Transcriptional Co-expression Research Articles

Pan-Cancer Analysis of Head-to-Head Gene Pairs in Terms of Transcriptional Activity, Co-expression and Regulation.

BackgroundHead-to-Head (H2H) gene pairs are regulated by bidirectional promoters and divergently transcribed from opposite DNA strands with transcription start sites (TSSs) separated within 1 kb. H2H organization is ancient and conserved, and H2H pairs tend to exhibit similar expression patterns. Although some H2H genes have been reported to be associated with disease and cancer, there is a lack of systematic studies on H2H organization in the scenario of cancer development.MethodsHuman H2H gene pairs were identified based on GENCODE hg19 and the functional relevance of H2H pairs was explored through function enrichment and semantic similarity analysis. To investigate the association between H2H organization and carcinogenesis, pan-cancer differential analysis of H2H genes about transcriptional activity, co-expression and transcriptional regulation by transcription factors and enhancers were performed based on data from The Cancer Genome Atlas. Cox proportional hazards regression model and log-rank test were used to determine the prognostic powers of H2H pairs.ResultsIn the present study, we first updated H2H genes from 1,447 to 3,150 pairs, from which the peak group with TSS distance of 1–100 was observed as expected in our previous work. It was found that housekeeping genes, mitochondrial-functional associated genes and cancer genes tend to be organized in H2H arrangement. Pan-cancer analysis indicates that H2H genes are transcriptionally active than random genes in both normal and cancer tissues, but H2H pairs display higher correlation in cancer than in normal. Particularly, housekeeping H2H pairs are differentially correlated much more significantly than non-housekeeping H2H pairs are. Some of differentially correlated H2H pairs were found to be associated with prognosis. The alteration of TF similarity seems to contribute to differential co-expression of H2H pairs during carcinogenesis; meanwhile remote enhancers also at least partly explain the differential co-expression and co-regulation of H2H pairs.ConclusionH2H pairs tend to show much stronger positive expression correlation in cancer than in normal due to differential regulation of bidirectional promoters. The study provides insights into the significance of H2H organization in carcinogenesis and the underlying dysfunctional regulation mechanisms. Those differentially correlated H2H pairs associated with survival have the potential to be prognostic biomarkers and therapeutic targets for cancer.

A simple approach to identify functional antibody variable genes in murine hybridoma cells that coexpress aberrant kappa light transcripts by restriction enzyme digestion

Specific binding to target protein epitopes by a mouse monoclonal antibody (mAb) relies on its variable domains. However, the isolation of functional variable gene transcripts is sometimes hindered by co-expression of aberrant transcripts in hybridoma cells.To develop general strategies for identifying the functional variable transcripts of both heavy (VH) and kappa light (Vκ) chains from mouse hybridomas.VH and Vκ genes of anti-dengue hybridoma clones were PCR-amplified using set of degenerate primers covering all mouse immunoglobulin families. Vκ amplicons were additionally digested with BciVI to eliminate aberrant Vκ transcripts. The productive VH and Vκ sequences were identified by Immunogenetics (IMGT) database analysis and cloned into a dual human IgG expression vector to generate chimeric antibodies (chAbs) in mammalian cells. The reactivity of chAbs was tested by immunoblot and immunofluorescent assays.Among 17 tested hybridoma clones, 400 bp Vκ amplicons were obtained using eight different Vκ primers. Amplicons from productive Vκ transcripts are resistant to BciVI digestion, whereas BciVI-digested amplicons indicated aberrant Vκ transcripts. 500-bp productive VH amplicons could be obtained from all clones using a set of five VH primers. The productive VH/Vκ genes of three anti-dengue NS1 mAbs (m2G6, m1F11 and m1A4) were cloned and mouse-human chAbs were generated. The binding reactivities of the chAbs to dengue NS1 were similar to the original mAbs.A general protocol to identify productive/functional VH and Vκ genes was demonstrated. The method is useful for producing chAbs and genetic archiving of valuable hybridoma cell lines.

NEK1 Phosphorylation of YAP Promotes Its Stabilization and Transcriptional Output.

