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Background: Adenosine triphosphate-binding cassette subfamily G member 1 (ABCG1) has the function of transporting free intracellular cholesterol to extracellular high-density lipoprotein (HDL) particles, which play a crucial role in atherosclerosis. The goal of this study is to examine the relationship between the polymorphisms of the ABCG1 gene promoter region and ischemic stroke., Methods: In the present study, a case-control association study was designed to identify 3 single-nucleotide polymorphisms (SNPs; rs5713919, rs1378577, and rs1893590), which were located in the promoter region of ABCG1 gene by kompetitive allele-specific polymerase chain reaction genotyping approach. The in vitro luciferase assay was done to estimate the effect of rs5713919 on gene expression. Finally, the relationships of 3 SNPs of ABCG1 gene with plasma lipids and lipoproteins were investigated in this Chinese cohort., Results: The correlation analysis between lipids and genotypes showed that the rs57137919 locus genotype was significantly associated with HDL cholesterol (HDL-C) and low-density lipoprotein cholesterol (LDL-C) levels (P = 0.021 and P = 0.017, respectively), and the GA and AA genotypes had higher HDL-C levels than the GG genotype., Conclusions: Our study provides evidence that ABCG1 promoter region polymorphism rs57137919 has an influence on plasma HDL-C and LDL-C levels in Chinese Han population., (Copyright © 2020 Elsevier Inc. All rights reserved.)

To build a biomechanical numerical model of the nasopharynx, construct an accurate computerized numerical description of its specific anatomical structures, analyze the distribution of air flow field, starting with the anatomical structure of the pharyngeal recess, correlate its anatomical characteristics with the occurrence and development of nasopharyngeal carcinoma from the perspective of biomechanics. In this study, the nasal and nasopharyngeal cavities of healthy male adult, with the pharyngeal recess in an open state, were scanned by CT to obtain DICOM imaging data. Then, they were imported into Mimics 20.0 to build a model which was recorded in binary STL format. Each file was imported into Geomagic studio 12.0 to construct a 3D model saved in an IGES format. Then, it was imported into ANSYS Workbench for numerical simulation of air flow field. The authors found that:Above all, the causes and pathogenesis of nasopharyngeal carcinoma can be identified from the perspective of biomechanics through the construction of a 3D model and analysis of the characteristics of air flow field. With more in-depth research, it is expected that a more solid scientific foundation will be created for related quantitative analysis.

Methicillin-resistant Staphylococcus aureus (MRSA) has become a worrisome superbug, due to its wide distribution and multidrug resistance. To characterize effects of a newly identified plantaricin GZ1-27 on MRSA, transcriptomic and proteomic profiling of MRSA strain ATCC43300 was performed in response to sub-MIC (16 μg/mL) plantaricin GZ1-27 stress. In total, 1090 differentially expressed genes (padj < 0.05) and 418 differentially expressed proteins (fold change > 1.2, p < 0.05) were identified. Centralized protein expression clusters were predicted in biological functions (biofilm formation, DNA replication and repair, and heat-shock) and metabolic pathways (purine metabolism, amino acid metabolism, and biosynthesis of secondary metabolites). Moreover, a capacity of inhibition MRSA biofilm formation and killing biofilm cells were verified using crystal violet staining, scanning electron microscopy, and confocal laser-scanning microscopy. These findings yielded comprehensive new data regarding responses induced by plantaricin and could inform evidence-based methods to mitigate MRSA biofilm formation.

Background: Diverse and circumstantial evidence suggests that schizophrenia is a neurodevelopmental disorder. Genes contributing to neurodevelopment may be potential candidates for schizophrenia. The human SOX11 gene is a member of the developmentally essential SOX (Sry-related HMG box) transcription factor gene family and mapped to chromosome 2p, a potential candidate region for schizophrenia., Methods: Our previous genome-wide association study (GWAS) implicated an involvement of SOX11 with schizophrenia in a Chinese Han population. To further investigate the association between SOX11 polymorphisms and schizophrenia, we performed an independent replication case-control association study in a sample including 768 cases and 1348 controls., Results: After Bonferroni correction, four SNPs in SOX11 distal 3'UTR significantly associated with schizophrenia in the allele frequencies: rs16864067 (allelic P = .0022), rs12478711 (allelic P = .0009), rs2564045 (allelic P = .0027), and rs2252087 (allelic P = .0025). The haplotype analysis of the selected SNPs showed different haplotype frequencies for two blocks (rs4371338-rs7596062-rs16864067-rs12478711 and rs2564045-rs2252087-rs2564055-rs1366733) between cases and controls. Further luciferase assay and electrophoretic mobility shift assay (EMSA) revealed the schizophrenia-associated SOX11 SNPs may influence SOX11 gene expression, and the risk and non-risk alleles may have different affinity to certain transcription factors and can recruit divergent factors., Conclusions: Our results suggest SOX11 as a susceptibility gene for schizophrenia, and SOX11 polymorphisms and haplotypes in the distal 3'UTR of the gene might modulate transcriptional activity by serving as cis-regulatory elements and recruiting transcriptional activators or repressors. Also, these SNPs may potentiate as diagnostic markers for the disease., (© 2020 The Authors. Journal of Clinical Laboratory Analysis published by Wiley Periodicals, Inc.)

Purpose: A previous study reported a novel c.544&#95;618del75bp mutation in exon 7 of the PRPF31 gene in a Chinese family with autosomal dominant retinal pigmentosa (ADRP). However, the selected pedigree was a small part of the whole family and the function of the c.544&#95;618del75bp mutation was not explored deeply. The aim of the present study was to validate the previous results and explore the functional significance of the c.544&#95;618del75bp mutation., Methods: We extended the size of the ADRP pedigree and sequenced DNA and cDNA of the PRPF31 gene for all members of the family and 100 healthy controls. Real-time quantitative polymerase chain reaction (PCR) analysis was performed on the cDNA of patients in the family and cell culture, plasmids transfection and western blot analysis were done to evaluate the functional effect of the mutation in vitro., Results: Sanger sequencing showed that the mutation was present in all patients and absent in all normal individuals, except for participant III-9. Bioinformatics analysis revealed that the c.544&#95;618del75bp mutation caused a 25 amino acid deletion in the PRPF31 protein. In addition, the mRNA expression assay revealed that the mRNA expression level of the PRPF31 and RP9 genes were significantly lower in RP patients than controls (p < 0.05). Finally, the in vitro transfection assay demonstrated that the mRNA expression level of the mutant transfection group was significantly lower than the wild-type transfection group (p < 0.05)., Conclusions: Our study suggested that the c.544&#95;618del75bp mutation in the PRPF31 gene was a causative mutation in this ADRP family and affected the expression of RP9 gene by influencing the formation of U4/U6-U5 tri-snRNP, eventually leading to the occurrence of RP., (© 2020 The Authors Ophthalmic & Physiological Optics © 2020 The College of Optometrists.)

Malassezia globosa is an opportunistic pathogen that causes various skin disorders, which disturbs people's life all the time, and conventional drugs are not completely satisfactory. Bacillomycin D (BD), an antifungal lipopeptide, could inhibit various fungi growth. However, the reports about its effect on M. globosa were not found yet. In this study, we showed that BD and BD-C16 (fatty acid chain had sixteen carbon atoms) completely inhibited growth of M. globosa at concentration of 64 μg/ml in 15 h, which was confirmed with the observation of irregular morphological change of M. globosa treated with BD. Significantly, the study on the working mechanism showed that BD induced cell death by changing cell membrane permeability and thus promoting the release of cellular contents, which may be mediated by the interaction between BD and ergosterol from membrane. Further study showed that BD reduced the overall content of cellular sterol, and interestingly, the expression of some genes involved in membrane and ergosterol synthesis were significantly upregulated, which was likely to be a feedback regulation. Besides, we found that BD had additive and synergistic effects with ketoconazole and amphotericin B, respectively, on inhibition of M. globosa, suggesting that combination use of BD with other commercial drugs could be a promising strategy to relieve skin disorders caused by M. globosa. KEY POINTS: • BD could efficiently inhibit the growth of M. globosa. • BD increases cell membrane permeability and thus promotes the release of cellular contents. • BD has additive or synergistic effect with other antifungal drugs.

