FAX-RIC enables robust profiling of dynamic RNP complex formation in multicellular organisms in vivo.

RNA-protein interaction is central to post-transcriptional gene regulation. Identification of RNA-binding proteins relies mainly on UV-induced crosslinking (UVX) followed by the enrichment of RNA-protein conjugates and LC-MS/MS analysis. However, UVX has limited applicability in tissues of multicellular organisms due to its low penetration depth. Here, we introduce formaldehyde crosslinking (FAX) as an alternative chemical crosslinking for RNA interactome capture (RIC). Mild FAX captures RNA-protein interaction with high specificity and efficiency in cell culture. Unlike UVX-RIC, FAX-RIC robustly detects proteins that bind to structured RNAs or uracil-poor RNAs (e.g. AGO1, STAU1, UPF1, NCBP2, EIF4E, YTHDF proteins and PABP), broadening the coverage. Applied to Xenopus laevis oocytes and embryos, FAX-RIC provided comprehensive and unbiased RNA interactome, revealing dynamic remodeling of RNA-protein complexes. Notably, translation machinery changes during oocyte-to-embryo transition, for instance, from canonical eIF4E to noncanonical eIF4E3. Furthermore, using Mus musculus liver, we demonstrate that FAX-RIC is applicable to mammalian tissue samples. Taken together, we report that FAX can extend the RNA interactome profiling into multicellular organisms.

Integrated Analysis of Transcriptome and Proteome of the Human Cornea and Aqueous Humor Reveal Novel Biomarkers for Corneal Endothelial Cell Dysfunction.

Earlier studies have reported that elevated protein levels in the aqueous humor (AH) are associated with corneal endothelial cell dysfunction (CECD), but the details of the underlying mechanism as well as specific biomarkers for CECD remain elusive. In the present study, we aimed to identify protein markers in AH directly associated with changes to corneal endothelial cells (CECs), as AH can be easily obtained for analysis. We carried out an in-depth proteomic analysis of patient-derived AH as well as transcriptomic analysis of CECs from the same patients with bullous keratopathy (BK) resulting from CECD. We first determined differentially expressed genes (DEGs) and differentially expressed proteins (DEPs) from CECs and AH in CECD, respectively. By combining transcriptomic and proteomic analyses, 13 shared upregulated markers and 22 shared downregulated markers were observed between DEGs and DEPs. Among these 35 candidates from biomarker profiling, three upregulated markers were finally verified via data-independent acquisition (DIA) proteomic analysis using additional individual AH samples, namely metallopeptidase inhibitor 1 (TIMP1), Fc fragment of IgG binding protein (FCGBP), and angiopoietin-related protein 7 (ANGPTL7). Furthermore, we confirmed these AH biomarkers for CECD using individual immunoassay validation. Conclusively, our findings may provide valuable insights into the disease process and identify biofluid markers for the assessment of CEC function during BK development.

Proteomic analysis of human synovial fluid reveals potential diagnostic biomarkers for ankylosing spondylitis.

BACKGROUND: Ankylosing spondylitis (AS) is a chronic inflammatory rheumatic disease affecting the axial skeleton and peripheral joints. The etiology of this disease remains poorly understood, but interactions between genetic and environmental factors have been implicated. The present study identified differentially expressed proteins in the synovial fluid (SF) of AS patients to elucidate the underlying cause of AS. METHODS: A cohort of 40 SF samples from 10 AS and 10 each of rheumatoid arthritis (RA), gout, and osteoarthritis (OA) patients were analyzed by liquid chromatography tandem mass spectrometry (LC-MS/MS) to identify differentially expressed proteins specific to AS. The label-free LC-MS/MS results were verified by western blotting. RESULTS: We identified 8 proteins that were > 1.5-fold upregulated in the SF of AS patients compared to that of the disease control groups, including HP, MMP1, MMP3, serum amyloid P-component (APCS), complement factor H-related protein 5 (CFHR5), mannose-binding lectin 2 (MBL2), complement component C9 (C9), and complement C4-A (C4A). CFHR5 and C9 were previously found in serum from AS patients, while APCS was previously found in SF as well as in serum. However, the present study has identified C4A, and MBL2 as potential AS biomarkers for the first time. The expression levels of MMP3, C9, and CFHR5 were verified in AS SF using western blotting. CONCLUSION: We performed quantitative comparative proteomic analysis using by LC-MS/MS of the SF from four disease states: RA, gout, and OA. This systematic comparison revealed novel differentially expressed proteins in AS SF, as well as two previously reported candidate biomarkers. We further verified the expression of MMP3, C9 and CFHR5 by western blot. These proteins may serve as diagnostic or prognostic biomarkers in patients with AS, and may thus improve the clinical outcomes of this serious disease.

