Search

Plant extracellular vesicles (EVs) play critical roles in the cross-kingdom trafficking of molecules from hosts to interacting microbes, most notably in plant defense responses. However, the isolation of pure, intact EVs from plants remains challenging. A variety of methods have been utilized to isolate plant EVs from apoplastic washing fluid (AWF). Here, we compare published plant EV isolation methods, and provide our recommended method for the isolation and purification of plant EVs. This method includes a detailed protocol for clean AWF collection from Arabidopsis thaliana leaves, followed by EV isolation via differential centrifugation. To further separate and purify specific subclasses of EVs from heterogeneous vesicle populations, density gradient ultracentrifugation and immunoaffinity capture are then utilized. We found that immunoaffinity capture is the most precise method for specific EV subclass isolation when suitable specific EV biomarkers and their corresponding antibodies are available. Overall, this study provides a guide for the selection and optimization of EV isolation methods for desired downstream applications. This article is protected by copyright. All rights reserved.

Carbonyl compounds play a critical role in the pathogenesis of diabetic nephropathy (DN). Pueraria lobata (PL), also known as "Kudzu", is a widely consumed functional food or nutraceutical and has shown promise in the prevention of diabetes and complications such as DN. To explore the beneficial effects and the underlying mechanisms of PL against DN, a new strategy for in-depth metabolic profiling of carbonyl compounds in DN mice plasma by chemical derivatization combined with UPLC-Q-Orbitrap high-resolution mass spectrometry (HRMS)/MS analysis was developed for the first time. Pharmacological evaluation revealed that PL extracts containing a total of 73 identified compounds could ameliorate kidney injury and regulate abnormal glycolipid metabolism. In metabolomics analysis, 19 carbonyl compounds with significant differences were identified between DN mice and normal mice. Moreover, 12 metabolites had a tendency to return to normal levels after PL treatment. Overall, PL exerts beneficial effects on DN by regulating abnormal glycolipid metabolism and carbonyl stress, and endogenous carbonyl compounds might serve as potential biomarkers for DN.

Plantaricin Q7 is a bacteriocin produced by Lactobacillus plantarum Q7 with food preservation potential. Low yield is one of the bottlenecks of the wide application of plantaricin Q7. Nontargeted metabolomics was performed to reveal the mechanism of plantaricin Q7 biosynthesis. The results showed that the composition and abundance of intracellular metabolites varied significantly at key time points of plantaricin Q7 synthesis. Differential metabolic pathways were purine metabolism; pyrimidine metabolism; alanine, aspartate, and glutamate metabolism; amino acid biosynthesis; aminoacyl-tRNA biosynthesis; and ABC transporters. Differential metabolites were xanthine, deoxyadenosine, uracil, 5-methylcytosine, α-ketoglutarate, γ-aminobutyric acid, glutamate, glutamine, and tryptophan. Based on metabolomics information, the putative metabolic synthesis pathway of plantaricin Q7 was proposed. Glutamine, glutamate, and 5-methylcytosine could be critical metabolites and simulate plantaricin Q7 biosynthesis significantly (P < 0.05). Bacteriocin production was investigated by comparative metabolomics in this report, which could help to achieve higher plantaricin Q7 yield by metabolic regulation.

Plants use extracellular vesicles (EVs) to transport small RNAs (sRNAs) into their fungal pathogens and silence fungal virulence-related genes through a phenomenon called ‘cross-kingdom RNAi’. It remains unknown, however, how sRNAs are selectively loaded into EVs. Here, we identified several RNA-binding proteins in Arabidopsis, including Argonaute 1 (AGO1), RNA helicases (RHs) and annexins (ANNs), which are secreted by exosome-like EVs. AGO1, RH11 and RH37 selectively bind to EV-enriched sRNAs but not to non-EV-associated sRNAs, suggesting that they contribute to the selective loading of sRNAs into EVs. Conversely, ANN1 and ANN2 bind to sRNAs non-specifically. The ago1, rh11 rh37 and ann1 ann2 mutants showed reduced secretion of sRNAs in EVs, demonstrating that these RNA-binding proteins play an important role in sRNA loading and/or stabilization in EVs. Furthermore, rh11 rh37 and ann1 ann2 showed increased susceptibility to Botrytis cinerea, suggesting that RH11, RH37, ANN1 and ANN2 positively regulate plant immunity against B. cinerea. Plants use extracellular vesicles to deliver small RNAs that could silence fungal virulence genes to their fungal pathogens. In this study, the authors profile the components of these extracellular vesicles and investigate regulators contributing to the specific RNA loading and stabilization.

Diabetes seriously endangers public health and brings a heavy economic burden to the country. Inflammation is one of the main inducing factors of type-2 diabetes (T2D) and may cause some complications of diabetes, such as diabetic encephalopathy and peripheral neuropathy. In-depth research and development of drugs to cure diabetes and complications are of great significance. Pueraria lobate is a medicinal herb used in several countries to treat many diseases. Here, two new polysaccharides (PLB-1-1 and PLB-1-2) were isolated and purified from the root of Pueraria lobata with molecular weights of 9.1 × 103 Da and 3.8 × 103 Da, respectively. The structure was evaluated by monosaccharide composition, GC-MS and NMR spectroscopy. It was determined that PLB-1-1 comprised →4)-α-D-Glcp-(1→, α-D-Glcp-(1→, →6)-β-D-Galp-(1→, →3)-α-L-Araf-(1→, →3,6)-β-D-Manp-(1→ and →4,6)-β-D-Manp-(1→, and PLB-1-2 consisted of →4)-α-D-Glcp-(1→, β-D-Glcp-(1→, →4,6)-β-D-Glcp-(1→, →3,6)-β-D-Manp-(1→ and α-L-Fucp-(1→. Furthermore, both PLB-1-1 and PLB-1-2 showed anti-inflammatory and inhibitory activities of α-glucosidase and α-amylase in vitro. Therefore, the new polysaccharides, i.e., PLB-1-1 and PLB-1-2, may be considered candidates for the treatment of diabetes and its related complications.

It is urgent to obtain targeted drugs that selectively bind to pathological targets rather than physiological targets in the early stage of drug screening. G-Quadruplex has become one of the important targets in the development of anti-tumor drugs. However, drugs that target quadruplexes may also bind to dsDNA, which may lead to adverse reactions. In this study, a new three-phase laminar flow chip was constructed to enable the multi-components of a traditional Chinese medicine extract to dynamically and competitively bind with G-quadruplex DNA (on target) and double-stranded DNA (off target), so as to select high-efficiency and low-toxicity anti-tumor drugs. The results showed that there were five compounds in the extracts of Macleaya cordata seeds that exhibited obvious differences in binding to the two targets. Furthermore, the binding constants and modes of four identified alkaloids as they bound to two DNA targets were verified by fluorescence spectra and molecular docking methods. The toxicity to HepG2 and LO2 cells from the four alkaloids was also compared. The results showed that sanguinarine and chelerythrine could be used as candidate drugs with stronger binding to HT24 than DNA26. The chip can also be used for other types of double-target screening of other traditional Chinese medicine extracts or compound libraries.

