Cell Projection Organization Research Articles

Overlapping peri-implantation phenotypes of ZNHIT1 and ZNHIT2 despite distinct functions during early mouse development†.

Mammalian preimplantation development culminates in the formation of a blastocyst that undergoes extensive gene expression regulation to successfully implant into the maternal endometrium. Zinc-finger HIT domain-containing (ZNHIT) 1 and 2 are members of a highly conserved family, yet they have been identified as subunits of distinct complexes. Here, we report that knockout of either Znhit1 or Znhit2 results in embryonic lethality during peri-implantation stages. Znhit1 and Znhit2 mutant embryos have overlapping phenotypes, including reduced proportion of SOX2-positive inner cell mass cells, a lack of Fgf4 expression, and aberrant expression of NANOG and SOX17. Furthermore, we find that the similar phenotypes are caused by distinct mechanisms. Specifically, embryos lacking ZNHIT1 likely fail to incorporate sufficient H2A.Z at the promoter region of Fgf4 and other genes involved in cell projection organization resulting in impaired invasion of trophoblast cells during implantation. In contrast, Znhit2 mutant embryos display a complete lack of nuclear EFTUD2, a key component of U5 spliceosome, indicating a global splicing deficiency. Our findings unveil the indispensable yet distinct roles of ZNHIT1 and ZNHIT2 in early mammalian embryonic development.

Sex‐specific gene expression in eastern oyster, Crassostrea virginica, gonad and mantle tissues

AbstractThe eastern oyster (Crassostrea virginica) is a protandrous hermaphrodite of commercial importance. As with many marine invertebrates, little is known about sex determination and differentiation systems in this species. Such knowledge has important implications not only for understanding the evolution of sex but also for applied questions in aquaculture. In order to examine mechanistic differences in reproductive development between the sexes, we compared the transcriptomes of gonad and mantle tissues from six male and six female oysters. A total of 7675 transcripts were differentially expressed between male and female gonads (3936 and 3739 were upregulated in males and females, respectively). Transcripts identified include those associated with sex in other invertebrate and vertebrate species such as Dmrt1, Sox‐30, Bindin, Dpy‐30, and Histone H4 in males and Foxl2, Vitellogenin, and Bystin in females. GO terms associated with transcripts upregulated in male gonads include protein modification, reproductive process, and cell projection organization, whereas RNA metabolic process and amino acid metabolic process were associated with transcripts upregulated in females. Far fewer transcripts were differentially expressed between male and female mantle tissues, with 87 transcripts upregulated in females and 16 upregulated in males. However, 41% of transcripts identified as differentially expressed between mantle tissues were also differentially expressed between male and female gonads including Histone H4 and Bystin. This study represents the first characterization of eastern oyster male and female gonad transcriptomes. We further identify differing expression profiles between male and female mantle tissues, which provides evidence for sex‐specific functions of the mantle and suggests that this tissue could harbor biomarkers for identifying oyster sex non‐destructively.

Bioinformatics and Next-Generation Data Analysis for Identification of Genes and Molecular Pathways Involved in Subjects with Diabetes and Obesity

Background and Objectives: A subject with diabetes and obesity is a class of the metabolic disorder. The current investigation aimed to elucidate the potential biomarker and prognostic targets in subjects with diabetes and obesity. Materials and Methods: The next-generation sequencing (NGS) data of GSE132831 was downloaded from Gene Expression Omnibus (GEO) database. Functional enrichment analysis of DEGs was conducted with ToppGene. The protein-protein interactions network, module analysis, target gene-miRNA regulatory network and target gene-TF regulatory network were constructed and analyzed. Furthermore, hub genes were validated by receiver operating characteristic (ROC) analysis. A total of 872 DEGs, including 439 up-regulated genes and 433 down-regulated genes were observed. Results: Second, functional enrichment analysis showed that these DEGs are mainly involved in the axon guidance, neutrophil degranulation, plasma membrane bounded cell projection organization and cell activation. The top ten hub genes (MYH9, FLNA, DCTN1, CLTC, ERBB2, TCF4, VIM, LRRK2, IFI16 and CAV1) could be utilized as potential diagnostic indicators for subjects with diabetes and obesity. The hub genes were validated in subjects with diabetes and obesity. Conclusion: This investigation found effective and reliable molecular biomarkers for diagnosis and prognosis by integrated bioinformatics analysis, suggesting new and key therapeutic targets for subjects with diabetes and obesity.

HAND1 knockdown disrupts trophoblast global geneexpression.

