The transition of antimicrobial peptides (AMPs) from the laboratory to market has been severely hindered by their instability toward proteases in biological systems. In the present study, we synthesized derivatives of the cationic AMP Pep05 (KRLFKKLLKYLRKF) by substituting L -amino acid residues with D - and unnatural amino acids, such as D- lysine, D- arginine, L- 2,4-diaminobutanoic acid (Dab), L- 2,3-diaminopropionic acid (Dap), L- homoarginine, 4-aminobutanoic acid (Aib), and L- thienylalanine, and evaluated their antimicrobial activities, toxicities, and stabilities toward trypsin, plasma proteases, and secreted bacterial proteases. In addition to measuring changes in the concentration of the intact peptides, LC-MS was used to identify the degradation products of the modified AMPs in the presence of trypsin and plasma proteases to determine degradation pathways and examine whether the amino acid substitutions afforded improved proteolytic resistance. The results revealed that both D- and unnatural amino acids enhanced the stabilities of the peptides toward proteases. The derivative DP06, in which all of the L- lysine and L- arginine residues were replaced by D -amino acids, displayed remarkable stability and mild toxicity in vitro but only slight activity and severe toxicity in vivo , indicating a significant difference between the in vivo and in vitro results. Unexpectedly, we found that the incorporation of a single Aib residue at the N-terminus of compound UP09 afforded remarkably enhanced plasma stability and improved activity in vivo . Hence, this derivative may represent a candidate AMP for further optimization, providing a new strategy for the design of novel AMPs with improved bioavailability.
Global hypomethylation, gene-specific methylation, and genome instability are common events in tumorigenesis. To date, few studies have examined the aberrant DNA methylation patterns in coke oven workers, who are highly at risk of lung cancer by occupational exposure to polycyclic aromatic hydrocarbons (PAHs). We recruited 82 PAH-exposed workers and 62 unexposed controls, assessed exposure levels by urinary 1-hydroxypyrene, and measured genetic damages by comet assay, bleomycin sensitivity, and micronucleus assay. The PAHs in coke oven emissions (COE) were estimated based on toxic equivalency factors. We used bisulfite-PCR pyrosequencing to quantitate DNA methylation in long interspersed nuclear element-1 (LINE-1) and O(6)-methylguanine-DNA methyltransferase (MGMT). Further, the methylation alteration was also investigated in COE-treated human bronchial epithelial (16HBE) cells. We found there are higher levels of PAHs in COE. Among PAH-exposed workers, LINE-1 and MGMT methylation levels (with CpG site specificity) were significantly lowered. LINE-1, MGMT, and its hot CpG site-specific methylation were negatively correlated with urinary 1-hydroxypyrene levels (r = -0.329, p < 0.001; r = -0.164, p = 0.049 and r = -0.176, p = 0.034, respectively). In addition, LINE-1 methylation was inversely associated with comet tail moment and micronucleus frequency, and a significant increase of micronucleus in low MGMT methylation group. In vitro study revealed that treatment of COE in 16HBE cells resulted in higher production of BPDE-DNA adducts, LINE-1 hypomethylation, hypomethylation, and suppression of MGMT expression. These findings suggest hypomethylation of LINE-1 and MGMT promoter could be used as markers for PAHs exposure and merit further investigation.
Background: Sufficient epidemiologic evidence shows an etiologic link between polycyclic aromatic hydrocarbons (PAH) exposure and lung cancer risk. While the genetic modifications have been found in PAH-exposed population, it is unclear whether gene-specific methylation involves in the process of PAHassociated biologic consequence.Methods: Sixty-nine PAH-exposed workers and 59 control subjects were recruited. Using bisulfite sequencing, we examined the methylation status of p16INK4a promoter in peripheral blood lymphocytes (PBL) from
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