Two new phenylpropanoids, named stytonkinol A (1) and stytonkinol B (2), have been isolated from the resin of Styrax tonkinensis (Pierre) Craib ex Hartw. Their structures were determined by spectroscopic analysis, including 1D and 2D NMR, and HR-ESI–MS. Two isolated compounds were assayed for cytotoxic activities against five tumor cell lines (HepG-2, A549, Hela, MCF-7, and PC-3) by Cell Counting Kit-8 (CCK-8) test in vitro. The cytotoxic effectiveness observed against Hela, and MCF-7 cell lines of compound 1 were superior or similar to the positive control cisplatin (IC50 values of 40.95 and 47.36 μM), with IC50 values of 26.75 and 45.16 μM, respectively, while it showed moderate cytotoxic activities against the HepG-2 and PC-3 cell lines. Compound 2 showed moderate cytotoxic activities on cells MCF-7 with IC50 values of 57.1 μM.

Near infrared (NIR) spectroscopy is an effective technique for adulteration detection in traditional Chinese medicine. The aim is to develop a discriminant model with the aid of chemometrics tools. The discriminant model is conventionally established by the means of stepwise optimization. This approach is often limited to trial-and-error and considered as a burden. In this study, a systematic optimization approach was proposed to develop the discriminant model with the aid of the design of experiment tools and applied to a case study of differentiating Pinellia ternata from Pinellia pedatisecta and adulterated Pinellia ternata using NIR spectroscopy. Spectral pretreatment, variable selection, and discriminant methods were identified as critical factors. The classification accuracy and no-error rate of the calibration set, cross-validation, and the prediction set were calculated to evaluate the performance of discriminant models. A full factorial design was applied to analyze the effect of critical factors at different levels on the model performance and optimize these factors. Three discriminant models including discriminant analysis coupled with principal component analysis (PCA-DA), partial least squares – discriminant analysis (PLS-DA), and k-nearest neighbors (KNN) were obtained by systematic optimization. The performance of PCA-DA and PLS-DA models obtained by systematic optimization was very good, and no samples were misclassified, which were better than those obtained by stepwise optimization. The performance of the KNN model obtained by systematic optimization was not desired and it was equal to that obtained by stepwise optimization. The results showed that Pinellia ternata could be successfully discriminated from Pinellia pedatisecta and adulterated Pinellia ternata by the PCA-DA and PLS-DA models. Compared to the stepwise optimization approach, the systematic optimization approach can improve the PCA-DA and PLS-DA model performance for differentiating Pinellia ternata from Pinellia pedatisecta and adulterated Pinellia ternata.

The milk fat globule membrane (MFGM) is a complex, highly conserved structure surrounding fat droplets secreted into mammalian milk. This study evaluated the impact of MFGM on Lactobacillus rhamnosus GG (LGG). MFGM-10 (2.5 g/L, 5 g/L, and 10 g/L) did not affect LGG growth in MRS medium but enhanced the ability of LGG to survive in the presence of 0.5% porcine bile. In the presence of MFGM-10 (5 g/L) and bile (0.5%), there were less complex polysaccharides in the media and less capsular polysaccharides associated with the LGG cells compared to the bile exposure alone (p < 0.05). The expression of four EPS genes was modulated by bile stress and MFGM. Biofilm thickness was increased (p < 0.05) during bile stress with MFGM compared to other treatments. Furthermore, MFGM increased LGG survival during transit in the murine GI tract. Future experiments will determine the impact of MFGM on LGG probiotic functionality.

Ulcerative colitis (UC) is considered an immune disease, which is related to the dysbiosis of intestinal microbiota and disorders of the host immune system and metabolism. Sophora flavescens Aiton has been used for the clinical treatment of UC in China and East Asia for thousands of years. It has many traditional prescriptions and modern preparations, and its curative effects are definite. We are the first to report that the flavonoids in Sophora flavescens (S. flavescens) Aiton EtOAc extract (SFE) could potentially attenuate the dextran sodium sulfate–induced UC in mice, which changed the current understanding of considering alkaloids as the only anti-UC pharmacological substances of S. flavescens Aiton. Based on the 16S rRNA gene sequencing and metabolomic analysis, it was found that the anti-UC effects of SFE were due to the regulation of gut microbiota, reversing the abnormal metabolisms, and regulation of the short-chain fatty acids synthesis. Notably, according to the interaction networks of specific bacteria and “bacteria and metabolites” co-expression network, the SFE could enrich the abundance of the commensal bacterium Lactobacillus, Roseburia, norank_f__Muribaculaceae, Anaerotruncus, Candidatus_Saccharimona, and Parasutterella, which are proposed as potentially beneficial bacteria, thereby playing vital roles in the treatment of UC.

Much effort has been devoted to studying the production of Streptomyces transglutaminase (TGase). However, more exploration into the mechanism of TGase biosynthesis is necessary to enhance its production further. The effect of excessive metal stress on Streptomyces mobaraensis's TGase activity, growth rate, and mycelium differentiation were evaluated. To elucidate the regulatory mechanism of TGase production and cell differentiations, a proteomic analysis and qRT-PCR of S. mobaraensis was performed. This study showed that the TGase biosynthesis was enhanced while the cell growth was inhibited under MgCl2 stress at the earlier stage of incubation. Furthermore, MgCl2 stress resulted in early cell differentiation compared to the control group. The proteomic analysis indicated that both the nucleotide metabolism and primary metabolism were repressed at the onset of TGase production, explaining the observed decrease in cell growth rate. Several enriched enzymes in the nitrogen metabolic pathways confirmed that the metabolic fluxes for the syntheses of glycine and serine were increased. Furthermore, some stress or stress-related proteins were expressed at a low level in the strain cultivated in normal medium but were highly expressed at the onset of TGase production.

Communication between plants and interacting microorganisms requires functional molecule trafficking, which is essential for host defense and pathogen virulence. Extracellular vesicles (EVs) are single membrane-bound spheres that carry complex cargos, including lipids, proteins, and nucleic acids. They mediate cell-to-cell communication via the transfer of molecules between cells. Plant EVs have been isolated from many plant species and play a prominent role in immune system modulation and plant defense response. Recent studies have shown that plant EVs are emerging players in cross-kingdom regulation and contribute to plant immunity by mediating the trafficking of regulatory small RNA into pathogens, leading to the silencing of pathogen virulence-related genes. This review summarizes the current understanding of plant EV isolation technologies, the role of plant EVs in plant immunity, and the mechanism of plant EV biogenesis, as well as approaches for how these findings can be developed into innovative strategies for crop protection.

