Characterization of lymphocyte-rich hepatocellular carcinoma and the prognostic role of tertiary lymphoid structures.

BACKGROUND & AIMS: Lymphocyte-rich hepatocellular carcinoma (LR-HCC) is largely unknown and a rare subtype of HCC with immune-rich stroma. Tertiary lymphoid structures (TLS), frequently observed in LR-HCC, are known to be prognostically significant in various malignancies; however, their significance in HCC remains unevaluated. METHODS: Clinicopathologic data of 191 cases of surgically resected conventional HCC (C-HCC, n = 160) and LR-HCC (n = 31) were retrieved. Immunohistochemistry, multiplex immunofluorescence staining, RNA sequencing and proteomic analysis were conducted. Differences between the subtypes were statistically evaluated. RESULTS: LR-HCC was significantly correlated to larger tumour size, higher Edmondson-Steiner grade, presence of TLS and higher CD3-, CD8- and FOXP3-positive T cell, high PD-1 and PD-L1 expression (p < .001 for all) compared to C-HCC. Patients with LR-HCC exhibited significantly better overall survival (OS) (p = .044) and recurrence-free survival (RFS) (p = .025) than C-HCC. LR-HCC demonstrated TLS signatures with significantly higher proteomic-based immune scores in 14 of 17 types of tumour-infiltrating immune cells. Furthermore, C-HCC with secondary follicles, the most mature form of TLS, exhibited significantly better OS (p = .031) and RFS (p = .033) than those without. Across the global proteome, LR-HCC was well-differentiated from C-HCC and a map of protein-protein interactions between tumour-infiltrating lymphocytes and HCC in tumour microenvironment was completed. CONCLUSION: LR-HCC is clinicopathologically and molecularly distinct and shows better prognosis compared to C-HCC. Also, the presence of secondary follicle can be an important prognostic marker for better prognosis in both LR-HCC and C-HCC.

Highly secreted tryptophanyl tRNA synthetase 1 as a potential theranostic target for hypercytokinemic severe sepsis.

Despite intensive clinical and scientific efforts, the mortality rate of sepsis remains high due to the lack of precise biomarkers for patient stratification and therapeutic guidance. Secreted human tryptophanyl-tRNA synthetase 1 (WARS1), an endogenous ligand for Toll-like receptor (TLR) 2 and TLR4 against infection, activates the genes that signify the hyperinflammatory sepsis phenotype. High plasma WARS1 levels stratified the early death of critically ill patients with sepsis, along with elevated levels of cytokines, chemokines, and lactate, as well as increased numbers of absolute neutrophils and monocytes, and higher Sequential Organ Failure Assessment (SOFA) scores. These symptoms were recapitulated in severely ill septic mice with hypercytokinemia. Further, injection of WARS1 into mildly septic mice worsened morbidity and mortality. We created an anti-human WARS1-neutralizing antibody that suppresses proinflammatory cytokine expression in marmosets with endotoxemia. Administration of this antibody into severe septic mice attenuated cytokine storm, organ failure, and early mortality. With antibiotics, the antibody almost completely prevented fatalities. These data imply that blood-circulating WARS1-guided anti-WARS1 therapy may provide a novel theranostic strategy for life-threatening systemic hyperinflammatory sepsis.

Migrasomal autophagosomes relieve endoplasmic reticulum stress in glioblastoma cells.

BACKGROUND: Glioblastoma (GBM) is more difficult to treat than other intractable adult tumors. The main reason that GBM is so difficult to treat is that it is highly infiltrative. Migrasomes are newly discovered membrane structures observed in migrating cells. Thus, they can be generated from GBM cells that have the ability to migrate along the brain parenchyma. However, the function of migrasomes has not yet been elucidated in GBM cells. RESULTS: Here, we describe the composition and function of migrasomes generated along with GBM cell migration. Proteomic analysis revealed that LC3B-positive autophagosomes were abundant in the migrasomes of GBM cells. An increased number of migrasomes was observed following treatment with chloroquine (CQ) or inhibition of the expression of STX17 and SNAP29, which are involved in autophagosome/lysosome fusion. Furthermore, depletion of ITGA5 or TSPAN4 did not relieve endoplasmic reticulum (ER) stress in cells, resulting in cell death. CONCLUSIONS: Taken together, our study suggests that increasing the number of autophagosomes, through inhibition of autophagosome/lysosome fusion, generates migrasomes that have the capacity to alleviate cellular stress.

