Tubulin beta 3 and 4 are involved in the generation of early fibrotic stages.
Wawro Marta E,Sobierajska Katarzyna,Ciszewski Wojciech M,Wagner Waldemar,Frontczak Marta,Wieczorek Katarzyna,Niewiarowska Jolanta
The endothelial-mesenchymal transition (EndMT) is a fundamental cellular mechanism that occurs under both physiological and pathological conditions and includes the fibrotic stages of numerous organs, namely, the skin, kidneys, heart, lungs and liver. Endothelial cells that undergo EndMT are one of the main source of (myo)fibroblasts in fibrotic tissues. A critical step in cellular transdifferentiation is morphological change, which is engineered by the reorganization of cytoskeletal elements such as microtubules. These dynamic structures consist of αβ-tubulin heterodimers that are also involved in cellular movement and intracellular trafficking, processes modulated during EndMT. One fundamental mechanism that underlies microtubule stabilization is the regulation of the levels of α and β-tubulin. However, little is known about the roles of specific tubulin isotypes in the development of EndMT-based diseases. This study provides the first evidence that the upregulation of TUBB3 and TUBB4 is coupled with increased cell migration in EndMT-induced HMEC-1 cells. Immunochemical analysis reveals that these tubulins are upregulated in the early stages of EndMT, and siRNA analysis indicates that they are engaged in the generation of mesenchymal behavior via the enhancement of cell migration. This modulation seems to be especially important in wound healing. Finally, cell surface analysis reveals that TUBB3 and TUBB4 are necessary for the transport and proper localization of N-cadherin within the plasma membrane. We believe that our results will be valuable for the development of effective new anti-fibrotic therapies.
Genes targeted by the Hedgehog-signaling pathway can be regulated by Estrogen related receptor β.
Lu Yuan,Li Jilong,Cheng Jianlin,Lubahn Dennis B
BMC molecular biology
BACKGROUND:Nuclear receptor family member, Estrogen related receptor β, and the Hedgehog signal transduction pathway are both reported to relate to tumorigenesis and induced pluripotent stem cell reprogramming. We hypothesize that Estrogen related receptor β can modulate the Hedgehog signaling pathway and affect Hedgehog driven downstream gene expression. RESULTS:We established an estrogen related receptor β-expressing Hedgehog-responsive NIH3T3 cell line by Esrrb transfection, and performed mRNA profiling using RNA-Seq after Hedgehog ligand conditioned medium treatment. Esrrb expression altered 171 genes, while Hedgehog signaling activation alone altered 339 genes. Additionally, estrogen related receptor β expression in combination with Hedgehog signaling activation affects a group of 109 Hedgehog responsive mRNAs, including Hsd11b1, Ogn, Smoc2, Igf1, Pdcd4, Igfbp4, Stmn1, Hp, Hoxd8, Top2a, Tubb4b, Sfrp2, Saa3, Prl2c3 and Dpt. CONCLUSIONS:We conclude that Estrogen related receptor β is capable of interacting with Hh-signaling downstream targets. Our results suggest a new level of regulation of Hedgehog signaling by Estrogen related receptor β, and indicate modulation of Estrogen related receptor β can be a new strategy to regulate various functions driven by the Hedgehog signaling pathway.
A Study of Differential Expression of Testicular Genes in Various Reproductive Phases of Hemidactylus flaviviridis (Wall Lizard) to Derive Their Association with Onset of Spermatogenesis and Its Relevance to Mammals.
Sarkar Hironmoy,Arya Satyapal,Rai Umesh,Majumdar Subeer S
Testis of Hemidactylus flaviviridis, commonly known as Indian wall lizard, displays a lack of cellular and metabolic activity in regressed phase of testis during non-breeding season of the year. Retracted Sertoli cells (Sc), fibroid myoid cells and pre-meiotic resting spermatogonia are observed in such testis. This situation is akin to certain forms of infertility in men where hormone supplementation fails to generate sperm despite the presence of Sc and germ cells (Gc) in testis. In testis of lizard, spermatogenesis is reinitiated upon increased level of hormones during appropriate season (phase of recrudescence). Study of genes associated with generation of sperm, from regressed adult testis in lizard, may provide valuable information for understanding certain forms of male idiopathic infertility. Subtractive hybridization using testicular RNA obtained from the regressed and active phases of lizard reproductive cycle led to identify eight partial mRNA sequences that showed sequence homology with mice genes. We further evaluated the gene expression prolife by real-time PCR in three different reproductive phases of H. flaviviridis: regressed (pre-meiotic), recrudescent (meiotic) and active (post meiotic), for comparison with the corresponding testicular phases found in testis of 5 days (pre-meiotic), 20 days (meiotic) and 60 days (post-meiotic) old mouse. This is the first report where genes associated with progression of spermatogenesis during active phase, which follows a regressed state of adult testis, were identified in lizard and found to be conserved in mouse. Six important genes, Hk1, Nme5, Akap4, Arih1, Rassf7 and Tubb4b were found to be strictly associated with active spermatogenesis in both mouse and lizard. Factors interfering with the expression of any of these genes may potentially abrogate the process of spermatogenesis leading to infertility. Such information may shed light on unknown causes of idiopathic male infertility.
Comparative proteomic analyses of two reovirus T3D subtypes and comparison to T1L identifies multiple novel proteins in key cellular pathogenic pathways.
Berard Alicia R,Severini Alberto,Coombs Kevin M
Viruses induce changes in the host to facilitate replication and evade the immune response. These changes are reflected by the host's proteome, including differences in protein abundance. Focusing on up and down regulated proteins after a virus infects the cell will lead to a characterization of the host response to infection, and may give insight into how viruses modulate proteins to evade host defense responses. We previously used SILAC to examine host proteomic changes in protein abundance in HEK293 cells infected with reovirus type 1, strain Lang (T1L). For the present study, we extended this analysis by determining cell protein alterations induced by two different reovirus subtypes, a less pathogenic type 3 Dearing (T3D(F)) isolate, and a more pathogenic isolate named T3D(C) that is presently in clinical trials as an anti-cancer oncolytic agent. This comparison of host proteome regulation showed that T3D(C) had a more marked effect on DNA replication proteins, recombination and repair, as well as immunological, apoptotic, and survival cell functions. We also identified several proteins not previously identified in any virus infection; branched chain amino-acid transaminase 2 (BCAT), paternally expressed 10 (PEG10), target of myb1 (TOM1), histone cluster 2 H4b (HIST2H4B) and tubulin beta 4B (TUBB4B).
Proteomic analysis of mucopolysaccharidosis I mouse brain with two-dimensional polyacrylamide gel electrophoresis.
