A Duplex PCR Assay for Rapid Detection of Klebsiella pneumoniae and Chryseobacterium in Large Yellow Croaker Fish.

Both Klebsiella pneumoniae and Chryseobacterium cause an increasing number of diseases in fish, resulting in great economic losses in aquaculture. In addition, the disease infected with Klebsiella pneumoniae or Chryseobacterium exhibited the similar clinical symptoms in aquatic animals. However, there is no effective means for the simultaneous detection of co-infection and discrimination them for these two pathogens. Here, we developed a duplex polymerase chain reaction (PCR) method based on the outer membrane protein A (ompA) gene of Klebsiella pneumoniae and Chryseobacterium. The specificity and validity of the designed primers were confirmed experimentally using simplex PCR. The expected amplicons for Klebsiella pneumoniae and Chryseobacterium had a size of 663 and 1404 bp, respectively. The optimal condition for duplex PCR were determined to encompass a primer concentration of 0.5 µM and annealing temperature of 57°C. This method was analytical specific with no amplification being observed from the genomic DNA of Escherichia coli, Vibrio harveyi, Pseudomonas plecoglossicida, Aeromonas hydrophila and Acinetobacter johnsonii. The limit of detection was estimated to be 20 fg of genomic DNA for Chryseobacterium and 200 fg for Klebsiella pneumoniae, or 100 colony-forming units (CFU) of bacterial cells in both cases. The duplex PCR was capable of simultaneously amplifying target fragments from genomic DNA extracted from the bacteria and fish liver. For practical validation of the method, 20 diseased fish were collected from farms, among which 4 samples were PCR-positive for Klebsiella pneumoniae and Chryseobacterium. The duplex PCR method developed here is time-saving, specific, convenient, and may prove to be an invaluable tool for molecular detection and epidemiological investigation of Klebsiella pneumoniae and Chryseobacterium in the field of aquaculture.

Enhanced catalytic activity and stability of lactate dehydrogenase for cascade catalysis of D-PLA by rational design.

Serving as a vital medical intermediate and an environmentally-friendly preservative, D-PLA exhibits substantial potential across various industries. In this report, the urgent need for efficient production motivated us to achieve the rational design of lactate dehydrogenase and enhance catalytic efficiency. Surprisingly, the enzymatic properties revealed that a mutant enzyme, LrLDHT247I/D249A/F306W/A214Y (LrLDH-M1), had a viable catalytic advantage. It demonstrated a 3.3-fold increase in specific enzyme activity and approximately a 2.08-fold improvement of Kcat. Correspondingly, molecular docking analysis provided a supporting explanation for the lower Km and higher Kcat/Km of the mutant enzyme. Thermostability analysis exhibited increased half-lives and the deactivation rate constants decreased at different temperatures (1.47-2.26-fold). In addition, the mutant showed excellent resistance abilities in harsh environments, particularly under acidic conditions. Then, a two-bacterium (E. coli/pET28a-lrldh-M1 and E. coli/pET28a-ladd) coupled catalytic system was developed and realized a significant conversion rate (77.7%) of D-phenyllactic acid, using 10 g/L L-phenylalanine as the substrate in a two-step cascade reaction.

Comparing the Volatile and Soluble Profiles of Fermented and Integrated Chinese Bayberry Wine with HS-SPME GC-MS and UHPLC Q-TOF.

To evaluate the flavor characteristics of Chinese bayberry alcoholic beverages, fermented bayberry wine (FBW) and integrated bayberry wine (IBW) were investigated for their volatile and soluble profiles using HS-SPME GC-MS and UHPLC Q-TOF and were analyzed with multidimensional statistical analysis, including PCA and OPLS-DA. The volatile compounds 1-pentanol, ß-caryophyllene and isopentanol were only detected in IBW. ß-caryophyllene, the key flavor component of bayberry, was found to be the most abundant volatile compound in IBW (25.89%) and was 3.73 times more abundant in IBW than in FBW. The levels of ethyl octanoate, ethyl nonanoate, and ethyl decanoate were also several times higher in IBW than in FBW. These compounds contributed to the strong bayberry aroma and better fruity flavor of IBW. On the other hand, high levels of ethyl acetate and octanoic acid in FBW, representing pineapple/overripe or sweat odor, were key contributors to the fermented flavor of FBW. Soluble sugars, such as sucrose, D-glucose, and D-tagatose, as well as amino acids, such as L-glutamate and L-aspartate, had much higher levels in IBW. The anthocyanin pigment cyanidin 3-glucoside, which generates red color, was also higher in IBW. On the other hand, most of the differentially expressed alcohols, acids, amino acids, purines/pyrimidines and esters were present in higher concentrations in FBW compared to IBW. This demonstrated that IBW has a much sweeter and more savory taste as well as a better color generated by more anthocyanins, while FBW presents a more acidic and drier taste as well as a complex formation of alcohols and esters. The study also prompts the need for further research on the flavor profiles of IBW and its potential application and market value.

