Identification of early diagnostic biomarkers via WGCNA in gastric cancer.

BACKGROUND: Gastric cancer (GC) is the world's second-leading cause of cancer-related mortality, continuing to make it a serious healthcare concern. Even though the prevalence of GC reduces, the prognosis for GC patients remains poor in terms of a lack of reliable biomarkers to diagnose early GC and predict chemosensitivity and recurrence. METHODS AND MATERIAL: We integrated the gene expression patterns of gastric cancers from four RNAseq datasets (GSE113255, GSE142000, GSE118897, and GSE130823) from Gene Expression Omnibus (GEO) database to recognize differentially expressed genes (DEGs) between normal and GC samples. A gene co-expression network was built using weighted co-expression network analysis (WGCNA). Furthermore, RT-qPCR was performed to validate the in silico results. RESULTS: The red modules in GSE113255, Turquoise in GSE142000, Brown in GSE118897, and the green-yellow module in GSE130823 datasets were found to be highly correlated with the anatomical site of GC. ITGAX, CCL14, ADHFE1, and HOXB13) as the hub gene are differentially expressed in tumor and non-tumor gastric tissues in this study. RT-qPCR demonstrated a high level of the expression of this gene. CONCLUSION: The expression levels of ITGAX, CCL14, ADHFE1, and HOXB13 in GC tumor tissues are considerably greater than in adjacent normal tissues. Systems biology approaches identified that these genes could be possible GC marker genes, providing ideas for other experimental studies in the future.

D-2-hydroxyglutarate dehydrogenase in breast carcinoma as a potent prognostic marker associated with proliferation.

BACKGROUND: D-2-hydroxyglutarate dehydrogenase (D2HGDH) catalyzes D-2-hydroxyglutarate to α-ketoglutarate and is involved in the regulation of cellular energy and biosynthetic intermediates. Previously, D2HGDH was reported to decrease 2-hydroxyglutarate level in breast carcinoma cells, but no other report has examined D2HGDH in breast carcinoma, and its significance remains unknown. METHODS: We first immunolocalized D2HGDH in 224 invasive breast carcinomas and evaluated its clinicopathological significance. We next examined associations between gene expression of D2HGDH and α-ketoglutarate-dependent dioxygenases in 23 breast carcinoma tissues using the gene expression profile data. Finally, we examined the effects of D2HGDH on the proliferation in three breast carcinoma cells. RESULTS: D2HGDH immunoreactivity was detected in 49% of invasive breast carcinomas, and the immunohistochemical D2HGDH status was positively associated with histological grade, HER2 and Ki-67, while it was inversely associated with estrogen receptor. Moreover, it was significantly associated with worse prognosis of the breast cancer patients, and it turned out to be an independent prognostic factor for both the disease-free and breast cancer-specific survival in these patients. Gene expression profile data revealed that D2HGDH expression was positively associated with the expression of 6 α-ketoglutarate-dependent dioxygenases (KDM3A, PLOD1, EGLN2, ALKBH1, ASPH and ALKBH7). Consequent in vitro experiments demonstrated that D2HGDH overexpression significantly increased the cell proliferation activity of MCF-7, T47D and MDA-MB-231 cells. CONCLUSION: These results suggest that D2HGDH plays an important role in the growth of breast carcinoma, possibly through regulating functions of α-ketoglutarate-dependent dioxygenases, and that D2HGDH status is a potent worse prognostic factor in breast cancer patients.

C-terminal binding protein-2 is a prognostic marker for lung adenocarcinomas.

C-terminal binding protein-2 (CtBP2) a transcriptional corepressor, has been reported to involve in tumorigenesis and progression and predict a poor prognosis in several human cancers. However, few studies on CtBP2 in lung cancer tissues have been performed. In the present study, we first explored the CtBP2 gene expression profile from the the cancer genome atlas (TCGA) datasets, then western blot analysis and immunohistochemistry were performed to investigate and verified whether lung adenocarcinoma (LUAD) tissues exhibit deregulated CtBP2 expression. We evaluated the correlations between CtBP2 expression and the clinicopathological characteristics, and Kaplan-Meier survival analyses were performed to estimate the effect of CtBP2 expression on prognosis of LUAD patients. The results revealed that CtBP2 expression was significantly upregulated in LUAD tissues compared with normal lung tissues. Furthermore, increasing CtBP2 expression in LUAD was significantly associated with tumor differentiation (P = .028), tumor node metastasis (TNM) stage (P = .042). CtBP2 expression was significantly correlated with LUAD patients' survival (P = .028). In conclusion, the present study revealed that CtBP2 protein is a novel prognostic marker for LUAD. A further large-scale study is needed to confirm the present results.