Generating Detailed Spectral Libraries for Canine Proteomes Obtained from Serum and Urine.

Domestic dogs (Canis lupus familiaris) are popular companion animals. Increase in medical expenses associated with them and demand for extending their lifespan in a healthy manner has created the need to develop new diagnostic technology. Companion dogs also serve as important animal models for non-clinical research as they can provide various biological phenotypes. Proteomics have been increasingly used on dogs and humans to identify novel biomarkers of various diseases. Despite the growing applications of proteomics in liquid biopsy in veterinary medicine, no publicly available spectral assay libraries have been created for the proteome of canine serum and urine. In this study, we generated spectral assay libraries for the two-representative liquid-biopsy samples using mid-pH fractionation that allows in-depth understanding of proteome coverage. The resultant canine serum and urine spectral assay libraries include 1,132 and 4,749 protein groups and 5,483 and 25,228 peptides, respectively. We built these complimentary accessible resources for proteomic biomarker discovery studies through ProteomeXchange with the identifier PXD034770.

Magnetic transferrin nanoparticles (MTNs) assay as a novel isolation approach for exosomal biomarkers in neurological diseases.

BACKGROUND: Brain-derived exosomes released into the blood are considered a liquid biopsy to investigate the pathophysiological state, reflecting the aberrant heterogeneous pathways of pathological progression of the brain in neurological diseases. Brain-derived blood exosomes provide promising prospects for the diagnosis of neurological diseases, with exciting possibilities for the early and sensitive diagnosis of such diseases. However, the capability of traditional exosome isolation assays to specifically isolate blood exosomes and to characterize the brain-derived blood exosomal proteins by high-throughput proteomics for clinical specimens from patients with neurological diseases cannot be assured. We report a magnetic transferrin nanoparticles (MTNs) assay, which combined transferrin and magnetic nanoparticles to isolate brain-derived blood exosomes from clinical samples. METHODS: The principle of the MTNs assay is a ligand-receptor interaction through transferrin on MTNs and transferrin receptor on exosomes, and electrostatic interaction via positively charged MTNs and negatively charged exosomes to isolate brain-derived blood exosomes. In addition, the MTNs assay is simple and rapid (< 35 min) and does not require any large instrument. We confirmed that the MTNs assay accurately and efficiently isolated exosomes from serum samples of humans with neurodegenerative diseases, such as dementia, Parkinson's disease (PD), and multiple sclerosis (MS). Moreover, we isolated exosomes from serum samples of 30 patients with three distinct neurodegenerative diseases and performed unbiased proteomic analysis to explore the pilot value of brain-derived blood protein profiles as biomarkers. RESULTS: Using comparative statistical analysis, we found 21 candidate protein biomarkers that were significantly different among three groups of neurodegenerative diseases. CONCLUSION: The MTNs assay is a convenient approach for the specific and affordable isolation of extracellular vesicles from body fluids for minimally-invasive diagnosis of neurological diseases.

TGFß1 and POSTN as biomarkers of postinfectious bronchiolitis obliterans and asthma in children.