Ou Li,Przybilla Michael J,Whitley Chester B
Molecular genetics and metabolism
Mucopolysaccharidosis type I (MPS I) is due to deficiency of α-l-iduronidase (IDUA) and subsequent storage of undegraded glycosaminoglycans (GAG). The severe form of the disease, known as Hurler syndrome, is characterized by mental retardation and neurodegeneration of unknown etiology. To identify potential biomarkers and unveil the neuropathology mechanism of MPS I disease, two-dimensional polyacrylamide gel electrophoresis (PAGE) and nanoliquid chromatography-tandem mass spectrometry (nanoLC-MS/MS) were applied to compare proteome profiling of brains from MPS I and control mice (5-month old). A total of 2055 spots were compared, and 25 spots (corresponding to 50 different proteins) with a fold change ≥3.5 and a p value <0.05 between MPS I and control mice were further analyzed by nanoLC-MS/MS. These altered proteins could be divided into three major groups based on Gene Ontology (GO) terms: proteins involved in metabolism, neurotransmission and cytoskeleton. Cytoskeletal proteins including ACTA1, ACTN4, TUBB4B and DNM1 were significantly downregulated. STXBP1, a regulator of synaptic vesicle fusion and docking was also downregulated, indicating impaired synaptic transmission. Additionally, proteins regulating Ca and H homeostasis including ATP6V1B2 and RYR3 were downregulated, which may be related to disrupted autophagic and endocytotic pathways. Notably, there is no altered expression in proteins associated with cell death, ubiquitin or inflammation. These results for the first time highlight the important role of alterations in metabolism pathways, intracellular ionic homeostasis and the cytoskeleton in the neuropathology of MPS I disease. The proteins identified in this study would provide potential biomarkers for diagnostic and therapeutic studies of MPS I.
Bioinformatics analysis of differentially expressed proteins in prostate cancer based on proteomics data.
Chen Chen,Zhang Li-Guo,Liu Jian,Han Hui,Chen Ning,Yao An-Liang,Kang Shao-San,Gao Wei-Xing,Shen Hong,Zhang Long-Jun,Li Ya-Peng,Cao Feng-Hong,Li Zhi-Guo
OncoTargets and therapy
We mined the literature for proteomics data to examine the occurrence and metastasis of prostate cancer (PCa) through a bioinformatics analysis. We divided the differentially expressed proteins (DEPs) into two groups: the group consisting of PCa and benign tissues (P&b) and the group presenting both high and low PCa metastatic tendencies (H&L). In the P&b group, we found 320 DEPs, 20 of which were reported more than three times, and DES was the most commonly reported. Among these DEPs, the expression levels of FGG, GSN, SERPINC1, TPM1, and TUBB4B have not yet been correlated with PCa. In the H&L group, we identified 353 DEPs, 13 of which were reported more than three times. Among these DEPs, MDH2 and MYH9 have not yet been correlated with PCa metastasis. We further confirmed that DES was differentially expressed between 30 cancer and 30 benign tissues. In addition, DEPs associated with protein transport, regulation of actin cytoskeleton, and the extracellular matrix (ECM)-receptor interaction pathway were prevalent in the H&L group and have not yet been studied in detail in this context. Proteins related to homeostasis, the wound-healing response, focal adhesions, and the complement and coagulation pathways were overrepresented in both groups. Our findings suggest that the repeatedly reported DEPs in the two groups may function as potential biomarkers for detecting PCa and predicting its aggressiveness. Furthermore, the implicated biological processes and signaling pathways may help elucidate the molecular mechanisms of PCa carcinogenesis and metastasis and provide new targets for clinical treatment.
Insulin during in vitro oocyte maturation has an impact on development, mitochondria, and cytoskeleton in bovine day 8 blastocysts.
Laskowski Denise,Båge Renée,Humblot Patrice,Andersson Göran,Sirard Marc-André,Sjunnesson Ylva
Insulin is a key metabolic hormone that controls energy homeostasis in the body, including playing a specific role in regulating reproductive functions. Conditions associated with hyperinsulinemia can lower developmental rates in bovine in vitro embryo production and are linked to decreased fertility in humans, as in cases of obesity or type 2 diabetes. Embryo quality is important for fertility outcome and it can be assessed by choosing scoring standards for various characteristics, such as developmental stage, quality grade, cell number, mitochondrial pattern or actin cytoskeleton structure. Changes in the embryo's gene expression can reflect environmental impacts during maturation and may explain morphological differences. Together with morphological evaluation, this could enable better assessment and possibly prediction of the developmental potential of the embryo. The aim of this study was to use a bovine model to identify potential gene signatures of insulin-induced changes in the embryo by combining gene expression data and confocal microscopy evaluation. Bovine embryos were derived from oocytes matured in two different insulin concentrations (10 µg mL and 0.1 µg mL), then stained to distinguish f-Actin, DNA and active mitochondria. The total cell number of the embryo, quality of the actin cytoskeleton and mitochondrial distribution were assessed and compared to an insulin-free control group. A microarray-based transcriptome analysis was used to investigate key genes involved in cell structure, mitochondrial function and cell division. Our results indicate that insulin supplementation during oocyte maturation leads to lower blastocyst rates and a different phenotype, characterised by an increased cell number and different actin and mitochondrial distribution patterns. These changes were reflected by an up-regulation of genes involved in cell division (MAP2K2; DHCR7), cell structure (LMNA; VIM; TUBB2B; TUBB3; TUBB4B) and mitochondrial activation (ATP5D; CYP11A1; NDUFB7; NDUFB10; NDUFS8). Taken together, we hypothesise that the increased proliferation in the insulin-treated groups might impair the developmental potential of the embryos by inducing metabolic stress on the molecular level, which could be detrimental for the survival of the embryo.
Analysis of Post-Traumatic Brain Injury Gene Expression Signature Reveals Tubulins, Nfe2l2, Nfkb, Cd44, and S100a4 as Treatment Targets.
Lipponen Anssi,Paananen Jussi,Puhakka Noora,Pitkänen Asla
We aimed to define the chronically altered gene expression signature of traumatic brain injury (TBI-sig) to discover novel treatments to reverse pathologic gene expression or reinforce the expression of recovery-related genes. Genome-wide RNA-sequencing was performed at 3 months post-TBI induced by lateral fluid-percussion injury in rats. We found 4964 regulated genes in the perilesional cortex and 1966 in the thalamus (FDR < 0.05). TBI-sig was used for a LINCS analysis which identified 11 compounds that showed a strong connectivity with the TBI-sig in neuronal cell lines. Of these, celecoxib and sirolimus were recently reported to have a disease-modifying effect in in vivo animal models of epilepsy. Other compounds revealed by the analysis were BRD-K91844626, BRD-A11009626, NO-ASA, BRD-K55260239, SDZ-NKT-343, STK-661558, BRD-K75971499, ionomycin, and desmethylclomipramine. Network analysis of overlapping genes revealed the effects on tubulins (Tubb2a, Tubb3, Tubb4b), Nfe2l2, S100a4, Cd44, and Nfkb2, all of which are linked to TBI-relevant outcomes, including epileptogenesis and tissue repair. Desmethylclomipramine modulated most of the gene targets considered favorable for TBI outcome. Our data demonstrate long-lasting transcriptomics changes after TBI. LINCS analysis predicted that these changes could be modulated by various compounds, some of which are already in clinical use but never tested in TBI.
Comparative membrane proteomics analyses of breast cancer cell lines to understand the molecular mechanism of breast cancer brain metastasis.
Peng Wenjing,Zhang Yu,Zhu Rui,Mechref Yehia
Breast cancer is the leading type of cancer in women. Breast cancer brain metastasis is currently considered an issue of concern among breast cancer patients. Membrane proteins play important roles in breast cancer brain metastasis, involving cell adhesion and penetration of blood-brain barrier. To understand the mechanism of breast cancer brain metastasis, liquid chromatography-tandem mass spectrometry (LC-MS/MS) was employed in conjunction with enrichment of membrane proteins to analyze the proteomes from five different breast cancer and a brain cancer cell lines. Quantitative proteomic data of all cell lines were compared with MDA-MB-231BR which is a brain seeking breast cancer cell line, thus representing brain metastasis characteristics. Label-free proteomics of the six cell lines facilitates the identification of 1238 proteins and the quantification of 899 proteins of which more than 70% were membrane proteins. Unsupervised principal component analysis (PCA) of the label-free proteomics data resulted in a distinct clustering of cell lines, suggesting quantitative differences in the expression of several proteins among the different cell lines. Unique protein expressions in 231BR were observed for 28 proteins. The up-regulation of STAU1, AT1B3, NPM1, hnRNP Q, and hnRNP K and the down-regulation of TUBB4B and TUBB5 were noted in 231BR relative to 231 (precursor cell lines from which 231BR is derived). These proteins might contribute to the breast cancer brain metastasis. Ingenuity pathway analysis (IPA) supported the great brain metastatic propensity of 231BR and suggested the importance of the up-regulation of integrin proteins and down-regulation of EPHA2 in brain metastasis.
