Quantitative trait loci and genomic prediction for grain sugar and mineral concentrations of cowpea [Vigna unguiculata (L.) Walp.].

Development of high yielding cowpea varieties coupled with good taste and rich in essential minerals can promote consumption and thus nutrition and profitability. The sweet taste of cowpea grain is determined by its sugar content, which comprises mainly sucrose and galacto-oligosaccharides (GOS) including raffinose and stachyose. However, GOS are indigestible and their fermentation in the colon can produce excess intestinal gas, causing undesirable bloating and flatulence. In this study, we aimed to examine variation in grain sugar and mineral concentrations, then map quantitative trait loci (QTLs) and estimate genomic-prediction (GP) accuracies for possible application in breeding. Grain samples were collected from a multi-parent advanced generation intercross (MAGIC) population grown in California during 2016-2017. Grain sugars were assayed using high-performance liquid chromatography. Grain minerals were determined by inductively coupled plasma-optical emission spectrometry and combustion. Considerable variation was observed for sucrose (0.6-6.9%) and stachyose (2.3-8.4%). Major QTLs for sucrose (QSuc.vu-1.1), stachyose (QSta.vu-7.1), copper (QCu.vu-1.1) and manganese (QMn.vu-5.1) were identified. Allelic effects of major sugar QTLs were validated using the MAGIC grain samples grown in West Africa in 2017. GP accuracies for minerals were moderate (0.4-0.58). These findings help guide future breeding efforts to develop mineral-rich cowpea varieties with desirable sugar content.

Mutation of Basic Residues R283, R286, and K288 in the Matrix Protein of Newcastle Disease Virus Attenuates Viral Replication and Pathogenicity.

The matrix (M) protein of Newcastle disease virus (NDV) contains large numbers of unevenly distributed basic residues, but the precise function of most basic residues in the M protein remains enigmatic. We previously demonstrated that the C-terminus (aa 264-313) of M protein interacted with the extra-terminal (ET) domain of chicken bromodomain-containing protein 2 (chBRD2), which promoted NDV replication by downregulating chBRD2 expression and facilitating viral RNA synthesis and transcription. However, the key amino acid sites determining M's interaction with chBRD2/ET and their roles in the replication and pathogenicity of NDV are not known. In this study, three basic residues-R283, R286, and K288-in the NDV M protein were verified to be responsible for its interaction with chBRD2/ET. In addition, mutation of these basic residues (R283A/R286A/K288A) in the M protein changed its electrostatic pattern and abrogated the decreased expression of endogenic chBRD2. Moreover, a recombinant virus harboring these mutations resulted in a pathotype change of NDV and attenuated viral replication and pathogenicity in chickens due to the decreased viral RNA synthesis and transcription. Our findings therefore provide a better understanding of the crucial biological functions of M's basic residues and also aid in understanding the poorly understood pathogenesis of NDV.

The matrix protein of Newcastle disease virus inhibits inflammatory response through IRAK4/TRAF6/TAK1/NF-κB signaling pathway.

The matrix (M) protein of several cytoplasmic RNA viruses has been reported to be an NF-κB pathway antagonist. However, the function and mechanism of NDV M protein antagonizing NF-κB activation remain largely unknown. In this study, we found that the expression levels of IRAK4, TRAF6, TAK1, and RELA/p65 were obviously reduced late in NDV infection. In addition, the cytoplasmic M protein rather than other viral proteins decreased the expression of these proteins in a dose-dependent manner. Further indepth analysis showed that the N-terminal 180 amino acids of M protein were not only responsible for the reduced expression of these proteins, but also responsible for the inhibition of NF-κB activation and nuclear translocation of RELA/p65, as well as the production of inflammatory cytokines. Moreover, small interference RNA-mediated knockdown of IRAK4 or overexpression of IRAK4 markedly enhanced or reduced NDV replication by decreasing or increasing inflammatory cytokines production through the IRAK4/TRAF6/TAK1/NF-κB signaling pathway. Strangely, there were no interactions detected between NDV M protein and IRAK4, TRAF6, TAK1 or RELA/p65. Our findings described here contribute to a better understanding of the innate immune antagonism function of M protein and the molecular mechanism underlying the replication and pathogenesis of NDV.

Heterologous expression and physicochemical characteristics identification of Kunitz protease inhibitor in Brassica napus.

