Risk assessment for biopharmaceutical single-use manufacturing: A case study of upstream continuous processing.

In the current transition to intensified upstream processing, the risks of adopting traditional single-use systems for high-titer, long-duration perfusion cultures, have thus far not been considered. This case study uses the Failure Modes and Effects Analysis (FMEA) method to evaluate the risks associated with implementing upstream single-use technology. The simulated model process was used to compare the risk level of single-use technology for a traditional fed-batch cell culture with that for perfusion culture, under the same annual protein production conditions. To provide a reasonable source of potential risk for FMEA, all single-use upstream operations for both fed-batch and perfusion processes were investigated using an analytical method developed to quantify the impact of process parameters and operating conditions on single-use system specifications and to ensure objectivity. Many of the risks and their levels, were similar in long-duration perfusion cultures and fed-batch cultures. However, differences were observed for high-risk components such as daily sampling and installation. The result of this analysis indicates that the reasons for risk are different for fed-batch cultures and perfusion cultures such as larger bioreactors in fed-batch and longer runs in perfusion, respectively. This risk assessment method could identify additional control measures and be part of a holistic contamination control strategy and help visualize their effectiveness.

Response of nutrient loss to natural erosive rainfall events under typical tillage practices of contour ridge system in the rocky mountain areas of Northern China.

Changes in natural rainfall characterized by heavy precipitation and high rainfall intensity would increase the risks and uncertainty of nutrients losses. Losses of nitrogen (N) and phosphorus (P) with water erosion from agriculture-related activities has become the principal nutrients resulting the eutrophication of water bodies. However, a little attention has been paid to the loss characteristic of N and P responding to natural rainfall in widely used contour ridge systems. To explore the loss mechanism of N and P in contour ridge system, nutrient loss associated with runoff and sediment yield was observed in in situ runoff plots of sweet potato (SP) and peanut (PT) contour ridges under natural rainfall. Rainfall events were divided into light rain, moderate rain, heavy rain, rainstorm, large rainstorm, and extreme rainstorm level, and rainfall characteristics for each rainfall level were recorded. Results showed that rainstorm, accounting for 46.27% of the total precipitation, played a destructive role in inducing runoff, sediment yield, and nutrient loss. The average contribution of rainstorm to sediment yield (52.30%) was higher than that to runoff production (38.06%). Rainstorm respectively generated 43.65-44.05% of N loss and 40.71-52.42% of P loss, although light rain induced the greatest enrichment value for total nitrogen (TN, 2.44-4.08) and PO4-P (5.40). N and P losses were dominated by sediment, and up to 95.70% of the total phosphorus and 66.08% of TN occurred in sediment. Nutrient loss exhibited the highest sensitivity to sediment yield compared to runoff and rainfall variables, and a significant positive linear relationship was observed between nutrient loss and sediment yield. SP contour ridge presented higher nutrient loss than that in PT contour ridge, especially for P loss. Findings gained in this study provide references for the response strategies of nutrient loss control to natural rainfall change in contour ridge system.

The study on the feasibility of dietary supplementation with dimethyl silicone oil to prevent frothy rumen bloat in goats fed with high concentrate diets.

The current study was conducted to investigate the feasibility of high concentration diet (HCD) supplementation with Dimethyl Silicone Oil (DSO) to prevent frothy rumen bloat in goats. The treatments were control group (group C, feeding HCD) and test group (group T, feeding HCD supplemented with 0.1%DSO). The results showed that compared with the group C, the ruminal pH value, Microbial Crude Protein content of group T was extremely significantly higher (p < 0.01), the levels of acetic acid and propionic acid were significantly (p < 0.05) and extremely significantly (p < 0.01) lower in group T, respectively. The foam production and foam strength of the rumen fluid in the group T was extremely significantly lower (p < 0.01), the viscosity was extremely significantly (p < 0.01) higher than those of group C. The total gastrointestinal apparent digestibility of various nutrients, the rumen microbial relative abundance at the phylum level and genus level were not significantly different (p > 0.05). The results indicated that the supplementation of 0.1% DSO in HCD can significantly eliminate foam of the rumen fluid, and didn't disturb the ruminal microorganisms, no negatively affect on digestibility of nutrients in goats, thereby has the application prospect of preventing frothy rumen bloat.

