CHTOP in Chemoresistant Epithelial Ovarian Cancer: A Novel and Potential Therapeutic Target.

Objective: Chemoresistance is a major challenge in epithelial ovarian cancer (EOC) treatment. Chromatin target of protein arginine methyltransferase (CHTOP) was identified as a potential biomarker in chemoresistant EOC cell lines using label-free LC-MS/MS quantitative proteomics. Thus, the aim of this study is to investigate the role of CHTOP in chemoresistant EOC and the underlying mechanism. Methods: The expression of CHTOP in human ovarian cancer cells and tissues was detected using immunofluorescence (IF), western blot (WB), and immunohistochemistry (IHC), respectively. Flow cytometry and TUNEL assay were employed to detect the effect of CHTOP knockdown (KD) in chemoresistant EOC cell apoptosis, while colony and sphere formation assays were used to evaluate its effect on cell stemness. The association of CHTOP with cell metastasis was determined using Matrigel invasion and wound-healing assays. Results: The higher level expression of CHTOP protein was found in chemoresistant EOC cells as compared to their sensitive parental cells or normal epithelial ovarian cells. Results from IHC and bioinformatic analysis showed CHTOP was highly expressed in human ovarian cancer tissues and associated with a poor progression-free survival in patients. In addition, CHTOP KD significantly enhanced cisplatin-induced apoptosis, reduced the stemness of chemoresistant EOC cells, and decreased their metastatic potential. Conclusion: Our findings suggest that CHTOP is associated with apoptosis, stemness, and metastasis in chemoresistant EOC cells and might be a promising target to overcome chemoresistance in EOC treatment

Activation of the eIF2α/ATF4 axis drives triple-negative breast cancer radioresistance by promoting glutathione biosynthesis.

Triple-negative breast cancer (TNBC) is the most aggressive breast cancer subtype. Radiotherapy is an effective option for the treatment of TNBC; however, acquired radioresistance is a major challenge to the modality. In this study, we show that the integrated stress response (ISR) is the most activated signaling pathway in radioresistant TNBC cells. The constitutive phosphorylation of eIF2α in radioresistant TNBC cells promotes the activation of ATF4 and elicits the transcription of genes implicated in glutathione biosynthesis, including GCLC, SLC7A11, and CTH, which increases the intracellular level of reduced glutathione (GSH) and the scavenging of reactive oxygen species (ROS) after irradiation (IR), leading to a radioresistant phenotype. The cascade is significantly up-regulated in human TNBC tissues and is associated with unfavorable survival in patients. Dephosphorylation of eIF2α increases IR-induced ROS accumulation in radioresistant TNBC cells by disrupting ATF4-mediated GSH biosynthesis and sensitizes them to IR in vitro and in vivo. These findings reveal ISR as a vital mechanism underlying TNBC radioresistance and propose the eIF2α/ATF4 axis as a novel therapeutic target for TNBC treatment

A Genome-Scale Metabolic Reconstruction of Mycoplasma genitalium, iPS189

With a genome size of ∼580 kb and approximately 480 protein coding regions, Mycoplasma genitalium is one of the smallest known self-replicating organisms and, additionally, has extremely fastidious nutrient requirements. The reduced genomic content of M. genitalium has led researchers to suggest that the molecular assembly contained in this organism may be a close approximation to the minimal set of genes required for bacterial growth. Here, we introduce a systematic approach for the construction and curation of a genome-scale in silico metabolic model for M. genitalium. Key challenges included estimation of biomass composition, handling of enzymes with broad specificities, and the lack of a defined medium. Computational tools were subsequently employed to identify and resolve connectivity gaps in the model as well as growth prediction inconsistencies with gene essentiality experimental data. The curated model, M. genitalium iPS189 (262 reactions, 274 metabolites), is 87% accurate in recapitulating in vivo gene essentiality results for M. genitalium. Approaches and tools described herein provide a roadmap for the automated construction of in silico metabolic models of other organisms

Proteomics Characterization of Cytoplasmic and Lipid-Associated Membrane Proteins of Human Pathogen Mycoplasma fermentans M64

