[Screening and identification of key enzyme genes for steroidal saponin biosynthesis in Dioscorea zingiberensis under low phosphorus stress].

To investigate the responses of key enzymes involved in steroidal saponin biosynthesis of Dioscorea zingiberensis to low phosphorus stress, we designed three treatments of severe phosphorus stress, moderate phosphorus stress, and normal phosphorus level. The D. zingiberensis plants were collected at the early, middle, and late stages of treatment. The content of total steroidal saponins in different tissues of D. zingiberensis was determined by spectrophotometry for the identification of the critical stage in response to low phosphorus stress. BGI 500 sequencing platform was employed to obtain the transcript information of D. zingiberensis samples at the critical stage of low phosphorus stress, and then a transcriptome library was constructed. The correlation between the expression of genes involved in steroidal saponin biosynthesis and the content of total steroidal saponins was analyzed for the screening of the key enzyme genes in response to low phosphorus stress. Further, the expression patterns of these genes were analyzed by real-time fluorescence PCR(qRT-PCR). The content of total steroidal saponins in D. zingiberensis had obvious tissue specificity under low phosphorus stress, and the early stage of stress was particularly important for D. zingiberensis to respond to low phosphorus stress. A total of 101 593 unigenes were obtained by transcriptome sequencing, of which 77.35% were annotated in NT, NR, SwissProt, KOG, GO, and KEGG. A total of 256 transcripts of known key enzyme genes in the biosynthetic pathway of steroidal saponins were identified. The expression levels of 69 transcripts encoding 18 catalytic enzymes were significantly correlated with the content of total steroidal saponins. The qRT-PCR results showed that several key enzyme genes presented different expression patterns in four tissues under low phosphorus stress. The results indicated that the content of total steroidal saponins and the expression of key enzyme genes regulating steroidal saponin biosynthesis in D. zingensis changed under low phosphorus stress. This study provides the biological information for elucidating the molecular mechanism of steroidal saponin biosynthesis in D. zingensis exposed to low phosphorus stress.

Metal-Free ß-Amino Alcohol Synthesis: A Two-step Smiles Rearrangement.

A novel method for the synthesis of ß-amino alcohols has been demonstrated under mild reaction conditions with a broad scope via a two-step Smiles rearrangement. What is more, theoretical calculations have been performed to confirm the rationality of the mechanism. The method has been proved to be notably effective for N-arylated amino alcohols, which are difficult to synthesize by traditional methods.

Docosahexaenoic acid inhibits Ca2+ influx and downregulates CaSR by upregulating microRNA-16 in pulmonary artery smooth muscle cells.

Docosahexaenoic acid (DHA) is reported to have the potential to ameliorate pulmonary arterial hypertension (PAH), while the specific mechanism is still obscure. This study aims to investigate the function of DHA in pulmonary artery smooth muscle cells (PASMCs) and explore the underlying mechanism. In our study, DHA was used to incubate PASMCs. Cytosolic-free Ca2+ concentration ([Ca2+ ]cyt) was measured using Fluo-3 AM method. Real-time polymerase chain reaction was used to detect microRNA-16 (miR-16) and calcium-sensing receptor (CaSR) messenger RNA expression levels. CCK-8 assay, BrdU assay, and Transwell assay were employed to detect the effects of DHA on proliferation and migration of PASMCs. CaSR was confirmed as a direct target of miR-16 using dual-luciferase assay, polymerase chain reaction, and Western blot analysis. It was found that DHA significantly inhibited PASMC proliferation and migration and decreased [Ca2+ ]cyt. After transfection of miR-16 mimics, proliferation and migration ability of PASMCs were significantly inhibited, whereas opposite effects were observed after miR-16 inhibition. [Ca2+ ]cyt was also inhibited by miR-16 transfection. DHA then promoted the expression of miR-16, and the effects of DHA on PASMCs were annulled when miR-16 was inhibited. CaSR was identified as a direct target of miR-16. CaSR was inhibited directly by miR-16 and indirectly by DHA. In conclusion, DHA inhibits the proliferation and migration of PASMCs, and probably ameliorates PAH via regulating miR-16/CaSR axis.

Mechanisms and the role of probiotic Bacillus in mitigating fish pathogens in aquaculture.

