Insight into the inhibitory activity and mechanism of bovine cathelicidin BMAP 27 against Salmonella Typhimurium.

This study synthesized an antimicrobial peptide based on the bovine cathelicidin BMAP 27 sequence. It was found to have a broad spectrum of antibacterial activity, with exceptionally high activity against Salmonella. However, the antibacterial mechanism of BMAP 27 against Salmonella remains unclear. The minimum inhibitory concentrations (MIC) and minimum bactericidal concentrations (MBC) of BMAP 27 against Salmonella enterica serovar Typhimurium were determined to be 2 µM and 4 µM, respectively. After treatment with 2 MIC of BMAP 27, the absorbance of DNA in centrifugal supernatant increased from 0.244 to 1.464, and that of protein rose from 0.174 to 0.774, respectively. BMAP 27 has compromised the cell membrane as observed through field emission scanning electron microscope (FESEM) and transmission electron microscopy (TEM), and confirmed by the propidium iodide (PI) test. The alkaline phosphatase (AKP) enzyme activity in the supernatant of the 2 MIC treatment group was 2.15 times higher than the control group, indicating extracellular membrane damage. BMAP 27 treatment increased intracellular ROS levels as tested by dichlorofluorescein diacetate (DCFH) staining. DNA interaction analysis revealed that BMAP 27 has a binding affinity towards DNA, causing its characteristic bands to disappear and peak intensity at 260 nm to reduce. Molecular docking identified its potential binding mode with DNA. The crystal violet biofilm staining results demonstrated that BMAP 27 inhibited S. Typhimurium biofilm formation by 43.1 % and cleared mature biofilms by 53.62 %. Confocal Laser scanning electron microscopy (CLSM) observed that BMAP 27 could kill bacteria within the biofilm and dislodge bacteria from the surface of glasses. Swimming tests identified that the motor capacity of S. Typhimurium was diminished by BMAP 27. By counting the total bacteria, BMAP 27 was revealed to exert bacteriostatic effects in chilled pork and orange juice, which might provide a basis for its application in the inhibition of Salmonella.

Analysis of treatment methods and prognostic factors in 354 cases of hilar cholangiocarcinoma: A cohort study.

CONTEXT: Better management strategies are needed to improve the survival of patients with hilar cholangiocarcinoma (HCCA). AIMS: This study was designed to examine the effects of different treatment methods on survival and prognostic factors in HCCA. SETTINGS AND DESIGN: We retrospectively analyzed the clinical data of 354 patients with HCCA treated at our institution from 2003 to 2013. MATERIALS AND METHODS: Patients were divided into three groups according to the treatment: the radical resection group, the nonradical resection group, and the biliary drainage-only group. STATISTICAL ANALYSIS USED: The Kaplan-Meier method was used to compare survival rates between the groups, and the independent prognostic factors were assessed using the Cox proportional hazards model. RESULTS: There were 110 patients in the radical resection group, 93 patients in the nonradical resection group, and 151 patients in the biliary drainage-only group, and they showed differing survival rates: 1-year survival rates of 70.7%, 49.5%, and 31.3%; 2-year survival rates of 62.9%, 24.7%, and 9.0%; 3-year survival rates of 34.7%, 4.0%, and 0%; and median survival of 21.7 months, 13.6 months, and 8.7 months, respectively. The radical resection group had the longest overall survival (P< 0.001). Treatment method, albumin (ALB), total bilirubin (TBIL), postoperative pathological T-stage, and distant metastasis were identified as independent prognostic indicators of survival. CONCLUSIONS: Radical resection significantly increases survival in patients with HCCA, and an increase in ALB and a decrease in TBIL improve the prognosis of patients with HCCA.

Micro-Computed Tomography to Assess the Processing Quality of the Additive Manufacturing of Spiral Microcoils.

