Potential Biomarker Candidates Research Articles

Integrative Analysis Developing and Validating Potential Candidate Biomarkers for Cancer Stemness Features of Pan-Renal Cell Carcinoma

In our study, 49 key genes significantly associated with renal cell carcinoma (RCC) stemness were obtained. Next, we developed a molecular prognostic signature associated with stemness features of pan-RCC. The difference in overall survival (OS) between the high- and low-risk groups was statistically significant (p < .05). The area under the receiver operating characteristic curve for 1-year OS, 5-year OS, and 10-year OS was 0.759, 0.712, and 0.918, respectively. The results of validation in The Cancer Genome Atlas cohort and International Cancer Genome Consortium cohort revealed the predictive capability of this signature. Furthermore, we selected three genes and further validation showed that these three hub genes were potential hub biomarkers for pan-RCC stemness features.

Detection of cytotoxins by sandwich-ELISA for discrimination of cobra envenomation and indication of necrotic severity

Snake envenoming is both a healthcare and socioeconomic problem for developing countries and underserved communities. In Taiwan, clinical management of Naja atra envenomation is a major challenge, since cobra venom-induced symptoms are usually confused with hemorrhagic snakebites and current antivenom treatments do not effectively prevent venom-induced necrosis for which early surgical debridement should be administered. Identification and validation of biomarkers of cobra envenomation is critical for progress in setting a realistic goal for snakebite management in Taiwan. Previously, cytotoxin (CTX) was determined as one of potential biomarker candidates; however, its ability to discriminate cobra envenoming remains to be verified, especially in clinical practice. In this study, we selected a monoclonal single-chain variable fragment (scFv) and a polyclonal antibody to develop a sandwich enzyme-linked immunosorbent assay (ELISA) for CTX detection, which successfully recognized CTX from N. atra venom over that from other snake species. Using this specific assay, the CTX concentration in envenoming mice was shown to remain consistent in about 150 ng/mL during the 2-hour post-injection period. The measured concentration was highly correlated with the size of local necrosis in mouse dorsal skin, which the correlation coefficient is about 0.988. Furthermore, our ELISA method displayed 100 % of specificity and sensitivity in discriminating cobra envenoming among snakebite victims through CTX detection and the level of CTX in victim plasma was ranged from 5.8 to 253.9 ng/mL. Additionally, patients developed tissue necrosis at plasma CTX concentrations higher than 150 ng/mL. Thus, CTX not only serves as a verified biomarker for discrimination of cobra envenoming but also a potential indicator of severity of local necrosis. In this context, detection of CTX may facilitate reliable identification of envenoming species and improve snakebite management in Taiwan.

The HDAC2/YY1/c-Myc signaling axis regulates lung cancer cell migration and proliferation.

Lung cancer is among the most aggressive types of malignant tumors that contributes to cancer-associated deaths worldwide with a high occurrence and fatality rate. Histone deacetylase 2 (HDAC2), prevent the aberrant transcription of a number of genes that are primarily responsible for controlling the cell cycle, cell proliferation, and signaling pathways in numerous cancers. Previous studies reported the role of HDACs and YY1 in the growth and development of several cancers. Although, it is noteworthy that remarkable efforts have been taken for the treatment of lung cancer using molecularly targeted therapies and chemotherapeutic agents, but the outcome is still poor for this critically persistent cancer. Therefore, the aim of the present study is to identify an efficacious, novel therapeutic biomarkers for the successful diagnosis of lung cancer at the early stage of the disease and the molecular insights involved. In the present study, qPCR and western bot data revealed that the expression level of HDAC2 and YY1 were upregulated in the cell lines and tumor samples of lung cancer patients. Moreover, MTT, qPCR, western blot, cell cycle analysis, and migration assays showed that inhibition of HDAC2 reduced YY1 expression, similarly, depletion of YY1 using knockdown approach inhibited the proliferation, migration, invasion, and blockage of the cell cycle by suppressing c-Myc in lung cancer cell lines. In conclusion, the current study findings support the notion that HDAC2's anticancer role was attributed through YY1 regulation by targeting c-Myc and could act as potential novel candidate biomarker for the lung cancer diagnosis.

Blood-Based Proteomic Profiling Identifies Potential Biomarker Candidates and Pathogenic Pathways in Dementia.