Simple SummaryWe earlier described the involvement of the TLK1>NEK1>ATR>Chk1 axis as a key determinant of cell cycle arrest in androgen-dependent prostate cancer (PCa) cells after androgen deprivation. We now report that the TLK1>NEK1 axis is also involved in stabilization of yes-associated protein 1 (YAP1), the transcriptional co-activator in the Hippo pathway, presumably facilitating reprogramming of the cells toward castration-resistant PCa (CRPC). NEK1 interacts with YAP1 physically resulting in its phosphorylation of 6 residues, which enhance its stability and activity. Analyses of cancer Protein Atlas and TCGA expression panels revealed a link between activated NEK1 and YAP1 expression and several YAP transcription targets.Most prostate cancer (PCa) deaths result from progressive failure in standard androgen deprivation therapy (ADT), leading to metastatic castration-resistant PCa (mCRPC); however, the mechanism and key players leading to this are not fully understood. While studying the role of tousled-like kinase 1 (TLK1) and never in mitosis gene A (NIMA)-related kinase 1 (NEK1) in a DNA damage response (DDR)-mediated cell cycle arrest in LNCaP cells treated with bicalutamide, we uncovered that overexpression of wt-NEK1 resulted in a rapid conversion to androgen-independent (AI) growth, analogous to what has been observed when YAP1 is overexpressed. We now report that overexpression of wt-NEK1 results in accumulation of YAP1, suggesting the existence of a TLK1>NEK1>YAP1 axis that leads to adaptation to AI growth. Further, YAP1 is co-immunoprecipitated with NEK1. Importantly, NEK1 was able to phosphorylate YAP1 on six residues in vitro, which we believe are important for stabilization of the protein, possibly by increasing its interaction with transcriptional partners. In fact, knockout (KO) of NEK1 in NT1 PCa cells resulted in a parallel decrease of YAP1 level and reduced expression of typical YAP-regulated target genes. In terms of cancer potential implications, the expression of NEK1 and YAP1 proteins was found to be increased and correlated in several cancers. These include PCa stages according to Gleason score, head and neck squamous cell carcinoma, and glioblastoma, suggesting that this co-regulation is imparted by increased YAP1 stability when NEK1 is overexpressed or activated by TLK1, and not through transcriptional co-expression. We propose that the TLK1>NEK1>YAP1 axis is a key determinant for cancer progression, particularly during the process of androgen-sensitive to -independent conversion during progression to mCRPC.

Involvement of Cxcl12a/Cxcr4b Chemokine System in Mediating the Stimulatory Effect of Embryonic Ethanol Exposure on Neuronal Density in Zebrafish Hypothalamus.

Embryonic exposure to ethanol (EtOH) produces marked disturbances in neuronal development and alcohol-related behaviors, with low-moderate EtOH doses stimulating neurogenesis without producing apoptosis and high doses having major cytotoxic effects while causing gross morphological abnormalities. With the pro-inflammatory chemokine system, Cxcl12, and its main receptor Cxcr4, known to promote processes of neurogenesis, we examined here this neuroimmune system in the embryonic hypothalamus to test directly if it mediates the stimulatory effects low-moderate EtOH doses have on neuronal development. We used the zebrafish (Danio rerio) model, which develops externally and allows one to investigate the developing brain in vivo with precise control of dose and timing of EtOH delivery in the absence of maternal influence. Zebrafish were exposed to low-moderate EtOH doses (0.1, 0.25, 0.5% v/v), specifically during a period of peak hypothalamic development from 22 to 24hours postfertilization, and in some tests were pretreated from 2 to 22 hpf with the Cxcr4 receptor antagonist, AMD3100. Measurements in the hypothalamus at 26 hpf were taken of cxcl12a and cxcr4b transcription, signaling, and neuronal density using qRT-PCR, RNAscope, and live imaging of transgenic zebrafish. Embryonic EtOH exposure, particularly at the 0.5% dose, significantly increased levels of cxcl12a and cxcr4b mRNA in whole embryos, number of cxcl12a and cxcr4b transcripts in developing hypothalamus, and internalization of Cxcr4b receptors in hypothalamic cells. Embryonic EtOH also caused an increase in the number of hypothalamic neurons and coexpression of cxcl12a and cxcr4b transcripts within these neurons. Each of these stimulatory effects of EtOH in the embryo was blocked by pretreatment with the Cxcr4 antagonist AMD3100. These results provide clear evidence that EtOH's stimulatory effects at low-moderate doses on the number of hypothalamic neurons early in development are mediated, in part, by increased transcription and intracellular activation of this chemokine system, likely due to autocrine signaling of Cxcl12a at its Cxcr4b receptor within the neurons.