Congenital heart disease (CHD) affects nearly 1% of births annually, and CHD pregnancies carry increased risk of developing pathologies of abnormal placentation. We previously reported significant developmental impacts of disrupting Hand1, a gene associated with CHD, expression in placenta trophoblast and endothelial cells in multiple mouse models. In this study, we aimed to build upon this knowledge and characterize the mechanistic impacts of disrupting HAND1 on human placenta trophoblast and vascular endothelial cell gene expression. HAND1 gene expression was silenced in BeWo cells, a choriocarcinoma model of human cytotrophoblasts, (n=3-9 passages) and isolated human placental microvascular endothelial cells (HPMVEC; n=3 passages), with HAND1 siRNA for 96 h. Cells were harvested, mRNA isolated and RNA sequencing performed using the Illumina NextSeq 550 platform. Normalization and differential gene expression analyses were conducted using general linear modeling in edgeR packages. Statistical significance was determined using a log2 fold change of >1.0 or < -1.0 and unadjusted p-value ≤0.05. Panther DB was used for overrepresentation analysis, and String DB for protein association network analysis. There was downregulation of 664 genes, and upregulation of 59 genes in BeWo cells with direct HAND1 knockdown. Overrepresentation analysis identified disruption to pathways including cell differentiation, localization, and cell projection organization. In contrast, only seven genes were changed with direct HAND1 knockdown in HPMVECs. Disruption to HAND1 expression significantly alters gene expression profile in trophoblast but not endothelial cells. This data provides further evidence that future studies on genetic perturbations in CHDs should consider the extra-embryonic tissue in addition to the fetal heart.

Downregulation of zinc finger protein 71 expression in oral squamous cell carcinoma tissues and its underlying molecular mechanism.

Patients with oral squamous cell carcinoma (OSCC) have poor prognoses due to a limited understanding of the pathogenesis of OSCC. Zinc finger protein (ZNF) is the largest transcription factor family in the human genome and exert diverse and important functions. Nevertheless, the exact expression status and molecular mechanism of ZNF71 have not been described in OSCC. Therefore, this study aimed to identify the specific expression level of ZNF71 in OSCC tissues and to further interpret the potential molecular mechanism of ZNF71 in the pathogenesis of OSCC. In-house immunohistochemical staining of 116 OSCC samples and 29 non-OSCC samples was employed to detect the expression status of ZNF71 at the protein level of OSCC tissues. Single-cell RNA sequencing data from 7 OSCC samples was used to explore the expression landscape of ZNF71 in different cell types from OSCC tissues. High-throughput RNA sequencing data and gene chips data from 893 OSCC samples and 301 non-OSCC samples were utilized to identify the specific expression level of ZNF71 at the bulk mRNA level of OSCC tissues. Here, standardized mean difference (SMD) value was applied to calculate the expression differences between OSCC group and non-OSCC group. Multiple datasets were included; hence, the results were considered to be more reliable. Sensitivity analysis was conducted to evaluate the stability of the results. Enrichment analysis and immune infiltration analysis were used to explore the underlying molecular mechanism of ZNF71 in OSCC. ZNF71 was significantly downregulated in OSCC tissues at the protein level (SMD = -1.96, 95 % confidence interval [95 % CI]: -2.43 to -1.50). ZNF71 was absent in various cell types from OSCC tissues including cancerous epithelial cells and tumor-infiltrating immune cells. ZNF71 was downregulated in OSCC tissues at the bulk mRNA level (SMD = -0.38, 95 % CI: -0.75 to -0.02). Enrichment analysis showed that positively and differentially co-expressed genes mainly concentrated on "herpes simplex virus 1 infection" and "regulation of plasma membrane bounded cell projection organization", and negatively and differentially co-expressed genes mainly participated in "cell cycle" and "DNA metabolic process". Moreover, the putative target genes of ZNF71 mainly participated in "cellular respiration" and "protein catabolic process". Finally, immune infiltration analysis revealed that ZNF71 expression was positively correlated with multiple immune cells including activated B cells, memory B cells, and natural killer (NK) cells, and negatively correlated with various immune cells, including CD56 bright NK cells, neutrophil, and immature dendritic cells. The downregulation of ZNF71 may influence the initiation and promotion of OSCC by reducing immune infiltration, accelerating cell cycle progression, and affecting metabolic process, and this requires further research.

The Plasmodium falciparum CCCH Zinc Finger Protein ZNF4 Plays an Important Role in Gametocyte Exflagellation through the Regulation of Male Enriched Transcripts.

