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.

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.

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.

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.

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.

Proteome alterations in the aqueous humor reflect structural and functional phenotypes in patients with advanced normal-tension glaucoma.

Previous reports have shown possible association between altered protein levels in aqueous humor (AH) and normal-tension glaucoma (NTG), but the underlying pathogenetic mechanism as well as specific molecular biomarkers for NTG remains still elusive. Here, we aimed to identify novel biomarkers for advanced NTG by analyzing the proteome of patient-derived AH and their correlation with various functional and structural parameters from the visual field test (VF), optical coherence tomography (OCT), and OCT angiography (OCTA). We determined differentially expressed proteins (DEPs) of the AH of patients with advanced NTG (n = 20) using label-free quantitative (LFQ) proteomics with pooled samples and data-independent acquisition (DIA) analysis with individual samples, and the roles of AH DEPs in biological pathways were evaluated using bioinformatics. We identified 603 proteins in the AH of patients with advanced NTG, and 61 of them were selected as DEPs via global proteome LFQ profiling. Individual DIA analyses identified a total of 12 DEPs as biomarker candidates, seven of which were upregulated, and five were downregulated. Gene ontology enrichment analysis revealed that those DEPs were mainly involved in the immune response. Moreover, IGFBP2, ENO1, C7, B2M, AMBP, DSP, and DCD showed a significant correlation with the mean deviation of VF and with peripapillary and macular parameters from OCT and OCTA. The present study provides possible molecular biomarkers for the diagnosis of advanced NTG.

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.

Rapid Androgen-Responsive Proteome Is Involved in Prostate Cancer Progression.

Androgen exerts its functions by binding with an androgen receptor (AR). It can activate many signaling pathways that are important to the progression of castration-resistant prostate cancer (CRPC). Here, we characterized the rapid proteomic changes seen at 5, 15, 30, and 60 min after the androgen treatment of VCaP cells via the tandem mass tag (TMT) labeling strategy. A total of 5529 proteins were successfully identified and quantified. Dynamic time profiling of protein expression patterns allowed us to identify five protein clusters involved in various stages of androgen-initiated signal transmission and processing. More details of protein functions and localization patterns, and our elucidation of an AR-interacting protein network, were obtained. Finally, we validated the expression level of AR-regulated proteins known to be significantly regulated in CRPC patients using the mouse xenograft model and patient samples. Our work offers a systematic analysis of the rapid proteomic changes induced by androgen and provides a global view of the molecular mechanisms underlying CRPC progression.

Maternal serum lipidomics identifies lysophosphatidic acid as a predictor of small for gestational age neonates.

To discover lipidomic alterations during pregnancy in mothers who subsequently delivered small for gestational age (SGA) neonates and identify predictive lipid markers that can help recognize and manage these mothers, we carried out untargeted lipidomics on maternal serum samples collected between 24-28 weeks of gestation. We used a nested case-control study design and serum from mothers who delivered SGA and appropriate for gestational age babies. We applied untargeted lipidomics using mass spectrometry to characterize lipids and discover changes associated with SGA births during pregnancy. Multivariate pattern recognition software Collaborative Laboratory Integrated Reports (CLIR) was used for the post-analytical recognition of range differences in lipid ratios that could differentiate between SGA and control mothers and their integration for complete separation between the two groups. Here, we report changes in lipids from serum collected during pregnancy in mothers who delivered SGA neonates. In contrast to normal pregnancies where lysophosphatidic acid increased over the course of the pregnancy owing to increased activity of lysophospholipase D, we observed a decrease (32%; P = 0.05) of 20:4-lysophosphatidic acid in SGA mothers, which could potentially compromise fetal growth and development. Integration of lipid ratios in an interpretive tool (CLIR) could completely separate SGA mothers from controls demonstrating the power of untargeted lipidomic analyses for identifying novel predictive biomarkers. Additional studies are required for further assessment of the lipid biomarkers identified in this report.

Correlation between the Level of Social Distancing and Activity of Influenza Epidemic or COVID-19 Pandemic: A Subway Use-Based Assessment.