A Kunitz protease inhibitor gene (RTI; rti) was cloned from rapeseed and expressed in a Pichia pastoris expression system for the first time. After isolation and purification, the physical and chemical characteristics of the inhibitor were analyzed. The results showed that the induced expression level of the recombinant RTI reached 628 mg/L, and the specific activity of the inhibitor reached 69.6 TIU/mg protein at the shake flask fermentation level; the recombinant RTI retained more than 70% inhibitory activity between 30 and 90 °C and more than 80% inhibitory activity between pH 2.0-11.0. The metal ions Cu2+ and CO2+ and the organic reagents methanol, ethanol, acetone, and chloroform inhibit its activity. The recombinant RTI interacts with trypsin in a noncompetitive manner and has a strong and specific inhibitory effect on trypsin, a typical Kunitz trypsin inhibitor from plants. Combined with its good physical and chemical properties, recombinant RTI has the potential to be developed into an insect resistance protein.

Cloning, expression and characterization of metalloproteinase HypZn from Aspergillus niger.

A predicted metalloproteinase gene, HypZn, was cloned from Aspergillus niger CGMCC 3.7193 and expressed in Pichia pastoris GS115, and the physicochemical characteristics of recombinant HypZn were investigated after separation and purification. The results showed that the specific activity of the purified HypZn reached 1859.2 U/mg, and the optimum temperature and pH value of HypZn were 35°C and 7.0, respectively. HypZn remained stable both at 40°C and at pH values between 5.0 and 8.0. The preferred substrate of HypZn was soybean protein isolates, and the Km and Vmax values were 21.5 µmol/mL and 4926.6 µmol/(mL∙min), respectively. HypZn was activated by Co2+ and Zn2+ and inhibited by Cu2+ and Fe2+. The degree of soybean protein isolate hydrolysis reached 14.7%, and the hydrolysates were of uniform molecular weight. HypZn could tolerate 5000 mM NaCl and completely lost its activity after 30 min at 50°C. The enzymological characterizations indicated that HypZn has great application potential in the food industry, especially in fermented food processing.

A new carboxypeptidase from Aspergillus niger with good thermostability, pH stability and broad substrate specificity.

A new serine carboxypeptidase gene, capA, was identified in Aspergillus niger CBS 513.88 by reading genomic information and performing sequence alignment, and the gene was cloned and expressed in Pichia pastoris GS115. In a shake flask, the enzyme activity of the recombinant strain GS115 (pPIC9K-capA) reached 209.3 U mg-1. The optimal temperature and pH for enzyme activity were determined to be 45 °C and 6.0, respectively. After incubation at 40-50 °C or at pH 4.0-8.0 for 1 h, the enzyme retained more than 80% or 60% of its initial activity. The presence of 1-10 mmol L-1 Mg2+ enhanced the activity of CapA, whereas 1-10 mmol L-1 Cu2+, Fe2+, or Co2+, 10 mmol L-1 Mn2+, or 1-10 mmol L-1 phenylmethylsulfonyl fluoride (PMSF) significantly inhibited its activity. CapA had a broad substrate specificity and preferred the hydrophobic amino acids Leu and Lys at the C terminus of proteins, and N-benzyloxycarbonyl-L-phenylalanyl-L-leucine (Cbz-Phe-Leu) was the optimal substrate, for which CapA exhibited Km 0.063 mmol L-1 and kcat/Km 186.35 mmol L-1 s-1. The good thermostability, pH stability and hydrolysis characteristics of CapA provide a solid foundation for application in the food and biotechnology fields.

Coexpression of VHb and MceG genes in Mycobacterium sp. Strain LZ2 enhances androstenone production via immobilized repeated batch fermentation.

Androstenone production is limited by low-efficiency substrate transport and dissolved oxygen levels during fermentation. In this study, the coexpression of the optimized Vitreoscilla hemoglobin (VHb) and sterol transporter ATPase (MceG) genes in Mycobacterium sp. LZ2 (Msp) was investigated to alleviate dissolved oxygen and mass transfer limitations. Results revealed that Msp-vgb/mceG effectively improved the growth, production, and adaptation to dissolved oxygen compared with those of Msp. The increased catalase activity and reduced intracellular ROS levels enhanced cell viability and promoted transcription of genes critical for phytosterol metabolism. Bagasse as an immobilization carrier increased the productivity of Msp-vgb/mceG by 56%. Immobilized repeat batch fermentation reduced the biotransformation period from 60 days to 37 days and improved the productivity from 0.039 g/L/h to 0.069 g/L/h. To the best of our knowledge, this work is the first study on the immobilization of recombinant mycobacteria on bagasse for androstenone production.

Application of serum markers in diagnosis and staging of ovarian endometriosis.

