Transcriptomics and metabolomics reveal improved performance of Hu sheep on hybridization with Southdown sheep.

Consumers are increasingly demanding high-quality mutton. Cross breeding can improve meat quality and is widely used in sheep breeding. However, little is known about the molecular mechanism of cross breeding sheep meat quality. In this study, male Southdown and female Hu sheep were hybridized. The slaughter performance and longissimus dorsi quality of the 6-month-old hybrid offspring were measured, and the longissimus dorsi of the hybrid offspring was analyzed by transcriptomics and metabolomics to explore the effect of cross breeding on meat quality. The results showed that the production performance of Southdown × Hu F1 sheep was significantly improved, the carcass fat content was significantly decreased, and the eating quality of Southdown × Hu F1 sheep were better. Compared with the HS group (Hu × Hu), the NH group (Southdown × Hu) had 538 differentially expressed genes and 166 differentially expressed metabolites (P < 0.05), which were significantly enriched in amino acid metabolism and other related pathways. Up-regulated genes METTL21C, PPARGC1A and down-regulated gene WFIKKN2 are related to muscle growth and development. Among them, the METTL21C gene, which is related to muscle development, was highly correlated with carnosine, a metabolite related to meat quality (correlation > 0.6 and P < 0.05). Our results provide further understanding of the molecular mechanism of cross breeding for sheep muscle growth and meat quality optimization.

Effects of planting density on photosynthetic characteristics of flag leaf and senescence of leaf and root under wide-width sowing condition.

To determine the optimal planting density under wide-width sowing condition, we investigated the effects of different planting densities on photosynthetic characteristics of flag leaves, senescence characteristics of flag lea-ves and roots, grain yield, and water use efficiency under four planting density levels, 90×104 plants·hm-2 (D1), 180×104 plants·hm-2 (D2), 270×104 plants·hm-2 (D3) and 360×104 plants·hm-2 (D4), in field condition set in Yanzhou, Shandong during the growing season of 2018-2019 and 2019-2020. The results showed that compared with D1 and D4 treatments, D2 treatment significantly improved photosynthetic characteristics of wheat flag leaves during grain filling, increased the activity of superoxide dismutase (SOD) and soluble protein content, reduced the malondialdehyde (MDA) content, and delayed the senescence of flag leaves and roots. Compared with other treatments, D2 treatment significantly increased root length, root surface area and root volume in 0-40 cm soil layer. Compared with D1, D3 and D4 treatments, the grain yield of D2 treatment was increased by 11.8%, 2.5%, 6.4% in 2018-2019 and 22.7%, 5.7%, 17.1% in 2019-2020, respectively. In addition, water use efficiency was increased by 9.2%, 8.8%, 14.2% in 2018-2019 and 21.1%, 6.2%, 21.5% in 2019-2020, respectively. The planting density at 180×104 plants·hm-2 improved photosynthetic characteristics of flag leaves and root morphology during filling stage, delayed plant senescence, increased grain number per spike and grain weight. Consequently, the highest grain yield and water use efficiency were obtained under D2 treatment, which was the optimal treatment under the experimental wide-width sowing condition.

Investigating Historical Baseflow Characteristics and Variations in the Upper Yellow River Basin, China.

The baseflow of the Yellow River is vital and important for water resource management and for understanding the hydrological cycle and ecohydrology setting in this arid and semi-arid basin. This study uses a Lyne and Hollick digital filtering technique to investigate the behaviors of the baseflow and the baseflow index in the upper reaches of the Yellow River Basin (China). The observed streamflow discharges along the river were used to analyze the baseflow trend, persistence, and periodic characteristics during the period of 1950-2000. The results show that the average baseflow and BFI in the upper reaches of the Yellow River exhibit a decreasing trend and will continue to decline in the future. Generally, the annual average baseflow and BFI for the most upstream areas of the Yellow River show little difference, while the baseflow and BFI exhibit significant differences for the downstream areas. The filtered annual baseflow varied between 128 × 108 m3/year and 193 × 108 m3/year for the Yellow River. The BFI ranged from 0.54 to 0.65, with an average of 0.60. This indicates that on average, 60% of the long-term streamflow is likely controlled by groundwater discharge and shallow subsurface flow. Statistics show that two periodic variations were observed in the baseflow evolution process. The results indicate that on average, the first and second main cycles of baseflow behaviors occur at 28 years and 12-17 years, respectively. Correspondingly, the estimation indicates that the abrupt change points tend to appear in the 1960s, the 1980s, and the 1990s. An improved understanding of baseflow behaviors can help guide future strategies to manage the river regime, its water resources, and water quality.