Dementia is a progressive and debilitating neurological disease that affects millions of people worldwide. Identifying the minimally invasive biomarkers associated with dementia that could provide insights into the disease pathogenesis, improve early diagnosis, and facilitate the development of effective treatments is pressing. Proteomic studies have emerged as a promising approach for identifying the protein biomarkers associated with dementia. This pilot study aimed to investigate the plasma proteome profile and identify a panel of various protein biomarkers for dementia. We used a high-throughput proximity extension immunoassay to quantify 1090 proteins in 122 participants (22 with dementia, 64 with mild cognitive impairment (MCI), and 36 controls with normal cognitive function). Limma-based differential expression analysis reported the dysregulation of 61 proteins in the plasma of those with dementia compared with controls, and machine learning algorithms identified 17 stable diagnostic biomarkers that differentiated individuals with AUC = 0.98 ± 0.02. There was also the dysregulation of 153 plasma proteins in individuals with dementia compared with those with MCI, and machine learning algorithms identified 8 biomarkers that classified dementia from MCI with an AUC of 0.87 ± 0.07. Moreover, multiple proteins selected in both diagnostic panels such as NEFL, IL17D, WNT9A, and PGF were negatively correlated with cognitive performance, with a correlation coefficient (r2) ≤ -0.47. Gene Ontology (GO) and pathway analysis of dementia-associated proteins implicated immune response, vascular injury, and extracellular matrix organization pathways in dementia pathogenesis. In conclusion, the combination of high-throughput proteomics and machine learning enabled us to identify a blood-based protein signature capable of potentially differentiating dementia from MCI and cognitively normal controls. Further research is required to validate these biomarkers and investigate the potential underlying mechanisms for the development of dementia.

Investigation of conceptus stimulated gene expression in buffalo peripheral blood mononuclear cells as potential diagnostic markers of early pregnancy

Exploration of novel strategies for early pregnancy diagnosis is pivotal in enhancing the reproductive potential and monetary gains from dairy herds. In buffalo, the trophectoderm cells of the elongating conceptus secrete interferon-tau that stimulates the transcription of various genes in peripheral blood mononuclear cells (PBMC) during the peri-implantation period. We explored the differential expression of classical (ISG15) and novel (LGALS3BP and CD9) early pregnancy markers in PBMC of buffaloes during various stages of pregnancy. Natural heat was detected in buffaloes by assessing the vaginal fluid, and artificial insemination (AI) was done. Whole blood was collected from the jugular vein in EDTA-containing vacutainers for PBMC isolation before AI (0-day) and 20, 25 and 40 d post-AI. On day 40, transrectal ultrasonography examination was performed to confirm pregnancy. The inseminated non-pregnant animals served as control. Total RNA was extracted using the TRIzol method. The temporal abundance of ISG15, LGALS3BP and CD9 genes in PBMC was compared between pregnant and non-pregnant groups (n = 9 per group) using real time-qPCR. Results showed transcripts of ISG15 and LGALS3BP were more abundant at 20 d in the pregnant group compared to the 0 d and 20 d values of the non-pregnant group. However, due to variability in expression, threshold (Ct) cycle of RT-qPCR alone could not distinguish pregnant and non-pregnant animals. In conclusion, ISG15 and LGALS3BP transcripts abundance in PBMCs are potential candidate biomarkers for early prediction of buffalo pregnancy 20-days post-AI, but further work is required to allow the development of a reliable new methodology.

Abstract 6507: E3 ubiquitin-protein ligase, tetratricopeptide repeat domain 3 (TTC3), H4 clustered histone 5 (H4C5), and epithelial cell adhesion molecule (EpCAM) are novel urine-enriched liquid biopsy biomarkers to detect prostate cancer in men

Abstract Background: Prostate Cancer (PCa) is one of the leading causes of cancer deaths among American men; however, the estimated number of diagnoses represents a small fraction of disease-related biopsies performed yearly. Although the Prostate-Specific Antigen (PSA) test is widely used for screening, several advisory groups recommend against using PSA because of its suboptimal performance. Thus, there is an urgent unmet need for novel and more accurate diagnostic biomarkers for PCa detection and to differentiate aggressive cancer from its indolent form. Methods: In our study, we used voided urine (50ml) from pre and post-prostatectomy men with PCa and urine from normal healthy men as control. We isolated RNA from exfoliated cells and debris shed into urine and performed RNA-sequencing using the Illumina Next-seq 550 platform. Advanced computational and machine-learning approaches were employed to identify candidate biomarkers in men with PCa. We examined the TCGA database to validate the PCa-specific expression of the identified RNA in tumor tissues. Two RNA markers were further tested by qPCR, and one urinary soluble protein marker was measured by immunoassays. Results: Our study included 106 men with PCa and 88 control men. We identified and validated the presence of ≥1 RNA markers (TTC3, H4C5) and a protein marker (EpCAM) in urine as potential candidate biomarkers for PCa detection. We tested and developed these markers using qPCR for TTC3, H4C5, and ELISA assay for EpCAM with higher specificity and sensitivity (Table 1). Our results outperformed known urinary markers, PCA3 and SPDEF (Figure 1). TTC3, H4C5, and EpCAM markers diminished to low or undetectable levels in post-prostatectomy compared to pre-prostatectomy men with PCa. shRNA knockdown of TTC3 and EpCAM in androgen-sensitive and insensitive cells induced biological changes, suggesting their relevance to Prostate Cancer. Conclusions: We report a highly accurate panel of 3 urine-based biomarkers that detect PCa consisting of EpCAM (protein) and TTC3 and H4C5 (RNA). To identify the advantage of using individual or combinatorial markers is an ongoing study in our laboratory. Citation Format: Ranjan Joseph Perera, Marcio Moschovas, Roshane Perera, Bongyong Lee, Alexandra Miller, Ernest K. Amankwah, Rudramani Pokhrel, Christian Pavlovich, Guru Sonpavde, Vipul Patel. E3 ubiquitin-protein ligase, tetratricopeptide repeat domain 3 (TTC3), H4 clustered histone 5 (H4C5), and epithelial cell adhesion molecule (EpCAM) are novel urine-enriched liquid biopsy biomarkers to detect prostate cancer in men [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6507.