Additive manufactured polymers and metallized microstructures are widely used in the production of electronic components. However, such three-dimensional printed metallized electrical components inevitably have processing errors that affect their performance. It is vital to understand defects' effects on the performance of the final product. In this study, we simulated micro-computed tomography (CT) data. A spiral cavity is printed by stereolithography and spiral inductors with different numbers of turns are fabricated by injecting Gallium Indium liquid metal (EGaIn). Through the theoretical simulation of the spiral inductor and the characterization of the electrical performance, we found that the relative error in the simulation of 2.5-turn, 4.5-turn, and 6.5-turn spiral inductors is +30.6%, +13%, and +6%, respectively, compared with the experimental data. The CT data are obtained by a CT scanning microcoil and a reconstruction model with real structural features is established based on the data. The results show that the relative error between the reconstructed model with real defects and the experimental data is +10.4%, -3.7%, and -1.5%, respectively, which is closer to the experimental data. According to the CT data simulation that provides a more accurate theoretical prediction, the actual effect of a defect on the final product can be assessed. The difference between the experimental results and the theoretical simulation can be inferred from the reconstruction model.

Optimization and purification of glucansucrase produced by Leuconostoc mesenteroides DRP2-19 isolated from Chinese Sauerkraut.

Strain DRP2-19 was detected to produce high yield of glucansucrase in MRS broth, which was identified to be Leuconostoc mesenteroides. In order for industrial glucansucrase production of L. mesenteroides DRP2-19, a one-factor test was conducted, then response surface method was applied to optimize its yield and discover the best production condition. Based on Plackett-Burman (PB) experiment, sucrose, Ca2+, and initial pH were found to be the most significant factors for glucansucrase production. Afterwards, effects of the three main factors on glucansucrase activity were further investigated by central composite design and the optimum composition was sucrose 35.87 g/L, Ca2+ 0.21 mmol/L, and initial pH 5.56. Optimum results showed that glucansucrase activity was increased to 3.94 ± 0.43 U/mL in 24 hr fermentation, 2.66-fold higher than before. In addition, the crude enzyme was purified using ammonium sulfate precipitation, ion-exchange chromatography, and gel filtration. The molecular weight of glucansucrase was determined as approximately 170 kDa by Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The enzyme was purified 15.77-fold and showed a final specific activity of 338.56 U/mg protein.

Optimization, chain conformation and characterization of exopolysaccharide isolated from Leuconostoc mesenteroides DRP105.

Response surface methodology (RSM) was used to optimize the fermentation condition of exopolysaccharide (EPS) producing strain Leuconostoc mesenteroides DRP105. Result showed that the optimum condition was sucrose 86.83g/L, tryptone 15.47g/L, initial pH7.18 and maximum yield was 53.79±0.78g/L in 36h fermentation. Chain conformation was characterized by Congo red test, ß-elimination and circular dichroism (CD), which indicated that the EPS was O-linkage and exhibited random coil structure in aqueous solution. CD results concluded hydrogen-bond interaction and chirality might be connected with concentration. Purified EPS has a higher degradation temperature of 279.42°C, suggesting high thermal stability of the EPS. The absolute value of zeta potential and particle size were enhanced with increasing concentration. Crude EPS and its purified fraction were found to have moderate DPPH, hydroxyl, superoxide anion radicals scavenging activities and reducing power. This study provided a high yield EPS with unique characteristics for industrial applications.

Characterization of highly branched dextran produced by Leuconostoc citreum B-2 from pineapple fermented product.

A strain Leuconostoc citreum B-2 was isolated from homemade fermentation product of pineapple and its polysaccharide yield was 28.3g/L after cultivating the strain in Man-Rogosa-Sharpe (MRS) medium with 75g/L sucrose. The exopolysaccharide (EPS) was characterized by gas chromatography (GC), Fourier-transform infrared (FT-IR) spectra, high-performance size-exclusion chromatography (HPSEC), nuclear magnetic resonance (NMR) spectroscopy and scanning electron microscope (SEM) analysis in present study. The monosaccharide composition of EPS was glucose and molecular weight was 3.77×106Da. FT-IR and NMR spectra revealed that the B-2 EPS was composed of 75% α-(1→6) linked d-glucopyranose units existing in the main chain with 19% α-(1→3) branching and only a few α-(1→2) branching. The SEM of the dried EPS appeared irregular sheets with glittering surface and compact structure. Water solubility index and water holding capacity of B-2 EPS were 80% and 450%, respectively. All the mentioned characteristics suggested that the EPS has a potential application in the food, cosmetic and pharmaceuticals industry.