The gas produced by rumen fermentation is wrapped in foam and cannot be discharged is the root cause of frothy bloat induced by a high concentration diet. In the present study, the feasibility of dietary supplementation with Dimethyl Silicone Oil (DSO) to prevent frothy bloat was preliminarily evaluated. The results indicated that DSO can significantly eliminate foam of the rumen fluid, and has not negatively effect on the ruminal microorganisms and the digestibility of nutrients in goats, thereby has the application prospect of preventing frothy bloat.

Influence of Potamogeton crispus harvesting on phosphorus composition of Lake Yimeng.

Harvesting is an important method used to control the overproduction of Potamogeton crispus in lakes. A three-year comparative field study was performed in a eutrophic lake (harvested area) and its connected lake (non-harvested area) to determine the effects of harvesting on the phosphorus (P) composition and environmental factors in the water and sediment. Results revealed that harvesting significantly reduced the dissolved total P and dissolved organic P (DOP) and increased the alkaline phosphatase activity and particulate P (PP) in the water. No significant differences were detected in the water total P (TP), soluble reactive P, chlorophyll-a, pH, and dissolved oxygen between the harvested and non-harvested areas. Sediment TP and organic P (OP) were significantly reduced in the harvested area. Harvesting changed the P composition in the water. In the non-harvested area, P was mainly formed by DOP (40%) in the water body, while in the harvested area, PP was the main water component (47%). Harvesting increased the proportion of inorganic P (IP) in the sediment and decreased the proportion of OP. In the water, the IP to TP ratio in the non-harvested and harvested areas were 58.26% and 63.51%, respectively. Our results showed that harvesting changed the P composition in the water and sediment. In the harvesting of submerged vegetation, our results can serve as a reference for the management of vegetation-rich lakes.

The replacement of bacitracin methylene disalicylate with Bacillus subtilis PB6 in the diet of male Cherry Valley Ducks reduces the feed conversion ratio by improving intestinal health and modulating gut microbiota.

In this study, we compared the impacts of Bacillus subtilis PB6 (BS) and bacitracin methylene disalicylate (BMD) on the growth performance, intestinal morphology, expression of tight connection protein, and cecal microbiota community of male ducks through a 42-d trial. Three-hundred and sixty male Cherry Valley meat-type ducklings (1-day-old) were distributed into 3 groups of 6 replicates: CON group (control, basal diet), BMD group (basal diet + 45 mg/kg BMD, active ingredient dose in the feed), and BS group (basal diet + 2 × 107 CFU/kg BS in the feed). Results showed that supplementing the diet with BS reduced the average daily feed intake (ADFI) during d 15 to 42 and d 1 to 42 compared with the CON group (P = 0.032). It also reduced feed conversion ratio (FCR) during d 15 to 42 and d 1 to 42 (P < 0.05) relative to the other groups. The ileal villus height (VH) and villus height /crypt depth ratio (V/C) were increased (P < 0.05) in both the BS and BMD groups, and the jejunal VH and V/C ratio were increased in the BS group (P < 0.05). Relative to the CON, BS supplementation was associated with numerical augmentation of goblet cells in the jejunal mucosa and upregulation of jejunal zonula occludens (ZO-1) and ileal mucin2 (P < 0.05) mRNA levels. Analysis showed a negative correlation between FCR (d 0-42) and VH, V/C, and the number of goblet cells in the jejunum (P < 0.05). Additionally, BMD or BS supplementation altered the alpha diversity of colonic microbiota (P < 0.05). Correlation analysis revealed that Butyricimonas, Enterobacteriaceae, Clostridiaceae, and Tannerellaceae were positively associated with the acetic acid and butyrate concentrations (P < 0.05). Taken together, the supplementation of BS in the diet of male ducks was conducive to reducing FCR by meliorating intestinal morphology, upregulating ZO-1 and mucin2 mRNA levels, regulating the abundance of microbiota, and metabolites, and having a greater effect than BMD supplementation.