Evaluation of the value of fasting plasma glucose in the first trimester for the prediction of adverse pregnancy outcomes.

AIMS: To evaluate the importance and usefulness of fasting plasma glucose (FPG) in the first trimester in predicting adverse pregnancy outcomes. METHODS: A retrospective study of 22,398 singleton pregnancies was conducted. Participants were divided into subgroups according to first-trimester FPG (low FPG, FPG < 5.1 mmol/L; medium FPG, 5.1 mmol/L ≤ FPG < 5.6 mmol/L; high FPG, 5.6 ≤ FPG < 7.0 mmol/L) and oral glucose tolerance test(OGTT) results (normal and abnormal) during pregnancy. Patient characteristics and risk of adverse pregnancy outcomes were compared. Then, the whole population of women with abnormal OGTT served as a reference, and the relative risks of maternal and neonatal complications in normal OGTT women were analyzed by categorical analyses and logistic regression. Subgroup analyses were performed according to pre-pregnancy body mass index (BMI). RESULTS: The frequency of adverse pregnancy outcomes increased with increasing FPG levels during the first trimester, regardless of OGTT results. High FPG + Abnormal OGTT had the worst outcome. Compared to the whole population of women with abnormal OGTT, Normal OGTT + Medium FPG showed the same risk of PIH and macrosomia. Normal OGTT + High FPG showed the same risk of PIH, macrosomia as well as LGA and preterm birth. Additionally, Normal OGTT + Medium FPG + BMI ≥ 24 kg/m2 showed significantly higher risk of PIH (OR = 1.867, 1.245-2.800), macrosomia (OR = 1.748, 1.304-2.344) and LGA (OR = 1.274, 1.019-1.593). Furthermore, the OR value for PIH was 3.759 (1.680-8.412) in Normal OGTT + High FPG + BMI ≥ 24 kg/m2 compared to women with abnormal OGTT. CONCLUSIONS: First-trimester FPG values can help identify women at increased risk for adverse pregnancy outcomes. Increased attention and management should be given to women with early pregnancy FPG ≥ 5.10 mmol/L despite a normal OGTT, especially if their BMI ≥ 24 kg/m2.

Cancer-testis antigen lactate dehydrogenase C4 in hepatocellular carcinoma: a promising biomarker for early diagnosis, efficacy evaluation and prognosis prediction.

Expressions and clinical implications of cancer-testis antigen (CTA) lactate dehydrogenase (LDH)-C4 in hepatocellular carcinoma (HCC) have not been fully elucidated. Herein, expressions of LDHC mRNA in the serum and serum-derived exosomes of early-stage HCC patients were determined using qRT-PCR, and the expression of LDH-C4 protein in HCC tissues was detected using high-throughput tissue microarray analysis. It was found that positive rates of LDHC mRNA expressions in the serum and serum exosomes of HCC patients were 68% and 60%, respectively. The AUCs of serum and exosomal LDHC in differentiating HCC patients from healthy controls were 0.8382 and 0.9451, respectively. The serum and exosomal LDHC levels in HCC patients in the treatment group were higher than the levels in the preliminary diagnosis group, but lower than those in the recurrence group. Survival analysis showed that the expression of LDH-C4 was negatively correlated with the prognosis of HCC. The Cox regression analysis showed that an LDH-C4 level was an independent risk factor for the prognosis of HCC patients. Therefore, serum and exosomal LDHC can be used as a biomarker for early diagnosis, efficacy evaluation and recurrence prediction of HCC. Moreover, LDH-C4 can be used as an important reference indicator for monitoring the prognosis of HCC.