Phytophthora species cause some of the most serious diseases of trees and threaten forests in many parts of the world. Despite the generation of genome sequence assemblies for over 10 tree‐pathogenic Phytophthora species and improved detection methods, there are many gaps in our knowledge of how these pathogens interact with their hosts. To facilitate cell biology studies of the infection cycle we examined whether the tree pathogen Phytophthora kernoviae could infect the model plant Nicotiana benthamiana. We transformed P. kernoviae to express green fluorescent protein (GFP) and demonstrated that it forms haustoria within infected N. benthamiana cells. Haustoria were also formed in infected cells of natural hosts, Rhododendron ponticum and European beech (Fagus sylvatica). We analysed the transcriptome of P. kernoviae in cultured mycelia, spores, and during infection of N. benthamiana, and detected 12,559 transcripts. Of these, 1,052 were predicted to encode secreted proteins, some of which may function as effectors to facilitate disease development. From these, we identified 87 expressed candidate RXLR (Arg‐any amino acid‐Leu‐Arg) effectors. We transiently expressed 12 of these as GFP fusions in N. benthamiana leaves and demonstrated that nine significantly enhanced P. kernoviae disease progression and diversely localized to the cytoplasm, nucleus, nucleolus, and plasma membrane. Our results show that N. benthamiana can be used as a model host plant for studying this tree pathogen, and that the interaction likely involves suppression of host immune responses by RXLR effectors. These results establish a platform to expand the understanding of Phytophthora tree diseases., Tree pathogen Phytophthora kernoviae forms digit‐shaped haustoria during infection of model and natural hosts, and secretes RXLR effectors that promote infection and localize to diverse cellular locations. ​

BackgroundDengue fever is a mosquito-borne febrile illness. Southeast Asia experienced severe dengue outbreaks in 2019, and over 1000 cases had been reported in Jiangxi, a previously known low-epidemic region in China. However, the emergence of a dengue virus epidemic in a non-epidemic region remains unclear.MethodsWe enrolled 154 dengue fever patients from four hospitals in Jiangxi, from April 2019 to September 2019. Real-time PCR, NS1 antigen rapid test, and IgM, IgG tests were performed, and 14 samples were outsourced to be sequenced metagenomically.ResultsAmong the 154 cases, 42 were identified as imported and most of them returned from Cambodia. A total of 113 blood samples were obtained and 106 were identified as DENV-1, two as DENV-2, and five were negative through RT-PCR. All DENV-1 strains sequenced in this study were all classified to one cluster and owned a high similarity with a Cambodia strain isolated in 2019. The evolutionary relationships of amino acid were consistent with that of nucleotide genome result. The sequence-based findings of Jiangxi strains were consistent with epidemiological investigation.ConclusionEpidemiological analysis demonstrated that the emergence of dengue cases led to autochthonous transmission in several cities in Jiangxi, a low-epidemic region before. This study emphasized future prevention and control of dengue fever in both epidemic and non-epidemic regions.

Immune receptors at the plant cell surface can recognize invading microbes. The perceived microbial molecules are typically widely conserved and therefore the matching surface receptors can detect a broad spectrum of pathogens. Here we describe a family of Phytophthora small extracellular proteins that consists of conserved subfamilies that are widely recognized by solanaceous plants. Remarkably, one subclass of SCR74 proteins is highly diverse, restricted to the late blight pathogen Phytophthora infestans and is specifically detected in wild potato plants. The diversification of this subfamily exhibits signatures of a coevolutionary arms race with surface receptors in potato. Insights into the molecular interaction between these potato-specific receptors and the recognized Phytophthora proteins are expected to contribute to disease resistance breeding in potato., Plants deploy cell surface receptors known as pattern-recognition receptors (PRRs) that recognize non-self molecules from pathogens and microbes to defend against invaders. PRRs typically recognize microbe-associated molecular patterns (MAMPs) that are usually widely conserved, some even across kingdoms. Here, we report an oomycete-specific family of small secreted cysteine-rich (SCR) proteins that displays divergent patterns of sequence variation in the Irish potato famine pathogen Phytophthora infestans. A subclass that includes the conserved effector PcF from Phytophthora cactorum activates immunity in a wide range of plant species. In contrast, the more diverse SCR74 subclass is specific to P. infestans and tends to trigger immune responses only in a limited number of wild potato genotypes. The SCR74 response was recently mapped to a G-type lectin receptor kinase (G-LecRK) locus in the wild potato Solanum microdontum subsp. gigantophyllum. The G-LecRK locus displays a high diversity in Solanum host species compared to other solanaceous plants. We propose that the diversification of the SCR74 proteins in P. infestans is driven by a fast coevolutionary arms race with cell surface immune receptors in wild potato, which contrasts the presumed slower dynamics between conserved apoplastic effectors and PRRs. Understanding the molecular determinants of plant immune responses to these divergent molecular patterns in oomycetes is expected to contribute to deploying multiple layers of disease resistance in crop plants.

Background: Understanding of the restriction of HIV-1 transcription in resting CD4+ Tcells is critical to find a cure for AIDS. Although many negative factors causing HIV-1 transcription blockage in resting CD4+ T-cells have been found, there are still unknown mechanisms to explore. Objective: To explore the mechanism for the suppression of de novo HIV-1 transcription in resting CD4+ T-cells. Methods: In this study, a short isoform of Per-1 expression plasmid was transfected into 293T cells with or without Tat's presence to identify Per-1 as a negative regulator for HIV-1 transcription. Silencing of Per-1 was conducted in resting CD4+ T-cells or monocyte-derived macrophages (MDMs) to evaluate the antiviral activity of Per-1. Additionally, we analyzed the correlation between Per-1 expression and viral loads in vivo, and silenced Per-1 by siRNA technology to investigate the potential anti-HIV-1 roles of Per-1 in vivo in untreated HIV-1-infected individuals. Results: We found that short isoform Per-1 can restrict HIV-1 replication and Tat ameliorates this inhibitory effect. Silencing of Per-1 could upregulate HIV-1 transcription both in resting CD4+ Tcells and MDMs. Moreover, Per-1 expression is inversely correlated with viral loads in Rapid progressors (RPs) in vivo. Conclusion: These data together suggest that Per-1 is a novel negative regulator of HIV-1 transcription. This restrictive activity of Per-1 to HIV-1 replication may contribute to HIV-1 latency in resting CD4+ T-cells.

Stir-heating is a common method to process Gardenia jasminoides Ellis. The processing of the G. jasminoides Ellis which do not meet the regulated requirements can result in the unqualified products...

Fifty-two infection-induced Phytophthora infestans RXLR effectors have diverse subcellular localizations, and the majority significantly enhance infection when expressed in leaves. Combining effectors targeting different defence pathways additively enhances colonization., Oomycetes such as the potato blight pathogen Phytophthora infestans deliver RXLR effectors into plant cells to manipulate host processes and promote disease. Knowledge of where they localize inside host cells is important in understanding their function. Fifty-two P. infestans RXLR effectors (PiRXLRs) up-regulated during early stages of infection were expressed as fluorescent protein (FP) fusions inside cells of the model host Nicotiana benthamiana. FP–PiRXLR fusions were predominantly nucleo-cytoplasmic, nuclear, or plasma membrane-associated. Some also localized to the endoplasmic reticulum, mitochondria, peroxisomes, or microtubules, suggesting diverse sites of subcellular activity. Seven of the 25 PiRXLRs examined during infection accumulated at sites of haustorium penetration, probably due to co-localization with host target processes; Pi16663 (Avr1), for example, localized to Sec5-associated mobile bodies which showed perihaustorial accumulation. Forty-five FP–RXLR fusions enhanced pathogen leaf colonization when expressed in Nicotiana benthamiana, revealing that their presence was beneficial to infection. Co-expression of PiRXLRs that target and suppress different immune pathways resulted in an additive enhancement of colonization, indicating the potential to study effector combinations using transient expression assays. We provide a broad platform of high confidence P. infestans effector candidates from which to investigate the mechanisms, singly and in combination, by which this pathogen causes disease.

