Stepwise identification of potent antimicrobial peptides from human genome

The increasing incidence of hospital acquired infections caused by antibiotic resistant pathogens has led to an increase in morbidity and mortality, finding alternative antibiotics unaffected by resistance mechanisms is fundamentally important for treating this problem. Naturally occurring proteins usually carry short peptide fragments that exhibit noticeable biological activity against a wide variety of microorganisms such as bacteria, fungi and protozoa. Traditional discovery of such antimicrobially active fragments (i.e. antimicrobial peptides, AMPs) from protein repertoire is either random or led by chance. Here, we report the use of a rational protocol that combines in silico prediction and in vitro assay to identify potential AMPs with high activity and low toxicity from the entire human genome. In the procedure, a three-step inference strategy is first proposed to perform genome-wide analysis to infer AMPs in a high-throughput manner. By employing this strategy we are able to screen more than one million peptide candidates generated from various human proteins, from which we identify four highly promising samples, and subsequently their antibacterial activity on five strains as well as cytotoxicity on human myoblasts are tested experimentally. As a consequence, two high-activity, low-toxicity peptides are discovered, which could be used as the structural basis to further develop new antibiotics. In addition, from 1491 known AMPs we also derive a quantitative measure called antibacterial propensity index (API) for 20 naturally occurring amino acids, which shows a significant allometric correlation with the theoretical minimal inhibitory concentration of putative peptides against Gram-positive and Gram-negative bacteria. This study may provide a proof-of-concept paradigm for the genome-wide discovery of novel antimicrobial peptides by using a combination of in silico and in vitro analyses.     

VKORC1 rs2359612C allele is associated with increased risk of coronary artery disease in the presence of coronary calcification

VKORC1 genetic polymorphisms affect warfarin dose response, aortic calcification, and the susceptibility of coronary artery disease as shown in our previous study. Little is known regarding the association of VKORC1 polymorphisms with coronary artery calcification (CAC) and the role of CAC in the association with coronary artery disease (CAD). Due to a natural haplotype block in the VKORC1 gene in Chinese, polymorphism rs2359612 was analyzed in a case–control study and a prospective study. The case–control study included 464 CAD patients with non-calcified plaque (NCP), 562 CAD patients with mixed calcified plaque (MCP), 492 subjects with calcified plaque (CP), and 521 controls. The rs2359612C was only associated with increased risk of MCP, the CAD in the presence of CAC; the odds ratio was 1.397 (95 % CI 1.008–1.937, P < 0.05), which was replicated in the second independent population. On the contrary, a negative correlation was observed between rs2359612 and log-transformed Agatston score, and rs2359612 was negatively associated with the number of calcified vessels. Moreover, in a prospective study including 849 CAD patients undergoing revascularization, rs2359612C predicted a higher incidence of cardiovascular events in MCP subgroup; the relative risk was 1.435 (95 % CI 1.008–2.041, P = 0.045), which was not observed in the NCP subgroup. We conclude that the rs2359612C was associated with a higher risk of CAD in the presence of CAC and a higher incidence of cardiovascular events in CAD patients with CAC, but a lower coronary calcification. VKORC1 polymorphisms may be associated with the endophenotype of CAD, calcification-related atherosclerosis.     

Lack of association between EGF + 61A>G polymorphism and melanoma susceptibility in Caucasians: A HuGE review and meta-analysis

Emerging evidence showed that the common polymorphism (+ 61A>G, rs4444903) in the promoter region of epidermal growth factor (EGF) gene might be associated with melanoma susceptibility in humans. But individually published results are inconclusive. The aim of this Human Genome Epidemiology (HuGE) review and meta-analysis is to derive a more precise estimation of the association between EGF + 61A>G polymorphism and melanoma risk. The PubMed, Embase, Web of Science and CBM databases were searched for all articles published up to July 1st, 2012. Seven case–control studies were included with a total of 2367 melanoma cases and 4184 healthy controls. Meta-analysis results showed that there was no significant relationship between EGF + 61A>G polymorphism and the risk of melanoma (G vs A: odds ratio [OR] = 1.08, 95% confidence interval [CI]: 0.91–1.28, P = 0.386; GG + AG vs AA: OR = 1.05, 95%CI: 0.88–1.26, P = 0.580; GG vs AA + AG: OR = 1.10, 95%CI: 0.81–1.49, P = 0.552; GG vs AA: OR = 1.06, 95%CI: 0.80–1.41, P = 0.700; GG vs AG: OR = 1.12, 95%CI: 0.81–1.56, P = 0.494). Further subgroup analyses based on source of controls, country, detection samples, genotype methods, and Breslow thickness of tumor, we also found no significant association between EGF + 61A>G polymorphism and melanoma risk. In conclusion, this meta-analysis indicates that EGF + 61A>G polymorphism might not be a primary determinant in melanoma development and progression; EGF gene might be expected to interact with other genes in different signaling pathways to initiate and promote the carcinogenic process.     

Association between rs10118757(A/G) in methylthioadenosine phosphorylase gene and coronary artery disease in Chinese Hans

Studies focusing on the association of gene methylthioadenosine phosphorylase (MTAP) with the risk of coronary artery disease (CAD) and myocardial infarction (MI) are limited.     

