Jianying Tang

In Vitro and In Vivo Activities of Antimicrobial Peptides Developed Using an Amino Acid-Based Activity Prediction Method

ABSTRACT To design and discover new antimicrobial peptides (AMPs) with high levels of antimicrobial activity, a number of machine-learning methods and prediction methods have been developed. Here, we present a new prediction method that can identify novel AMPs that are highly similar in sequence to known peptides but offer improved antimicrobial activity along with lower host cytotoxicity. Using previously generated AMP amino acid substitution data, we developed an amino acid activity contribution matrix that contained an activity contribution value for each amino acid in each position of the model peptide. A series of AMPs were designed with this method. After evaluating the antimicrobial activities of these novel AMPs against both Gram-positive and Gram-negative bacterial strains, DP7 was chosen for further analysis. Compared to the parent peptide HH2, this novel AMP showed broad-spectrum, improved antimicrobial activity, and in a cytotoxicity assay it showed lower toxicity against human cells. The in vivo antimicrobial activity of DP7 was tested in a Staphylococcus aureus infection murine model. When inoculated and treated via intraperitoneal injection, DP7 reduced the bacterial load in the peritoneal lavage solution. Electron microscope imaging and the results indicated disruption of the S. aureus outer membrane by DP7. Our new prediction method can therefore be employed to identify AMPs possessing minor amino acid differences with improved antimicrobial activities, potentially increasing the therapeutic agents available to combat multidrug-resistant infections.

X-ray Irradiated Vaccine Confers protection against Pneumonia caused by Pseudomonas Aeruginosa

Pseudomonas aeruginosa is a gram-negative bacterium and one of the leading causes of nosocomial infection worldwide, however, no effective vaccine is currently available in the market. Here, we demonstrate that inactivation of the bacteria by X-ray irradiation inhibits its replication capability but retained antigenic expression functionally thus allowing its use as a potential vaccine. Mice immunized by this vaccine were challenged by the parental strain, the O-antigen-homologous strain PAO-1 (O2/O5) and heterologous strain PAO-6 (O6) in an acute pneumonia model. We further measured the protective effect of the vaccine, as well as host innate and cellular immunity responses. We found immunized mice could protect against both strains. Notably, the antiserum only had significant protective role against similar bacteria, while adoptive transfer of lymphocytes significantly controlled the spread of the virulent heterologous serogroup PAO-6 infection, and the protective role could be reversed by CD4 rather than CD8 antibody. We further revealed that vaccinated mice could rapidly recruit neutrophils to the airways early after intranasal challenge by PAO-6, and the irradiated vaccine was proved to be protective by the generated CD4+ IL-17+ Th17 cells. In conclusion, the generation of inactivated but metabolically active microbes is a promising strategy for safely vaccinating against Pseudomonas aeruginosa.

Efficacy of the novel oxazolidinone compound FYL-67 for preventing biofilm formation by Staphylococcus aureus

OBJECTIVES Infections of hospitalized patients caused by biofilms formed by Staphylococcus aureus represent a major problem. Using in vitro and in vivo biofilm models, we evaluated the efficacy of the novel oxazolidinone FYL-67, by using linezolid (the only clinically approved oxazolidinone antibiotic) as a control, for inhibiting S. aureus biofilm formation. METHODS Antibiofilm activity was determined using strains of methicillin-susceptible S. aureus and methicillin-resistant S. aureus. We studied the mechanism(s) and pharmacodynamics of antibiofilm activity as follows: (i) effects of pre- and post-exposure to FYL-67 or linezolid on biofilm formation; (ii) the effect of FYL-67 on biofilm structure; (iii) the role of FYL-67 in biofilm composition; (iv) effects on cell morphology; and (v) efficacy of FYL-67 and linezolid using an in vivo murine model of catheter infection. RESULTS FYL-67 effectively inhibited biofilm formation using in vitro and in vivo assays. CONCLUSIONS Our data suggest that oxazolidinone compounds, such as FYL-67, may serve as antibiofilm agents.

Discovery of a Teraryl Oxazolidinone Compound (S)-N-((3-(3-Fluoro-4-(4-(pyridin-2-yl)-1H-pyrazol-1-yl)phenyl)-2-oxooxazolidin-5-yl)methyl)acetamide Phosphate as a Novel Antimicrobial Agent with Enhanced Safety Profile and Efficacies

A series of novel teraryl oxazolidinone compounds was designed, synthesized, and evaluated for their antimicrobial activity and toxicities. The compounds with aromatic N-heterocyclic substituents at the 4-position of pyrazolyl ring showed better antibacterial activity against the tested bacteria than other compounds with different patterns of substitution. Among all potent compounds, 10f exhibited promising safety profile in MTT assays and in hERG K(+) channel inhibition test. Furthermore, its phosphate was found to be highly soluble in water (47.1 mg/mL), which is beneficial for the subsequent in vivo test. In MRSA systemic infection mice models, 10f phosphate exerted significantly improved survival protection compared with linezolid. The compound also demonstrated high oral bioavailability (F = 99.1%). Moreover, from the results of in vivo toxicology experiments, 10f phosphate would be predicted to have less bone marrow suppression.

Allele Frequencies of 18 STR Loci in Chinese Population

Specimens of 100 unrelated individuals were collected from Han ethnic group in Sichuan Province of China. DNA was extracted from blood specimens using Chelex 100 method (1).