Increased DHRS12 expression independently predicts poor survival in patients with high-grade serous ovarian cancer.

AIM: To explore the expression profile of some DHRS genes in high-grade serous ovarian cancer (SOVC) and to study their prognostic values. PATIENTS & METHODS: A retrospective bioinformatic analysis was performed using data in the Gene Expression Omnibus, the Human Protein Atlas and the Cancer Genome Atlas-Ovarian Cancer. RESULTS: Increased DHRS12 expression was an independent indicator of poor overall survival (hazard ratio [HR]: 1.265, 95% CI: 1.075-1.488; p = 0.005) and recurrence-free survival (RFS; HR: 2.242, 95%CI: 1.464-3.432; p < 0.001) in patients with high-grade SOVC. DNA deletion was associated with decreased DHRS12 expression, as well as the best overall survival and RFS among the three copy number alteration groups. CONCLUSION: DHRS12 might serve as a valuable prognostic biomarker in high-grade SOVC.

A rapid procedure for the in situ assay of periplasmic, PQQ-dependent methanol dehydrogenase in intact single bacterial colonies.

Mechanistic details of methanol oxidation catalyzed by the periplasmically-located pyrroloquinoline quinone-dependent methanol dehydrogenase of methylotrophs can be elucidated using site-directed mutants. Here, we present an in situ colony assay of methanol dehydrogenase, which allows robotic screening of large populations of intact small colonies, and regrowth of colonies for subsequent analysis.

Prostaglandin pathway gene expression in human placenta, amnion and choriodecidua is differentially affected by preterm and term labour and by uterine inflammation.

BACKGROUND: Elucidation of the biochemical pathways involved in activation of preterm and term human labour would facilitate the development of effective management and inform judgements regarding the necessity for preterm tocolysis and post-term induction. Prostaglandins act at all stages of human reproduction, and are potentially activators of labour. METHODS: Expression of 15 genes involved in prostaglandin synthesis, transport and degradation was measured by qPCR using tissue samples from human placenta, amnion and choriodecidua at preterm and full-term vaginal and caesarean delivery. Cellular localisation of eight prostaglandin pathway proteins was determined by immunohistochemistry. RESULTS: Expression of prostaglandin pathway genes was differentially affected by factors including gestational age at delivery, and the incidence and duration of labour. Chorioamnionitis/deciduitis was associated with upregulation of PTGS2 (prostaglandin-endoperoxide synthase 2 (prostaglandin G/H synthase and cyclooxygenase)), along with the inflammatory genes IL8 (interleukin 8), S100A8 (S100 calcium binding protein A8) and TLR2 (toll-like receptor 2), in amnion and choriodecidua, and with downregulation of CBR1 (carbonyl reductase 1) and HPGD (hydroxyprostaglandin dehydrogenase 15-(NAD)) in choriodecidua. Protein localisation differed greatly between the various maternal and fetal cell types. CONCLUSIONS: Preterm and term labour are associated with distinct prostaglandin pathway expression profiles; inflammation provokes specific changes, unrelated to the presence of labour; spontaneous and induced term labour are indistinguishable.

Interindividual variability in the cardiac expression of anthracycline reductases in donors with and without Down syndrome.

