Meyerozyma guilliermondii promoted the deposition of GSH type lignin by activating the biosynthesis and polymerization of monolignols at the wounds of potato tubers.

Lignin is a crucial component in the wound tissue of tubers. The biocontrol yeast Meyerozyma guilliermondii increased the activities of phenylalanine ammonia lyase, cinnamate-4-hydroxylase, 4-coenzyme coenzyme A ligase, and cinnamyl alcohol dehydrogenase, and elevated the levels of coniferyl, sinapyl, and p-coumaryl alcohol. The yeast also enhanced the activities of peroxidase and laccase, as well as the content of hydrogen peroxide. The lignin promoted by the yeast was identified as guaiacyl-syringyl-p-hydroxyphenyl type using Fourier transform infrared spectroscopy and two-dimensional heteronuclear single quantum coherence nuclear magnetic resonance. Furthermore, a larger signal area for G2, G5, G'6, S2, 6, and S'2, 6 units was observed in the treated tubers, and the G'2 and G6 units were only detected in the treated tuber. Taken together, M. guilliermondii could promote deposition of guaiacyl-syringyl-p-hydroxyphenyl type lignin by activating the biosynthesis and polymerization of monolignols at the wounds of potato tubers.

Flavonoid Extract from Seed Residues of Hippophae rhamnoides ssp. sinensis Protects against Alcohol-Induced Intestinal Barrier Dysfunction by Regulating the Nrf2 Pathway.

Alcohol has been demonstrated to disrupt intestinal barrier integrity. Some flavonoid compounds that exert antioxidant activity have a protective effect on intestinal barrier function. As an important medicinal and edible plant, sea buckthorn (Hippophae) seeds are rich in flavonoids, but their protective effect on the intestinal barrier has not been reported. In our research, 76 kinds of flavonoids were identified in Hippophae rhamnoides ssp. sinensis seed residue flavonoids (HRSF) by ultra-performance liquid chromatography-tandem mass spectrometry. Kaempferol-3-O-rutinoside, isorhamnetin-3-O-rutinoside, kaempferol-3-O-robinoside-7-O-rhamnoside, isorhamnetin-3-O-2G-rhamnosylrutinoside, quercetin-3-O-rutinoside, (-)-epigallocatechin, and B type of procyanidin were the most abundant substances, accounting for 15.276%, 15.128%, 18.328%, 10.904%, 4.596%, 5.082%, and 10.079% of all identified flavonoids, respectively. Meanwhile, pre-treatment with HRSF was able to prevent alcohol-induced disruption of intestinal barrier integrity through elevating the transepithelial monolayer resistance value, inhibiting the flux of fluorescein isothiocyanate-dextran, and upregulating the mRNA and protein level of TJs (occludin and ZO-1). Furthermore, it was also able to reverse alcohol-induced oxidative stress through suppressing the accumulation of reactive oxygen species and malondialdehyde, improving the glutathione level and superoxide dismutase activity. Finally, the results showed that HRSF pre-treatment effectively elevated the erythroid-related factor 2 mRNA and protein level compared with the alcohol-alone treatment group. Our research was the first to demonstrate that HRSF could prevent alcohol-induced intestinal barrier dysfunction through regulating the Nrf2-mediated pathway in order to attenuate oxidative stress and enhance TJ expression.

Natural fermentation quality, bacteria, and functional profiles of three cuttings of alfalfa silage in a year in Inner Mongolia, China.

Alfalfa is harvested two or three times a year in central and western Inner Mongolia, China. However, the variations in bacterial communities as affected by wilting and ensiling, and the ensiling characteristics of alfalfa among the different cuttings, are not fully understood. To enable a more complete evaluation, alfalfa was harvested three times a year. At each time of cutting, alfalfa was harvested at early bloom, wilted for 6 h, and then ensiled in polyethylene bags for 60 days. The bacterial communities and nutritional components of fresh alfalfa(F), wilted alfalfa(W) and ensiled alfalfa(S), and the fermentation quality and functional profile of bacterial communities of the three cuttings alfalfa silage, were then analyzed. Functional characteristics of silage bacterial communities were evaluated according to the Kyoto Encyclopedia of Genes and Genomes. The results showed that all nutritional components, fermentation quality, bacterial communities, carbohydrate, amino acid metabolism and key enzymes of bacterial communities were influenced by cutting time. The species richness of F increased from the first cutting to the third cutting; it was not changed by wilting, but was decreased by ensiling. At phylum level, Proteobacteria were more predominant than other bacteria, followed by Firmicutes (0.063-21.39%) in F and W in the first and second cuttings. Firmicutes (96.66-99.79%) were more predominant than other bacteria, followed by Proteobacteria (0.13-3.19%) in S in the first and second cuttings. Proteobacteria, however, predominated over all other bacteria in F, W, or S in the third cutting. The third-cutting silage showed the highest levels of dry matter, pH and butyric acid (p < 0.05). Higher levels of pH and butyric acid were positively correlated with the most predominant genus in silage, and with Rosenbergiella and Pantoea. The third-cutting silage had the lowest fermentation quality as Proteobacteria were more predominant. This suggested that, compared with the first and second cutting, the third cutting is more likely to result in poorly preserved silage in the region studied.