Background: Constructing a three-dimensional (3D) model through non-invasive techniques will greatly benefit the diagnosis and treatment of otitis media. However, such a model should reflect the physiological characteristics of the middle ear; in particular, the pressure-flow responses determine the validity of the model. Objectives: A 3D model of the middle ear was constructed by digital scanning and simulation. The pressure-flow properties in the model were measured to evaluate whether the model could reflect the real middle ear under physiological and pathological conditions. Materials and methods: Computed tomography (CT) scanning data from a healthy woman were input into Mimics 20.0 to construct 3D images of the middle ear. The 3D images were treated with Ansys 15.0 for finite element mesh generation. Msc Nastran 2014 were used for the fluid-solid coupling calculations. Results: The pressure-flow rate in the model resembled a Venturi tube, namely, the pressure decreased with increasing flow velocity, especially in the Eustachian tube. In the absence of the right mastoid process, the differences in air pressure and the maximal velocity in the model were reduced. Conclusions: This numerical model based on CT images of the middle ear recapitulates the biomechanical characteristics of the real middle ear. Significance: This study provides an easy and rapid approach to constructing a middle ear model for diagnosis and treatment of otitis media.

The purpose of this study was to assemble two types of loop-mediated isothermal amplification (LAMP) kit that have the ability to visually detect Salmonella in ready-to-eat fruits and vegetables. The reaction results were obtained within 20-40 min after addition of DNA and can be discerned by the naked eye or an amplification plot. The stability of the LAMP wet kit was evident after multiple freezing and thawing cycles, and the one-step LAMP lyophilized kit was further evolved to allow ambient temperature transport for deployment in resource-limited settings. The cost-effective wet kit had the ability to detect minimum amounts of 1.8 CFU/ml Salmonella DNA without enrichment, while the sensitivity of the one-step LAMP lyophilized kit was only 9.8 × 10 3  CFU/ml. They both have good anti-interference, as they were both able to detect 2.1 × 10 2  CFU/ml Salmonella mixed with 10 6  CFU/ml four non-Salmonella strain mixture. Moreover, cucumber and lettuce that were contaminated with an initial inoculation of 1.7 CFU of Salmonella/10 g showed detection within a reaction time of 30 min after 10 h enrichment. The present research setup is a convenient and practical kit for Salmonella rapid detection that has good application prospects in food safety monitoring., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Increasing cases of infections by foodborne pathogenic bacteria resulted in a great demand to find safe and novel antimicrobial compounds that can be used in the food industry. The isolation and application of antimicrobial peptides including lipopeptides has been increasing tremendously in the past years. In this study, a new bacterial strain called Brevibacillus laterosporus fmb70 (fmb70) was isolated and exhibited strong antimicrobial activities against Gram-positive, Gram-negative bacteria, and fungi. Two major antimicrobial components produced by fmb70 were respectively identified as lipopeptide: brevibacillin V (MW: 1570.12 Da) and brevibacillin (MW: 1583.75 Da), of which brevibacillin V was a new compound. Both of them consisted of 13 amino acids and C 6 fatty acyl (FA) chain. Brevibacillin V and brevibacillin showed significant antimicrobial activities against most foodborne pathogenic bacteria and phytopathogenic fungi. They stayed activity at 100 °C and remained 50% of their antimicrobial activities at pH 3 for 22 h. Hemolytic activities of them were lower than 8%. They effectively eliminated the S. aureus GIM 1.142 and L. monocytogenes ATCC 21633 in skim milk. In conclusion, the Brevibacillus laterosporus fmb70 and its major antimicrobial components has remarkable potentials in the food industry.

S. enterica is an important foodborne pathogen worldwide. As some strains can form biofilms which may offer protection against antimicrobials, it is of interest to explore ways to prevent biofilm formation by S. enterica. In this study, we engineered a short β-sheet peptide WK2 (WKWKCTKSGCKWKW-NH 2 ) and examined its antimicrobial and anti-biofilm activities against various S. enterica strains, including the multidrug-resistant S. Typhimurium DT104. WK2 displayed bacteriostatic activity with a geometric mean (GM) minimum inhibitory concentration (MIC) of 4.17 μg/mL, and bactericidal activity, with a GM lethal concentration (LC) of 7.51 μg/mL. Crystal violet staining and fluorescence measurements demonstrated that WK2 inhibited S. Typhimurium DT104 biofilm formation at 0.5 μg/mL and killed the sessile cells in biofilms at 8 μg/mL. Real-time polymerase chain reaction (qPCR) and microscopic observation revealed that the anti-biofilm activity of WK2 likely arises through the formation of complexes with bacterial DNA, inhibition of surface organelle biosynthesis and interference with autoinducer-2 (AI-2)-mediated quorum sensing (QS). Therefore, WK2 is a promising antimicrobial agent for the prevention and control of biofilms produced by multidrug-resistant S. enterica., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Millet bran was fermented with Bacillus natto and the changes of structural, physicochemical and functional properties of its dietary fiber were investigated. Results showed that B. natto fermentation enhanced soluble DF content from 2.3% to 13.2%, and soluble DF/insoluble DF ratio from 3.1% to 19.9%. SEM and FTIR assay indicated that fermentation led to the degradation of cellulose and hemicellulose, thereby forming more porous and loose structure and polysaccharides. The binding capacities such as water and oil holding capacity, swelling capacity as well as cholesterol, bile salts, nitrite ion and glucose adsorption capacity were improved, while cation exchange capacity was not significantly changed. The total phenolic content and DPPH free radical scavenging capacity increased significantly. Overall, fermentation of millet bran by B. natto improved the structural and functional properties of its DF, which could be applied as a functional ingredient in food products., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Deoxynivalenol (DON) is a widely distributed mycotoxin that frequently occurs in various agricultural raw materials and feeds. DON acts as a virulence factor that accelerates the spread of plant diseases; moreover, its accumulation in grains causes yield loss and serious health problems to humans and livestock. Biodegradation of DON into less- or non-toxic substances using naturally existing microorganisms is considered the best approach for DON detoxification. Although various single isolates and mixed cultures capable of detoxifying DON have been reported, details of the metabolic pathways and the degrading enzymes/coding genes involved are scarce. In this study, we aimed to isolate DON-degrading bacteria from soil samples and explore the mechanisms. Toward this end, 85 soil samples collected from different provinces in China were enriched under aerobic conditions with mineral media containing 50 μg/ml of DON as the sole carbon source. The bacterial consortium LZ - N1 exhibited highly efficient and steady DON-transforming activity. High-throughput sequencing was used to characterize the composition of the involved microflora, and analysis of 16S rRNA sequences indicated that LZ - N1 was composed of at least 11 bacterial genera, with Pseudomonas accounting for nearly half the relative abundance. Coincubation of a mixed culture of two novel strains from the LZ-N1 consortium, namely Pseudomonas sp. Y1 and Lysobacter sp. S1, showed sustained transformation of DON into the metabolite 3- epi -deoxynivalenol, with no degradation products detected after 72 h. The cell-free supernatant, lysate, and cell debris of the mixed culture possessed DON-degrading ability, with the supernatant reaching a DON degradation rate of 100% within 48 h with 50 μg/ml of DON. This is the first report of two-step enzymatic epimerization of DON by a mixed culture, which may provide a new insight into this pathway for future applications in detoxification of DON-contaminated cereals and feed., (Copyright © 2019 Zhai, Zhong, Gao, Lu, Bie, Zhao, Zhang and Lu.)

