High‐yield recombinant expression of the chicken antimicrobial peptide fowlicidin‐2 in Escherichia coli

The antimicrobial peptide fowlicidin‐2 identified in chicken is a member of the cathelicidins family. The mature fowlicidin‐2 possesses high antibacterial efficacy and lipopolysaccharide (LPS) neutralizing activity, and also represents an excellent candidate as an antimicrobial agent. In the present study, the recombinant fowlicidin‐2 was successfully produced by Escherichia coli (E. coli) recombinant expression system. The gene encoding fowlicidin‐2 with the codon preference of E. coli was designed through codon optimization and synthesized in vitro. The gene was then ligated into the plasmid pET‐32a(+), which features fusion protein thioredoxin at the N‐terminal. The recombinant plasmid was transformed into E. coli BL21(DE3) and cultured in Luria‐Bertani (LB) medium. After isopropyl‐β‐D‐thiogalactopyranoside (IPTG) induction, the fowlicidin‐2 fusion protein was successfully expressed as inclusion bodies. The inclusion bodies were dissolved and successfully released the peptide in 70% formic acid solution containing cyanogen bromide (CNBr) in a single step. After purification by reverse‐phase high‐performance liquid chromatography (RP‐HPLC), ～6.0 mg of fowlicidin‐2 with purity more than 97% was obtained from 1 litre of bacteria culture. The recombinant peptide exhibited high antibacterial activity against the Gram‐positive and Gram‐negative bacteria, and even drug‐resistant strains. This system could be used to rapidly and efficiently produce milligram quantities of a battery of recombinant antimicrobial peptides as well as for large‐scale production. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:369–374, 2015     

Active Site Loop Conformation Regulates Promiscuous Activity in a Lactonase from Geobacillus kaustophilus HTA426

Enzyme promiscuity is a prerequisite for fast divergent evolution of biocatalysts. A phosphotriesterase-like lactonase (PLL) from Geobacillus kaustophilus HTA426 (GkaP) exhibits main lactonase and promiscuous phosphotriesterase activities. To understand its catalytic and evolutionary mechanisms, we investigated a “hot spot” in the active site by saturation mutagenesis as well as X-ray crystallographic analyses. We found that position 99 in the active site was involved in substrate discrimination. One mutant, Y99L, exhibited 11-fold improvement over wild-type in reactivity (k_cat/K_m) toward the phosphotriesterase substrate ethyl-paraoxon, but showed 15-fold decrease toward the lactonase substrate δ-decanolactone, resulting in a 157-fold inversion of the substrate specificity. Structural analysis of Y99L revealed that the mutation causes a ∼6.6 Å outward shift of adjacent loop 7, which may cause increased flexibility of the active site and facilitate accommodation and/or catalysis of organophosphate substrate. This study provides for the PLL family an example of how the evolutionary route from promiscuity to specificity can derive from very few mutations, which promotes alteration in the conformational adjustment of the active site loops, in turn draws the capacity of substrate binding and activity.     

Preparation and Characterization of Self-Reinforced Antibacterial and Oil-Resistant Paper Using a NaOH/Urea/ZnO Solution

This paper describes self-reinforced antibacterial and oil-resistant properties that were successfully prepared by surface selective dissolution of filter paper in a NaOH/Urea/ZnO (weight ratio of 8:12:0.25) aqueous solution. The effect of the processing time on the mechanical properties of this paper was evaluated at -12°C. The paper morphologies were characterized using Scanning Electron Microscopy (SEM), X-ray Diffraction (XRD), Fourier Transform Infrared Spectroscopy (FT-IR) and X-ray photoelectron spectroscopy (XPS). The oil-resistance and antibacterial properties of the produced paper were also investigated. Excellent mechanical properties were observed for an optimized handling time. The tensile and burst strengths of the modified paper were in excess of 100% of the original. Meanwhile, the treated paper was completely oil-resistant within 24 h and demonstrated good antibacterial properties when exposed to Staphylococcus aureus. The traces of residual zinc oxide were found to be safe for food.     