PURPOSE: The intracardiac synthesis of anthracycline alcohol metabolites (e.g., daunorubicinol) contributes to the pathogenesis of anthracycline-related cardiotoxicity. Cancer patients with Down syndrome (DS) are at increased risk for anthracycline-related cardiotoxicity. We profiled the expression of anthracycline metabolizing enzymes in hearts from donors with- and without- DS. METHODS: Cardiac expression of CBR1, CBR3, AKR1A1, AKR1C3 and AKR7A2 was examined by quantitative real time PCR, quantitative immunoblotting, and enzyme activity assays using daunorubicin. The CBR1 polymorphism rs9024 was investigated by allelic discrimination with fluorescent probes. The contribution of CBRs/AKRs proteins to daunorubicin reductase activity was examined by multiple linear regression. RESULTS: CBR1 was the most abundant transcript (average relative expression; DS: 81%, non-DS: 58%), and AKR7A2 was the most abundant protein (average relative expression; DS: 38%, non-DS: 35%). Positive associations between cardiac CBR1 protein levels and daunorubicin reductase activity were found for samples from donors with- and without- DS. Regression analysis suggests that sex, CBR1, AKR1A1, and AKR7A2 protein levels were significant contributors to cardiac daunorubicin reductase activity. CBR1 rs9024 genotype status impacts on cardiac CBR1 expression in non-DS hearts. CONCLUSIONS: CBR1, AKR1A1, and AKR7A2 protein levels point to be important determinants for predicting the synthesis of cardiotoxic daunorubicinol in heart.

Quantitative evaluation of aldo-keto reductase expression in hepatocellular carcinoma (HCC) cell lines.

The involvement of aldo-keto reductases (AKRs) in tumorigenesis is widely reported, but their roles in the pathological process are not generally recognized due to inconsistent measurements of their expression. To overcome this problem, we simultaneously employed real-time PCR to examine gene expression and multiple reaction monitoring (MRM) of mass spectrometry (MS) to examine the protein expression of AKRs in five different hepatic cell lines. These include one relatively normal hepatic cell line, L-02, and four hepatocellular carcinoma (HCC) cell lines, HepG2, HuH7, BEL7402 and SMMC7721. The results of real-time PCR showed that expression of genes encoding the AKR1C family members rather than AKR1A and AKR1B was associated with tumor, and most of genes encoding AKRs were highly expressed in HuH7. Similar observations were obtained through MRM. Different from HuH7, the protein abundance of AKR1A and AKR1B was relatively consistent among the other four hepatic cell lines, while protein expression of AKR1C varied significantly compared to L-02. Therefore, we conclude that the abundant distribution of AKR1C proteins is likely to be associated with liver tumorigenesis, and the AKR expression status in HuH7 is completely different from other liver cancer cell lines. This study, for the first time, provided both overall and quantitative information regarding the expression of AKRs at both mRNA and protein levels in hepatic cell lines. Our observations put the previous use of AKRs as a biomarker into question since it is only consistent with our data from HuH7. Furthermore, the data presented herein demonstrated that quantitative evaluation and comparisons within a protein family at both mRNA and protein levels were feasible using current techniques.

Glyoxylate reductase/hydroxypyruvate reductase: a novel prognostic marker for hepatocellular carcinoma patients after curative resection.

OBJECTIVE: Glyoxylate reductase/hydroxypyruvate reductase (GRHPR) is a key enzyme in the glyoxylate cycle. Its deficiency causes primary hyperoxaluria type 2. We first noticed that GRHPR was also lost in human hepatocellular carcinoma (HCC) and proliferative HCC cells. The aim of the present study was to investigate the potential clinical utility of GRHPR in HCC. METHODS: The expression of GRHPR in tissues and cells was detected by Western blotting. Immunohistochemistry was utilized to examine the expression patterns of GRHPR and Ki-67 in a surgical cohort of HCC and adjacent liver tissues. RESULTS: We demonstrated that GRHPR showed a lower expression in tumor tissues than in nontumoral tissues. GRHPR was negatively correlated with Ki-67 (R(2) = 0.771, p < 0.05) and GRHPR was reduced in proliferative Huh7 cells (p < 0.05). Patients with negative GRHPR both in tumor tissues and nontumoral tissues had a significantly shorter survival time than those with positive GRHPR (p < 0.001). Multivariate analysis established that GRHPR was detected in nontumoral tissues as an independent prognostic factor for patients with HCC. CONCLUSIONS: Our findings suggest that the GRHPR defect in noncancerous tissues may represent an independent predictor of poor survival for HCC patients after curative resection and that there may be a link between GRHPR and prognosis of HCC patients.

