = 0.001) in liver tissue. Conclusion: In mice with acute liver failure induced by D-GalN/LPS, the GSK3ß-PPARα-inflammatory factor signaling pathway may play an important role. GSK3ß inhibition has a protective effect in mice with acute liver failure possibly by activating the inhibitory inflammatory factor of PPARα.

Serum Proteomics Analysis in Rats of Immunosuppression Induced by Chronic Stress.

The immune system can be damaged by chronic stress. However, for this process, the involved molecular alterations and their regulatory roles played in immunosuppression still remain unclear. This study was aimed to identify the differences in serum protein expressions that are closely associated with the effect of chronic stress on immune function. Serum protein levels of rats in control group and chronic stress group were measured by iTRAQ analysis. Subsequently, among the 121 differentially expressed proteins screened between the two groups, 46 proteins were upregulated (>1.5-fold, P < 0.05), while 75 proteins were downregulated (<0.67-fold, P < 0.05). Bioinformatics analysis revealed that most of the differentially expressed proteins were in relation with the metabolic, cellular, response stimulus and immune system processes. The significantly differential expression of ceruloplasmin, haptoglobin, catalase and peroxiredoxin-1 were picked out for reconfirmation by ELISA analysis. The results were consistent with those obtained by iTRAQ. What is more, the roles of above-mentioned four proteins, apolipoprotein B-100 and heat-shock protein 90 in immunosuppression induced by chronic stress were discussed. Taken together, these findings may provide a new insight into better understanding the molecular mechanisms of immunosuppression induced by chronic stress.

[Expression pattern of beta-amyloid in the peri-infarct area and ipsilateral thalamus of rats after focal cerebral cortex infarction].

Objective: To investigate the expression pattern of beta-amyloid (Aß) in rats after focal cerebral cortex infarction, and to identify whether the Aß expression in the ipsilateral thalamus was directly related to focal cerebral ischemia. Methods: The distal middle cerebral artery occlusion (MCAO) was performed by electrocoagulation in rats. The rats were divided randomly into sham group (n=18) and MCAO group (n=30) . We used 2, 3, 5-triphenyltetrazolium chloride (TTC) staining and immunohistochemical staining to detect the location of cerebral infarction and Aß expression, respectively. Results: TTC staining showed that the cerebral infarction was consistently restricted to the frontal and temporoparietal cortex. In the peri-infarct area of the MCAO group, Aß expression began at day 2, reached the maximum level at day 7, and disappeared almost completely at day 28 after MCAO. The Aß appeared as diffuse small dots, and was located in neurons and astrocytes at day 2 and day 28, respectively. Meanwhile, in the ipsilateral thalamus, Aß expression began at day 3, increased markedly at day 7, and remained until day 28 after MCAO. The Aß was located constantly in the extracellular region of the ipsilateral thalamus, and aggregated gradually from small dots to dense plaque-like deposits with the time of ischemia. Conclusions: There are dynamic changes of Aß expression in both the peri-infarct area and the ipsilateral thalamus following MCAO. The Aß expression in the ipsilateral thalamus is not directly related to focal cerebral ischemia.

[Clinical analysis of laparoscopic surgery in patients with maintenance hemodialysis and colon cancer].

OBJECTIVE: To explore the safety of laparoscopic assisted radical resection of colon cancer in uremic patients. METHODS: Retrospective analysis of Zhejiang Jinhua Guangfu hospital 2012 March to 2015 March 5 cases of uremia patients complicated with colon cancer underwent laparoscopic assisted colorectal cancer radical resection, and compared with 10 cases, observing two groups of operative time, intraoperative bleeding volume, postoperative drainage volume, lymph nodes dissect, anal exhaust time, postoperative complications, postoperative hospitalization time and postoperative pathological staging. RESULTS: Before operation two groups of age, gender, tumor location was no significant difference (P>0.05). In preoperative comorbidities uremic patients were significantly more than that of ordinary patients, mainly in cardiovascular complications, and patients in the two groups in operation time, intraoperative bleeding, post operative drainage volume, lymph node dissection numbers were no significant difference, the postoperative gastrointestinal function recovery was almost no difference, but postoperative hospitalization time significantly prolonged (P<0.05). CONCLUSIONS: Patients with uremia as long as full preoperative preparation, perioperative management, plus timely hemodialysis, uremia patients undergoing laparoscopic assisted radical surgery is safe and feasible, and it is worth promoting.