Social distancing is an effective measure to mitigate the spread of novel viral infections in the absence of antiviral agents and insufficient vaccine supplies. Subway utilization density may reflect social activity and the degree of social distancing in the general population.; This study aimed to evaluate the correlations between subway use density and the activity of the influenza epidemic or coronavirus disease 2019 (COVID-19) pandemic using a time-series regression method. The subway use-based social distancing score (S-SDS) was calculated using the weekly ridership of 11 major subway stations. The temporal association of S-SDS with influenza-like illness (ILI) rates or the COVID-19 pandemic activity was analyzed using structural vector autoregressive modeling and the Granger causality (GC) test. During three influenza seasons (2017-2020), the time-series regression presented a significant causality from S-SDS to ILI (p = 0.0484). During the COVID-19 pandemic in January 2020, S-SDS had been suppressed at a level similar to or below the average of the previous four years. In contrast to the ILI rate, there was a negative correlation between COVID-19 activity and S-SDS. GC analysis revealed a negative causal relationship between COVID-19 and S-SDS (p = 0.0098).; S-SDS showed a significant time-series association with the ILI rate but not with COVID-19 activity. When public transportation use is sufficiently suppressed, additional social mobility restrictions are unlikely to significantly affect COVID-19 pandemic activity. It would be more important to strengthen universal mask-wearing and detailed public health measures focused on risk activities, particularly in enclosed spaces.

Vitamin D Status and Factors Associated with Vitamin D Deficiency during the First Year of Life in Preterm Infants.

We aimed to investigate the changes in vitamin D levels and factors associated with vitamin D deficiency (VDD) during the first year of life in Korean preterm infants. We enrolled 333 preterm infants who were born at Kyungpook National University Children's Hospital between March 2013 and December 2019. 25-hydroxyvitamin D (25-OHD) levels and medical records were collected at birth, 6 months, and 12 months of age. The mean gestational age was 33.4 ± 2.3 weeks and mean 25-OHD levels at birth were 18.2 ± 13.5 ng/mL. The incidence of VDD was 82.8%, 30.6%, and 27.0% at birth, 6 months, and 12 months, respectively. The incidence of severe VDD (25-OHD < 10 ng/mL) was 31.5%, 1.5%, and 0%, at birth, 6 months, and 12 months, respectively. Among infants with severe VDD, the deficiency persisted in 49.6% at 6 months, and 35.3% at 12 months. The strongest predictor of VDD during follow-up was 25-OHD concentration at birth. Vitamin D supplementation at 400 IU/day did not affect vitamin D levels during the first year of life. Therefore, it is important to prevent neonatal VDD through maternal vitamin D supplementation during pregnancy. Further research is needed to determine the optimal vitamin D supplementation dose for Korean preterm infants.

Cerebrospinal fluid lipidomics for biomarkers of Alzheimer's disease.

Alzheimer's disease (AD) is the most common cause of dementia and is associated with serious neurologic sequelae resulting from neurodegenerative changes. Identification of markers of early-stage AD could be important for designing strategies to arrest the progression of the disease. The brain is rich in lipids because they are crucial for signal transduction and anchoring of membrane proteins. Cerebrospinal fluid (CSF) is an excellent specimen for studying the metabolism of lipids in AD because it can reflect changes occurring in the brain. We aimed to identify CSF lipidomic alterations associated with AD, using untargeted lipidomics, carried out in positive and negative ion modes. We found CSF lipids that were significantly altered in AD cases. In addition, comparison of CSF lipid profiles between persons with mild cognitive impairment (MCI) and AD showed a strong positive correlation between the lipidomes of the MCI and AD groups. The novel lipid biomarkers identified in this study are excellent candidates for validation in a larger set of patient samples and as predictive biomarkers of AD through future longitudinal studies. Once validated, the lipid biomarkers could lead to early detection, disease monitoring and the ability to measure the efficacy of potential therapeutic interventions in AD.

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.

Inflammatory markers for predicting overall survival in gastric cancer patients: A systematic review and meta-analysis.

