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Rectal cancer patients can conceivably obtain relief from neoadjuvant concurrent chemoradiotherapy (CCRT) for downstaging before resection, but the stratification of risk and clinical outcomes remains challenging. Therefore, identifying effective predictive biomarkers offers clinicians the opportunity to individually tailor early interventions, which would help optimize therapy. Using a public rectal cancer transcriptome dataset (GSE35452), we focused on cytoskeletal protein binding (GO: 0008092)-related genes and identified FERM domain containing 3 (FRMD3) as the most significant differentially expressed gene associated with CCRT resistance. We gathered 172 tumor samples from rectal cancer patients treated with neoadjuvant CCRT accompanied by curative resection and estimated the expression level of FRMD3 using immunohistochemistry. The results revealed that high FRMD3 immunoexpression was remarkably associated with advanced pre-CCRT and post-CCRT tumor status (p = 0.004 and p

Melanoma is a highly aggressive malignant skin tumor with a high rate of metastasis and mortality. In this study, a comprehensive bioinformatics analysis was used to clarify the hub genes and potential drugs. Download the GSE3189, GSE22301, and GSE35388 microarray datasets from the Gene Expression Omnibus (GEO), which contains a total of 33 normal samples and 67 melanoma samples. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) approach analyze DEGs based on the DAVID. Use STRING to construct protein-protein interaction network, and use MCODE and cytoHubba plug-ins in Cytoscape to perform module analysis and identified hub genes. Use Gene Expression Profile Interactive Analysis (GEPIA) to assess the prognosis of genes in tumors. Finally, use the Drug-Gene Interaction Database (DGIdb) to screen targeted drugs related to hub genes. A total of 140 overlapping DEGs were identified from the three microarray datasets, including 59 up-regulated DEGs and 81 down-regulated DEGs. GO enrichment analysis showed that these DEGs are mainly involved in the biological process such as positive regulation of gene expression, positive regulation of cell proliferation, positive regulation of MAP kinase activity, cell migration, and negative regulation of the apoptotic process. The cellular components are concentrated in the membrane, dendritic spine, the perinuclear region of cytoplasm, extracellular exosome, and membrane raft. Molecular functions include protein homodimerization activity, calmodulin-binding, transcription factor binding, protein binding, and cytoskeletal protein binding. KEGG pathway analysis shows that these DEGs are mainly related to protein digestion and absorption, PPAR signaling pathway, signaling pathways regulating stem cells' pluripotency, and Retinol metabolism. The 23 most closely related DEGs were identified from the PPI network and combined with the GEPIA prognostic analysis, CDH3, ESRP1, FGF2, GBP2, KCNN4, KIT, SEMA4D, and ZEB1 were selected as hub genes, which are considered to be associated with poor prognosis of melanoma closely related. Besides, ten related drugs that may have therapeutic effects on melanoma were also screened. These newly discovered genes and drugs provide new ideas for further research on melanoma.

The development of selectively bred high and low alcohol-preferring mice (HAP and LAP, respectively) has allowed for an assessment of the polygenetic risk for pathological alcohol consumption and phenotypes associated with alcohol use disorder (AUD). Accumulating evidence indicates that the dorsal striatum (DS) is a central node in the neurocircuitry underlying addictive processes. Therefore, knowledge of differential gene, protein, and phosphorylated protein expression in the DS of HAP and LAP mice may foster new insights into how aberrant DS functioning may contribute to AUD-related phenotypes. To begin to elucidate these basal differences, a complementary and integrated analysis of DS tissue from alcohol-naïve male and female HAP and LAP mice was performed using RNA sequencing, quantitative proteomics, and phosphoproteomics. These datasets were subjected to a thorough analysis of gene ontology, pathway enrichment, and hub gene assessment. Analyses identified 2,108, 390, and 521 significant differentially expressed genes, proteins, and phosphopeptides, respectively between the two lines. Network analyses revealed an enrichment in the differential expression of genes, proteins, and phosphorylated proteins connected to cellular organization, cytoskeletal protein binding, and pathways involved in synaptic transmission and functioning. These findings suggest that the selective breeding to generate HAP and LAP mice may lead to a rearrangement of synaptic architecture which could alter DS neurotransmission and plasticity differentially between mouse lines. These rich data sets will serve as an excellent resource to inform future studies on how inherited differences in gene, protein, and phosphorylated protein expression contribute to AUD-related phenotypes.

Xin Wang,1,* Ya Cheng,1,* Liang-liang Yan,2 Ran An,1 Xing-yu Wang,1 Heng-yi Wang1 1Department of Emergency Surgery, The First Affiliated Hospital of Anhui Medical University, Hefei 230032, People’s Republic of China; 2Department of Rheumatology and Immunology, The First Affiliated Hospital of Anhui Medical University, Hefei 230032, People’s Republic of China*These authors contributed equally to this workCorrespondence: Heng-yi Wang Tel +86-156-0551-1180Email why00606@sina.comBackground: Hepatocellular carcinoma (HCC) includes cryptogenic hepatocellular carcinomas (CR-HCC) that lack a defined cause. Specific DNA methylation patterns and comparisons of the aberrant alterations in DNA methylation between CR-HCC and adjacent peritumor tissues (APTs) have not yet been reported.Methods: The SureSelectXT Methyl-Seq Target Enrichment System was used to sequence targeted DNA methylation in three paired CR-HCC tissues and APTs. Gene Ontology (GO) enrichment and KEGG pathway analysis were performed to investigate the DNA methylation mechanism of CR-HCC. The mRNA expression levels of HOXB-AS3, HOXB6, HOXB3, USP18, MAP3K6, TIRAP, TNNI2, SHC3, CTTN, and TFAP2A, selected from the identified signaling pathways, were evaluated by quantitative real-time PCR (qPCR).Results: A total of 1728 differentially methylated regions (DMRs) were identified in tumor tissues compared with non-tumor tissues, of which 868 DMRs were hypermethylated and 860 were hypomethylated. The DMRs were mapped within 2091 DMR-associated genes (DMGs). The mRNA expression of HOXB-AS3, HOXB3, and MAP3K6 was downregulated in CR-HCC tissues compared to the APTs. However, the mRNA expression of TIRAP, SHC3, and CTTN was upregulated in the CR-HCC tissues. Differences between the mRNA expression of HOXB6, USP18, TNNI2, and TFAP2A in the CR-HCC and APTS tissues were not statistically significant. GO analysis showed that the molecular functions of “binding”, “protein binding”, and “cytoskeletal protein binding” were the main categories for the hypermethylated DMGs. The hypomethylated DMGs were mostly enriched in the molecular functions “binding”, “protein binding”, “calcium ion binding”, among others. KEGG pathway analysis showed that the hypermethylated DMGs were enriched in several pathways such as “estrogen signaling pathway”, while hypomethylated DMGs were enriched in several pathways such as “proteoglycans in cancer”, suggesting that epigenetic modifications play important roles in the cryptogenic hepatocarcinogenesis.Conclusion: These results provide useful information for future work to characterize the functions of epigenetic mechanisms on CR-HCC.Keywords: cryptogenic hepatocellular carcinomas, DNA methylation, DMR

Muscle tissue is involved with every stage of life activities and has roles in biological processes. For example, the blood circulation system needs the heart muscle to transport blood to all parts, and the movement cannot be separated from the participation of skeletal muscle. However, the process of muscle development and the regulatory mechanisms of muscle development are not clear at present. In this study, we used bioinformatics techniques to identify differentially expressed genes specifically expressed in multiple muscle tissues of mice as potential candidate genes for studying the regulatory mechanisms of muscle development. Mouse tissue microarray data from 17 tissue samples was selected from the GEO database for analysis. Muscle tissue as the treatment group, and the other 16 tissues as the control group. Genes expressed in the muscle tissue were different to those in the other 16 tissues and identified 272 differential genes with highly specific expression in muscle tissue, including 260 up-regulated genes and 12 down regulated genes. is the genes were associated with the myofibril, contractile fibers, and sarcomere, cytoskeletal protein binding, and actin binding. KEGG pathway analysis showed that the differentially expressed genes in muscle tissue were mainly concentrated in pathways for AMPK signaling, cGMP PKG signaling calcium signaling, glycolysis, and, arginine and proline metabolism. A PPI protein interaction network was constructed for the selected differential genes, and the MCODE module used for modular analysis. Five modules with Score > 3.0 are selected. Then the Cytoscape software was used to analyze the tissue specificity of differential genes, and the genes with high degree scores collected, and some common genes selected for quantitative PCR verification. The conclusion is that we have screened the differentially expressed gene set specific to mouse muscle to provide potential candidate genes for the study of the important mechanisms of muscle development.