[Role of endoplasmic reticulum stress-mediated glycogen synthase kinase 3ß activity in pathogenesis of acute liver failure in mice].

Objective: To study the role of endoplasmic reticulum (ER) stress-mediated glycogen synthase kinase 3ß (GSK3ß) activity in the pathological process of liver injury in acute liver failure (ALF) mice. Methods: ALF model was established by intraperitoneal injection of D-galactosamine/lipopolysaccharide (D-GalN/LPS) in C57BL/6 mice. The mice were divided into control group (n=10), ALF model group (n=18), 4-phenylbutyrate (4-PBA, an ER stress inhibitor) group (n=18) and SB216763 (a specific inhibitor of GSK3ß) group (n=16). The serum alanine transaminase (ALT) and aspartate transaminase (AST) levels were measured to reflect the liver function, liver injury was assessed by observing pathological changes of liver tissue, the levels of proteins in liver tissue were analyzed by Western blotting, the activity of GSK3ß in liver tissue was detected using GSK3ß activity assay kit, and the survival rate of hepatocyte was measured by methyl thiazolyl tetrazolium (MTT) assay. Results: In in vivo experiment, the expression levels of ER stress markers, glucose-regulated protein 78 (GRP78) and CCAAT/enhancer-binding protein homologous protein (CHOP), were upregulated during the progression of D-GalN/LPS-induced ALF, indicating activation of severe ER stress and increased activity of GSK3ß. Compared with the model group, inhibition of ER stress by 4-PBA improved liver function[ALT: (365.4±58.6) U/L vs (1 094.5±201.5) U/L, P<0.05; AST: (555.1±60.8) U/L vs (1 444.3±533.7) U/L, P<0.05)and pathological injury, also decreased the activity of GSK3ß (2.6±0.3 vs 4.6±1.3, P<0.05). Inhibition of GSK3ß activity was shown to alleviate liver injury in ALF by reducing the expression levels of GRP78 and CHOP. The in vitro experiment of tunicamycin-induced hepatocyte apoptosis showed that inhibition of GSK3ß activity increased the cell survival rate. Conclusion: In ALF induced by D-GalN/LPS, severe ER stress may accelerate the development and progress of ALF by upregulating the activity of GSK3ß.

[Role of autophagy in acute liver failure induced by D-galactosamine/lipopolysaccharide in mice].

Objective: To investigate the expression and role of autophagy in the progression of acute liver failure (ALF) using the mouse model of ALF induced by D-galactosamine/LPS (D-GalN/LPS). Methods: The C57BL/6 mice were used, and intraperitoneal injection of D-galactosamine (D-GalN) and lipopolysaccharide (LPS) was performed to establish the mouse model of ALF. The mice were divided into control group and 2-, 4-, and 6-hour D-GalN/LPS-induced ALF model groups. The serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were measured to assess liver function, and the pathological changes in liver tissue were observed to evaluate the status of liver injury. Quantitative real-time PCR was used to measure the expression of autophagy-related genes, Western blot was used to measure the expression of autophagy-related proteins in liver tissue, and a fluorescence microscope was used to observe the expression of autophagosome in the progression of liver failure. A one-way ANOVA was used for comparison of means of multiple samples between any two groups (LSD-t test for data with homogeneity of variance and Games-Howell method for data with heterogeneity of variance).P< 0.05 was considered statistically significant. Results: The ALF model groups showed gradual liver impairment over the time of D-GalN/LPS stimulation. There were significant increases in ALT and AST after 4 hours; the pathological injury of liver tissue gradually aggravated over the time of D-GalN/LPS stimulation and fulfilled the criteria for ALF at 6 hours. The mRNA and protein expression of autophagy-related genes (ATG-7, ATG-5, Beclin-1, Lamp-1, and LC3a) increased in the early and medium stages of ALF (2 and 4 hours) and decreased after ALF progressed to liver failure (6 hours). As was observed via the fluorescence microscope, the 4-hour D-GalN/LPS-induced ALF model group showed the highest expression of autophagosome. Conclusion: The expression of autophagy gradually increases in the early and medium stages of ALF and decreases when ALF progresses to liver failure. Therefore, autophagy plays an important role in the pathogenesis of ALF.