Male- and female-specific microRNA expression patterns in a mouse model of methanol poisoning.

The aims of this study were to determine the miRNAs involved in the methanol poisoning, and identify the male- and female-specific miRNA expression patterns in mice. Methanol was applied orally at the doses of 4g/kg and 8g/kg to induce mild and severe methanol poisoning in Balb/c mice. miRNA expression levels were detected at 3 different time periods (30, 60, and 180min) following methanol exposure. miRNA expression profiles were determined using the high-throughput Fluidigm BioMark real-time PCR. We observed that serum miR-206 expression in male mice and miR-6357 expression in female mice could be an indicator of methanol poisoning. miR-9-3p downregulation and miR-1187 upregulation could be important for liver tissue. miR-3106-5p and miR-133a-5p upregulations and miR-122-3p downregulation could be poison biomarkers for ocular tissue in male mice. However, miR-194-5p downregulation could be a biomarker for ocular tissue in female mice. miR-122-5p and miR-124-3p downregulations and miR-499a-5p upregulation appeared to be important for kidney tissue in male mice. miR-543 and miR-6342 upregulations could be potential candidate biomarkers for kidney tissue in female mice. Our study is the first to report that differential miRNA expressions are involved in blood and tissues in male and female mice after methanol treatment.

MP44-18 E3 UBIQUITIN-PROTEIN LIGASE, TETRATRICOPEPTIDE REPEAT DOMAIN 3 (TTC3), H4 CLUSTERED HISTONE 5 (H4C5), AND EPITHELIAL CELL ADHESION MOLECULE (EPCAM) ARE NOVEL URINE-ENRICHED LIQUID BIOPSY BIOMARKERS TO DETECT PROSTATE CANCER IN MEN

