Gut bacterial communities in the freshwater snail Planorbella trivolvis and their modification by a non-herbivorous diet.

The freshwater pulmonate snail Planorbella trivolvis is a common species in various bodies of water but is not native to China. Planorbella trivolvis usually live on diets with high fiber content, such as water grasses, algae and fallen leaves. These snails can attach to the wall of a water tank or to water grass and can be transported overseas to China through the ornamental fish trade. There are few studies investigating the intestinal microbiota of freshwater snails. In this study, using culture-independent molecular analysis, we assessed for the first time the complexity of bacterial communities in the intestines of reared snails. The intestinal microbiota in the snails fed different diets, that is, herbivorous feed (HV) with high cellulose and non-herbivorous feed (NHV) with low cellulose, were analyzed by Illumina sequencing. The results showed that the NHV-based diet significantly increased the body mass, shell diameter and specific growth rate of the snails after 60 days of rearing (P < 0.05). Histological experiments showed that the fat droplets in the epithelium columnar cells of the intestines of the NHV snails increased, and the cilia on these cells fell off. The sequencing results identified 486 and 195 OTUs in HV and NHV, respectively. Lots of bacteria were not reported previously in snails. The intestinal microbiota diversity index (Shannon, Simpson, Ace and Chao) in the NHV snails was significantly lower than that in the HV snails. The gut microbiota in the HV snails were predominantly Proteobacteria (52.97%) and Bacteroidetes (28.75%), while the gut microbiota in NHV snails were predominantly Proteobacteria (95.23%). At the genus level, Cloacibacterium (24.60%), Pseudomonas (4.47%), OM6ON (6.12%), and Rhodobacter (5.79%) were observed to be abundant in HV snails. However, Aeromonas (85.4%) was determined to be predominant in NHV snails. Functional prediction of the gut microbiome in snails by PICRUSt demonstrated a significant difference between the two groups, and the HV snails exhibited higher lignocellulose enzyme activity than did the NHV snails. This study represents a first step in characterizing the gut microbiota of the freshwater snail. Most of these microbes can process plant biomass and digest cellulose and lignocellulose, and the enzymes of these bacteria may have potential biotechnological applications in a variety of industrial processes.

Enzyme additives influence bacterial communities of Medicago sativa silage as determined by Illumina sequencing.

The goal of the present study was to evaluate the effects of enzymes (cellulase combined with galactosidase) and their combination with Lactobacillus plantarum (LP) on bacterial diversity in alfalfa silages using high-throughput sequencing. Alfalfa forages were treated with or without cellulase + ɑ-galactosidase (CEGA), cellulase + LP (CELP), or ɑ-galactosidase + LP (GALP). After 56 days of ensiling, all treated silages exhibited improved fermentation quality, as reflected by decreased pH, ammonium-N and increased lactic acid levels compared to the control silage (P < 0.05). Enzymatic treatment improved nutrient value by increasing crude protein levels and decreasing neutral detergent fibre (NDF) levels (P < 0.05). Silage treatment significantly altered the bacterial community, as determined by PCoA (P < 0.05). Lactic acid bacteria (LAB) dominated the bacterial community of the treated silage after ensiling. The dominant bacteria changed from Garciella, Enterococcus, Lactobacillus and Pediococcus in the control silage to Lactobacillus and Pediococcus in the CEGA silage and Lactobacillus in the CELP and GALP silages. Collectively, these results suggest that treatment with both enzymes alone and in combination with inoculants greatly increased the abundance of LAB, with Enterococcus, Lactobacillus and Pediococcus observed in the silage treated with enzymes alone (CEGA) and Lactobacillus observed in the silage treated with a combination of enzymes and inoculants (CELP and GALP).

Resveratrol alleviates oxygen/glucose deprivation/reoxygenation­induced neuronal damage through induction of mitophagy.

