Gram‐negative bacteria constitutively secrete native outer membrane vesicles (OMVs) into the extracellular milieu. Although recent progress in this area has revealed that OMVs are essential for ...bacterial survival and pathogenesis, the mechanism of vesicle formation and the biological roles of OMVs have not been clearly defined. Using a proteomics approach, we identified 141 protein components of Escherichia coli‐derived native OMVs with high confidence; two separate analyses yielded identifications of 104 and 117 proteins, respectively, with 80 proteins overlapping between the two trials. In the group of identified proteins, the outer membrane proteins were highly enriched, whereas inner membrane proteins were lacking, suggesting that a specific sorting mechanism for vesicular proteins exists. We also identified proteins involved in vesicle formation, the removal of toxic compounds and attacking phage, and the elimination of competing organisms, as well as those involved in facilitating the transfer of genetic material and protein to other bacteria, targeting host cells, and modulating host immune responses. This study provides a global view of native bacterial OMVs. This information will help us not only to elucidate the biogenesis and functions of OMV from nonpathogenic and pathogenic bacteria but also to develop vaccines and antibiotics effective against pathogenic strains.
The N-glycosylation of proteins is one of the most important post-translational modifications relevant to various biological functions. The identification and quantification of N-glycoproteins in ...liquid chromatography–mass spectrometry (LC-MS) is challenging because of their low analytical sensitivity and selectivity. This is due to their microheterogeneity and the difficulty of synthesizing N-glycopeptides as an internal standard. Parallel reaction monitoring (PRM) is widely used in targeted LC-MS. The key advantage of LC-PRM is that it can identify N-glycopeptides using tandem mass spectrometry (MS/MS) fragmentation, even without an internal standard. We investigated the feasibility of analyzing N-glycoproteins using multiplex immunoprecipitation to improve sensitivity and selectivity. We targeted N-glycoproteins α-fetoprotein (AFP), vitronectin (VTN), and α-1-antichymotrypsin (AACT) that are abnormally glycosylated in hepatocellular carcinoma (HCC). Their tryptic N-glycopeptides were selected to determine the percentages of fucosylated N-glycopeptides using Y ions, which include glycopeptide fragments with amino acid sequences. Finally, we confirmed that the area under the receiver operating characteristic curve (AUC = 0.944) for the combination of AFP and VTN increased more so than for a single glycopeptide (AUC = 0.889 for AFP and 0.792 for VTN) with respect to discriminating between HCC and cirrhosis serum. This study shows that an LC-PRM method using multiplex N-glycoproteins immunoprecipitated from serum could be applied to develop and verify cancer biomarkers.
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Lichen sclerosus is a chronic inflammatory dermatosis presenting with significant sclerosis, atrophy and pruritus. The treatment for this condition remains unsatisfactory, with potent corticosteroids ...being the most effective therapy. In this study, we investigated the efficacy and safety of tacrolimus ointment in patients with genital and extragenital lichen sclerosus. Sixteen patients with active lichen sclerosus (10 with anogenital and six with extragenital localization) were treated with topical tacrolimus ointment twice daily. The therapeutic effects were evaluated according to 3 grades: complete response (>75% improvement), partial response (25–75% improvement), or no response (<25% improvement). Applications were continued until complete disappearance or stabilization of the cutaneous lesions. In addition, we conducted telephone surveys to determine the long‐term treatment outcome and relapse rate. Objective response to therapy occurred in nine of 10 patients (90%) with anogenital and one of six patients (16.7%) with extragenital lesions. Out of 10 patients with anogenital lichen sclerosus, five showed more than 75% improvement. Complete, partial and no response were achieved in five (50%), four (40%) and one (10%) patient, respectively. During the follow‐up period of a mean of 29.3 months, six of nine patients had a relapse of symptoms. However, most patients with extragenital involvement did not respond to tacrolimus, except one patient showing partial response. No significant adverse effects were observed. Topical tacrolimus ointment was a safe and effective treatment for genital lichen sclerosus and should be used for long‐term duration to prevent relapse. However, it was not useful for patients with extragenital lichen sclerosus.
Microvesicles (MV) are membrane vesicles secreted from the plasma and endosomal membrane compartment by various cell types such as hematopoietic, epithelial, and tumor cells. Actively growing tumor ...cells shed MV, and the rate of shedding increases in malignant tumors. Although recent progress in this area has revealed that tumor-derived MV play multiple roles in tumor growth and metastasis via immune escape, tumor invasion, and angiogenesis, the mechanism of vesicle formation and the biological roles of tumor-derived MV are not understood. Here, we report the first global proteomic analysis of highly purified MV from human colorectal cancer cells. Using 1D SDS gel electrophoresis and nano-LC–MS/MS analyses, we identified a total of 547 microvesicular proteins from three independent experiments with high confidence; 416 proteins were identified at least in two trials, including 181 as yet unreported proteins. We identified 49 proteins involved in the biogenesis of MV, including annexins, ADP-ribosylation factors, and Rab proteins. We also identified 28 proteins that may function in tumorigenesis via promotion of migration, invasion, and growth of tumor cells, immune modulation, metastasis, and angiogenesis. Taken together with previously reported results, our observations suggest that tumor-derived MV may act as communicasomes, that is, extracellular organelles that play diverse roles in intercellular communication. This information will help elucidate the biogenesis and functions of tumor-derived MV, and aid in the development of effective vaccines for various cancers, including colorectal cancer.
Rosacea is a common inflammatory skin disorder; the pathogenesis is unclear. Various treatment options for rosacea are available, but most have limited effectiveness. The aim of this study was to ...investigate the efficacy and safety of 1% pimecrolimus cream for the treatment of rosacea. Thirty patients with rosacea were enrolled in this 4‐week, single‐center, open‐label study of 1% pimecrolimus cream. Patients were instructed to apply the cream to their faces twice daily and were not permitted to use any other agents. Clinical efficacy was evaluated by a rosacea grading system using photographic documentation and a mexameter. The 26 patients who completed the study experienced significantly reduced rosacea clinical scores from 9.65 ± 1.79 at baseline to 7.27 ± 2.11 at the end of treatment (P < 0.05). The mexameter‐measured erythema index decreased significantly from 418.54 ± 89.56 at baseline to 382.23 ± 80.04 at week 4 (P < 0.05). The side‐effects were mostly transient local irritations. The results of this study suggest that 1% pimecrolimus cream is an effective and well‐tolerated treatment for patients with mild to moderate inflammatory rosacea.