You have accessJournal of UrologyCME1 Apr 2023MP44-18 E3 UBIQUITIN-PROTEIN LIGASE, TETRATRICOPEPTIDE REPEAT DOMAIN 3 (TTC3), H4 CLUSTERED HISTONE 5 (H4C5), AND EPITHELIAL CELL ADHESION MOLECULE (EPCAM) ARE NOVEL URINE-ENRICHED LIQUID BIOPSY BIOMARKERS TO DETECT PROSTATE CANCER IN MEN Menglang Yuan, Marcio Moschovas, Roshane Perera, Bongyong Lee, Alexandra Miller, Ernest Amankwah, Rudramani Pokhrel, Christian Pavlovich, Guru Sonpavde, Vipul Patel, and Ranjan Perera Menglang YuanMenglang Yuan More articles by this author , Marcio MoschovasMarcio Moschovas More articles by this author , Roshane PereraRoshane Perera More articles by this author , Bongyong LeeBongyong Lee More articles by this author , Alexandra MillerAlexandra Miller More articles by this author , Ernest AmankwahErnest Amankwah More articles by this author , Rudramani PokhrelRudramani Pokhrel More articles by this author , Christian PavlovichChristian Pavlovich More articles by this author , Guru SonpavdeGuru Sonpavde More articles by this author , Vipul PatelVipul Patel More articles by this author , and Ranjan PereraRanjan Perera More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003290.18AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Prostate Cancer (PCa) is one of the leading causes of cancer deaths among American men; however, the estimated number of diagnoses represents a small fraction of disease-related biopsies performed yearly. Although the Prostate-Specific Antigen (PSA) test is widely used for screening, several advisory groups recommend against using PSA because of its suboptimal performance. Thus, there is an urgent unmet need for novel and more accurate diagnostic biomarkers for PCa detection and to differentiate aggressive cancer from its indolent form. METHODS: In our study, we used voided urine (50ml) from pre and post-prostatectomy men with PCa and urine from normal healthy men as control. We isolated RNA from exfoliated cells and debris shed into urine and performed RNA-sequencing using the Illumina Next-seq 550 platform. Advanced computational and machine-learning approaches were employed to identify candidate biomarkers in men with PCa. We examined the TCGA database to validate the PCa-specific expression of the identified RNA in tumor tissues. Two RNA markers were further tested by qPCR, and one urinary soluble protein marker was measured by immunoassays. RESULTS: Our study included 106 men with PCa and 88 control men. We identified and validated the presence of ≥1 RNA markers (TTC3, H4C5) and a protein marker (EpCAM) in urine as potential candidate biomarkers for PCa detection. We tested and developed these markers using qPCR for TTC3, H4C5, and ELISA assay for EpCAM with higher specificity and sensitivity (Table 1). Our results outperformed known urinary markers, PCA3 and SPDEF (Figure 1). TTC3, H4C5, and EpCAM markers diminished to low or undetectable levels in post-prostatectomy compared to pre-prostatectomy men with PCa. shRNA knockdown of TTC3 and EpCAM in androgen-sensitive and insensitive cells induced biological changes, suggesting their relevance to Prostate Cancer. CONCLUSIONS: We report a highly accurate panel of 3 urine-based biomarkers that detect PCa consisting of EpCAM (protein) and TTC3 and H4C5 (RNA). To identify the advantage of using individual or combinatorial markers is an ongoing study in our laboratory. Source of Funding: Florida Department of Health, Bankhead-Coley Cancer Research Program 5BC08 to RJP. International Prostate Cancer Foundation (IPCF) to R.J.P and VP. National Institutes of Health grant NCI 5P30CA030199 to RJP © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e619 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Menglang Yuan More articles by this author Marcio Moschovas More articles by this author Roshane Perera More articles by this author Bongyong Lee More articles by this author Alexandra Miller More articles by this author Ernest Amankwah More articles by this author Rudramani Pokhrel More articles by this author Christian Pavlovich More articles by this author Guru Sonpavde More articles by this author Vipul Patel More articles by this author Ranjan Perera More articles by this author Expand All Advertisement PDF downloadLoading ...

ATPergic signaling disruption in human sepsis as a potential source of biomarkers for clinical use.

Sepsis is a life-threatening organ dysfunction caused by a dysregulated inflammatory response to infection. To date, there is no specific treatment established for sepsis. In the extracellular compartment, purines such as adenosine triphosphate (ATP) and adenosine play essential roles in the immune/inflammatory responses during sepsis and septic shock. The balance of extracellular levels among ATP and adenosine is intimately involved in the signals related to immune stimulation/immunosuppression balance. Specialized enzymes, including CD39, CD73, and adenosine deaminase (ADA), are responsible to metabolize ATP to adenosine which will further sensitize the P2 and P1 purinoceptors, respectively. Disruption of the purinergic pathway had been described in the sepsis pathophysiology. Although purinergic signaling has been suggested as a potential target for sepsis treatment, the majority of data available were obtained using pre-clinical approaches. We hypothesized that, as a reflection of deregulation on purinergic signaling, septic patients exhibit differential measurements of serum, neutrophils and monocytes purinergic pathway markers when compared to two types of controls (healthyand ward). It was observed that ATP and ADP serum levels were increased in septic patients, as well as the A2a mRNA expression in neutrophils and monocytes. Both ATPase/ADPase activities were increased during sepsis. Serum ATP and ADP levels, and both ATPase and ADPase activities were associated with the diagnosis of sepsis, representing potential biomarkers candidates. In conclusion, our results advance the translation of purinergic signaling from pre-clinical models into the clinical setting opening opportunities for so much needed new strategies for sepsis and septic shock diagnostics and treatment.

Polyunsaturated and Saturated Oxylipin Plasma Levels Allow Monitoring the Non-Alcoholic Fatty Liver Disease Progression to Severe Stages.