Limonoids from the Fresh Young Leaves and Buds of Toona sinensis and Their Potential Neuroprotective Effects.

Toona sinensis, popularly known as Chinese toon or Chinese mahogany, is a perennial deciduous arbor belonging to the genus Toona in the Meliaceae family, which is widely distributed and cultivated in eastern and southeastern Asia. Its fresh young leaves and buds have been consumed as a very popular nutritious vegetable in China and confirmed to display a wide variety of biological activities. To investigate the chemical constituents and their potential health benefits from the fresh young leaves and buds of T. sinensis, a phytochemical study on its fresh young leaves and buds was therefore undertaken. In our current investigation, 16 limonoids (1-16), including four new limonoids, toonasinenoids A-D (1-4), and a new naturally occurring limonoid, toonasinenoid E (5), were isolated and characterized from the fresh young leaves and buds of T. sinensis. The chemical structures and absolute configurations of limonoids 1-5 were elucidated by comprehensive spectroscopic data analyses. All known limonoids (6-16) were identified via comparing their experimental spectral data containing mass spectrometry data, 1H and 13C nuclear magnetic resonance data, and optical rotation values to the data reported in the literature. All known limonoids (6-16) were isolated from T. sinensis for the first time. Furthermore, the neuroprotective effects of all isolated limonoids 1-16 against 6-hydroxydopamine-induced cell death in human neuroblastoma SH-SY5Y cells were assessed in vitro. Limonoids 1-16 exhibited notable neuroprotective activities, with EC50 values in the range from 0.27 ± 0.03 to 17.28 ± 0.16 µM. These results suggest that regular consumption of the fresh young leaves and buds of T. sinensis might prevent the occurrence and development of Parkinson's disease (PD). Moreover, the isolation and characterization of these limonoids that exhibit notable neuroprotective activities from the fresh young leaves and buds of T. sinensis could be very significant for researching and developing new neuroprotective drugs used for the prevention and treatment of PD.

Diagnostic and prognostic value of the cancer-testis antigen lactate dehydrogenase C4 in breast cancer.

BACKGROUND: Lactate dehydrogenase C4 (LDH-C4) as a cancer/testis antigen (CTA) is abnormally expressed in some malignant tumors. However, the expression and clinical significance of LDH-C4 in breast cancer (BC) has not been characterized. METHODS: We determined LDHC mRNA expression in serum and serum-derived exosomes of BC patients by quantitative RT-PCR. We also evaluated the protein expression of LDH-C4 in BC tissues using high-throughput tissue microarray analysis and immunohistochemistry. RESULTS: Our results showed high mRNA expression level of LDHC in serum and serum-derived exosomes of BC patients. The LDHC level in serum and exosomes could distinguish BC cases from healthy individuals based on their AUCs of 0.9587 and 0.9464, respectively. Besides, the LDHC level in exosomes of BC patients associated with tumor size, and positively correlated with HER2 and Ki-67 expressions (all with P < 0.05). Serum and exosomal level of LDHC negatively correlated with medical treatment and positively with the recurrence of BC. Survival analysis showed that LDH-C4 expression negatively correlated with BC prognosis. CONCLUSION: Serum and exosomal LDHC may be an effective indicator for the diagnosis, efficacy evaluation, and monitoring the recurrence of BC. LDH-C4 may act as a biomarker that predicts BC prognosis.

Recommended reference values for serum lipids during early and middle pregnancy: a retrospective study from China.