Objective Crude Leonuri Fructus (CLF), the fruits of the Leonurus japonicus Houtt, and processed Leonuri Fructus (PLF) by stir-baking as the important Chinese herbal medicines, have been used in China and other Asian countries for thousands of years. The objective of this research is to reveal the difference between CLF and PLF. Methods The sensory technologies of the colorimetry, sensitive and validated HPLC-ELSD and GC combined with flame ionization detector (GC-FID) were employed to discriminate CLF and its processed product PLF. The color parameters of the samples were determined by colorimetric instrument CR-410. Moreover, the content of stachydrine and six fatty acids were determined by HPLC and GC. Subsequently, analysis of variance (ANOVA), principal components analysis (PCA), hierarchical cluster analysis (HCA), and Kendall's correlation test were performed for data analysis. Results The CLF and PLF were divided into two categories by PCA and HCA in terms of their component content and color. The results distinctly demonstrated significant changes in color and the content of indicative components between CLF and PLF. Conclusion The study revealed that HPLC, GC, and colorimetric method in combination with chemometric method could be used as comprehensive quality evaluation for CLF and PLF.

A bioarray chip was developed for the rapid and sensitive detection of the antibody (Ab) in nanoliter volumes of biological fluids. The chip was used to detect the anti-hemagglutinin (anti-HA) Ab f...

MicroRNAs (miRNAs) are a novel class of diagnostic and therapeutic target in cancer. Here, we aimed to explore the effects and mechanism of miR-195 regulation in colon cancer. The expressions of several putative miRNAs in colon tumors, compared to those in normal tissues, were investigated by bioinformatical analysis of a Gene Expression Omnibus database. Quantitative real-time PCR analysis (qRT-PCR) was used to validate the identified changes in normal tissues, primary tumors, and metastatic tumors. MTT, soft agar colony formation, and transwell assays were used to evaluate the effects of miR-195 overexpression or inhibition on cell viability, proliferation, migration, and invasion. Targets of miR-195 were identified by TargetScan, and subsequently verified by qRT-PCR and Western blot. The role of miR-195 in the β-catenin pathway was also studied using RT-PCR and Western blot. MiR-195 expression was downregulated in colon carcinoma tissues and negatively correlated with the metastatic potential. While transfecting miR-195 mimics decreased the proliferation, migration, and invasion of colon cancer cells, miR-195 inhibition exerted opposing effects. WNT3A was identified as a direct target of miR-195. β-catenin was also downregulated by miR-195 in colon cancers. MiR-195 downregulation is associated with the enhanced proliferation, migration, and invasion of colon cancer. MiR-195 directly downregulates WNT3A. Our results indicate that miR-195 is a potential diagnostic marker and therapeutic target for improving the clinical management of colon cancer.

Exopolysaccharides (EPSs), produced by lactic acid bacteria (LAB), have been used primarily to improve the quality and taste of food, also possess a variety of unique biological functions, such as immunoregulation and anti-tumor activities. The diversity of molecular structural characteristics of LAB-generated EPSs represents one of the main factors responsible for this plethora of functions. Accordingly, the structural analysis of the EPSs produced by LAB is the prerequisite and basis for examining its functional and structure-activity relationships. In this report, we summarized the current progress of key methodologies involved in the structural analysis of LAB-generated EPSs, including their isolation, purification, primary structure and advances in structural research. A comprehensive discussion regarding the application of chemical analysis, instrument analysis and computer aided technology in the structure analysis of LAB-generated EPSs was provided. Further, the future development of the research on the structure of LAB-generated EPSs was also presented.

Emerging evidences have linked the gut microbiota to poultry physiology. Gut microbiota composition in Shaoxing ducks were profiled under different rearing conditions: raised on the litter floor and the plastic mesh floor. A total of 46 and 39 luminal content samples from the duodenum, ileum, and cecum of the ducks reared under the two conditions were analyzed by 16S rRNA gene amplicon sequencing analysis. Proteobacteria (48.66%), Proteobacteria (33.38%), and Bacteroidetes (55.35%) were the dominant phyla in the duodenum, ileum, and cecum of the ducks reared on the litter floor respectively, while Firmicutes (30.80%), Firmicutes (66.62%) and Bacteroidetes (47.15%) were the topmost phyla in the duodenum, ileum, and cecum of the ducks reared on the plastic mesh floor. Physiologically, the height of villi and the ratio of villus height to crypt depth in the ileum and duodenum were significantly greater in the ducks reared on plastic mesh floor. Furthermore, our results demonstrate that the gut microbiota was significantly associated with the duck phenotypes, such as chest depth and serum estradiol levels (p

Epidemiological studies have revealed that ambient fine particulate matter (PM2.5) exposure is closely associated with autism spectrum disorder (ASD). However, there is a relative paucity of laboratory data to support this epidemic finding. In order to assess the relationship between PM2.5 exposure and ASD, neonatal male Sprague-Dawley rats were chosen and exposed to PM2.5 (2 or 20 mg/kg body weight, once a day) by intranasal instillation from postnatal day 8 to 22. It was found that when exposed to PM2.5 in the early neonatal period for two weeks, both groups of the exposure rats manifested typical behavioral features of autism, including communication deficits, poor social interaction, and novelty avoidance. And, we further found, among five ASD candidate genes we chose, both the mRNA level and protein expression of SH3 and multiple ankyrin repeat domains 3 (Shank3) decreased significantly in the rat hippocampus after high dose of PM2.5 exposure. Moreover, results showed that PM2.5-exposure significantly increased the levels of proinflammatory cytokines, interleukin 1β, interleukin 6, and tumor necrosis factor alpha in the hippocampus and prefrontal cortex. The expression of glial fibrillary acidic protein and ionized calcium-binding adapter molecule, markers of astrocytes and microglial cell activation, respectively, also increased in the exposed animals. Our work provides new data on the link between postnatal exposure to ambient PM2.5 and the onset of ASD-like symptoms in human beings, and the increased inflammatory response and abnormalities in Shank3 expression in the brain may contribute to the mechanisms of PM2.5 exposure-induced ASD.