Enzymatic assay for quantitative analysis of (D)-2-hydroxyglutarate.

Levels of (D)-2-hydroxyglutarate [D2HG, (R)-2-hydroxyglutarate] are increased in some metabolic diseases and in neoplasms with mutations in the isocitrate dehydrogenase 1 (IDH1) and isocitrate dehydrogenase 2 (IDH2) genes. Determination of D2HG is of relevance to diagnosis and monitoring of disease. Standard detection methods of D2HG levels are liquid-chromatography-mass spectrometry or gas-chromatography-mass spectrometry. Here we present a rapid, inexpensive and sensitive enzymatic assay for the detection of D2HG levels. The assay is based on the conversion of D2HG to α-ketoglutarate (αKG) in the presence of the enzyme (D)-2-hydroxyglutarate dehydrogenase (HGDH) and nicotinamide adenine dinucleotide (NAD(+)). Determination of D2HG concentration is based on the detection of stoichiometrically generated NADH. The quantification limit of the enzymatic assay for D2HG in tumor tissue is 0.44 µM and in serum 2.77 µM. These limits enable detection of basal D2HG levels in human tumor tissues and serum without IDH mutations. Levels of D2HG in frozen and paraffin-embedded tumor tissues containing IDH mutations or in serum from acute myeloid leukemia patients with IDH mutations are significantly higher and can be easily identified with this assay. In conclusion, the assay presented is useful for differentiating basal from elevated D2HG levels in tumor tissue, serum, urine, cultured cells and culture supernatants.

A system-wide investigation of the dynamics of Wnt signaling reveals novel phases of transcriptional regulation.

Aberrant Wnt signaling has been implicated in a wide variety of cancers and many components of the Wnt signaling network have now been identified. Much less is known, however, about how these proteins are coordinately regulated. Here, a broad, quantitative, and dynamic study of Wnt3a-mediated stimulation of HEK 293 cells revealed two phases of transcriptional regulation: an early phase in which signaling antagonists were downregulated, providing positive feedback, and a later phase in which many of these same antagonists were upregulated, attenuating signaling. The dynamic expression profiles of several response genes, including MYC and CTBP1, correlated significantly with proliferation and migration (P<0.05). Additionally, their levels tracked with the tumorigenicity of colon cancer cell lines and they were significantly overexpressed in colorectal adenocarcinomas (P<0.05). Our data highlight CtBP1 as a transcription factor that contributes to positive feedback during the early phases of Wnt signaling and serves as a novel marker for colorectal cancer progression.

Differential expression of prostaglandin (PG) synthesis enzymes in conceptus during peri-implantation period and endometrial expression of carbonyl reductase/PG 9-ketoreductase in the pig.

Prostaglandins (PGs) play a pivotal role in luteolysis, maternal recognition of pregnancy, and implantation. In many species, including pigs, both conceptus (embryo and associated membranes) and endometrium synthesize PGE(2), which may antagonize PGF(2alpha) by playing a luteotropic/antiluteolytic role. Previously, we have reported expression profiles of PG G/H synthases (PGHS-1 and PGHS-2), PGE synthase (mPGES-1), and PGF synthase (PGFS) in the endometrium of cyclic and pregnant pigs. In the present study, expression of above-mentioned PG synthesis enzymes and PG 9-ketoreductase (CBR1), which converts PGE(2) into PGF(2alpha), and the PGE(2)/PGF(2alpha) ratios were investigated in porcine peri- and post-implantation conceptuses. Furthermore, expression of CBR1 was examined in the endometrium. PGHS-2 and mPGES-1 were upregulated, and PGHS-1, PGFS, and CBR1 were downregulated in conceptuses during trophoblastic elongation. A second increase of mPGES-1 mRNA occurred after days 20-21 of pregnancy. After initiation of implantation, expression of PGHS-1, PGFS, and CBR1 in conceptuses increased and remained higher until days 24-25 of pregnancy. Comparison of the endometrial CBR1 protein expression in cyclic and pregnant gilts revealed upregulation on days 16-17 of the cycle and downregulation on days 10-11 of pregnancy. In conclusion, reciprocal expression of PGHS-2, mPGES-1, PGFS, and CBR1 in day 10-13 conceptuses and decrease of endometrial CBR1 may be important in increasing the PGE(2)/PGF(2alpha) ratio during maternal recognition of pregnancy. This study indicates that PGE(2) produced via PGHS-2 and mPGES-1 in conceptus may be involved in corpus luteum control. Moreover, high expression of conceptus PGHS-1, mPGES-1, PGFS, and CBR1 after initiation of implantation suggests their significant role in placentation.