BACKGROUND: Disturbances in maternal lipid metabolism have been shown to increase the risk of adverse pregnancy outcomes. However, there is no consensus as to what constitutes normal maternal lipid values during pregnancy. Thus, the aim of this study was to establish serum lipid reference ranges during early and middle pregnancy. METHODS: We conducted a retrospective survey in Beijing from 2013 to 2014. A total of 17,610 singleton pregnancies with lipid data from early and middle pregnancy were included. First, after excluding women with adverse pregnancy outcomes, we performed a descriptive analysis of total cholesterol (TC), triglycerides (TG), high-density lipid cholesterol (HDL-C) and low-density lipid cholesterol (LDL-C) levels using means and standard deviations to determine appropriate percentiles. Second, in the total population, we examined the lipid levels in different trimesters with the risk of adverse pregnancy outcomes using categorical analyses and logistic regression models. Third, we determined the lipid reference range in early and middle pregnancy based on the first two results. Finally, based on the reference ranges we determined, we assessed whether the number of abnormal lipid values affected the risk of adverse pregnancy outcomes. RESULTS: (1) Serum levels of TC, TG, LDL-C and HDL-C all increased significantly from early to middle pregnancy, with the greatest increase in TG. (2) A trend towards an increasing incidence of adverse pregnancy outcomes was observed with increasing levels of TC, TG, and LDL-C and decreasing levels of HDL-C in both early and middle pregnancy. (3) We recommend that serum TC, TG and LDL-C reference values in early and middle pregnancy should be less than the 95th percentiles, whereas that of HDL-C should be greater than the 5th percentile, i.e., in early pregnancy, TC < 5.64 mmol/L, TG < 1.95 mmol/L, HDL-C > 1.23 mmol/L, and LDL-C < 3.27 mmol/L, and in middle pregnancy, TC < 7.50 mmol/L, TG < 3.56 mmol/L, HDL-C > 1.41 mmol/L, and LDL-C < 4.83 mmol/L. (4) Higher numbers out-of-range lipids during early and middle pregnancy were correlated with a higher risk of adverse pregnancy outcomes. CONCLUSIONS: The reference ranges recommended in this paper can identify pregnant women with unfavourable lipid values.

A pooled analysis of the diagnostic efficacy of plasmic methylated septin-9 as a novel biomarker for colorectal cancer.

The methylation status of septin-9 gene in plasma has been developed as a promising biomarker to aid in the diagnosis of colorectal cancer (CRC). In this study, we aimed to evaluate the overall diagnostic ability of septin-9 methylation for detection of CRC. Studies on the diagnostic performance of plasma septin-9 in CRC were searched from the online databases up to January 31st, 2017. Risk of bias among the studies was estimated according to the Quality Assessment of Studies of Diagnostic Accuracy included in the Systematic Reviews (QUADAS) II checklist. The aggregation of the effect sizes was enabled by utilizing a bivariate analysis model. A meta-regression test and influence analysis were conducted to determine the underlying sources of heterogeneity. According to the predefined criteria, 1,462 patients with CRC from 14 eligible trials were included. The quantitative meta-analyses showed that methylated septin-9 in plasma sustained a pooled sensitivity of 0.67 (95% CI, 0.61-0.73) and specificity of 0.89 (95% CI, 0.86-0.92) in discriminating CRC patients from cancer-free individuals, along with an area under the curve of 0.87. Moreover, the stratified analyses grouped by ethnicity demonstrated that methylayted septin-9 testing achieved a better sensitivity of 0.72 (95% CI, 0.68-0.76) in the European-based population group and a higher specificity of 0.90 (95% CI, 0.88-0.92) in the Asian-based population group. Plasmic methylated septin-9 suggests a promising diagnostic efficacy in confirming CRC. However, more studies are required to confirm our findings.

Expression of lactate dehydrogenase C in MDA­MB­231 cells and its role in tumor invasion and migration.

The cancer/testis antigen (CTA) lactate dehydrogenase C (LDHC) is a unique LDH isoenzyme associated with glucose and adenosine triphosphate production in mammalian germ cells. However, the role of LDHC in cancer has thus far largely remained elusive. The present study described the expression status of LDHC in human MDA­MB­231 breast cancer cells as well as its role in tumor invasion and migration. Immunohistochemical analysis revealed endogenous LDHC expression in the cytoplasm and nuclei of MDA­MB­231 cells yielded. In addition, in vitro cell invasion and migration assays revealed that when LDHC expression was blocked by its specific inhibitor, cell invasion and migration were compromised in MDA­MB­231 cells. Of note, inhibition of LDHC was unable to induce apoptosis in MDA­MB­231 cells. The present study provided evidence that the LDHC enzyme acts as a CTA in breast carcinoma and exerts an essential role in tumor invasion and migration.