CCCH zinc finger proteins (ZFPs) function mainly as RNA-binding proteins (RBPs) and play a central role in the mRNA metabolism. Over twenty seven CCCH-ZFPs are encoded in the genome of the human malaria parasite Plasmodium falciparum, the causative agent of malaria tropica. However, little is known about their functions. In this study, we characterize one member of the PfCCCH-ZFP named ZNF4. We show that ZNF4 is highly expressed in mature gametocytes, where it predominantly localizes to the cytoplasm. Targeted gene disruption of ZNF4 showed no significant effect in asexual blood stage replication and gametocyte development while male gametocyte exflagellation was significantly impaired, leading to reduced malaria transmission in the mosquito. Comparative transcriptomics between wildtype (WT) and the ZNF4-deficient line (ZNF4-KO) demonstrated the deregulation of about 473 genes (274 upregulated and 199 downregulated) in mature gametocytes. Most of the downregulated genes show peak expression in mature gametocyte with male enriched genes associated to the axonemal dynein complex formation, and cell projection organization is highly affected, pointing to the phenotype in male gametocyte exflagellation. Upregulated genes are associated to ATP synthesis. Our combined data therefore indicate that ZNF4 is a CCCH zinc finger protein which plays an important role in male gametocyte exflagellation through the regulation of male gametocyte-enriched genes.

Ionizing radiations induce shared epigenomic signatures unraveling adaptive mechanisms of cancerous cell lines with or without methionine dependency

BackgroundAlthough radiation therapy represents a core cancer treatment modality, its efficacy is hampered by radioresistance. The effect of ionizing radiations (IRs) is well known regarding their ability to induce genetic alterations; however, their impact on the epigenome landscape in cancer, notably at the CpG dinucleotide resolution, remains to be further deciphered. In addition, no evidence is available regarding the effect of IRs on the DNA methylome profile according to the methionine dependency phenotype, which represents a hallmark of metabolic adaptation in cancer.MethodsWe used a case–control study design with a fractionated irradiation regimen on four cancerous cell lines representative of HCC (HepG2), melanoma (MeWo and MeWo-LC1, which exhibit opposed methionine dependency phenotypes), and glioblastoma (U251). We performed high-resolution genome-wide DNA methylome profiling using the MethylationEPIC BeadChip on baseline conditions, irradiated cell lines (cumulative dose of 10 Gy), and non-irradiated counterparts. We performed epigenome-wide association studies to assess the effect of IRs and methionine-dependency-oriented analysis by carrying out epigenome-wide conditional logistic regression. We looked for epigenome signatures at the locus and single-probe (CpG dinucleotide) levels and through enrichment analyses of gene ontologies (GO). The EpiMet project was registered under the ID#AAP-BMS_003_211.ResultsEWASs revealed shared GO annotation pathways associated with increased methylation signatures for several biological processes in response to IRs, including blood circulation, plasma membrane-bounded cell projection organization, cell projection organization, multicellular organismal process, developmental process, and animal organ morphogenesis. Epigenome-wide conditional logistic regression analysis on the methionine dependency phenotype highlighted several epigenome signatures related to cell cycle and division and responses to IR and ultraviolet light.ConclusionsIRs generated a variation in the methylation level of a high number of CpG probes with shared biological pathways, including those associated with cell cycle and division, responses to IRs, sustained angiogenesis, tissue invasion, and metastasis. These results provide insight on shared adaptive mechanisms of the epigenome in cancerous cell lines in response to IR. Future experiments should focus on the tryptic association between IRs, the initiation of a radioresistance phenotype, and their interaction with methionine dependency as a hallmark of metabolic adaptation in cancer.Graphical abstract

Transcriptome analysis of lncRNA expression patterns in human congenital lung malformations