Comparative Assessment of Functional Components and Antioxidant Activities between Hippophae rhamnoides ssp. sinensis and H. tibetana Berries in Qinghai-Tibet Plateau.

The Qinghai-Tibet Plateau is the main production area of Hippophae rhamnoides ssp. sinensis (Rha) and H. tibetana (Tib), but studies on the types and contents of soluble sugars, organic acids, free phenolics, bound phenolics, vitamin C (VC), tocopherol (VE) and carotenoids of the two sea buckthorn berries from this region have not been reported. In this paper, we found that the soluble sugars in Rha and Tib were mainly glucose and fructose; Rha exhibited a higher content of total sugar and fructose compared to Tib. The organic acids were mainly quinic acid and malic acid; Rha exhibited a higher content of total acids and quinic acid, but lower tartaric acid and citric acid compared to Tib. Rha also possessed a higher total (free and bound) phenolic as well as total (free and bound) flavonoid content than those in Tib; twelve phenolic compounds were analyzed, among which flavonols were dominant. Catechin, isorhamnetin and quercetin were the main phenolic substances. VC and VE (γ-tocopherol (γ-VE) and δ-tocopherol (δ-VE)) were higher in Rha than Tib. The total carotenoid, lutein, ß-carotene and lycopene content of Tib was remarkably higher than that in Rha. Moreover, both Rha and Tib showed good in vitro and cellular antioxidant activities, and Rha had a stronger antioxidant activity. Taken together, Rha had a higher antioxidant activity, which may be due to its higher content of phenolics, flavonoids, VC and VE.

Phenolic compositions and antioxidant activities of Hippophae tibetana and H. rhamnoides ssp. sinensis berries produced in Qinghai-Tibet Plateau.

Phenolic ingredients of Hippophae tibetana (Tib) and H. rhamnoides ssp. sinensis (Rha) berry from Qinghai-Tibet Plateau were identified by Ultra Performance Liquid Chromatography-triple Quadrupole Tandem Mass Spectrometry. Results demonstrated that both of them possessed high levels of total phenolic and flavonoid, and compared to Tib, Rha berry exhibited higher contents. Moreover, flavonols was the most predominant subclass in Rha berry, flavonols and flavanols were the two most abundant subclasses in Tib berry. Among them, rutin and narcissin were present in the most abundant amounts in Rha berry, while (-)-epigallocatechin was the richest substance in Tib berry. Furthermore, both phenolic extracts of sea buckthorn berry exhibited strong in vitro and cellular antioxidant properties. Rha berry extract exhibited much stronger effects because of its higher levels of phenolic and flavonoid profiles. This finding proved that the Rha berry could serve as a food source for better health with great potential antioxidant activity.

Analysis and estimation of nonpoint source pollution under different land use in Anjiagou watershed, Gansu, China.

