A fluorimetric study on the interaction between a Trp-containing beta-strand peptide and amphiphilic polymer-coated gold nanoparticles.

Owing to the inevitability of nanoparticles encountering proteins/peptides in current bio-nano-medicine development, it is important to know how they interact with each other in vitro before developing in vivo applications. To this end, a model de novo ß-sheet-forming peptide and typical biocompatible nanoparticles were selected to study thermodynamic aspects of their interactions via a fluorescence quenching method. The results showed that Pep11 and AuNPs spontaneously formed conjugates, mainly driven by a coulombic interaction with a binding affinity of ~ 0.1 µM(-1); the physical adsorption process was cooperative. These results deepen our quantitative understanding of nanoparticle-peptide interactions. The results may also be helpful in further nanoparticle-peptide hybrid nanofabrication and also useful for the application of nanoparticles in the treatment of amyloid diseases.

Genome-wide identification of the MADS-box gene family in Avena sativa and its role in photoperiod-insensitive oat.

Background: Traditional spring-summer sown oat is a typical long-day crop that cannot head under short-day conditions. The creation of photoperiod-insensitive oats overcomes this limitation. MADS-box genes are a class of transcription factors involved in plant flowering signal transduction regulation. Previous transcriptome studies have shown that MADS-box genes may be related to the oat photoperiod. Methods: Putative MADS-box genes were identified in the whole genome of oat. Bioinformatics methods were used to analyze their classification, conserved motifs, gene structure, evolution, chromosome localization, collinearity and cis-elements. Ten representative genes were further screened via qRT‒PCR analysis under short days. Results: In total, sixteen AsMADS genes were identified and grouped into nine subfamilies. The domains, conserved motifs and gene structures of all AsMADS genes were conserved. All members contained light-responsive elements. Using the photoperiod-insensitive oat MENGSIYAN4HAO (MSY4) and spring-summer sown oat HongQi2hao (HQ2) as materials, qRT‒PCR analysis was used to analyze the AsMADS gene at different panicle differentiation stages under short-day conditions. Compared with HQ2, AsMADS3, AsMADS8, AsMADS11, AsMADS13, and AsMADS16 were upregulated from the initial stage to the branch differentiation stage in MSY4, while AsMADS12 was downregulated. qRT‒PCR analysis was also performed on the whole panicle differentiation stages in MSY4 under short-day conditions, the result showed that the expression levels of AsMADS9 and AsMADS11 gradually decreased. Based on the subfamily to which these genes belong, the above results indicated that AsMADS genes, especially SVP, SQUA and Mα subfamily members, regulated panicle development in MSY4 by responding to short-days. This work provides a foundation for revealing the function of the AsMADS gene family in the oat photoperiod pathway.

Metabolite profiling of camel milk and the fermentation bacteria agent TR1 fermented two types of sour camel milk using LC-MS in relation to their probiotic potentials.

Camel milk is a nutrient-rich diet and fermentation affects its nutritional value and probiotic function. In this study, sour camel milk and oat jujube sour camel milk were prepared using fermentation bacteria agent TR1, and the metabolites of camel milk, sour camel milk and oat jujube sour camel milk were detected using a non-targeted metabolomics approach using liquid chromatography-mass spectrometry (LC-MS).The results showed that the partial least squares discriminant analysis (PLS-DA) with 100 % accuracy and good predictive power detected 343 components in positive ion mode and 220 components in negative ion mode. The differential metabolites were mainly organic acids, amino acids, esters, vitamins and other substances contained in camel milk.It showed that there were significant differences in the metabolites of camel milk, sour camel milk and oat jujube sour camel milk. Based on the pathway enrichment analysis of the three dairy products in the KEGG database, 12 metabolic pathways mainly involved in the positive ion mode and 20 metabolic pathways mainly involved in the negative ion mode were identified. The main biochemical metabolic pathways and signal transduction pathways of the differential metabolites of the three dairy products were obtained. This study provides theoretical support for improving the nutritional quality and probiotic function of camel milk and fermented camel milk products and provides a basis for the development of relevant processing technologies and products for camel milk and fermented camel milk.

Production and secretion of Lactobacillus crispatus ß-galactosidase in Pichia pastoris.