The fragile alpine vegetation in the Tibetan Plateau (TP) is very sensitive to environmental changes, making TP one of the hotspots for studying the response of vegetation to climate change. Existing studies lack detailed description of the response of vegetation to different climatic factors using the method of multiple nested time series analysis and the method of grey correlation analysis. In this paper, based on the Normalized Difference Vegetation Index (NDVI) of TP in the growing season calculated from the MOD09A1 data product of Moderate-resolution Imaging Spectroradiometer (MODIS), the method of multiple nested time series analysis is adopted to study the variation trends of NDVI in recent 17 years, and the lag time of NDVI to climate change is analyzed using the method of Grey Relational Analysis (GRA). Finally, the characteristics of temporal and spatial differences of NDVI to different climate factors are summarized. The results indicate that: (1) the spatial distribution of NDVI values in the growing season shows a trend of decreasing from east to west, and from north to south, with a change rate of -0.13/10° E and -0.30/10° N, respectively. (2) From 2001 to 2017, the NDVI in the TP shows a slight trend of increase, with a growth rate of 0.01/10a. (3) The lag time of NDVI to air temperature is not obvious, while the NDVI response lags behind cumulative precipitation by zero to one month, relative humidity by two months, and sunshine duration by three months. (4) The effects of different climatic factors on NDVI are significantly different with the increase of the study period.

The inhibitory effect of Bacillomycin D, a cyclic lipopeptide, on Rhizopus stolonifer colonization of cherry tomato was studied, and its possible mechanism of action was explored. Bacillomycin D showed a direct inhibitory effect on R. stolonifer spore germination and mycelial growth in vitro. It conferred both a direct inhibitory effect on R. stolonifer growth in cherry tomato in vivo and induced host resistance in cherry tomato. Moreover, Bacillomycin D treatment significantly increased the activities of plant defense-related enzymes, including chitinase (CHI), β-1,3-glucanase (GLU), phenylalanine ammonia-lyase (PAL), and peroxidase (POD). Real-time PCR (RT-PCR) showed that defense-related genes involved in the salicylic acid defense signaling pathway and genes encoding pathogenesis-related proteins were up-regulated in Bacillomycin D treatment. Furthermore, Bacillomycin D-C16 resulted in direct inhibition and a remarkable induced resistance to R. stolonifer which was higher than as induced by Bacillomycin D-C14. Together, the data indicated that Bacillomycin D can control the growth of R. stolonifer through both the direct inhibition of the fungus and the activation of defense-related genes and enzymes in cherry tomato.

Deoxynivalenol (DON) is a potent mycotoxin produced by many Fusarium spp. that invade grains during the growth and storage seasons. Lactic acid bacteria have been reported to be capable of removing several toxins, thereby providing an effective detoxification method for possible contaminated substrates. The present study mainly focused on investigating the detoxification characteristics of DON by a Lactobacillus paracasei LHZ-1 strain, which was recently isolated from yogurt with a strong promise of removing DON from liquid culture. The results obtained showed that the cell wall of L. paracasei LHZ-1 can remove up to 40.7% of 50 μg/mL DON, whereas only 10.5 and 8.9% are removed by the culture supernatant or cellular lysate, respectively. Laser scanning confocal microscopy helped to identify the mechanism of DON detoxification by L. paracasei LHZ-1 through cellular adsorption, where DON was found to bind to the surface of bacterial cells to form complexes. In stability tests, about 39 or 99% of bound DON, either to viable bacterial cells or heat-inactivated cells, respectively, was released by methanol extractions, which indicated that the binding force between viable cells and DON could be stronger than it is in heat-inactivated cells. Adsorption kinetics demonstrated that approximately 33% of DON was removed within 20 h, with a maximum adsorption capacity of approximately 50.5 μg/mL in phosphate-buffered solution.

The use of natural preservatives has attracted considerable attention owing to their generally safe and environmentally friendly properties. In this study, we investigated the effects of the preservative A1, composed of plantaricin 163, thymol, and surfactin, on bacterial communities and storage quality of refrigerated crucian carp. A total of 522 operational taxonomic units belonging to 20 phyla and 272 genera were identified by high-throughput sequencing, showing a comprehensive coverage of bacterial composition of crucian carp. In untreated samples after spoilage, Brochothrix was the predominant genus, followed by Aeromonas and Pseudomonas . After treatment with A1, the growth of these spoilage bacteria was significantly inhibited according to high-throughput sequencing and plate counts, and Lactococcus became the most abundant organism at the end of storage. Meanwhile, compared with control samples, the shelf life of A1-treated samples extended from 3 to 12 days on the basis of the sensory evaluation and the total viable counts. Furthermore, the total volatile basic nitrogen, thiobarbituric acid, and pH values for A1-treated samples were significantly lower than that of control samples. The results indicate that preservative A1 has potential commercial application in the preservation of refrigerated crucian carp.

Salmonella enterica serovar Heidelberg causes foodborne infections and is a major threat to the food chain and public health. In this study, we aimed to develop a rapid molecular typing approach to identify Salmonella enterica serovar Heidelberg. Using comparative genomics, four serovar-specific gene fragments were identified, and a real-time polymerase chain reaction (PCR) combined with a propidium monoazide (PMA) pretreatment method was developed for simultaneous detection of viable Salmonella sp. ( invA ) and Salmonella Heidelberg ( SeHA&#95;C3258 ). The assay showed 100% specificity for all strains tested. The assay was able to distinguish effectively viable or dead cells with the PMA. The detection limit was 2.4 CFU/mL following 6 h of incubation in enrichment Luria-Bertani medium, and the assay could detect 1.7 × 10 2 CFU/mL in the presence of pork background flora. In artificially contaminated pork, real-time PCR detected inoculum levels of 1.15 CFU/25 g of pork after a 6 h enrichment. Thus, our findings indicated that this comparative genomics approach could be used to screen for serovar-specific fragments and that real-time PCR with PMA was a simple and reliable method for detecting viability of Salmonella species and Salmonella Heidelberg.

Nasopharyngeal carcinoma (NPC) is one of the most severe types of malignant cancer of the head and neck as it is difficult to treat. Ezrin is highly expressed in numerous types of cancer. However, the role of Ezrin in NPC has not been fully investigated and further studies are required in order to uncover its therapeutic potential in the treatment of NPC. The aim of the present study was to investigate the expression of Ezrin in human NPC and to evaluate the effect of knockdown of Ezrin using small interfering (si)-RNA on NPC cell migration and invasion. The expression levels of Ezrin were determined using reverse transcription-quantitative polymerase chain reaction, immunohistochemical staining and western blotting. Following transfection of Ezrin-siRNA into NPC cells, cell invasion and migration were analyzed and the mRNA expression levels of matrix metalloproteinase(MMP)-2 and MMP9 were determined. The results revealed that the expression of Ezrin was markedly increased in human NPC tissue samples compared with normal adjacent nasopharyngeal tissue samples. Ezrin was also highly expressed in the NPC cell lines 6-10B and C6661 when compared with the normal nasopharyngeal cell line NP69. Transfection of NPC cell lines with siRNA targeting Ezrin significantly inhibited NPC cell migration and invasion, and downregulated the mRNA expression level of MMP2; however, no effect was observed on MMP9 mRNA expression. At the same time, knockdown of Ezrin significantly decreased the expression levels of phosphatidylinositol 3-kinase (PI3K) and phosphorylated protein kinase B (Akt), which downregulated the mRNA expression of MMP2. In conclusion, the results revealed that knockdown of Ezrin suppressed NPC migration and invasion by reducing the mRNA expression of MMP2 via the PI3K/Akt signaling pathway. These results highlight the important role of Ezrin in NPC cell migration and invasion. In addition, they indicate that silencing of Ezrin may serve as a potential therapeutic strategy to treat human NPC.