RATIONALE: Asthma and postinfectious bronchiolitis obliterans (PIBO) are common chronic lung diseases in company with wheezing in children. However, it is not clear what is common and unique mechanisms between the two diseases. Thus, we used proteomic analysis to compare differences in biomarkers between children with asthma and PIBO. METHODS: Overall, 30 healthy children without respiratory underlying diseases, 18 children with asthma and 15 with PIBO were included for this study. Sequential window acquisition of all theoretical mass spectra (SWATH)-mass spectrometry (MS) was used to measure proteins in plasma samples. To identify specific pathways of each groups, we used the ingenuity pathway analysis (IPA) software. RESULTS: We identified and quantified 354 proteins across all 63 samples in the SWATH-MS analysis. Forty eight proteins were significantly different among 3 groups. The upstream analysis of IPA suggested that inhibitor of nuclear factor kappa B kinase subunit beta (IKBKB) was the upstream inhibitor of 4 differentially expressed proteins (DEPs) in asthma, while the upstream activator in PIBO subjects. Among 4 DEPs, TGF-ß1 in PIBO and periostin in asthma were negatively correlated with forced expiratory volume in 1 s (FEV1), FEV1/forced vital capacity, maximal med-expiratory flow, and PC20, respectively. CONCLUSION: These findings demonstrate that transforming growth factor ß1 and periostin were unique biomarkers of PIBO and asthma in children, respectively. The mechanism regulated by IKBKB may be therapeutically relevant for PIBO and asthma.

Antibody-mediated blockade for galectin-3 binding protein in tumor secretome abrogates PDAC metastasis.

The major challenges in pancreatic ductal adenocarcinoma (PDAC) management are local or distant metastasis and limited targeted therapeutics to prevent it. To identify a druggable target in tumor secretome and to explore its therapeutic intervention, we performed a liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based proteomic analysis of tumors obtained from a patient-derived xenograft model of PDAC. Galectin-3 binding protein (Gal-3BP) is identified as a highly secreted protein, and its overexpression is further validated in multiple PDAC tumors and primary cells. Knockdown and exogenous treatment of Gal-3BP showed that it is required for PDAC cell proliferation, migration, and invasion. Mechanistically, we revealed that Gal-3BP enhances galectin-3-mediated epidermal growth factor receptor signaling, leading to increased cMyc and epithelial-mesenchymal transition. To explore the clinical impact of these findings, two antibody clones were developed, and they profoundly abrogated the metastasis of PDAC cells in vivo. Altogether, our data demonstrate that Gal-3BP is an important therapeutic target in PDAC, and we propose its blockade by antibody as a therapeutic option for suppressing PDAC metastasis.

Comparative Analysis of Proteomes and Phosphoproteomes in Patients with Prostate Cancer Using Different Surgical Conditions.

PURPOSE: To establish the standard of procedure in preparing benign and cancerous prostate tissues and evaluate the quality of proteomics and phosphoproteomics during transurethral resection of the prostate (TUR-P) with different surgical conditions. MATERIALS AND METHODS: TUR-P tissue samples from three patients, two diagnosed with prostate cancer and one with benign prostatic hyperplasia, were each analyzed under three different conditions, based on differences in energy values, tissue locations, and surgical techniques. Global- and phosphorylated proteomic profiles of prostate tissues were analyzed by liquid chromatography-tandem mass spectrometry. RESULTS: A total of 6,019 global proteins and 4,280 phosphorylated peptides were identified in the nine tissues. The quantitative distributions of proteins and phosphorylation in tissues from the same patient were not affected by changes in the surgical conditions, but indirect relative comparisons differed among patients. Phosphorylation levels, especially of proteins involved in the androgen receptor pathway, important in prostate cancer, were preserved in each patient. CONCLUSIONS: Proteomic profiles of prostate tissue collected by TUR-P were not significantly affected by energy levels, tissue location, or surgical technique. In addition, since protein denaturation of samples through TUR-P is rarely confirmed in this study, we think that it will be an important guide for tissue samples in castration resistant prostate cancer patients, where it is difficult to obtain tissue. This result is the first report about proteomic and phosphoproteomic results with TUR-P samples in prostate cancer and will be theoretical basis in protein analysis research with prostate cancer tissues.

Plasma Proteome Signature to Predict the Outcome of Breast Cancer Patients Receiving Neoadjuvant Chemotherapy.