Hepatic fat accumulation is the hallmark of non-alcoholic fatty liver disease (NAFLD). Our aim was to determine the plasma levels of oxylipins, free polyunsaturated fatty acids (PUFA) and markers of lipid peroxidation in patients with NAFLD in progressive stages of the pathology. Ninety 40-60-year-old adults diagnosed with metabolic syndrome were distributed in without, mild, moderate or severe NAFLD stages. The free PUFA and oxylipin plasma levels were determined by the UHPLC-MS/MS system. The plasma levels of oxylipins produced by cyclooxygenases, lipoxygenases and cytochrome P450, such as prostaglandin 2α (PGF2α), lipoxinB4 and maresin-1, were higher in severe NAFLD patients, pointing to the coexistence of both inflammation and resolution processes. The plasma levels of the saturated oxylipins 16-hydroxyl-palmitate and 3-hydroxyl-myristate were also higher in the severe NAFLD patients, suggesting a dysregulation of oxidation of fatty acids. The plasma 12-hydroxyl-estearate (12HEST) levels in severe NAFLD were higher than in the other stages, indicating that the hydroxylation of saturated fatty acid produced by reactive oxygen species is more present in this severe stage of NAFLD. The plasma levels of 12HEST and PGF2α are potential candidate biomarkers for diagnosing NAFLD vs. non-NAFLD. In conclusion, the NAFLD progression can be monitored by measuring the plasma levels of free PUFA and oxylipins characterizing the different NAFLD stages or the absence of this disease in metabolic syndrome patients.

An extensive evaluation of hepatic markers of damage and regeneration in controlled and uncontrolled donation after circulatory death.

Livers from donations after circulatory death (DCDs) are very sensitive to ischemia/reperfusion injury and thus need careful reconditioning, such as normothermic regional perfusion (NRP). So far, its impact on DCDs has not been thoroughly investigated. This pilot cohort study aimed to explore the NRP impact on liver function by evaluating dynamic changes of circulating markers and hepatic gene expression in 9 uncontrolled DCDs (uDCDs) and 10 controlled DCDs. At NRP start, controlled DCDs had lower plasma levels of inflammatory and liver damage markers, including α-glutathione s-transferase, sorbitol-dehydrogenase, malate dehydrogenase 1, liver-type arginase-1, and keratin-18, but higher levels of osteopontin, sFas, flavin mononucleotide, and succinate than uDCDs. During 4-hour NRP, some damage and inflammatory markers increased in both groups, while IL-6, HGF, and osteopontin increased only in uDCDs. At the NRP end, the tissue expression of early transcriptional regulators, apoptosis, and autophagy mediators was higher in uDCDs than in controlled DCDs. In conclusion, despite initial differences in liver damage biomarkers, the uDCD group was characterized by a major gene expression of regenerative and repair factors after the NRP procedure. Correlative analysis among circulating/tissue biomarkers and the tissue congestion/necrosis degree revealed new potential candidate biomarkers.

Quantitative proteomic analysis of bronchoalveolar lavage fluids from patients with small cell lung cancers.

Small cell lung cancer (SCLC) is one of the malignant cancers with aggressive progression and poor prognosis. Bronchoalveolar lavage fluid (BALF) has been arising recently as a potential source of biomarkers for lung cancers. In this study, we performed quantitative BALF proteomic analysis to identify potential biomarkers for SCLC. BALF were collected from tumor-bearing lungs and non-tumor lungs of five SCLC patients. Then, BALF proteomes were prepared for a TMT-based quantitative mass spectrometry analysis. Differentially expressed proteins (DEP) were identified when considering individual variation. Potential SCLC biomarker candidates were validated by immunohistochemistry (IHC). A public database of multiple SCLC cell lines was used to evaluate the correlation of these markers with SCLC subtypes and chemo-drug responses. We identified 460 BALF proteins in SCLC patients and observed considerable individual variation among the patients. Immunohistochemical analysis and bioinformatics resulted in the identification of CNDP2 and RNPEP as potential subtype markers for ASCL1 and NEUROD1, respectively. In addition, CNDP2 was found to be positively correlated with responses to etoposide, carboplatin, and irinotecan. BALF is an emerging source of biomarkers, making it useful for the diagnosis and prognosis of lung cancers. We characterized the proteomes of paired BALF samples collected from tumor-bearing and non-tumor lungs of SCLC patients. Several proteins were found elevated in tumor-bearing BALF, and especially CNDP2 and RNPEP appeared to be potential indicators for ASLC1-high and NEUROD1-high subtypes of SCLC, respectively. The positive correlation of CNDP2 with chemo-drug responses would help to make decisions for treatment of SCLC patients. These putative biomarkers could be comprehensively investigated for a clinical use towards precision medicine.

Biomarker discovery for tuberculosis using metabolomics.