Diseases are natural components of the environment, and many have economic implications for aquaculture and fisheries. Aquaculture is a fast-growing industry with the aim to meet the high protein demand of the ever-increasing global population; however, the emergence of diseases is a major setback to the industry. Probiotics emerged as a better solution to curb the disease problem in aquaculture among many alternatives. Probiotic Bacillus has been proven to better combat a wide range of fish pathogens relative to other probiotics in aquaculture; therefore, understanding the various mechanisms used by Bacillus in combating diseases will help improve their mode of action hence yielding better results in their combat against pathogens in the aquaculture industry. Thus, an overview of the mechanisms (production of bacteriocins, suppression of virulence gene expression, competition for adhesion sites, production of lytic enzymes, production of antibiotics, immunostimulation, competition for nutrients and energy, and production of organic acids) used by Bacillus probiotics in mitigating fish pathogens ranging from Aeromonas, Vibrio, Streptococcus, Yersinia, Pseudomonas, Clostridium, Acinetobacter, Edwardsiella, Flavobacterium, white spot syndrome virus, and infectious hypodermal and hematopoietic necrosis virus proven to be mitigated by Bacillus have been provided.

Effects of three host-associated Bacillus species on mucosal immunity and gut health of Nile tilapia, Oreochromis niloticus and its resistance against Aeromonas hydrophila infection.

Skin and intestinal mucosa lymphoid tissues are known to be the fish's first line of defence since they serve as the first point of contact for pathogens. Only few studies have investigated the influence of host-associated Bacillus on mucosal immunity. In this study, the effects of three host-associated Bacillus species on mucosal immunity, intestinal morphology, intestinal digestive enzymes activity, intestinal microbiome and resistance of Nile tilapia against Aeromonas hydrophila infection was evaluated. The fish were divided into five treatment groups and fed with diets containing no bacteria denoted as Control, Bacillus velezensis TPS3N denoted as group V, Bacillus subtilis TPS4 denoted as group S, Bacillus amyloliquefaciens TPS17 denoted as group A and a 5th group containing the three Bacillus species at a ratio 1:1:1 denoted as group CB. At the end of the feeding trial, significant enhancement of both skin mucus and intestinal immune titres were recorded in terms of nitric oxide (NO) (except in the mucus of V and S groups), immunoglobulin M (IgM) (except in the intestine of group V), lysozyme (LZM), and alkaline phosphatase (AKP) in all fish fed the Bacillus supplemented groups relative to the untreated group. Intestinal antioxidant enzymes (catalase (CAT) (except in the intestine of group S) and superoxide dismutase (SOD)) capacity of Nile tilapia were higher in the Bacillus groups. Intestinal lipase activity was elevated in the Bacillus supplemented groups. The intestinal morphological parameters (villus height, villus width, goblet cells count (except in group S and A), and intestinal muscle thickness) were significantly enhanced in the Bacillus supplemented groups relative to the Control group. Dietary probiotic supplementation also influenced the intestinal microflora composition of Nile tilapia. Proteobacteria recorded the highest abundance followed by Firmicutes, Fusobacteria, and Bacteroidetes at the phylum level in this study. At the genus level, the abundance of pathogenic bacteria viz Staphylococcus and Aeromonas were reduced in the Bacillus supplemented groups in comparison to the Control group. A challenge test with A. hydrophila resulted in lower mortalities (%) in the Bacillus treated groups thus 86.67%, 50.00%, 43.33%, 63.33%, and 30.00% for Nile tilapia fed Control, V, S, A, and CB diets respectively. In conclusion, the inclusion of B. velezensis TPS3N, B. subtilis TPS4, and B. amyloliquefaciens TPS17 in the diet of Nile tilapia singularly or in combination, could enhance the mucosal immunity, intestinal health, and resistance of Nile tilapia against A. hydrophila infection.

[Molecular cloning and expression analysis of iridoid synthase genes from Rehmannia glutinosa].