Relationship between the coprecipitation of phosphorus-on-calcite by submerged macrophytes and the phosphorus cycle in water.

To assess potential phosphorus removal, we utilized Potamogeton crispus to determine the effects of calcium addition on phosphorus removal. Plastic film was used to block material exchange between the overlying water and the sediment, and we compared the experimental results with long-term monitoring results of Yimeng Lake, which contained a dense population of P. crispus. The results revealed that the first 10-40 days constituted a period of rapid P decrease, as P. crispus could effectively remove the phosphorus in the water through coprecipitation of CaCO3-P. The treatment groups indicated that P. crispus released calcium into the overlying water, and after the addition of calcium ions, P. crispus showed increased phosphorus removal efficiency in the water. Total phosphorus (TP) and P/Ca content increased with increasing pH in the treatment groups, and the TP and pH declined as the calcium content increased in the treatment groups. Long-term field observations showed that the calcium-to-phosphorus ratio in the coprecipitates was dependent on the pH during the crystallization process. Thus, water calcium driven by P. crispus plays an important role in the phosphorus cycle of water, due to P. crispus assisted precipitation. This study revealed the effect of P. crispus on the water purification, the migration and transformation of Ca and P in sediment and overlying water under the condition of sediment calcium addition, so as to provide a theoretical basis for the ecological restoration of shallow lakes eutrophication.

Nutrients and Environmental Factors Cross Wavelet Analysis of River Yi in East China: A Multi-Scale Approach.

The accumulation of nutrients in rivers is a major cause of eutrophication, and the change in nutrient content is affected by a variety of factors. Taking the River Yi as an example, this study used wavelet analysis tools to examine the periodic changes in nutrients and environmental factors, as well as the relationship between nutrients and environmental factors. The results revealed that total phosphorus (TP), total nitrogen (TN), and ammonia nitrogen (NH4+-N) exhibit multiscale oscillation features, with the dominating periods of 16-17, 26, and 57-60 months. The continuous wavelet transform revealed periodic fluctuation laws on multiple scales between nutrients and several environmental factors. Wavelet transform coherence (WTC) was performed on nutrients and environmental factors, and the results showed that temperature and dissolved oxygen (DO) have a strong influence on nutrient concentration fluctuation. The WTC revealed a weak correlation between pH and TP. On a longer period, however, pH was positively correlated with TN. The flow was found to be positively correct with N and P, while N and P were found to be negatively correct with DO and electrical conductance (EC) at different scales. In most cases, TP was negatively correlated with 5-day biochemical oxygen demand (BOD5) and permanganate index (CODMn). The correlation between TN and CODMn and BOD5 was limited, and no clear dominant phase emerged. In a nutshell, wavelet analysis revealed that water temperature, pH, DO, flow, EC, CODMn, and BOD5 had a pronounced influence on nutrient concentration in the River Yi at different time scales. In the case of the combination of environmental factors, pH and DO play the largest role in determining nutrient concentration.

Comparison of coated and uncoated trace elements on growth performance, apparent digestibility, intestinal development and microbial diversity in growing sheep.

The suitable supplement pattern affects the digestion and absorption of trace minerals by ruminants. This study aimed to compare the effects of coated and uncoated trace elements on growth performance, apparent digestibility, intestinal development and microbial diversity in growing sheep. Thirty 4-month-old male Yunnan semi-fine wool sheep were randomly assigned to three treatments (n = 10) and fed with following diets: basal diet without adding exogenous trace elements (CON), basal diet plus 400 mg/kg coated trace elements (CTE, the rumen passage rate was 65.87%) and basal diet plus an equal amount of trace elements in uncoated form (UTE). Compared with the CON group, the average daily weight gain and apparent digestibility of crude protein were higher (P < 0.05) in the CTE and UTE groups, while there was no difference between the CTE and UTE groups. The serum levels of selenium, iodine and cobalt were higher (P < 0.05) in the CTE and UTE groups than those in the CON group, the serum levels of selenium and cobalt were higher (P < 0.05) in the CTE group than those in the UTE group. Compared with the CON and UTE groups, the villus height and the ratio of villus height to crypt depth in duodenum and ileum were higher (P < 0.05) in the CTE groups. The addition of trace minerals in diet upregulated most of the relative gene expression of Ocludin, Claudin-1, Claudin-2, ZO-1, and ZO-2 in the duodenum and jejunum and metal ion transporters (FPN1 and ZNT4) in small intestine. The relative abundance of the genera Christensenellaceae R-7 group, Ruminococcus 1, Lachnospiraceae NK3A20 group, and Ruminococcaceae in ileum, and Ruminococcaceae UCG-014 and Lactobacillus in colon was higher in the CTE group that in the CON group. These results indicated that dietary trace mineral addition improved the growth performance and intestinal development, and altered the structure of intestinal bacteria in growing sheep. Compared to uncoated form, offering trace mineral elements to sheep in coated form had a higher absorption efficiency, however, had little effect on improving growth performance of growing sheep.