Previous evidence pointed out a role for the striatal-enriched protein Rhes in modulating dopaminergic transmission. Based on the knowledge that cocaine induces both addiction and motor stimulation, through its ability to enhance dopaminergic signaling in the corpus striatum, we have now explored the involvement of Rhes in the effects associated with this psychostimulant. Our behavioral data showed that a lack of Rhes in knockout animals caused profound alterations in motor stimulation following cocaine exposure, eliciting a significant leftward shift in the dose-response curve and triggering a dramatic hyperactivity. We also found that Rhes modulated either short- or long-term motor sensitization induced by cocaine, since lack of this protein prevents both of them in mutants. Consistent with this in vivo observation, we found that lack of Rhes in mice caused a greater increase in striatal cocaine-dependent D1R/cAMP/PKA signaling, along with considerable enhancement of Arc, zif268, and Homer1 mRNA expression. We also documented that lack of Rhes in mice produced cocaine-related striatal alterations in proteomic profiling, with a differential expression of proteins clustering in calcium homeostasis and cytoskeletal protein binding categories. Despite dramatic striatal alterations associated to cocaine exposure, our data did not reveal any significant changes in midbrain dopaminergic neurons as a lack of Rhes did not affect: (i) DAT activity; (ii) D2R-dependent regulation of GIRK; and (iii) D2R-dependent regulation of dopamine release. Collectively, our results strengthen the view that Rhes acts as a pivotal physiological “molecular brake” for striatal dopaminergic system overactivation induced by psychostimulants, thus making this protein of interest in regulating the molecular mechanism underpinning cocaine-dependent motor stimulatory effects.

This study, for the first time in fish, compared the transcriptome of fresh and frozen-thawed sperm, and would help to better understand the effect of cryopreservation on fish sperm and then better preserve the aquatic germplasm resources. Here, we employed high-throughput sequencing technology to obtain the transcriptome of yellow catfish from fresh sperm, cryopreserved sperm with and without cryoprotectant. When cryoprotectant (Me2SO) was excluded, down-regulated genes were significantly enriched into calcium ion binding, cytoskeletal protein binding, microfilament motor activity, calmodulin binding and carnitine O-acyltransferase activity, which affected Ca2+ regulation, cellular morphology, motility and metabolism. Moreover, heat shock proteins and genes associated with regulation of cholesterol, HCO3- and protein tyrosine phosphorylation (PTP) were down-regulated, and thus would impair ability against stress, membrane rigidity, pH regulation and signal transduction of cryopreserved sperm. After Me2SO was added, the amounts of DEGs decreased significantly and down-regulation of genes were found mainly in cytoskeleton and heat shock proteins, thereby suggesting that Me2SO effectively reduced the impact caused by low temperature on gene expression. Whether adding Me2SO or not, the up-regulated genes were mainly found in ribosomal proteins genes. However, when Me2SO was added, over-expression of some genes might contribute to maintain normal function of cryopreserved sperm.

In total, ~25% of familial breast cancer (BC) is attributed to germline mutations of the BRCA1 and BRCA2 genes, while the rest of the cases are included in the BRCAX group. BC is also known to affect men, with a worldwide incidence of 1%. Epigenetic alterations, including DNA methylation, have been rarely studied in male breast cancer (MBC) on a genome-wide level. The aim of the present study was to examine the global DNA methylation profiles of patients with BC to identify differences between familial female breast cancer (FBC) and MBC, and according to BRCA1, BRCA2 or BRCAX mutation status. The genomic DNA of formalin-fixed paraffin-embedded tissues from 17 women and 7 men with BC was subjected to methylated DNA immunoprecipitation and hybridized on human promoter microarrays. The comparison between FBC and MBC revealed 2,846 significant differentially methylated regions corresponding to 2,486 annotated genes. Gene Ontology enrichment analysis revealed molecular function terms, such as the GTPase superfamily genes (particularly the GTPase Rho GAP/GEF and GTPase RAB), and cellular component terms associated with cytoskeletal architecture, such as ‘cytoskeletal part’, ‘keratin filament’ and ‘intermediate filament’. When only FBC was considered, several cancer-associated pathways were among the most enriched KEGG pathways of differentially methylated genes when the BRCA2 group was compared with the BRCAX or BRCA1+BRCAX groups. The comparison between the BRCA1 and BRCA2+BRCAX groups comprised the molecular function term ‘cytoskeletal protein binding’. Finally, the functional annotation of differentially methylated genes between the BRCAX and BRCA1+BRCA2 groups indicated that the most enriched molecular function terms were associated with GTPase activity. In conclusion, to the best of our knowledge, the present study was the first to compare the global DNA methylation profile of familial FBC and MBC. The results may provide useful insights into the epigenomic subtyping of BC and shed light on a possible novel molecular mechanism underlying BC carcinogenesis.

Burkholderia mallei, the causative agent of glanders, is a gram-negative intracellular bacterium. Depending on different routes of infection, the disease is manifested by pneumonia, septicemia, and chronic infections of the skin. B. mallei poses a serious biological threat due to its ability to infect via aerosol route, resistance to multiple antibiotics and to date there are no US Food and Drug Administration (FDA) approved vaccines available. Induction of innate immunity, inflammatory cytokines and chemokines following B. mallei infection, have been observed in in vitro and small rodent models; however, a global characterization of host responses has never been systematically investigated using a non-human primate (NHP) model. Here, using a liquid chromatography-tandem mass spectrometry (LC-MS/MS) approach, we identified alterations in expression levels of host proteins in peripheral blood mononuclear cells (PBMCs) originating from naïve rhesus macaques (Macaca mulatta), African green monkeys (Chlorocebus sabaeus), and cynomolgus macaques (Macaca fascicularis) exposed to aerosolized B. mallei. Gene ontology (GO) analysis identified several statistically significant overrepresented biological annotations including complement and coagulation cascade, nucleoside metabolic process, vesicle-mediated transport, intracellular signal transduction and cytoskeletal protein binding. By integrating an LC-MS/MS derived proteomics dataset with a previously published B. mallei host-pathogen interaction dataset, a statistically significant predictive protein-protein interaction (PPI) network was constructed. Pharmacological perturbation of one component of the PPI network, specifically ezrin, reduced B. mallei mediated interleukin-1β (IL-1β). On the contrary, the expression of IL-1β receptor antagonist (IL-1Ra) was upregulated upon pretreatment with the ezrin inhibitor. Taken together, inflammasome activation as demonstrated by IL-1β production and the homeostasis of inflammatory response is critical during the pathogenesis of glanders. Furthermore, the topology of the network reflects the underlying molecular mechanism of B. mallei infections in the NHP model.

BackgroundNew noninvasive and affordable molecular approaches that will complement current practices and increase the accuracy of PD diagnosis are urgently needed. CircRNAs are highly stable non-coding RNAs that accumulate with aging in neurons and are increasingly shown to regulate all aspects of neuronal development and function.ObjectivesThe aims of the present study were to identify differentially expressed circRNAs in PBMCs of idiopathic PD patients and explore the competing endogenous RNA networks affected.MethodsEighty-seven circRNAs were initially selected based on relatively high gene expression in the human brain. Over half of these were readily detectable in PBMCs using RT-qPCR. Comparative expression analysis was then performed in PBMCs from sixty controls and sixty idiopathic subjects with PD.ResultsSix circRNAs derived from MAPK9, HOMER1, SLAIN1, DOP1B, REPS1, and PSEN1 transcripts were significantly downregulated in PD patients. The classifier that best distinguished PD consisted of four circRNAs with an AUC of 0.84. CLIP-Seq data revealed that the RNA binding proteins bound by most of the deregulated circRNAs include the neurodegeneration-associated FUS, TDP43, FMR1 and ATXN2. MicroRNAs predicted to be sequestered by most deregulated circRNAs had the GOslim categories ‘Protein modification’, ‘Transcription factor activity’ and ‘Cytoskeletal protein binding’ mostly enriched.ConclusionsThis is the first study that identifies circRNAs deregulated in the peripheral blood of PD patients. They may serve as diagnostic biomarkers and since they are highly expressed in the brain and are derived from genes with essential brain functions, they may also hint on the PD pathways affected.