Maternal Choline Supplementation Alters Basal Forebrain Cholinergic Neuron Gene Expression in the Ts65Dn Mouse Model of Down Syndrome.
Kelley Christy M,Ginsberg Stephen D,Alldred Melissa J,Strupp Barbara J,Mufson Elliott J
Down syndrome (DS), trisomy 21, is marked by intellectual disability and a premature aging profile including degeneration of the basal forebrain cholinergic neuron (BFCN) projection system, similar to Alzheimer's disease (AD). Although data indicate that perinatal maternal choline supplementation (MCS) alters the structure and function of these neurons in the Ts65Dn mouse model of DS and AD (Ts), whether MCS affects the molecular profile of vulnerable BFCNs remains unknown. We investigated the genetic signature of BFCNs obtained from Ts and disomic (2N) offspring of Ts65Dn dams maintained on a MCS diet (Ts+, 2N+) or a choline normal diet (ND) from mating until weaning, then maintained on ND until 4.4-7.5 months of age. Brains were then collected and prepared for choline acetyltransferase (ChAT) immunohistochemistry and laser capture microdissection followed by RNA extraction and custom-designed microarray analysis. Findings revealed upregulation of select transcripts in classes of genes related to the cytoskeleton (Tubb4b), AD (Cav1), cell death (Bcl2), presynaptic (Syngr1), immediate early (Fosb, Arc), G protein signaling (Gabarap, Rgs10), and cholinergic neurotransmission (Chrnb3) in Ts compared to 2N mice, which were normalized with MCS. Moreover, significant downregulation was seen in select transcripts associated with the cytoskeleton (Dync1h1), intracellular signaling (Itpka, Gng3, and Mlst8), and cell death (Ccng1) in Ts compared to 2N mice that was normalized with MCS. This study provides insight into genotype-dependent differences and the effects of MCS at the molecular level within a key vulnerable cell type in DS and AD.
Antibodies Against ARHGDIB and ARHGDIB Gene Expression Associate With Kidney Allograft Outcome.
Senev Aleksandar,Otten Henny G,Kamburova Elena G,Callemeyn Jasper,Lerut Evelyne,Van Sandt Vicky,Kuypers Dirk,Emonds Marie-Paule,Naesens Maarten
BACKGROUND:The impact of donor-specific anti-HLA antibodies (DSA) on antibody-mediated rejection (AMR) and kidney allograft failure is well established. However, the relevance of non-HLA antibodies remains unclear. METHODS:We investigated 13 pretransplant non-HLA antibodies and their association with histology of AMR (AMRh) and kidney allograft failure. We included single kidney recipients (n = 203) with AMRh, according to the Banff 2017 classification and matched AMRh-free controls (n = 219). Non-HLA antibodies were assessed using multiplex Luminex assay. RESULTS:Of the selected non-HLA antibodies (against agrin, adipocyte plasma membrane-associated protein, Rho GDP-dissociation inhibitor 2 [ARHGDIB], Rho guanine nucleotide exchange factor 6, angiotensin-II type 1 receptor, endothelin type A receptor, lamin B1, BPI fold-containing family B member 1, peroxisomal trans-2-enoyl-coenzyme A reductase, phospholipase A2 receptor, protein kinase C zeta type, tubulin beta-4B class IVb, vimentin), only antibodies against ARHGDIB (adjusted median fluorescence intensity [aMFI] ≥ 1000), a minor histocompatibility antigen, associated with graft failure, in univariate and multivariate models (hazard ratio = 2.7; 95% confidence interval [CI],1.3-5.4; P = 0.007). There was a 19.5-fold (95% CI, 6.0-63.9; P < 0.0001) increased risk of graft failure in patients positive for both DSA and anti-ARHGDIB antibodies (aMFI ≥ 1000) versus patients negative for both DSA and anti-ARHGDIB antibodies, compared with a 4.4-fold (95% CI, 2.4-8.2; P < 0.0001) increased risk in patients with only DSA, and a 4.1-fold (95% CI, 1.4-11.7; P = 0.009) increased risk in patients with only anti-ARHGDIB antibodies above 2000 aMFI. AMRh associated with increased intrarenal expression of the ARHGDIB gene. In the absence of AMRh and DSA, anti-ARHGDIB antibodies were not clearly associated with graft failure. CONCLUSIONS:The presence of pretransplant anti-ARHGDIB antibodies has an additive effect in patients with DSA on the risk of graft failure via AMRh. Other investigated non-HLA antibodies, including antibodies against angiotensin-II type 1 receptor, did not contribute to risk stratification and could not explain the histology of AMR in the absence of DSA.
Acute epididymitis induces alterations in sperm protein composition.
Pilatz Adrian,Lochnit Guenter,Karnati Srikanth,Paradowska-Dogan Agnieszka,Lang Tali,Schultheiss Dirk,Schuppe Hans-Christian,Hossain Hamid,Baumgart-Vogt Eveline,Weidner Wolfgang,Wagenlehner Florian
Fertility and sterility
OBJECTIVE:To use a proteomic approach to evaluate possible postinflammatory alterations in the protein composition of motile sperm in patients 3 months after acute epididymitis. DESIGN:Prospective case-control study. SETTING:University medical school research laboratory. PATIENT(S):Eight patients 3 months after acute unilateral epididymitis and 10 healthy controls. INTERVENTION(S):None. MAIN OUTCOME MEASURE(S):Proteome analysis of sperm samples collected by swim-up from control and acute epididymitis patients analyzed by two-dimensional gel electrophoresis and subsequent protein identification by matrix-assisted laser desorption/ionization-time-of-flight (MALDI-TOF) mass spectrometry; immunofluorescence staining for mitochondrial ATP synthase subunit β (ATP5B), α-tubulin (TUBA1A), and tubulin-β2c (TUBB4B) for validation purposes. RESULT(S):Proteome analysis identified 35 proteins in sperm from epididymitis patients that were down-regulated, irrespective of subcellular localization and biologic function. Furthermore, immunofluorescence microscopy confirmed ATP5B, TUBA1A, and TUBB4B were less abundantly expressed in epididymitis samples compared with controls. CONCLUSION(S):Despite normal semen parameters observed by conventional semen analysis in patients after epididymitis, significant changes to sperm protein composition were observed. These changes may be implicated as additional factors contributing to subfertility/infertility in men after episodes of epididymitis.
Predicting Outcomes of Hormone and Chemotherapy in the Molecular Taxonomy of Breast Cancer International Consortium (METABRIC) Study by Biochemically-inspired Machine Learning.