Tuberculosis (TB) is the leading cause of death among infectious diseases, and the ratio of cases in which its pathogen Mycobacterium tuberculosis (Mtb) is drug resistant has been increasing worldwide, whereas latent tuberculosis infection (LTBI) may develop into active TB. Thus it is important to understand the mechanism of drug resistance, find new drugs, and find biomarkers for TB diagnosis. The rapid progress of metabolomics has enabled quantitative metabolite profiling of both the host and the pathogen. In this context, we provide recent progress in the application of metabolomics toward biomarker discovery for tuberculosis. In particular, we first focus on biomarkers based on blood or other body fluids for diagnosing active TB, identifying LTBI and predicting the risk of developing active TB, as well as monitoring the effectiveness of anti-TB drugs. Then we discuss the pathogen-based biomarker research for identifying drug resistant TB. While there have been many reports of potential candidate biomarkers, validations and clinical testing as well as improved bioinformatics analysis are needed to further substantiate and select key biomarkers before they can be made clinically applicable.

Differences of gut microbiota and behavioral symptoms between two subgroups of autistic children based on γδT cells-derived IFN-γ Levels: A preliminary study.

Autism Spectrum Disorders (ASD) are defined as a group of pervasive neurodevelopmental disorders, and the heterogeneity in the symptomology and etiology of ASD has long been recognized. Altered immune function and gut microbiota have been found in ASD populations. Immune dysfunction has been hypothesized to involve in the pathophysiology of a subtype of ASD. A cohort of 105 ASD children were recruited and grouped based on IFN-γ levels derived from ex vivo stimulated γδT cells. Fecal samples were collected and analyzed with a metagenomic approach. Comparison of autistic symptoms and gut microbiota composition was made between subgroups. Enriched KEGG orthologues markers and pathogen-host interactions based on metagenome were also analyzed to reveal the differences in functional features. The autistic behavioral symptoms were more severe for children in the IFN-γ-high group, especially in the body and object use, social and self-help, and expressive language performance domains. LEfSe analysis of gut microbiota revealed an overrepresentation of Selenomonadales, Negatiyicutes, Veillonellaceae and Verrucomicrobiaceae and underrepresentation of Bacteroides xylanisolvens and Bifidobacterium longum in children with higher IFN-γ level. Decreased metabolism function of carbohydrate, amino acid and lipid in gut microbiota were found in the IFN-γ-high group. Additional functional profiles analyses revealed significant differences in the abundances of genes encoding carbohydrate-active enzymes between the two groups. And enriched phenotypes related to infection and gastroenteritis and underrepresentation of one gut-brain module associated with histamine degradation were also found in the IFN-γ-High group. Results of multivariate analyses revealed relatively good separation between the two groups. Levels of IFN-γ derived from γδT cell could serve as one of the potential candidate biomarkers to subtype ASD individuals to reduce the heterogeneity associated with ASD and produce subgroups which are more likely to share a more similar phenotype and etiology. A better understanding of the associations among immune function, gut microbiota composition and metabolism abnormalities in ASD would facilitate the development of individualized biomedical treatment for this complex neurodevelopmental disorder.

Comprehensive characterization of mesothelin expression in colorectal cancer.

245 Background: Mesothelin (MSLN) is highly expressed in several malignancies including colorectal cancer (CRC) compared to normal tissue and is associated with tumor cell proliferation and worse patient survival. MSLN could be a potential target for antigen-specific therapy. Here, we characterized the MSLN expression in CRC and its association with molecular and immune-related markers. Methods: 15,673 CRC samples were tested by NGS (592, NextSeq; WES, NovaSeq), WTS (NovaSeq) (Caris Life Sciences, Phoenix, AZ). Microsatellite-instability (MSI) was tested by fragment analysis, IHC, and NGS. Tumor mutational burden (TMB) totaled somatic mutations per tumor (high&gt;10 mt/MB). Tumors with MSLN-high(H) and MSLN-low(L) expression were classified by top and bottom quartile, respectively. Real world overall survival (OS) was extracted from insurance claims and calculated using Kaplan-Meier estimates for molecularly defined cohorts from tissue collection to last contact. Statistical significance was determined using chi-square and Mann-Whitney U test with p-values adjusted for multiple comparisons (q&lt;0.05). Results: There was no difference in median age or gender distribution between MSLN-H and MSLN-L CRC tumors. Right-sided tumors had increased median MSLN expression compared to rectal, transverse, and left-sided tumors (6.12 vs 5.29 vs 5.35 vs 4.81 TPM, q&lt;0.05). Median MSLN expression was higher in metastatic compared to primary tumors (1.21-fold, q&lt;0.05). Median MSLN expression was highest in CMS4 (10.17 TPM), but, in MSS CRC, MSLN expression was highest in CMS1 (10.46 TPM, all q&lt;0.05). MSLN-H tumors had higher frequency of KRAS (59.8% vs 36.4% ) and BRAF (13.1% vs 9.2% ) mutations but lower frequency of APC mutations (67.5% vs 77.2%), TMB High (9.8% vs 12%) and dMMR/MSI-H (6.6% vs 8.9%) compared to MSLN-L tumors (q&lt;0.05). Further, MSLN-H tumors had higher MAPK activation score (MPAS) compared to MSLN-L tumors (1.72-fold, q&lt;0.05). Similarly, in the MSS only cohort, MSLN-H tumors had increased frequency of KRAS (62.2% vs 37.9%) BRAF (10.5% vs 5.0%) mutations but decreased APC mutations (70% vs 80.9%) (q&lt;0.05). MSLN-H tumors had higher expression of immune checkpoint genes ( CD274, PDCD1, PDCD1LG2, CTLA4, LAG3, HAVC2 and FOXP3, FC: 1.1-1.8) and T cell inflamed score (3.3-fold) (all q&lt;0.05). MSLN-H was associated with worse OS compared to MSLN-L in all CRC (934 vs 1253 days; HR: 1.21, 95% CI (1.10-1.32), p&lt;0.0001) and MSS CRC (972 vs 1168 days; HR: 1.15, 95% CI (1.06-1.23), p&lt;0.001) but not in MSI-H CRC (p=0.67). Conclusions: Our data suggest a strong association between MSLN expression and increased mutations in the RAS pathway and MAPK activation score, decreased mutations in WNT pathway genes, a differentially modulated immune tumor microenvironment, and poor overall survival. These findings support MSLN as a potential prognostic biomarker candidate in colorectal cancers pending further prospective validation.