Orthogonal lipid sensors identify transbilayer asymmetry of plasma membrane cholesterol.

Controlled distribution of lipids across various cell membranes is crucial for cell homeostasis and regulation. We developed an imaging method that allows simultaneous in situ quantification of cholesterol in two leaflets of the plasma membrane (PM) using tunable orthogonal cholesterol sensors. Our imaging revealed marked transbilayer asymmetry of PM cholesterol (TAPMC) in various mammalian cells, with the concentration in the inner leaflet (IPM) being ∼12-fold lower than that in the outer leaflet (OPM). The asymmetry was maintained by active transport of cholesterol from IPM to OPM and its chemical retention at OPM. Furthermore, the increase in the IPM cholesterol level was triggered in a stimulus-specific manner, allowing cholesterol to serve as a signaling lipid. We found excellent correlation between the IPM cholesterol level and cellular Wnt signaling activity, suggesting that TAPMC and stimulus-induced PM cholesterol redistribution are crucial for tight regulation of cellular processes under physiological conditions.

Effect of Cyclophilin from Pyropia Yezoensis on the Proliferation of Intestinal Epithelial Cells by Epidermal Growth Factor Receptor/Ras Signaling Pathway.

Cyclophilin (Cyp) is peptidyl-prolyl isomerase (PPIase), and it has many biological functions, including immune response regulation, antioxidants, etc. Cyp from red algae is known for its antioxidant and antifungal activity. However, the other biological effects of Cyp from Pyropia yezoensis are unclear. In this study, we synthesized Cyp from P. yezoensis (pyCyp) and examined its biological activity on IEC-6 cells. First, the MTS assay showed that pyCyp increased cell proliferation in a dose-dependent manner. pyCyp activated the EGFR signaling pathway that regulates cell growth, proliferation, and survival. It induced intracellular signaling pathways, including the Ras signaling pathway. In addition, we observed cell cycle-related proteins. pyCyp increased the expression of cyclin A, cyclin E, and Cdk2, and decreased the expression of p27 and p21 proteins. These results indicate that pyCyp stimulates cell proliferation via the EGFR signaling pathway and promotes cell cycle progression in intestinal epithelial cells. Therefore, we suggest pyCyp as a potential material to promote the proliferation of intestinal epithelial cells.

Discovery of gastric cancer specific biomarkers by the application of serum proteomics.

Current diagnostic markers for gastric cancer are not sufficiently specific or sensitive for use in clinical practice. The aims of this study are to compare the proteomes of serum samples from patients with gastric cancers and normal controls, and to develop useful tumor markers of gastric cancer by quantitative proteomic analysis. We identified a total of 388 proteins with a ≤1% FDR and with at least two unique peptides from the sera of each group. Among them, 215, 251, and 260 proteins were identified in serum samples of patients in an advanced cancer group, early cancer group, and normal control group, respectively. We selected differentially expressed proteins in cancer patients compared with those of normal controls via semiquantitative analyses comparing the spectral counts of identified proteins. These differentially expressed proteins were successfully verified using an MS-based quantitative assay, multiple reactions monitoring analysis. Four proteins (vitronectin, clusterin isoform 1, thrombospondin 1, and tyrosine-protein kinase SRMS) were shown to have significant changes between the cancer groups and the normal control group. These four serum proteins were able to discriminate gastric cancer patients from normal controls with sufficient specificity and selectivity.