With the widespread use of invasive surgery, immunosuppressive therapy and broad-spectrum antibiotics, there has resulted a corresponding increase in severe systemic infections as produced by Candida albicans (C.albicans), as it combines with bacterial infections. Such infections often result in high rates of mortality. In this report, we examined the effects of the C. albicans cell wall mannoprotein (MP) on macrophage immunity. The MTS assay was used to detect cell proliferation activity and neutral red staining to observe cell phagocytosis. The Griess method was used to detect NO secretion in culture supernatants and apoptosis of macrophages were determined with use of FITC-Annexin V and PI staining. mRNA and protein expressions of JAK2, STAT3, IL-1β, IL-6, TNF-α and iNOS in RAW264.7 cells were determined with use of RT-PCR and western blot. MP significantly promoted the proliferation of RAW264.7 cells, inhibited their phagocytic capacity, but exerted no significant effects on apoptosis of macrophages. In addition, MP not only up-regulated the expression of cytokines, but also the expressions of p-stat3 and p-jak2. Interestingly, when MP was combined with lipopolysaccharide (LPS) a markedly accentuated release of inflammatory cytokines was observed. MP promotes macrophage inflammation induced by LPS and participates in the inflammatory response. One of the potential mechanisms of this effect involves MP activation of the JAK2/STAT3 signaling pathway in RAW264.7 cells, which enables macrophages to transform from M0 to M1 and promote the occurrence of inflammation., (Copyright © 2019. Published by Elsevier Ltd.)

Forming biofilm is a strategy utilized by Shiga toxin-producing Escherichia coli (STEC) to survive and persist in food processing environments. We investigated the biofilm-forming potential of STEC strains from 10 clinically important serogroups on stainless steel at 22 °C or 13 °C after 24, 48, and 72 h of incubation. Results from crystal violet staining, plate counts, and scanning electron microscopy (SEM) identified a single isolate from each of the O113, O145, O91, O157, and O121 serogroups that was capable of forming strong or moderate biofilms on stainless steel at 22 °C. However, the biofilm-forming strength of these five strains was reduced when incubation time progressed. Moreover, we found that these strains formed a dense pellicle at the air-liquid interface on stainless steel, which suggests that oxygen was conducive to biofilm formation. At 13 °C, biofilm formation by these strains decreased (P < 0.05), but gradually increased over time. Overall, STEC biofilm formation was most prominent at 22 °C up to 24 h. The findings in this study identify the environmental conditions that may promote STEC biofilm formation in food processing facilities and suggest that the ability of specific strains to form biofilms contributes to their persistence within these environments., Competing Interests: The authors declare no conflict of interest.

Salmonella spp. is one of the most common foodborne infectious pathogen. This study aimed to develop a real-time nucleic acid sequence-based amplification (NASBA) assay for detecting Salmonella in foods. Primers and a molecular beacon targeting the Salmonella-specific xcd gene were designed for mRNA transcription, and 48 Salmonella and 18 non-Salmonella strains were examined. The assay showed a high specificity and low detection limit for Salmonella (7 × 10 -1  CFU/mL) after 12 h of pre-enrichment. Importantly, it could detect viable cells. Additionally, the efficacy of the NASBA assay was examined in the presence of pork background microbiota; it could detect Salmonella cells at 9.5 × 10 3  CFU/mL. Lastly, it was successfully used to detect Salmonella in pork, beef, and milk, and its detection limit was as low as 10 CFU/25 g (mL). The real-time NASBA assay developed in this study may be useful for rapid, specific, and sensitive detection of Salmonella in food of animal origin.

Salmonella spp. are health-threatening foodborne pathogens. The increasingly common spread of antibiotic-resistant Salmonella spp. is a major public healthcare issue worldwide. In this study, we wished to explore (1) antibiotic or polypeptide combinations to inhibit multidrug-resistant Salmonella bredeney and (2) the regulation of cross-resistance and collateral sensitivity of antibiotics and polypeptides. We undertook a study to select antibiotic combinations. Then, we promoted drug-resistant strains of S. bredeney after 15 types of antibiotic treatment. From each evolving population, the S. bredeney strain was exposed to a particular single drug. Then, we analyzed how the evolved S. bredeney strains acquired resistance or susceptibility to other drugs. A total of 105 combinations were tested against S. bredeney following the protocols of CLSI-2016 and EUCAST-2017. The synergistic interactions between drug pairings were diverse. Notably, polypeptides were more likely to be linked to synergistic combinations: 56% (19/34) of the synergistic pairings were relevant to polypeptides. Simultaneously, macrolides demonstrated antagonism toward polypeptides. The latter were more frequently related to collateral sensitivity than the other drugs because the other 13 drugs sensitized S. bredeney to polypeptides. In an experimental evolution involving 15 drugs, single drug-evolved strains were examined against the other 14 drugs, and the results were compared with the minimal inhibitory concentration of the ancestral strain. Single drug-evolved S. bredeney strains could alter the sensitivity to other drugs, and S. bredeney evolution against antibiotics could sensitize it to polypeptides.

Pullulanase plays an important role as a starch hydrolysis enzyme in the production of bio-fuels and animal feed, and in the food industry. Compared to the methods currently used for pullulanase production, synthesis by Bacillus subtilis would be safer and easier. However, the current yield of pullulanase from B. subtilis is low to meet industrial requirements. Therefore, it is necessary to improve the yield of pullulanase by B. subtilis . In this study, we mined 10 highly active promoters from B. subtilis based on transcriptome and bioinformatic data. Individual promoters and combinations of promoters were used to improve the yield of pullulanase in B. subtilis BS001. Four recombinant strains with new promoters (Phag, PtufA, PsodA, and PfusA) had higher enzyme activity than the control (PamyE). The strain containing PsodA+fusA (163 U/mL) and the strain containing PsodA+fusA+amyE (336 U/mL) had the highest activity among the analyzed dual- and triple-promoter construct stains in shake flask, which were 2.29 and 4.73 times higher than that of the strain with PamyE, respectively. Moreover, the activity of the strain containing PsodA+fusA+amyE showed a maximum activity of 1,555 U/mL, which was 21.9 times higher than that of the flask-grown PamyE strain in a 50-liter fermenter. Our work showed that these four strong promoters mined from transcriptome data and their combinations could reliably increase the yield of pullulanase in quantities suitable for industrial applications.

The concept of encoding metasurfaces makes a connection between the physical metamaterial particles and the digital code so the digital coding can be performed on the coding surface for signal processing to achieve unusual physical phenomena. Here, a complete 2π transmission phase with high efficiency at the target frequency can be manipulated by a multilayer metasurface structure. Different period sequence codes are designed to obtain the deflection angles, and then a new sequence is obtained through a four-bit system operation sequence according to the Fourier convolution principle. This study shows that Fourier calculation provides an efficient way to optimize the coding to achieve a pre-designed transmitted beam. The advantage of this method over previous solutions in generating an anomalous single transmission beam is its flexibility and continuous control of arbitrary directions, and it is possible to transmit a normally incident terahertz beam to an abnormal arbitrary direction with cross-polarization. This work opens up a new digital perspective on the new approach to metamaterials, predicting the possibility of combining electromagnetic wave operations with the convolution theorems in digital signal processing and the transcoding surface.

