Bioinformatics and expression analysis of proline metabolism-related gene families in alfalfa under saline-alkali stress.

Regulation of the proline metabolic pathway is essential for the accumulation of proline under abiotic stress and for the amelioration of plant stress resistance. Δ1-pyrroline-5-carboxylate synthase (P5CS), pyrroline-5-carboxylate reductase (P5CR), ornithine transaminase (δ-OAT), proline dehydrogenase (PDH), pyrroline-5-carboxylate dehydrogenase (P5CDH), and proline transporter (ProT) are the key enzymes in the proline metabolic pathway. However, the gene families responsible for proline metabolism have not yet been identified or reported in alfalfa. In this study, a total of 12 MsP5CSs, 4 MsP5CRs, 3 MsOATs, 6 MsPDHs, 2 MsP5CDHs, and 5 MsProTs were identified in the genome of alfalfa, and the members of the same subfamily had similar gene structures and conserved motifs. Analysis of cis-regulatory elements revealed the presence of light-responsive, hormone-regulated, and stress-responsive elements in the promoter regions of alfalfa proline metabolism-related genes. Following treatment with saline-alkali, the expression of MsP5CSs, MsP5CRs, MsOATs, and MsProTs was significantly upregulated, whereas the expression of MsPDH1.1, MsPDH1.3, and MsP5CDH was significantly downregulated. The proline content and enzyme activity of P5CS gradually increased, whereas the enzyme activity of PDH gradually decreased as the duration of stress increased. Root growth rates decreased upon MsP5CS1a suppression (MsP5CS1a-RNAi) in the hairy roots of alfalfa compared to the empty vector line under saline-alkali stress. These results show that proline metabolism-related genes play an important role in the saline-alkali stress tolerance of alfalfa and provide a theoretical basis for further research on the functions of proline metabolism-related genes in alfalfa in response to saline-alkali stress.

Alfalfa MsATG13 Confers Cold Stress Tolerance to Plants by Promoting Autophagy.

Autophagy is a conserved cellular process that functions in the maintenance of physiological and metabolic balance. It has previously been demonstrated to improve plant tolerance to abiotic stress. Numerous autophagy-related genes (ATGs) that regulate abiotic stress have been identified, but there have been few functional studies showing how ATGs confer cold stress tolerance. The cold transcriptome data of the crown buds that experienced overwintering of the alfalfa (Medicago sativa L.) showed that MsATG13 is upregulated in response to cold stress. In the present study, we found that MsATG13 transgenic tobacco enhanced cold tolerance compared to wild-type (WT) plants. Transmission electron microscopy demonstrated that transgenic tobacco overexpressing MsATG13 formed more autophagosomes than WT plants in response to cold stress conditions. The transgenic tobacco increased autophagy levels due to upregulation of other ATGs that were necessary for autophagosome production under cold stress conditions. MsATG13 transgenic tobacco also increased the proline contents and antioxidant enzyme activities, enhancing the antioxidant defense capabilities under cold stress conditions. Furthermore, MsATG13 overexpression decreased levels of superoxide anion radicals and hydrogen peroxide under cold stress conditions. These findings demonstrate the role of MsATG13 in enhancing plant cold tolerance through modulation of autophagy and antioxidant levels.

Genome-wide identification of the myo-inositol oxygenase gene family in alfalfa (Medicago sativa L.) and expression analysis under abiotic stress.