ObjectivesTo explore the long non-coding RNA (lncRNA) expression pattern of congenital lung malformations on a genome-wide scale and investigate their potential biological function in four subtypes of congenital lung malformations.MethodsWe obtained both lesions and normal lung control tissues from the patients diagnosed with CPAM-I, CPAM-II, ILS, and ILS-CPAM, and underwent lobectomy (i.e., surgical removal of the whole lobe which contains the localized lesion as well as normal lung tissue). Then, we performed lncRNA transcriptome profiling in these tissues by RNA sequencing (RNA-seq). A comprehensive bioinformatics analysis was conducted to characterize the expression profiles and relevant biological functions and for multiple comparisons of lncRNA expression in the different subtypes of congenital lung malformation tissues. Furthermore, the lncRNA-mRNA co-expression network was constructed, and dysregulated mRNAs were functionally analyzed. Finally, gene set enrichment analysis (GSEA) was used to predict the potential molecular mechanism of the identified lncRNAs.ResultsA total of 5921 lncRNA transcripts were identified between congenital lung malformations tissues and normal lung control tissues. Compared with normal lung control, 481of these expressed lncRNAs were upregulated and 142 were downregulated in CPAM-I, 91 were upregulated and 14 were downregulated in CPAM-II, 39 were upregulated and 38 were downregulated in ILS, and 201 were upregulated and 38 were downregulated in ILS-CPAM. Unsupervised clustering and principal component analysis of the expressed lncRNAs visualized the differences between normal lung control and different subtypes of congenital lung malformations samples. We also confirmed significant differences in the composition of differentially expressed genes (DEGs) and the differentially expressed lncRNAs (DE lncRNAs) between CPAM-I and other subtypes of congenital lung malformations, as well as in normal lung control tissues, and observed enrichment of DEGs in the regulation of the immune system, cell projection organization, and inflammatory pathways. Finally, we identified the lncRNA FLJ26850 might be related to congenital lung malformations via ZNF473.ConclusionsSignificant differences in lncRNAs expression patterns were observed between different subtypes of congenital lung malformations and normal control. The lncRNA FLJ26850 might be related to congenital lung malformations via ZNF473.

STAU2 binds a complex RNA cargo that changes temporally with production of diverse intermediate progenitor cells during mouse corticogenesis.

STAU2 is a double-stranded RNA-binding protein enriched in the nervous system. During asymmetric divisions in the developing mouse cortex, STAU2 preferentially distributes into the intermediate progenitor cell (IPC), delivering RNA molecules that can impact IPC behavior. Corticogenesis occurs on a precise time schedule, raising the hypothesis that the cargo STAU2 delivers into IPCs changes over time. To test this, we combine RNA-immunoprecipitation with sequencing (RIP-seq) over four stages of mouse cortical development, generating a comprehensive cargo profile for STAU2. A subset of the cargo was 'stable', present at all stages, and involved in chromosome organization, macromolecule localization, translation and DNA repair. Another subset was 'dynamic', changing with cortical stage, and involved in neurogenesis, cell projection organization, neurite outgrowth, and included cortical layer markers. Notably, the dynamic STAU2 cargo included determinants of IPC versus neuronal fates and genes contributing to abnormal corticogenesis. Knockdown of one STAU2 target, Taf13, previously linked to microcephaly and impaired myelination, reduced oligodendrogenesis in vitro. We conclude that STAU2 contributes to the timing of corticogenesis by binding and delivering complex and temporally regulated RNA cargo into IPCs.

Identification of key pathways and genes in polycystic ovary syndrome via integrated bioinformatics analysis and prediction of small therapeutic molecules

To enhance understanding of polycystic ovary syndrome (PCOS) at the molecular level; this investigation intends to examine the genes and pathways associated with PCOS by using an integrated bioinformatics analysis. Based on the expression profiling by high throughput sequencing data GSE84958 derived from the Gene Expression Omnibus (GEO) database, the differentially expressed genes (DEGs) between PCOS samples and normal controls were identified. We performed a functional enrichment analysis. A protein-protein interaction (PPI) network, miRNA- target genes and TF - target gene networks, were constructed and visualized, with which the hub gene nodes were identified. Validation of hub genes was performed by using receiver operating characteristic (ROC) and RT-PCR. Small drug molecules were predicted by using molecular docking. A total of 739 DEGs were identified, of which 360 genes were up regulated and 379 genes were down regulated. GO enrichment analysis revealed that up regulated genes were mainly involved in peptide metabolic process, organelle envelope and RNA binding and the down regulated genes were significantly enriched in plasma membrane bounded cell projection organization, neuron projection and DNA-binding transcription factor activity, RNA polymerase II-specific. REACTOME pathway enrichment analysis revealed that the up regulated genes were mainly enriched in translation and respiratory electron transport and the down regulated genes were mainly enriched in generic transcription pathway and transmembrane transport of small molecules. The top 10 hub genes (SAA1, ADCY6, POLR2K, RPS15, RPS15A, CTNND1, ESR1, NEDD4L, KNTC1 and NGFR) were identified from PPI network, miRNA - target gene network and TF - target gene network. The modules analysis showed that genes in modules were mainly associated with the transport of respiratory electrons and signaling NGF, respectively. We find a series of crucial genes along with the pathways that were most closely related with PCOS initiation and advancement. Our investigations provide a more detailed molecular mechanism for the progression of PCOS, detail information on the potential biomarkers and therapeutic targets.