Immunogenic characteristics of the outer membrane phosphoporin as a vaccine candidate against Klebsiella pneumoniae.

In recent years, Klebsiella pneumoniae (KP) has caused disease outbreaks in different animals, resulting in serious economic losses and biosafety concerns. Considering the broad antibiotic resistance of KP, vaccines are the most effective tools against infection. However, there is still no KP vaccine available in the veterinary field. Our results indicate that the highly conserved outer membrane phosphoporin (PhoE) of KP is immunogenic in mice and elicits high titers of antibodies that were shown to be specific for PhoE by immunoblotting. Immunization with PhoE also induced robust cell-mediated immunity and elicited the secretion of high levels of IFN-γ and IL-4, suggesting the induction of mixed Th1 and Th2 responses. Sera from PhoE-immunized mice induced significantly higher complement-mediated lysis of KP cells than did sera from the PBS control mice. Finally, mice immunized with PhoE were significantly protected against KP challenge, with better survival and a reduced visceral bacterial load. Our data underscore the great potential of PhoE as a novel candidate antigen for a vaccine against KP infection.

Metabolic changes of Issatchenkia orientalis under acetic acid stress by transcriptome profile using RNA-sequencing.

Issatchenkia orientalis (I. orientalis) is tolerant to various environmental stresses especially acetic acid stress in wine making. However, limited literature is available on the transcriptome profile of I. orientalis under acetic acid stress. RNA-sequence was used to investigate the metabolic changes due to underlying I. orientalis 166 (Io 166) tolerant to acetic acid. Transcriptomic analyses showed that genes involved in ergosterol biosynthesis are differentially expressed under acetic acid stress. Genes associated with ribosome function were downregulated, while energy metabolism-related genes were upregulated. Moreover, Hsp70/Hsp90 and related molecular chaperones were upregulated to recognize and degrade misfolded proteins. Compared to Saccharomyces cerevisiae, transcriptomic changes of Io 166 showed many similarities under acetic acid stress. There were significant upregulation of genes in ergosterol biosynthesis and for the application of wine production.

A Method of Constructing Measurement Matrix for Compressed Sensing by Chebyshev Chaotic Sequence.

In this paper, the problem of constructing the measurement matrix in compressed sensing is addressed. In compressed sensing, constructing a measurement matrix of good performance and easy hardware implementation is of interest. It has been recently shown that the measurement matrices constructed by Logistic or Tent chaotic sequences satisfy the restricted isometric property (RIP) with a certain probability and are easy to be implemented in the physical electric circuit. However, a large sample distance that means large resources consumption is required to obtain uncorrelated samples from these sequences in the construction. To solve this problem, we propose a method of constructing the measurement matrix by the Chebyshev chaotic sequence. The method effectively reduces the sample distance and the proposed measurement matrix is proved to satisfy the RIP with high probability on the assumption that the sampled elements are statistically independent. Simulation results show that the proposed measurement matrix has comparable reconstruction performance to that of the existing chaotic matrices for compressed sensing.

Identification of a novel cell-penetrating peptide derived from the capsid protein of chicken anemia virus and its application in gene delivery.

Cell membranes are a great obstacle for entrance of gene therapeutic agents. Cell-penetrating peptides (CPPs) have been proven as a promising gene delivery tool. However, the early TAT peptide derived from the HIV transcription activator protein has been proven that the sequence contains Furin protease cleaved motifs which limited the TAT application in delivery of exogenous active molecules. In the present study, through the bioinformatics and experimental approach, we have identified a novel CPP derived from the N terminus of VP1 protein of chicken anemia virus (CAV), designated as CVP1-N2, which is rich in arginine residues and contains α-helical structure. Then, the ability of CVP1-N2 cell penetrating was detected using confocal imaging and flow cytometry. FITC-labeled CVP1-N2 peptide could rapidly internalize into different types of live cells with dose dependence and without cytotoxic effects by MTT assay. Surprisingly, CVP1-N2 with a pattern of nuclear sub-location has shown the higher uptake efficiency than TAT. At 10, 1, and 0.1 µM, the mean relative internalization of CVP1-N2 was respectively 1.08-, 12-, and 75-fold higher than that of CVP1, as well as 1.6-, 56-, and 75-fold higher than that of TAT. Moreover, using endocytic inhibitors along with low-temperature stress validated that the CVP1-N2 internalization route is direct translocation pathway. Finally, the capacity of CVP1-N2 for delivery of gene into cells was determined, where it was able to carry red fluorescent protein (RFP) and apoptin genes into cells respectively and induce the apoptosis. All these data indicate that CVP1-N2 could be used as a novel gene delivery vehicle for gene therapy in the future. KEY POINTS: • 1CVP1-N2 was identified as a novel more efficient cell-penetrating peptide. • 2. CVP1-N2 localized to the nucleus through the direct transduction pathway. • 3. CVP1-N2 was able to deliver the apoptin gene into HCT116 cells and induce apoptosis.

