Novel approaches are needed to combat antibiotic resistance. Here, we describe a computational-experimental framework for the discovery of novel cryptic antimicrobial peptides (AMPs). The computational platform, based on previously validated antimicrobial scoring functions, indicated the activation peptide of pepsin A, the main human stomach protease, and its N- and C-terminal halves as antimicrobial peptides. The three peptides from pepsinogen A3 isoform were prepared in a recombinant form using a fusion carrier specifically developed to express toxic peptides in Escherichia coli. Recombinant pepsinogen A3-derived peptides proved to be wide-spectrum antimicrobial agents with MIC values in the range 1.56-50 μM (1.56-12.5 μM for the whole activation peptide). Moreover, the activation peptide was bactericidal at pH 3.5 for relevant foodborne pathogens, suggesting that this new class of previously unexplored AMPs may contribute to microbial surveillance within the human stomach. The peptides showed no toxicity toward human cells and exhibited anti-infective activity in vivo, reducing by up to 4 orders of magnitude the bacterial load in a mouse skin infection model. These peptides thus represent a promising new class of antibiotics. We envision that computationally guided data mining approaches such as the one described here will lead to the discovery of antibiotics from previously unexplored sources.
Peptides with an N-terminal cysteine residue allow site-specific modification of proteins and peptides and chemical synthesis of proteins. They have been widely used to develop new strategies for imaging, drug discovery, diagnostics, and chip technologies. Here we present a method to produce recombinant peptides with an N-terminal cysteine residue as a convenient alternative to chemical synthesis. The method is based on the release of the desired peptide from a recombinant fusion protein by mild acid hydrolysis of an Asp-Cys sequence. To test the general validity of the method we prepared four fusion proteins bearing three different peptides (20-37 amino acid long) at the C-terminus of a ketosteroid isomerase-derived and two Onconase-derived carriers for the production of toxic peptides in E. coli. The chosen peptides were (C)GKY20, an antimicrobial peptide from the C-terminus of human thrombin, (C)ApoB, an antimicrobial peptide from an inner region of human Apolipoprotein B, and (C)p53pAnt, an anticancer peptide containing the C-terminal region of the p53 protein fused to the cell penetrating peptide Penetratin. Cleavage efficiency of Asp-Cys bonds in the four fusion proteins was studied as a function of pH, temperature, and incubation time. In spite of the differences in the amino acid sequence (GTGDCGKY, GTGDCHVA, GSGTDCGSR, SQGSDCGSR) we obtained for all the proteins a cleavage efficiency of about 70-80% after 24 h incubation at 60 °C and pH 2. All the peptides were produced with very good yield (5-16 mg/L of LB cultures), high purity (>96%), and the expected content of free thiol groups (1 mol per mole of peptide). Furthermore, (C)GKY20 was modified with PyMPO-maleimide, a commercially available fluorophore bearing a thiol reactive group, and with 6-hydroxy-2-cyanobenzothiazole, a reagent specific for N-terminal cysteines, with yields of 100% thus demonstrating that our method is very well suited for the production of fully reactive peptides with an N-terminal cysteine residue.
By relying on the photonic immobilization\ud technique of antibodies onto surfaces, we realized portable\ud biosensors for light molecules based on the use of quartz\ud crystal microbalances, given the linear dependence of the\ud method on the laser pulse intensity. Here, we compare the\ud quality of the anchoring method when using nanosecond\ud (260 nm, 25 mJ/pulse, 5 ns, 10 Hz rep. rate) and femtosecond\ud (258 nm, 25 lJ/pulse, 150 fs, 10 kHz rep. rate) laser\ud source, delivering the same energy to the sample with the\ud same average power. As a reference, we also tethered\ud untreated antibodies by means of the passive adsorption. The\ud results are striking: When the antibodies are irradiated with\ud the femtosecond pulses, the deposition on the gold plate is\ud much more ordered than in the other two cases. The effects\ud of UV pulses irradiation onto the antibodies are also analyzed\ud by measuring absorption and fluorescence and suggest\ud the occurrence of remarkable degradation when nanosecond\ud pulses are used likely induced by a larger thermal coupling.\ud In view of the high average power required to activate th
Background Many studies have reported that the information women receive about the risk-to-benefit ratio of breast cancer screening is still scarce and biased toward benefit. In a study we conducted in 2014, we analysed online documents about breast cancer screening that were addressed to the general female public. In the present study, we used the same methodology to verify if the information provided to women was improved. Methods We evaluated documents addressed to the general female public and posted on the Internet by the Italian national and regional public health services. False-positive and false-negative screening results, biopsy-proven false-positive results, interval cancer, overdiagnosis, radiation exposure, and decrease in risk of mortality were analysed. In addition, quantitative data were searched. Results In 2021, the most frequently reported information was reduction in breast cancer mortality (58.2%). The most frequently reported risk was a false-positive mammogram (42.5%). Similar frequency rates were reported for interval cancer, false-negative result, and radiation exposure (35.8%, 31.3%, and 28.3%, respectively). Overdiagnosis and biopsy-proven false-positive result were the less reported risks (20.1% and 10.4%). Thirteen documents provided quantitative data about reduction of mortality risk (16.7%), and only 19 provided quantitative data about risks or harms (8.4%). Almost all organisations sent letters of invitation to women (92.5%) and provided screening free of charge (92.5%). The most recommended was biennial screening for women aged between 50 and 69 years (48.5%). Compared with the information in 2014, that in 2021 showed some improvements. The most marked improvements were in the numbers of reports on overdiagnosis, which increased from 8.0 to 20.1%, and biopsy-proven false-positive result, which increased from 1.4 to 10.4%. Regarding the benefits of breast cancer screening, reduced mortality risk became increasingly reported from 2014 (34.5%) to 2021 (58.2%). Conversely, quantitative data remained scarce in 2021. Conclusions Moderate improvements in information were observed from 2014 to 2021. However, the information on breast cancer screening in documents intended for women published on Italian websites remain scarce.
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