Isolation and characterization of dextran produced by Leuconostoc citreum NM105 from manchurian sauerkraut.

A water-soluble exopolysaccharide (EPS) was produced by Leuconostoc ctireum NM105 from homemade manchurian sauerkraut. After culturing the strain in Man-Rogosa-Sharpe medium containing 5% sucrose at 25°C for 48h, the EPS was purified and a yield of 23.5g/L was achieved. The EPS consisted exclusively of glucose and the weight-average molecular weight was 1.01×10(8)Da. The structural characterization of the purified EPS determined by FT-IR, (1)H, (13)C and two-dimensional NMR spectroscopy demonstrated that the glucan contained α-(1→6)-linked d-glucopyranose units, 2,6-linked d-glucopyranose units and terminal α-d-glucopyranose units at a ratio of 1:1:1. The microstructure of the dried dextran appeared a sheet-like smooth glittering and highly branched surface. The NM105 dextran showed high water solubility and excellent water retention. All the results suggested that the highly α-(1→2) branched dextran might have the potential to serve as valuable polymers applied in foods, cosmetics and other fields.

Encoded protein from ycbR gene of enterohemorrhagic Escherichia coli O157:H7 associated with adherence to HEp-2 cells.

Adhesion is one of the significant virulence factors in enterohemorrhagic Escherichia coli (EHEC) O157:H7 pathogenesis. It is regulated by specific loci in the genome sequence. This study mainly focused on investigating the influence of ycbR gene and its encoded YCBR protein on the adhesion of EHEC O157:H7 to HEp-2 cells. In the first part, mutants of EHEC O157:H7 were constructed through TnphoA mutagenesis and assayed for adherent ability. Six mutant strains with lost adherence to HEp-2 cells were isolated and then sequenced using a primer that hybridized to phoA sequence downstream of the fusion joint. The sequencing results indicated that the gene of eae and ycbR, between the initiation codon and the -10 sequence of Z4182, yciI, ARAC-type regulator protein, and high-affinity gluconate transporter of EHEC were all possibly related to adhesion. Of the six genes, the ycbR gene was cloned to the pET28a vector to analyze its function further. Recombinant YCBR protein fused to a His tag (YCBR-His) was expressed under IPTG induction and purified by Ni-NTA column. The purified protein was subcutaneously injected to rabbits to prepare antisera. The results of an adherence assay in the presence of anti-YCBR-His antibodies indicated that antibodies blocked the adherence of EHEC O157:H7 to HEp-2 cells. These observations suggested that ycbR encoded a novel adherence-associated determinant of EHEC O157:H7, which could contribute to the adhesive capacity of the bacteria.

Isolation and identification of antifungal peptides from Bacillus BH072, a novel bacterium isolated from honey.

A bacterial strain BH072 isolated from a honey sample showed antifungal activity against mold. Based on morphological, biochemical, physiological tests, and analysis of 16S rDNA sequence, the strain was identified to be a new subspecies of Bacillus sp. It had a broad spectrum of antifungal activity against various mold, such as Aspergillus niger, Pythium, and Botrytis cinerea. Six pairs of antifungal genes primers were designed and synthesized, and ituA, hag, tasA genes were detected by PCR analysis. The remarkable antifungal activity could be associated with the co-production of these three peptides. One of them was purified by 30-40% ammonium sulfate precipitation, Sephadex G-75 gel filtration and anion exchange chromatography on D201 resin. The purified peptide was estimated to be 35.615 kDa and identified to be flagellin by micrOTOF-Q II. By using methanol extraction, another substance was isolated from fermentation liquor, and determined to be iturin with liquid chromatography-mass spectrometry (LC-MS) method. The third possible peptide encoded by tasA was not isolated in this study. The culture liquor displayed antifungal activity in a wide pH range (5.0-9.0) and at 40-100°C. The result of the present work suggested that Bacillus BH072 might be a bio-control bacterium of research value.