Agriculture-related nonpoint source pollution has been a worldwide issue in the past few decades. Estimating pollutant sources at the basin scale remains a challenge due to the complexity of pollutant transport mechanisms affected by land use, variable climatic condition, geological formation, and lack of long-term observation data. This study was based on the long-term plot observational data of surface runoff and water quality and used principal component analysis and other statistical analyses to analyze the variation of water quality affected by different land uses (cropland, tree forest, shrub, grassland, and wildland). The mean concentration method with the local calibrated/verified SWAT (soil and water assessment tool) model was used to quantify the load of nonpoint source (NPS) pollutants on slope areas under different land uses in the Anjiagou Watershed. Our research results determined permanganate index (CODMn), ammonia nitrogen (NH3-N), total nitrogen (TN), fluoride (F-), nitrite nitrogen (NO2-N), total phosphorus (TP), and hexavalent chromium (Cr6+) as the significant pollutants while 5-day biochemical oxygen demand (BOD5) was identified to be below the water quality standards of Grade V (water for agricultural and general landscape use) in the studied watershed. Pollutants were discharged through either hillslope at a total rate of 2.4 kg ha-1, accounting for 67.6% of the total, or through waterway channels (32.4%). The pollutant concentrations were from 23.5 mg L-1 to 37.4 mg L-1, varying with pollutants and land uses. All examined water quality indicators exceeded the minimum safety standards defined by the regulations of the Gansu provincial government by averaging 3.5 times higher than the safety threshold. The pollutants from hillslopes exceeded water quality standards by a factor of 3.4-4.4 times compared with from the waterway channel by 1.9. Implementing soil and water conservation measures can mitigate pollutants to some extent, particularly during the process of highland runoff converging into waterways. At the watershed level, between 33 and 38% of the runoff and pollutants were discharged from croplands, between 59 and 66% from forest land, < 2% from grassland, and 1% from wildland. This study also demonstrates a simple but novel method to estimate NPS pollutants using long-term plot observations in conjunction with SWAT models, which can be used in other watersheds with similar conditions.

Effects of a Furrow-Bed Seeding System on Stand Establishment, Soil Bacterial Diversity, and the Yield and Quality of Alfalfa Under Saline Condition.

Salt stress account for large decreases in crop yield all over the world. Furrow-bed system is an efficient practice to promote plant growth in saline soil. However, the effects of Furrow-bed system on the soil environment and the growth of alfalfa (Medicago sativa L.) in salinity are not clear. For a wider and more detail evaluation, alfalfa were planted in saline sandy loam soil in fall, the effects of two plant systems (FU, furrow-bed seeding system; FL, flat-bed seeding system) on soil moisture, root zone salinity, soil microbial community structure, seedling emergence number in the early stage of the growth period and soil nutrient contents, alfalfa production characteristics in the second growth year were determined in a 2-year field experiment. The result showed that, compared with FL, FU resulted in increased soil moisture content and seedling emergence, and significantly reduced relative abundance of Actinobacteria and Choroflexi in soil, but it did not affect root zone salinity at the seedling stage. In April of second growth year, the soil salinity was lower, and the soil available phosphorus, potassium, nitrogen, and soil organic matter contents of the root zone were higher in FU than in FL. Compared with FL, FU resulted in increased yield (by 37.5%), protein content (by 3.6%), and potassium concentration (by 33.2%), and decreased ash content (by 7.7%), and sodium concentration (by 19.0%) in alfalfa plants. Pearson's correlation analysis indicated that the increased yield was positively correlated with seedling emergence, soil available potassium, total nitrogen, and organic matter contents, and shoot potassium content and negatively correlated with shoot sodium content. The relative abundance of Actinobacteria was negatively correlated with alfalfa ash, calcium, and sodium concentrations, and positively correlated with shoot potassium content. Taken together, the results indicate that Furrow-bed seeding in early fall alleviated salt stress of alfalfa and have the potential to enhance the yield and quality of alfalfa cultivated in saline soils by improving the soil environment and regulating the growth and physiology of alfalfa. Graphical Abstract.

Nutrient compositions and antioxidant capacity of kiwifruit (Actinidia) and their relationship with flesh color and commercial value.

The nutritional quality and antioxidant capacity of China domestic kiwifruit and imported kiwifruit with different prices and the relationships of retail price among cultivars, nutritional qualities and flesh color were investigated. Results showed that cultivar was the main factor influencing nutritional quality, though the product region could also affect the cultivar characteristics to some extent. Additionally, nutritional quality and antioxidant capacity of kiwifruit had no relationship to the product region. What's more, there was no correlation among the nutrient compositions, antioxidant capacity and retail price of kiwifruit. At the same time, the flesh color was associated with greater commercial value but was not higher nutritional quality or antioxidant capacity. Thus indicates to consumers that imported kiwifruit were not all superior to local versions, and the priciest kiwifruit did not equal to the most nutritious kiwifruit, also the color kiwifruit. There still need more research to help consumers to choose kiwifruit.

A novel wheat-Dasypyrum breviaristatum substitution line with stripe rust resistance.