Gene expression profiling of the spinal cord at the chronic pain phase identified CDKL5 as a candidate gene for neural remodeling

BackgroundChronic pain is a highly refractory and complicated condition that persists even without nociception. Several genome-wide gene expression analyses have shown that the immune response and inflammatory cytokines affect chronic pain establishment in the acute pain phase. However, compared with the acute phase, the chronic phase has a poorly elucidated gene expression profile. This study aimed to determine the gene expression profile in the spinal cord of a neuropathic pain mouse model in the chronic phase to elucidate the chronic pain characteristics. MethodsWe established a sciatic nerve cuff mouse model as a neuropathic pain model by placing a 2-mm section of a split PE-20 polyethylene tube around the sciatic nerve. The spinal cord was harvested at the L4-6 level at 28 postoperative days. Next, we examined differentially expressed genes (DEGs) through RNA sequencing (RNA-seq) compared with the sham group; moreover, we conducted enrichment analyses of the expressed genes. To reveal the chronic pain characteristics, we compared the gene expression profiles of the spinal cord between the acute and chronic phases in the neuropathic pain model. Among the chronic pain-related genes categorized in the dendrites, we focused on cyclin-dependent kinase-like 5 (CDKL5). We analyzed CDKL5 expression and function using real-time polymerase chain reaction (PCR), immunohistochemistry, and neurite extension assay in Neuro 2a (N2a) cells. We used three types of CDKL5 plasmids: wild type, nuclear localization signal-attached, and K42R kinase-dead CDKL5. ResultsWe identified 403 DEGs, including 104 upregulated and 43 downregulated genes (false discovery rate < 0.01). Rather than inflammation or immune response, the most enriched terms in the chronic phase were "regulation of plasma membrane-bounded cell projection organization" and "dendrite." Real-time PCR assay confirmed increased CDKL5 expression in the ipsilateral dorsal horn. CDKL5 was broadly expressed in the ipsilateral dorsal horn across all layers. The neurite extension assay revealed that the cytoplasmic kinase function of CDKL5 was necessary for neurite outgrowth in N2a cells. ConclusionRNA-seq of the spinal cord revealed that the most enriched genes during the chronic pain phase were involved in regulating axon and dendrite morphogenesis, including CDKL5. Our findings suggest that neural remodeling affects chronic pain establishment. Since patients with CDKL5 mutations have shown reduced pain perception, our findings suggest that CDKL5 in the spinal cord could result in neural remodeling during the chronic pain phase through cytoplasmic kinase activity.

1112HAND1 knockdown disrupts trophoblast but not placental endothelial global gene expression

We previously reported disrupted placental structure, and vascular changes in placentas from fetuses with Congenital Heart Defects. Signaling between trophoblast and villous endothelium is proposed for proper placental development and function. We hypothesize that disruption of genes associated with CHD within the trophoblast impacts fetal endothelium and results in dysregulated placental vasculature in CHD fetuses. A model of human cytotrophoblast, BeWo choriocarcinoma cells and isolated human placental microvascular endothelial cells (HPMVEC) were treated for 48 hrs with HAND1 siRNA/lipofectamine. Additionally, control HPMVECs were exposed to conditioned BeWo media (+/- HAND1) for 48 hrs. Cells were harvested and total RNA isolated. RNA was sequenced on an Illumina NextSeq 550 platform (single end reads 85bp, read depth 25M). The Galaxy Bioinformatic Platform was used for quality control, alignment (Bowtie2) and generation of read counts (Feature Counts). Normalization and differential gene expression analyses were conducted using general linear modeling in edgeR package from Bioconductor. Significance was determined using a fold change cut off of >1.5 and raw p<0.05. Overrepresentation analysis was performed using Panther DB for Reactome pathways and GO terms Direct HAND1 knockdown in the BeWo choriocarcinoma cell line caused downregulation of 632 genes, and upregulation of 102 genes. Overrepresentation analysis identified disruption to pathways including cell differentiation, neurogenesis, localization, and cell projection organization. In contrast, direct HAND1 knockdown in HPMVECs resulted in minimal disruption to global gene expression with differential expression in just 18 genes. Indirect HAND1 disruption did not alter HPMVEC gene expression. Disruption to HAND1 expression significantly alters gene expression profile in trophoblast but not HPMVEC, either directly or indirectly. Disruption to the structure and vasculature of the placenta in cases of CHD is likely primarily due to abnormal trophoblast development.