AIMS: Laparoscopic surgery is widely used for diagnosing ovarian endometriosis but it has medical risks. This study explored the application of blood indicators in diagnosis and staging of ovarian endometriosis, aiming to develop a noninvasive diagnostic method. METHODS: A total of 190 ovarian endometriosis patients were included in observation group, among these participants, 77 patients among them were stages I-II, and the rest 113 patients were stages III-IV, and a total of 103 healthy women as control group. Serum biochemical indexes, tumor markers, and cytokines levels in two groups were used for the diagnosis and staging of the disease. Area under the receiver operating characteristic (ROC) curve (AUC) predicted the value of individual and joint tests for indicators. RESULTS: Biochemical indexes, namely, alkaline phosphatase (ALP), total protein (TP), and glucose (Glu) could distinguish patients from normal women; and that ALP and Glu could indicate disease staging. In tumor markers, alpha fetoprotein (AFP), carcinoembryonic antigen (CA) 125, CA199 and human epididymis protein 4 (HE4) helped to diagnose endometriosis; CA125, HE4, and cytokeratin 19 fragment (CYFRA21-1) could differentiate stages. In cytokines, vascular endothelial growth factor (VEGF), tumor necrosis factor (TNF)-α, interleukin (IL)-6, soluble fms-like tyrosine kinase receptor 1 (sflt-1), monocyte chemoattractant protein (MCP)-1 therefore, have values to diagnose endometriosis; VEGF, TNF-α, IL-6, and sflt-1 helped to differentiate disease staging. CONCLUSION: Serological indicators in ovarian endometriosis patients were different from healthy women, which were of certain differential values in diagnosis and disease staging. The current study provided a novel strategy for endometriosis diagnosis.

A Bayesian model for detection of high-order interactions among genetic variants in genome-wide association studies.

BACKGROUND: A central question for disease studies and crop improvements is how genetics variants drive phenotypes. Genome Wide Association Study (GWAS) provides a powerful tool for characterizing the genotype-phenotype relationships in complex traits and diseases. Epistasis (gene-gene interaction), including high-order interaction among more than two genes, often plays important roles in complex traits and diseases, but current GWAS analysis usually just focuses on additive effects of single nucleotide polymorphisms (SNPs). The lack of effective computational modelling of high-order functional interactions often leads to significant under-utilization of GWAS data. RESULTS: We have developed a novel Bayesian computational method with a Markov Chain Monte Carlo (MCMC) search, and implemented the method as a Bayesian High-order Interaction Toolkit (BHIT) for detecting epistatic interactions among SNPs. BHIT first builds a Bayesian model on both continuous data and discrete data, which is capable of detecting high-order interactions in SNPs related to case--control or quantitative phenotypes. We also developed a pipeline that enables users to apply BHIT on different species in different use cases. CONCLUSIONS: Using both simulation data and soybean nutritional seed composition studies on oil content and protein content, BHIT effectively detected some high-order interactions associated with phenotypes, and it outperformed a number of other available tools. BHIT is freely available for academic users at http://digbio.missouri.edu/BHIT/.

Identification and characterization of a stachyose synthase gene controlling reduced stachyose content in soybean.

KEY MESSAGE: We identified and characterized a mutant of soybean stachyose synthase gene controlling reduced stachyose content which benefit the soybean seed composition breeding program in the future. It has been shown that in soybean, increased sucrose and reduced raffinose family oligosaccharides would have a positive impact on the world's feed industry by improving digestibility and feed efficiency. We searched for new sources of modified oligosaccharide content in a subset of the USDA Soybean Germplasm Collection and then identified plant introduction (PI) 603176A as having ultra-low stachyose content (0.5%). We identified a 33-bp deletion mutant in the putative stachyose synthase gene (STS gene, Glyma19g40550) of PI 603176A. A co-dominate indel marker was successfully developed from this 33-bp deletion area and was genetically mapped into two F 2:3 populations and a F 4:5 population, which associated with low stachyose content in the progeny lines. These observations provided strong evidence that the STS gene is responsible for stachyose biosynthesis in the soybean plant. Expression of the sts gene remained at the normal level, suggesting the loss of function in the gene is due to defective protein function. This gene-based perfect genetic marker for low stachyose content can be useful for marker-assisted selection in soybean molecular breeding programs.

[Observation on both Na(+)-K+ Atpase activity and expression of Na(+)-K+ AtPase alpha1 mRNA in lung tissues of rats acute poisoned with nickel carbonyl].