Resveratrol confers neuroprotective effects in cerebral ischemia; however, the involvement of mitophagy in the neuroprotective function of resveratrol remains unclear. The aim of the present study was to investigate whether resveratrol exerts neuroprotective effects on primary cortical neurons subjected to oxygen/glucose deprivation/reoxygenation (OGD/R) via modulating mitophagy. The data demonstrated that resveratrol at 1­10 µM during reoxygenation improved cell viability and suppressed apoptosis following OGD/R in a concentration­dependent manner. Moreover, resveratrol alleviated OGD/R­induced loss of mitochondrial membrane potential and excessive oxidative stress. Confocal imaging of LC3 and TOM20 antibody­labeled mitochondria, as well as western blot analysis, demonstrated that mitophagy was further enhanced following resveratrol treatment. In addition, resveratrol was revealed to stimulate the phosphatase and tensin homolog­induced kinase 1/Parkin pathway. Mitophagy inhibition then inhibited the protective effects of resveratrol. These results indicated that resveratrol exerts its protective effects against OGD/R damage, at least in part, by promoting mitophagy.

The Microbiota Dynamics of Alfalfa Silage During Ensiling and After Air Exposure, and the Metabolomics After Air Exposure Are Affected by Lactobacillus casei and Cellulase Addition.

Both inoculants treatment and enzyme treatment promote the reproduction of lactic acid bacteria (LAB) to produce enough lactic acid to lower pH in silage. The present study investigated the microbial community and metabolome in cellulase, Lactobacillus casei, and air treated alfalfa silage. Chopped and wilted alfalfa (first cutting, 29% dry matter) was ensiled without (CON) or with L. casei (1 × 106 cfu g-1 fresh matter) (LC) or cellulase (20,000IU, 0.5% of fresh matter) (CE) for 56 days, then exposed to air for 3 days (PO). Greater ensiling quality was observed in LC and CE, which had lower pH and higher lactic acid content than CON at 56 days of ensiling and 3 days post-oxygen exposure. Air exposure was associated with decreased lactic acid concentrations and increased yeast and mold counts in all silages. SEM showed that the structure of leaf epicuticular wax crystals were intact in fresh alfalfa, totally decomposed in CON silage, and partly preserved in CE and LC silage. Gas chromatography mass spectrometry revealed that 196 metabolites and 95 differential concentration were present in the 3 days air exposure samples. Most of these metabolites, mainly organic acids, polyols, ketones, aldehydes, are capable of antimicrobial activity. The bacterial communities were obviously different among groups and Lactobacillus developed to a dominant status in all silages. Lactobacillus became dominant in bacterial communities of LC and CE silages from days 7 to 56, and their relative abundances reached 94.17-83.93% at day 56, respectively. For CON silage, until day 56, Lactobacillus dominated the bacterial community with abundance of 75.10%. After 3 days of oxygen exposure, Lactobacillus and Enterococcus were predominant in CON, and Lactobacillus remained dominant in LC and CE silages. The results indicated that, compared to untreated silages, L. casei could be a priority inoculant for alfalfa silage to boost Lactobacillus abundance and improve fermentation quality. Our high-throughput sequencing and gas chromatography mass spectrometry results provide a deep insight into the bacterial community and metabolites in alfalfa silage.

[High-throughput sequencing analysis of intestinal flora diversity of two freshwater snails (Radix auricularia and Planorbella trivolvis)].

Freshwater snail is an important biological group in aquatic ecosystem and an intermediate host of many parasites. Intestinal flora plays an important role in animal energy metabolism and resistance to pathogens. We analyzed the intestinal microbiota diversity of Radix auricularia (RA) and Planorbella trivolvis (PL) by 16S rRNA high-throughput sequencing. At the phylum level, RA had 23 phyla, including Proteobacteria (33.63%), Cyanobacteria (15.33%), Chloroflexi (13.95%), and Actinomycetes (12.99%). PL had 13 phyla, including Proteobacteria (54.88%), Bacteroidetes (28.49%), and Actinomycetes (7.65%). At the genus level, there were 445 genera in RA, including Pleurocapsa, Thiodictyon, Leptotrichia, and Nocardioides. There were 238 genera in PL, including Cloacibacterium, OM60NOR5_clade, Pseudomonas, and Rhodobacter. Ninety-three genera were the common core flora of the two snail species (all the samples were present), and 27 genera had an abundance greater than 0.5%. The structure of intestinal microbiota was significantly different between the two groups (P=0.027). We performed the functional prediction of intestinal microbiota using Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUSt), and the results show that the KEGG functional composition of the intestinal flora of the two snails was similar, and the abundance of the amino acid metabolism, carbohydrate metabolism and membrane transport were large. In summary, the intestinal microbiota of the two snails was high in diversity and significantly different, but there were a large number of common core flora.