Background The scale degeneration is thought to be related to the adaptation to the extreme environment with cold climate and high-altitude in schizothoracine fishes. Gymnocypris eckloni, a schizothoracine fish living in plateau waters with the elevation above 2500 m, is nearly esquamate and only covered with shoulder scales and anal scales, making it a good model species to study the molecular mechanism of scale degeneration. Results The transcriptomes of shoulder scaled skins (SSS), anal scaled skins (ASS) and scaleless skins (NSS) were sequenced and analyzed in G. eckloni at the age of 1 year. Histological examination showed that shoulder scale had completed its differentiation and anal scale just initiated the differentiation. A total of 578,046 unigenes were obtained from the transcriptomes, with 407,799 unigenes annotated in public databases. A total of 428 and 142 differentially expressed unigenes (DEUs) were identified between SSS and NSS, and between ASS and NSS, respectively, with 45 DEUs that were overlapped. Annotation analysis indicated that these DEUs were mainly enriched in Gene Ontology (GO) terms and KEGG pathways associated with bone and muscle formation, such as myofibril, contractile fiber, cytoskeletal protein binding, muscle structure development, cardiac muscle contraction, hypertrophic cardiomyopathy (HCM) and calcium signaling pathway. Conclusions Our results would provide insights into the molecular mechanisms of scale degeneration in G. eckloni and other congeneric fishes. In addition, the transcriptome data provides candidate genes and markers for future studies.

Background and objectives Dental cementum (DC) is a mineralized tissue covering tooth roots that plays a critical role in dental attachment. Differences in deciduous vs. permanent tooth DC have not been explored. We hypothesized that proteomic analysis of DC matrix would identify compositional differences in deciduous (DecDC) vs. permanent (PermDC) cementum that might reflect physiological or pathological differences, such as root resorption that is physiological in deciduous teeth but can be pathological in the permanent dentition. Methods Protein extracts from deciduous (n = 25) and permanent (n = 12) teeth were pooled (five pools of DecDC, five teeth each; four pools of PermDC, three teeth each). Samples were denatured, and proteins were extracted, reduced, alkylated, digested, and analyzed by liquid chromatography-mass spectrometry (LC-MS/MS). The beta-binomial statistical test was applied to normalized spectrum counts with 5% significance level to determine differentially expressed proteins. Immunohistochemistry was used to validate selected proteins. Results A total of 510 proteins were identified: 123 (24.1%) exclusive to DecDC; 128 (25.1%) exclusive to PermDC; 259 (50.8%) commonly expressed in both DecDC and PermDC. Out of 60 differentially expressed proteins, 17 (28.3%) were detected in DecDC, including myeloperoxidase (MPO), whereas 43 (71.7%) were detected in PermDC, including decorin (DCN) and osteocalcin (BGLAP). Overall, Gene Ontology (GO) analysis indicated that all expressed proteins were related to GO biological processes that included localization and response to stress, and the GO molecular function of differentially expressed proteins was enriched in cell adhesion, molecular binding, cytoskeletal protein binding, structural molecular activity, and macromolecular complex binding. Immunohistochemistry confirmed the trends for selected differentially expressed proteins in human teeth. Conclusions Clear differences were found between the proteomes of DecDC and PermDC. These findings may lead to new insights into developmental differences between DecDC and PermDC, as well as to a better understanding of physiological/pathological events such as root resorption.

Background Circular RNA (circRNA) is produced during the splicing of mRNA (in addition to linear splicing) and is part of the gene regulatory network. The temporal expression patterns the different developmental stages were inseparable from these molecules’ function. Results Skeletal muscles of Anhui white goat (AWG) across seven fetal to postnatal development stages were sequenced and 21 RNA sequencing libraries were constructed. We thereby identified 9090 circRNAs and analyzed their molecular properties, temporal expression patterns, and potential functions at the different stages. CircRNAs showed complexities and diversity of formation as the same host gene produces multiple isoforms of these nucleic acids with different expression profiles. The differential expression of 2881 circRNAs (DECs, P FC| > 2 and P-adj value Conclusion Our current study provides a catalog of goat muscle-related circRNAs that can stratify skeletal muscle development fetus 45 days to newborn 90 days into three developmental stages. These findings better our understanding of functional transitions during mammalian muscle development.

The dentin-enamel junction (DEJ) is the border where two different mineralized structures - enamel and dentin - meet. The protein-rich DEJ, together with the inner enamel region of mature teeth, is known to exhibit higher fracture toughness and crack growth resistance than bulk phase enamel. However, an explanation for this behavior has been hampered by the lack of compositional information for the DEJ and the adjacent enamel organic matrix (EOM). We studied proteomes of the DEJ and EOM of healthy human molars and compared them with dentin and enamel proteomes from the same teeth. These tissues were cut out of tooth sections by laser capture microdissection, proteins were extracted and cleaved by trypsin, then processed by liquid chromatography coupled to tandem mass spectrometry to analyze the proteome profiles of these tissues. This study identified 46 proteins in DEJ and EOM. The proteins identified have a variety of functions, including calcium ion-binding, formation of extracellular matrix, formation of cytoskeleton, cytoskeletal protein binding, cell adhesion, and transport. Collagens were identified as the most dominant proteins. Tissue-specific proteins, such as ameloblastin and amelogenin, were also detected. Our findings reveal new insight into proteomics of DEJ and EOM, highly mineralized tissues that are obviously difficult to analyze.

Large yellow croaker (Larimichthys crocea), one of the most important marine fish species in China, can change its abdominal skin color when it is shifted from light to dark or from dark to light, providing us an opportunity of investigating the molecular responding mechanism of teleost in light stress. The gene expression profile of fish under light stress is rarely documented. In this research, the transcriptome profiles of the abdominal skin of L. crocea exposed to light or dark for 0 h, 0.5 h and 2 h were produced by next-generation sequencing (NGS). The cluster results demonstrated that stress period, rather than light intensity (e.g., light or dark), is the major influencing factor. Differently expressed genes (DEGs) were identified between 0 h and 0.5 h groups, between 0 h and 2 h groups, between 0.5 h light and 0.5 h dark, and between 2 h light and 2 h dark, respectively. The gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) annotation revealed that the genes relating to immunity, energy metabolism, and cytoskeletal protein binding were significantly enriched. The detailed analysis of transcriptome profiles also revealed regular gene expression trends, indicating that the elaborate gene regulation networks underlined the molecular responses of the fish to light stress. This transcriptome analysis suggested that systematic and complicated regulatory cascades were functionally activated in response to external stress, and coloration change caused by light stress was mainly attributed to the change in the density of chromatophores for L. crocea. This study also provided valuable information for skin coloration or light stress research on other marine fish species.