Lactobacillus ß-galactosidases are mostly heterodimeric proteins, which are encoded by the two overlapping genes, lacL and lacM, and produced in recombinant prokaryotic systems for higher yield. This is the first report on the expression of a heterodimeric ß-galactosidase from Lactobacillus crispatus B470 in Pichia pastoris. The overlapping consecutive genes, lacL and lacM, that shared 17 nucleotides were cloned from the genomic DNA of L. crispatus. A recombinant plasmid harboring both expression cassettes of lacL and lacM was constructed and transformed into P. pastoris GS115 competent cells. Two recombinant P. pastoris strains (GSLac01 and GSLac02) showed the highest ß-galactosidase activities of 24.5 and 31.0 U/ml in the culture supernatants, respectively. The recombinant ß-galactosidase (LcLacLM) from GSLac02 was purified to electrphoretic homogeneity by ion-exchange chromatography and molecular sieve chromatography. Similar to most Lactobacillus ß-galactosidases that operate at moderately thermophilic and weak acid to neutral conditions, LcLacLM showed optimal activity at 50°C and pH 5.5-6.5. It's the first report on functional and secretory expression of LacLM-type ß-galactosidase in eukaryotic system. This strategy might be applied to the expression of other overlapping genes.

Analysis of metabolites of bactrain camel milk in Alxa of China before and after fermentation with fermenting agent TR1 based on untargeted LC-MS/MS based metabolomics.

Camel milk produces many beneficial functional compounds and affects body health through metabolism. The differential metabolites of bactrain camel milk in Alxa before and after fermentation were identified by liquid chromatography-tandem mass spectrometry based metabolomics (LC-MS/MS). The differential metabolite pathway types were also identified in this paper. We obtained the following results that 148 and 82 differential metabolites were detected in positive and negative ion mode respectively, 85 differential metabolites were shown a significant upward trend and 63 with downward trend after fermentation in positive ion mode. Meanwhile, 32 differential metabolites characterized upward trend and 50 characterized downward trend in negative ion mode. The differential metabolites were mainly organic acids, amino acids, esters, vitamins and other substances contained in camel milk. Among them, most up-regulated substances had the functions of lowering blood pressure, lowering blood sugar, treatment of inflammation, antibiosis and other effects. Many harmful substances were significantly down-regulated after camel milk fermentation. However, there were also some metabolites whose prebiotic functions have been weakened by camel milk fermentation, which may provide reference values for healthcare function, exploitation and application of camel milk.

Characterization and functional of four mutants of hydroxy fatty acid dehydrogenase from Lactobacillus plantarump-8.

In this study, the hydroxy fatty acid dehydrogenase CLA-DH from Lactobacillus plantarump-8 and its four mutant variants were expressed in Escherichia coli Rosetta (DE3). UV spectrophotometry was employed to verify the catalytic power of the purified CLA-DH to convert ricinoleic acid into 12-oxo-cis-9-octadecenoic acid in the presence of oxidized nicotinamide adenine dinucleotide (NAD+). The optimum reaction temperature for CLA-DH was 45°C, with a maintained stability between 20°C and 40°C. The optimal pH for CLA-DH catalytic activity was 6.0-7.0, with a maintained stability at a pH range of 6.0-8.0. In addition, Fe3+ promoted enzyme activity, whereas Cu2+, Zn2+, and Fe2+ inhibited enzyme activity (P < 0.05). The Km, Vmax, Kcat, and Kcat/Km of CLA-DH were determined as 2.19 ± 0.34 µM, 2.06 ± 0.28 µM min-1, 2.00 ± 0.27 min-1, and 0.92 ± 0.02 min-1µM-1, respectively. Site-directed mutagenesis and molecular dynamics simulations demonstrated that both Tyr156 and Ser143 residues play significant roles in the catalysis of CLA-DH, and its solubility is affected by Lys160 and Asp63. Moreover, Gas chromatography determined that recombinant CLA-DH could be successfully applied to Conjugated linoleic acids production.

Probiotic Bifidobacterium lactis V9 attenuates hepatic steatosis and inflammation in rats with non-alcoholic fatty liver disease.

Both steatosis and inflammation are key pathological events in the progression of non-alcoholic fatty liver disease (NAFLD). Probiotics are beneficial for the prevention and treatment of NAFLD. Bifidobacterium animalis subsp. lactis V9 (V9) is a newly isolated strain with favorable probiotic properties. The study aims to evaluate the effects and mechanisms of V9 on the hepatic steatosis and inflammatory responses in a rat model of NAFLD induced by high-fat diets (HFD). Our results showed that administration of V9 significantly attenuated the HFD-induced increases in alanine transaminase (ALT) and aspartate aminotransferase (AST) levels, resulting in alleviated hepatic steatosis. V9 supplementation reduced the accumulation of hepatic triglyceride and free fatty acid,while increasing the levels of glycogen. Serum levels of glucose were also decreased in HFD rats administrated with V9. Meanwhile, the transcription of SREBP-1c and FAS was reduced, and the hepatic expression of phosphorylated-AMPK and PPAR-α was restored after V9 administration. V9 suppressed the production of inflammatory cytokines (e.g. IL-6, IL-1ß, and TNF-α) in HFD-fed rats. The anti-inflammatory effects of V9 was found to be associated with the inhibition of hepatic expression of TLR4, TLR9, NLRP3, and ASC mRNA. Furthermore, the activation of ERK, JNK, AKT and NF-κB were suppressed by V9 treatment. These results indicate that Bifidobacterium lactis V9 improves NAFLD by regulating de novo lipid synthesis and suppressing inflammation through AMPK and TLR-NF-κB pathways, respectively.