Caramel Color in Soft Drinks and Exposure to 4-Methylimidazole: A Quantitative Risk Assessment

Caramel color is added to many widely-consumed beverages as a colorant. Consumers of these beverages can be exposed to 4-methylimidazole (4-MEI), a potential carcinogen formed during its manufacture. California’s Proposition 65 law requires that beverages containing 4-MEI concentrations corresponding to exposures that pose excess cancer risks > 1 case per 100,000 exposed persons (29 μg 4-MEI/day) carry warning labels. Using ultrahigh-performance liquid chromatography-tandem mass spectrometry, we assessed 4-MEI concentrations in 12 beverages purchased in California and a geographically distant metropolitan area (New York) in which warning labels are not required. In addition, we characterized beverage consumption by age and race/ethnicity (using weighted means calculated from logistic regressions) and assessed 4-MEI exposure and resulting cancer risks and US population cancer burdens attributable to beverage consumption. Data on beverage consumption were obtained from the National Health and Nutrition Examination Survey, dose-response data for 4-MEI were obtained from the California Environmental Protection Agency Office of Environmental Health Hazards Assessment, and data on population characteristics were obtained from the U.S. Census Bureau. Of the 12 beverages, Malta Goya had the highest 4-MEI concentration (915.8 to 963.3μg/L), lifetime average daily dose (LADD - 8.04x10^-3 mg/kgBW-day), lifetime excess cancer risk (1.93x10^-4) and burden (5,011 cancer cases in the U.S. population over 70 years); Coca-Cola had the lowest value of each (4-MEI: 9.5 to 11.7μg/L; LADD: 1.01x10^-4 mg/kgBW-day; risk: 1.92x10^-6; and burden: 76 cases). 4-MEI concentrations varied considerably by soda and state/area of purchase, but were generally consistent across lots of the same beverage purchased in the same state/area. Routine consumption of certain beverages can result in 4-MEI exposures > 29 μg/day. State regulatory standards appear to have been effective in reducing exposure to carcinogens in some beverages. Federal regulation of 4-MEI in caramel color may be appropriate.     

Establishment of Hairy Root Cultures by Agrobacterium Rhizogenes Mediated Transformation of Isatis Tinctoria L. for the Efficient Production of Flavonoids and Evaluation of Antioxidant Activities

In this work, Isatis tinctoria hairy root cultures (ITHRCs) were established as an alternative source for flavonoids (FL) production. I. tinctoria hairy root line V was found to be the most efficient line and was further confirmed by the PCR amplification of rolB, rolC and aux1 genes. Culture parameters of ITHRCs were optimized by Box-Behnken design (BBD), and eight bioactive FL constituents (rutin, neohesperidin, buddleoside, liquiritigenin, quercetin, isorhamnetin, kaempferol and isoliquiritigenin) were quali-quantitatively determined by LC-MS/MS. Under optimal conditions, the total FL accumulation of ITHRCs (24 day-old) achieved was 438.10 μg/g dry weight (DW), which exhibited significant superiority as against that of 2 year-old field grown roots (341.73 μg/g DW). Additionally, in vitro antioxidant assays demonstrated that ITHRCs extracts exhibited better antioxidant activities with lower IC₅₀ values (0.41 and 0.39, mg/mL) as compared to those of field grown roots (0.56 and 0.48, mg/mL). To the best of our knowledge, this is the first report describing FL production and antioxidant activities from ITHRCs.     