Proteomic Analysis Reveals Distinct Metabolic Differences Between Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF) and Macrophage Colony Stimulating Factor (M-CSF) Grown Macrophages Derived from Murine Bone Marrow Cells.

Macrophages are crucial in controlling infectious agents and tissue homeostasis. Macrophages require a wide range of functional capabilities in order to fulfill distinct roles in our body, one being rapid and robust immune responses. To gain insight into macrophage plasticity and the key regulatory protein networks governing their specific functions, we performed quantitative analyses of the proteome and phosphoproteome of murine primary GM-CSF and M-CSF grown bone marrow derived macrophages (GM-BMMs and M-BMMs, respectively) using the latest isobaric tag based tandem mass tag (TMT) labeling and liquid chromatography-tandem mass spectrometry (LC-MS/MS). Strikingly, metabolic processes emerged as a major difference between these macrophages. Specifically, GM-BMMs show significant enrichment of proteins involving glycolysis, the mevalonate pathway, and nitrogen compound biosynthesis. This evidence of enhanced glycolytic capability in GM-BMMs is particularly significant regarding their pro-inflammatory responses, because increased production of cytokines upon LPS stimulation in GM-BMMs depends on their acute glycolytic capacity. In contrast, M-BMMs up-regulate proteins involved in endocytosis, which correlates with a tendency toward homeostatic functions such as scavenging cellular debris. Together, our data describes a proteomic network that underlies the pro-inflammatory actions of GM-BMMs as well as the homeostatic functions of M-BMMs.

Global changes of phospholipids identified by MALDI imaging mass spectrometry in a mouse model of Alzheimer's disease.

Alzheimer's disease (AD) is the most common form of dementia; however, at the present time there is no disease-modifying drug for AD. There is increasing evidence supporting the role of lipid changes in the process of normal cognitive aging and in the etiology of age-related neurodegenerative diseases. AD is characterized by the presence of intraneuronal protein clusters and extracellular aggregates of ß-amyloid (Aß). Disrupted Aß kinetics may activate intracellular signaling pathways, including tau hyperphosphorylation and proinflammatory pathways. We analyzed and visualized the lipid profiles of mouse brains using MALDI-TOF MS. Direct tissue analysis by MALDI-TOF imaging MS (IMS) can determine the relative abundance and spatial distribution of specific lipids in different tissues. We used 5XFAD mice that almost exclusively generate and rapidly accumulate massive cerebral levels of Aß-42 (1). Our data showed changes in lipid distribution in the mouse frontal cortex, hippocampus, and subiculum, where Aß plaques are first generated in AD. Our results suggest that MALDI-IMS is a powerful tool for analyzing the distribution of various phospholipids and that this application might provide novel insight into the prediction of disease.

Phospholipids of tumor extracellular vesicles stratify gefitinib-resistant nonsmall cell lung cancer cells from gefitinib-sensitive cells.

Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) such as gefitinib are one of gold standard treatment options for nonsmall-cell lung cancer (NSCLC) patients, which eventually fail due to the acquired resistance and relapse because of the development of secondary activating mutations such as T790M in EGFR. Predicting chemo-responsiveness of cancer patients provides a major challenge in chemotherapy. The goal of the present study is to determine whether phospholipid signatures of tumor extracellular vesicles (EV) are associated with gefitinib-resistance of NSCLC. A sophisticated MS-based shotgun lipidomic assays were performed for in-depth analysis of the lipidomes of gefitinib-resistant (PC9R) and responsive (PC9) NSCLC cells and their shed EV from these cell lines (PC9EV or PC9REV). Lipid MALDI-MS analysis showed that EV phospholipid composition was significantly distinct in PC9R, compared to PC9 cells. Following statistical analyses has identified 35 (20 positive and 15 negative ion mode) differentially regulated lipids, which are significantly over- or underexpressed in PC9R EV, compared to PC9 EV (p value < 0.01, fold change > 1.5). Our phospholipid signatures suggest that EV associates with drug sensitivity, which is worthy of additional investigation to assess chemoresistance in patients with NSCLC treated with anti-EGFR TKIs.