Integrated transcriptomic and proteomic analysis of the acetic acid stress in Issatchenkia orientalis.

Issatchenkia orientalis known as a multi-tolerant non-Saccharomyces yeast, which tolerant environmental stresses, exhibits potential in wine making and bioethanol production. It is essential for the growth of I. orientalis to tolerant acetic acid in the mixed cultures with Saccharomyces cerevisiae. In this work, RNA-sequence and TMT (Tandem Mass Tag) were used to examine the comprehensive transcriptomic and proteomic profiles of I. orientalis in response to acetic acid. The results showed that 876 genes were identified differentially transcribed in I. orientalis genome and 399 proteins expressed in proteome after 4 hr acetic acid (90 mM, pH 4.5). The comprehensive analysis showed a series of determinants of acetic acid tolerance: Glycolysis and TCA cycle provide enough nicotinamide adenine dinucleotide to effectively convert acetic acid. Genes associated with potassium, iron, zinc, and glutathione synthesis were upregulated. The same changes of differentially expressed genes and proteins were mainly concentrated in chaperones, coenzyme, energy production, and transformation. PRACTICAL APPLICATIONS: In addition to the main fermentation products, wine yeast also produces metabolite acetic acid in the fermentation process, and yeast cells are exposed to acetic acid stress, which restrains cell proliferation. Issatchenkia orientalis exhibits great potential in winemaking and bioethanol production. The yeast is known as a multi-tolerant non-Saccharomyces yeast that can tolerate a variety of environmental stresses. In this study, RNA-Seq and TMT were conducted to investigate the changes in transcriptional and proteomic profile of I. orientalis under acetic acid stress. The knowledge of the transcription and expression changes of the I. orientalis is expected to understand the tolerance mechanisms in I. orientalis and to guide traditional fermentation processes by Saccharomyces cerevisiae improving its high resistance to acetic acid stress.

Integrated transcriptomic and proteomic analysis of the ethanol stress response in Saccharomyces cerevisiae Sc131.

The molecular mechanism of Saccharomyces cerevisiae tolerant to ethanol stress remains to be further elucidated. In this study, a comprehensive analysis based on RNA-seq and iTRAQ LC-MS/MS was used to investigate the global mechanism of S. cerevisiae strain Sc131 in response to ethanol stress at transcriptomic and proteomic levels. Totally, 937 differentially expressed genes (DEGs) and 457 differentially expressed proteins (DEPs) were identified in Sc131 under ethanol stress. Results revealed that 4-h ethanol stress (10% v/v) can induce filamentous growth, sexual reproduction. Mitochondria and endoplasmic reticulum (ER) were proved to be two important organelles in resisting ethanol stress. Signal transduction such as G-protein coupled receptor signaling and metal ion regulation were remarkably activated at the presence of ethanol. Moreover, silent information regulator (Sir) proteins and aromatic amino acids especially tryptophan were involved in response to ethanol and might be helpful for cell survival. This study provides a global perspective on the mechanism of S. cerevisiae tolerant to ethanol stress and sheds light on the potential application of Sc131 in Chinese bayberry wine brewing. BIOLOGICAL SIGNIFICANCE: It is of great importance for S. cerevisiae to tolerate high levels of ethanol during wine fermentation. However, the molecular mechanism of S. cerevisiae tolerant to ethanol stress remains to be further elucidated at transcriptomic and proteomic levels. In present study, we employed a comprehensive analysis based on RNA-seq and iTRAQ and found several potential pathways involving in the response of Sc131 to ethanol stress. To our knowledge, this is the first integrated analysis combining transcriptomic and proteomic technologies to study the mechanism of Sc131 under ethanol stress.

Transcriptome profiling of Issatchenkia orientalis under ethanol stress.