Myo-inositol oxygenase (MIOX), a pivotal enzyme in the myo-inositol oxygenation pathway, catalyzes the cleavage of myo-inositol to UDP-glucuronic acid and plays a major role in plant adaptation to abiotic stress factors. However, studies pertaining to the MIOX gene family in alfalfa (Medicago sativa L.) are lacking. Therefore, this study characterized ten MsMIOX genes in the alfalfa genome. These genes were divisible into two classes distributed over three chromosomes and produced 12 pairs of fragment repeats and one pair of tandem repeats. Physicochemical properties, subcellular location, protein structure, conserved motifs, and gene structure pertinent to these MsMIOX genes were analyzed. Construction of a phylogenetic tree revealed that similar gene structures and conserved motifs were present in the same MsMIOX groups. Analysis of cis-acting elements revealed the presence of stress- and hormone-induced expression elements in the promoter regions of the MsMIOX genes. qRT-PCR analysis revealed that MsMIOX genes could be induced by various abiotic stress factors, such as salt, saline-alkali, drought, and cold. Under such conditions, MIOX activity in alfalfa was significantly increased. Heterologous MsMIOX2 expression in yeast enhanced salt, saline-alkali, drought, and cold tolerance. Overexpression of MsMIOX2 in the hairy roots of alfalfa decreased O2- and H2O2 content and enhanced the abiotic stress tolerance. This study offers comprehensive perspectives on the functional features of the MsMIOX family and provides a candidate gene for improving the abiotic stress tolerance of alfalfa.

Combined application of allantoin and strain JIT1 synergistically or additively promotes the growth of rice under 2, 4-DCP stress by enhancing the phosphate solubility, improving soil enzyme activities and photosynthesis.

Environmental pollution by 2, 4 dichlorophenol (2, 4-DCP) has become a widespread concern due to its detrimental influence on human and natural ecosystem. With the increasing accumulation of 2, 4-DCP in soil, it is of great significance to explore some appropriate approaches for enhancing plant tolerance to 2, 4-DCP stress. In the current study, a strain resistant to 2, 4-DCP was obtained from the tall fescue rhizosphere soil and named as Pseudomonas sp. JIT1. The strain JIT1 exhibited several remarkable plant growth-promoting traits, including the production of IAA, fixation of biological nitrogen and solubilization of phosphate. The inoculation of strain JIT1 significantly increased biomass, photosynthesis, antioxidant levels, chlorophyll contents and the osmotic substance contents in rice seedlings exposed to 2, 4-DCP. Meanwhile, inoculation of strain JIT1 also enhanced activities of soil alkaline phosphatase, urease, sucrase and cellulase. Moreover, under 2, 4-DCP stress, the content of allantoin in seedlings significantly increased and the pretreatment of exogenous allantoin noticeably ameliorated the negative effects caused by 2, 4-DCP stress in rice seedlings. Interesting, allantoin treatment also enhanced phosphate solubilization properties of strain JIT1. The chlorophyll contents, photosynthesis and osmotic substance further increased by combination use of strain JIT1 and allantoin, which improved the growth of seedlings, most likely to be attributed to the synergistic or additive effect between allantoin and strain JIT1. The results of this study highlight the important roles of combined use of strain JIT1 and allantoin for improving the tolerance of rice to 2, 4-DCP to stress.

The combined use of a plant growth promoting Bacillus sp. strain and GABA promotes the growth of rice under salt stress by regulating antioxidant enzyme system, enhancing photosynthesis and improving soil enzyme activities.

Salt stress poses a global challenge for agriculture, crop growth, and food production. In this study, a strain of rhizobacteria with both plant growth-promoting (PGP) traits and salt tolerance was isolated. The strain was identified as Bacillus pumilus via 16 S rDNA sequencing and was named B. pumilus JIZ13. This strain had the potential to solubilize phosphates and produce 1-aminocyclopropane-1-carboxylic acid deaminase, siderophores, and indole-3-acetic acid. After 35 days of salt stress exposure, the root length, plant height, dry weight, fresh weight, and relative water content of rice plants inoculated with strain JIZ13 were significantly higher than those without inoculation. Interestingly, the PGP properties of strain JIZ13 were significantly improved by the exogenous addition of gamma-aminobutyric acid (GABA). Moreover, GABA also enhanced the growth and development of rice plants under salt stress by providing substrates for the tricarboxylic acid cycle. Furthermore, the synergistic roles of GABA and strain JIZ13 in mitigating the damage caused by salt stress in rice plants was investigated. The results showed that the co-application of GABA and JIZ13 significantly increased photosynthetic efficiency, chlorophyll accumulation, antioxidant levels, levels of osmotic adjustment substances, and biomass of rice under salinity stress. In addition, the activities of urease, protease, invertase, and catalase enzymes in soil significantly improved under the combination of strain JIZ13 and GABA and increased by 39.65%, 36.88%, 70.21%, and 65.23%, respectively, compared to those without rhizobacterial and GABA additions. The enhancement of these four soil enzyme activities might thus improve soil quality and increase root elongation and biomass in rice plants. The results of this study provide the first evidence that PGP-rhizobacterial strain JIZ13 along with GABA can attenuate the negative effects of salt stress in rice plants.