Extracellular vesicles shed from gefitinib-resistant nonsmall cell lung cancer regulate the tumor microenvironment.

Epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitors (TKIs), including gefitinib, are the first-line treatment of choice for nonsmall cell lung cancer patients who harbor activating EGFR mutations, however, acquired resistance to EGFR-TKIs is inevitable. The main objective of this study was to identify informative protein signatures of extracellular vesicles (EV) derived from gefitinib-resistant nonsmall cell lung cancer cells using proteomics analysis. Nano-LC-MS/MS analysis identified with high confidence (false discovery rate < 0.05, fold change ≥2) 664 EV proteins enriched in PC9R cells, which are resistant to gefitinib due to EGFR T790M mutation. Computational analyses suggested components of several signal transduction mechanisms including the AKT (also PKB, protein kinase B)/mTOR (mechanistic target of rapamycin) pathway are overrepresented in EV from PC9R cells. Treatment of recipient cells with EV harvested from PC9R cells increased phosphorylation of signaling molecules, and enhanced proliferation, invasion, and drug resistance to gefitinib-induced apoptosis. Dose- and time-dependent pharmaceutical inhibition of AKT/mTOR pathway overcame drug resistance of PC9R cells and those of H1975 exhibiting EGFR T790M mutation. Our findings provide new insight into an oncogenic EV protein signature regulating tumor microenvironment, and will aid in the development of novel diagnostic strategies for prediction and assessment of gefitinib resistance.

Identification and characterization of proteins isolated from microvesicles derived from human lung cancer pleural effusions.

Microvesicles (MVs, also known as exosomes, ectosomes, microparticles) are released by various cancer cells, including lung, colorectal, and prostate carcinoma cells. MVs released from tumor cells and other sources accumulate in the circulation and in pleural effusion. Although recent studies have shown that MVs play multiple roles in tumor progression, the potential pathological roles of MV in pleural effusion, and their protein composition, are still unknown. In this study, we report the first global proteomic analysis of highly purified MVs derived from human nonsmall cell lung cancer (NSCLC) pleural effusion. Using nano-LC-MS/MS following 1D SDS-PAGE separation, we identified a total of 912 MV proteins with high confidence. Three independent experiments on three patients showed that MV proteins from PE were distinct from MV obtained from other malignancies. Bioinformatics analyses of the MS data identified pathologically relevant proteins and potential diagnostic makers for NSCLC, including lung-enriched surface antigens and proteins related to epidermal growth factor receptor signaling. These findings provide new insight into the diverse functions of MVs in cancer progression and will aid in the development of novel diagnostic tools for NSCLC.

Evaluation of the Microbiological Status of Cattle Carcasses in Mongolia: Considering the Hygienic Practices of Slaughter Establishments.

The meat industry has received great attention in Mongolia, having over 70 million livestock, and is important to the nation's economy. Systematic microbiological testing of carcasses has not been mandatorily regulated in all abattoir premises, and the efficacy of the introduction of the Good Hygiene Practice and Hazard Analysis Critical Control Points (HACCP) to some plants has not yet been tested microbiologically in Mongolia. Therefore, samples were collected from two establishments: plant A with an HACCP certificate from a third party and plant B without an HACCP certificate. The rates and levels of the total bacterial count (TBC) as overall hygiene indicators, the Enterobacteriaceae count (EBC) as fecal contamination indicators, and the Staphylococcus spp. count (SC) as personal hygiene indicators were determined on different parts of beef carcasses. The contamination rates in most parts were lower in plant A than in plant B (e.g., TBC in the rump and flank: 103-105 and 105-107, in plant A vs. 104-106 and 105-108 in plant B, respectively). Plant A also had a lower EBC and SC (p < 0.001). Furthermore, 2 out of 100 beef carcasses (2%) were positive for enterohemorrhagic Escherichia coli as a foodborne pathogen indicator in plant A.

Characterization of Lipid Alterations by Oncogenic PIK3CA Mutations Using Untargeted Lipidomics in Breast Cancer.