Issatchenkia orientalis, a non-Saccharomyces yeast that can resist a wide variety of environmental stresses, has potential use in winemaking and bioethanol production. Little is known about gene expression or the physiology of I. orientalis under ethanol stress. In this study, high-throughput RNA sequencing was used to investigate the transcriptome profile of I. orientalis in response to ethanol. 502 gene transcripts were differentially expressed, of which 451 were more abundant, and 51 less abundant, in cells subjected to 4 h of ethanol stress (10% v/v). Annotation and statistical analyses suggest that multiple genes involved in ergosterol biosynthesis, trehalose metabolism, and stress response are differentially expressed under these conditions. The up-regulation of molecular chaperones HSP90 and HSP70, and also genes associated with the ubiquitin-proteasome proteolytic pathway suggests that ethanol stress may cause aggregation of misfolded proteins. Finally, ethanol stress in I. orientalis appears to have a nitrogen starvation effect, and many genes involved in nutrient uptake were up-regulated.

Investigating the underlying mechanism of Saccharomyces cerevisiae in response to ethanol stress employing RNA-seq analysis.

Saccharomyces cerevisiae has been widely used for wine fermentation and bio-fuels production. A S. cerevisiae strain Sc131 isolated from tropical fruit shows good fermentation properties and ethanol tolerance, exhibiting significant potential in Chinese bayberry wine fermentation. In this study, RNA-sequence and RT-qPCR was used to investigate the transcriptome profile of Sc131 in response to ethanol stress. Scanning Electron Microscopy were carried out to observe surface morphology of yeast cells. Totally, 937 genes were identified differential expressed, including 587 up-regulated and 350 down-regulated genes, after 4-h ethanol stress (10% v/v). Transcriptomic analysis revealed that, most genes involved in regulating filamentous growth or pseudohyphal growth were significantly up-regulated in response to ethanol stress. The complex protein quality control machineries, Hsp90/Hsp70 and Hsp104/Hsp70/Hsp40 based chaperone system combining with ubiquitin-proteasome proteolytic pathway were both activated to recognize and degrade misfolding proteins. Genes related to biosynthesis and metabolism of two well-known stress-responsive substances trehalose and ergosterol were generally up-regulated, while genes associated with amino acids biosynthesis and metabolism processes were differentially expressed. Moreover, thiamine was also important in response to ethanol stress. This research may promote the potential applications of Sc131 in the fermentation of Chinese bayberry wine.

The modulatory effect of (-)-epigallocatechin 3-O-(3-O-methyl) gallate (EGCG3″Me) on intestinal microbiota of high fat diet-induced obesity mice model.

(-)-Epigallocatechin 3-O-(3-O-methyl) gallate (EGCG3″Me) has exhibited multiple beneficial effects on the prevention of obesity in oolong tea. However, its absorption is relatively low, and the potential to be fully utilized is not completely elucidated. Therefore, with human flora-associated (HFA) mice model, the effect of EGCG3″Me on high fat diet-induced obesity was investigated by high-throughput sequencing. The shifts in relative abundance of the dominant taxa at the phylum, family and genus levels showed the dramatically effects of EGCG3″Me. Despite significant inter-individual variation, a large increase in Bacteroidetes with concomitant decrease of Firmicutes was observed after the administration of EGCG3″Me for 8weeks, with a corresponding decrease in the Firmicutes/Bacteroidetes ratio, which reflect the modulatory effect of EGCG3″Me on intestinal microbiota. The results showed that EGCG3″Me may have prebiotic-like activity and can be used as a functional food component with potential therapeutic utility in manipulating intestinal microbiota, contributing to the prevention of gut dysbiosis.

Metabolic Engineering of Wheat Provitamin A by Simultaneously Overexpressing CrtB and Silencing Carotenoid Hydroxylase (TaHYD).

Increasing the provitamin A content in staple crops via carotenoid metabolic engineering is one way to address vitamin A deficiency. In this work a combination of methods was applied to specifically increase ß-carotene content in wheat by metabolic engineering. Endosperm-specific silencing of the carotenoid hydroxylase gene (TaHYD) increased ß-carotene content 10.5-fold to 1.76 µg g(-1) in wheat endosperm. Overexpression of CrtB introduced an additional flux into wheat, accompanied by a ß-carotene increase of 14.6-fold to 2.45 µg g(-1). When the "push strategy" (overexpressing CrtB) and "block strategy" (silencing TaHYD) were combined in wheat metabolic engineering, significant levels of ß-carotene accumulation were obtained, corresponding to an increase of up to 31-fold to 5.06 µg g(-1). This is the first example of successful metabolic engineering to specifically improve ß-carotene content in wheat endosperm through a combination of methods and demonstrates the potential of genetic engineering for specific nutritional enhancement of wheat.