Influence of dietary linseed oil as substitution of fish oil on whole fish fatty acid composition, lipid metabolism and oxidative status of juvenile Manchurian trout, Brachymystax lenok.

In this study, juvenile Manchurian trout, Brachymystax lenok (initial weight: 6.43 ± 0.02 g, mean ± SE) were received for nine weeks with five types of diets prepared by gradually replacing the proportion of fish oil (FO) with linseed oil (LO) from 0% (LO0) to 25% (LO25), 50% (LO50), 75% (LO75), and 100% (LO100). The eicosapentaenoic (EPA) and docosahexaenoic (DHA) composition decreased with increasing inclusion level of LO (P < 0.05). With increasing LO inclusion level, triglyceride (TAG) content of serum increased significantly, however, there was a decrease in high-density lipoprotein cholesterol (HDL) (P < 0.05). LO substitution of FO up-regulated the gene expression level of lipid metabolism-related genes Fatty Acid Desaturases 6 (FAD6), Acetyl-Coa Carboxylase (ACCα), Sterol Regulatory Element Binding Protein 1 (SREBP-1), and Sterol O- Acyl Transferase 2 (SOAT2), and down-regulated the gene expression level of Peroxisome Proliferator-Activated Receptor a (PPARα) (P < 0.05). The SOD activities of both serum and liver in LO100 were significantly lower than in LO25 (P < 0.05). The CAT activity of the liver in LO100 was significantly lower than in LO0 and LO25 (P < 0.05). This study indicates that the Manchurian trout may have the ability to synthesize LC-PUFAs from ALA, and an appropriate LO in substitution of FO (<75%) could improve both the lipid metabolism and the oxidation resistance.

Comparison of two malnutrition risk screening tools with nutritional biochemical parameters, BMI and length of stay in Chinese geriatric inpatients: a multicenter, cross-sectional study.

OBJECTIVES: The aims of this study were to assess malnutrition risk in Chinese geriatric inpatients using Nutritional Risk Screening 2002 (NRS2002) and Mini-Nutritional Assessment (MNA), and to identify the most appropriate nutritional screening tool for these patients. DESIGN: Cross-sectional study. SETTING: Eight medical centres in Hubei Province, China. PARTICIPANTS: A total of 425 inpatients aged ≥70 years were consecutively recruited between December 2014 and May 2016. PRIMARY AND SECONDARY OUTCOME MEASURES: Nutritional risk was assessed using NRS2002, MNA, anthropometric measurements and biochemical parameters within 24 hours of admission. Comorbidities and length of hospitalisation were recorded. Nutritional parameters, body mass index (BMI) and length of hospital stay (LOS) were employed to compare MNA and NRS2002. Kappa analysis was used to evaluate the consistency of the two tools. RESULTS: The average age was 81.2±5.9 years (range, 70-98). The prevalence of undernutrition classified by NRS2002 and MNA was 40.9% and 58.6%, respectively. Patients undergoing malnutrition had lower BMI, haemoglobin, albumin and prealbumin (p<0.05), and longer LOS (p<0.05). The NRS2002 showed moderate agreement (κ=0.521, p<0.001) with MNA. Both tools presented significant correlation with age, BMI and laboratory parameters (p<0.001). In addition, a significant association between both tools and LOS was found (p<0.05). In addition, the NRS2002 was not different from MNA in predicting nutritional risk in terms of the area under the receiver operating characteristic curve (p>0.05). CONCLUSIONS: The results show a relatively high prevalence of malnutrition risk in our sample cohort. We found that NRS2002 and MNA were both suitable in screening malnutrition risk among Chinese geriatric inpatients.