Kinetics and function of two mutants of CLA hydrase from Lactobacillus plantarum p-8.

In this work, the gene of conjugated linoleic acid hydrase (CLA-HY) was cloned from L. plantarum p-8, and the protein of CLA-HY was expressed in Escherichia coli BL21. Gas chromatography-mass spectrometry was employed to verify that the purified CLA-HY can convert linoleic acid (LA) into 10-hydroxy-cis-12-octadecenoic acid (10-HOE) in the presence of flavin adenine dinucleotide (FAD). The optimal pH and temperature for maximizing CLA-HY catalytic activity were found to be 6.0 and 35°C, respectively. In addition, the catalytic ability of CLA-HY can be inhibited by a number of cations such as Mg2+, Mn2+, Zn2+, Cu2+, Fe2+, Fe3+, Ni2+ and Ca2+. Finally, the Km,Vmax, Kcat and Kcat/Km of CLA-HY were determined as 7.62 mM, 2.59 mM h-1, 8.33 × 103 h-1 and 1.09 × 103 mM-1 h-1, respectively. Moreover, it was demonstrated that both M76 and G74 residues played significant roles in catalysing the conversion of LA into 10-HOE using site-directed mutation technology and molecular simulations.

Correlation of the rumen fluid microbiome and the average daily gain with a dietary supplementation of Allium mongolicum Regel extracts in sheep1.

Plant extracts can affect the rumen microbiome and ADG in ruminants, and studies of the association between the rumen microbiome and ADG provide information applicable to improving ruminant growth performance. The objectives were to investigate the effects of Allium mongolicum Regel extracts on the rumen microbiome and ADG and their association in sheep. Forty healthy, male, small-tailed Han sheep (6 mo, 34 ± 3.5 kg body weight) were randomly assigned to 1 of the following 4 dietary treatments: basal diet as control group (CK, n = 10), basal diet supplemented with 3.4 g·sheep-1·d-1A. mongolicum Regel powder extract as PAM group (PAM, n = 10), basal diet supplemented with 10 g·sheep-1·d-1A. mongolicum Regel powder as AM group (AM, n = 10), and basal diet supplemented with 10 g·sheep-1·d-1A. mongolicum Regel powder extract residue as RAM group (RAM, n = 10). The ADG for individual sheep was calculated using the sum of the ADGs observed during the experimental period divided by the number of days in the experimental period. At the end of the experiment, sheep were randomly selected from each treatment for slaughter (n = 6), and the rumen fluids were collected and stored immediately at -80 °C. Illumina HiSeq was subsequently used to investigate the changes in the rumen microbiome profile, and the associations with ADG were analyzed by Spearman correlation coefficient analysis. The results demonstrated that, compared with that in CK group, the ADG in AM and RAM significantly increased (P = 0.0171). The abundances of Tenericutes and Mollicutes ([ρ] = 0.5021, P = 0.0124) were positively correlated with ADG. Within Mollicutes, the abundances of Anaeroplasmatales ([ρ] = 0.5458, P = 0.0058) and Anaeroplasmataceae ([ρ] = 0.5458, P = 0.0058) were positively correlated with ADG. The main negatively correlated bacteria were Saccharibacteria ([ρ] = -0.4762, P = 0.0187) and Betaproteobacteria ([ρ] = -0.5669, P = 0.039). Although Anaeroplasmatales and Anaeroplasmataceae were positively correlated with ADG, Saccharibacteria and Betaproteobacteria were negatively correlated with ADG. In conclusion, supplementation with A. mongolicum Regel powder and extracts will influence the rumen microbiome and increase the ADG.

Discrete nanoparticle-BSA conjugates manipulated by hydrophobic interaction.

Nanoparticle-protein conjugates are promising probes for biological diagnostics as well as versatile building blocks for nanotechnology. Here we demonstrate a facile method to prepare nanoparticles bearing discrete numbers of BSA simply by physical adsorption and electrophoretic isolation, in which the specific amphiphilic properties of BSA play important roles and the number of adsorbed BSA molecules can also be manipulated by tuning the coating extent of nanoparticles by amphiphilic polymer.

Has the rate of extensive surgery to treat idiopathic clubfoot declined in the United States?