Combined application of H2S and a plant growth promoting strain JIL321 regulates photosynthetic efficacy, soil enzyme activity and growth-promotion in rice under salt stress.

Salinity stress is one of the most harmful abiotic stresses that inhibit crop growth and grain yield. In this study, a salt-tolerant bacterium was isolated from the soil of the rice rhizosphere and named Myroides sp. JIL321, based on the results of the phylogenetic tree analysis. The strain JIL321 tolerated up to 1, 283.37 mM of NaCl and exhibited positive plant growth-promoting traits, such as the production of indole acetic acid (IAA) and 1-aminocyclopropane-1-carboxylate (ACC) deaminase. Therefore, the effects of JIL321 on rice (Oryza sativa L.) under salinity stress were determined. The inoculation of strain JIL321 significantly increased the chlorophyll content and the accumulation of osmotic adjustment substances, such as proline and soluble sugars, in rice expose to salt stress. Additionally, strain JIL321 inoculation significantly enhanced the activities of some enzymes commonly found in soil, such as urease, invertase and catalase. Moreover, the production of hydrogen sulfide (H2S), a pivotal signaling molecule, was also induced in rice by salt stress. Treatment with sodium hydrogen sulfide (NaHS, H2S donor) improved salt stress tolerance of the rice, while treatment with hypotaurine (HT, H2S scavenger) significantly suppressed it. Interestingly, NaHS treatment also improved the production of IAA and ACC deaminase in strain JIL321 under 0 mM and 150 mM salt concentrations. The combined treatment of JIL321 and NaHS could further improve the growth of salt-stressed rice seedlings, most likely due to the interaction effect between H2S and strain JIL321. To our knowledge, this study is the first to demonstrate that the combined use of H2S and plant growth-promoting bacteria could alleviate the adverse effects of salt stress on rice plants, and further verifies the novel role of H2S as a signaling molecule that enhance the tolerance of plant to abiotic stresses.

Alfalfa MsCBL4 enhances calcium metabolism but not sodium transport in transgenic tobacco under salt and saline-alkali stress.

KEY MESSAGE: MsCBL4 expression in tobacco enhanced its salt and saline-alkali stress tolerance by regulating calcium accumulation in roots, indicating the important role of calcium metabolism in plant saline-alkali stress tolerance The calcineurin B-like (CBL) family of proteins play important roles in plant abiotic stress tolerance and signal transduction. CBL4 is known to participate in the Salt Overly Sensitive pathway; however, little is currently known regarding the mechanisms underlying the response of CBL4 to saline-alkali stress. In this study, we cloned and characterized the alfalfa MsCBL4 gene. We found that MsCBL4 showed the highest expression in root tissues and was induced by salt and saline-alkali stress, with the latter causing higher induction. Overexpression of MsCBL4 in tobacco enhanced salt and saline-alkali stress tolerance and reduced the Na+/K+ ratio in roots of transgenic lines. Salt (30 and 300 mM NaCl) and saline-alkali (30 mM NaHCO3) stress assays performed for MsCBL4 transgenic tobacco lines revealed a substantial influx of sodium ions in roots under saline-alkali stress and indicated that the expression of MsCBL4 had little influence on sodium ion content reduction. In contrast, in roots subjected to saline-alkali stress, calcium accumulation occurred and was significantly enhanced by the overexpression of MsCBL4. Physiological and biochemical analyses indicated that MsCBL4 plays an important role in saline-alkali stress tolerance via its influence on the regulation of calcium transport and accumulation. These results provide novel insights into the saline-alkali stress tolerance mechanisms of plants.