Compositional and predicted functional analysis of the gut microbiota of Radix auricularia (Linnaeus) via high-throughput Illumina sequencing.

Due to its wide distribution across the world, the snail Radix auricularia plays a central role in the transferal of energy and biomass by consuming plant biomass in freshwater systems. The gut microbiota are involved in the nutrition, digestion, immunity, and development of snails, particularly for cellulolytic bacteria, which greatly contribute to the digestion of plant fiber. For the first time, this study characterized the gut bacterial communities of R. auricularia, as well as predicted functions, using the Illumina Miseq platform to sequence 16S rRNA amplicons. Both juvenile snails (JS) and adult snails (AS) were sampled. The obtained 251,072 sequences were rarefied to 214,584 sequences and clustered into 1,196 operational taxonomic units (OTUs) with 97% sequence identity. The predominant phyla were Proteobacteria (JS: 36.0%, AS: 31.6%) and Cyanobacteria (JS: 16.3%, AS: 19.5%), followed by Chloroflexi (JS: 9.7%, AS: 13.1%), Firmicutes (JS: 14.4%, AS: 6.7%), Actinobacteria (JS: 8.2%, AS: 12.6%), and Tenericutes (JS: 7.3%, AS: 6.2%). The phylum Cyanobacteria may have originated from the plant diet instead of the gut microbiome. A total of 52 bacterial families and 55 genera were found with >1% abundance in at least one sample. A large number of species could not be successfully identified, which could indicate the detection of novel ribotypes or result from insufficient availability of snail microbiome data. The core microbiome consisted of 469 OTUs, representing 88.4% of all sequences. Furthermore, the predicted function of bacterial community of R. auricularia performed by Phylogenetic Investigation of Communities by Reconstruction of Unobserved States suggests that functions related to metabolism and environmental information processing were enriched. The abundance of carbohydrate suggests a strong capability of the gut microbiome to digest lignin. Our results indicate an abundance of bacteria in both JS and AS, and thus the bacteria in R. auricularia gut form a promising source for novel enzymes, such as cellulolytic enzymes, that may be useful for biofuel production. Furthermore, searching for xenobiotic biodegradation bacteria may be a further important application of these snails.

The inhibitory effect of kokusaginine on the growth of human breast cancer cells and MDR-resistant cells is mediated by the inhibition of tubulin assembly.

The emergence of multidrug resistance (MDR) is a significant challenge in breast carcinoma chemotherapy. Kokusaginine isolated from Dictamnus dasycarpus Turcz. has been reported to show cytotoxicity in several human cancer cell lines including breast cancer cells MCF-7. In this study, kokusaginine showed the potent inhibitory effect on MCF-7 multidrug resistant subline MCF-7/ADR and MDA-MB-231 multidrug resistant subline MDA-MB-231/ADR. Kokusaginine markedly induced apoptosis in a concentration-dependent manner in MCF-7/ADR cells. Furthermore, kokusaginine reduced P-gp mRNA and protein levels, and suppressed P-gp function especially in MCF-7/ADR cells. In addition, kokusaginine showed to inhibit tubulin assembly and the binding of colchicine to tubulin by binding directly to tubulin and affects tubulin formation in vitro. Taken together, these results support the potential therapeutic value of kokusaginine as an anti-MDR agent in chemotherapy for breast carcinoma.

Diversity of bacterial community during ensiling and subsequent exposure to air in whole-plant maize silage.