Two high molecular weight fractions (LW1 and LW2) of exopolysaccharides (EPSs) produced by Lactobacillus casei SB27 were isolated from yak milk obtained from the Gansu Tibetan area of China. GC-MS, FTIR spectroscopy, methylation analysis and FE-SEM analysis were performed to elucidate the physicochemical characterization of these two fractions, and their in vitro antitumour activities were also evaluated. The molecular weights (Mws) of LW1 and LW2 as determined by HPGPC were 25.10 and 12.34kDa, respectively. Monosaccharide composition analysis revealed that LW1 and LW2 were mainly composed of galactose (52.4% and 57.4%, mol%) and glucose (29.1% and 22.2%, mol%), respectively. Methylation results showed that the main chain of LW1 likely involves (1→4)-linked Galp and (1→4)-linked Glcp with its side chains being (1→4,6)-linked Galp through the O-6 position connected to the backbone, whereas the main chain of LW2 likely involves (1→4)-linked Galp and (1→4)-linked Glcp with its side chains being (1→3)-Galp through the O-6 position of (1→3,6)-Galp linked to the main chain. Evaluation of the microcosmic morphology, as revealed by FE-SEM analysis of the two EPS fractions, showed a sheet-like appearance with a folded surface and a compact structure. The results from in vitro antitumour tests indicated that both LW1 and LW2 could significantly inhibit the proliferation of HT-29 colorectal cancer cells and up-regulated the expressions of Bad, Bax, Caspase-3 and -8 genes. Finally, TEM images revealed the apoptotic morphological changes of HT-29 cells induced by LW1 and LW2. Our results suggested that LW1 and LW2 possess potential not only for use in functional food products but also as a source of natural antitumour drugs.

The objectives of this study were to evaluate the anti-diabetic and anti-hyperlipidemic effects and relative mechanisms of guava polysaccharides (GPs) in rats with type 2 diabetic mellitus (T2DM). The chemical characterization and monosaccharide compositions of GPs, named as GP-1, GP-2, GP-3, and GP-4, were determined by PMP-HPLC and FT-IR. The results revealed that all GPs had the typical saccharide absorptions, and all were heteropolysaccharides. In addition, GPs efficiently decreased levels of fasting blood glucose, glucosylated serum protein, serum insulin, homeostasis model assessment of insulin resistance, total cholesterol, triglyceride and serum alanine transaminase, improved oral glucose tolerance, and increased insulin sensitivity in rats with T2DM. Histopathological observations suggested that GP-1, GP-3, and GP-4 could alleviate injury in pancreatic islet cells, and Western blot analysis showed that these GPs upregulated gene expression of the insulin receptor, insulin receptor substrate 2, Akt, and glucose transporter type 4. Taken together, these data suggest that GPs may be beneficial in treating T2DM and reducing the risk of hyperlipidemia, vascular disease, and cirrhosis via the PI3K/Akt signaling pathway.

Bromodomain-containing protein 9 (BRD9), an epigenetic "reader" of acetylated lysines on post-translationally modified histone proteins, is upregulated in multiple cancer cell lines. To assess the functional role of BRD9 in cancer cell lines, we identified a small-molecule inhibitor of the BRD9 bromodomain. Starting from a pyrrolopyridone lead, we used structure-based drug design to identify a potent and highly selective in vitro tool compound 11, (GNE-375). While this compound showed minimal effects in cell viability or gene expression assays, it showed remarkable potency in preventing the emergence of a drug tolerant population in EGFR mutant PC9 cells treated with EGFR inhibitors. Such tolerance has been linked to an altered epigenetic state, and 11 decreased BRD9 binding to chromatin, and this was associated with decreased expression of ALDH1A1, a gene previously shown to be important in drug tolerance. BRD9 inhibitors may therefore show utility in preventing epigenetically-defined drug resistance.

Compared with estrogens, estrogen 3 β -glucosides display more superiority in uptake and metabolism, which are thought to be potential replacement therapy agents for female. In the present research, various estrogen 3 β -glucosides ( 1a – 6a ) were obtained using the biotransformation experiment of estrogen substrates mediated by Rhizopus oryzae AS 3.2380. The characteristics of estrogens glucosylation with high specificity and regio-selectivity were observed. An optimal incubation medium was determined as the pH 7.0 (sodium phosphate buffer), 20 g/L ( d -glucose) and 24 h incubation time with the yields > 70%. A environment friendly preparative method for these glucosides was also established using MCI (Middle chromatogram isolated gel) column chromatography eluted with ethanol aqueous, which could give estrogen 3 β -glucosides with the purity > 95%. This study suggested that R. oryzae AS 3.2380 could be an efficient engineering bacteria for the glucosylation of chemical substance. Glucosyltransferase also could be prepared from R. oryzae AS 3.2380.

The biological function of bromodomains, epigenetic readers of acetylated lysine residues, remains largely unknown. Herein we report our efforts to discover a potent and selective inhibitor of the bromodomain of cat eye syndrome chromosome region candidate 2 (CECR2). Screening of our internal medicinal chemistry collection led to the identification of a pyrrolopyridone chemical lead, and subsequent structure-based drug design led to a potent and selective CECR2 bromodomain inhibitor (GNE-886) suitable for use as an in vitro tool compound.

Ethnopharmacological relevance The genus Melastoma consists of approximately 100 species distributed widely in tropical and subtropical countries, and Melastoma species are often used for medicinal purposes, such as treatment for bleeding, diarrhea, diabetes, and gynecological tumors by local people, mostly in Southeast Asian countries. Aim of the review: The present review summarizes the traditional uses, phytochemistry and pharmacology of species belonging to Melastoma to suggest further research strategies and to facilitate the exploitation of the therapeutic potential of Melastoma species for the treatment of human disorders. Materials and methods Information related to the traditional uses, phytochemistry and pharmacological activities was systematically collected by searching for the word “Melastoma” in electronic databases, including SciFinder, Web of Science, PubMed, and Google Scholar, from Apr. 1968 until Dec. 2019. Results A systematic literature survey revealed that Melastoma spp. are widely distributed in southern Asia to northern Oceania and the Pacific Islands and are traditionally used to treat bleeding, diarrhea, swelling, and gynecological tumors. Approximately 142 compounds, including flavonoids, tannins, phenylpropanoids, organic acids, terpenoids, and steroids, have been reported from Melastoma spp. Different extracts have been evaluated for their pharmacological activities, including anti-inflammatory, hemostatic, anticoagulant, cytotoxic, antibacterial, antioxidant, hepatoprotective, gastroprotective and hypoglycemic activities. Conclusions Melastoma spp. are popularly used in Southeast Asian countries as effective herbs and are rich in flavonoids, tannins and organic acids with valuable medicinal properties. However, additional studies of the chemical constituents and the mechanism-based pharmacological activities of many members of Melastoma are still needed for developing new plant-derived drugs. In addition, studies on the clinical safety and efficacy of Melastoma are also needed.

Surface-layer proteins are located in a paracrystalline layer outside the bacterial cell wall and are thought to play a role in the adherence. However, the functions of S-layer proteins are not yet completely revealed and it has been proposed that these structures protect the microbe from hostile environmental agents and aid in maintaining cellular integrity. In this study, the Lactobacillus paracasei M7 strains showed to inhibit the adhesion of Salmonella to epithelial cells, a process which may be related to specific component of the bacterial surface. Human epithelial HT-29 cells were treated with S-layer proteins after Salmonella infection, or were pretreated with S-layer proteins prior to infection to determine their importance in the inhibition of pathogen adherence.