OBJECTIVE: To examine the change of Na(+)-K+ ATPase activity and the expressions of Na(+)-K+ ATPase alpha1 mRNA in lung tissues of rats poisoned by nickel carbonyl and to discuss the mechanism of lung injury. METHODS: One hundred seventy healthy rats (85 male and 85 female) were exposed by inhalation of 20,135 and 250 mg/m3 nickel carbonyl for 30 min. Rats poisoned by chlorine gas with a concentration 250 mg/m3 served as positive group and healthy SD rats served as no-treatment negative group. The rats were euthanized on 1, 2, 3 and 7 d after the administration of nickel carbonyl or chlorine gas. In various treatment groups, Na(+)-K+ ATPase activity was studied by colorimetric method and the expressions of Na(+)-K+ ATPase alpha1 mRNA were determined by RT-PCR. RESULTS: Na(+)-K+ ATPase activity and expressions of Na(+)-K+ ATPase alpha1 mRNA in lung tissues decreased in all treatment groups and chlorine gas-poisoned group, especially it was obvious decreased on the 2ed and 3rd day (P < 0.05). CONCLUSION: Nickel carbonyl could induce lung damage and decrease Na(+)-K+ ATPase activity and expressions of Na(+)-K+ ATPase alpha1 mRNA in lung.

[Application of pulse-coupled neural network combined with genetic algorithm on MR images of hypoxic-ischemic encephalopathy].

This paper is to provide a basis for the establishment of an early diagnostic system for hypoxic-ischemic encephalopathy (HIE) by performing segmentation and feature extraction of lesions on the MR images of neonatal babies with HIE. The segmentation on MR images of HIE based on the genetic algorithm (GA) combined with a pulse-coupled neural network (PCNN) were carried out. There were better segmentation results by using PCNN segmentation based on GA than PCNN segmentation with fixed parameters. The data suggested that a PCNN based on GA could provide effective assistance for diagnosis and research.

[Effect of alpha-terthienyl on protein, esterase and lipid peroxidation of Aedes albopictus larvae].

OBJECTIVE: To study the effect of alpha-terthienyl (alpha-T) on protein, esterase and lipid peroxidation of Aedes albopictus larvae. METHODS: Sensitive and resistant strains of Aedes albopictus stage IV larvae were used. Bradford method was used to detect protein content. The breeding fluid of experiment group contained alpha-T (5.34 microg/L), and control group contained only acetone (3.95 microg/L). Histochemistry method was used to detect esterase activity. Larvae in the experiment group were cultured in fluid containing alpha-T (6.24 microg/L) but no alpha-T in the control group. TBA method was used to detect malondialdehyde (MDA). Larvae of the sensitive strain were divided into 5 sub-groups: A - acetone control (containing acetone 3.95 microg/L), B - ultra-violet irradiation (UV) control (same with A but treated by UV), C, D, E - experiment groups with alpha-T (4.58, 5.34 and 6.24 microg/L respectively). All groups were kept in dark condition for 1 hour, followed by UV for 1 hour (except group A), then fed under normal condition. RESULTS: With UV and alpha-T, the protein content of experiment group (1.225 mg/ml) was higher than that of control (1.120 mg/ml) (P<0.05) in sensitive strain; that of experiment group (1.199 mg/ml) was higher than that of control (1.114 mg/ml) (P<0.05) in the resistant strain. After 2, 4, 6, and 8 hour treated by both alpha-T and UV, the esterase activity all decreased in experiment group, and reached to the lowest 8 hours later (P<0.05). MDA contents was 2.286 nmol/mg protein in acetone control group and 2.322 nmol/mg protein in UV control, but 3.156, 4.188 and 4.684 nmol/mg protein respectively in the 3 experiment groups after treated by alpha-T and UV. The higher dose of alpha-T, the higher content of MDA (P<0.05). CONCLUSION: Under UV, alpha-T can increase the protein and MDA content of the larvae of Ae. albopictus but decrease the esterase activity.

Determination of benzyltriethyl ammonium chloride from polymeric media by capillary electrophoresis with ultraviolet absorbance detection.

The importance of benzyltriethyl ammonium chloride (BTEAC) in industrial applications has stimulated the development of a number of methods for its determination. In this paper, a high performance capillary electrophoresis (CE) method, coupled with an extraction technique for determining BTEAC in organic matrices, was developed. BTEAC was extracted from organic samples with a 20 mM sodium phosphate solution. Sonication was used to improve extraction efficiency. The repeatability and recovery of the technique have been studied and it was proven that the technique is satisfactory for quantitative determination of BTEAC in organic matrices. Separation was achieved within 6 min in 20 mM sodium phosphate buffer at pH 5.0. The recovery was above 92%. The detection limit for BTEAC is 5 mg L(-1) with a signal-to-noise ratio of 3. The linear range of the technique is 5-100 mg L(-1). This method is simple, fast, low-cost, and can be easily used for product quality control in industrial laboratories.