Effects of dry yeast supplementation on growth performance, rumen fermentation characteristics, slaughter performance and microbial communities in beef cattle.

This study aimed to investigate the effects of active dry yeast (ADY) on growth performance, rumen microbial composition and carcass performance of beef cattle. Thirty-two finishing beef cattle (yak ♂ × cattle-yaks ♀), with an average body weight of 110 ± 12.85 kg, were randomly assigned to one of four treatments: the low plane of nutrition group (control), low plane of nutrition group + ADY 2 g/head daily (ADY2), low plane of nutrition group + ADY 4 g/head daily (ADY4) and the high plane of nutrition group (HPN). Supplementation of ADY increased average daily gain compared to the control group. The neutral detergent fiber and acid detergent fiber apparent digestibility in HPN group was greater than that in control group. The propionic acid concentration in the rumen in ADY2, ADY4, and HPN groups was greater than that in control group. The Simpson and Shannon indexes in control and HPN groups were higher than that in ADY4 group. At the phylum level, the relative abundance of Firmicutes in the HPN group was higher than that in ADY4 group. The relative abundance of Ruminococcaceae UCG-002 in ADY4 group was higher than that in control and HPN groups. In conclusion, supplementation ADY 4 g/head daily shift the rumen microbial composition of beef cattle fed low plane of nutrition to a more similar composition with cattle fed with HPN diet and produce the similar carcass weight with HPN diet.HighlightsThe ADY can improve the utilization of nitrogen and decrease the negative impact on the environment in beef cattle.Cattle fed low plane of nutrition diet supplemented with ADY 4 g/head daily increased growth performance.Supplementation ADY 4 g/head daily in low plane of nutrition diet might be produced comparable carcass weight to HPN diet.

Chemometric Analyses for the Characterization of Raw and Stir-Frying Processed Drynariae Rhizoma Based on HPLC Fingerprints.

The processing of traditional Chinese medicine (TCM) is a necessary practice and usually occurs before most herbs are prescribed. According to Chinese medicine theory, raw (RDR) and stir-frying processed (PDR) Drynariae Rhizoma have different clinical applications. The purpose of this study was to establish HPLC fingerprints coupled with chemometric methods to evaluate the differences between RDR and PDR. Multivariate chemometric methods were applied to analyze the obtained HPLC fingerprints, including hierarchical cluster analysis (HCA), principle components analysis (PCA), and partial least squares discriminant analysis (PLS-DA). The results indicated that RDR and PDR samples showed a clear classification of the two groups, and seven chemical markers having great contributions to the differentiation were screened out. The findings suggested that 5-hydroxymethyl-2-furaldehyde (5-HMF) with a variable importance in the project (VIP > 1) can be used to differentiate between RDR and PDR. Moreover, 5-HMF, naringin, and neoeriocitrin were determined to evaluate their contents in RDR and PDR. The chemometrics combined with the quantitative analysis based on HPLC fingerprint results indicated that stir-frying processing may change the contents and types of components in Drynariae Rhizoma. These changes are probably responsible for the various pharmacological effects of RDR and PDR.

Lower Somatic Mutation Levels in the λ Light-Chain Repertoires with Chronic HBV Infection.