Microarray data analysis reveals gene expression changes in response to ionizing radiation in MCF7 human breast cancer cells

BackgroundThe aim of this study was to identify potential therapeutic target genes for breast cancer (BC) by the investigation of gene expression changes after ionizing radiation (IR) in BC cells. Gene expression profile GSE21748, including BC cell line MCF-7 samples at different time points after IR treatment, were downloaded from Gene Expression Omnibus. Differentially expressed genes (DEGs) were identified in different time points following IR compared with cell samples before IR, respectively. Gene ontology functions and The Kyoto Encyclopedia of Genes and Genomes pathways of the overlapping DEGs were enriched using DAVID. Transcription factor (TFs)-encoding genes were identified from the overlapping DEGs, followed by construction of transcriptional regulatory network and co-expression network.ResultsA total of 864 overlapping DEGs were identified, which were significantly enriched in regulation of cell proliferation and apoptosis, and cell cycle process. We found that FOXD1, STAT6, XBP1, STAT2, LMO2, TFAP4, STAT3, STAT1 were hub nodes in the transcriptional regulatory network of the overlapping DEGs. The co-expression network of target genes regulated by STAT3, STAT1, STAT6 and STAT2 included some key genes such as BCL2L1.ConclusionSTAT1, STAT2, STAT3, STAT6, XBP1, BCL2L1, CYB5D2, ESCO2, and PARP2 were significantly affected by IR and they may be used as therapeutic gene targets in the treatment of BC.

Functional expression of P2X1, P2X4 and P2X7 purinergic receptors in human monocyte-derived macrophages

This study sought to examine the co-expression of the following purinergic receptor subunits: P2X1, P2X1del, P2X4, and P2X7 and characterize the P2X response in human monocyte-derived macrophages (MDMs). Single-cell RT-PCR shows the presence of P2X1, P2X1del, P2X4, and P2X7 mRNA in 40%, 5%, 20%, and 90% of human MDMs, respectively. Of the studied human MDMs, 25% co-expressed P2X1 and P2X7 mRNA; 5% co-expressed P2X4 and P2X7; and 15% co-expressed P2X1, P2X4, and P2X7 mRNA. In whole-cell patch clamp recordings of human MDMs, rapid application of ATP (0.01 mM) evoked fast current activation and two different desensitization kinetics: 1. a rapid desensitizing current antagonized by PPADS (1 μM), reminiscent of the P2X1 receptor's current; 2. a slow desensitizing current, insensitive to PPADS but potentiated by ivermectin (3 μM), similar to the P2X4 receptor's current. Application of 5 mM ATP induced three current modalities: 1. slow current activation with no desensitization, similar to the P2X7 receptor current, present in 69% of human macrophages and antagonized by A-804598 (0.1 μM); 2. fast current activation and fast desensitization, present in 15% of human MDMs; 3. fast activation current followed by biphasic desensitization, observed in 15% of human MDMs. Both rapid and biphasic desensitization kinetics resemble those observed for the recombinant human P2X1 receptor expressed in oocytes. These data demonstrate, for the first time, the co-expression of P2X1, P2X4, and P2X7 transcripts and confirm the presence of functional P2X1, P2X4, and P2X7 receptors in human macrophages.

Xylem systems genetics analysis reveals a key regulator of lignin biosynthesis in Populus deltoides.