Deregulation of microRNAs (miRNAs) contributes to nontraumatic osteonecrosis of the femoral head (ONFH-N), but the differentially expressed circulating miRNAs in patients with ONFH-N receiving nonsurgical therapy are unknown. This study aimed to determine the miRNAs expression profile of patients with ONFH-N receiving conservative treatments. This was a case-control prospective study of 43 patients with ONFH-N and 43 participants without ONFH-N, enrolled from 10/2014 to 10/2016 at the Department of Orthopedics of the Linyi People’s Hospital (China). The two groups were matched for age, gender, and living area. Microarray analysis and quantitative RT-PCR were used to examine the differentially expressed miRNAs. Bioinformatics was used to predict miRNA target genes and signaling pathways. Microarray and quantitative RT-PCR revealed that nine miRNAs were downregulated and five miRNAs were upregulated in ONFH-N(n=3)compared with controls(n=3). Bioinformatics showed that calcium-mediated signaling pathway, regulation of calcium ion transmembrane transporter activity, cytoskeletal protein binding, and caveolae macromolecular signaling complex were probably regulated by the identified differentially expressed miRNAs. In the remaining 80 subjects (n=40/group), miR-335-5p was downregulated(P=0.01)and miR-100-5p was upregulated(P=0.02)in ONFH-N compared with controls. In conclusion, some miRNAs are differentially expressed in conservatively treated ONFH-N compared with controls. Those miRNAs could contribute to the pathogenesis of ONFH-N.

Abnormal expression of noncoding RNA molecules such as circRNA plays an important role in the development of malignant tumors. circRNAs are stable in structure and can be useful as ideal tumor markers. Advanced bladder cancer has poor treatment options and prognosis. Thus, we examined circRNAs to further understand the pathogenesis and development of bladder cancer and to identify molecular markers for the early diagnosis of bladder carcinoma. We found that hsa_circ_0018069 was differentially expressed in our RNA sequencing data. We used qRT-PCR to detect its expression in T24 and Biu-87 cell lines and in 41 paired samples of bladder cancer and adjacent normal tissue and analyzed the correlation between expression of hsa_circ_0018069 and the clinical characteristics of patients with bladder cancer. We then performed a bioinformatics analysis to reveal the mechanism of hsa_circ_0018069 in tumorigenesis of bladder cancer. The expression of hsa_circ_0018069 was significantly reduced in T24 and Biu-87 cells and was also significantly downregulated in bladder cancer tissues. Decreased expression of hsa_circ_0018069 was related to the grade stage (P=0.024), T stage (P=0.027), and muscular invasion depth (P=0.022) of bladder cancer. Bioinformatics analysis showed that hsa_circ_0018069 was coexpressed with protein-coding mRNAs that participate in cytoskeletal protein binding and cell-substrate junction assembly and play an anticancer role through focal adhesion and calcium signaling pathways. ceRNA analysis showed that hsa_circ_0018069 functions in ErbB, Ras, FoxO, and the focal adhesion signaling pathway by harboring miR-23c, miR-34a-5p, miR-181b-5p, miR-454-3p, and miR-3666. hsa_circ_0018069 may thus play an important role in the occurrence and progression of bladder cancer and serve as a valuable biomarker for the early diagnosis of this disease.

Heritable symbioses are thought to be important for the maintenance of mutually beneficial relationships (1), and for facilitating major transitions in individuality, such as the evolution of the eukaryotic cell (2, 3). In stony corals, vertical transmission has evolved repeatedly (4), providing a unique opportunity to investigate the genomic basis of this complex trait. We conducted a comparative analysis of 25 coral transcriptomes to identify orthologous genes exhibiting both signatures of positive selection and convergent amino acid substitutions in vertically transmitting lineages. The frequency of convergence events tends to be higher among vertically transmitting lineages, consistent with the proposed role of natural selection in driving the evolution of convergent transmission mode phenotypes (5). Of the 10,774 total orthologous genes identified, 403 exhibited at least one molecular convergence event and evidence of positive selection in at least one vertically transmitting lineage. Functional enrichments among these top candidate genes include processes previously implicated in mediating the coral-Symbiodiniaceae symbiosis including endocytosis, immune response, cytoskeletal protein binding and cytoplasmic membrane-bounded vesicles (6). We also identified 100 genes showing evidence of positive selection at the particular convergence event. Among these, we identified several novel candidate genes, highlighting the value of our approach for generating new insight into the mechanistic basis of the coral symbiosis, in addition to uncovering host mechanisms associated with the evolution of heritable symbioses.

Human dental follicle cells (DFCs) and periodontal ligament cells (PDLCs) derived from the ectomesenchymal tissue, have been shown to exhibit stem/progenitor cell properties and the ability to induce tissue regeneration. Stem cells in dental follicle differentiate into cementoblasts, periodontal ligament fibroblasts and osteoblasts, these cells form cementum, periodontal ligament and alveolar bone, respectively. While stem cells in dental follicle are a precursor to periodontal ligament fibroblasts, the molecular changes that distinguish cultured DFCs from PDLCs are still unknown. In this study, we have compared the immunophenotypic features and cell cycle status of the two cell lines. The results suggest that DFCs and PDLCs displayed similar features related to immunophenotype and cell cycle. Then we employed an isobaric tag for relative and absolute quantitation (iTRAQ) proteomics strategy to reveal the molecular differences between the two cell types. A total of 2138 proteins were identified and 39 of these proteins were consistently differentially expressed between DFCs and PDLCs. Gene ontology analyses revealed that the protein subsets expressed higher in PDLCs were related to actin binding, cytoskeletal protein binding, and structural constituent of muscle. Upon validation by real-time PCR, western blotting, and immunofluorescence staining. Tropomyosin 1 (TPM1) and caldesmon 1 (CALD1) were expressed higher in PDLCs than in DFCs. Our results suggested that PDLCs display enhanced actin cytoskeletal dynamics relative to DFCs while DFCs may exhibit a more robust antioxidant defense ability relative to PDLCs. This study expands our knowledge of the cultured DFCs and PDLCs proteome and provides new insights into possible mechanisms responsible for the different biological features observed in each cell type.

Meat quality is a complex trait influenced by many factors, including genetics, nutrition, feeding environment, animal handling, and their interactions. To elucidate relevant factors affecting pork quality associated with oxidative stress and muscle development, we analyzed protein expression in high quality longissimus dorsi muscles (HQLD) and low quality longissimus dorsi muscles (LQLD) from Duroc pigs by liquid chromatographytandem mass spectrometry (LC-MS/MS)-based proteomic analysis. Between HQLD (n = 20) and LQLD (n = 20) Duroc pigs, 24 differentially expressed proteins were identified by LC-MS/MS. A total of 10 and 14 proteins were highly expressed in HQLD and LQLD, respectively. The 24 proteins have putative functions in the following seven categories: catalytic activity (31%), ATPase activity (19%), oxidoreductase activity (13%), cytoskeletal protein binding (13%), actin binding (12%), calcium ion binding (6%), and structural constituent of muscle (6%). Silver-stained image analysis revealed significant differential expression of lactate dehydrogenase A (LDHA) between HQLD and LQLD Duroc pigs. LDHA was subjected to in vitro study of myogenesis under oxidative stress conditions and LDH activity assay to verification its role in oxidative stress. No significant difference of mRNA expression level of LDHA was found between normal and oxidative stress condition. However, LDH activity was significantly higher under oxidative stress condition than at normal condition using in vitro model of myogenesis. The highly expressed LDHA was positively correlated with LQLD. Moreover, LDHA activity increased by oxidative stress was reduced by antioxidant resveratrol. This paper emphasizes the importance of differential expression patterns of proteins and their interaction for the development of meat quality traits. Our proteome data provides valuable information on important factors which might aid in the regulation of muscle development and the improvement of meat quality in longissimus dorsi muscles of Duroc pigs under oxidative stress conditions.

Osteoporosis is known to be highly heritable. However, to date, the findings from more than 20 genome-wide association studies (GWASs) have explained less than 6% of genetic risks. Studies suggest that the missing heritability data may be because of joint effects among genes. To identify novel heritability for osteoporosis, we performed a system-level study on bone mineral density (BMD) by weighted gene coexpression network analysis (WGCNA), using the largest GWAS data set for BMD in the field, Genetic Factors for Osteoporosis Consortium (GEFOS-2), and a transcriptomic gene expression data set generated from transiliac bone biopsies in women. A weighted gene coexpression network was generated for 1574 genes with GWAS nominal evidence of association (p ≤ 0.05) based on dissimilarity measurement on the expression data. Twelve distinct gene modules were identified, and four modules showed nominally significant associations with BMD (p ≤ 0.05), but only one module, the yellow module, demonstrated a good correlation between module membership (MM) and gene significance (GS), suggesting that the yellow module serves an important biological role in bone regulation. Interestingly, through characterization of module content and topology, the yellow module was found to be significantly enriched with contractile fiber part (GO:044449), which is widely recognized as having a close relationship between muscle and bone. Furthermore, detailed submodule analyses of important candidate genes (HOMER1, SPTBN1) by all edges within the yellow module implied significant enrichment of functional connections between bone and cytoskeletal protein binding. Our study yielded novel information from system genetics analyses of GWAS data jointly with transcriptomic data. The findings highlighted a module and several genes in the model as playing important roles in the regulation of bone mass in females, which may yield novel insights into the genetic basis of osteoporosis. © 2016 American Society for Bone and Mineral Research.