To investigate the characteristics of the immunoglobulin light-chain repertoires with chronic HBV infection, the high-throughput sequencing and IMGT/HighV-QUEST were adapted to analyze the κ (IgK) and λ (IgL) light-chain repertoires from the inactive HBV carriers (IHB) and the healthy adults (HH). The comparative analysis revealed high similarity between the κ light-chain repertoires of the HBV carriers and the healthy adults. Nevertheless, the proportion of IGLV genes with ≥90% identity as the germline genes was higher in the IgL light-chain repertoire of the IHB library compared with that of HH library (74.6% vs. 69.1%). Besides, the frequency of amino acid mutations in the CDR1 regions was significantly lower in the IgL light-chain repertoire of the IHB library than that of the HH library (65.52% vs. 56.0%). These results suggested the lower somatic mutation level in the IgL repertoire of IHB library, which might indicate the biased selection of IGLV genes in the IgL repertoire with chronic HBV infection. These findings might lead to a better understanding of the characteristics of the light-chain repertoires of HBV chronically infected individuals.

Glutamine supplementation affected the gut bacterial community and fermentation leading to improved nutrient digestibility in growth-retarded yaks.

This study evaluated the effects of glutamine supplementation on nutrient digestibility, immunity, digestive enzyme activity, gut bacterial community and fermentation of growth-retarded yaks. A total of 16 growth-retarded yaks were randomly allocated to two groups: negative control (GRY) and glutamine supplementation group (GLN). Another eight growth-normal yaks were used as a positive control (GNY). Compared with GRY group, the crude protein digestibility was higher in GLN and GNY animals and the neutral detergent fiber digestibility was increased in GLN yaks. The concentrations of serum IgA, IgG, IgM and IL-10, as well as butyrate concentration and cellulase activity in the rumen and cecum were higher in GLN yaks compared to those in GRY animals. Supplementation with glutamine enhanced the chymotrypsin activity and increased the relative abundances of unclassified Peptostreptococcaceae and Romboutsia while decreased the relative abundances of unclassified Chitinophagaceae and Bacteroides in the jejunum and ileum of growth-retarded yaks. In the cecum, the relative abundance of unclassified Muribaculaceae was higher in GLN group than that in GRY group. The findings in this study suggest that the improved nutrient digestibility and immunity of growth-retarded yaks with glutamine supplementation may be through its potential impact on the lower gut host and microbial functions.

Metabonomic Responses of Grazing Yak to Different Concentrate Supplementations in Cold Season.

Supplementation plays an important role in reversing the weight loss of grazing yaks during cold season. However, little is known about the effect of supplementation on the serum metabolites of grazing yaks. The objective of this study was to explore the effects of supplementary feeding on average daily gain (ADG) and serum metabolites with nuclear magnetic resonance (NMR)-based metabolomics method in growing yaks during cold season on the Qinghai-Tibetan plateau. Twenty 1.5-year-old female yaks (91.38 ± 10.43 kg LW) were evenly divided into three treatment groups and a control group (CON) (n = 5 per group). All the yaks were released to graze during daytime, whereas the yaks in the treatment groups were supplemented with highland barley (HLB), rapeseed meal (RSM), and highland barley plus rapeseed meal (HLB + RSM) at night. The whole experiment lasted for 120 days. Results indicated that the ADG of growing yak heifers was increased by concentrate supplementations, and ADG under HLB and HLB + RSM group was 37.5% higher (p < 0.05) than that with RSM supplementation. Supplementary feeding increased the plasma concentrations of total protein (TP), albumin (ALB), and blood urea nitrogen (BUN) of those in the CON group, and concentrations of BUN were higher in the RSM group than in the HLB and HLB + RSM group. Compared with the CON group, serum levels of glutamine, glycine, ß-glucose were lower and that of choline was higher in the HLB group; serum levels of lactate were lower and that of choline, glutamate were higher in the HLB + RSM group. Compared with the HLB + RSM group, serum levels of glycerophosphoryl choline (GPC) and lactate were higher, and those of choline, glutamine, glutamate, leucine, N-acetyaspartate, α-glucose, and ß-glucose were lower in the HLB group; serum levels of citrate, GPC and lactate were higher, and those of 3-Hydroxybutyrate, betaine, choline, glutamate, glutamine, N-acetylglycoprotein, N-acetyaspartate, α-glucose, and ß-glucose were lower in the RSM group. It could be concluded that concentrate supplementations significantly improved the growth performance of growing yaks and supplementation with HBL or HLB plus RSM was better than RSM during the cold season. Supplementation with HBL or HLB plus RSM affected the serum metabolites of grazing yaks, and both treatments promoted lipid synthesis. Supplementation of yaks with HBL plus RSM could improve energy-supply efficiency, protein and lipid deposition compared with HLB and RSM.