[Role of glycogen synthase kinase-3ß in development of severe hepatitis liver failure caused by hepatitis B virus].

OBJECTIVE: To investigate the role of glycogen synthase kinase-3ß (GSK3ß) in the development of severe hepatitis liver failure (SHLF) caused by the hepatitis B virus. METHODS: Twelve patients with chronic hepatitis B (CHB) (CHB group), 12 patients with SHLF caused by hepatitis B virus (SHLF group), and 8 normal subjects (control group), who were admitted to Beijing You'an Hospital from January 2009 to December 2011, were included in this study. Their liver tissues were collected to do some clinical examinations. The GSK3ß activity in the liver tissue was detected with a GSK3ß activity assay kit. Western blot was used to determine the expression of p-GSK3, total GSK3, and -actin. The paraffin sections of liver tissue were prepared for immunofluorescence assay. All data were expressed as mean±standard deviation, and comparison between groups was made by least significant difference t test. P < 0.05 was considered statistically significant. RESULTS: Western blot results showed that compared with the control group, the CHB group had a higher level of p-GSK3ß and the SHLF group had a significantly lower level of p-GSK3ß (P = 0.0342). The immunofluorescence assay results showed that the SHLF group had a significantly lower level of p-GSK3ß than the control group. GSK3ß activity assay results showed that compared with the control group, the CHB group had a significantly lower GSK3ß activity and the SHLF group had a significantly higher GSK3ß activity (P = 0.0289), which were consistent with the results of Western blot and immunofluorescence assay. CONCLUSION: GSK3 is activated in the development of SHLF, so it is an important signaling molecule in the pathogenesis of SHLF. Inhibiting its activity may play a role in the prevention and treatment of SHLF.

Genetic structure and genetic diversity of single-variety Lonicera macranthoides populations in China, as indicated by SCoT markers.

Lonicera macranthoides is an important traditional Chinese herb. The lack of information regarding the genetic structure and genetic relationships among its cultivars has hindered the conservation and utilization of this resource. This study used start codon targeted markers to assess the genetic diversity and other genetic characteristics of five single-variety L. macranthoides populations in China. Using 22 primers produced a total of 266 bands, of which 227 were polymorphic, indicating a high level of polymorphism. At the species level, genetic diversity was high: percentage of polymorphic loci (PPB) = 85.34%, effective number of alleles (NE) = 1.3479, Nei's gene diversity (H) = 0.2075, and Shannon's information index (Hsp, species level) = 0.3198. However, at the varietal population level, genetic diversity was lower, with averages of: PPB = 19.74%, NE = 1.0946, H = 0.0561, Hpop = 0.0850 (population level). Nei's genetic differentiation coefficient was 0.7319, which is consistent with Shannon's population genetic differentiation coefficient (0.7324). This indicates that most of the genetic variation in this species exists among the varietal populations. The differentiation among varieties may have been caused by artificial selection, mode of reproduction, and barriers to gene flow (0.1831). The genetic similarity coefficient ranged from 0.7222 to 0.9419. Phylogenetic analysis showed the five varieties to form two major clades. Results suggest that cultivar breeders should strengthen the exchange of germplasm and increase the mutual penetration of useful genes, which would broaden the hereditary basis of L. macranthoides.