The long non-coding RNA PANDAR regulates cell proliferation and epithelial-to-mesenchymal transition in glioma.

Glioma is one of the most aggressive intracranial tumors in the central nervous system. The long non-coding RNA P21 associated ncRNA DNA damage activated (PANDAR) has been reported to be an oncogene or tumor suppressor in several cancers. However, the prognostic value and biological function of PANDAR in glioma have not been described. Here, we report that expression of PANDAR is significantly up-regulated in glioma tissues and cell lines. PANDAR expression was correlated with tumor size (p=0.044) and World Health Organization (WHO) grades (p=0.005), as shown by chi-squared test. Moreover, significant upregulation of PANDAR was found to correlate with poor prognosis in glioma, as shown using Kaplan-Meier method and Cox multivariate survival analysis. Furthermore, PANDAR knockdown suppressed cell proliferation, G1/S transition, migration and invasion, and promoted apoptosis in glioma cell lines (U251 and U87). PANDAR knockdown decreased expression of CDK4, Bcl-2, N-cadherin and Vimentin, but increased E-cadherin expression in glioma cells. In conclusion, our data suggest PANDAR as a potential prognostic biomarker and therapeutic candidate for glioma.

A characteristic N-glycopeptide signature associated with diabetic cognitive impairment identified in a longitudinal cohort study

BackgroundIdentifying a biomarker for the decline in cognitive function in patients with diabetes is important. Therefore, we aimed to identify the N-glycopeptides on plasma proteins associated with diabetic cognitive impairment in participants in a longitudinal study using N-glycoproteomics. MethodsWe used samples from the 3-year SONIC (Septuagenarians, Octogenarians, Nonagenarians Investigation with Centenarians) longitudinal cohort study of older Japanese people in the general population. First, we placed the participants with diabetes into two groups: those that did or did not have cognitive decline over a 6-year period. Next, their plasma protein profiles were compared between baseline and the 6-year time point using two-dimensional fluorescence difference gel electrophoresis. Finally, an N-glycoproteomic study of the focused proteins was performed using an enrichment technique and liquid chromatography-tandem mass spectrometry. ResultsApproximately 500 N-glycopeptides, derived from 18 proteins, were identified in each sample, from among which we identified the N-glycopeptides that were associated with diabetic cognitive impairment using multivariate analysis. We found that N-glycopeptides with sialylated tri- or tetra-antennary glycans on alpha-2-macroglobulin, clusterin, serum paraoxonase/arylesterase 1, and haptoglobin were less abundant, whereas 3-sialylated tri-antennary N-glycopeptides on serotransferrin were more abundant. ConclusionN-glycopeptides with sialylated multi-antennary glycans comprise a characteristic signature associated with diabetic cognitive impairment. General significanceThe characterized N-glycopeptides represent potential biomarker candidates for diabetic cognitive impairment.