DNA methylation and MeCP2 regulation of PTCH1 expression during rats hepatic fibrosis

Hepatic stellate cell (HSC) activation plays an important role in liver fibrogenesis. Transdifferentiation of quiescent hepatic stellate cells into myofibroblastic-HSCs is a key event in liver fibrosis. The methyl-CpG-binding protein MeCP2 which promotes repressed chromatin structure is selectively detected in myofibroblasts of diseased liver. MeCP2 binds to methylated CpG dinucleotides, which are abundant in the promoters of many genes. Treatment of HSCs with DNA methylation inhibitor 5-aza-2′- deoxycytidine (5-azadC) prevented proliferation and activation. Treatment with 5-azadC prevented loss of Patched (PTCH1) expression that occurred during HSCs activation. In a search for underlying molecular medchanisms, we investigated whether the targeting of epigenetic silencing mechanisms could be useful in the treatment of PTCH1-associated fibrogenesis. It was indicated that hypermethylation of PTCH1 is associated with the perpetuation of fibroblast activation and fibrosis in the liver. siRNA knockdown of MeCP2 increased the expressions of PTCH1 mRNA and protein in hepatic myofibroblasts. These data suggest that DNA methylation and MeCP2 may provide molecular mechanisms for silencing of PTCH1.     

Comprehensive Review of Ocular Angiostrongyliasis with Special Reference to Optic Neuritis

Angiostrongyliasis, caused by Angiostrongylus cantonensis infection, is a food-borne parasitic disease. Its larvae evoke eosinophilic inflammation in the central nervous system, but can also cause pathological changes in the eyes. Among ocular angiostrongyliasis cases, the incidence of optic neuritis is low and only few sporadic reports exist. Some patients with optic neuritis developed obvious hypopsia or even vision loss, which would seriously influence the quality of life of patients. Prompt treatment of optic neuritis caused by A. cantonensis is the key factor for minimizing the incidence of serious complications of this disease. In this review, we first provide a comprehensive overview of ocular angiostrongyliasis, and then focus on the clinical features of optic neuritis caused by A. cantonensis.     

Liquid chromatography tandem mass spectrometry in study of the pharmacokinetics of six steroidal saponins in rats

A rapid, simple and sensitive high-performance liquid chromatography tandem mass spectrometry method was developed and validated for simultaneous determination of six main steroidal saponins in Paris polyphylla in rat plasma. Ginsenoside Rg3 was selected as the internal standard (IS). Plasma samples were pretreated with protein precipitation, and the separation was achieved on a reverse phase Agilent poroshell120 EC-C18 column using a gradient mobile phase system of acetonitrile–water containing 0.1% formic acid. The triple quadruple mass spectrometer was set in negative electrospray ionization mode and multiple reaction monitoring (MRM) was used for six steroidal saponins quantification. The precursors to produce ion transitions monitored for polyphyllin I, polyphyllin II, polyphyllin VI, polyphyllin VII, dioscin, gracillin and IS were m/z 899.5 > 853.4, 1059.5 > 1013.5, 783.4 > 737.4, 1075.5 > 1029.5, 913.5 > 867.4, 929.5 > 883.4 and 819.5 > 783.4, respectively. The intra- and inter-day precisions (RSD%) were less than 13% and the average extraction recoveries ranged from 85% to 97.0% for each analyte. Six steroidal saponins were proved to be stable during sample storage, preparation and analytical procedures. The established method was employed for simultaneous quantification and successfully used for the first time for the pharmacokinetics evaluation of the six main compounds after intragastric administration of P. polyphylla extract in Sprague–Dawley rats.     

Specificity of miR-378a-5p targeting rodent fibronectin

One criterion for microRNA identification is based on their conservation across species, and prediction of miRNA targets by empirical approaches using computational analysis relies on the presence of conservative mRNA 3′UTR. Because most miRNA target sites identified are highly conserved across different species, it is not clear whether miRNA targeting is species-specific. To predict miRNA targeting, we aligned all available fibronectin 3′UTRs and observed significant conservation of all 20 species. Twelve miRNAs were predicted to target most fibronectin 3′UTRs, but rodent fibronectin showed potential binding sites specific for five different miRNAs. One of them, the miR-378a-5p, contained a complete matching seed-region for all rodent fibronectin, which could not be found in any other species. We designed experiments to test whether the species-specific targeting possessed biological function and found that expression of miR-378a-5p decreased cancer cell proliferation, migration, and invasion, resulting in inhibition of tumor growth. Silencing fibronectin expression produced similar effects as miR-378a-5p, while transfection with a construct targeting miR-378-5p produced opposite results. Tumor formation assay showed that enhanced expression of fibronectin in the stromal tissues as a background environment suppressed tumor growth, while increased fibronectin expression inside the tumor cells promoted tumor growth. This was likely due to the different signaling direction, either inside-out or outside-in signal. Our results demonstrated that species-specific targeting by miRNA could also exert functional effects. Thus, one layer of regulation has been added to the complex network of miRNA signaling.