Genetic characterization of red-colored heartwood genotypes of Chinese fir using simple sequence repeat (SSR) markers.

The present study investigated the genetic characterization of red-colored heartwood Chinese fir [Cunninghamia lanceolata (Lamb.) Hook.] in Guangxi using 21 simple sequence repeat (SSR) markers and analyzes of the genetic variation (N = 149) in samples obtained from five sites in Guangxi Province, China. The number of different alleles and the Shannon's information index per locus ranged from 3 to 12 and from 0.398 to 2.258 with average values of 6 and 1.211, respectively, indicating moderate levels of genetic diversity within this germplasm collection. The observed and expected heterozygosity ranged from 0.199 to 0.827 and from 0.198 to 0.878 with an average of 0.562 and 0.584, respectively. Although, the mean fixation index was 0.044, indicative of a low level of genetic differentiation among germplasms, analysis of molecular variance revealed considerable differentiation (99%) within the samples. The neighbor-joining dendrogram revealed that the majority of red-colored Chinese fir genotypes were apparently not associated with their geographic origins. Further analysis by STRUCTURE showed that this Guangxi germplasm collection could be divided into three genetic groups comprising 76, 37, and 36 members, respectively; these were classified into mixed groups with no obvious population structure. These results were consistent with those of the cluster analysis. On the whole, our data provide a starting point for the management and conservation of the current Guangxi germplasm collection as well as for their efficient use in Chinese fir-breeding programs.

Genetic diversity and population structure of wild Dipsacus asperoides in China as indicated by ISSR markers.

In order to evaluate the genetic diversity and genetic structure of wild Dipsacus asperoides, we surveyed genetic polymorphisms in 288 individuals from 12 populations using ISSR. A total of 240 bands were amplified, among which 190 were polymorphic loci. At the species level, genetic diversity was found to be abundant: PPB = 79.17%, NE = 1.2152, H = 0.1361, and Hsp = 0.2213. At the population level, genetic diversity was lower: PPB = 30.76 %, NE = 1.0786, H = 0.0897, and Hpop = 0.1375. The calculated Nei genetic differentiation coefficient was 0.3406, which is consistent with the calculated Shannon population genetic differentiation coefficient of 0.3787. This is commonly taken to indicate that most of the genetic variation existed within the populations. Gene flow was calculated as Nm = 0.9679, suggesting that gene exchange only occurred at a low level. Based on the Nei genetic distance, the 12 populations were divided into 4 categories. Our results suggest that D. asperoides has abundant genetic diversity and provides a foundation for the protection and improvement of germplasm resources.

Clinical features of diabetes mellitus cases complicated by Burkholderia pseudomallei septicemia.

The aim of this study was to analyze the clinical characteristics of diabetes mellitus patients with Burkholderia pseudomallei septicemia and evaluate strategies of diagnosis and treatment. The clinical characteristics, diagnosis, treatment, and prognosis of 39 diabetes mellitus patients with B. pseudomallei septicemia were retrospectively analyzed. Farmers, fishermen and workers were found to be high-risk groups. The clinical manifestations of patients were diverse without specific features, but mainly presented manifestations of acute fulminant septicemia, diabetic ketoacidosis, and abscesses in tissues or/and organs. Patients showed high mortality and misdiagnosis rates and were prone to relapses and long treatment duration as there are currently few effective and sensitive antibiotics for the disease. Consequently, the cost of treatment for the disease was high. Early diagnosis, a prolonged course of heavy doses of sensitive intravenous antibiotics, drainage of abscesses, intensive insulin therapy, and supportive treatment are the keys for successful management of the disease. Regular follow-ups combined with long-term blood glucose control can help reduce the disease recurrence.