The introduction of genetic variation from wild and cultivated Triticeae species has been a long-standing approach for wheat improvement. Dasypyrum breviaristatum species harbor novel and agronomically important genes for resistance against multi-fungal diseases. The development of new wheat-D. breviaristatum introgression lines offers chances for the identification of stripe rust resistance gene(s). A wheat line, D11-5, was selected from a cross between wheat line MY11 and wheat-D. breviaristatum partial amphiploid TDH-2. It was characterized by FISH and PCR-based molecular markers. Chromosome counting revealed that the D11-5 line shows a hexaploid set of 2n = 6x = 42 chromosomes. FISH analysis using the Dasypyrum repetitive sequence pDb12H as a probe demonstrated that D11-5 contained a pair of D. breviaristatum chromosomes, while FISH with wheat D-genomic repetitive sequences revealed that the chromosome 2D was absent in D11-5. The functional molecular markers confirmed that the introduced D. breviaristatum chromosomes belong to the homoeologous group 2, indicating that D11-5 was a 2V(b) (2D) disomic substitution line. Field resistance showed that the introduced D. breviaristatum chromosomes 2V(b) were responsible for the stripe rust resistance at the adult plant stage. FISH, C-banding, and PCR-based molecular marker analysis indicated that the chromosome 2V(b) of D. breviaristatum was completely different from the chromosome 2V of D. villosum. The identified wheat-D. breviaristatum chromosome substitution line D11-5 may be applied to produce agronomically desirable stripe rust resistance germplasm.

Identification of small non-coding RNAs in the planarian Dugesia japonica via deep sequencing.

Freshwater planarian flatworm possesses an extraordinary ability to regenerate lost body parts after amputation; it is perfect organism model in regeneration and stem cell biology. Recently, small RNAs have been an increasing concern and studied in many aspects, including regeneration and stem cell biology, among others. In the current study, the large-scale cloning and sequencing of sRNAs from the intact and regenerative planarian Dugesia japonica are reported. Sequence analysis shows that sRNAs between 18nt and 40nt are mainly microRNAs and piRNAs. In addition, 209 conserved miRNAs and 12 novel miRNAs are identified. Especially, a better screening target method, negative-correlation relationship of miRNAs and mRNA, is adopted to improve target prediction accuracy. Similar to miRNAs, a diverse population of piRNAs and changes in the two samples are also listed. The present study is the first to report on the important role of sRNAs during planarian Dugesia japonica regeneration.

Differential expression of microRNA patterns in planarian normal and regenerative tissues.

MicroRNAs (miRNAs) are ~22-nt small non-coding RNAs that regulate the expression of specific target genes in many eukaryotes. miRNAs have been shown to play important roles in stem cell maintenance, cell fate determination, and differentiation. Planarians are capable of regenerating entire body plans from tiny fragments; this regenerative capacity is facilitated by a population of pluripotent stem cells known as neoblasts. Planarians have been a classic model system for the study of many aspects of stem cell biology. However, very limited knowledge on miRNA involved in this regulatory mechanism exists. This study profiles the expression of miRNAs in the normal and regenerative tissues of planarians using miRCURY LNA array technology. Thirteen miRNAs showed significant differences in expression between these two tissues. To further confirm our results, we examined the expression of two miRNAs by qRT-PCR. Results show that some known miRNAs may play key roles in the regulatory mechanisms of regeneration. Our findings can be utilized in future research on miRNA function.

Modular design, expression and characterization of novel bifunctional mutants of fibrolase with combined platelet aggregation-inhibition and fibrinolytic activity.

Fibrolase is a non-hemorrhagic zinc metalloproteinase found in southern copperhead snake (Agkistrodon contortrix contortrix) venom that acts directly on fibrin clots and does not require plasminogen or any other blood-borne intermediate for activity. Chimeras of fibrolase with RGD peptides conferring antiplatelet activity have been synthesized covalently, but we describe a simpler, cheaper and less toxic method, using site-directed mutagensis. Fibrolase variants that constitute the arginine-glycine-aspartic acid (Arg-Gly-Asp, RGD) motif were constructed using site-directed mutagenesis. Chimeric genes of fibrolase were expressed in Escherichia coli to obtain the bifunctional chimeric molecule of fibrolase that can inhibit platelet aggregation. After refolding and purification, platelet-targeted thrombolysis and antiplatelet aggregation of the target chimeric protein were determined. The mutant RGD-F2, using the GPRGDWRMLG peptide to replace the TSVSHD sequence between sites 69 and 72, not only had almost the same catalytic ability as wild-type fibrolase but also a strong ability to inhibit platelet aggregation.