Ten New Dammarane-Type Saponins with Hypolipidemia Activity from a Functional Herbal Tea-Gynostemma pentaphyllum.

Gynostemma pentaphyllum (thumb.) Makino is a functional herbal tea commonly used in Asian countries and regions to reduce blood lipid levels. G. pentaphyllum saponin is the main component, but there are still a large number of components with lipid-lowering activity that have not been found. In this study, 10 novel dammarane-type saponins, (1-10) and a known one (11) were isolated from G. pentaphyllum. Ten new compounds were identified and named as yunnangypenosides A-J (1-10), and another known one (11) was also obtained. Their chemical structures were determined by MS, NMR spectroscopic analyses. Moreover, the cytotoxicities on human HepG-2 hepatocellular carcinoma cells of these isolates were evaluated, and the results showed that compounds 1-11 had no obvious cytotoxicity. Finally, all these compounds were evaluated for their lipid-lowering effect by means of the oil red O staining method. Ten compounds could significantly reduce lipid levels except of 2, especially 8 exhibite the strongest hypolipidemia activity.

Combining DNA Barcoding and HPLC Fingerprints to Trace Species of an Important Traditional Chinese Medicine Fritillariae Bulbus.

Fritillariae Bulbus is a precious Chinese herbal medicine that is grown at high elevation and used to relieve coughs, remove phlegm, and nourish the lungs. Historically, Fritillariae Bulbus has been divided into two odourless crude drugs: Fritillariae Cirrhosae Bulbus and Fritillariae Thunbergii Bulbus. However, now the Chinese Pharmacopoeia has described five Fritillariae Bulbus-the new additions include Fritillariae Pallidiflorae Bulbus, Fritillariae Ussuriensis Bulbus, and Fritillariae Hupehensis Bulbus. Because the morphology of dried Fritillariae Bulbus is similar, it is difficult to accurately identify the different types of Fritillariae Bulbus. In the current study, we develop a method combining DNA barcoding and high-performance liquid chromatography (HPLC) to help distinguish Fritillariae Cirrhosae Bulbus from other Fritillariae Bulbus and guarantee species traceability of the five types of Fritillariae Bulbus. We report on the validation of an integrated analysis method for plant species identification using DNA barcoding that is based on genetic distance, identification efficiency, inter- and intra-specific variation, calculated nearest distance, neighbour-joining tree and barcoding gap. Our results show that the DNA barcoding data successfully identified the five Fritillariae Bulbus by internal transcribed spacer region (ITS) and ITS2, with the ability to distinguish the species origin of these Fritillariae Bulbus. ITS2 can serve as a potentially useful DNA barcode for the Fritillaria species. Additionally, the effective chemical constituents are identified by HPLC combined with a chemical identification method to classify Fritillaria. The HPLC fingerprint data and HCA (hierarchical clustering analysis) show that Fritillariae Cirrhosae Bulbus is clearly different from Fritillariae Thunbergii Bulbus and Fritillariae Hupehensis Bulbus, but there is no difference between Fritillariae Cirrhosae Bulbus, Fritillariae Ussuriensis Bulbus, and Fritillariae Pallidiflorae Bulbus. These results show that DNA barcoding and HPLC fingerprinting can discriminate between the five Fritillariae Bulbus types and trace species to identify related species that are genetically similar.

Hybrid sequencing of the Gynostemma pentaphyllum transcriptome provides new insights into gypenoside biosynthesis.