We report a case of acute massive cerebellar infarction associated with craniocervical junction (CVJ) complex malformation in a 21-year-old male. Timely surgical intervention prevented the deterioration of his neurological status.

Background: Lipoxygenase (LOX) from Anabaena sp. PCC 7120 (Ana-rLOX) offers important applications in the food industry, especially for improving aroma and dough rheological properties. However, industrial applications of LOXs have been limited by their poor thermostability. Herein, we report a bioinformatics-based consensus concept approach for the engineering of thermostable Ana-rLOX., Results: A series of mutations (N130D, G260A, S437T, N130D/G260Q, N130D/S437Y) showed higher thermostability and activity than the wild-type enzyme. Thus, N130D/G260Q exhibited a 6.6-fold increase in half-life and 2.45 °C increase in unfolding temperature; N130D/S437Y showed a 10 °C increase in optimal temperature. The secondary structure did not change much that contributed to improved thermostability were investigated in detail using circular dichroism. Homology modeling suggested that enhanced thermostability and specific activity may result from favorable hydrophobic interactions., Conclusions: A series of mutations were achieved, showing higher thermostability and activity than the wild-type enzyme by semi-rational mutagenesis with limited structure information. Our findings provide important new insights into molecular modifications aimed at improving Ana-rLOX thermostability and activity.

To improve the antibacterial and antioxidant properties of chitosan (CS), CS grafted with gallic acid (GA) using recombinant bacterial laccase from Bacillus vallismortis fmb-103 (fmb-rL103) as a catalyst. The structures of grafted chitosans were identified using Fourier transform infrared spectroscopy (FT-IR) and UV visible spectrum (UV-Vis spectroscopy). After gallic acid grafting, the antibacterial properties of chitosans against Pseudomonas , Acinetobacter , Brochothrix thermosphacta , Escherichia coli , Staphylococcus aureus , Salmonella , and Listeria monocytogenes were significantly improved. Meanwhile, 1,1-Diphenyl-2-picrylhydrazyl (DPPH) radical scavenging results showed that the antioxidant properties of grafted CS increased as well. The preservative effects of the grafted chitosan on chilled meat were then investigated. For this purpose, the quality indexes of the chilled meat during the storage were monitored, including total bacterial count, total basic volatile nitrogen (TVB-N) content, pH value, color and thiobarbituric acid reactive substances (TBARS) and so on. The results showed that coating with the grafted chitosan retarded the growth of spoilage bacteria, and decreased TVB-N and TBARS values of meat. The shelf life of chilled meat coated by CS grafted with GA (GA-g-CS) also extended from 6 days to 18 days at 4°C. These results provided a theoretical basis for the future application of the GA-g-CS in the preservation of chilled meat. Highlights: (1)The temperature and pH-stable bacterial laccase was used to synthesize gallic acid grafted chitosan.(2)Antioxidant and antibacterial properties of chitosan were improved through grafting gallic acid.(3)Storage properties of chilled meat were improved by coating with gallic acid grafted chitosan.

Background: Plipastatin, an antifungal lipopeptide, is synthesized by a non-ribosomal peptide synthetase (NRPS) in Bacillus subtilis. However, little information is available on the combinatorial biosynthesis strategies applied in plipastatin biosynthetic pathway. In this study, we applied module or individual domain deletion strategies to engineer the plipastatin biosynthetic pathway, and investigated the effect of deletions on the plipastatin assembly line, as well as revealed the synthetic patterns of novel lipopeptides., Results: Module deletion inactivated the entire enzyme complex, whereas individual domain (A/T domain) deletion within module 7 truncated the assembly line, resulting in truncated linear hexapeptides (C 16~17 β-OHFA-Glu-Orn-Tyr-Thr-Glu-Ala/Val). Interestingly, within the module 6 catalytic unit, the effect of thiolation domain deletion differed from that of adenylation deletion. Absence of the T 6 -domain resulted in a nonproductive strain, whereas deletion of the A 6 -domain resulted in multiple assembly lines via module-skipping mechanism, generating three novel types of plipastatin derivatives, pentapeptides (C 16~17 β-OHFA-Glu-Orn-Tyr-Thr-Glu), hexapeptides (C 16~17 β-OHFA-Glu-Orn-Tyr-Thr-Glu-Ile), and octapeptides (C 16~17 β-OHFA-Glu-Orn-Tyr-Thr-Glu-Gln-Tyr-Ile)., Conclusions: Notably, a unique module-skipping process occurred following deletion of the A 6 -domain, which has not been previously reported for engineered NRPS systems. This finding provides new insight into the lipopeptides engineering. It is of significant importance for combinatorial approaches and should be taken into consideration in engineering non-ribosomal peptide biosynthetic pathways for generating novel lipopeptides.

Bacillus cereus is an opportunistic pathogen that causes foodborne diseases. We isolated a novel bacteriocin, designated plantaricin GZ1-27, and elucidated its mode of action against B. cereus. Plantaricin GZ1-27 was purified using ammonium sulfate precipitation, gel-filtration chromatography, and RP-HPLC. MALDI-TOF/MS revealed that its molecular mass was 975 Da, and Q-TOF-MS/MS analysis predicted the amino acid sequence as VSGPAGPPGTH. Plantaricin GZ1-27 showed thermostability and pH stability. The antibacterial mechanism was investigated using flow cytometry, confocal laser-scanning microscopy, scanning and transmission electron microscopy, and RT-PCR, which revealed that GZ1-27 increased cell membrane permeability, triggered K + leakage and pore formation, damaged cell membrane integrity, altered cell morphology and intracellular organization, and reduced the expression of genes related to cytotoxin production, peptidoglycan synthesis, and cell division. These results suggest that plantaricin GZ1-27 effectively inhibits B. cereus at both the cellular and the molecular levels and is a potential natural food preservative targeting B. cereus.

Bacillus amyloliquefaciens, a Gram-positive and soil-dwelling bacterium, could produce secondary metabolites that suppress plant pathogens. In this study, we provided the whole genome sequence results of B. amyloliquefaciens fmbJ, which had one circular chromosome of 4 193 344 bp with 4249 genes, 87 tRNA genes, and 27 rRNA genes. In addition, fmbJ was found to contain several gene clusters of antimicrobial lipopeptides (bacillomycin D, surfactin, and fengycin), and bacillomycin D homologues were further comprehensively identified. To clarify the influence of rapC regulating the synthesis of lipopeptide on the yield of bacillomycin D, rapC gene in fmbJ was successfully deleted by the marker-free method. Finally, it was found that the deletion of rapC gene in fmbJ significantly improved bacillomycin D production from 240.7 ± 18.9 to 360.8 ± 30.7 mg/L, attributed to the increased the expression of bacillomycin D synthesis-related genes through enhancing the transcriptional level of comA, comP, and phrC. These results showed that the production of bacillomycin D in B. amyloliquefaciens fmbJ might be regulated by the RapC-PhrC system. The findings are expected to advance further agricultural application of Bacillus spp. as a promising source of natural bioactive compounds.