Iridoid synthase( IS),the key enzyme in the natural biosynthesis of vegetal iridoids,catalyzes the irreversible cyclization of 10-oxogeranial to epi-iridodial. In this study,we screened the Rehmannia glutinosa transcriptome data by BLASTn with Catharanthus roseus CrIS cDNA,and found four c DNA fragments with length of 1 527,1 743,1 425,1 718 bp,named RgIS1,RgIS2,RgIS3 and RgIS4,respectively. Bioinformatics analysis revealed that the four iridoid synthase genes encoding proteins with 389-392 amino acid residues,protein molecular weights were between 44. 30-44. 74 k Da,and theoretical isoelectric points were between 5. 30 and 5. 87. Subcellular localization predictions showed that the four iridoid synthase were distributed in the cytoplasm. Structure analysis revealed that R. glutinosa iridoid synthases contain six conserved short-chain dehydrogenase/reductase( SDR) motifs,and their 3 D models were composed typical dinucleotide-binding " Rossmann" folds covered by helical C-terminal extensions. Using the amino acid sequences of four R. glutinosa iridoid synthases,phylogenetic analysis was performed,the result indicated that RgIS3,CrIS and Olea europaea OeIS were grouped together,the other R. glutinosa iridoid synthases and fifteen proteins in other plants had close relationship. Real-time fluorescent quantitative PCR revealed that RgIS1 and RgIS3 highly expressed in unfold leaves,however,RgIS2 and RgIS4 highly expressed in stems and tuberous roots,respectively. RgIS3 showed higher expression levels in non-radial striations( nRS) of the two cultivars,and RgIS1 and RgIS2 had higher expression levels in nRS of QH,while RgIS4 had less expression levels in nRS of QH1. RgIS1,RgIS2 and RgIS3 were up-regulated by Me JA treatment,although the time and degree of response differed. Our findings are helpful to reveal molecular function of R. glutinosa iridoid synthases and provide a clue for studing the molecular mechanism of iridoid biosynthesis.

[Progress on germplasm enhancement and breeding of Rehmannia glutinosa].

The history of Rehmannia glutinosa breeding has already beyond 100 years. There are rich cultivated varieties and wild germplasm resources in R. glutinosa. However, there also exist a lot of problems, such as, the pedigree of the existing varieties is not clear, the genetic basis is narrow, backward method of germplasm enhancement and breeding. Breeding of new varieties has been unable to meet the demand of R. glutinosa production in the new era. This paper summarizes the species of Rehmannia and their distribution, the diversity of plant morphology and the quality of R. glutinosa germplasm resources, as well as the progress of R. glutinosa breeding in recent 100 years. For ensuring the orderly, effective and safe production of R. glutinosa, the authors suggest to establish the wild resources protection area and germplasm resources garden, deeply study the genetic base of quality, strengthen application of new breeding method such as mutation breeding, haploid breeding and gene editing.

[Effect of different initial processing methods on quality of gardenia].

To investigate the effect of different initial processing methods on the quality of Gardenia and determine the best cooking time in gardenia processing through the determination of index components content. The contents of geniposide, crocetin Ⅰ and total iridoid glycosides in Gardenia were determined before storage, six months after storage and one year after storage. During storage, the contents of geniposide, crocetin Ⅰ and total iridoid glycosides in directly dried Gardenia were 1.68%, 0.45% and 6.45% respectively. The contents of geniposide, crocetin Ⅰ and total iridoid glycosides in Gardenia with different steaming time were 1.34%-0.5%, 0.28%-0.06% and 6.09%-1.59% respectively. The contents of geniposide, crocetin Ⅰ and total iridoid glycosides in Gardenia with different boiling time (adding alum)were 1.42%-0.41%, 0.35%-0.07% and 6.40%-1.65% respectively. The direct drying of Gardenia samples could not achieve the function of killing enzyme and protecting glycosides. The enzymes from degradation of the index components were basically destroyed after steaming time of 13 min or boiling (adding alum) time of 8 min, achieving the function of killing enzyme and protecting glycosides.

Effect of neuromuscular electrical stimulation for endometriosis-associated pain: A retrospective study.