The plasma proteome of 51 non-metastatic breast cancer patients receiving neoadjuvant chemotherapy (NCT) was prospectively analyzed by high-resolution mass spectrometry coupled with nano-flow liquid chromatography using blood drawn at the time of diagnosis. Plasma proteins were identified as potential biomarkers, and their correlation with clinicopathological variables and survival outcomes was analyzed. Of 51 patients, 20 (39.2%) were HR+/HER2-, five (9.8%) were HR+/HER2+, five (9.8%) were HER2+, and 21 (41.2%) were triple-negative subtype. During a median follow-up of 52.0 months, there were 15 relapses (29.4%) and eight deaths (15.7%). Four potential biomarkers were identified among differentially expressed proteins: APOC3 had higher plasma concentrations in the pathological complete response (pCR) group, whereas MBL2, ENG, and P4HB were higher in the non-pCR group. Proteins statistically significantly associated with survival and capable of differentiating low- and high-risk groups were MBL2 and P4HB for disease-free survival, P4HB for overall survival, and MBL2 for distant metastasis-free survival (DMFS). In the multivariate analysis, only MBL2 was a consistent risk factor for DMFS (HR: 9.65, 95% CI 2.10-44.31). The results demonstrate that the proteomes from non-invasive sampling correlate with pCR and survival in breast cancer patients receiving NCT. Further investigation may clarify the role of these proteins in predicting prognosis and thus their therapeutic potential for the prevention of recurrence.

Comparative analysis of therapeutic effects between medium cut-off and high flux dialyzers using metabolomics and proteomics: exploratory, prospective study in hemodialysis.

In this single-center prospective study of 20 patients receiving maintenance hemodialysis (HD), we compared the therapeutic effects of medium cut-off (MCO) and high flux (HF) dialyzers using metabolomics and proteomics. A consecutive dialyzer membrane was used for 15-week study periods: 1st HF dialyzer, MCO dialyzer, 2nd HF dialyzer, for 5 weeks respectively. 1H-nuclear magnetic resonance was used to identify the metabolites and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis was used to identify proteins. To compare the effects of the HF and MCO dialyzers, orthogonal projection to latent structure discriminant analysis (OPLS-DA) was performed. OPLS-DA showed that metabolite characteristics could be significantly classified by 1st HF and MCO dialyzers. The Pre-HD metabolites with variable importance in projection scores ≥ 1.0 in both 1st HF versus MCO and MCO versus 2nd HF were succinate, glutamate, and histidine. The pre-HD levels of succinate and histidine were significantly lower, while those of glutamate were significantly higher in MCO period than in the HF period. OPLS-DA of the proteome also substantially separated 1st HF and MCO periods. Plasma pre-HD levels of fibronectin 1 were significantly higher, and those of complement component 4B and retinol-binding protein 4 were significantly lower in MCO than in the 1st HF period. Interestingly, as per Ingenuity Pathway Analysis, an increase in epithelial cell proliferation and a decrease in endothelial cell apoptosis occurred during the MCO period. Overall, our results suggest that the use of MCO dialyzers results in characteristic metabolomics and proteomics profiles during HD compared with HF dialyzers, which might be related to oxidative stress, insulin resistance, complement-coagulation axis, inflammation, and nutrition.

CRP immunodeposition and proteomic analysis in abdominal aortic aneurysm.