Biofilms are communities of bacterial cells that serve to protect them from external adverse influences and enhance bacterial resistance to antibiotics and sanitizers. Here, we studied the regulatory effects of glucose and sodium chloride on biofilm formation in Salmonella serovar Dublin (S. Dublin). To analyze expression levels of the quorum sensing gene luxS, we created a luxS knockout mutant. Also, antimicrobial resistance, hydrophobicity and autoinducer-2 (AI-2) activity of both the wild-type (WT) and the mutant strain were investigated. Our results revealed that glucose was not essential for S. Dublin biofilm formation but had an inhibitory effect on biofilm formation when the concentration was over 0.1%. NaCl was found to be indispensable in forming biofilm, and it also exerted an inhibitory effect at high concentrations (>1.0%). Both the WT and the mutant strains displayed significant MIC growth after biofilm formation. An increase of up to 32,768 times in the resistance of S. Dublin in biofilm phonotype against antibiotic (ampicillin) compared to its planktonic phonotype was observed. However, S. Dublin luxS knockout mutant only showed slight differences compared to the WT strain in the antimicrobial tests although it displayed better biofilm-forming capacity than the WT strain. The mutant strain also exhibited higher hydrophobicity than the WT strain, which was a feature related to biofilm formation. The production of the quorum sensing autoinducer-2 (AI-2) was significantly lower in the mutant strain than in the WT strain since the LuxS enzyme, encoded by the luxS gene, plays an essential role in AI-2 synthesis. However, the limited biofilm-forming ability in the WT strain indicated AI-2 was not directly related to S. Dublin biofilm formation. Furthermore, gene expression analysis of the WT and mutant strains revealed upregulation of genes related to biofilm stress response and enhanced resistance in the luxS mutant strain, which may provide evidence for the regulatory role of the luxS gene in biofilm formation., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

LI-F type peptides are a family of cyclic lipodepsipeptide antibiotics isolated from Paenibacillus polymyxa and display potent activities against positive bacteria including methicillin-resistant S. aureus (MRSA). In this study, we investigated the mechanism of action of LI-F type peptide AMP-jsa9 against a MRSA (S. aureus CICC10790), which is resistant to ciprofloxacin, gentamicin, kanamycin, chloramphenicol, methicillin, and tetracycline. It was found that AMP-jsa9 mainly targets the cell membrane of MRSA and is able to inhibit biofilm formation through killing planktonic bacteria cells. Moreover, AMP-jsa9 can bind to DNA in vitro, which represents another pathway for the action on MRSA. Furthermore, in vivo treatment of scalded mice with AMP-jsa9 resulted in inhibiting MRSA infections and healing of the scalded wound. In addition, it was demonstrated that AMP-jsa9 can effectively inhibit MRSA infections in scalded murine epidermis and that inflammatory cytokines including IL-8, IL-6, tumor necrosis factor alpha (TNF-α), and monocyte chemotactic factor-1 (MCP-1) were reduced; moreover, both protein and gene expression levels of vascular endothelial growth factor (VEGF) and endothelial nitric oxide synthase (e-NOS) were enhanced, which promote neovascularization and proliferation of new granulation tissue.

Mg-Li hybrid batteries have attracted wide interest in recent years because of their potential safety as well as their cost benefit and high volumetric capacity. However, slow kinetic properties strongly hinder their commercial application. In this study, we have prepared spinel LiCrTiO 4 by a solid-state reaction and have conducted a comprehensive study aimed at improving the performance of Mg-Li hybrid batteries by optimizing the dual-salt electrolyte. LiCrTiO 4 has been found to show reversible discharge/charge capacities of 178 and 169 mA h g -1 in electrolytes of 1 m LiCl and 0.3 m APC (all-phenyl-complex), respectively. When the concentration of APC was increased to 0.4 m, LiCrTiO 4 showed a high capacity retention of 95 % after 30 cycles. In addition, no phase transition could be observed for an LiCrTiO 4 electrode in a dual-salt system, suggesting high electrochemical reversibility. Ex situ EDX and SEM studies have indicated that only Li + ions are inserted into the cathode side, while Mg 2+ ions are reversibly deposited on the surface of Mg metal without dendrite-like growth, indicative of good safety of the Mg-Li hybrid batteries., (© 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Methicillin-resistant Staphylococcus aureus (MRSA) has become increasingly prevalent in hospitals, clinics, and the community. MRSA can cause significant and even lethal infections, especially in skin burn wounds. The currently available topical agents have largely failed to eliminate MRSA infections due to resistance. Therefore, there is an urgent need for new and effective approaches for treating MRSA. Here, we show that a novel engineered amphipathic peptide, WRL3 (WLRAFRRLVRRLARGLRR-NH2), exhibits potent antimicrobial activity against MRSA, even in the presence of various salts or serum. The cell selectivity of WRL3 was demonstrated by its ability to specifically eliminate MRSA cells over host cells in a coculture model. Additionally, WRL3 showed a synergistic effect against MRSA when combined with ceftriaxone and effectively inhibited sessile biofilm bacteria growth leading to a reduction in biomass. Fluorescent measurements and microscopic observations of live bacterial cells and artificial membranes revealed that WRL3 exerted its bactericidal activity possibly by destroying the bacterial membrane. In vivo studies indicate that WRL3 is able to control proliferation of MRSA in wound tissue and reduce bioburden and provides a more favorable environment for wound healing. Collectively, our data suggest that WRL3 has enormous potential as a novel antimicrobial agent for the treatment of clinical MRSA infections of skin burn wounds.

The gene encoding a novel acidic lipoxygenase from Myxococcus xanthus DK1622 (accession: WP&#95;011551853.1) was cloned into vector pET-28a and expressed in Escherichia coli BL21(DE3). The recombinant enzyme (rMxLOX), with a molecular weight of approximately 80 kDa, was purified to homogeneity using one-step nickel-affinity chromatography and showed an activity of 5.6 × 10 4 U/mg. The optimum pH and temperature for rMxLOX activity were found to be 3.0 and 30 °C, respectively. Purified rMxLOX exhibited activity towards linoleic acid and arachidonic acid as substrates, with linoleic acid being the better substrate (K m and k cat values of 0.048 mM and 13.3/s, respectively). The synthetic dye aniline blue was decolorized 69.7 ± 3.5%, following incubation with rMxLOX for 35 min. These results reveal the potential for the use of rMxLOX in the pulp, textile, and wastewater treatment industries., (Copyright © 2017 Elsevier Inc. All rights reserved.)

To convert the lipopeptide non-producer strain Bacillus subtilis pB2 into a plipastatin and surfactin coproducer, a gene expression cassette composed of a constitutive promoter (P43), functional gene sfp, and pleiotropic regulatory gene degQ was integrated into the chromosomal amyE locus of strain B. subtilis pB2 by homologous recombination, which generated a plipastatin and surfactin co-producer. Thirteen plipastatins and fifteen surfactins were identified in lipopeptide extracts using analytical techniques, and their effects on microorganisms were described by microscopic, cytoskeleton analysis and flow-cytometry, respectively. Plipastatins isolated from the engineered strain pB2-L exhibited strong antifungal activity (MIC 16 μg ml -1 ) by disrupting the cell walls, membranes and cytoskeleton of Fusarium oxysporum f. sp. cucumerinum hyphae. Surfactins affected the cell membrane of Staphylococcus aureus (MIC 20 μg ml -1 ), resulting in nucleic acid leakage and ultimately, cell death. Based on the convenience of genetic manipulation in the engineering strain, this work could be useful for the rational design of lipopeptide synthetases via the recombination of gene fragments to generate arrays of peptide derivatives and thus expand the diversity of microbial-produced lipopeptides.