Mucaki Eliseos J,Baranova Katherina,Pham Huy Q,Rezaeian Iman,Angelov Dimo,Ngom Alioune,Rueda Luis,Rogan Peter K
Genomic aberrations and gene expression-defined subtypes in the large METABRIC patient cohort have been used to stratify and predict survival. The present study used normalized gene expression signatures of paclitaxel drug response to predict outcome for different survival times in METABRIC patients receiving hormone (HT) and, in some cases, chemotherapy (CT) agents. This machine learning method, which distinguishes sensitivity vs. resistance in breast cancer cell lines and validates predictions in patients; was also used to derive gene signatures of other HT (tamoxifen) and CT agents (methotrexate, epirubicin, doxorubicin, and 5-fluorouracil) used in METABRIC. Paclitaxel gene signatures exhibited the best performance, however the other agents also predicted survival with acceptable accuracies. A support vector machine (SVM) model of paclitaxel response containing genes and was 78.6% accurate in predicting survival of 84 patients treated with both HT and CT (median survival ≥ 4.4 yr). Accuracy was lower (73.4%) in 304 untreated patients. The performance of other machine learning approaches was also evaluated at different survival thresholds. Minimum redundancy maximum relevance feature selection of a paclitaxel-based SVM classifier based on expression of genes and was 81.1% accurate in 53 CT patients. In addition, a random forest (RF) classifier using a gene signature ( and ) predicted >3-year survival with 85.5% accuracy in 420 HT patients. A similar RF gene signature showed 82.7% accuracy in 504 patients treated with CT and/or HT. These results suggest that tumor gene expression signatures refined by machine learning techniques can be useful for predicting survival after drug therapies.
Using RNA-Seq with 11 marker genes to evaluate 1,4-dioxane compared with typical genotoxic and non-genotoxic rat hepatocarcinogens.
Furihata Chie,Toyoda Takeshi,Ogawa Kumiko,Suzuki Takayoshi
Mutation research. Genetic toxicology and environmental mutagenesis
It has long been unclear whether 1,4-dioxane (DO) is a genotoxic hepatocarcinogen (GTHC). Therefore, the present study aimed to evaluate rat GTHCs and non-genotoxic hepatocarcinogens (NGTHCs) via selected gene expression patterns in the liver, as determined by next generation sequencing-targeted mRNA sequencing (RNA-Seq) and principal component analysis (PCA). Previously, we selected 11 marker genes (Aen, Bax, Btg2, Ccnf, Ccng1, Cdkn1a, Lrp1, Mbd1, Phlda3, Plk2, and Tubb4b) to discriminate GTHCs and NGTHCs. In the present study, we quantified changes in the expression of these genes following DO treatment, and compared them with treatment with two typical rat GTHCs, N-nitrosodiethylamine (DEN) and 3,3'-dimethylbenzidine·2HCl (DMB), and a typical rat NGTHC, di(2-ethylhexyl)phthalate (DEHP). RNA-Seq was conducted on liver samples from groups of five male, 10-week-old F344 rats after 4 weeks' feeding of chemicals in the water or the food. Rats in the control group were given water and a basal diet. Significant changes in gene expression in experimental groups compared with the control group were observed in eight genes (Aen, Bax, Btg2, Ccnf, Ccng1, Cdkn1a, Phlda3 and Plk2), as shown by Tukey's test. Gene expression profiles of the 11 genes under DO treatment differed significantly from those with DEN and DMB, as well as DEHP. Gene expression profiles with DO treatment differed partially from those with typical GTHCs for five genes (Bax, Btg2, Cdkn1a, Lrp1 and Plk2) and were substantially different from treatment with a typical NGTHC (DEHP) for nine genes (Aen, Bax, Btg2, Ccnf, Ccng1, Cdkn1a, Mbd1, Phlda3 and Tubb4b) as determined by Tukey's test. Finally, PCA successfully differentiated GTHCs from DEHP and DO with the 11 genes. The present results suggest that RNA-Seq and PCA are useful to evaluate rat typical GTHCs and typical NGTHCs. DO was suggested to result in a different intermediate gene expression profile from typical GTHCs and NGTHC.
Evaluation of 12 mouse marker genes in rat toxicogenomics public data, Open TG-GATEs: Discrimination of genotoxic from non-genotoxic hepatocarcinogens.
Furihata Chie,Suzuki Takayoshi
Mutation research. Genetic toxicology and environmental mutagenesis
Previously, we proposed 12 marker genes (Aen, Bax, Btg2, Ccnf, Ccng1, Cdkn1a, Gdf15, Lrp1, Mbd1, Phlda3, Plk2 and Tubb4b) to discriminate mouse genotoxic hepatocarcinogens (GTHC) from non-genotoxic hepatocarcinogens (NGTHC). This was determined by qPCR and principal component analysis (PCA), as the aim of an in vivo short-term screening for genotoxic hepatocarcinogens. For this paper, we conducted an application study of the 12 mouse marker genes to rat data, Open TG-GATEs (public data). We analyzed five typical rat GTHC (2-acetamodofluorene, aflatoxin B1, 2-nitrofluorene, N-nitrosodiethylamine and N-nitrosomorpholine), and not only seven typical rat NGTHC (clofibrate, ethanol, fenofibrate, gemfibrozil, hexachlorobenzene, phenobarbital and WY-14643) but also 11 non-genotoxic non-hepatocarcinogens (NGTNHC; allyl alcohol, aspirin, caffeine, chlorpheniramine, chlorpropamide, dexamethasone, diazepam, indomethacin, phenylbutazone, theophylline and tolbutamide) from Open TG-GATEs. The analysis was performed at 3, 6, 9 and 24 h after a single administration and 4, 8, 15 and 29 days after repeated administrations. We transferred Open TG-GATEs DNA microarray data into log data using the "R Project for Statistical Computing". GTHC-specific dose-dependent gene expression changes were observed and significance assessed with the Williams test. Similar significant changes were observed during 3-24 h and 4-29 days, assessed with Welch's t-test, except not for NGTHC or NGTNHC. Significant differential changes in gene expression were observed between GTHC and NGTHC in 11 genes (except not Tubb4b) and between GTHC and NGTNHC in all 12 genes at 24 h and 10 genes (except Ccnf and Mbd1) at 29 days, per Tukey's test. PCA successfully discriminated GTHC from NGTHC and NGTNHC at 24 h and 29 days. The results demonstrate that 12 previously proposed mouse marker genes are useful for discriminating rat GTHC from NGTHC and NGTNHC from Open TG-GATEs.
Mutations in TUBB4B Cause a Distinctive Sensorineural Disease.
Luscan Romain,Mechaussier Sabrina,Paul Antoine,Tian Guoling,Gérard Xavier,Defoort-Dellhemmes Sabine,Loundon Natalie,Audo Isabelle,Bonnin Sophie,LeGargasson Jean-François,Dumont Julien,Goudin Nicolas,Garfa-Traoré Meriem,Bras Marc,Pouliet Aurore,Bessières Bettina,Boddaert Nathalie,Sahel José-Alain,Lyonnet Stanislas,Kaplan Josseline,Cowan Nicholas J,Rozet Jean-Michel,Marlin Sandrine,Perrault Isabelle
American journal of human genetics
Leber congenital amaurosis (LCA) is a neurodegenerative disease of photoreceptor cells that causes blindness within the first year of life. It occasionally occurs in syndromic metabolic diseases and plurisystemic ciliopathies. Using exome sequencing in a multiplex family and three simplex case subjects with an atypical association of LCA with early-onset hearing loss, we identified two heterozygous mutations affecting Arg391 in β-tubulin 4B isotype-encoding (TUBB4B). Inspection of the atomic structure of the microtubule (MT) protofilament reveals that the β-tubulin Arg391 residue contributes to a binding pocket that interacts with α-tubulin contained in the longitudinally adjacent αβ-heterodimer, consistent with a role in maintaining MT stability. Functional analysis in cultured cells overexpressing FLAG-tagged wild-type or mutant TUBB4B as well as in primary skin-derived fibroblasts showed that the mutant TUBB4B is able to fold, form αβ-heterodimers, and co-assemble into the endogenous MT lattice. However, the dynamics of growing MTs were consistently altered, showing that the mutations have a significant dampening impact on normal MT growth. Our findings provide a link between sensorineural disease and anomalies in MT behavior and describe a syndromic LCA unrelated to ciliary dysfunction.