P0131HIGH-DOSE URIC ACID ALTERS CELLULAR PROTEOME, INCREASES INTRACELLULAR ATP, ENHANCES TISSUE REPAIR CAPABILITY AND INCREASES CALCIUM OXALATE CRYSTAL-BINDING CAPABILITY OF RENAL TUBULAR CELLS: IMPLICATIONS TO HYPERURICOSURIA-INDUCED KIDNEY STONE DISEASE

Abstract Background and Aims Hyperuricosuria is well documented to get involved in the pathogenesis of kidney stone disease, especially uric acid (UA) and calcium oxalate (CaOx) types. Nevertheless, hyperuricosuria-induced alterations in renal tubular cells and cascade mechanisms that subsequently trigger kidney stone formation remained largely unknown. This study aimed to examine changes in cellular proteome and function in renal tubular cells after treatment with high-dose UA. Method MDCK cells were incubated with 3.5 mM uric acid for 48-h. Cellular proteins were extracted and subjected to quantitative proteomics analysis using 2-D PAGE followed by nanoLC-ESI-ETD MS/MS. The proteome data were confirmed by Western blotting, whereas functional analyses were performed using various assays. Results Quantitative proteomics analysis revealed significant changes in levels of 22 proteins in the UA-treated cells that were related to ATPase activity, mitochondrial transport, cell adhesion and molecule binding, cell projection organization and maintenance of location network, metabolic process, response to unfold proteins, and vesicle-mediated transport. Western blotting confirmed changes in levels of cellular proteins identified by quantitative proteomics. Functional assays revealed an increase in intracellular ATP level and enhancement of tissue repair capability in the UA-treated cells. Interestingly, isolation/purification of apical membranes followed by Western blotting showed the increased levels of HSP70 and HSP90 (the known receptors for CaOx crystals) on apical membranes of the UA-treated cells. CaOx crystal-cell adhesion assay revealed significant increase in CaOx-binding capability of the UA-treated cells, whereas neutralization of the surface HSP70 and HSP90 using their specific monoclonal antibodies caused significant reduction in such binding capability (even below the basal level). Conclusion These findings highlighted changes in renal tubular cells in response to high-dose UA that might be related to the pathogenic mechanisms of kidney stone disease associated with hyperuricosuria.

Functional analysis and signaling pathway enrichment analysis of genes associated with Alzheimer’s disease and Parkinson’s disease

We identified significant functions of susceptibility-genes and performed an analysis of pathway enrichment for Alzheimer’s disease, Parkinson’s disease and for both of them. Genes were extracted from a Catalog of Published Genome-Wide Association Studies (GWAS). We uploaded genes into Cytoscape version 3.2.1. ClueGO plugin was used for functional and pathway enrichment analysis of genes based on the hypergeometric test. Two databases, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway and REACTOME, were selected for analysis. The identified susceptibility genes are involved in the synthesis regulation and accumulation of toxic proteins, β -amyloid and α -synuclein, and lead to apoptosis of neurons. We have defined 14 shared functions: collagen catabolic process, cellular response to retinoic acid, regulation of calcium-mediated signaling, negative regulation of cell projection organization, negative regulation of neuron projection development, glial cell activation, microglial cell activation, macrophage activation, regulation of cholesterol metabolism, clathrin-mediated endocytosis, regulation of protein oligomerization, regulation of dendritic spine development, kinesin binding and clathrin binding. Also, we have defined 3 shared signaling pathways: trans-Golgi Network Vesicle Budding, Clathrin derived vesicle budding, Intestinal immune network for IgA production. These pathways contain genes susceptible to Alzheimer’s disease and Parkinson’s disease. The results suggest the metabolic, neuronal and immunological factors participate in the development of Parkinson’s disease and Alzheimer’s disease.

In silico approach to calculate the transcript capacity.

We sought the novel concept, transcript capacity (TC) and analyzed TC. Our approach to estimate TC was through an in silico method. TC refers to the capacity that a transcript exerts in a cell as enzyme or protein function after translation. We used the genome-wide association study (GWAS) beta effect and transcription level in RNA-sequencing to estimate TC. The trait was body fat percent and the transcript reads were obtained from the human protein atlas. The assumption was that the GWAS beta effect is the gene’s effect and TC was related to the corresponding gene effect and transcript reads. Further, we surveyed gene ontology (GO) in the highest TC and the lowest TC genes. The most frequent GOs with the highest TC were neuronal-related and cell projection organization related. The most frequent GOs with the lowest TC were wound-healing related and embryo development related. We expect that our analysis contributes to estimating TC in the diverse species and playing a benevolent role to the new bioinformatic analysis.