This paper is the first public report that Streptomyces flavogriseus can produce both actinomycin D and holomycin. The actinomycete strain NJ-4 isolated from the soil of Nanjing Agricultural University was identified as S. flavogriseus . This S. flavogriseus strain was found for the first time to produce two antimicrobial compounds that were identified as actinomycin D and holomycin. GS medium, CS medium and GSS medium were used for the production experiments. All three media supported the production of actinomycin D, while holomycin was detected only in GS medium and was undetectable by HPLC in the CS and GSS media. The antimicrobial activity against B. pumilus , S. aureus , Escherichia coli , F. moniliforme , F. graminearum and A. niger was tested using the agar well diffusion method. Actinomycin D exhibited strong antagonistic activities against all the indicator strains. Holomycin exhibited strong antagonistic activities against B. pumilus , S. aureus and E. coli and had antifungal activity against F. moniliforme and F. graminearum but had no antifungal activity against A. niger . The cell viability was determined using an MTT assay. Holomycin exhibited cytotoxic activity against A549 lung cancer cells, BGC823 gastric cancer cells and HepG2 hepatocellular carcinoma cells. The yield of actinomycin D from S. flavogriseus NJ-4 was 960 mg/l. S. flavogriseus NJ-4 exhibits a distinct capability and has the industrial potential to produce considerable yields of actinomycin D under unoptimized conditions., Competing Interests: The authors declare there are no competing interests.

LI-F type peptides (AMP-jsa9) are a group of cyclic lipodepsipeptides that exhibit broad antimicrobial spectrum against Gram-positive bacteria and filamentous fungi. We sought to assess the toxicity of AMP-jsa9 and the mechanism of AMP-jsa9 action against Fusarium moniliforme. AMP-jsa9 exhibited weak hemolytic activity and weak cytotoxicity at antimicrobial concentrations (32μg/ml). Confocal laser microscopy, SEM, and TEM indicated that AMP-jsa9 primarily targets the cell wall, plasma membrane, and cytoskeleton, increases membranepermeability, and enhances cytoplasm leakage (e.g., K + , protein). Quantitative proteomic analysis using isobaric tags for relative and absolute quantitation (iTRAQ) detected a total of 162 differentially expressed proteins (59 up-regulated and 103 down-regulated) following treatment of F. moniliforme with AMP-jsa9. AMP-jsa9 treatment also led to reductions in chitin, ergosterol, NADH, NADPH, and ATP levels. Moreover, fumonisin B 1 expression and biosynthesis was suppressed in AMP-jsa9-treated F. moniliforme. Our results provide a theoretical basis for the application of AMP-jsa9 as a natural and effective antifungal agent in the agricultural, food, and animal feed industries., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Sodium-ion batteries operating at ambient temperature hold great promise for use in grid energy storage owing to their significant cost advantages. However, challenges remain in the development of suitable electrode materials to enable long lifespan and high rate capability. Here we report a sodium super-ionic conductor structured electrode, sodium vanadium titanium phosphate, which delivers a high specific capacity of 147 mA h g -1 at a rate of 0.1 C and excellent capacity retentions at high rates. A symmetric sodium-ion full cell demonstrates a superior rate capability with a specific capacity of about 49 mA h g -1 at 20 C rate and ultralong lifetime over 10,000 cycles. Furthermore, in situ synchrotron diffraction and X-ray absorption spectroscopy measurement are carried out to unravel the underlying sodium storage mechanism and charge compensation behaviour. Our results suggest the potential application of symmetric batteries for electrochemical energy storage given the superior rate capability and long cycle life.

The aim of this study was to find a virulent bacteriophage for the biocontrol of Salmonella in duck meat. A broad host-spectrum virulent phage, fmb-p1, was isolated and purified from an duck farm, and its host range was determined to include S. Typhimurium, S. Enteritidis, S. Saintpaul, S. Agona, S. Miami, S. Anatum, S. Heidelberg and S. Paratyphi-C. Electron microscopy and genome sequencing showed that fmb-p1 belongs to the family Siphoviridae. The genome of fmb-p1 is composed of a 43,327-bp double-stranded DNA molecule with 60 open reading frames and a total G+C content of 46.09%. There are no deleterious sequences or genes encoding known harmful products in the phage fmb-p1 genome. Phage fmb-p1 was stable under different temperature (40-75°C), pH (4-10) and NaCl solutions (1-11%). The phage treatment (9.9×10 9 PFU/cm 2 ) caused a peak reduction in S. Typhimurium of 4.52 log CFU/cm 2 in ready-to-eat (RTE) duck meat, whereas potassium sorbate treatment (PS, 2mg/cm 2 ) resulted in a 0.05-0.12 log reduction. Compared to PS treatment, there was significant difference in the S. Typhimurium reduction (P˂0.05) by phage treatment at both 4°C and 25°C. The results suggested that phage could be applied to reduce Salmonella, on commercial poultry products., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Plantaricins are a group of ribosomally synthesized antimicrobial peptides in Lactobacillus plantarum that exert antimicrobial activities against some foodborne pathogens. In this study, we observed that plantaricin E in L. plantarum 163 was missing 19 amino acids (plnE mutant amino acid sequence: FNRGGYNFGKSVRH, plnE amino acid sequence: FNRGGYNFGKSVRHVVDAIGSVAGIRGILKSIR). In order to study the effects of mutant plnE, plnE mutant genes with and without the signal peptide were cloned from the L. plantarum 163 genome, linked to the pET32a vector, and expressed via a fusion protein (thioredoxin) in Escherichia coli BL21 (DE3). All target proteins were purified using Ni-NTA, SP FF columns, and RP-HPLC. The purified proteins were stable in an acidic environment and at temperatures below 80 °C, but they were easily degraded under alkaline conditions and by protease treatment. They showed antimicrobial activity against gram-positive bacteria such as Micrococcus luteus, Staphylococcus epidermidis, Lactococcus lactis, Lactobacillus paracasei, and Listeria innocua. In addition, SP-plnE and PlnE exerted stronger activity than nisin. The signal peptide had a positive effect on the activities of PlnE and PlnEm. Thus, these purified proteins may have potential applications in the food industry to control foodborne pathogens.

Salmonella enterica serovar Heidelberg (S. Heidelberg) is one of the pathogens most frequently detected in recent years in food products from animals; here, we examine the acid tolerance of this strain. Mild acid (pH5.5-5.0) induced a strong acid resistance in S. Heidelberg; the induced acid resistance improved within 0.5-6h and resulted in >95% cell survival following challenge in pH3.0 medium. Addition of lysine or arginine to pH2.5 acid challenge medium significantly improved the survival of S. Heidelberg; lysine induced the largest increase in survival. The lysine and arginine decarboxylase-related genes (i.e., cadA, cadB, adiA, and adiY) are acid induced genes, and they play an important role in S. Heidelberg acid resistance. RT-PCR showed that the genes expression levels increased as acid adaptation pH decreased. The increased expression was maintained for at least 4h during adaptation. Moreover, acid adaptation may have increased the production of cellulose and swimming in S. Heidelberg; pH5.5 acid-adapted cells showed a red, dry, and rough morphotype on Congo red plates. The transcriptional levels of Enterotoxin gene (stn) increased three times following acid adaptation; however, the expression of Salmonella pathogenicity island 1 virulence genes significantly decreased., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Cronobacter spp. have been linked to clinical cases of infection in both adults and infants. Enrichment of Cronobacter spp. before detection has been necessary but is quite time consuming. Hence, we sought to develop an immunomagnetic separation (IMS) PCR method that could shorten the time of enrichment before the detection of Cronobacter spp. The polyclonal antibody used in this immunomagnetic separation was prepared based on the outer membrane protein A of Cronobacter sakazakii China Center of Industrial Culture Collection 21560 and had high specificity to the target. The primers used in the IMS-PCR method also showed high specificity. The detection limit of IMS-PCR for pure C. sakazakii culture was 5.2 × 10 2 cfu/mL. Cronobacter sakazakii in artificially contaminated powdered infant formula (PIF) was also detected at a detection limit of 5.2 × 10 2 cfu/mL. After 8 h of enrichment, the detection limit in PIF was lower than 5.2 × 10 1 cfu/mL. An interference test using Escherichia coli in artificially contaminated PIF showed that the IMS-PCR method developed in this study had a good ability to resist interference. Finally, the IMS-PCR method was applied to the detection of Cronobacter in food samples and was shown to be reliable. Thus, this newly developed IMS-PCR detection method was quite sensitive, rapid, and reliable and could be applied to the detection of Cronobacter in foods., (Copyright © 2017 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