Synthesis and antibacterial activity evaluation of C-5 side chain modified analogues of FYL-66, a potential agent against methicillin-resistant Staphylococcus aureus

FYL-66 and its hydrochloride FYL-67 have been identified as new chemical entities (NCE) with pronounced in vitro and in vivo activities against MRSA and MSSA. Aiming to explore the structure–activity relationship at the C-5 side chain of FYL-66 and find novel potential antibacterial agents, a series of analogues were designed and synthesized by the introduction of various substituents at the C-5 position of the oxazolidinone ring. Their in vitro antibacterial activities were also evaluated by the microdilution method. Novel compounds 31, 33, 37, 39 and 40 demonstrated potent antibacterial activities with MIC values in the range of 2–4 μg mL−1. Difluoro-substituted analogue 40 was found to possess a good balance between antibacterial efficacy, physicochemical properties and safety profile. In a murine systemic infection model, analogue 40 showed comparable protection rates with FYL-66. The absolute bioavailability of 40 was 89.6% with half-lives of 8.87 ± 3.25 h (p.o.) and 5.40 ± 1.40 h (i.v.), respectively. Meanwhile, our findings show the importance of the C-5 side chain of FYL-66 and imply compounds with small C-5 substituents mimicking the acetamide group display better activities. It is also quite intriguing that different antibacterial effects are presented by analogues of FYL-66, Linezolid and other oxazolidinones with the same fluoro-substitution patterns of the acetyl group at the C-5 position.

Phosphoinositide 3-kinase δ/γ inhibition does not prevent concanavalin A-induced hepatitis.

An increasing number of studies have suggested that phosphoinositide 3-kinase-γ (PI3Kγ) and PI3Kδ are involved in the pathogenesis of autoimmune and inflammatory diseases, such as asthma and atherosclerosis. However, the underlying mechanism of acute hepatitis remains unknown. The present study aimed to determine the effect of PI3Kδ/γ inhibition on hepatic injury in a murine model of hepatitis induced by concanavalin A (ConA). It was demonstrated that the pharmacological inhibition of PI3Kδ/γ by TG100-115 did not prevent liver damage following ConA challenge. Furthermore, the PI3Kδ/γ inhibition resulted in elevated transaminase activity in the serum, aggravated hepatic lesions characterized by hepatic necrosis, increased inflammatory cell infiltration and apoptosis of hepatocytes. Survival tests demonstrated that TG100-115 significantly increased the death rate of mice following ConA challenge. In addition, TG100-115 increased the serum levels of the proinflammatory cytokine IL-2 following ConA injection. These results may oppose the development of PI3Kδ/γ inhibitors as therapeutic agents, particularly for the treatment of human hepatitis.

Typing of pentanucleotide STR polymorphisms

Pentanucleotide tandem repeat markers are of interest for forensic science because they may present less stutter in electrophoretic patterns. We focused on the analysis of the DNA sequence for each allele at two pentanucleotide STR loci, D6S957 and D10S2325, in order to understand their structures in the human genome and to construct allelic ladders, necessary for forensic DNA typing. In order to evaluate the forensic applicability of both pentanucleotide tandem repeat loci and to construct a preliminary database, the genotype distributions and allele frequencies in different ethnic groups were investigated. The population samples included Caucasians (Germans) and Asians (Chinese). The Amp-FLP technique was employed for DNA typing. An example of each allele and new alleles were sequenced. Allele determination for each pentanucleotide STR locus was carried out by comparison with a sequenced allelic ladder made in-house. Both pentanucleotide STR markers provided easily interpretable results. No evidence of deviation from Hardy–Weinberg equilibrium was observed. In 64 confirmed father/mother/child trios, no mutation event was observed. Using a maximum likelihood method, the mutation rate at both STR loci was indirectly estimated as 2.510 5 , suggesting both pentanucleotide STR markers would be useful for

Allele Frequencies for Two STR Loci D11S1977 and D22S444 in Chinese Population

Blood samples were collected from unrelated individuals of Chinese Han ethnic group in Chengdu of China. DNA was extracted using Chelex method (1). Reaction condition of PCR amplification can be accessed at http://www.legalmed.org/ dna/D11S1977.htm. The volume of PCR reaction for each locus was 37.5 µL. The PCR products were analyzed by horizontal non-denaturing polyacrylamide gel electrophoresis with discontinuous buffer system, and visualized by silver staining (2,3). Data of population genetics and forensic science were analyzed using POWERSTATS program (4). The genotype distribution was analyzed for Hardy-Weinberg equilibrium according to Hous method (5) and no deviation from Hardy-Weinberg equilibrium was observed. The complete data can be accessed at http://www.legalmed.org/dna/D11S1977.htm.

Polymorphisms of six STR loci on chromosome 22 in a Chinese population

Abstract To develop more STR markers for forensic DNA typing in Chinese populations, six STR loci on chromosome 22 were investigated. An Amp-FLP technique was employed for genotyping individuals from a Han population sample from North and South China. Hardy–Weinberg equilibrium was checked using the chi-square test and no significant deviation was found at any of the six loci. The results showed that the differences of genotype distributions at two loci were significant between two populations, but the distributions of genotypes at the other four loci were similar in both populations. Five of the six loci showed good levels of polymorphism in both populations, with heterozygosities of more than 0.59. The discrimination power and the exclusion probability were more than 0.82 and 0.40, respectively, making them suitable candidate markers for forensic applications. At another locus, only two alleles were found. The heterozygosity of this locus was less than 0.36 and the discrimination power and exclusion probability were less than 0.62 and 0.12, respectively. A two-step mutation was observed at one of six loci.