Glycoproteomic Analysis of Urinary Extracellular Vesicles for Biomarkers of Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) accounts for the most common form of primary liver cancer cases and constitutes a major health problem worldwide. The diagnosis of HCC is still challenging due to the low sensitivity and specificity of the serum α-fetoprotein (AFP) diagnostic method. Extracellular vesicles (EVs) are heterogeneous populations of phospholipid bilayer-enclosed vesicles that can be found in many biological fluids, and have great potential as circulating biomarkers for biomarker discovery and disease diagnosis. Protein glycosylation plays crucial roles in many biological processes and aberrant glycosylation is a hallmark of cancer. Herein, we performed a comprehensive glycoproteomic profiling of urinary EVs at the intact N-glycopeptide level to screen potential biomarkers for the diagnosis of HCC. With the control of the spectrum-level false discovery rate ≤1%, 756 intact N-glycopeptides with 154 N-glycosites, 158 peptide backbones, and 107 N-glycoproteins were identified. Out of 756 intact N-glycopeptides, 344 differentially expressed intact N-glycopeptides (DEGPs) were identified, corresponding to 308 upregulated and 36 downregulated N-glycopeptides, respectively. Compared to normal control (NC), the glycoproteins LG3BP, PIGR and KNG1 are upregulated in HCC-derived EVs, while ASPP2 is downregulated. The findings demonstrated that specific site-specific glycoforms in these glycoproteins from urinary EVs could be potential and efficient non-invasive candidate biomarkers for HCC diagnosis.

Identification of radiation responsive RBC membrane associated proteins (RMAPs) in whole-body γ-irradiated New Zealand white rabbits

This study is aimed to identify radiation-responsive RBC Membrane Associated Proteins (RMAPs) in Rabbits in vivo. Male New Zealand White rabbits were exposed to a single acute total body γ-radiation dose of 2 Gy at a dose rate of 0.746 Gy/min. Following this, at early time points of 6 h till the 7 d, RMAPs were collected and analyzed by MALDI-TOF-MS. Bioinformatics analysis was conducted to explore the biological functions of these proteins. Based on fold change, radiation responsiveness, GO, pathway enrichment, and hub position in the PPI network, we identified seven RMAPs as potential biomarker candidates viz., PVALB, PRKCB, GPD1, CP2G1, CSNK2B, ATP1B1, TPI1. As per KEGG enrichment, most of the proteins were implicated in cellular radiation response, oxidative damage, DNA repair, apoptosis, immune response, and cell signaling. This study forms the foundation for RMAPs-based Proteomic strategies for high throughput radiation bio-dosimetry for triage in the case of a radiological/nuclear incident.

Identification and validation of oxidative stress and immune-related hub genes in Alzheimer’s disease through bioinformatics analysis

Alzheimer’s disease (AD) is the leading cause of dementia in aged population. Oxidative stress and neuroinflammation play important roles in the pathogenesis of AD. Investigation of hub genes for the development of potential therapeutic targets and candidate biomarkers is warranted. The differentially expressed genes (DEGs) in AD were screened in GSE48350 dataset. The differentially expressed oxidative stress genes (DEOSGs) were analyzed by intersection of DEGs and oxidative stress-related genes. The immune-related DEOSGs and hub genes were identified by weighted gene co-expression network analysis (WGCNA) and protein–protein interaction (PPI) analysis, respectively. Enrichment analysis was performed by Gene Ontology and Kyoto Encyclopedia of Genes and Genomes. The diagnostic value of hub genes was assessed by receiver operating characteristic analysis and validated in GSE1297. The mRNA expression of diagnostic genes was determined by qRT-PCR analysis. Finally, we constructed the drug, transcription factors (TFs), and microRNA network of the diagnostic genes. A total of 1160 DEGs (259 up-regulated and 901 down-regulated) were screened in GSE48350. Among them 111 DEOSGs were identified in AD. Thereafter, we identified significant difference of infiltrated immune cells (effector memory CD8 T cell, activated B cell, memory B cell, natural killer cell, CD56 bright natural killer cell, natural killer T cell, plasmacytoid dendritic cell, and neutrophil) between AD and control samples. 27 gene modules were obtained through WGCNA and turquoise module was the most relevant module. We obtained 66 immune-related DEOSGs by intersecting turquoise module with the DEOSGs and identified 15 hub genes through PPI analysis. Among them, 9 hub genes (CCK, CNR1, GAD1, GAP43, NEFL, NPY, PENK, SST, and TAC1) were identified with good diagnostic values and verified in GSE1297. qRT-PCR analysis revealed the downregulation of SST, NPY, GAP43, CCK, and PENK and upregulation of NEFL in AD. Finally, we identified 76 therapeutic agents, 152 miRNAs targets, and 91 TFs regulatory networks. Our study identified 9 key genes associated with oxidative stress and immune reaction in AD pathogenesis. The findings may help to provide promising candidate biomarkers and therapeutic targets for AD.