An anamorph of the white-rot fungus Bjerkandera adusta capable of colonizing and degrading compact disc components.

A Geotrichum-like fungus isolated from a biodeteriorated compact disc (CD) was able to degrade in vitro the components of different CD types. The fungal hyphae inside the CD fragments grew through the aluminium layer and produced the solubilization of this metal. Furthermore, examination of CDs by scanning electron microscopy showed that the fungus was able to destroy the pits and lands structures grooved in the polycarbonate layer, confirming degradation of this aromatic polymer. The fungus secretes aryl-alcohol oxidase and Mn2+-oxidizing peroxidase, two kinds of oxidoreductases characteristic of ligninolytic basidiomycetes. Analysis of the ITS region of ribosomal DNA, as well as the morphological characteristics, the lack of sexual forms and the profile of enzymes secreted in liquid medium identified the fungus as a Geotrichum-like anamorph of Bjerkandera adusta (Willd.) P. Karst.

Proteome analysis of Sulfolobus solfataricus P2 propanol metabolism.

Sulfolobus solfataricus P2 is able to metabolize n-propanol as the sole carbon source. An average n-propanol consumption rate of 9.7 and 3.3 mg/L/hr was detected using GC-MS analysis from S. solfataricus cultures grown in 0.40 and 0.16% w/v n-propanol, respectively. The detection of propionaldehyde, the key intermediate of n-propanol degradation, produced at a rate of 1.3 and 1.0 mg/L/hr in 0.40 and 0.16% w/v n-propanol cultures, further validated the ability of S. solfataricus to utilize n-propanol. The translational and transcriptional responses of S. solfataricus grown on n-propanol versus glucose were also investigated using quantitative RT-PCR and iTRAQ approaches. Approximately 257 proteins with > or =2 MS/MS spectra were identified and quantified via iTRAQ. The global quantitative proteome overview obtained showed significant up-regulation of acetyl-CoA synthetases, propionyl-CoA carboxylase, and methylmalonyl-CoA mutase enzymes. This led to the proposition that the propionyl-CoA formed from n-propanol degradation is catabolised into the citrate cycle (central metabolism) via succinyl-CoA intermediates. In contrast, evidence obtained from these analysis approaches and in vivo stable isotope labeling experiments, suggests that S. solfataricus is only capable of converting isopropyl alcohol to acetone (and vice versa) but lacks the ability to further metabolize these compounds.

Role of the unique N-terminal domain of CtBP2 in determining the subcellular localisation of CtBP family proteins.

BACKGROUND: CtBP1 and CtBP2 are transcriptional co-repressors that modulate the activity of a large number of transcriptional repressors via the recruitment of chromatin modifiers. Many CtBP-regulated proteins are involved in pathways associated with tumorigenesis, including TGF-beta and Wnt signalling pathways and cell cycle regulators such as RB/p130 and HDM2, as well as adenovirus E1A. CtBP1 and CtBP2 are highly similar proteins, although evidence is emerging that their activity can be differentially regulated, particularly through the control of their subcellular localisation. CtBP2s from diverse species contain a unique N-terminus, absent in CtBP1 that plays a key role in controlling the nuclear-cytoplasmic distribution of the protein. RESULTS: Here we show that amino acids (a.a.) 4-14 of CtBP2 direct CtBP2 into an almost exclusively nuclear distribution in cell lines of diverse origins. Whilst this sequence contains similarity to known nuclear localisation motifs, it cannot drive nuclear localisation of a heterologous protein, but rather has been shown to function as a p300 acetyltransferase-dependent nuclear retention sequence. Here we define the region of CtBP2 required to co-operate with a.a. 4-14 to promote CtBP2 nuclear accumulation as being within a.a. 1-119. In addition, we show that a.a. 120-445 of CtBP2 can also promote CtBP2 nuclear accumulation, independently of a.a. 4-14. Finally, CtBP1 and CtBP2 can form heterodimers, and we show that the interaction with CtBP2 is one mechanism whereby CtBP1 can be recruited to the nucleus. CONCLUSION: Together, these findings represent key distinctions in the regulation of the functions of CtBP family members that may have important implications as to their roles in development, and cell differentiation and survival.