TUBB4B Downregulation Is Critical for Increasing Migration of Metastatic Colon Cancer Cells.
Sobierajska Katarzyna,Ciszewski Wojciech M,Wawro Marta E,Wieczorek-Szukała Katarzyna,Boncela Joanna,Papiewska-Pajak Izabela,Niewiarowska Jolanta,Kowalska M Anna
Tumor metastasis, the major problem for clinical oncology in colon cancer treatment, is linked with an epithelial-mesenchymal transition (EMT). The observed cellular transformation in this process is manifested by cell elongation, enhanced cell migration and invasion ability, coordinated by cytoskeleton reorganization. In the present study, we examined the role of tubulin-β4 (TUBB4B) downregulation that occurs during EMT in colon cancer cells, in the modulation of the function of microtubules. Based on biochemical and behavioral analysis (transmigration) we posit that the decrease of the TUBB4B level is critical for microtubule-vimentin interaction and contributes to the maintenance of polarity in migrating cells. The microscopic studies revealed that TUBB4B decrease is accompanied by cell elongation and increased number of matured focal adhesion sites, which is a characteristic of the cell metastatic stage. We also demonstrated faster polymerization of microtubules in cells with a lower level of TUBB4B. Simultaneous TUBB3 upregulation, reported during EMT, acts additively in this process. Our studies suggest that the protein level of TUBB4B could be used as a marker for detection of the preinvasive stages of the colon cancer cells. We also concluded that chemotherapy enriched to increase TUBB4B level and/or to stabilize microtubule polymerization might more effectively prevent metastasis in colon cancer development.
Tumoral and tissue-specific expression of the major human beta-tubulin isotypes.
Leandro-García Luis J,Leskelä Susanna,Landa Iñigo,Montero-Conde Cristina,López-Jiménez Elena,Letón Rocío,Cascón Alberto,Robledo Mercedes,Rodríguez-Antona Cristina
Cytoskeleton (Hoboken, N.J.)
The beta-tubulins are microtubule components encoded by a multigene family, which produces slightly different proteins with complex expression patterns. Several widely used anticancer drugs base their activity on beta-tubulin binding, microtubule dynamics alteration, and cell division blockage. The expression of these drug targets in tumoral and normal cells could be of crucial importance for therapy outcome, unfortunately, the complex beta-tubulin expression patterns have been poorly characterized in human. In this study, we developed a quantitative RT-PCR technique that accurately determines the mRNA expression of the eight human beta-tubulin isotypes, encoding class I, IIa, IIb, III, IVa, IVb, V, and VI and applied it to 21 nontumoral tissues and 79 tumor samples belonging to seven cancer types. In the nontumoral tissues, we found that, overall, TUBB (I), TUBB2C (IVb), and TUBB6 (V) were ubiquitous, TUBB1(VI) was hematopoietic cell-specific, and TUBB2A (IIa), TUBB2B (IIb), TUBB3 (III), and TUBB4 (IVa) had high expression in brain; however, the contribution of the different isotypes to the total beta-tubulin content varied for each tissue and had a complex pattern. In tumoral tissues, most isotypes exhibited an altered expression in specific tumor types or related to tumoral characteristics. In general, TUBB3 showed a great increase in expression while TUBB6 expression was largely decreased in most tumors. Thus, normal tissues showed a complex beta-tubulin isotype distribution, which could contribute to the toxicity profile of the microtubule-binding drugs. In addition, the specific isotypes significantly altered in tumors might represent markers for drug response.
Transcriptional profile analysis of RPGRORF15 frameshift mutation identifies novel genes associated with retinal degeneration.
Genini Sem,Zangerl Barbara,Slavik Julianna,Acland Gregory M,Beltran William A,Aguirre Gustavo D
Investigative ophthalmology & visual science
PURPOSE:To identify genes and molecular mechanisms associated with photoreceptor degeneration in a canine model of XLRP caused by an RPGR exon ORF15 microdeletion. Methods. Expression profiles of mutant and normal retinas were compared by using canine retinal custom cDNA microarrays. qRT-PCR, Western blot analysis, and immunohistochemistry (IHC) were applied to selected genes, to confirm and expand the microarray results. RESULTS:At 7 and 16 weeks, respectively, 56 and 18 transcripts were downregulated in the mutant retinas, but none were differentially expressed (DE) at both ages, suggesting the involvement of temporally distinct pathways. Downregulated genes included the known retina-relevant genes PAX6, CHML, and RDH11 at 7 weeks and CRX and SAG at 16 weeks. Genes directly or indirectly active in apoptotic processes were altered at 7 weeks (CAMK2G, NTRK2, PRKCB, RALA, RBBP6, RNF41, SMYD3, SPP1, and TUBB2C) and 16 weeks (SLC25A5 and NKAP). Furthermore, the DE genes at 7 weeks (ELOVL6, GLOD4, NDUFS4, and REEP1) and 16 weeks (SLC25A5 and TARS2) are related to mitochondrial functions. qRT-PCR of 18 genes confirmed the microarray results and showed DE of additional genes not on the array. Only GFAP was DE at 3 weeks of age. Western blot and IHC analyses also confirmed the high reliability of the transcriptomic data. CONCLUSIONS:Several DE genes were identified in mutant retinas. At 7 weeks, a combination of nonclassic anti- and proapoptosis genes appear to be involved in photoreceptor degeneration, whereas at both 7 and 16 weeks, the expression of mitochondria-related genes indicates that they may play a relevant role in the disease process.
Gene expression profiling of oral squamous cell carcinoma by differential display rt-PCR and identification of tumor biomarkers.
Chakraborty Sanjukta,Nagashri M N,Mohiyuddin S M Azeem,Gopinath K S,Kumar Arun
Indian journal of surgical oncology
Oral squamous cell carcinoma (OSCC) is the sixth most common cancer worldwide. Despite progress in therapeutic and surgical treatments, its survival period at 5 years is the lowest among major cancers, and remains unchanged in the last two decades. The growing epidemiological relevance of oral cancer emphasizes the need to better understand the molecular mechanisms underlying this disease and identify predictive tumor markers and therapeutic targets. To this end, we have used the DDRT-PCR analysis to profile the oral tumor transcriptome and identify differentially regulated genes that may be used as potential biomarkers and therapeutic targets. Our DDRT-PCR analysis identified 51 differentially expressed fragments, of which 25 were revalidated by reverse Northern analysis. Northern blot analysis further corroborated these findings for a few genes. In order to ascertain the utility of some of the identified genes as molecular markers and therapeutic targets, semi-quantitative RT-PCR analysis was carried out in a panel of matched oral normal and tumor samples, that confirmed GLTP, PCNA, RBM28, C17orf75 and DIAPH1 as significantly upregulated, whereas TNKS2, PAM and TUBB2C showed significant downregulation in tumor samples. Taken together, our DDRT-PCR analysis has revealed several genes, belonging to diverse cellular pathways, that have been associated with OSCC for the first time. Thus, these genes could be investigated as biomarkers and therapeutic targets for OSCC.