This retrospective study evaluated the effect of neuromuscular electrical stimulation (NMES) for the treatment of endometriosis-associated pain (EAP).A total of 154 patients with EAP were included and were divided into 2 groups in this retrospective study. Eighty-three patients were assigned a treatment group, and underwent NMES therapy, while 71 subjects in the control group were at waiting list. The primary outcome of pain was measured by the numerical rating scale (NRS) and the Endometriosis Symptom Severity scale (ESSS). The secondary outcome was quality of life, measured by the 36-Item Short Form Health Survey (SF-36). All outcomes were measured before and after 5-week and 10-week treatment. Moreover, we also recorded the adverse events in this study.After 5-week treatment, no significant differences in all outcome measurements were found between the 2 groups. However, after 10-week treatment, NMES therapy exerted better outcomes in NRS (P = .02), ESSS (P = .04), and SF-36 [Physical Component Summary (PCS), P < .01; Mental Component Summary (MCS), P < .01], compared with the patients at the waiting list. Moreover, no significant differences of all adverse events were found between the 2 groups, although mild and acceptable adverse events occurred in the treatment group.This study demonstrated that NMES is effective for treating patients with EAP.

[Comparison of chemical quality characteristics between radial striations and non-radial striations in tuberous root of Rehmannia glutinosa].

An HPLC method was established to determine the contents of catalpol, acteoside, rehmaionoside A, rehmaionoside D, leonuride in three part of Rehmanni glutinosa in Beijing No.1 variety R. glutinosa during the growth period, This method, in combination with its HPLC fingerprint was used to evaluate its overall quality characteristics.The results showed that：① the content of main components of R. glutinosa varied in different growth stages ;② there was a great difference of the content of main components between theradial striations and the non-radial striations; ③ the two sections almost have the same content distribution of catalpol, acteoside and rehmaionoside D; ④the content of rehmaionoside A in non-radial striations was higher than that in radial striations,while the content of leonuride in radial striations was higher than that in non-radial striations.; ⑤the HPLC fingerprint of radial striations, non-radial striations and whole root tuber were basically identical, except for the big difference in the content of chemical components. The result of clustering displayed that the radial striations, non-radial striations, and whole root were divided into two groups. In conclusion, there was a significant difference in the quality characteristics of radial striations and non-radial striations of R. glutinosa. This research provides a reference for quality evaluation and geoherbalism of R. glutinosa.

[Cloning and expression analysis of a tyrosine decarboxylase gene from Rehmannia glutinosa].

Tyrosine decarboxylase (TyrDC) is an important enzyme in the secondary metabolism of several plant species, and was hypothesized to play a key role in the biosynthesis of phenylethanoid glycosides. Based on the transcriptome data, we cloned the full-length cDNA (GenBank accession NO. KU640395) of RgTyDC gene from Rehmannia glutinosa, and then performed bioinformatic analysis of the sequence. Further, we detected the expression pattern in different organs and hair roots treated with four elicitors by qRT-PCR. The results showed that the full length of RgTyDC cDNA was 1 530 bp encoding 509 amino acids. The molecular weight of the putative RgTyDC protein was about 56.6 kDa and the theoretical isoelectric point was 6.25. The RgTyDC indicated the highest homology with Sesamum indicum SiTyDC and Erythranthe guttata EgTyDC, both of them were reached 88%. RgTyDC highly expressed in R. glutinosa leaf, especially in senescing leaf, and rarely expressed in tuberous root. After the treatment of SA and MeJA, the relative expression level of RgTyDC mRNA was substantially increased. The results provide a foundation for exploring the molecular function of RgTyDC involved in phenylethanoid glycosides biosynthesis.

[Interactions between Hemoglobin and Other Proteins were Proved by the Electrophoresis Release Test and Co-immunoprecipitation].

OBJECTIVE: To study the interactions between hemoglobin (Hb) and other proteins within human erythrocytes by using the electrophoresis release test (ERT) and co-immunoprecipitation. METHODS: First, the fresh normal adult anti-coagulated whole blood was washed to prepare packed RBCs, which were further prepared to erythrocyte suspension and hemolysate.The erythrocyte suspension and hemolysate were analyzed by the electrophoresis release test (ERT) at the same time, and then the band of HbA of erythrocyte sample (RA) and the corresponding band of hemolysate sample (HA) were cut out from the gel and were enriched by freeze-thaw method. Then, the samples were bound with hemoglobin ß antibody (37-8) AC, the complexes were separated through 5%-12% SDS-PAGE followed by Q-TOF. RESULTS: Five bands were found in the gel, each of which was treated by hemoglobin ß antibody (37-8) AC, the protein bands of 16,20,22,28 and 50 kD were emerged in RA, HA and RBC lysis.The bands were identified by MS, and the results showed that these bands were hemoglobin, band 3, peroxiredoxin2 (Prx2), band 3 and ß-actin, band 3 respectively. CONCLUSION: HbA may interact with Prx2, Band 3 and ß-actin, and then the complexes are formed with each other within erythrocytes.