OBJECTIVE: To describe in-depth sequencing, the bacterial community diversity and its succession during ensiling of whole-plant maize and subsequent exposure to air. METHODS: The microbial community dynamics of fermented whole-plant maize for 60 days (sampled on day 5, 10, 20, 40, 60) and subsequent aerobic exposure (sampled on day 63 after exposure to air for 3 days) were explored using Illumina Miseq sequence platform. RESULTS: A total of 227,220 effective reads were obtained. At the genus level, there were 12 genera with relative abundance >1%, Lactobacillus, Klebsiella, Sporolactobacillus, Norank-c-cyanobacteria, Pantoea, Pediococcus, Rahnella, Sphingomonas, Serratia, Chryseobacterium, Sphingobacterium, and Lactococcus. Lactobacillus consistently dominated the bacterial communities with relative abundance from 49.56% to 64.17% during the ensiling process. Klebsiella was also an important succession bacterium with a decrease tendency from 15.20% to 6.41% during the ensiling process. The genus Sporolactobacillus appeared in late-ensiling stages with 7.70% abundance on day 40 and 5.32% on day 60. After aerobic exposure, the Lactobacillus decreased its abundance from 63.2% on day 60 to 45.03% on d 63, and Klebsiella from 5.51% to 5.64%, while Sporolactobacillus greatly increased its abundance to 28.15%. These bacterial genera belong to 5 phyla: Firmicutes (relative abundance: 56.38% to 78.43%) was dominant, others were Proteobacteria, Bacteroidetes, Cyanobacteria, and Actinobacteria. The bacterial communities clearly clustered into early-ensiling (d 5), medium-ensiling (d 10, d 20), late-ensiling (d 40, d 60), and aerobic exposure (d 63) clusters, with early- and late-ensiling communities more like each other than to the aerobic exposure communities. CONCLUSION: High-throughput sequencing based on 16S rRNA genes proved to be a useful method to explore bacterial communities of silage. The results indicated that the bacterial communities varied during fermentation and more dramatically during aerobic exposure. The study is valuable for understanding the mechanism of population change and the relationship between bacteria and ensilage characteristics.

RNA interference and ischemic injury.

In the early period of 21st century, RNA interference (RNAi) had emerged as one of the most important discoveries. This highly conserved endogenous gene silencing mechanism has been largely exploited as a powerful tool to determine biological functions of each gene. Both direct introduction of chemically synthesized small interference RNA (siRNA) and a plasmid or viral vectors encoding for siRNA can allow especially stable RNA knockdown. Recently, it has been widely used in the production of therapeutic drugs against hepatitis or immuno-deficiency viruses in human beings. Here, we provide a brief overview of the RNAi mechanism and the technology of RNAi on ischemic injury.

A new photoelectrochemical aptasensor for the detection of thrombin based on functionalized graphene and CdSe nanoparticles multilayers.

A photoelectrochemical sensing strategy for highly sensitive detection of thrombin was developed based on a layer-by-layer (LBL) assembly of functionalized graphene and CdSe nanoparticles.

Aptamer based photoelectrochemical cytosensor with layer-by-layer assembly of CdSe semiconductor nanoparticles as photoelectrochemically active species.

A label-free photoelectrochemical cytosensor for highly sensitive and specific detection of Ramos cell was developed based on photoactive films. The films were fabricated by a layer-by-layer (LBL) assembly of positively charged poly(dimethyldiallylammonium chloride) (PDDA) and negatively charged CdSe semiconductor nanoparticles (NPs) capped with mercaptoacetic acid. The resulting modified electrodes were tested as sensors for Ramos cell through the recognition of DNA aptamer which was covalently bound to the electrode using the classic coupling reactions between -COOH groups on the surfaces of CdSe NPs and -NH(2) groups of the aptamer. The newly developed cytosensor exhibited excellent sensitivity and selectivity. The linear range was from 160 to 1600 cells/mL and the detection limit was 84 cells/mL.

The cardioprotective effect of fluvastatin on ischemic injury via down-regulation of toll-like receptor 4.