BACKGROUND: Gypenosides are a group of triterpene saponins from Gynostemma pentaphyllum that are the same as or very similar to ginsenosides from the Panax species. Several enzymes involved in ginsenoside biosynthesis have been characterized, which provide important clues for elucidating the gypenoside biosynthetic pathway. We suppose that gypenosides and ginsenosides may have a similar biosynthetic mechanism and that the corresponding enzymes in the two pathways may have considerable similarity in their sequences. To further understand gypenoside biosynthesis, we sequenced the G. pentaphyllum transcriptome with a hybrid sequencing-based strategy and then determined the candidate genes involved in this pathway using phylogenetic tree construction and gene expression analysis. RESULTS: Following the PacBio standard analysis pipeline, 66,046 polished consensus sequences were obtained, while Illumina data were assembled into 140,601 unigenes with Trinity software. Then, these output sequences from the two analytical routes were merged. After removing redundant data with CD-HIT software, a total of 140,157 final unigenes were obtained. After functional annotation, five 2,3-oxidosqualene cyclase genes, 145 cytochrome P450 genes and 254 UDP-glycosyltransferase genes were selected for the screening of genes involved in gypenoside biosynthesis. Using phylogenetic analysis, several genes were divided into the same subfamilies or closely related evolutionary branches with characterized enzymes involved in ginsenoside biosynthesis. Using real-time PCR technology, their expression patterns were investigated in different tissues and at different times after methyl jasmonate induction. Since the genes in the same biosynthetic pathway are generally coexpressed, we speculated that GpOSC1, GpCYP89, and GpUGT35 were the leading candidates for gypenoside biosynthesis. In addition, six GpWRKYs and one GpbHLH might play a possible role in regulating gypenoside biosynthesis. CONCLUSIONS: We developed a hybrid sequencing strategy to obtain longer length transcriptomes with increased accuracy, which will greatly contribute to downstream gene screening and characterization, thus improving our ability to elucidate secondary metabolite biosynthetic pathways. With this strategy, we found several candidate genes that may be involved in gypenoside biosynthesis, which laid an important foundation for the elucidation of this biosynthetic pathway, thus greatly contributing to further research in metabolic regulation, synthetic biology and molecular breeding in this species.

The Oleaceae family: A source of secoiridoids with multiple biological activities.

In the quest to search and discover bioactive compounds from nature, terpenoids have emerged as one of the most interesting and researched classes of compounds. Secoiridoid, a type of the terpenoid, has also been extensively studied, especially their chemical structures and pharmacological effects. Oleaceae is a family of woody dicotyledonous plants with broad economic and medicinal values. This family contains a large number of flavonoids, monoterpenoids, iridoids, secoiridoids and phenylethyl alcohols, of which the secoiridoids have various biological activities. The purpose of this review is to summarize the phytochemical and pharmacological of the secoiridoids (glycosides, aglycones, derivatives and dimers) in the Oleaceae family from 1987 to 2018. This review will also serve as a reference for further studies.

[Effects of Sediment Burial and Exogenous Cd Input on Biomass Allocation and Antioxidative Enzyme Activities of Suaeda salsa in the Coastal Wetland of the Yellow River Delta].

The Yellow River Delta has been facing the threat of functional degradation during the recent years. The Water-Sediment Regulation Project not only supplements abundant freshwater, but also alters the sediment burial and heavy metal levels, which affects vegetation growth. Thus, we selected the pioneer species Suaeda salsa, to study the effects of different sediment burial depths (0, 3, 6, 12 cm) and exogenous Cd inputs (0, 0.5, 1.0, 1.5 mg·kg-1) on biomass allocation and activities of antioxidative enzymes in the coastal wetlands of the Yellow River delta. The results showed that a shallow or moderate burial depth had a stimulatory effect on chlorophyll content, while an excessive burial depth inhibited the growth of Suaeda salsa and chlorophyll content. With increasing Cd input, chlorophyll content and dry mass decreased. At a lower Cd input and moderate burial depth, activities of CAT and SOD increased, and at high levels, SOD activities decreased, while activities of CAT at a 12 cm burial depth and 1.0 mg·kg-1, 1.5 mg·kg-1 Cd input were higher than those for the control (62.66% and 58.56%). CAT activities reached high values (15.76 U·mg-1) at a high Cd input (1.5 mg·kg-1) and burial depth (12 cm). Analysis of variance showed that Cd input had a significant effect on protein content, and CAT and SOD activities, and sediment burial depth had a significant effect on the protein content and SOD activities. Interaction between Cd input and sediment burial depth had a significant effect on CAT and SOD activities (P<0.05). These results demonstrated that sediment burial depth and Cd input had a great influence on the growth of Suaeda salsa, and to some extent, Suaeda salsa could change its biomass allocation and antioxidative enzyme activities to adapt to severe environments.