LI-F type peptides (AMP-jsa9) produced by Paenibacillus polymyxa JSa-9 are a group of cyclic lipodepsipeptide antibiotics that exhibit a broad antimicrobial spectrum against Gram-positive bacteria and filamentous fungi, especially Bacillus cereus and Fusarium moniliforme. In this study, to better understand the antibacterial mechanism of AMP-jsa9 against B. cereus, the ultrastructure of AMP-jsa9-treated B. cereus cells was observed by both atomic force microscopy and transmission electron microscopy, and quantitative proteomic analysis was performed on proteins extracted from treated and untreated bacterial cells by using isobaric tag for relative and absolute quantitation (iTRAQ) labeling and LC-MS/MS analysis to access differentially expressed proteins. Furthermore, multiple experiments were conducted to validate the results of the proteomic analysis, including determinations of ATP, NAD (+) H, NADP (+) H, reactive oxygen species (ROS), the activities of catalase (CAT) and superoxide dismutase (SOD), and the relative expression of target genes by quantitative real-time PCR. Bacterial cells exposed to AMP-jsa9 showed irregular surfaces with bleb projections and concaves; we hypothesize that AMP-jsa9 penetrated the cell wall and was anchored on the cytoplasmic membrane and that ROS accumulated in the cell membrane after treatment with AMP-jsa9, modulating the bacterial membrane properties and increasing membrane permeability. Consequently, the blebs were formed on the cell wall by the impulsive force of the leakage of intercellular contents. iTRAQ-based proteomic analysis detected a total of 1317 proteins, including 176 differentially expressed proteins (75 upregulated (fold >2) and 101 downregulated (fold <0.5)). Based on proteome analysis, the putative pathways of AMP-jsa9 action against B. cereus can be summarized as: (i) inhibition of bacterial sporulation, thiamine biosynthesis, energy metabolism, DNA transcription and translation, and cell wall biosynthesis, through direct regulation of protein levels; and (ii) indirect effects on the same pathways through the accumulation of ROS and the consequent impairment of cellular functions, resulting from downregulation of antioxidant proteins, especially CAT and SOD., Biological Significance: The mode of action of LI-F type antimicrobial peptides (AMP-jsa9) against B. cereus was elucidated at the proteomic level. Two pathways of AMP-jsa9 action upon B. cereus cells were identified and the mechanism of bleb formation on the surfaces of bacterial cells was predicted based on the results of ultrastructural observation and proteomic analysis. These results are helpful in understanding the mechanism of LI-F type peptides and in providing the theoretical base for applying AMP-jsa9 or its analogs to combat Gram-positive pathogenic bacteria in the food and feed industries., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Non-ribosomal peptide synthetases (NRPSs) are large enzymatic complexes that catalyse the synthesis of biologically active peptides in microorganisms. Genetic engineering has recently been applied to reprogram NRPSs to produce lipopeptides with a new sequence. The carboxyl-terminal thioesterase (TE) domains from NRPSs catalyse cleavage products by hydrolysis or complex macrocyclization. In this study, we modified plipastatin synthetase by moving the intrinsic TE region to the end of the internal thiolation (T) domains, thus generating Bacillus subtilis strains that could produce new truncated cyclic or linear peptides of the predicted sequence, which further provided an important insight into the regioselectivity of plipastatin TE. The TE was capable of recognizing and catalysing the lactone formation between L -Try 3 with the last few residues L -Pro7 and L -Gln8 at the C-terminus. Additionally, the unmatched linkers connecting the TE region and T domain resulted in nonproduction strains, suggesting that the native T-TE linker is necessary and sufficient for the TE domain to release the products from the hybrid enzymes. This is the first report to demonstrate truncated cyclic lipopeptides production and module skipping by simply moving the TE domain forward in an NRPS system.

Antimicrobial peptides (AMPs) have gained increasing attention, as they can overcome recurring microbial invasions. However, their poor antimicrobial activity and potential cytotoxicity remain impediments to their clinical applications as novel therapeutic agents. To enhance the antimicrobial activity and cell selectivity of AMPs, a series of amphiphilic peptides based on leucocin A were designed by substituting noncharged hydrophilic residues with arginine and leucine. Of the engineered peptides, peptide 7 (WRL3) (WLRAFRRLVRRLARGLRR-NH2) exhibited the highest cell selectivity toward bacterial cells over erythrocytes and macrophages. Fluorescent measurements and microscopic observations demonstrated that 7 increased cell membrane permeability and disrupted membrane envelope integrity, and eventually led to whole cell lysis. Additionally, flow cytometry analysis and subcellular localization studies revealed that 7 showed potent cytotoxicity against human hepatoma cells HepG2. In summary, the data indicate that these engineered peptides, in particular 7, have enormous promise for antibacterial and/or antitumor therapeutics.

Both donors and acceptors of communication-mediating (COM) domains are essential for coordinating intermolecular communication within nonribosomal peptides synthetases (NRPSs) complexes. Different sets of COM domains provide selectivity, allowing NRPSs to utilize different natural biosynthetic templates. In this study, novel lipopeptides were synthesized by reprogramming the plipastatin biosynthetic machinery. A Thr-to-Asp point mutation was sufficient to shift the selectivity of the donor COM domain of ppsB toward that of ppsD. Deletion and/or interchangeability established donor and acceptor function. Variations in acceptor COM domain did not result in novel product formation in the presence of its partner donor, whereas plipastatin formation was completely abrogated by altering donor modules. Five novel lipopeptides (cyclic pentapeptide, linear hexapeptide, nonapeptide, heptapeptide, and cyclic octapeptide) were identified and verified by high-resolution LC-ESI-MS/MS. In addition, we demonstrated the potential to generate novel strains with the antimicrobial activity by selecting compatible COM domains, and the novel lipopeptides exhibited antimicrobial activity against five of the fungal species at a contention of 31.25-125 μg/ml.

In the present study, thirty autosomal insertion and deletion polymorphic loci were simultaneously amplified and genotyped in a multiplex system, and their allelic frequencies as well as several forensic parameters were obtained in a sample of 236 unrelated healthy Tujia individuals. All the loci were in Hardy-Weinberg equilibrium after applying a Bonferroni correction and all pair-wise loci showed no significant linkage disequilibrium. These loci were observed to be relatively informative and discriminating, quite efficient for forensic applications. Allelic frequencies of 30 loci were compared between the Tujia group and other reference populations, and the results of analysis of molecular variance indicated the Tujia group showed the least significant differences with the Shanghai Han at one locus, and the most with Central Spanish population at 22 loci. We analyzed the population genetic structure by the principal component analysis, the clustering of STRUCTURE program and a Neighbor-Joining tree, and then evaluated the genetic relationships among Tujia and other 15 populations.