To determine whether the cardioprotection effect of fluvastatin mediates by toll-like receptor 4 (TLR4) signaling pathway, fifty Sprague-Dawley rats were randomly divided into five groups: sham operation group, ischemia/reperfusion (I/R) group, fluvastatin groups (high-dosage, medium-dosage, low-dosage, n = 10 in each group). Except sham operation group, the rest four groups of rats were artificially afflicted with coronary occlusion for 30 min, then reperfusion 2 h. Light microscope and transmission electronic microscope were used to observe structural changes of myocardium. RT-PCR was used to measure TLR4 mRNA expression level, TLR4 protein expression was detected by immunohistochemistry. Western blot was used to measure myocardial NF-κB protein level; ELISA was used to measure the level of TNF-α in myocardium. The results demonstrated that fluvastatin treatment markedly decreased ischemic injury caused by ischemia/reperfusion, and inhibited the expression levels of TLR4, TNF-α and NF-κB, all of which up-regulated by ischemia/reperfusion. Taken together, our results suggest that proper dosage of fluvastatin may have protective effect on the ischemic injury mediated by ischemia/reperfusion in the hearts, which might be associated with inhibition of TLR4 signaling pathway and inflammatory response during ischemia/reperfusion.

Photoelectrochemical biosensor for detection of adenosine triphosphate in the extracts of cancer cells.

A photoelectrochemical sensing strategy for highly sensitive detection of small molecules was developed based on the recognition interaction between aptamer and target molecule-ATP.

DNA-based amplified electrical bio-barcode assay for one-pot detection of two target DNAs.

A sensitive label-free bio-barcode assay provided a PCR-free method for quantitative detection of two nucleic acid targets (HTLV-I and HTLV-II) simultaneously. This DNA biosensor was fabricated with two-component oligonucleotide-modified gold nanoparticles (AuNPs) and two-component oligonucleotide-modified magnetic beads (MBs), which can sandwich a specific target. After liberating the adsorbed thiolated barcode DNA strands (poly A and poly G) from the AuNPs surface with dithiothreitol (DTT) and acidic dipurinization, the electrochemical measurements were directly performed based on the redox activity of guanine (G) and adenine (A) nucleobases. Under the optimal assembling and detection conditions, a good linearity for simultaneous detection was obtained in the range from 4.4x10(-11) to 2.0x10(-9) M, and the detection limit (3sigma) was estimated to be 1.71x10(-12) M for T(1)-DNA and 1.55x10(-12) M for T(2)-DNA.

Adipose tissue stromal cells transplantation in rats of acute myocardial infarction.

OBJECTIVE: This study was to investigate the proliferation and differentiation of rat adipose stromal cells when implanted into ischemic myocardium and the improvement of heart function. METHODS: Sprague-Dawley rat adipose tissue was digested with collagenase type I solution and adipose stromal cells were derived by culture. The cells' surface phenotype was examined by flow cytometry. Adipose stromal cells labeled with 4'6-diamidino-2-phenylindole (adipose stromal cells group) or Dulbecco's modified Eagle medium (control group) was transplanted into the ischemic myocardium, which was produced by ligation of left descending branch of coronary artery. At 1 and 4 weeks after transplantation, specimens were acquired from infarcted area and also echocardiography was done to detect the effects on heart function. Then, cell morphology and capillary density were measured, and vascular endothelial growth factor expression levels were assayed by reverse transcription-PCR and enzyme-linked immunosorbent assay. RESULT: Adipose stromal cells derived by culture expressed CD44 and CD90 but not CD31 or CD45. Adipose stromal cells were alive at 1 and 4 weeks after transplantation and had a trend toward differentiation into vascular endothelial cells. The number of capillary vessels in peri-infarct area in adipose stromal cells group increased significantly compared with control group (P<0.01). The levels of vascular endothelial growth factor mRNA and protein expression at 1 week increased significantly in adipose stromal cells group compared with control group (P<0.01). Left ventricular function, including ejection fraction and fractional shortening, was higher in adipose stromal cells group when compared with control group at 4 weeks (P<0.01). CONCLUSION: Adipose stromal cells transplantation can accelerate angiogenesis in infarcted area after rat myocardial infarction and can improve heart function.