Despite the growing resources and tools for high-throughput characterization and analysis of genomic information, the discovery of the genetic elements that regulate complex traits remains a challenge. Systems genetics is an emerging field that aims to understand the flow of biological information that underlies complex traits from genotype to phenotype. In this study, we used a systems genetics approach to identify and evaluate regulators of the lignin biosynthesis pathway in Populus deltoides by combining genome, transcriptome, and phenotype data from a population of 268 unrelated individuals of P. deltoides. The discovery of lignin regulators began with the quantitative genetic analysis of the xylem transcriptome and resulted in the detection of 6706 and 4628 significant local- and distant-eQTL associations, respectively. Among the locally regulated genes, we identified the R2R3-MYB transcription factor MYB125 (Potri.003G114100) as a putative trans-regulator of the majority of genes in the lignin biosynthesis pathway. The expression of MYB125 in a diverse population positively correlated with lignin content. Furthermore, overexpression of MYB125 in transgenic poplar resulted in increased lignin content, as well as altered expression of genes in the lignin biosynthesis pathway. Altogether, our findings indicate that MYB125 is involved in the control of a transcriptional coexpression network of lignin biosynthesis genes during secondary cell wall formation in P. deltoides.

A Combined Analysis of Genetically Correlated Traits Identifies Genes and Brain Regions for Insomnia.

Previous studies have inferred that there is a strong genetic component in insomnia. However, the etiology of insomnia is still unclear. This study systematically analyzed multiple genome-wide association study (GWAS) data sets with core human pathways and functional networks to detect potential gene pathways and networks associated with insomnia. We used a novel method, multitrait analysis of genome-wide association studies (MTAG), to combine 3 large GWASs of insomnia symptoms/complaints and sleep duration. The i-Gsea4GwasV2 and Reactome FI programs were used to analyze data from the result of MTAG analysis and the nominally significant pathways, respectively. Through analyzing data sets using the MTAG program, our sample size increased from 113,006 subjects to 163,188 subjects. A total of 17 of 1,816 Reactome pathways were identified and showed to be associated with insomnia. We further revealed 11 interconnected functional and topologically interacting clusters (Clusters 0 to 10) that were associated with insomnia. Based on the brain transcriptome data, it was found that the genes in Cluster 4 were enriched for the transcriptional coexpression profile in the prenatal dorsolateral prefrontal cortex (P = 7 × 10-5), inferolateral temporal cortex (P = 0.02), medial prefrontal cortex (P < 1 × 10-5), and amygdala (P < 1 × 10-5), and detected RPA2, ORC6, PIAS3, and PRIM2 as core nodes in these 4 brain regions. The findings provided new genes, pathways, and brain regions to understand the pathology of insomnia.

An Insight into Oligopeptide Transporter 3 (OPT3) Family Proteins.

OPT3s are involved in the transport of Fe from xylem to phloem, in loading Fe into phloem, and in the transmission of shoot-to-root iron signaling. Yet, apart from Arabidopsis, little is known about these transporters'functions in other plant species. OPT3 proteins of several plant species were characterized using bioinformatical tools. Also, a probable Fe chelating protein, GSH, was used in docking analyses to shed light on the interactions of ligand binding sites of OPT3s. The multiple sequence alignment (MSA) analysis, protein secondary and tertiary structure analyses, molecular phylogeny analysis, transcription factor binding site analyses, co-expression and docking analyses were performed using up-to-date bioinformatical tools. All OPT3s in this study appear to be transmembrane proteins. They appear to have broad roles and substrate specificities in different metabolic processes. OPT3 gene structures were highly conserved. Promoter analysis showed that bZIP, WRKY, Dof and AT-Hook Transcription factors (TFs) may regulate the expression of OPT3 genes. Consequently, they seemed to be taking part in both biotic and abiotic stress responses as well as growth and developmental processes. The results showed that OPT3 proteins are involved in ROS regulation, plant stress responses, and basal pathogen resistance. They have species-specific roles in biological processes. Lastly, the transport of iron through OPT3s may occur with GSH according to the binding affinity results of the docking analyses.