One out of ten patients with pheochromocytoma (PCC) and paraganglioma (PGL) develop malignant disease. Today there are no reliable pathological methods to predict malignancy at the time of diagnosis. Tumors harboring mutations in the succinate dehydrogenase subunit B (SDHB) gene often metastasize but the sequential genetic events resulting in malignant progression are not fully understood. The aim of this study was to identify somatic mutations that contribute to the malignant transformation of PCC/PGL. We performed pair-wise (tumor-normal) whole-exome sequencing to analyze the somatic mutational landscape in five malignant and four benign primary PCC/sympathetic PGL (sPGL), including two biological replicates from each specimen. In total, 225 unique somatic mutations were identified in 215 genes, with an average mutation rate of 0.54 mutations/megabase. Malignant tumors had a significantly higher number of mutations compared to benign tumors (p

High-throughput sequencing (HTS) was recently applied to detect microRNA (miRNA) regulation in age-related osteoporosis. However, miRNA regulation has not been reported in glucocorticoid-induced osteoporosis (GIOP) patients and the mechanism of GIOP remains elusive. To comprehensively analyze the role of miRNA regulation in GIOP based on human vertebrae and to explore the molecular mechanism, a high-throughput sequencing strategy was employed to identify miRNAs involved in GIOP. Twenty-six patients undergoing spinal surgery were included in this study. Six vertebral samples were selected for miRNA sequencing (miRNA-seq) analysis and 26 vertebral samples were verified by qRT-PCR. Bioinformatics was utilized for target prediction, to investigate the regulation of miRNA-mRNA networks, and Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses. Six significantly up-regulated miRNAs (including one novel miRNA) and three significantly down-regulated miRNAs were verified via miRNA-seq and verified in the vertebrae of GIOP patients. Up-regulated miRNAs included hsa-miR-214-5p, hsa-miR-10b-5p, hsa-miR-21-5p, hsa-miR-451a, hsa-miR-186-5p, and hsa-miR-novel-chr3_49,413 while down-regulated miRNAs included hsa-let-7f-5p, hsa-let-7a-5p, and hsa-miR-27a-3p. Bioinformatics analysis revealed 5983 and 23,463 predicted targets in the up-regulated and down-regulated miRNAs respectively, using the miRanda, miRBase and TargetScan databases. The target genes of these significantly altered miRNAs were enriched to 1939 GO terms and 84 KEGG pathways. GO terms revealed that up-regulated targets were most enriched in actin filament-based processes (BP), anchoring junction (CC), and cytoskeletal protein binding (MF). Conversely, the down-regulated targets were mostly enriched in multicellular organismal development (BP), intracellular membrane-bounded organelles (CC), and protein binding (MF). Top-10 pathway analysis revealed that the differentially expressed miRNAs in GIOP were closely related to bone metabolism-related pathways such as FoxO, PI3K-Akt, MAPK and Notch signaling pathway. These results suggest that significantly altered miRNAs may play an important role in GIOP by targeting mRNA and regulating biological processes and bone metabolism-related pathways such as MAPK, FoxO, PI3K-Akt and Notch signaling, which provides novel insight into the mechanism of GIOP and lays a good foundation for the prevention and treatment of GIOP.

Stable single-alpha helices (SAH-domains) function as rigid connectors and constant force springs between structural domains, and can provide contact surfaces for protein-protein and protein-RNA interactions. SAH-domains mainly consist of charged amino acids and are monomeric and stable in polar solutions, characteristics which distinguish them from coiled-coil domains and intrinsically disordered regions. Although the number of reported SAH-domains is steadily increasing, genome-wide analyses of SAH-domains in eukaryotic genomes are still missing. Here, we present Waggawagga-CLI, a command-line tool for predicting and analysing SAH-domains in protein sequence datasets. Using Waggawagga-CLI we predicted SAH-domains in 24 datasets from eukaryotes across the tree of life. SAH-domains were predicted in 0.5 to 3.5% of the protein-coding content per species. SAH-domains are particularly present in longer proteins supporting their function as structural building block in multi-domain proteins. In human, SAH-domains are mainly used as alternative building blocks not being present in all transcripts of a gene. Gene ontology analysis showed that yeast proteins with SAH-domains are particular enriched in macromolecular complex subunit organization, cellular component biogenesis and RNA metabolic processes, and that they have a strong nuclear and ribonucleoprotein complex localization and function in ribosome and nucleic acid binding. Human proteins with SAH-domains have roles in all types of RNA processing and cytoskeleton organization, and are predicted to function in RNA binding, protein binding involved in cell and cell-cell adhesion, and cytoskeletal protein binding. Waggawagga-CLI allows the user to adjust the stabilizing and destabilizing contribution of amino acid interactions in i,i+3 and i,i+4 spacings, and provides extensive flexibility for user-designed analyses.

Bone marrow mesenchymal stem cells (BMSCs) nowadays are regarded as promising candidates in cell-based therapy for the regeneration of damaged bone tissues that are either incurable or intractable due to the insufficiency of current therapies. Recent studies suggest that BMSCs differentiate into osteoblasts, and that this differentiation is regulated by some specific patterns of epigenetic modifications, such as DNA methylation. However, the potential role of DNA methylation modification in BMSC osteogenic differentiation is unclear. In this study, we performed a genome-wide study of DNA methylation between the noninduced and induced osteogenic differentiation of BMSCs at day 7. We found that the majority of cytosines in a CpG context were methylated in induced BMSCs. Our results also revealed that, along with the induced osteogenic differentiation in BMSCs, the average genomic methylation levels and CpG methylation in transcriptional factor regions (TFs) were increased, the CpG methylation level of various genomic elements was mainly in the medium-high methylation section, and CpG methylation levels in the repeat element had highly methylated levels. The GO analysis of differentially methylated region- (DMR-) associated genes (DMGs) showed that GO terms, including cytoskeletal protein binding (included in Molecular Function GO terms), skeletal development (included in Biological Process GO terms), mesenchymal cell differentiation (included in Biological Process GO terms), and stem cell differentiation (included in Biological Process), were enriched in the hypermethylated DMGs. Then, the KEGG analysis results showed that the WNT pathway, inositol phosphate metabolism pathway, and cocaine addiction pathway were more correlative with the DMRs during the induced osteogenic differentiation in BMSCs. In conclusion, this study revealed the difference of methylated levels during the noninduced and induced osteogenic differentiation of BMSCs and provided useful information for future works to characterize the important function of epigenetic mechanisms on BMSCs’ differentiation.