Ionic liquid-induced structural and activity changes in hen egg white lysozyme.

Lysozyme crystals in the presence of 1-butyl-3-methylimidazolium tetrafluoroborate ([C(4)mim]BF(4)), 1-butyl-3-methylimidazolium chloride ([C(4)mim]Cl), 1-butyl-3-methylimidazolium bromide([C(4)mim]Br), and 1,3-dimethylimidazolium iodine([dmim]I) were prepared, and the influence of ionic liquids (ILs) on the structure and activity change of lysozyme was investigated. Fourier transform infrared spectroscopy revealed the major secondary structures of α-helix and ß-sheet for lysozyme. It was interesting to note that increases of the band near 2,935 and 1,656 cm(-1) from Raman spectroscopy are attributed to the unfolding of lysozyme molecules. A shift in amide III from 1,230 to 1,270 cm(-1) in adding [dmim]I occurs, indicating a transformation from ß-sheet to random coil. With regard to adding [C(4)mim]BF(4), [C(4)mim]Cl, and [C(4)mim]Br, α-helix and ß-sheet are the predominant structures for lysozyme. The activity study showed that the ILs used brought a positive effect. Especially, [dmim]I leads to a drastic increase in relative activity, and its value reaches 50 %.

Development of a loop-mediated isothermal amplification method for rapid detection of caprine arthritis-encephalitis virus proviral DNA.

A rapid detection assay based on loop-mediated isothermal amplification (LAMP) has been developed for detecting caprine arthritis-encephalitis (CAEV) proviral DNA. The LAMP assay utilized a set of five primers designed against highly conserved sequences located within the p25 gene region. The assay successfully detected CAEV proviral DNA in total DNA extracts originating from cell culture, whole blood samples and separated PBMCs. There was no cross-reaction with the negative control. Amplification was monitored using a Loopamp real-time turbidimeter; turbidity and the corresponding time were recorded. Amplification from CAEV-Shanxi DNA was detected as early as 17 min, with a maximum sensitivity of 0.0001 TCID(50), reached at 32 min. Sixty-eight animal blood samples were tested using AGID, PCR and LAMP assay, and the positive rates were 30.9 %, 33.8 % and 47.1 %, respectively. Whole blood can be used directly, eliminating the need for separation of PBMCs and nucleic acid extraction, reducing the overall procedure time to approximately 80 min. Therefore, the LAMP assay provides a specific and sensitive means for detecting CAEV proviral DNA in a simple, fast, and cost-effective manner and should be useful in eradication programs and epidemiological studies. Furthermore, the LAMP assay can be performed in less-well-equipped laboratories as well as in the field.

Effects of ripening, 1-methylcyclopropene and ultra-high-pressure pasteurisation on the change of volatiles in Chinese pear cultivars.

BACKGROUND: Aroma is one important fruit sensory attribute influenced by the volatile constituents related to species, variety and technological treatments. We analysed the variations of volatile compounds in five pear cultivars and investigated their changes related to different pear organs, different ripening stages, 1-MCP treatment and ultra-high-pressure pasteurisation. RESULTS: Considerable variations exist in the quantity of 10 volatile compounds among five pear cultivars. Their levels generally showed an increasing trend when collected at later harvest time in Ya pear. In Whangkeumbae pear, most volatile compounds reached their maximum levels in skin and pulp. After treating pears with 42 µmol L(-1) 1-methylcyclopropene (1-MCP), the levels of volatiles remained basically unchanged or only slightly increased in Ya pear during a shelf life of 21 days. When Huangguan pear juice was pasteurised by using ultra-high pressure, the levels of volatiles significantly changed during the shelf life. CONCLUSION: The volatile compositions of five different Chinese pear cultivars differ considerably. The levels of these volatiles vary along with ripening stages and pear tissues. A moderate concentration of 1-MCP could keep the levels of volatile compounds basically unchanged during storage and ultra-high-pressure pasteurisation could change the levels of volatiles significantly during the following shelf life.