Identification of Associated SSR Markers for Yield Component and Fiber Quality Traits Based on Frame Map and Upland Cotton Collections

Detecting QTLs (quantitative trait loci) that enhance cotton yield and fiber quality traits and accelerate breeding has been the focus of many cotton breeders. In the present study, 359 SSR (simple sequence repeat) markers were used for the association mapping of 241 Upland cotton collections. A total of 333 markers, representing 733 polymorphic loci, were detected. The average linkage disequilibrium (LD) decay distances were 8.58 cM (r² > 0.1) and 5.76 cM (r² > 0.2). 241 collections were arranged into two subgroups using STRUCTURE software. Mixed linear modeling (MLM) methods (with population structure (Q) and relative kinship matrix (K)) were applied to analyze four phenotypic datasets obtained from four environments (two different locations and two years). Forty-six markers associated with the number of bolls per plant (NB), boll weight (BW), lint percentage (LP), fiber length (FL), fiber strength (FS) and fiber micornaire value (FM) were repeatedly detected in at least two environments. Of 46 associated markers, 32 were identified as new association markers, and 14 had been previously reported in the literature. Nine association markers were near QTLs (at a distance of less than 1–2 LD decay on the reference map) that had been previously described. These results provide new useful markers for marker-assisted selection in breeding programs and new insights for understanding the genetic basis of Upland cotton yields and fiber quality traits at the whole-genome level.     

N-Acetyl Cysteine (NAC)-Directed Detoxification of Methacryloxylethyl Cetyl Ammonium Chloride (DMAE-CB)

Methacryloxylethyl cetyl ammonium chloride (DMAE-CB) is a polymerizable antibacterial monomer and has been proved as an effective strategy to achieve bioactive bonding with reliable bacterial inhibitory effects. However, the toxicity of DMAE-CB may hamper its wide application in clinical situations. Thus, this study was designed to investigate the toxicity of DMAE-CB and explore the possible protective effects of N-acetyl cysteine (NAC). High performance liquid chromatography (HPLC) and liquid chromatography-mass spectrometry (LC-MS) analysis showed that chemical binding of NAC and DMAE-CB occurred in a time dependent manner. Pre-incubation of fourty-eight hours is required for adequate reaction between DMAE-CB and NAC. DMAE-CB reduced human dental pulp cells (hDPCs) viability in a dose-dependent manner. The toxic effects of DMAE-CB were accompanied by increased reactive oxygen species (ROS) level and reduced glutathione (GSH) content. NAC alleviated DMAE-CB-induced oxidative stress. Annexin V/ Propidium Iodide (PI) staining and Hoechst 33342 staining indicated that DMAE-CB induced apoptosis. Collapsed mitochondrial membrane potential (MMP) and activation of caspase-3 were also observed after DMAE-CB treatment. NAC rescued hDPCs from DMAE-CB-induced apoptosis, accompanied by lower level of MMP loss and caspase-3 activity. This study assists to elucidate the mechanism underlying the cytotoxic effects of DMAE-CB and provides theoretical supports for the searching of effective strategies to reduce toxicity of quaternary ammonium dental monomers.     

Chinese Herbal Therapy and Western Drug Use,Belief and Adherence for Hypertension Management in the Rural Areas of Heilongjiang Province,China

Background

Traditional Chinese medicine (TCM) including Chinese herbal therapy has been widely practiced in China. However, little is known about Chinese herbal therapy use for hypertension management, which is one of the most prevalent chronic conditions in China. Thus we described Chinese herbal therapy and western drug users, beliefs, hypertension knowledge, and Chinese herbal and western drug adherence and determinants of Chinese herbal therapy use among patients with hypertension in rural areas of Heilongjiang Province, China.

Methodology and Principal Findings

This face-to-face cross sectional survey included 665 hypertensive respondents aged 30 years or older in rural areas of Heilongjiang Province, China. Of 665 respondents, 39.7% were male, 27.4% were aged 65 years or older. At the survey, 14.0% reported using Chinese herbal therapy and 71.3% reported using western drug for hypertension management. A majority of patients had low level of treatment adherence (80.6% for the Chinese herbal therapy users and 81.2% for the western drug users). When respondents felt that their blood pressure was under control, 72.0% of the Chinese herbal therapy users and 69.2% of the western drug users sometimes stopped taking their medicine. Hypertensive patients with high education level or better quality of life are more likely use Chinese herbal therapy.

Conclusions and Significance

Majority of patients diagnosed with hypertension use western drugs to control blood pressure. Chinese herbal therapy use was associated with education level and quality of life.