BACKGROUND CONTEXT High-throughput sequencing (HTS) was recently applied to detect microRNA (miRNA) regulation in age-related osteoporosis. However, miRNA regulation has not been reported in glucocorticoid-induced osteoporosis (GIOP) patients and the mechanism of GIOP remains elusive. PURPOSE To comprehensively analyze the role of miRNA regulation in GIOP based on human vertebrae and to explore the molecular mechanism. STUDY DESIGN/SETTING A high-throughput sequencing strategy was employed to identify miRNAs involved in GIOP. Twenty-six patients undergoing spinal surgery were included in this study. Six vertebral samples were selected for miRNA sequencing (miRNA-seq) analysis and 26 vertebral samples were verified by qRT-PCR. Bioinformatics was utilized for target prediction, to investigate the regulation of miRNA-mRNA networks, and Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses. PATIENT SAMPLE Twenty-six patients undergoing spinal surgery were included in this study. Six vertebral samples were selected for miRNA sequencing (miRNA-seq) analysis and 26 vertebral samples were verified by qRT-PCR. OUTCOME MEASURES A high-throughput sequencing strategy was employed to identify miRNAs involved in GIOP. Twenty-six patients undergoing spinal surgery were included in this study. Six vertebral samples were selected for miRNA sequencing (miRNA-seq) analysis and 26 vertebral samples were verified by qRT-PCR. Bioinformatics was utilized for target prediction, to investigate the regulation of miRNA-mRNA networks, and Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses. METHODS A high-throughput sequencing strategy was employed to identify miRNAs involved in GIOP. Twenty-six patients undergoing spinal surgery were included in this study. Six vertebral samples were selected for miRNA sequencing (miRNA-seq) analysis and 6 vertebral samples were verified by qRT-PCR. Bioinformatics was utilized for target prediction, to investigate the regulation of miRNA-mRNA networks, and Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses. RESULTS Six significantly up-regulated miRNAs (including one novel miRNA) and three significantly down-regulated miRNAs were verified via miRNA-seq and verified in the vertebrae of GIOP patients. Up-regulated miRNAs included hsa-miR-214-5p, hsa-miR-10b-5p, hsa-miR-21-5p, hsa-miR-451a, hsamiR-186-5p, and hsa-miR-novel-chr3_49,413 while down-regulated miRNAs included hsa-let-7f-5p, hsa-let-7a-5p, and hsa-miR-27a-3p. Bioinformatics analysis revealed 5,983 and 23,463 predicted targets in the up-regulated and down-regulated miRNAs respectively, using the miRanda, miRBase and TargetScan databases. The target genes of these significantly altered miRNAs were enriched to 1,939 GO terms and 84 KEGG pathways. GO terms revealed that up-regulated targets were most enriched in actin filament-based processes (BP), anchoring junction (CC), and cytoskeletal protein binding (MF). Conversely, the down-regulated targets were mostly enriched in multicellular organismal development (BP), intracellular membrane-bounded organelles (CC), and protein binding (MF). Top-10 pathway analysis revealed that the differentially expressed miRNAs in GIOP were closely related to bone metabolism-related pathways such as FoxO, PI3K-Akt, MAPK and Notch signaling pathway. CONCLUSIONS These results suggest that significantly altered miRNAs may play an important role in GIOP by targeting mRNA and regulating biological processes and bone metabolism-related pathways such as MAPK, FoxO, PI3K-Akt and Notch signaling, which provides novel insight into the mechanism of GIOP and lays a good foundation for the prevention and treatment of GIOP. FDA DEVICE/DRUG STATUS This abstract does not discuss or include any applicable devices or drugs.

Teleosts have more types of chromatophores than other vertebrates and the genetic basis for pigmentation is highly conserved among vertebrates. Therefore, teleosts are important models to study the mechanism of pigmentation. Although functional genes and genetic variations of pigmentation have been studied, the mechanisms of different skin coloration remains poorly understood. The koi strain of common carp has various colors and patterns, making it a good model for studying the genetic basis of pigmentation. We performed RNA-sequencing for red skin and white skin and identified 62 differentially expressed genes (DEGs). Most of them were validated with RT-qPCR. The up-regulated DEGs in red skin were enriched in Kupffer’s vesicle development while the up-regulated DEGs in white skin were involved in cytoskeletal protein binding, sarcomere organization and glycogen phosphorylase activity. The distinct enriched activity might be associated with different structures and functions in erythrophores and iridophores. The DNA methylation levels of two selected DEGs inversely correlated with gene expression, indicating the participation of DNA methylation in the coloration. This expression characterization of red—white skin along with the accompanying transcriptome-wide expression data will be a useful resource for further studies of pigment cell biology.

Adeno-associated viruses (AAVs) are attractive gene therapy vectors due to their low toxicity, high stability, and rare integration into the host genome. Expressing ligands on the viral capsid can re-target AAVs to new cell types, but limited sites have been identified on the capsid that tolerate a peptide insertion. Here, we incorporated a site-specific tetracysteine sequence into the AAV serotype 9 (AAV9) capsid, to permit labelling of viral particles with either a fluorescent dye or biotin. We demonstrate that fluorescently labelled particles are detectable in vitro, and explore the utility of the method in vivo in mice with time-lapse imaging. We exploit the biotinylated viral particles to generate two distinct AAV interactomes, and identify several functional classes of proteins that are highly represented: actin/cytoskeletal protein binding, RNA binding, RNA splicing/processing, chromatin modifying, intracellular trafficking and RNA transport proteins. To examine the biological relevance of the capsid interactome, we modulated the expression of two proteins from the interactomes prior to AAV transduction. Blocking integrin αVβ6 receptor function reduced AAV9 transduction, while reducing histone deacetylase 4 (HDAC4) expression enhanced AAV transduction. Our method demonstrates a strategy for inserting motifs into the AAV capsid without compromising viral titer or infectivity.

Hypercalcemia can cause renal dysfunction such as nephrogenic diabetes insipidus (NDI), but the mechanisms underlying hypercalcemia-induced NDI are not well understood. To elucidate the early molecular changes responsible for this disorder, we employed mass spectrometry-based proteomic analysis of inner medullary collecting ducts (IMCD) isolated from parathyroid hormone-treated rats at onset of hypercalcemia-induced NDI. Forty-one proteins, including the water channel aquaporin-2, exhibited significant changes in abundance, most of which were decreased. Bioinformatic analysis revealed that many of the downregulated proteins were associated with cytoskeletal protein binding, regulation of actin filament polymerization, and cell-cell junctions. Targeted LC-MS/MS and immunoblot studies confirmed the downregulation of 16 proteins identified in the initial proteomic analysis and in additional experiments using a vitamin D treatment model of hypercalcemia-induced NDI. Evaluation of transcript levels and estimated half-life of the downregulated proteins suggested enhanced protein degradation as the possible regulatory mechanism. Electron microscopy showed defective intercellular junctions and autophagy in the IMCD cells from both vitamin D- and parathyroid hormone-treated rats. A significant increase in the number of autophagosomes was confirmed by immunofluorescence labeling of LC3. Colocalization of LC3 and Lamp1 with aquaporin-2 and other downregulated proteins was found in both models. Immunogold electron microscopy revealed aquaporin-2 in autophagosomes in IMCD cells from both hypercalcemia models. Finally, parathyroid hormone withdrawal reversed the NDI phenotype, accompanied by termination of aquaporin-2 autophagic degradation and normalization of both nonphoshorylated and S256-phosphorylated aquaporin-2 levels. Thus, enhanced autophagic degradation of proteins plays an important role in the initial mechanism of hypercalcemic-induced NDI.

Cell cultures are widely used model systems. Some immortalized cell lines can be grown in either two-dimensional (2D) adherent monolayers or in three-dimensional (3D) multicellular aggregates, or spheroids. Here, the quantitative proteome and phosphoproteome of colon carcinoma HT29 cells cultures in 2D monolayers and 3D spheroids were compared with a stable isotope labeling of amino acids (SILAC) labeling strategy. Two biological replicates from each sample were examined and notable differences in both the proteome and the phosphoproteome were determined by nano-liquid chromatography tandem mass spectrometry (nLC-MS/MS) to assess how growth configuration affects molecular expression. A total of 5867 protein groups, including 2523 phosphoprotein groups and 8733 phosphopeptides were identified in the samples. The Gene Ontology analysis revealed enriched GO terms in the 3D samples for RNA binding, nucleic acid binding, enzyme binding, cytoskeletal protein binding, and histone binding for their molecular functions (MF) and in the process of cell cycle, cytoskeleton organization, and DNA metabolic process for the biological process (BP). The KEGG pathway analysis indicated that 3D cultures are enriched for oxidative phosphorylation pathways, metabolic pathways, peroxisome pathways, and biosynthesis of amino acids. In contrast, analysis of the phosphoproteomes indicated that 3D cultures have decreased phosphorylation correlating with slower growth rates and lower cell-to extracellular matrix interactions. In sum, these results provide quantitative assessments of the effects on the proteome and phosphoproteome of culturing cells in 2D versus 3D cell culture configurations.