Electrosynthesized poly(pyrrole)/poly(2-naphthol) bilayer membrane as an effective anti-interference layer for simultaneous determination of acethylcholine and choline by a dual electrode amperometric biosensor.

Several neural diseases appear related to the neurotransmitter acethylcholine (ACh) and its metabolite choline (Ch) brain levels so that their simultaneous determination is essential. A cross-talk and interference free dual electrode amperometric biosensor for the simultaneous determination of both analytes has been developed. Acetylcholinesterase (AChE) and choline oxidase (ChO) were immobilized by glutaraldehyde co-crosslinking with bovine serum albumin. A very efficient rejection of electroactive interferents has been achieved by a novel electrosynthesized polymeric bilayer membrane composed by overoxidised poly(pyrrole) and poly(2-naphthol) films. Sensitivities towards several electroactive interferents ranged from ca. 0.04% (e.g. ascorbate) to ca. 0.3% (e.g. dopamine) of those relevant to ACh and Ch (11 and 15 microA/microM, respectively). Detection limits (at S/N=3) in flow injection analysis were ca. 100 nM for both ACh and Ch at the ChO-AChE electrode and ca. 40 nM for Ch at the ChO sensor. Biosensor performances appear more than adequate for brain tissue homogenates and cerebrospinal fluids analysis where average levels in the low micromolar range are typically found.

Biosensing approach for alcohol determination using immobilized alcohol oxidase.

Alcohol oxidase (AOD) was immobilized in polypyrrole (PPy) and a random copolymer containing 3-methylthienyl methacrylate and p-vinylbenzyloxy poly(ethyleneoxide) matrices. Immobilization of enzyme was performed via entrapment in conducting polymers during electrochemical polymerization of pyrrole through the thiophene moiety of the copolymer. Three different alcohols, namely methanol, ethanol and n-propanol, were used as substrates. Maximum reaction rates, Michaelis-Menten constants, optimum temperature and pH values, operational stabilities and shelf life of the enzyme electrodes were investigated.

Detection and identification of tumor-associated protein variants in human hepatocellular carcinomas.

The proteomic approach is a valuable tool to detect and identify proteins that are associated with cancer. In previous investigations on experimentally induced rat hepatomas, we detected aldose reductase-like protein (ARLP) as a highly significant marker protein. Our present study was intended to look for the presence of similar tumor-associated marker proteins on human hepatocellular carcinomas (HCC). We found several novel tumor-associated protein variants that represent members of the aldo-keto reductase (AKR) superfamily. Human aldose reductase-like protein-1 (hARLP-1) was the most prominent tumor-associated AKR member detected in HCC by 2-dimensional electrophoresis (2-DE) and identified by mass spectrometric fingerprinting. The enzyme was found in 4 distinct forms (hARLP-1, 36/7.4 (kd/pI); hARLP-2, 36/7.2; hARLP-3, 36/6.4; and hARLP-4, 33/7.35). In addition, a human aldose reductase-like protein (hARLP-5, 36/7.6) was identified that differed from hARLP-1 by 1 amino acid (D313N), indicating 2 allelic forms of the human aldose reductase-like gene. A novel antibody directed against common parts of the hARLPs revealed hARLP reactivity in human HCC by immunohistochemistry. Furthermore, aldose reductase (AR) was identified and characterized as a tumor-associated variant. In conclusion, in all investigated human HCCs at least one of the various types of the described tumor-associated proteins of the AKR superfamily was clearly present. Of these HCC samples, 95% were positive for hARLPs as proven by 2-DE analysis and/or by use of the antibody directed against hARLP. Thus, hARLP is a strong candidate for use as an immunohistochemical diagnostic marker of human HCC.