Dihydromyricetin (DMY) has a protective effect on neural function under central nervous system dysfunction conditions. There is growing interest concerning the beneficial effects of DMY on treating diabetic neuropathy (DN). This study was carried to detect protective effects of DMY on high glucose (HG)-induced cell damage and related mechanisms. The effect of DMY on cell survival was detected by MTT assay. Caspase-3 and phosphorylated AMP-activated protein kinase (AMPK) was evaluated by Western blotting. The effects of DMY and AMPK agonist AICAR on ROS production was determined. Our results showed that DMY treatment protect against HG-induced cell damage. DMY treatment significantly reduced the expression of caspase-3 and phosphorylated AMPK. ROS production was inhibited by DMY or AMPK agonist AICAR treatment. These studies demonstrate that DMY may inhibit ROS production, caspase-3 expression through AMPK pathway. Keywords: dihydromyricetin, caspase, oxidative stress

Ethnopharmacological relevance Type 2 diabetes mellitus (T2DM) is becoming a serious threat to human health. The fruit of Morus alba L. is widely used as a traditional Chinese medicine for the treatment of DM, dizziness, tinnitus, insomnia, and premature graying, as well as to protect the liver and kidneys. Several studies have demonstrated that the aqueous extracts of the roots bark, leaves, and ramuli of mulberry, which are known to contain polyphenols and polysaccharides, have antihyperglycemic and antihyperlipidemic activities. The aim of the present study was to further investigate the active polysaccharides from M. alba fruit by evaluating the antidiabetic activities of different fractions on T2DM rats and elucidate the mechanism underlying these activities. Materials and methods Diabetic rats were treated with two fractions of M. alba fruit polysaccharides (MFP50 and MFP90). The disease models were induced by a high-fat diet and low dose injection of streptozotocin and were compared to normal rats and metformin-treated diabetic rats. After seven weeks, the fasting blood glucose (FBG), oral glucose tolerance test (OGTT), fasting serum insulin (FINS) levels, homeostasis model of assessment-insulin resistance (HOMA-IR), glycated serum protein (GSP), and serum alanine transaminase (ALT) levels, as well as serum lipid profiles and histopathological changes in the pancreas were measured. Next, the expressions of the insulin signaling pathway were measured by western blot analysis to elucidate the potential mechanism underlying these antidiabetic activities. Results After seven weeks of treatment, a significant reduction in the FBG levels, OGTT-area under the curve (OGTT-AUC), FINS, HOMA-IR, ALT, and triglyceride (TG) values of the MFP50 group was observed. On the other hand, in the MFP90 group, the FBG, OGTT-AUC, FINS, HOMA-IR, GSP, and TG levels were significantly reduced. The level of high-density lipoprotein cholesterol (HDL-c) and the proportion of HDL-c to total cholesterol (TC) significantly increased in the MFP50 group. Moreover, MFP50 and MFP90 induced repair of damaged pancreatic tissues of the diabetic rats. The hypoglycemic effect of MFP50 was more stable than MFP90, whereas the hypolipidemic effect of MFP90 was slightly better than MFP50. Moreover, the expression levels of InsR, IRS-2, Akt and GLUT4 in the MFP90 group significantly increased relative to that of the T2DM group. Conclusions MFP50 and MFP90 have markedly antihyperglycemic and antihyperlipidemic effects and can clearly relieve diabetes symptoms in the T2DM rat model. The M. alba fruit polysaccharides may potentially be utilized as an effective treatment for T2DM. Further research into the structures of active M. alba fruit polysaccharides and their mechanisms in promoting antidiabetic effects are underway.

Traditional Chinese medicines (TCMs) have been widely used in Asian countries for thousands of years due to their supreme quality and good clinical efficacy. However, the increasing demand for TCMs in recent decades warrants effective quality control methodology to avoid clinical problems. Therefore, comprehensive quality evaluation systems should be established for ensuring TCM's quality, in terms of chemical components, as well as bioactivity for identifying quality markers in TCM and developing suitable analytical methods for quality control. In this study, we selected Sophora flavescens (S. flavescens) as the research object and developed a novel integrated strategy combining metabolomics and network pharmacology to explore the quality markers. Firstly, we determined the targeted metabolomic profiles of seventy-four batches of S. flavescens (aged from 1 to 6 years) by UHPLC/QE-MS. Six potential markers were successfully screened, quantified and reverse-verified as the most influential effective compounds by UHPLC/QE-MS and multivariate statistical analysis. Secondly, the network of "components-targets-pathways" was constructed, and the pharmacological activities of six potential markers were predicted. Finally, we determined the anti-tumor activity of six flavonoids (kurarinone, norkurarinone, kuraridin, kushenol N, trifolirhizin, and genistein) as the quality markers for Sophora flavescens, evaluated their pharmacokinetic profiles and reviewed their existing pharmacological researches. Thus, integrated metabolomics and network pharmacology technology were applied for the effective discovery of quality markers of Chinese material medica.

Ulcerative colitis (UC), an immune system disease, is characterized by long duration and easy relapse. Sophora flavescens (S. flavescens), also named "Kushen", is a traditional Chinese medicine, widely used to treat UC in clinics. Alkaloids and flavonoids are the main constituents of S. flavescens. Previous studies indicated that the effects of S. flavescens against UC mainly attribute to its alkaloids. In view of the clinical applications of its flavonoids and our preliminary experiments on the effects of S. flavescens treatment, we speculated that flavonoids also could exert an anti-UC effect, but its efficacy and mechanism are still not yet to be revealed. Herein, we examined the pharmacodynamic effects of the ethyl acetate (EtOAc) extract of S. flavescens EtOAc (SFE) against dextran sodium sulfate-induced UC rats for the first time. Pharmacodynamic effects indicated that SFE could significantly alleviate the loss in the body weight and shortening of the colon length, reduce colon bleeding and improve colon tissue damage of UC rats. A total of 28 prototypes and 41 metabolites were unambiguously or tentatively detected in rat's plasma and urine. Among them, 28 prototypes and 3 phase I metabolites shared 40 UC targets, the targets contributed to 51 metabolic pathways in 5 modules. Additionally, genistein, formononetin, isokurarinone, kurarinone, maackiain, kushenol N, trifolirnizin, kuraridin and norkurarinone were suggested to be potential active compounds in SFE for treating UC by comprehensively investigating the results of network pharmacology analysis, metabolic analysis in vivo, and previous researches. Finally, a combination of metabolic analysis in vivo with network pharmacology can elucidate the material basis and pharmacodynamic effect of traditional Chinese medicines, and lay the foundation for further clarify the anti-UC mechanism of SFE.