BACKGROUND: In the late 1990s, renewed interest emerged in less invasive treatment options, most notably the Ponseti method, to correct idiopathic clubfoot deformity. Recently, reports from several centers have demonstrated that such minimally invasive techniques may be used reliably to correct this complex deformity. The present study sought to determine whether the rate of extensive surgical releases to treat idiopathic clubfoot in the United States has decreased. METHODS: We used data from the Centers for Disease Control and Prevention and the Nationwide Inpatient Sample to determine the number of live births, the number of patients diagnosed with clubfoot, and the number of extensive surgical releases that were performed each year from 1996 to 2006. The trends over time were evaluated with use of regression analysis, and changes in frequency were analyzed with use of time series analysis. The percentage of clubfeet that were treated with surgery in each year was calculated by dividing the number of surgical release procedures by the number of clubfoot diagnoses. RESULTS: Between 1996 and 2006, the estimated number of patients under six months of age diagnosed with clubfoot remained fairly constant, averaging 2140 infants per year. The linear equation estimated a slight decrease of approximately thirty-one infants with clubfoot per year (R(2) = 0.51, p < 0.05). In contrast, in the same decade, the estimated number of surgical releases performed in patients less than twelve months of age decreased substantially, from 1641 releases in 1996 to 230 releases in 2006. The linear equation estimated a decrease of approximately 157 surgical releases per year (R(2) = 0.83, p < 0.05). The trend analysis indicated that the percentage of clubfeet treated with surgical release generally decreased over time at a rate of 6.7% per year, decreasing from just over 70% in 1996 to just over 10% in 2006 (R(2) = 0.81, p < 0.05). CONCLUSIONS: In the United States between 1996 and 2006, the rate of extensive surgery to treat idiopathic clubfoot in patients less than twelve months old decreased substantially. This trend is likely due to an increased use of less invasive techniques, such as the Ponseti method, which a growing body of evidence has shown to be a viable treatment option for clubfoot.

Levels of evidence at the AAOS meeting: can authors rate their own submissions, and do other raters agree?

BACKGROUND: A hierarchy of levels of evidence is commonly used to categorize the methodology of scientific studies in order to assist in their critical analysis. Organizers of large scientific meetings are faced with the problem of whether and how to assign levels of evidence to studies that are presented. The present study was performed to investigate two hypotheses: (1) that session moderators and others can consistently assign a level of evidence to papers presented at national meetings, and (2) that there is no difference between the level of evidence provided by the author of a paper and the level of evidence assigned by independent third parties (e.g., members of the Program Committee). METHODS: A subset of papers accepted for presentation at the 2007 American Academy of Orthopaedic Surgeons (AAOS) Annual Meeting was used to evaluate differences in the levels of evidence assigned by the authors, volunteer graders who had access to only the abstract, and session moderators who had access to the full paper. The approved AAOS levels of evidence were used. Statistical tests of interrater correlation were done to compare the various raters to each other, with significance appropriately adjusted for multiple comparisons. RESULTS: Interrater agreement was better than chance for most comparisons between different graders; however, the level of agreement ranged from slight to moderate (kappa=0.16 to 0.46), a finding confirmed by agreement coefficient statistics. In general, raters had difficulty in agreeing whether a study comprised Level-I or Level-II evidence and authors graded the level of evidence of their own work more favorably than did others who graded the abstract. CONCLUSIONS: When abstracts submitted to the AAOS Annual Meeting were rated, there was substantial inconsistency in the assignments of the level of evidence to a given study by different observers and there was some evidence that authors may not rate their own work the same as independent reviewers. This has important implications for the use of levels of evidence in scientific meetings.

[Structure characteristics of ORF2a gene of potato leafroll virus Chinese isolate].

According to the genomic sequence of foreign four PLRV isolates, three pairs of specific primer were designed and synthesized. The cDNA of the ORF2a gene of PLRV-Ch was synthesized by reverse transcription and followed by Polymerase Chain Reaction amplication. The synthesized 3' and 5' cDNA fragment of the PLRV-Ch ORF2a gene were inserted into pUC19 and cloned in E. coli JM109 and were sequenced respectively. The middle cDNA fragment were directly sequenced. The homology of nucleotide sequence of PLRV-Ch compared with PLRV-S (Scotland, UK), PLRV-N(Netherlands), PLRV-A(Australia) and PLRV-C(Canada) were 98.96%, 98.70%, 94.79%, 97.5%, the homology of putative amino acid sequence are 97.97%, 97.97%, 89.69%, 95.94%. In 3' region of ORF2a gene a slippery sequence for-1 frameshift and its downstream "stem-loop" or "pseudoknot" and upstream nucleotide sequence repeats were found. Authors suggested that the nucleotide repeat sequences characteristic for PLRV could form a tight successively folded complementary double stranded regions and hairpins. This structure possibly has something to do with-1 frameshift. The amino acid sequence of C terminus region of 70 kD protein translated by motif IV has a protease characteristic motif and a helicase motif IV. The amino acid sequence of polypeptide translated by ORF2a gene undergoing frameshift has a single-stranded nucleic acid binding protein-like characteristic motif.