Lipids play crucial biological roles in health and disease, including in cancers. The phosphatidylinositol 3-kinase (PI3K) signaling pathway is a pivotal promoter of cell growth and proliferation in various types of cancer. The somatic mutations in PIK3CA, the gene coding for the catalytic subunit p110α of PI3K, are frequently present in cancer cells, including breast cancer. Although the most prominent mutants, represented by single amino acid substitutions in the helical domain in exon 9 (E545K) and the kinase domain in exon 20 (H1047R) are known to cause a gain of PI3K function, activate AKT signaling and induce oncogenic transformation, the effect of these mutations on cellular lipid profiles has not been studied. We carried out untargeted lipidomics using liquid chromatography-tandem mass spectrometry to detect the lipid alterations in mammary gland epithelial MCF10A cells with isogenic knockin of these mutations. A total of 536 species of lipids were analyzed. We found that the levels of monosialogangliosides, signaling molecules known to enhance cell motility through PI3K/AKT pathway, were significantly higher in both mutants. In addition, triglycerides and ceramides, lipid molecules known to be involved in promoting lipid droplet production, cancer cell migration and invasion, were increased, whereas lysophosphatidylcholines and phosphatidylcholines that are known to inhibit cancer cell motility were decreased in both mutants. Our results provide novel insights into a potential link between altered lipid profile and carcinogenesis caused by the PIK3CA hotspot mutations. In addition, we suggest untargeted lipidomics offers prospects for precision/personalized medicine by unpacking new molecular substrates of cancer biology.

Improvement of optical extraction efficiency of organic light-emitting diode using a large area nano-pattern.

In order to improve optical extraction efficiencies, we propose a nano-patterned organic light-emitting diode (OLED) which doesn't rely on high precision lithography nor rigorous periodicity. The nano-pattern is fabricated by spin-coating PS (poly-styrene) spheres on the substrate and carrying out reactive ion etching and flattening using dielectric material. The verification of the optical properties of the nano patterns was carried out by fabricating OLED-like structures using quantum dot and distributed Bragg reflector (DBR). As a result, the nano-patterned structure showed a 28% increase in optical efficiency compared to the non-patterned sample. In addition, the use of a prism sheet on the backside of a glass substrate also showed a 23% increase in optical efficiency by disturbing the total internal reflection between glass and air. In this way, the large area nano-patterns can be fabricated and applied to increasing the optical extraction in OLED.

Synthesis of Thermoplastic Polyurethanes Containing Bio-Based Polyester Polyol and Their Fiber Property.

Among the starting materials of thermoplastic polyurethanes (TPUs), it was confirmed that succinic acid-based polyester biopolyols having different molecular weights (Mn = 1000, 2000, and 4000) affect the physicochemical properties of the final polymer significantly. Bio-TPUs synthesized through a solvent-free one-shot polymerization process were synthesized with a polyester polyol, 1,4 butanediol (BDO), and 4,4'-methylene diphenyl diisocyanate (MDI) in a molar ratio of 1:1:2. As a control group, one typical petroleum-based TPU was synthesized and characterized along with other bio-based TPUs. Representative petroleum-based and bio-based TPUs synthesized were manufactured as monofilaments with a diameter of about 0.2 mm through an extrusion process with different draw ratios (4, 5, and 6 times). The molecular weight and structural properties of the TPUs were characterized by GPC and FT-IR analysis and thermal characterization by DSC and TGA analysis. Petroleum-based TPU and bio-based TPU having the same molecular weight soft segment (SS) tended to have similar molecular weight and hard segment (HS) content. TPUs with high HS content had excellent thermal stability, enabling stable extrusion of TPUs. In addition, it was confirmed that the bio-based TPU fibers produced in this way had a tensile strength corresponding to the physical properties of petroleum-based TPU fibers and an excellent elastic recovery rate of almost 100 %. These results indicate the application potential of bio-TPU.

Emerging role of LETM1/GRP78 axis in lung cancer.