Scope: Carnitine improves protein accretion, muscle mass, and protein:fat accretion in piglets. The underlying mechanisms, however, are largely unknown. Methods and results: To gain insight into mechanisms through which carnitine exerts these effects, we fed piglets either a control or a carnitine-supplemented diet, and analyzed the transcriptome in skeletal muscle. Carnitine concentrations in plasma and muscle were about four-fold higher in the carnitine group when compared to the control group. Transcript profiling revealed 211 genes to be differentially expressed in muscle by carnitine supplementation. The identified genes were mainly involved in molecular processes such as cytoskeletal protein binding, insulin-like growth factor (IGF) binding, transcription factor activity, and insulin receptor binding. Identified genes with the molecular function transcription factor activity encoded primarily transcription factors, most of which were down-regulated by carnitine, including pro-apoptotic transcription factors such as proto-oncogene c-fos, proto-oncogene c-jun and activating transcription factor 3. Furthermore, atrophy-related genes such as atrogin-1, MuRF1, and DRE1 were significantly down-regulated by carnitine. IGF signalling and insulin signalling were identified as significantly up-regulated regulatory pathways in the carnitine group. Conclusion: Carnitine may have beneficial effects on skeletal muscle mass through stimulating the anabolic IGF-1 pathway and suppressing pro-apoptotic and atrophy-related genes, which are involved in apoptosis of muscle fibers and proteolysis of muscle proteins, respectively.

The large amounts of microarray data provide us a great opportunity to identify gene expression profiles (GEPs) in different tissues or disease states. Disease-specific biomarker genes likely share GEPs that are distinct in disease samples as compared with normal samples. The similarity of the GEPs may be evaluated by Pearson Correlation Coefficient (PCC) and the distinctness of GEPs may be assessed by Kolmogorov-Smirnov distance (KSD). In this study, we used the PCC and KSD metrics for GEPs to identify disease-specific (cancer-specific) biomarkers. We first analyzed and compared GEPs using microarray datasets for smoking and lung cancer. We found that the number of genes with highly different GEPs between comparing groups in smoking dataset was much larger than that in lung cancer dataset; this observation was further verified when we compared GEPs in smoking dataset with prostate cancer datasets. Moreover, our Gene Ontology analysis revealed that the top ranked biomarker candidate genes for prostate cancer were highly enriched in molecular function categories such as ‘cytoskeletal protein binding’ and biological process categories such as ‘muscle contraction’. Finally, we used two genes, ACTC1 (encoding an actin subunit) and HPN (encoding hepsin), to demonstrate the feasibility of diagnosing and monitoring prostate cancer using the expression intensity histograms of marker genes. In summary, our results suggested that this approach might prove promising and powerful for diagnosing and monitoring the patients who come to the clinic for screening or evaluation of a disease state including cancer.

The present study aimed to screen several differentially expressed genes (DEGs) and differentially expressed microRNAs (miRNAs) for two types of mesenchymal stem cell (MSC) differentiation. Bone morphogenetic protein 6 (BMP‑6) and dexamethasone were used to induce MSCs towards osteoblastic differentiation or adipocytic differentiation. The t‑test in the Bioconductor bioinformatics software tool was used to screen DEGs and differentially expressed miRNAs in the two samples. Subsequent gene ontology (GO) and pathway analyses on the DEGs were performed using the GO and Kyoto Encyclopedia of Genes and Genomes databases, respectively; potential target genes for the screened miRNAs were predicted using the TargetScan database. In addition, an interaction network between the DEGs and miRNAs was constructed. Numerous DEGs and miRNAs were screened during osteoblastic and adipocytic differentiation of MSCs. Important pathways, such as glutathione metabolism, pathogenic Escherichia coli infection and Parkinson's disease, and GO terms, including cytoskeletal protein binding and phospholipase inhibitor activity, were enriched in the screened DEGs from MSCs undergoing osteogenic differentiation and adipocytic differentiation. miRNAs, including miRNA (miR)‑382 and miR‑203, and DEGs, including neuronal growth regulator 1 (NEGR1), phosphatidic acid phosphatase 2B (PPAP2B), platelet‑derived growth factor receptor alpha (PDGFRA), interleukin 6 signal transducer (IL6ST) and sortilin 1 (SORT1), were demonstrated to be involved in osteoblastic differentiation. In addition, the downregulated miRNAs (including miR‑495, miR‑376a and miR‑543), the upregulated miR‑106a, the upregulated DEGs, including enabled homolog (ENAH), polypeptide N‑acetylgalactosaminyltransferase 1 and acyl‑CoA synthetase long‑chain family member 1, and the downregulated repulsive guidance molecule family member B and semaphorin SEMA7A were demonstrated to be involved in adipocytic differentiation. The results of the present study suggested that miRNAs (miR‑203 and miR‑382) and DEGs (NEGR1, PPAP2B, PDGFRA, IL6ST and SORT1) may serve pivotal functions in the osteoblastic differentiation of MSCs, whereas miR‑495, which is also involved in osteoblast differentiation and had four targets, including NEGR1, miR‑376a, miR‑543 and ENAH may have crucial roles in adipocytic differentiation of MSCs.

To explore the molecular mechanisms of diabetic nephropathy (DN) progression and provide the theoretical basis for treating DN, GSE1009 microarray data were downloaded from Gene Expression Omnibus database. Microarray data were obtained from glomeruli isolated from normal kidneys (n=3) and kidneys from patients with DN (n=3). We first screened the differentially expressed genes (DEGs) in kidneys by the Linear Models for Microarray Data package in R. Then the function of DEGs in DN was explored through Gene Ontology (GO) and KEGG pathway enrichment analyses. Critical DEGs for DN progression were investigated by constructing PPI network and mining significant modules. Afterwards, enriched protein domains of modules were analyzed by Interpro and DAVID. At last, the regulatory miRNAs for DEGs were calculated by WebGestalt, and DEGs-miRNAs network was visualized with Cytoscape. A total of 666 DEGs including 384 up- and 282 down-regulated genes were screened out. The up-regulated DEGs were significantly enriched in plasma membrane and signal transmission, and mainly participated in pathways of cytokine-cytokine receptor and neuroactive ligand-receptor interaction. The down-regulated DEGs significantly enriched in extracellular region and cytoskeletal protein binding, and mainly participated in ECM-receptor interaction and dilated cardiomyopathy. 2 PPI networks were constructed with confidence score>0.4. One significant module obtained from PPI network for up-regulated DEGs mainly enriched in protein domain of rhodopsin-like G protein-coupled receptors. The down-regulated DEGs were mainly regulated by 10 miRNAs clusters. Together, we constructed a comprehensive molecular network for DN progression and miR-1 and miR-25 might be theoretical targets for DN.

Purpose Collapsin response mediator protein-2 (CRMP2) is a CNS protein involved in neuronal development, axonal and neuronal growth, cell migration, and protein trafficking. Recent studies have linked perturbations in CRMP2 function to neurodegenerative disorders such as Alzheimer's disease, neuropathic pain, and Batten disease, and to psychiatric disorders such as schizophrenia. Like most proteins, CRMP2 functions though interactions with a molecular network of proteins and other molecules. Experimental design Here, we have attempted to identify additional proteins of the CRMP2 interactome to provide further leads about its roles in neurological functions. We used a combined co-immunoprecipitation and shotgun proteomic approach in order to identify CRMP2 protein partners. Results We identified 78 CRMP2 protein partners not previously reported in public protein interaction databases. These were involved in seven biological processes, which included cell signaling, growth, metabolism, trafficking, and immune function, according to Gene Ontology classifications. Furthermore, 32 different molecular functions were found to be associated with these proteins, such as RNA binding, ribosomal functions, transporter activity, receptor activity, serine/threonine phosphatase activity, cell adhesion, cytoskeletal protein binding and catalytic activity. In silico pathway interactome construction revealed a highly connected network with the most overrepresented functions corresponding to semaphorin interactions, along with axon guidance and WNT5A signaling. Conclusions and clinical relevance Taken together, these findings suggest that the CRMP2 pathway is critical for regulating neuronal and synaptic architecture. Further studies along these lines might uncover novel biomarkers and drug targets for use in drug discovery.