PAX1 Methylation Hallmarks Promising Accuracy for Cervical Cancer Screening in Asians: Results from a Meta-Analysis.

BACKGROUND: DNA methylation has been proposed as a potential biomarker for cervical cancer detection. This study aimed to evaluate the diagnostic role of paired boxed gene 1 (PAX1) methylation for cervical cancer screening in Asians. METHODS: Eligible studies were retrieved by searching the electronic databases, and the quality of the enrolled studies was assessed via the quality assessment for studies of diagnostic accuracy (QUADAS) tool. The bivariate meta-analysis model was employed to generate the summary receiver operator characteristic (SROC) curve using Stata 12.0 software. Cochran's Q test and I2 statistics were applied to assess heterogeneity among studies. Publication bias was evaluated by the Deeks' funnel plot asymmetry test. RESULTS: A total of 9 articles containing 15 individual studies were included. The SROC analysis showed that single PAX1 methylation allowed for the discrimination between cancer/high-grade squamous intraepithelial lesion (HSIL) patients and normal individuals with a sensitivity (95% confidence interval) of 0.80 (0.70 - 0.87) and specificity of 0.89 (0.86 - 0.92), corresponding to an area under curve (AUC) of 0.92. Notably, our subgroup analysis suggested that combing parallel testing of PAX1 methylation and HPV DNA (AUC, sensitivity, and specificity of 0.90, 0.82, and 0.84, respectively) seemed to harbor higher accuracy than single HPV DNA testing (AUC, sensitivity, and specificity of 0.81, 0.86, and 0.67, respectively). CONCLUSIONS: PAX1 methylation hallmarks a potential diagnostic value for cervical cancer screening in Asians, and parallel testing of PAX1 methylation and HPV in cervical scrapings confers an improved accuracy than single HPV DNA testing.

Effects and mechanism of recombinant human erythropoietin on the growth of human breast cancer MDA-MB-231 cells in nude mice.

This study aimed to explore the effects of recombinant human erythropoietin (rhEPO) on the growth of human breast cancer MDA-MB-231 cells in nude mice, and investigate its functions in regulating tumor growth, angiogenesis and apoptosis. A tumor-bearing nude mice model was established by subcutaneous injection of human breast cancer MDA-MB-231 cells. Two weeks later, the mice were randomly divided into four groups (n=6 for each group): negative control group, rhEPO group, EPO antibody group and EPO+EPO antibody group. Drugs were administered to the corresponding mice once every 3 days for five times. The size and weight of tumors were measured after the mice were sacrificed by cervical dislocation. The expression levels of EPO/EPOR, TNF-α, IL-10, and Bcl-2 in the tumor tissues were determined using RT-PCR and Western blot. The microvessel density (MVD) and expression of VEGF in the tumors were detected using immunohistochemistry. TUNEL assay was used to determine apoptosis in tumors. Results show that rhEPO significantly promoted the growth of MDA-MB-231 cells in nude mice (P<0.05). Compared with the negative control group, the expression levels of EPO, EPOR, TNF-α, IL-10, and VEGF, as well as the MVD values, were significantly elevated in the rhEPO group. However, the apoptotic index was significantly reduced (P<0.05). The ability of rhEPO to promote tumor growth may be associated with its functions in promoting microvessel formation and inhibiting tumor cell apoptosis.

Structural view and substrate specificity of papain-like protease from avian infectious bronchitis virus.