Prospective nested case-control study of feature genes related to leukemic evolution of myelodysplastic syndrome.
Ma Yan,Chen Bobin,Xu Xiaoping,Lin Guowei
Molecular biology reports
We established a nested case-control study cohort of myelodysplastic syndrome patients (n = 435). And 41 patients had conditions progressing to leukemia (case group = 41), 342 patients had no leukemic transformation (control group = 342), and 52 patients died. Bone marrow mononuclear cell of the patients in the case group and after the evolution were analyzed for the gene expression microarray test (self-control study), whereas the bone marrow mononuclear cell of the paired patients extracted at diagnosis were analyzed for the gene expression microarray test (case-control study). By incorporating the results of above two studies, we identified the genes related to the transformation of myelodysplastic syndrome to acute leukemia. A total of 958 deregulated genes were identified via bioinformatics analysis. Further analyses identified a subset of six genes that help distinguish between the case and control groups. These genes are TUBB, PSMD1, SLC7A5, ATG3, TUBB2C, and TIMM10. The combined gene expression microarray test and nested case-control study method identified a subset of six genes that help distinguish between the case and control groups. The six genes may play critical roles in the evolution of myelodysplastic syndrome to acute leukemia.
Differential gene expression profiling between genotoxic and non-genotoxic hepatocarcinogens in young rat liver determined by quantitative real-time PCR and principal component analysis.
Suenaga Kazuya,Takasawa Hironao,Watanabe Takashi,Wako Yumi,Suzuki Takayoshi,Hamada Shuichi,Furihata Chie
We recently successfully discriminated mouse genotoxic hepatocarcinogens from non-genotoxic hepatocarcinogens via selected gene expression profiling in the mouse liver based on quantitative real-time PCR (qPCR) and statistical analysis using principal component analysis (PCA). In the present study, we applied these candidate marker genes to rat hepatocarcinogens in the rat liver. qPCR analysis of 33 genes was conducted on liver samples from groups of 4 male 4-week-old F344 rats at 4 and 48 h after a single oral administration of chemicals [2 genotoxic hepatocarcinogens: diethylnitrosamine and 2,6-dinitrotoluene; a non-genotoxic hepatocarcinogen: di(2-ethylhexyl)phthalate; and a non-genotoxic non-hepatocarcinogen: phenacetin]. Thirty-two genes exhibited significant changes in their gene expression ratios (experimental group/control group) according to statistical analysis using the Williams' test and the Dunnett's test. The changes appeared to be greater at 4h than at 48 h. Finally, statistical analysis via PCA successfully differentiated the genotoxic hepatocarcinogens from the non-genotoxic hepatocarcinogen and the non-genotoxic non-hepatocarcinogen at 4h based on 16 genes (Ccnf, Ccng1, Cyp4a10, Ddit4l, Egfr, Gadd45g, Gdf15, Hspb1, Igfbp1, Jun, Myc, Net1, Phlda3, Pml, Rcan1 and Tubb2c) and at 48 h based on 10 genes (Aen, Ccng1, Cdkn1a, Cyp21a1, Cyp4a10, Gdf15, Igfbp1, Mdm2, Phlda3 and Pmm1). Eight major biological processes were extracted from a gene ontology analysis: apoptosis, the cell cycle, cell proliferation, DNA damage, DNA repair, oxidative stress, oncogenes and tumor suppression. The major, biologically relevant gene pathway suggested was the DNA damage response, which signals through a Tp53-mediated pathway and leads to the induction of apoptosis. Immunohistochemical analyses for the expression of Cdkn1a and Hmox1 proteins and the level of apoptosis measured by the TUNEL assay in the liver confirmed the aforementioned results. The present results showed that mouse candidate marker genes are applicable for differentiating genotoxic hepatocarcinogens from non-genotoxic hepatocarcinogens examined in this paper in the rat liver.
Sperm superoxide dismutase is associated with bull fertility.
Grant Kamilah E,de Oliveira Rodrigo V,Hennington Bettye Sue,Govindaraju Aruna,Perkins Andy,Stokes John,Rowe Dennis,Topper Einko,Kaya Abdullah,Moura Arlindo,Memili Erdogan
Reproduction, fertility, and development
Decreasing mammalian fertility and sperm quality have created an urgent need to find effective methods to distinguish non-viable from viable fertilising spermatozoa. The aims of the present study were to evaluate expression levels of ?-tubulin 2C (TUBB2C), heat shock protein 10 (HSP10), hexokinase 1 (HXK1) and superoxide dismutase 1 (SOD1) in spermatozoa from Holstein bulls with varying fertility using western blotting and to analyse the biological networks of these key sperm proteins using a bioinformatics software (Metacore; Thomson-Reuters, Philadelphia, PA, USA). The rationales behind this study were that the sperm proteins play crucial roles in fertilisation and early embryonic development in mammals and ascertaining the biological networks of the proteins helps us better understand sperm physiology and early mammalian development. The results showed that expression of SOD1 was higher in spermatozoa from high fertility bulls (PPin vivo bull fertility. The findings are important because they illuminate molecular and cellular determinants of sperm viability and the identified protein markers can be used to determine bull fertility.
Differential proteomic analysis of endemic and sporadic Epstein-Barr virus-positive and negative Burkitt lymphoma.
El-Mallawany Nader Kim,Day Nancy,Ayello Janet,Van de Ven Carmella,Conlon Kevin,Fermin Damian,Basrur Venkatesha,Elenitoba-Johnson Kojo,Lim Megan,Cairo Mitchell S
European journal of cancer (Oxford, England : 1990)
BACKGROUND:Burkitt lymphoma (BL) is the most common non-Hodgkin lymphoma in children worldwide and the most common paediatric malignancy in sub-Saharan Africa. The endemic (eBL) and sporadic (sBL) variants have distinct epidemiologic and virologic characteristics. Although gene expression studies have defined the transcriptional profiles of both, their proteomic signatures have not been studied. METHODS:We compared the proteomic expression profiles using differential mass spectrometry-based isotope tag for relative and absolute quantitation (iTRAQ) analysis of a cell line representing Epstein-Barr virus (EBV)+ eBL, EBV+ and EBV- sBL, and EBV+/- normal B cells from healthy donors. RESULTS:In total, there were 144 differentially expressed proteins with a statistically significant false discovery rate (FDR) of ⩽0.2. Results revealed over-expression of specific proteins with well-established links to lymphomagenesis such as TUBB2C (FDR 0.05), UCHL1 (FDR 0.05) and HSP90AB1 (FDR 0.1). Distinct characteristics based upon the epidemiologic and virologic subtypes of BL were also identified. In sBL, PCNA (FDR 0.05) and SLC3A2 (FDR 0.1) were significantly over-expressed. In eBL, C1QBP (FDR 0.1) and ENO1 (FDR 0.25) were significantly over-expressed. Comparison of EBV+ to EBV- BL cell lines and B cells revealed significant over-expression of DDX3X (FDR 0.1). Proteins were validated using Western blot analysis. CONCLUSION:Our results suggest unique signal transduction pathways associated with EBV infection and epidemiological subtype of BL that may contribute to lymphomagenesis. These proteomic findings provide potential diagnostic, prognostic and therapeutic links to BL.
Dose-dependent alterations in gene expression in mouse liver induced by diethylnitrosamine and ethylnitrosourea and determined by quantitative real-time PCR.