OBJECTIVE: The molecular mechanisms of the degeneration of the aortic wall in abdominal aortic aneurysm (AAA) are poorly understood. The monomeric form of C-reactive protein (mCRP) is deposited in damaged cardiovascular organs and aggravates the prognosis; however, it is unknown whether mCRP is deposited in the degenerated aorta of abdominal aortic aneurysm (AAA). We investigated whether mCRP is deposited in AAA and examined the associated pathogenic signaling pathways. METHODS: Twenty-four cases of AAA were analyzed and their histological features were compared according to the level of serum CRP and the degree of mCRP deposition. Proteomic analysis was performed in AAA cases with strong and diffuse CRP immunopositivity (n = 7) and those with weak, focal, and junctional CRP immunopositivity (n = 3). RESULTS: mCRP was deposited in the aortic specimens of AAA in a characteristic pattern that coincided with the lesion of the diminished elastic layer of the aortic wall. High serum CRP level was associated with stronger mCRP immunopositivity and a larger maximal diameter of aortic aneurysm. Proteomic analysis in AAA showed that multiple proteins were differentially expressed according to mCRP immunopositivity. Also, ingenuity pathway analysis showed that pathways associated with atherosclerosis, acute phase response, complement system, immune system, and coagulation were enriched in AAA cases with high mCRP immunopositivity. CONCLUSIONS: AAA showed a characteristic deposition of mCRP, and multiple potentially pathologic signaling pathways were upregulated in AAA cases with strong CRP immunopositivity. mCRP and the aforementioned pathological pathways may serve as targets for managing the progression of AAA.

Plasma Protein Biomarkers Associated with Higher Ovarian Cancer Risk in BRCA1/2 Carriers.

Ovarian cancer (OC) is the most lethal gynecologic malignancy and in-time diagnosis is limited because of the absence of effective biomarkers. Germline BRCA1/2 genetic alterations are risk factors for hereditary OC; risk-reducing salpingo-oophorectomy (RRSO) is pursued for disease prevention. However, not all healthy carriers develop the disease. Therefore, identifying predictive markers in the BRCA1/2 carrier population could help improve the identification of candidates for preventive RRSO. In this study, plasma samples from 20 OC patients (10 patients with BRCA1/2 wild type (wt) and 10 with the BRCA1/2 variant (var)) and 20 normal subjects (10 subjects with BRCA1/2wt and 10 with BRCA1/2var) were analyzed for potential biomarkers of hereditary OC. We applied a bottom-up proteomics approach, using nano-flow LC-MS to analyze depleted plasma proteome quantitatively, and potential plasma protein markers specific to the BRCA1/2 variant were identified from a comparative statistical analysis of the four groups. We obtained 1505 protein candidates from the 40 subjects, and SPARC and THBS1 were verified by enzyme-linked immunosorbent assay. Plasma SPARC and THBS1 concentrations in healthy BRCA1/2 carriers were found to be lower than in OC patients with BRCA1/2var. If plasma SPARC concentrations increase over 337.35 ng/mL or plasma THBS1 concentrations increase over 65.28 µg/mL in a healthy BRCA1/2 carrier, oophorectomy may be suggested.

A Proteomics-Based Analysis of Blood Biomarkers for the Diagnosis of COPD Acute Exacerbation.

PURPOSE: The identification of blood biomarkers to diagnose acute exacerbation of chronic obstructive pulmonary disease (AECOPD) will have clinical utility. Here, we used a proteomics-based approach to identify biomarkers capable of identifying AECOPD. PATIENTS AND METHODS: This prospective, single-center pilot study enrolled 12 patients who came to Asan Medical Center (South Korea) via the outpatient clinic or emergency department with symptoms of AECOPD and were follow-up in the outpatient clinic during convalescence between 2015 and 2017. Paired blood samples collected from each patient during the treatment naïve AECOPD and convalescence stages were analyzed. A sequential window acquisition of all theoretical fragmentation spectra-mass spectrometry (SWATH-MS)-based proteome analysis was performed and a subset of the data were verified by ELISA. RESULTS: The SWATH-MS analysis identified 226 plasma proteins across all samples examined. The median coefficient of variation for triplicate technical replicates of each sample was 1.13 ± 1.38%, indicating high precision of the technique. Fold-change and paired t-test analyses revealed that 14 proteins were present at higher levels in the AECOPD samples than in the convalescence samples. A gene ontology analysis revealed that these proteins are involved in the acute-phase response. A total of 15 proteins were present at higher levels during the recovery (convalescence) stage than during the acute exacerbation phase, and gene ontology analysis revealed that these proteins are related to lipid metabolism and transport. Verification of the SWATH-MS data was performed using ELISAs for three proteins that were up-regulated in AECOPD, namely, LBP, ORM2, and SERPINA3. Among them, SERPINA3 (p = 0.005) was up-regulated significantly in AECOPD compared with the convalescence state. CONCLUSION: Potential plasma biomarkers of AECOPD were discovered using the SWATH-MS proteomics method, and functional molecular associations were investigated. SERPINA3 could be a promising diagnostic biomarker for the early identification and tracking of AECOPD.