Systemic inflammatory biomarkers have begun to be used in clinical practice to predict prognosis and survival of cancer patients, but the approach remains controversial. We conducted a meta-analysis to determine the predictive value of the c-reactive protein (CRP), neutrophil-to-lymphocyte ratio (NLR), and Glasgow prognostic score (GPS)/modified Glasgow prognostic score (mGPS) in the clinical outcome of gastric cancer (GC) patients. We searched literature databases to identify relevant studies. All articles identified in the search were independently reviewed based on predetermined selection criteria. Meta-analysis was conducted to calculate the hazard ratio (HR) and 95% confidence intervals (CI) of overall survival of the included studies. A total of 41 eligible cohort studies, involving a total of 18,348 patients meeting the inclusion criteria, were considered for meta-analysis. Increases in CRP (HR = 1.654, 95% CI: 1.272-2.151), NLR (HR = 1.605, 95% CI: 1.449-1.779), and GPS/mGPS (HR = 1.648, 95% CI: 1.351-2.011) were significantly associated with poorer survival in patients with GC. Substantial heterogeneities were noted in all three markers (I2 = 86.479%, 50.799%, 69.774%, in CRP, NLR, and GPS/mGPS, respectively). Subgroup analysis revealed a significant positive correlation between each marker and poor survival, regardless of country, study quality, cancer stage, study design, or the inclusion of patients undergoing chemotherapy. This meta-analysis demonstrates that CRP, NLR, and GPS/mGPS are associated with poor survival in patients with GC. Further prospective studies using standardized measurements are warranted to conclude the prognostic value of various inflammatory markers.

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.

Evaluation of COVID-19 epidemic outbreak caused by temporal contact-increase in South Korea.

OBJECTIVES: On March 15, 2020, 61.3% of the confirmed cases of COVID-19 infection in South Korea are associated with the worship service that was organized on February 9 in the Shincheonji Church of Jesus in Daegu. We aim to evaluate the effects of mass infection in South Korea and assess the preventive control intervention. METHOD: Using openly available data of daily cumulative confirmed cases and deaths, the basic and effective reproduction numbers was estimated using a modified susceptible-exposed-infected-recovered-type epidemic model. RESULTS: The basic reproduction number was estimated to be R0=1.77. The effective reproduction number increased approximately 20 times after the mass infections from the 31 st patient, which was confirmed on February 9 in the Shincheonji Church of Jesus, Daegu. However, the effective reproduction number decreased to less than unity after February 28 owing to the implementation of high-level preventive control interventions in South Korea, coupled with voluntary prevention actions by citizens. CONCLUSION: Preventive action and control intervention were successfully established in South Korea.

Comparative lipidomics of 5-Fluorouracil-sensitive and -resistant colorectal cancer cells reveals altered sphingomyelin and ceramide controlled by acid sphingomyelinase (SMPD1).

5-Fluorouracil (5-FU) is a chemotherapeutic drug widely used to treat colorectal cancer. 5-FU is known to gradually lose its efficacy in treating colorectal cancer following the acquisition of resistance. We investigated the mechanism of 5-FU resistance using comprehensive lipidomic approaches. We performed lipidomic analysis on 5-FU-resistant (DLD-1/5-FU) and -sensitive (DLD-1) colorectal cancer cells using MALDI-MS and LC-MRM-MS. In particular, sphingomyelin (SM) species were significantly up-regulated in 5-FU-resistant cells in MALDI-TOF analysis. Further, we quantified sphingolipids including SM and Ceramide (Cer) using Multiple Reaction Monitoring (MRM), as they play a vital role in drug resistance. We found that 5-FU resistance in DLD-1/5-FU colorectal cancer cells was mainly associated with SM increase and Cer decrease, which are controlled by acid sphingomyelinase (SMPD1). In addition, reduction of SMPD1 expression was confirmed by LC-MRM-MS analysis and the effect of SMPD1 in drug resistance was assessed by treating DLD-1 cells with siRNA-SMPD1. Furthermore, clinical colorectal cancer data set analysis showed that down-regulation of SMPD1 was associated with resistance to chemotherapy regimens that include 5-FU. Thus, from our study, we propose that SM/Cer and SMPD1 are new potential target molecules for therapeutic strategies to overcome 5-FU resistance.