[Network Meta-analysis of Yiqi Huoxue Chinese patent medicine for coronary heart disease with angina pectoris].

To assess the clinical efficacy of Yiqi Huoxue Chinese patent medicine for coronary heart disease with angina pectoris by using network Meta-analysis method. The relative randomized controlled trials( RCTs) of Yiqi Huoxue Chinese patent medicine for coronary heart disease with angina pectoris were retrieved from China National Knowledge Infrastructure( CNKI),Wan Fang,VIP and Chinese Biomedical Literature Database( CBM) in July 2018. Two researchers independently completed the literature screening,data extraction and quality evaluation according to the pre-determined inclusion and exclusion criteria,and the results were cross-checked.The data were analyzed by Win Bugs,and STATA software was used for plotting. Finally,114 RCTs were included,involving 7 Yiqi Huoxue Chinese patent medicines and 11 775 patients. Network Meta-analysis showed that the total effective rate for improvement in AP symptoms had 7 direct comparisons and 21 indirect comparisons,8 of which were statistically significant. The ECG improvement had 7 direct comparisons and 21 indirect comparisons,7 of which were statistically significant. In terms of the total effective rate of improvement in AP symptoms,the order of efficacy was as follows: Shensong Yangxin Capsules > Shexiang Baoxin Pills > Qishen Yiqi Dropping Pills > Tongxinluo Capsules > Wenxin Granules > Qishen Capsules > Naoxintong Capsules. In terms of ECG improvement,the order of efficacy was as follows: Shexiang Baoxin Pills > Tongxinluo Capsules > Naoxintong Capsules > Qishen Yiqi Dropping Pills> Wenxin Granules > Shensong Yangxin Capsules > Qishen Capsules. The results showed that Shensong Yangxin Capsules and Shexiang Baoxin Pills had certain advantages in the treatment of coronary heart disease with angina pectoris. Due to the small sample size,more studies were required to further verify the evidences.

Efficacy and Safety of Berberine Alone or Combined with Statins for the Treatment of Hyperlipidemia: A Systematic Review and Meta-Analysis of Randomized Controlled Clinical Trials.

To systematically evaluate the efficacy and safety of berberine for the treatment of hyperlipidemia, six electronic literature databases including SinoMed, CNKI, WanFang Data, PubMed, Embase and The Cochrane Library were searched to collect clinical randomized controlled trials (RCTs) of berberine alone or combined with statins for the treatment of hyperlipidemia from the inception to 8 March 2018. Two reviewers independently screened literature, extracted data and assessed the risk of bias of included RCTs. Then, meta-analysis was performed by using RevMan 5.3 software. A total of 11 RCTs involving 1386 patients were finally included. The results of meta-analysis showed that compared with the placebo group, berberine could significantly reduce the total cholesterol and low-density lipoprotein levels and elevate the high density lipoprotein level ( P<0.05 ). Compared with the simvastatin group, berberine was effective only in reducing the level of triglyceride ( MD=-0.37 , 95% CI: - 0.66, - 0.07, P=0.02 ). There, however, was no statistical significance between the BBR group and simvastatin group in the low density lipoprotein and high density lipoprotein levels. Compared with the simvastatin group, berberine plus simvastatin was more effective in reducing the level of triglyceride ( MD=-0.33 , 95% CI: - 0.46, - 0.20, P<0.00001 ) and total cholesterol ( MD=-0.36 , 95% CI: - 0.60, - 0.12, P=0.003 ). In terms of adverse reactions, the incidence of adverse reactions including transaminase elevation and muscle aches was lower in the berberine alone or combined with simvastatin group than that in the control group, while the instance of constipation was higher. This study suggests that berberine is effective for hyperlipidemia. The quality and quantity of included studies, however, were dissatisfactory, which might decrease the reliability of the results. Higher quality studies are needed to provide more high quality evidence.