Family-based exome sequencing and case-control analysis implicate CEP41 as an ASD gene

Autism Spectrum Disorder (ASD) is a complex neurodevelopmental disorder with a strong genetic component. Although next-generation sequencing (NGS) technologies have been successfully applied to gene identification in de novo ASD, the genetic architecture of familial ASD remains largely unexplored. Our approach, which leverages the high specificity and sensitivity of NGS technology, has focused on rare variants in familial autism. We used NGS exome sequencing in 26 families with distantly related affected individuals to identify genes with private gene disrupting and missense variants of interest (VOI). We found that the genes carrying VOIs were enriched for biological processes related to cell projection organization and neuron development, which is consistent with the neurodevelopmental hypothesis of ASD. For a subset of genes carrying VOIs, we then used targeted NGS sequencing and gene-based variant burden case-control analysis to test for association with ASD. Missense variants in one gene, CEP41, associated significantly with ASD (p = 6.185e−05). Homozygous gene-disrupting variants in CEP41 were initially found to be responsible for recessive Joubert syndrome. Using a zebrafish model, we evaluated the mechanism by which the CEP41 variants might contribute to ASD. We found that CEP41 missense variants affect development of the axonal tract, cranial neural crest migration and social behavior phenotype. Our work demonstrates the involvement of CEP41 heterozygous missense variants in ASD and that biological processes involved in cell projection organization and neuron development are enriched in ASD families we have studied.

In silico and Wet Bench Interactomics Sheds Light on the Similitudes and Differences between Human ROCO Proteins.

Defining a common and specific function for homologs of a novel protein family is not a trivial task. In their recent study, Tomkins and colleagues have addressed this challenge for the ROCO protein family by exploring interactomes of its four human members: MASL1, DAPK1, LRRK1, and LRRK2. ROCO proteins are characterized by a Ras-GTPase domain embedded in complex multidomain proteins and a functional descriptor for this protein family has been elusive despite accumulating research, particularly for LRRK2, a protein implicated in Parkinson's disease. Tomkins etal. have combined an in silico weighted literature mining approach with novel interactomics data obtained on protein chips for all four proteins under strictly comparable conditions. The combination of these approaches has allowed the prudent formulation of common functions for ROCO proteins, including their involvement in stress response and cell projection organization. In addition, the study also confirms functional specificity for the individual ROCOs with such functions as cell death and apoptosis assigned to DAPK1, cellular, and neuronal development associated with LRRK1 and intracellular transport and organization assigned to LRRK2.

Bioinformatics analysis of key differentially expressed genes in well and poorly differentiated endometrial carcinoma.

Endometrial carcinoma (EC) is one of the most common gynecological malignancies. The malignant degree increases between grade (G)1 and G3, and EC of G3 usually presents a high recurrence rate and poor prognosis. Therefore, the present study aimed to examine the principal genes associated with the degree of differentiation in EC. The microarrays GSE17025, GSE24537 and GSE35784, representing data of Type I EC samples of G1 and G3, were downloaded from the Gene Expression Omnibus. The differentially expressed genes (DEGs) and differentially expressed micro (mi)RNAs (DEMs) were identified, followed by functional enrichment analyses and interaction network construction. In total, 83upregulated and 130downregulated DEGs with the same expression trends in two mRNA datasets were screened. The upregulated DEGs were primarily enriched in 'mitotic cell cycle process', 'cell cycle process' and 'mitotic cell cycle'; while the downregulated DEGs were enriched in 'cellular component assembly involved in morphogenesis', 'cell projection organization' and 'microtubule‑based movement'. From the protein‑protein interaction network, DNA topoisomeraseIIα, kinesin family member11, cyclinB1 and BUB1 mitotic checkpoint serine/threonine were identified as foremost hub genes. One module was extracted and involved in 'mitotic cell cycle process' and 'cell cycle process'. Based on the analysis of DEMs and the miRNA‑target regulatory network, miRNA‑9 may be the most important upregulated DEM, and the DEGs forkhead boxP1 and cyclinE1 may serve vital roles in the differentiation of EC. In conclusion, principal genes were identified that may be determinants of the carcinogenesis of poorly differentiated EC, which may facilitate the examination of potential molecular mechanisms. These genes may additionally help identify candidate biomarkers and novel therapeutic targets for poorly differentiated EC.