Papain-like protease (PLpro) of coronaviruses (CoVs) carries out proteolytic maturation of non-structural proteins that play a role in replication of the virus and performs deubiquitination of host cell factors to scuttle antiviral responses. Avian infectious bronchitis virus (IBV), the causative agent of bronchitis in chicken that results in huge economic losses every year in the poultry industry globally, encodes a PLpro. The substrate specificities of this PLpro are not clearly understood. Here, we show that IBV PLpro can degrade Lys(48)- and Lys(63)-linked polyubiquitin chains to monoubiquitin but not linear polyubiquitin. To explain the substrate specificities, we have solved the crystal structure of PLpro from IBV at 2.15-Å resolution. The overall structure is reminiscent of the structure of severe acute respiratory syndrome CoV PLpro. However, unlike the severe acute respiratory syndrome CoV PLpro that lacks blocking loop (BL) 1 of deubiquitinating enzymes, the IBV PLpro has a short BL1-like loop. Access to a conserved catalytic triad consisting of Cys(101), His(264), and Asp(275) is regulated by the flexible BL2. A model of ubiquitin-bound IBV CoV PLpro brings out key differences in substrate binding sites of PLpros. In particular, P3 and P4 subsites as well as residues interacting with the ß-barrel of ubiquitin are different, suggesting different catalytic efficiencies and substrate specificities. We show that IBV PLpro cleaves peptide substrates KKAG-7-amino-4-methylcoumarin and LRGG-7-amino-4-methylcoumarin with different catalytic efficiencies. These results demonstrate that substrate specificities of IBV PLpro are different from other PLpros and that IBV PLpro might target different ubiquitinated host factors to aid the propagation of the virus.

The WWOX gene inhibits the growth of U266 multiple myeloma cells by triggering the intrinsic apoptotic pathway.

The role of the WW domain-containing oxidoreductase (WWOX) gene in multiple types of solid human cancers has been documented extensively. However, the functional role of WWOX in human multiple myeloma has not yet been fully elucidated. The present study aimed to investigate the effects of exogenous WWOX expression on the biological properties of U266 multiple myeloma cells, as well as the possible molecular mechanisms involved. In vitro experiments revealed that exogenous WWOX cDNA transfection resulted in marked growth arrest and the induction of apoptosis in the U266 multiple myeloma cells, accompanied by the activation of the intrinsic apoptotic pathway. Our data provide evidence that WWOX also plays a role as a tumor suppressor gene in multiple myeloma, possibly by suppressing cell proliferation and promoting apoptosis by triggering the intrinsic apoptotic pathway.

p73 participates in WWOX-mediated apoptosis in leukemia cells.

The WWOX gene is considered to be a tumor-suppressor gene which encodes a protein (Wwox) implicated in various types of solid human cancers. It has been shown that overexpression of WWOX in human tumors promotes apoptosis in vitro and suppresses tumor growth in vivo. Recently, we investigated the effects of WWOX overexpression in vitro and observed marked growth arrest in human leukemia cells; however, the underlying mechanism(s) for this effect is unknown. The present study aimed to elucidate the primary mechanism(s) underlying WWOX-mediated apoptosis in human leukemia. We traced the interactions between WWOX and its associated factors p73 and p53 after WWOX overexpression was induced in Jurkat and K562 cells. Our data revealed that p73 participates in WWOX-mediated apoptosis in Jurkat and K562 cells through binding with Wwox in the cytoplasm without a nuclear-cytoplasmic translocation.

The role of the WWOX gene in leukemia and its mechanisms of action.

The WW domain-containing oxidoreductase (WWOX) gene which encompasses the common human fragile site FRA16D has been proposed as a putative tumor suppressor gene, and loss of WWOX expression has been found in several types of solid cancer. As the role of WWOX in human leukemia has not yet been fully elucidated, the present study examined the expression of WWOX in patients with different types of leukemia as well as in leukemia-derived cell lines. Based on the data, WWOX mRNA (WWOX) and protein (Wwox) were significantly reduced or absent in the leukemia patients as well as in the cell lines. In addition, a recombinant expression vector, pGC-FU-WWOX, was constructed and transfected WWOX cDNA into Jurkat cells (acute T-lymphoblastic leukemia) and K562 cells (chronic myeloid leukemia in erythroid crisis) which all lack endogenous Wwox. In vitro experiments indicated that restoration of Wwox in Jurkat and K562 cells significantly suppressed proliferation and colony formation. Of note, apoptosis was also induced by Wwox restoration. Furthermore, we traced the mechanisms underlying this process and found that Wwox restoration could trigger the mitochondrial pathway in leukemia. Our data provide evidence that WWOX exerts a role as a tumor suppressor gene in leukemia, possibly by inhibiting proliferation and promoting apoptosis via the mitochondrial pathway.