Different Characteristics of Childhood Asthma Related to Polyhexamethylene Guanidine Exposure.

Rationale: Exposure to humidifier disinfectants (HDs) can increase the risk of asthma, but the characteristics of HD-related asthma are currently unclear. Polyhexamethylene guanidine (PHMG)-containing HD was the most commonly used and the most frequently associated with HD-associated lung injury. Objectives: To investigate the characteristics of PHMG-induced asthma. Methods: This general population-based birth cohort study used data from the Panel Study of Korean Children from 2008 (n = 846). Spirometry, bronchial provocation tests, detailed history recording, and physical examinations were performed on 7-year-old patients (n = 362). Exploratory analysis of plasma proteomics was performed. Results: Compared with the healthy control group, forced expiratory volume in 1 second was the lowest in PHMG-exposed asthma group (z-score = -0.806; 95% confidence interval, -1.492 to -0.119). The positive rate of bronchial hyperresponsiveness was lower in children with PHMG-exposed asthma compared with children with asthma without HD exposure (13.3% vs. 47.4%). Long-term exposure to low-intensity PHMG before the age of 3 years was associated with asthma symptoms. Periostin was higher in subjects with asthma without HD exposure compared with the healthy control subjects. The inducible T-cell costimulator ligand and hepatocyte growth factor activator were lower in PHMG-exposed asthma compared with asthma without exposure. Hepatocyte growth factor activator had a positive correlation with forced vital capacity (z-score) in asthma with PHMG exposure (r = 0.78; P < 0.01). Conclusions: The asthma associated with low-intensity exposure to PHMG is characterized by lower lung function, lower positive rates of bronchial hyperresponsiveness, and varied distributions of plasma proteins. These findings suggest that asthma related to PHMG exposure may constitute a different mechanism of asthma pathophysiology.

Convergence of Plasma Metabolomics and Proteomics Analysis to Discover Signatures of High-Grade Serous Ovarian Cancer.

The 5-year survival rate in the early and late stages of ovarian cancer differs by 63%. In addition, a liquid biopsy is necessary because there are no symptoms in the early stage and tissue collection is difficult without using invasive methods. Therefore, there is a need for biomarkers to achieve this goal. In this study, we found blood-based metabolite or protein biomarker candidates for the diagnosis of ovarian cancer in the 20 clinical samples (10 ovarian cancer patients and 10 healthy control subjects). Plasma metabolites and proteins were measured and quantified using mass spectrometry in ovarian cancer patients and control groups. We identified the differential abundant biomolecules (34 metabolites and 197 proteins) and statistically integrated molecules of different dimensions to better understand ovarian cancer signal transduction and to identify novel biological mechanisms. In addition, the biomarker reliability was verified through comparison with existing research results. Integrated analysis of metabolome and proteome identified emerging properties difficult to grasp with the single omics approach, more reliably interpreted the cancer signaling pathway, and explored new drug targets. Especially, through this analysis, proteins (PPCS, PMP2, and TUBB) and metabolites (L-carnitine and PC-O (30:0)) related to the carnitine system involved in cancer plasticity were identified.

An Exploratory Pilot Study with Plasma Protein Signatures Associated with Response of Patients with Depression to Antidepressant Treatment for 10 Weeks.