Enrichment of provitamin A content in wheat (Triticum aestivum L.) by introduction of the bacterial carotenoid biosynthetic genes CrtB and CrtI.

Carotenoid content is a primary determinant of wheat nutritional value and affects its end-use quality. Wheat grains contain very low carotenoid levels and trace amounts of provitamin A content. In order to enrich the carotenoid content in wheat grains, the bacterial phytoene synthase gene (CrtB) and carotene desaturase gene (CrtI) were transformed into the common wheat cultivar Bobwhite. Expression of CrtB or CrtI alone slightly increased the carotenoid content in the grains of transgenic wheat, while co-expression of both genes resulted in a darker red/yellow grain phenotype, accompanied by a total carotenoid content increase of approximately 8-fold achieving 4.76 µg g(-1) of seed dry weight, a ß-carotene increase of 65-fold to 3.21 µg g(-1) of seed dry weight, and a provitamin A content (sum of α-carotene, ß-carotene, and ß-cryptoxanthin) increase of 76-fold to 3.82 µg g(-1) of seed dry weight. The high provitamin A content in the transgenic wheat was stably inherited over four generations. Quantitative PCR analysis revealed that enhancement of provitamin A content in transgenic wheat was also a result of the highly coordinated regulation of endogenous carotenoid biosynthetic genes, suggesting a metabolic feedback regulation in the wheat carotenoid biosynthetic pathway. These transgenic wheat lines are not only valuable for breeding wheat varieties with nutritional benefits for human health but also for understanding the mechanism regulating carotenoid biosynthesis in wheat endosperm.

Expression, purification and antimicrobial activity of puroindoline A protein and its mutants.

Wheat puroindoline proteins, PINA and PINB, play key roles in determining wheat grain hardness as well as in defending the plant against pathogens. PINA has much greater membrane-binding property and antimicrobial activity because it contains more tryptophan residues in the unique tryptophan-rich domain (TRD). In order to obtain proteins with higher antimicrobial activity, mutants of PINA containing two or three copies of TRD, designated ABBC and ABBBC, respectively, were constructed and expressed in E. coli Rosetta-gami (DE3). Metal affinity chromatography was used to purify the soluble affinity-tagged recombinant proteins. The secondary structures of the recombinant proteins were predicted by the online program Protein Homology/analog Y Recognition Engine v2.0 and experimentally assessed using circular dichroism. Minimum inhibition concentration tests and fluorescence microscope analyses were employed to evaluate the antimicrobial activities of the mutants. The results showed that the purified recombinant ABBC was correctly folded and presented significantly higher antimicrobial activities against E. coli and S. aureus than wild-type PINA, suggesting its potential use as an antimicrobial agent. The results also confirmed that TRD is a determinant of the antimicrobial activity of PINA and demonstrated that it is feasible to enhance the antimicrobial activity of PINA by adding one copy of TRD.

Expression of the 1Ax1 transgene in an elite Chinese wheat variety and its effect on functional properties.

BACKGROUND: The transfer of transgenes from 'model' wheat genotypes into elite wheat cultivars using conventional plant breeding is an alternative strategy for improving the dough quality of wheat. Thus a cross was made between a popular Chinese elite wheat cultivar of the Yangzi River down-central area that expresses high-molecular-weight glutenin subunit (HMW-GS): 1Bx7 + 1By8 plus 1Dx2 + 1Dy12, and a model transgenic wheat line B102-1-2 which over-expresses HMW-GS 1Ax1 in an L88-31 genetic background that includes HMW-GS 1Bx17 + By18. RESULTS: F(1) to F(6) generations of crosses between B102-1-2 (paternal) and Emai (maternal) were analysed for their HMW-GS compositions, allowing the selection of pure F(6) lines over-expressing HMS-GS 1Ax1 in the presence of the endogenous HMW-GS: 1Bx7 + 1By8 with 1Dx2 + 1Dy12, and 1Bx17 + 1By18 with 1Dx2 + 1Dy12. Analysis of the F(6) lines showed changes in the extensograph parameters, with increases in peak area, resistance to extension, and extensibility and peak resistance to extension, similar to those observed in the transgenic paternal line B102-1-2. CONCLUSION: This work showed that the expression levels of the 1Ax1 transgene and the effects on dough properties were similar in the transgenic parental line B102-1-2 and the F(6) progeny generated from a cross with an elite Chinese cultivar Emai. The feasibility of using transgenic lines expressing HMW-GS subunits in conventional breeding programmes was demonstrated.