Fasting plasma glucose at 24-28 weeks to screen for gestational diabetes mellitus: new evidence from China.

OBJECTIVE: To evaluate the usefulness of a fasting plasma glucose (FPG) at 24-28 weeks' gestation to screen for gestational diabetes mellitus (GDM). RESEARCH DESIGN AND METHODS: The medical records and results of a 75-g 2-h oral glucose tolerance test (OGTT) of 24,854 pregnant women without known pre-GDM attending prenatal clinics in 15 hospitals in China were examined. RESULTS: FPG cutoff value of 5.1 mmol/L identified 3,149 (12.1%) pregnant women with GDM. FPG cutoff value of 4.4 mmol/L ruled out GDM in 15,369 (38.2%) women. With use of this cutoff point, 12.2% of patients with mild GDM will be missed. The positive predictive value is 0.322, and the negative predictive value is 0.928. CONCLUSIONS: FPG at 24-28 weeks' gestation could be used as a screening test to identify GDM patients in low-resource regions. Women with an FPG between ≥4.4 and ≤5.0 mmol/L would require a 75-g OGTT to diagnose GDM. This would help to avoid approximately one-half (50.3%) of the formal 75-g OGTTs in China.

Cloning and expression of a toxin gene from Pseudomonas fluorescens GcM5-1A.

Pseudomonas fluorescens GcM5-1A was isolated from the pine wood nematode (PWN), Bursaphelenchus xylophilus, obtained from wilted Japanese black pine, Pinus thumbergii, in China. In this paper, a genomic library of the GcM5-1A strain was constructed and a toxin-producing clone was isolated by bioassay. Nucleotide sequence analysis revealed an open reading frame of 1,290 bp encoding a protein of 429 amino acids with N-terminal putative signal peptide of 36 amino acids, which shared a similarity of 83, 82 and 80% identity with hypothetical protein PFLU2919 from P. fluorescens SBW25, Dyp-type peroxidase family protein from P. fluorescens Pf-5 and Tat-translocated enzyme from P. fluorescens Pf0-1, respectively. The gene encoding a full-length protein or without the putative signal peptide was cloned and expressed as a soluble protein in E. coli. The recombinant protein was purified to electrophoretic homogeneity by affinity chromatography using a Ni2+ matrix column. Its relative molecular weight was estimated to be 48.5 kDa by SDS-PAGE for full-length protein, and 45.0 kDa for the recombinant protein without putative signal peptide. Bioassay results showed that the recombinant protein with or without the putative signal peptide was toxic to both suspension cells and P. thunbergii seedlings. HPLC analysis demonstrated that components in branch extracts of P. thunbergii were significantly changed after addition of the recombinant full-length protein and hydrogen peroxide, which indicated that it is probably a peroxidase. This study offers information that can be used to determine the mechanism of pine wilt disease caused by the PWN.

Aligning the proteome and genome of the silkworm, Bombyx mori.

A technology of mass spectrometry (MS) was used in this study for the large-scale proteomic identification and verification of protein-encoding genes present in the silkworm (Bombyx mori) genome. Peptide sequences identified by MS were compared with those from an open reading frame (ORF) library of the B. mori genome and a cDNA library, to validate the coding attributes of ORFs. Two databases were created. The first was based on a 9x draft sequence of the silkworm genome and contained 14,632 putative proteins. The second was based on a B. mori pupal cDNA library containing 3,187 putative proteins of at least 30 amino acid residues in length. A total of 81,000 peptide sequences with a threshold score of 60% were generated by the MS/MS analysis, and 55,400 of these were chosen for a sequence alignment. By searching these two databases, 6,649 and 250 proteins were matched, which accounted for approximately 45.4% and 7.8% of the peptide sequences and putative proteins, respectively. Further analyses carried out by several bioinformatic tools suggested that the matches included proteins with predicted transmembrane domains (1,393) and preproteins with a signal peptide (976). These results provide a fundamental understanding of the expression and function of silkworm proteins.