Major depressive disorder (MDD) is a leading cause of global disability with a chronic and recurrent course. Recognition of biological markers that could predict and monitor response to drug treatment could personalize clinical decision-making, minimize unnecessary drug exposure, and achieve better outcomes. Four longitudinal plasma samples were collected from each of ten patients with MDD treated with antidepressants for 10 weeks. Plasma proteins were analyzed qualitatively and quantitatively with a nanoflow LC-MS/MS technique. Of 1153 proteins identified in the 40 longitudinal plasma samples, 37 proteins were significantly associated with response/time and clustered into six according to time and response by the linear mixed model. Among them, three early-drug response markers (PHOX2B, SH3BGRL3, and YWHAE) detectable within one week were verified by liquid chromatography-multiple reaction monitoring/mass spectrometry (LC-MRM/MS) in the well-controlled 24 patients. In addition, 11 proteins correlated significantly with two or more psychiatric measurement indices. This pilot study might be useful in finding protein marker candidates that can monitor response to antidepressant treatment during follow-up visits within 10 weeks after the baseline visit.

Differential Urinary Proteome Analysis for Predicting Prognosis in Type 2 Diabetes Patients with and without Renal Dysfunction.

Renal dysfunction, a major complication of type 2 diabetes, can be predicted from estimated glomerular filtration rate (eGFR) and protein markers such as albumin concentration. Urinary protein biomarkers may be used to monitor or predict patient status. Urine samples were selected from patients enrolled in the retrospective diabetic kidney disease (DKD) study, including 35 with good and 19 with poor prognosis. After removal of albumin and immunoglobulin, the remaining proteins were reduced, alkylated, digested, and analyzed qualitatively and quantitatively with a nano LC-MS platform. Each protein was identified, and its concentration normalized to that of creatinine. A prognostic model of DKD was formulated based on the adjusted quantities of each protein in the two groups. Of 1296 proteins identified in the 54 urine samples, 66 were differentially abundant in the two groups (area under the curve (AUC): p-value < 0.05), but none showed significantly better performance than albumin. To improve the predictive power by multivariate analysis, five proteins (ACP2, CTSA, GM2A, MUC1, and SPARCL1) were selected as significant by an AUC-based random forest method. The application of two classifiers-support vector machine and random forest-showed that the multivariate model performed better than univariate analysis of mucin-1 (AUC: 0.935 vs. 0.791) and albumin (AUC: 1.0 vs. 0.722). The urinary proteome can reflect kidney function directly and can predict the prognosis of patients with chronic kidney dysfunction. Classification based on five urinary proteins may better predict the prognosis of DKD patients than urinary albumin concentration or eGFR.

Transglutaminase 2 mediates hypoxia-induced selective mRNA translation via polyamination of 4EBPs.

Hypoxia selectively enhances mRNA translation despite suppressed mammalian target of rapamycin complex 1 activity, contributing to gene expression reprogramming that promotes metastasis and survival of cancer cells. Little is known about how this paradoxical control of translation occurs. Here, we report a new pathway that links hypoxia to selective mRNA translation. Transglutaminase 2 (TG2) is a hypoxia-inducible factor 1-inducible enzyme that alters the activity of substrate proteins by polyamination or crosslinking. Under hypoxic conditions, TG2 polyaminated eukaryotic translation initiation factor 4E (eIF4E)-bound eukaryotic translation initiation factor 4E-binding proteins (4EBPs) at conserved glutamine residues. 4EBP1 polyamination enhances binding affinity for Raptor, thereby increasing phosphorylation of 4EBP1 and cap-dependent translation. Proteomic analyses of newly synthesized proteins in hypoxic cells revealed that TG2 activity preferentially enhanced the translation of a subset of mRNA containing G/C-rich 5'UTRs but not upstream ORF or terminal oligopyrimidine motifs. These results indicate that TG2 is a critical regulator in hypoxia-induced selective mRNA translation and provide a promising molecular target for the treatment of cancers.