[Chinese patent medicines with function of resolving hard lump for cyclomastopathy: network Meta-analysis].

This systematic review aims to systematically evaluate the efficacy and safety of Chinese patent medicines with resolving hard lump function for the treatment of cyclomastopathy. We searched CNKI, WanFang, SinoMed, PubMed, EMbase, and The Cochrane Library from the inception to January 3 2018, to collect the randomized controlled trials（RCTs）on Chinese patent medicines with resolving hard lump function in treating cyclomastopathy. Two reviewers independently screened literature, extracted data and assessed the methodological quality of included studies. Then, statistical analysis was performed by using Stata 12.0 and WinBUGS 1.4.3 software. A total of 52 RCTs involving 9 605 patients were finally included. The inventions included 11 commercial Chinese patent medicines with functions of resolving hard lump, such as Rupi Sanjie Capsules, Xiaoru Sanjie Capsules, Ruhe Sanjie Pills, and Hongjin Xiaojie Capsules. The result of direct Meta-analysis showed that: as compared with the simple western medicine group, the Chinese patent medicines such as Hongjin Xiaojie Capsules, Ruhe Sanjie Pills, Rupi Sanjie Capsules, Xiaoru Sanjie Capsules and Xiakucao Oral Liquid could significantly improve the clinical efficacy. In addition, the incidence of adverse reactions of Chinese patent medicines with resolving hard lump function was lower than that in Western medicine group in gastrointestinal reactions, menstrual disorders, leucorrhea abnormalities, liver dysfunction and estrogen-like effect. The network Meta-analysis showed that: Xiaoru Sanjie Capsules, Ruhe Sanjie Pills, Yanlu Rukang Capsules, Quyu Sanjie Capsules, and Hongjin Xiaojie Capsules were the top five in terms of treatment effect. Chinese patent medicines with resolving hard lump function had better clinical efficacy. Due to the low quality of included studies， more high quality RCTs are needed to verify the above conclusion.

[Guizhi Fuling capsule / pill treatment for chronic pelvic inflammatory disease：a systematic review of randomized clinical trials].

This systematic review aims to systematically evaluate the efficacy and safety of Guizhi Fuling Capsule/Pill on the treatment of chronic pelvic inflammatory disease. We searched CNKI datebases,WanFang,SinoMed,PubMed,Embase,The Cochrane Library from the inception to February 2017,to collect randomized controlled trials（RCTs）of Guizhi Fuling Capsule/Pill in treating chronic pelvic inflammation disease.Two reviewers independently screened literature,extracted date and assessed the risk of bias of included studies.Then,the risk assessment of included references was evaluated according to criteria recommended by Cochrane Handbook 5.3.A total of 30 RCTs involving 3 586 patients were finally included.30 studies reported the clinical efficacy,the result of meta-analysis showed that：compared with the western medicine group,Guizhi Fuling (capsule,pill) combined with western medicine could significantly improve the clinical efficacy [RR=1.20,95%CI（1.16,1.23）];3 studies reported recurrence rate,the result of meta-analysis showed that：compared with the western medicine,Guizhi Fuling (capsule,pill) combined with western medicine could decrease the recurrence rate [RR=0.33,95%CI（0.18,0.62）]; Their secondary indicators mainly included hs-CRP, plasma viscosity ratio and tumor necrosis factor and fibrin,the result of meta-analysis showed that： Guizhi Fuling (capsule,pill) combined with western medicine was better than western medicine in terms of anti-inflammatory and improving blood circulation.17 studies reported adverse reactions,most of the adverse events were the irritation of gastrointestinal tract. The result showed that： compared with the western medicine group, the incidence of adverse reactions in Guizhi Fuling (capsule,pill) combined with western medicine group is low.Guizhi Fuling（capsule,pill） did not increase the adverse reactions. The available evidence suggests that： Guizhi Fuling（capsule, pill） compared with the western medicine group, Guizhi Fuling (capsule,pill) combined with western medicine was more effective than the western medicine group in terms of clinical efficacy, recurrence rate, anti-inflammatory and plasma viscosity.Due to the limited quality and quantity of included studies,more high quality RCTs are needed to verify the above conciusion.