Watanabe Takashi,Tanaka Gotaro,Hamada Shuichi,Namiki Chiaki,Suzuki Takayoshi,Nakajima Madoka,Furihata Chie
We examined the dose-dependency of gene expression changes for 51 genes in mouse liver treated with two N-nitroso genotoxic hepatocarcinogens, diethylnitrosamine (DEN) and ethylnitrosourea (ENU) by quantitative real-time PCR (qPCR). DEN (3, 9, 27 and 80mg/kg bw) or ENU (6, 17, 50 and 150mg/kg bw) was injected intraperitoneally into groups of five male 9-week-old B6C3F(1) mice and the livers were dissected after 4h and 28 days. Total RNA from pooled livers was reverse-transcribed to cDNA and the amount of each gene was quantified by qPCR. Results were analyzed by hierarchical and k-means clustering and ingenuity pathway analysis (IPA). The most characteristic result was a similar dose-dependency of gene expression changes with DEN and ENU. Twenty-one genes exhibited a distinct dose-dependent increase in expression at 4h for both carcinogens [Bax, Btg2, Ccng1, Cdkn1a, Cyp4a10, Cyp21a1, Fos, Gadd45b, Gdf15, Hmox1, Hspb1, Isg20l1, Jun, Mbd1, Mdm2, Myc, Net1, Plk2, Ppp1r3c, Rcan1 and Tubb2c], although the increase in gene expression due to ENU was generally weaker than that due to DEN. Only Gdf15 showed a dose-dependent increase in expression at 28 days for both carcinogens. The differences between DEN and ENU were in the expression of additional genes (7 for DEN and 8 for ENU). IPA extracted five gene networks: Network-1 included genes related to cancer and cell cycle arrest and associated with Bax, Btg2, Ccng1, Cdkn1a, Gadd45b, Gdf15, Hspb1, Mdm2 and Plk2 and Network-2 was related to DNA replication, recombination, repair and cell death and associated with Cyp21a1, Gdf15, Ppp1r3c, Rcan1 and Tubb2c. The present results show a distinct dose-dependency of gene expression changes induced by DEN and ENU. These changes were associated with cancer, cell cycle arrest, DNA replication, recombination, repair and cell death and were seen not only at 4h but also, for some, at 28 days after administration.
Evaluation of potential reference genes for real time RT-PCR studies in Atlantic halibut (Hippoglossus Hippoglossus L.); during development, in tissues of healthy and NNV-injected fish, and in anterior kidney leucocytes.
Øvergård Aina-Cathrine,Nerland Audun Helge,Patel Sonal
BMC molecular biology
BACKGROUND:Real time RT-PCR has become an important tool for analyzing gene expression in fish. Although several housekeeping genes have been evaluated in Atlantic halibut (Hippoglossus Hippoglossus L.), appropriate reference genes for low copy mRNA transcripts at the earliest developmental stages have not been identified. No attempts have been reported to identify suitable reference genes in halibut infected with NNV or in stimulated halibut leucocytes. In this study, beta-actin1 (ACTB1), elongation factor 1 alpha (EF1A1), hypoxanthine-guanine phosphoribosyltransferase 1 (HPRT1), ribosomal protein L7 (RPL7), tubulin beta 2C (Tubb2C), and ubiquitin-conjugating enzyme (UbcE) were evaluated as reference genes for normalization of real time RT-PCR data during Atlantic halibut development, in tissue of healthy and NNV-infected fish, and in in vivo and in vitro stimulated anterior kidney leucocytes. RESULTS:The expression of all six genes was relatively stable from the unfertilized egg until 12 day degrees post fertilization (ddpf). However, none of the selected genes were found to be stably expressed throughout halibut development. The mRNA levels of the six genes increased from 18 ddpf, when zygotic transcription is likely to be activated, and stabilized at different time points. The Excel-based software programs BestKeeper, geNorm, and NormFinder ranked EF1A1 and UbcE as the best candidate reference genes before activation of zygotic transcription, and RPL7 and EF1A1 as the best candidates after hatching. EF1A1 and RPL7 were also listed as the best reference genes when exploring the expression levels of the six genes in various halibut organs, both in non-injected fish and in mock- and NNV-injected fish. None of the reference genes were found optimal for normalization of real time RT-PCR data from in vitro stimulated anterior kidney leucocytes. CONCLUSION:Generally, it was found that EF1A1 and RPL7 were the genes that showed least variation, with HPRT1 and UbcE as intermediate genes, and ACTB1 and Tubb2C as the least stable ones. None of the six reference genes can be recommended as reference gene candidates in ConA-PMA stimulated leucocytes. However, UbcE can be a good candidate in other experimental setups. This study emphasizes the need for reference gene evaluation, as universal reference genes have not been identified.
Discrimination of genotoxic and non-genotoxic hepatocarcinogens by statistical analysis based on gene expression profiling in the mouse liver as determined by quantitative real-time PCR.
Watanabe Takashi,Suzuki Takayoshi,Natsume Masakatsu,Nakajima Madoka,Narumi Kazunori,Hamada Shuichi,Sakuma Tomohiro,Koeda Akiko,Oshida Keiyu,Miyamoto Yohei,Maeda Akihisa,Hirayama Michiasa,Sanada Hisakazu,Honda Hiroshi,Ohyama Wakako,Okada Emiko,Fujiishi Yohei,Sutou Shizuyo,Tadakuma Ayami,Ishikawa Yasuyoshi,Kido Mahoko,Minamiguchi Rina,Hanahara Izumi,Furihata Chie
The general aim of the present study is to discriminate between mouse genotoxic and non-genotoxic hepatocarcinogens via selected gene expression patterns in the liver as analyzed by quantitative real-time PCR (qPCR) and statistical analysis. qPCR was conducted on liver samples from groups of 5 male, 9-week-old B6C3F(1) mice, at 4 and 48h following a single intraperitoneal administration of chemicals. We quantified 35 genes selected from our previous DNA microarray studies using 12 different chemicals: 8 genotoxic hepatocarcinogens (2-acetylaminofluorene, 2,4-diaminotoluene, diisopropanolnitrosamine, 4-dimethylaminoazobenzene, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone, N-nitrosomorpholine, quinoline and urethane) and 4 non-genotoxic hepatocarcinogens (1,4-dichlorobenzene, dichlorodiphenyltrichloroethane, di(2-ethylhexyl)phthalate and furan). A considerable number of genes exhibited significant changes in their gene expression ratios (experimental group/control group) analyzed statistically by the Dunnett's test and Welch's t-test. Finally, we distinguished between the genotoxic and non-genotoxic hepatocarcinogens by statistical analysis using principal component analysis (PCA) of the gene expression profiles for 7 genes (Btg2, Ccnf, Ccng1, Lpr1, Mbd1, Phlda3 and Tubb2c) at 4h and for 12 genes (Aen, Bax, Btg2, Ccnf, Ccng1, Cdkn1a, Gdf15, Lrp1, Mbd1, Phlda3, Plk2 and Tubb2c) at 48h. Seven major biological processes were extracted from the gene ontology analysis: apoptosis, the cell cycle, cell proliferation, DNA damage, DNA repair, oncogenes and tumor suppression. The major, biologically relevant gene pathway suggested was the DNA damage response pathway, resulting from signal transduction by a p53-class mediator leading to the induction of apoptosis. Eight genes (Aen, Bax, Btg2, Ccng1, Cdkn1a, Gdf15, Phlda3 and Plk2) that are directly associated with Trp53 contributed to the PCA. The current findings demonstrate a successful discrimination between genotoxic and non-genotoxic hepatocarcinogens, using qPCR and PCA, on 12 genes associated with a Trp53-mediated signaling pathway for DNA damage response at 4 and 48 h after a single administration of chemicals.