Analysis of the polymorphisms in the caprine Gli3 gene and their associations with production traits in goats.

Gli3 is a zinc finger transcription factor which plays a critical role in regulating animal development, metabolism and energy partitioning and thus has the potential to influence economical important traits in farm animals. In this study, we screened the complete exons of the caprine Gli3 gene using PCR-SSCP methods in 430 individuals from three goat breeds to identify sequence variants that might be associated with growth traits. Six novel mutations (GU363952:g.739C>G, 749A>T, 1636C>A, 1982delT, 1983T>C, 2856T>C) were identified. Significant associations were observed between the mutations GU363952:g.739C>G and g.749A>T with body height, chest circumference and canon circumference. Individuals with genotype G4-CC/AA and G4-CG/AT were significantly higher than individuals with genotype G4-GG/TT in body height, chest circumference and canon circumference. The results of this study suggested that the Gli3-gene-specific SNP could be a useful marker for growth traits in future marker-assisted selection programs in goat.

Genetic diversity in wild Dipsacus chinensis populations from China based on ISSR markers.

Wild Dipsacus chinensis plants in China have become endangered owing to over-harvesting and habitat fragmentation. We examined the genetic diversity and genetic structure of 90 individuals from three populations using inter-simple sequence repeat markers and found that 106 of 173 bands amplified by 22 informative and reliable primers were polymorphic. These findings correspond to a medium level of genetic diversity. At the species level, the estimates of parameters of genetic diversity were as follows: polymorphic loci (61.27%); effective number of alleles (1.3873); Nei's genetic diversity (0.2202); Shannon's information index (0.3235). At the population level, the estimates were polymorphic loci (9.53%); effective number of alleles (1.0419); Nei's genetic diversity (0.0258); Shannon's information index (0.0402). Nei's coefficient of genetic differentiation was 0.8829, which is consistent with Shannon's coefficient of genetic differentiation (0.8757). Most of the genetic variation existed among populations, and some differentiation may have resulted from habitat fragmentation and barriers to gene flow (gene flow = 0.0663). Combining our results with those of on-site field investigation, we conclude that the present genetic diversity and genetic structure of natural populations of D. chinensis have been strongly affected by harvesting and habitat fragmentation. We also propose strategies for the conservation of this plant.

Molecular characterization and polymorphisms of the caprine Somatostatin (SST) and SST Receptor 1 (SSTR1) genes that are linked with growth traits.

Somatostatin (SST) and its receptors (SSTR1-5) appear to be important in central regulation of many metabolic systems that affect growth, adiposity and nutrient absorption. In this study, we investigated polymorphisms within the caprine SST and SSTR1 genes and determined their relationship with growth traits. As there were no sequence information of the caprine SST and SSTR1 genes, we explored their DNA sequence and genomic organizations. The caprine SST gene is organized in two exons and is transcribed into an mRNA containing 351 bp of sequence coding for a protein of 116 amino acids. Its protein sequences showed substantial similarity (97-99%) to its respective orthologs from cattle, human and mouse. We also cloned and sequenced a 1.2 kb DNA fragment which contained the major part of the coding region and 3' UTR of the caprine SSTR1 gene. We then detected the polymorphisms in these determined sequences by PCR-SSCP and DNA sequencing methods in 459 goats from four breeds. Four SNPs (GU014693:g.647T>C, GU014693:g.844A>C, GU014693:g.970T>C, GU014693:g.1039T>A), segregating as two haplotypes (T-A-T-T and C-C-C-A), were identified in intron 1 of the caprine SST gene and showed the associations to body length and body height (P < 0.05). Two SNPs (GU014695:g.801 C>T, GU014695:g.948 C>T) were identified in the caprine SSTR1 gene. Significant associations between the three genotypes of GU014695:801 C>T and body length, body height, and chest circumference was observed (P < 0.05). These results suggest that the caprine SST and SSTR1 genes are strong candidate genes that influence growth traits in goat.