Genome-Wide Association Study Identifies Novel Susceptibility Genes Associated with Coronary Artery Aneurysm Formation in Kawasaki Disease

Kawasaki disease (KD) or Kawasaki syndrome is known as a vasculitis of small to medium-sized vessels, and coronary arteries are predominantly involved in childhood. Generally, 20–25% of untreated with IVIG and 3–5% of treated KD patients have been developed coronary artery lesions (CALs), such as dilatation and aneurysm. Understanding how coronary artery aneurysms (CAAs) are established and maintained in KD patients is therefore of great importance. Upon our previous genotyping data of 157 valid KD subjects, a genome-wide association study (GWAS) has been conducted among 11 (7%) CAA-developed KD patients to reveal five significant genetic variants passed pre-defined thresholds and resulted in two novel susceptibility protein-coding genes, which are NEBL (rs16921209 (P = 7.44 × 10−9; OR = 32.22) and rs7922552 (P = 8.43 × 10−9; OR = 32.0)) and TUBA3C (rs17076896 (P = 8.04 × 10−9; OR = 21.03)). Their known functions have been reported to associate with cardiac muscle and tubulin, respectively. As a result, this might imply their putative roles of establishing CAAs during KD progression. Additionally, various model analyses have been utilized to determine dominant and recessive inheritance patterns of identified susceptibility mutations. Finally, all susceptibility genes hit by significant genetic variants were further investigated and the top three representative gene-ontology (GO) clusters were regulation of cell projection organization, neuron recognition, and peptidyl-threonine phosphorylation. Our results help to depict the potential routes of the pathogenesis of CAAs in KD patients and will facilitate researchers to improve the diagnosis and prognosis of KD in personalized medicine.

Abstract 811: Identifying genomic signatures within circulating breast cancer cells.

Abstract Introduction: Breast cancer is not a single disease but encompasses several subtypes, each with a distinct molecular profile and response to treatment. The proportion of cells that express prognostic markers (ER/PR/HER2) can vary greatly within tumors, affecting sensitivity or resistance to therapies. Levels of circulating tumor cells (CTCs) in blood have been found to be related to disease progression. They show genetic evolution from the primary tumors they originate from, especially with acquired HER2 expression. CTCs could contain genomic alterations that define them as metastatic intermediates, and may be identified in primary tumors as an aggressive component that responds differentially to chemotherapy. Array-CGH likely fails to detect alterations present at low frequencies within primary tumours. Profiling of CTCs will help identify occult changes occurring in breast cancer cells during progression to metastasis. Methodology: CTCs and matched normal white blood cells were isolated from the blood of 17/38 breast cancer patients, prior to commencement of treatment. Copy number analysis was performed with the Affymetrix Genome-Wide Human SNP 6.0 array using a paired tumor-normal approach (Partek Genomics Suite). Minimal common regions (MCRs) of genomic gain were extrapolated, followed by unsupervised clustering. Associations between MCRs and luminal/HER2/triple negative subtypes; as well as metastasis, were identified using the Fisher's Exact test. Results: CTC genomic profiles clustered into 2 groups independent of subtype. A 1.2Mb amplicon containing the ERBB2 gene was gained in 15/17 patients, irrespective of HER2 status of the primary tumor. Cluster A had significantly fewer gains with 13 MCRs (AKT2, COL18A1), and was heterogeneous with no overlap in Cluster B. Cluster B showed higher genomic alteration and homogeneity between samples with 665 MCRs. Chromosome 19 was most frequently altered across all patients with 54/113 (48%) MCRs. Analysis of 787 primary breast carcinomas showed similar MCRs in 3-4% of samples. Patients with distant metastases and younger age (&amp;lt;50) clustered together. Region 19q13 (genes involved in proliferation, adhesion and cell projection organization) was associated with HER2 positivity and triple negative status of primary tumors. Regions 20q13 and 15q24 (genes involved in survival and progression) were associated with distant metastasis. Conclusions: Genomic profiles of CTCs clustered into two groups: a heterogeneous group with minimal alterations that may be sufficient for dissemination; and a more homogenous group with extensive alteration (particularly on chromosome 19) that could define those CTCs that are capable of survival and metastasis. The dissimilarity between groups is suggestive of cancer cells transitioning through stages that may be reflected in their genomes. We are currently using FISH to detect cells within primary tumors with CTC-like signatures. Citation Format: Nisha Kanwar, Pingzhao Hu, Mark Clemons, Philippe Bedard, David McCready, Susan J. Done. Identifying genomic signatures within circulating breast cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 811. doi:10.1158/1538-7445.AM2013-811