Background: Alpinia officinarum Hance is both an herbal medicine and a condiment, and generally has different cultivars such as Zhutou galangal and Fengwo galangal. The appearance of these A. officinarum cultivars is similar, but their chemical composition and quality are different. It is therefore important to discriminate between different A. officinarum plants to ensure the consistency of the efficacy of the medicine. Therefore, we used an electronic nose (E-nose) to explore the differences in odor information between the two cultivars for fast and robust discrimination. Methods: Odor and volatile components of all A. officinarum samples were detected by the E-nose and gas chromatography-mass spectrometry (GC-MS), respectively. The E-nose sensors and GC-MS data were analyzed respectively by principal component analysis (PCA), the correlation between E-nose sensors and GC-MS data were analyzed by partial least squares (PLS). Results: It was found that Zhutou galangal and Fengwo galangal can be discriminated by combining the E-nose with PCA, and the E-nose sensors S2, S6, S7, S9 were important sensors for distinguishing different cultivars of A. officinarum. A total of 56 volatile components of A. officinarum were identified by the GC-MS analysis, and the composition and content of the volatile components from the two different A. officinarum cultivars were different, in particular the relative contents of 1,8-cineole and &alpha, farnesene. The classification result by PCA analysis based on GC-MS data was consistent with the E-nose results. The PLS analysis demonstrated that the volatile terpene, alcohol and ester components primarily interacted with the sensors S2 and S7, indicating that particular E-nose sensors were highly correlated with some aroma constituents. Conclusions: Combined with advanced chemometrics, the E-nose detection technology can discriminate two cultivars of A. officinarum, with GC-MS providing support to determine the material basis of the E-nose sensors&rsquo, response.

Cysestermerol A (1), a rare and new stilbene sestermer, was isolated from the whole herb of Cynodon dactylon. The planar and relative structures of 1 were elucidated based on HRESIMS, one- and two-dimensional NMR analyses, and its absolute configuration was further established by electronic circular dichroism calculations. Compound 1 obviously increased the glucose consumption in HepG2 cells equivalent to the positive control rosiglitazone and markedly inhibited the activity of α-glucosidase in vitro.

Spectroscopy techniques are powerful tools for the rapid identification of traditional Chinese medicine because they provide chemical information with no sample preparation. In this study, a rapid and reliable approach was proposed to differentiate Pinellia ternata from adulterated P. ternata, processed P. ternata, and adulterated processed P. ternata by mid-infrared (MIR) and near-infrared (NIR) spectroscopy coupled with a partial least squares-discriminant analysis (PLS-DA) algorithm. One-hundred sixty-five batches of P. ternata, adulterated P. ternata, processed P. ternata, and adulterated processed P. ternata samples were collected and prepared. All of the samples were characterized by MIR and NIR spectra. The PLS-DA was first applied to build the discriminant model on the individual data matrices. Next, the data matrices coming from MIR and NIR spectra were fused at the low-level and mid-level, and PLS-DA models were built on the fused data. The classification accuracy, sensitivity, and specificity were calculated to evaluate the PLS-DA models. The results showed the use of mid-level fusion strategy, in particular, integrating latent variables from different spectral data matrices, allowed the correct discrimination of all samples in the training and testing sets. In the case of mid-level fusion with latent variables, the accuracy of the PLS-DA model was 100%, and the sensitivity and specificity of the PLS-DA model were all 1. The present discriminant model can be successful to differentiate P. ternata from adulterated P. ternata, processed P. ternata, and adulterated processed P. ternata. This study first provides a new path for the quality control of P. ternata.

TheLactobacillus paracaseisubsp. paracaseiM5 strain exerted potential anticancer activity through the cell wall. In this study, whole peptidoglycan (WPG) was extracted from theLactobacillus paracaseisubsp. paracaseiM5 strain and was evaluated for anticancer effects as well as its properties. SDS-PAGE analysis confirmed the presence of WPG with dominant bands of approximately 14.4 kDa. Further analysis revealed that the amino acids present in the WPG consisted of alanine, glycine, glutamic acid, and lysine in a molar ratio of approximately 8 : 5 : 3 : 3.5. In addition, the cell viability of HT-29 cells with WPG addition was investigated with methyl thiazolyl tetrazolium (MTT) and trypan blue exclusion (TBE) assays, and cell apoptosis was analyzed with a transmission electron microscope, flow cytometry, and semiquantitative RT-PCR. These results showed that WPG exerted cytotoxic effects on colon cancer HT-29 cells in a dose-dependent manner and upregulated proapoptotic genes, while downregulating antiapoptotic genes. The gene expression study definitively revealed that WPG induced a mitochondria-mediated apoptotic pathway.

Diets rich in high fructose and salt are increasingly popular in our daily life. A combination consumption of excessive fructose and salt can induce insulin resistance (IR) and hypertension (HT), which are major public health problems around the world. However, the effects of high fructose and salt on systematic metabonome remain unknown, which is very important for revealing the molecular mechanism of IR and HT induced by this dietary pattern. The metabolic profiling in urine, plasma, and fecal extracts from high fructose and salt-fed rats was investigated by use of (1) H nuclear magnetic resonance (NMR)-based metabonomics approach in this study. Multivariate analysis of NMR data showed the effects of high fructose and salt on the global metabonome. The metabolite analysis in urine and fecal extracts showed the time-dependent metabolic changes, which displayed metabonomic progression axes from normal to IR and HT status. The changes of 2-oxoglutarate, creatine and creatinine, citrate, hippurate, trimethylamine N-oxide (TMAO), and betaine in urine, together with gut microbiota disorder in feces, were observed at the preliminary formation stage of IR and HT (fourth week). At the severe stage (eighth week), the previously mentioned metabolic changes were aggravated, and the changes of lipid and choline metabolism in plasma suggested the increased risk of cardiovascular diseases. These findings provide an overview of biochemistry consequences of high fructose and salt feeding and comprehensive insights into the progression of systematic metabonome for IR and HT induced by this dietary pattern.

AIM This study aimed to test a model which involved clans and health providers to increase antenatal care attendance in rural minority areas of China with high HIV prevalence. METHODS Formative research was conducted to determine barriers and facilitators to antenatal care use. A strategy involving clans in addressing the barriers identified was developed. Implementation of the new strategy was done through three plan-do-study-act (PDSA) cycles, lasting four months each. RESULTS Awareness and uptake of antenatal care increased significantly after the intervention. The proportion of post-partum women who used any antenatal care increased from 21.3% to 64.5% (p

Surface layer proteins had been proposed that it could protect human intestine from pathogens and aid in maintaining cellular integrity. However, the functions of S-layer proteins have not yet been fully revealed, and it has been proposed that these structures protected the intestinal microbes from hostile environmental agents. In this study, the ability of the Lactobacilli to inhibit adhesion of Salmonella to intestinal mucosa was studied on cultured HT-29 cells. Lactobacillus paracasei M7 was shown to inhibit the adhesion of Salmonella to epithelial cells, a process which may be related to the specific component of the bacterial surface. Human epithelial HT-29 cells were treated with S-layer proteins after Salmonella injection and pretreated with S-layer proteins to determine their importance in the inhibition of pathogen adherence. According to the analysis of cell cycle by flow cytometry, the HT-29 cells treated by Salmonella in S phase were 26.88 % compared with 36.70 % in the normal HT-29 cells. However, the addition of S-layer proteins made the HT-29 cells in S phase increase to 30.13 %. The activity of caspase-3 in the HT-29 cell was determined using a caspase-3 activity kit, which indicated the degree of apoptosis. Moreover, we also found that Lactobacillus S-layer proteins could protect Salmonella-induced apoptosis through reducing the caspase-3 activity. This mechanism might represent a novel approach for antagonizing Salmonella infection.