Transcriptome profiling and digital gene expression by deep-sequencing in normal/regenerative tissues of planarian Dugesia japonica.

Planarians exhibit an extraordinary ability to regenerate lost body parts which is attributed to an abundance of pluripotent somatic stem cells called neoblasts. In this article, we report a transcriptome sequence of a Planaria subspecies Dugesia japonica derived by high-throughput sequencing. In addition, we researched transcriptome changes during different periods of regeneration by using a tag-based digital gene expression (DGE) system. Consequently, 11,913,548 transcriptome sequencing reads were obtained. Finally, these reads were eventually assembled into 37,218 unique unigenes. These assembled unigenes were annotated with various methods. Transcriptome changes during planarian regeneration were investigated by using a tag-based DGE system. We obtained a sequencing depth of more than 3.5million tags per sample and identified a large number of differentially expressed genes at various stages of regeneration. The results provide a fairly comprehensive molecular biology background to the research on planarian development, particularly with regard to its regeneration progress.

[Detection of CCAAT/enhancer binding protein alpha gene mutations in acute myeloid leukemia].

OBJECTIVE: To explore the relationship between CCAAT/enhancer binding protein alpha (C/EBPalpha) gene mutations and the development of acute myeloid leukemia (AML). METHODS: The whole coding region of C/EBPalpha gene were screened in 48 cases of AML and 11 normal subjects by PCR-single strand conformation polymorphism (PCR-SSCP) and sequencing. RESULTS: C/EBPalpha mutations were detected in 5 of 48 AML patients. Four duplications and 1 deletion were confirmed by DNA sequencing. All of those are newly identified mutations. CONCLUSIONS: Different mutation types of C/EBPalpha gene exist in a small number of patients with AML and might be related to the pathogenesis of some leukemias.

[Ectopic osteogenesis of tissue engineered bone with slow-released bioactive factors].

OBJECTIVE: To study the ectopic osteogenesis of tissue engineered bone with recombinant human bone morphogenetic protein/transforming growth factor-beta (rhBMP/TGF-beta) or WO-1 slow-released factors. METHODS: Partial demineralized freeze-dried bone (PDFDB) of pig was used as scaffold material. rhBMP/TGF-beta or WO-1 were pre-coated on the surface of material by means of vacuum negative pressure absorption, and then coated with polylactic acid (PLA) to make slow-released material. There were six group: PDFDB material (group A); PDFDB combined with osteoblasts (group B); PDFDB material with rhBMP/TGF-beta slow-released system (group C); PDFDB material combined with rhBMP/TGF-beta slow-released system osteoblasts (group D); PDFDB with WO-1 slow-released system (group E); PDFDB material combined with WO-1 slow-released system and osteoblasts (group F) were implanted in bilateral lower limbs of 36 Newzealand rabbits respectively (6 rabbits in each groups). Histological, histochemical and biochemical analysis were detected 2, 4, 6, 8 weeks after operation. RESULTS: Within the observation periods, no osteogenesis was observed in group A. The osteogenesis in group B, D, F were superior to that of group C and E (P < 0.05). The osteogenetic activity in group C and E was delayed. The quantity and quality of osteogenesis in group D and F were 2 weeks ahead of time compared with group B, and 4 weeks to that of group C and E. The newborn calcification content was superior to that of group A, C, and E (P < 0.05). CONCLUSIONS: The osteogenesis of PDFDB materials with BMP/TGF-beta or WO-1 is slower than that of which combined with osteoblasts. Simple material PDFDB has no ectopic osteogenesis.

[Morphological study on three-dimensional culture of rat muscle satellite cells under cyclic mechanical stretch].

OBJECTIVE: To study the effect of mechanical stretch on the morphologic change of rat muscle satellite cell in the three-dimensional culture. METHODS: Based on the apparatus for three-dimensional cell cultures under a cyclic mechanical strain, a specific stretch pattern (10% elongation, 10 stretches/min for 10 min of each hour 48 h total) was applied to cell-scaffolding composites. RESULTS: Under the stretch pattern, the shape of satellite cells changed to oblate and spread to the direction of the stretch. Furthermore, the myotube was observed. On the contrary, the satellite cells spread to the all direction, and the formation of the myotube was not been found in the control group. CONCLUSION: Cyclic mechanical stretch is helpful for the formation of the ideal directivity and these results are compatible with a significant role for the stretch in tissue-engineered muscle construction.