The selective autophagy of damaged mitochondria is called mitophagy. Mitochondrial dysfunction, mitophagy, and apoptosis have been suggested to be interrelated in various human lung carcinomas. Leucine zipper EF-hand-containing transmembrane protein-1 (LETM1) was cloned in an attempt to identify candidate genes for Wolf-Hirschhorn syndrome. LETM1 plays a role in mitochondrial morphology, ion homeostasis, and cell viability. LETM1 has also been shown to be overexpressed in different human cancer tissues, including lung cancer. In the current study, we have provided clear evidence that LETM1 acts as an anchoring protein for the mitochondria-associated ER membrane (MAM). Fragmented mitochondria have been found in lung cancer cells with LETM1 overexpression. In addition, a reduction of mitochondrial membrane potential and significant accumulation of microtubule-associated protein 1 A/1B-light chain 3 punctate, which localizes with Red-Mito, was found in LETM1-overexpressed cells, suggesting that mitophagy is upregulated in these cells. Interestingly, glucose-regulated protein 78 kDa (GRP78; an ER chaperon protein) and glucose-regulated protein 75 kDa (GRP75) were posited to interact with LETM1 in the immunoprecipitated LETM1 of H460 cells. This interaction was enhanced in cells treated with carbonyl cyanide m-chlorophenylhydrazone, a chemical mitophagy inducer. Treatment of cells with honokiol (a GRP78 inhibitor) blocked LETM1-mediated mitophagy, and CRISPR/Cas9-mediated GRP75 knockout inhibited LETM1-induced autophagy. Thus, GRP78 interacts with LETM1. Taken together, these observations support the notion that the complex formation of LETM1/GRP75/GRP78 might be an important step in MAM formation and mitophagy, thus regulating mitochondrial quality control in lung cancer.

Tissue extracellular matrix hydrogels as alternatives to Matrigel for culturing gastrointestinal organoids.

Matrigel, a mouse tumor extracellular matrix protein mixture, is an indispensable component of most organoid tissue culture. However, it has limited the utility of organoids for drug development and regenerative medicine due to its tumor-derived origin, batch-to-batch variation, high cost, and safety issues. Here, we demonstrate that gastrointestinal tissue-derived extracellular matrix hydrogels are suitable substitutes for Matrigel in gastrointestinal organoid culture. We found that the development and function of gastric or intestinal organoids grown in tissue extracellular matrix hydrogels are comparable or often superior to those in Matrigel. In addition, gastrointestinal extracellular matrix hydrogels enabled long-term subculture and transplantation of organoids by providing gastrointestinal tissue-mimetic microenvironments. Tissue-specific and age-related extracellular matrix profiles that affect organoid development were also elucidated through proteomic analysis. Together, our results suggest that extracellular matrix hydrogels derived from decellularized gastrointestinal tissues are effective alternatives to the current gold standard, Matrigel, and produce organoids suitable for gastrointestinal disease modeling, drug development, and tissue regeneration.

FCH domain only 1 (FCHo1), a potential new biomarker for lung cancer.

Lung carcinoma is the main reason for cancer-associated deaths in the world. In a previous study, FCH domain only 1 (FCHo1) which is managed by protein kinase B (AKT), was shown to be activated in lung cancer. FCHo1 knockdown has previously been shown to cause cell death in lung cancer. However, the specific roles of FCHo1 in lung carcinoma remain elusive. Herein, we propose that FCHo1's intracellular mechanism targets the G1 to S phase transition, following the M phase. We demonstrated that F-BAR and mu homology domains exist separately in human lung tissues and that one truncated form is not detected in patients with lung cancer. Furthermore, quantitative global proteome analysis of FCHo1 indicated that the inhibition of G1/S phase transition and FCHo1 RNAi led to the death of cells in the G1/S phase. Noninvasive viral aerosol-mediated delivery of FCHo1 shRNA suppressed cancer progression in mice with non-small-cell lung cancer (NSCLC), suggesting that the delivery of FCHo1 shRNA could be a meaningful therapeutic strategy in lung cancer. Additional studies are needed to make clear the detailed mechanism of action of FCHo1.