Cardiac myocytes are sensitive to specific and distinct mechanical stimuli. We examined the effect of the major axis of biaxial mechanical stretch on cardiac myocyte gene expression and reconstructed a stretch signaling model to identify pathways and transcription factors regulating these responses. Neonatal cardiac myocytes were cultured on a micropatterned substrate, and the primary stretch axis was applied either parallel or transverse to the myofibril direction. RNA sequencing (RNA-Seq) after acute myocyte stretch showed a more robust response to longitudinal than to transverse stretch. After 30 minutes of stretch, 53 and 168 genes were significantly up- or down-regulated by transverse or longitudinal stretch, respectively. After 4 hours, the number of differentially expressed genes increased to 795 with longitudinal stretch but only 35 with transverse stretch. Genes regulated by both stretches showed significant enrichment of transcription factor activity and protein kinase activity, whereas longitudinal but not transverse stretch specifically activated genes involved in sarcomere organization and cytoskeletal protein binding. Network analysis using logic-based signaling model constructed from published data identified serum response factor (SRF) and myocyte enhancer factor-2 (MEF2) as critical regulators of longitudinal stretch-induced changes mediated by protein kinase C (PKC). cAMP response element-binding protein (CREB) was found to be activated by both longitudinal and transverse stretch. Inhibitor experiments were used to validate these model predictions. Cardiac myocytes activate different pathways in response to transverse and longitudinal mechanical strain; these responses may underlie differential whole organ responses to pressure versus volume overload.

We aimed to explore the potential genes or pathways related to venous thromboembolism (VTE) and expected our findings could contribute to the development of new target drugs for VTE. The gene expression profile of GSE19151 was downloaded from Gene Expression Omnibus (GEO) database. The bioinformatics methods were applied to screen the feature genes and pathways related with VTE. A total of 115 DEGs were identified, including 25 downregulated genes and 90 upregulated genes. Function enrichment analysis showed that upregulated genes of VTE were mainly enriched in ribosome and translation-related pathways, while downregulated genes were mainly enriched in cytoskeletal protein binding and non-membrane-bounded organelle-related pathways. MCL1, TP53, and RERE were three outstanding genes involved in the interaction network. The most significant pathways enriched by module genes were ribosome and oxidative phosphorylation. Moreover, all the products of the 18 genes enriched in ribosome (hsa03010) were ribosomal proteins. Ribosome, translation, actin binding, and non-membrane-bounded organelle pathways were closely related to the development of VTE. Moreover, MCL1, TP53, and RERE might play key roles in the process of VTE.

Proteomic analysis of the human body is a significant recent scientific endeavour. In this study, we investigated the proteomic profile of human dentin using modern analytical and mass spectrometric techniques. Five healthy permanent human molars from five adults were cut, pulverized, denaturated with guanidine buffer, and demineralized with EDTA buffer. The extracted proteins were analysed by gel electrophoresis (SDS-PAGE and two-dimensional gel electrophoresis), digested with trypsin, and separated by liquid chromatography/high-resolution tandem mass spectrometry. We identified 289 proteins with high confidence, 90 of which had not been previously detected in human dentin. Nine (currently hypothetical) proteins were identified for the first time in an actual human sample. The proteins have a variety of functions, including calcium-ion binding, formation of the extracellular matrix, formation of the cytoskeleton, cytoskeletal protein binding, immune response, and transport. In conclusion, this is the first use of two-dimensional electrophoresis for investigating human dentin.

The authors describe a high-throughput patterning process used to create arrays of molecular-scale features for the study of cytoskeletal protein binding interactions. The process uses a shadow-evaporated metal mask to facilitate lift-off of features defined by nanoimprint lithography. This simple and robust approach alleviates difficulties in pattern transfer of ultrasmall features and results in arrays of highly ordered sub-10 nm features which are then functionalized with extracellular matrix proteins. Application of these arrays is demonstrated in cell spreading assays.

Udgivelsesdato: 2009-Dec OBJECTIVE: The aim of this study was to identify changes in the cervical transcriptome in the human uterine cervix as a function of ripening before the onset of labor. STUDY DESIGN: Human cervical tissue was obtained from women at term not in labor with ripe (n = 11) and unripe (n = 11) cervices and profiled using Affymetrix GeneChip HGU133Plus2.0 arrays. Gene expression was analyzed using a moderated t-test (False Discovery Rate 5%). Gene ontology and pathway analysis were performed. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was used for confirmation of selected differentially expressed genes. RESULTS: (1) Ninety-one genes were differentially expressed between ripe and unripe groups. (2) Cervical ripening was associated with enrichment of specific biological processes (e.g. cell adhesion, regulation of anatomical structure), pathways and 11 molecular functions (e.g. extracelluar matrix (ECM)-structural constituent, protein binding, glycosaminoglycan binding). (3) qRT-PCR confirmed that 9 of 11 tested differentially expressed genes (determined by microarray) were upregulated in a ripe cervix (e.g. MYOCD, VCAN, THBS1, COL5A1). (4) Twenty-three additional genes related to ECM metabolism and adhesion molecules were differentially regulated (by qRT-PCR) in ripe cervices. CONCLUSION: (1) This is the first description of the changes in the human cervical transcriptome with ripening before the onset of labor. (2) Biological processes, pathways and molecular functions were identified with the use of this unbiased approach. (3) In contrast to cervical dilation after term labor, inflammation-related genes did not emerge as differentially regulated with cervical ripening. (4) Myocardin was identified as a novel gene upregulated in human cervical ripening.

We have designed a system engineered to mimic biological spatial order. The system consists of nanoscale dots organized into hierarchical arrays in which structural parameters, such as spacing and orientation, are systematically varied. These arrays are used to probe the spatial distributions of binding sites in biomolecules. In this paper, we describe the fabrication process, including lithography and biofunctionalization, and we discuss issues related to surface chemistry that are critical to creating robust, biologically active nanoarrays.

It has been hypothesized that nonspecific reversible binding of cytoskeletal proteins to lipids in cells may guide their binding to integral membrane anchor proteins. In a model system, we measured desorption rates k(off) (off-rates) of the erythrocyte cytoskeletal proteins spectrin and protein 4.1 labeled with carboxyfluorescein (CF), at two different compositions of planar phospholipid membranes (supported on glass), using the total internal reflection/fluorescence recovery after photobleaching (TIR/FRAP) technique. The lipid membranes consisted of either pure phosphatidylcholine (PC) or a 3:1 mixture of PC with phosphatidylserine (PS). In general, the off-rates were not single exponentials and were fit to a combination of fast, slow, and irreversible fractions, reported both separately and as a weighted average. By a variation of TIR/FRAP, we also measured equilibrium affinities (the ratio of surface-bound to bulk protein concentration) and thereby calculated on-rates, k(on). The average off-rate of CF-4.1 from PC/PS (approximately 0.008/s) is much slower than that from pure PC (approximately 1.7/s). Despite the consequent increase in equilibrium affinity at PC/PS, the on-rate at PC/PS is also substantially decreased (by a factor of 40) relative to that at pure PC. The simultaneous presence of (unlabeled) spectrin tends to substantially decrease the on-rate (and the affinity) of CF-4.1 at both membrane types. Similar experiments for CF-spectrin alone showed much less sensitivity to membrane type and generally faster off-rates than those exhibited by CF-4.1. However, when mixed with (unlabeled) 4.1, both the on-rate and off-rate of CF-spectrin decreased drastically at PC/PS (but not PC), leading to a somewhat increased affinity. Clearly, changes in affinity often involve countervailing changes in both on-rates and off-rates. In many of these studies, the effect of varying ionic strength and bulk concentrations was examined; it appears that the binding is an electrostatic attraction and is far from saturation at the concentrations employed. These results and the techniques implemented carry general implications for understanding the functional role of nonspecific protein binding to cellular lipid membranes.