Cell surfactomes of two endometrial epithelial cell lines that differ in their adhesiveness to embryonic cells.
Bhagwat Sonali R,Redij Tejashree,Phalnikar Kruttika,Nayak Sumeet,Iyer Swati,Gadkar Sushama,Chaudhari Uddhav,Kholkute Sanjeeva D,Sachdeva Geetanjali
Molecular reproduction and development
Adhesiveness of the endometrial epithelium to an embryo plays a critical role in the initiation of pregnancy. Loss or gain of adhesiveness also dictates the potential of endometrial epithelial cells to metastasize, an event that can result from certain genetic insults. A proteomics-based exploration of the "adhesiveness" these epithelial cells was employed that could identify targets that could disrupt embryo-endometrium interactions and/or metastasis of endometrial cancer cells. The present study defined the surfactomes of two human endometrial epithelial cell lines known for their differential adhesiveness to embryonic cells. Comparative, two-dimensional electrophoretic analysis of the surfactomes of RL95-2 (exhibiting higher adhesiveness to the embryonic cell line JAr) and HEC-1A (exhibiting reduced adhesiveness to JAr cells) revealed 55 differentially enriched proteins. Of these, 10 proteins were identified by MALDI-TOF/TOF or LC-MS/MS. TUBB2C, ADAMTS3, and elongation factor beta were more abundant on the HEC-1A cell surface whereas HSP27, HSPA9, GP96, CRT, Tapasin-ERP57, PDI, and β-actin were more abundant on the RL95-2 cell surface. Nano LC-MS/MS was also employed to generate a more comprehensive surfactomes of RL95-2 and HEC-1A. The study also demonstrated a pro-adhesive role of CRT and HSPA9 and an anti-adhesive role of TUBB2C populations found on the cell surface. In brief, this study identifies the cell-surface protein complements of two human endometrial epithelial cell lines, and reveals the role of three proteins in heterotypic cell adhesion.
Construction and analysis of protein-protein interaction networks based on proteomics data of prostate cancer.
Chen Chen,Shen Hong,Zhang Li-Guo,Liu Jian,Cao Xiao-Ge,Yao An-Liang,Kang Shao-San,Gao Wei-Xing,Han Hui,Cao Feng-Hong,Li Zhi-Guo
International journal of molecular medicine
Currently, using human prostate cancer (PCa) tissue samples to conduct proteomics research has generated a large amount of data; however, only a very small amount has been thoroughly investigated. In this study, we manually carried out the mining of the full text of proteomics literature that involved comparisons between PCa and normal or benign tissue and identified 41 differentially expressed proteins verified or reported more than 2 times from different research studies. We regarded these proteins as seed proteins to construct a protein-protein interaction (PPI) network. The extended network included one giant network, which consisted of 1,264 nodes connected via 1,744 edges, and 3 small separate components. The backbone network was then constructed, which was derived from key nodes and the subnetwork consisting of the shortest path between seed proteins. Topological analyses of these networks were conducted to identify proteins essential for the genesis of PCa. Solute carrier family 2 (facilitated glucose transporter), member 4 (SLC2A4) had the highest closeness centrality located in the center of each network, and the highest betweenness centrality and largest degree in the backbone network. Tubulin, beta 2C (TUBB2C) had the largest degree in the giant network and subnetwork. In addition, using module analysis of the whole PPI network, we obtained a densely connected region. Functional annotation indicated that the Ras protein signal transduction biological process, mitogen-activated protein kinase (MAPK), neurotrophin and the gonadotropin-releasing hormone (GnRH) signaling pathway may play an important role in the genesis and development of PCa. Further investigation of the SLC2A4, TUBB2C proteins, and these biological processes and pathways may therefore provide a potential target for the diagnosis and treatment of PCa.
Protein expression information of prostate infection based on data mining.
Abula Asimujiang,Shao Weimin,Tusong Hamulati,Wang Feng,Yasheng Anniwaer,Wang Yue,Wang Yujie
Journal of infection and public health
In order to deeply explore the interaction between prostate cancer (PCa)-related proteins and to screen out effective targets for clinical practice, data mining of PCa proteomics literature is conducted, 41 differentially expressed seed proteins are identified, and a protein interaction network is constructed. The extended network consists of a mega network and three separate small parts, which are used to find key nodes and build a backbone network through connectivity screening. Topological analysis of these networks reveals that solute carrier family 2 (glucose transporter) member 4 (SLC2A4) and tubulin β-2C (TUBB2C) are centrally located in the protein interaction network. In addition, by using the module analysis, the dense connection area is found. Functional annotations indicate that the biological processes of Ras protein signaling, mitogen-activated protein kinase (MAPK), and neurotrophin and gonadotropin-releasing hormone (GnRH) signaling pathways play important roles in the pathogenesis of PCa. Therefore, further studies of SLC2A4 and TUBB2C proteins, and these biological processes and pathways may provide potential targets for the diagnosis and treatment of PCa.
Normalization strategies for gene expression studies by real-time PCR in a marine fish species, Scophthalmus maximus.
Urbatzka R,Galante-Oliveira S,Rocha E,Castro L F C,Cunha I
Thorough evaluation of normalization approaches is a fundamental aspect in real-time quantitative RT-PCR experiments to avoid artificial introduced intergroup variations. In our study, we tested three normalization strategies in an experimental data set derived from a toxicological exposure of Scophthalmus maximus to the peroxisome proliferator-activated receptor alpha (PPARα) agonist WY-14643. Juvenile turbots were exposed by repeated injections to 5 mg or 50 mg WY-14643/kg, and liver samples were taken at day 1, 7 and 21. Specifically, the mRNA expression of peroxiredoxin 5 (prdx5) was normalized to the cDNA content, to the mRNA expression of single reference genes (b-actin, b-act; elongation factor 1 α, ef1a; glyceraldehyde-3-phosphate dehydrogenase, gapdh; ribosomal protein L8, rpl8; tata-box binding protein, tbp; tubulin beta 2C chain, tubb2c; ubiquitin-conjugating enzyme E2L 3, ub2l3) or to a combination of multiple reference genes using geNorm, BestKeeper or NormFinder algorithms. Four single reference genes (ef1a, rpl8, tubb2c, tbp) did not show any significant differences between the treatment groups over time, while significant intergroup variations were observed for cDNA content, gapdh, b-act and ub2l3. The normalization of prdx5 to the valid (not altered) single reference genes led to significant up-regulated (prdx5/rpl8), not-regulated (prdx5/ef1a; prdx5/tbp) or down-regulated (prdx5/tubb2c) mRNA expression pattern. The multiple reference gene approaches resulted in different rankings and combinations of the most stable expressed reference genes (geNorm: ef1a>rpl8>b-act; BestKeeper: ub2l3>gapdh>ef1a; NormFinder: b-act>ef1a). However, the normalization with the three multiple reference gene procedures demonstrated consistent expression pattern with a significant up-regulation of prdx5 in response to the higher concentration after 21 days. Concluding, even if not yet established as "gold" standard for expression profiling in environmental toxicology or physiology using freshwater or marine fish models, the multiple reference gene approach is recommended, since it eliminates any biased results, which represented the major flaw of single reference genes.