Mycoplasma fermentans is a potent human pathogen which has been implicated in several diseases. Notably, its lipid-associated membrane proteins (LAMPs) play a role in immunomodulation and development of infection-associated inflammatory diseases. However, the systematic protein identification of pathogenic M. fermentans has not been reported. From our recent sequencing results of M. fermentans M64 isolated from human respiratory tract, its genome is around 1.1 Mb and encodes 1050 predicted protein-coding genes. In the present study, soluble proteome of M. fermentans was resolved and analyzed using two-dimensional gel electrophoresis. In addition, Triton X-114 extraction was carried out to enrich amphiphilic proteins including putative lipoproteins and membrane proteins. Subsequent mass spectrometric analyses of these proteins had identified a total of 181 M. fermentans ORFs. Further bioinformatics analysis of these ORFs encoding proteins with known or so far unknown orthologues among bacteria revealed that a total of 131 proteins are homologous to known proteins, 11 proteins are conserved hypothetical proteins, and the remaining 39 proteins are likely M. fermentans-specific proteins. Moreover, Triton X-114-enriched fraction was shown to activate NF-kB activity of raw264.7 macrophage and a total of 21 lipoproteins with predicted signal peptide were identified therefrom. Together, our work provides the first proteome reference map of M. fermentans as well as several putative virulence-associated proteins as diagnostic markers or vaccine candidates for further functional study of this human pathogen

The Current State of Proteomics in GI Oncology

Proteomics refers to the study of the entire set of proteins in a given cell or tissue. With the extensive development of protein separation, mass spectrometry, and bioinformatics technologies, clinical proteomics has shown its potential as a powerful approach for biomarker discovery, particularly in the area of oncology. More than 130 exploratory studies have defined candidate markers in serum, gastrointestinal (GI) fluids, or cancer tissue. In this article, we introduce the commonly adopted proteomic technologies and describe results of a comprehensive review of studies that have applied these technologies to GI oncology, with a particular emphasis on developments in the last 3 years. We discuss reasons why the more than 130 studies to date have had little discernible clinical impact, and we outline steps that may allow proteomics to realize its promise for early detection of disease, monitoring of disease recurrence, and identification of targets for individualized therapy

In vivo localization of antibodies raised against Eimeria maxima wall forming bodies during sexual intracellular development

Copyright © 2014 Cambridge University Press. Apicomplexan parasites cause devastating diseases in humans and livestock. Previously we demonstrated that antibodies targeting transmissible forms of the apicomplexan parasite, Eimeria, are effective at reducing parasite shedding thus preventing the transmission of the disease. However, the mechanisms responsible have not been fully defined. Moreover, there is no direct evidence that the parasite-specific IgG antibodies can reach the parasite developing in the enterocytes of the infected chicken host. This study summarizes our efforts using host immunity, parasite proteomics and 3D microscopy to provide a step forward in our understanding of how this immune response works. Eimeria maxima is an important pathogen of poultry and used as a surrogate for a number of human pathogens including Toxoplasma and Plasmodium. Our studies demonstrate that immunization with the purified wall forming bodies (WFBs) results in a production of parasite-specific IgG antibodies, which have the ability to reach in situ gametocytes in the intestinal lumen and permeate the enterocyte/parasite membranes in order to bind to the cytoplasmic Type 1 and Type 2 WFBs. This raises the intriguing possibility that via this process antibodies block the development of Eimeria maxima in vivo

Preliminary identifcation of differentially expressed tear proteins in keratoconus

Purpose: To examine the proteins differentially expressed in the tear flm of people with keratoconus and normal subjects. Methods: Unstimulated tears from people with keratoconus (KC) and controls (C) were collected using a capillary tube. Tear proteins from people with KC and controls were partitioned using a novel in-solution electrophoresis, Microfow 10 (ProteomeSep), and analyzed using linear ion trap quadrupole fourier transform mass spectrometry. Spectral counting was used to quantify the individual tear proteins. Results: Elevated levels of cathepsin B (threefold) were evident in the tears of people with KC. Polymeric immunoglobu-lin receptor (ninefold), fbrinogen alpha chain (eightfold), cystatin S (twofold), and cystatin SN (twofold) were reduced in tears from people with KC. Keratin type-1 cytoskeletal-14 and keratin type-2 cytoskeletal-5 were present only in the tears of people with KC. Conclusions: The protein changes in tears, that is, the decrease in protease inhibitors and increase in proteases, found in the present and other previously published studies refect the pathological events involved in KC corneas. Further investigations into tear proteins may help elucidate the underlying molecular mechanisms of KC, which could result in better treatment options. © 2013 Molecular Vision

A standardized and reproducible urine preparation protocol for cancer biomarkers discovery.

A suitable and standardized protein purification technique is essential to maintain consistency and to allow data comparison between proteomic studies for urine biomarker discovery. Ultimately, efforts should be made to standardize urine preparation protocols. The aim of this study was to develop an optimal analytical protocol to achieve maximal protein yield and to ensure that this method was applicable to examine urine protein patterns that distinguish disease and disease-free states. In this pilot study, we compared seven different urine sample preparation methods to remove salts, and to precipitate and isolate urinary proteins. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) profiles showed that the sequential preparation of urinary proteins by combining acetone and trichloroacetic acid (TCA) alongside high speed centrifugation (HSC) provided the best separation, and retained the most urinary proteins. Therefore, this approach is the preferred method for all further urine protein analysis