Activating mutations within the epidermal growth factor receptor (EGFR) kinase domain, commonly L858R or deletions within exon 19, increase EGFR-driven cell proliferation and survival and are correlated with impressive responses to the EGFR inhibitors erlotinib and gefitinib in nonsmall cell lung cancer patients. Approximately 60% of acquired resistance to these agents is driven by a single secondary mutation within the EGFR kinase domain, specifically substitution of the gatekeeper residue threonine-790 with methionine (T790M). Due to dose-limiting toxicities associated with inhibition of wild-type EGFR (wtEGFR), we sought inhibitors of T790M-containing EGFR mutants with selectivity over wtEGFR. We describe the evolution of HTS hits derived from Jak2/Tyk2 inhibitors into selective EGFR inhibitors. X-ray crystal structures revealed two distinct binding modes and enabled the design of a selective series of novel diaminopyrimidine-based inhibitors with good potency against T790M-containing mutants of EGFR, high selectivity over wtEGFR, broad kinase selectivity, and desirable physicochemical properties.

A gene from Zea mays coding a TGase was expressed in E. coli and identified by Western blotting. Under optimal expression conditions, the production and specific activity of refolded TGZ were 1.41 mg/L and 0.34 U/mg. The activated TGZ was employed to cross-link milk proteins. The yoghurt treated with TGZ showed a lower syneresis, higher apparent viscosity and texture than untreated yoghurt. The properties of TGZ-treated sample were better than those of MTG-treated samples, so TGZ may have potential as an additive in yoghurt manufacture.

A total of 138 lactobacillus strains were obtained from the traditional fermented foods of minority nationalities or infant faeces, respectively. The lactobacillus strains were screened for anticancer effects and probiotic potential. The results showed that 10 strains exerted anti-proliferative activity and higher adhering capability on HT-29 cells. They were then screened for resistance to biological barriers (acid and bile salts), and the four most promising strains were selected. Further analysis revealed that the 4 strains (cell walls and cytoplasm extracts) displayed the high anti-proliferative activity and the large extent of DNA strand breakage in individual cells. Through the selected procedure, cell walls that were extracted from X12, M5 and K14 strains induced apoptosis in HT-29 cells. Further investigation confirmed that apoptosis-inducing ability of cell wall extracts was attributed to the breakdown of mitochondrial membrane potential, which is a known initiation of apoptotic mitochondrial pathway. Cell walls from X12, M5 and K14 strains, were determined to be less harmful to noncancerous Vero cells than to human colon cancer HT-29 cells. These findings suggested that X12, M5 and K14 strains opposing the ability to induce HT-29 cells apoptosis, and cell wall extracts were involved in this apoptosis induction.

In this study, five selected Lactobacillus of fecal origin were investigated for their probiotic properties. They were preliminarily identified by partial 16S rDNA gene sequencing and characterized in vitro for their resistance to low pH and bile salts. Here, we assessed the adhesive properties and the ability to inhibit the adhesion and to displace pathogens of five selected Lactobacillus strains using HT-29 cells model through a process which may be related to specific components of the baterial surface. Surface-layer proteins are located in a paracrystalline layer outside the bacterial cell wall and are thought to play a role in tissue adherence. Removal of S-layer proteins from the Lactobacillus (treated with 5 M LiCl) reduced adhesion to Caco-2 cells. Under such conditions, inhibition activity was also decreased in all five strains, as revealed in competition assays, which suggested that S-layer protein had involved in the adhesion of probiotics. SDS-PAGE analysis confirmed the presence of S-layer proteins with dominant bands of was approximately 60 kDa. Our findings also indicate that the surface layer proteins contributed to its increased adhesion to the cultured cells and competitive exclusion of pathogens.

Three Lactobacillus strains were screened on the basis of probiotic characteristics (i.e., resistance to low pH and bile salts, adhesion to the human gastrointestinal tract, inhibition of pathogenic strains). They further exhibited producing antimicrobial activities of non-acid molecule (s). In addition, antibacterial peptides were isolated and purified from the cell-free culture supernatants of these three probiotic strains. Based on TricineSDSPAGE, the antimicrobial peptide was approximately 10 kDa in size. After analyzing the sequence of the 16SrDNA regions of these three strains, they were identified asLactobacillus crispatus Lactobacillus rhamnosus and Lactobacillus rhamnosua GG.Using an in vitro system simulating gastric transit, our findings indicated that the three probiotic strains had the ability to tolerate gastroenteric environment and the adhesive capacity to HT-29 cells. It was demonstrated that the probiotic strains inhibited subsequent adhesion of E. coli to the HT-29 cell. Among the selected strains,L. rhamnosusF1333 showed a high probiotic potential and could be used in health-promoting food products.

BACKGROUND Transglutaminases catalyze post-translational modification of proteins by e-(γ-glutamyl) links and covalent amide bonds. Research on properties and applications of plant transglutaminases is less developed than in animals and micro-organisms. In a previous study, optimized Zea mays transglutaminase was purified from recombinant Pichia pastoris strain. The main objective of the present study was to characterize this enzyme and assess its effect on the properties of yoghurt. RESULTS The purified recombinant transglutaminase presented a Km of 3.98 µmol L−1 and a Vmax of 2711 min−1 by the fluorometric method. The enzyme was stable after incubation for 30 min below 50 °C and over a broad pH range of 5–8 at −20 °C for 12 h. The results showed that the crosslinking reaction catalyzed by this enzyme could effectively improve the properties of full and non-fat yoghurts. Also, the properties of non-fat yoghurt could be improved similar to the full-fat product by recombinant transglutaminase. CONCLUSION The application of recombinant transglutaminase in yoghurt indicated that this enzyme could be used as a substitute for microbial transglutaminase in the production of yoghurt, thus providing experimental evidence for the future application of plant transglutaminases in the food industry. © 2013 Society of Chemical Industry

Conjugated linoleic acid (CLA) refers to a group of positional and geometric isomers of octadecadienoic fatty acid with conjugated double bonds. CLA possesses many important physiological functions and it can be produced from linoleic acid (LA) by LA isomerases. In this report, we first cloned the genes encoding LA isomerases: C12 isomerases and C9 isomerase, then transformed the recombinant plasmids into Escherichia coli TOP10 and induced E. coli with IPTG (isopropylthio-β-D-galactoside) to express the recombinant proteins. Next, we purified the isomerases using a HisTrap™ HP column, followed with the analysis by SDS-PAGE or Western blot. Finally, we compared their enzymatic activity by biotransformation of LA into CLA. Plasmids containing LA isomerase genes were successfully constructed. LA isomerases were found expressed in E. coli, and the molecular weight was 64 KD for C12 LA isomerase and 55 KD for C9 LA isomerase. The enzyme activity (9.93 ± 0.01 U/ml for C12 LA isomerase and 8.12 ± 0.02 U/ml for C9 LA isomerase) of both LA isomerases reached the highest when IPTG concentration is 0.2 mM and the induction time is 18 h. After purification, C9 LA isomerase was enriched in peak 4 and C12 LA isomerase was enriched in peak 3. Optimum pH for C9 LA and C12 LA isomerases were 7.5 and 7.0 separately, and optimum temperatures was 37 °C for highest concentration of CLA. The work may provide theoretical significance for an effective production process of CLA for the medical and nutritional purposes.