Effects of dietary supplementation of active dried yeast on fecal methanogenic archaea diversity in dairy cows.

This study aimed to investigate the effects of dietary supplementation of different dosages of active dried yeast (ADY) on the fecal methanogenic archaea community of dairy cattle. Twelve multiparous, healthy, mid-lactating Holstein dairy cows (body weight: 584 ± 23.2 kg, milk produced: 26.3 ± 1.22 kg/d) were randomly assigned to one of three treatments (control, ADY2, and ADY4) according to body weight with four replicates per treatment. Cows in the control group were fed conventional rations without ADY supplementation, while cows in the ADY2 and ADY4 group were fed rations supplemented with ADY at 2 or 4 g/d/head. Real-time PCR analysis showed the populations of total methanogens in the feces were significantly decreased (P < 0.05) in the ADY4 group compared with control. High-throughput sequencing technology was applied to examine the differences in methanogenic archaea diversity in the feces of the three treatment groups. A total of 155,609 sequences were recovered (a mean of 12,967 sequences per sample) from the twelve fecal samples, which consisted of a number of operational taxonomic units (OTUs) ranging from 1451 to 1,733, were assigned to two phyla, four classes, five orders, five families and six genera. Bioinformatic analyses illustrated that the natural fecal archaeal community of the control group was predominated by Methanobrevibacter (86.9% of the total sequence reads) and Methanocorpusculum (10.4%), while the relative abundance of the remaining four genera were below 1% with Methanosphaera comprising 0.8%, Thermoplasma composing 0.4%, and the relative abundance of Candidatus Nitrososphaera and Halalkalicoccus being close to zero. At the genus level, the relative abundances of Methanocorpusculum and Thermoplasma were increased (P < 0.05) with increasing dosage of ADY. Conversely, the predominant methanogen genus Methanobrevibacter was decreased with ADY dosage (P < 0.05). Dietary supplementation of ADY had no significant effect (P > 0.05) on the abundances of genera unclassified, Candidatus Nitrososphaera, and Halalkalicoccus. In conclusion, supplementation of ADY to the rations of dairy cattle could alter the population sizes and composition of fecal methanogenic archaea in the feces of dairy cattle. The decrease in Methanobrevibacter happened with a commensurate increase in the genera Methanocorpusculum and Thermoplasma.

The Complete Chloroplast Genome Sequence of the Medicinal Plant Swertia mussotii Using the PacBio RS II Platform.

Swertia mussotii is an important medicinal plant that has great economic and medicinal value and is found on the Qinghai Tibetan Plateau. The complete chloroplast (cp) genome of S. mussotii is 153,431 bp in size, with a pair of inverted repeat (IR) regions of 25,761 bp each that separate an large single-copy (LSC) region of 83,567 bp and an a small single-copy (SSC) region of 18,342 bp. The S. mussotii cp genome encodes 84 protein-coding genes, 37 transfer RNA (tRNA) genes, and eight ribosomal RNA (rRNA) genes. The identity, number, and GC content of S. mussotii cp genes were similar to those in the genomes of other Gentianales species. Via analysis of the repeat structure, 11 forward repeats, eight palindromic repeats, and one reverse repeat were detected in the S. mussotii cp genome. There are 45 SSRs in the S. mussotii cp genome, the majority of which are mononucleotides found in all other Gentianales species. An entire cp genome comparison study of S. mussotii and two other species in Gentianaceae was conducted. The complete cp genome sequence provides intragenic information for the cp genetic engineering of this medicinal plant.