Poly[[diaqua-bis-(µ(3)-3,5-dicarb-oxy-benzo-ato-κ(3)O(1):O(3):O(5))bis-(µ(3)-5-carb-oxy-ben-zene-1,3-dicarboxyl-ato-κ(3)O(1):O(3):O(5))tetrakis(methylformamide-κO)tri-man-ganese(II)] dimethyl-formamide tetra-solvate].

In the title complex, {[Mn(3)(C(9)H(4)O(6))(2)(C(9)H(5)O(6))(2)(C(3)H(7)NO)(4)(H(2)O)(2)]·4C(3)H(7)NO}(n), one Mn(II) ion sits on an inversion center, and is six-coordinated by four O atoms from four anions (monoanionic and dianionic) derived from benzene-1,3,5-tricarboxylic acid and by two dimethyl-formamide (DMF) mol-ecules in a slightly distorted octa-hedral geometry. The other Mn(II) ion is six-coordinated by four O atoms from four monoanionic and dianionic ligands, one DMF mol-ecule and one water mol-ecule in a distorted octa-hedral geometry. The monoanionic and dianionic ligands bridge the Mn(II) ions, resulting in the formation of a layered structure parallel to (111) in which all of the carboxyl-ate groups of the anionic ligands coordinate the Mn(II) ions in a monodentate manner. Intra- and inter-molecular O-H⋯O hydrogen bonds are present in the structure.

Down-regulation of fecal miR-143 and miR-145 as potential markers for colorectal cancer.

OBJECTIVE: To detect 4 MicroRNA (miRNA) in the stool samples of colorectal cancer (CRC) patients to determine whether these miRNAs could be biomarkers in CRC screening or treatment. METHODS: A retrospective comparison study was carried out in the Department of Colorectal Surgery, Xinhua Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, China from September 2009 to March 2011. We detected 4 miRNAs (miR-143, miR-145, miR-21, and miR-106a) in the stool samples of 38 CRC patients and 13 healthy individuals. Total RNA from the stool samples was extracted using the EZNA TM stool RNA kit R6828-01. The miRNA quantification was carried out using TaqMan miRNA assays and the TaqMan Gene Expression Master Mix. RESULTS: The expression levels of miR-143 and miR-145 in the stool of the CRC patients were lower than in those of the healthy persons (p<0.005, median of 2-∆∆ct). No statistically significant difference was found in the expression levels of both miR-21 and miR-106a between the stool of CRC patients and those of the healthy persons (p>0.05). CONCLUSION: The detection of fecal miRNAs is a potential method for CRC diagnosis or screening. Particularly, the down-regulation of fecal miR-143 and miR-145 could be a potential marker for CRC.

Bis[2-(2-fur-yl)-1-(2-furylmeth-yl)-1H-benzimidazole-κN]diiodidocadmium.

In the title complex, [CdI(2)(C(16)H(12)N(2)O(2))(2)], the Cd(II) atom is located on a twofold rotation axis and is four-coordinated by two N atoms from symmetry-related 2-(2-fur-yl)-1-(2-furyl-meth-yl)-1H-benzimidazole ligands and two I atoms in a distorted tetra-hedral configuration. The benzimidazole rings in adjacent mol-ecules are parallel, with an average inter-planar distance of 3.486 Å. The I atom is disordered over two sites in a 0.85 (5):0.15 (5) ratio.

[Analysis of biochemical indices and efficient ingredients in autotetraploid of Dioscorea zingibiernsis].

OBJECTIVE: To improve the yield and quality of Dioscrea zingibiernsis. METHODS: The Biochemical indices and the diosgenin's content were analysed in the autotetraploid lines. RESULTS: The results showed that the activities of APX, SOD and POD in most of autotetraploid lines were higher than that in diploid line or close to it, there was also difference between autotetraploid lines and control lines in the SDS-PAGE of soluble proteins. The content of diosgenin in most autotetraploid plantlets were higher than that in the control. CONCLUSION: There were difference between autotetraploid and control lines in the content of diosgenin and Biochemical indices, therefore, inducing autotetraploid could be an effective way to breeding the superior varieties.