Improvement of the Stabilization and Activity of Protocatechuate 3,4-Dioxygenase Isolated from Rhizobium sp. LMB-1 and Immobilized on Fe3O4 Nanoparticles.

Protocatechuate 3,4-dioxygenase (P34O), which is isolated from Rhizobium sp. LMB-1, catalyzes the ring cleavage step in the metabolism of aromatic compounds, and has great potential for environmental bioremediation. However, its structure is very sensitive to different environmental factors, which weaken its activity. Immobilization of the enzyme can improve its stability, allow reusability, and reduce operation costs. In this work, the relative molecular mass of the native P34O enzyme was determined to be 500 kDa by gel filtration chromatography on Sephadex G-200, and the enzyme was immobilized onto (3-aminopropyl) triethoxysilane-modified Fe3O4 nanoparticles (NPs) by the glutaraldehyde method. The optimum pH of immobilized and free P34O was unaffected, but the optimum temperature of immobilized P34O increased from 60 to 70 °C, and the thermal stability of immobilized P34O was better than that of the free enzyme and showed higher enzymatic activity at 60 and 70 °C. In addition, with the exception of Fe3+, most metal ions and organic chemicals could not improve the activity of free and immobilized P34O. The kinetic parameters of the immobilized P34O were higher than those of the free enzyme, and immobilized P34O on Fe3O4 NPs could be reused ten times without a remarkable decrease in enzymatic activity.

A novel modified peptide derived from membrane-proximal external region of human immunodeficiency virus type 1 envelope significantly enhances retrovirus infection.

Efficient gene transfer is a critical goal in retroviral transduction. Several peptides capable of forming amyloid fibrils, such as the 39-residue semen-derived infection-enhancing peptide (SEVI), have demonstrated the ability to boost retroviral gene delivery. Here, a 13-residue peptide P13 (Ac-(671) NWFDITNWLWYIK(683)) derived from the membrane-proximal external region of the human immunodeficiency virus type 1 (HIV-1) gp41 transmembrane protein, together with its 16-residue peptide derivative (P16) were found to enhance HIV-1 infection significantly. Both peptides, P13 and P16, could form amyloid fibril structures to potently enhance HIV-1 infectivity. Further investigations showed that both aromatic Trp residues and cationic Lys residues contributed to the enhancement of HIV-1 infection by these two active peptides. P16 could more effectively augment HIV-1 YU-2 infection than SEVI, implying its potential applications as a tool in the lab to improve gene transfer rates.

Multiple antigen peptide mimetics containing gp41 membrane-proximal external region elicit broad neutralizing antibodies against human immunodeficiency virus type 1 in guinea pigs.

Eliciting a broadly neutralizing antibody response against the HIV-1 membrane-proximal external region (MPER) mimicking the activity of 4E10 and 2F5 monoclonal antibodies remains a major challenge. In this study, two novel tetra-branched peptide immunogens, 4E10- and 2F5-MAP4, were designed and synthesized using a MAP system. Guinea pigs were immunized with either of these two synthetic immunogens emulsified in an oil-phase adjuvant at 3-week intervals. After four immunizations, epitope-specific antibody responses were induced successfully, and moderate neutralizing activities against tier 1 (clades B, BC, AE) and tier 2 (clade C) HIV-1 pseudoviruses were detectable in unfractionated sera and purified IgGs. The synthetic gp41 membrane-proximal external region peptide mimetics, 4E10- and 2F5-MAP4, assisted by an appropriate adjuvant, are promising prophylactic vaccine candidates potentially capable of eliciting broadly neutralizing antibody responses against HIV-1.