Identification of long noncoding RNA RP11-89K21.1 and RP11-357H14.17 as prognostic signature of endometrial carcinoma via integrated bioinformatics analysis

BackgroundEndometrial carcinoma (EC) is one of the most common malignant tumors in gynecology. The potential functions and mechanisms of long noncoding RNAs (lncRNAs) in the occurrence and progression of EC remains unclear. It’s meaningful to explore lncRNAs signature for providing prognostic value of EC.MethodsThe differentially expressed lncRNAs and their prognostic values in EC were investigated based on The Cancer Genome Atlas (TCGA) database; the transcriptional factors (TFs), the competing endogenous RNA (ceRNA) mechanism, functional regulatory network and immune infiltration of RP11-89K21.1 and RP11-357H14.17 were further explored by various bioinformatics tools and databases.ResultsWe firstly identified high expression of RP11-89K21.1 and RP11-357H14.17 were closely associated with shorten overall survival (OS) and poor prognosis in patients with EC. We also elucidated the networks of transcription factor and co-expression genes associated with RP11-89K21.1 and RP11-357H14.17. Furthermore, the ceRNA network mechanism was successfully constructed through 2 lncRNAs (RP11-89K21.1 and RP11-357H14.17), 11 miRNAs and 183 mRNAs. Functional enrichment analysis revealed that the targeting genes of RP11-89K21.1 and RP11-357H14.17 were strongly associated with microRNAs in cancer, vessel development, growth regulation, growth factor and cell differentiation, and involved in pathways including pathways in cancer, microRNAs in cancer and apoptotic signaling pathway.ConclusionsWe demonstrated for the first time that RP11-89K21.1 and RP11-357H14.17 may play crucial roles in the occurrence, development and malignant biological behavior of EC, and can be regarded as potential prognostic biomarkers for EC.

Regional, cellular and species difference of two key neuroinflammatory genes implicated in schizophrenia

The transcription factor nuclear factor kappa B (NF-κB) regulates the expression of many inflammatory genes that are overexpressed in a subset of people with schizophrenia. Transcriptional reduction in one NF-κB inhibitor, Human Immunodeficiency Virus Enhancer Binding Protein 2 (HIVEP2), is found in the brain of patients, aligning with evidence of NF-κB over-activity. Cellular co-expression of HIVEP2 and cytokine transcripts is a prerequisite for a direct effect of HIVEP2 on pro-inflammatory transcription, and we do not know if changes in HIVEP2 and markers of neuroinflammation are occurring in the same brain cell type. We performed in situ hybridisation on postmortem dorsolateral prefrontal cortex tissue to map and compare the expression of HIVEP2 and Serpin Family A Member 3 (SERPINA3), one of the most consistently increased inflammatory genes in schizophrenia, between schizophrenia patients and controls. We find that HIVEP2 expression is neuronal and is decreased in almost all grey matter cortical layers in schizophrenia patients with neuroinflammation, and that SERPINA3 is increased in the dorsolateral prefrontal cortex grey matter and white matter in the same group of patients. We are the first to map the upregulation of SERPINA3 to astrocytes and to some neurons, and find evidence to suggest that blood vessel-associated astrocytes are the main cellular source of SERPINA3 in the schizophrenia cortex. We show that a lack of HIVEP2 in mice does not cause astrocytic upregulation of Serpina3n but does induce its transcription in neurons. We speculate that HIVEP2 downregulation is not a direct cause of astrocytic pro-inflammatory cytokine synthesis in schizophrenia but may contribute to neuronally-mediated neuroinflammation.

Co-Regulation of Clustered and Neo-Functionalized Genes in Plant-Specialized Metabolism.

Current findings of neighboring genes involved in plant specialized metabolism provide the genomic signatures of metabolic evolution. Two such genomic features, namely, (i) metabolic gene cluster and (ii) neo-functionalization of tandem gene duplications, represent key factors corresponding to the creation of metabolic diversity of plant specialized metabolism. So far, several terpenoid and alkaloid biosynthetic genes have been characterized with gene clusters in some plants. On the other hand, some modification genes involved in flavonoid and glucosinolate biosynthesis were found to arise via gene neo-functionalization. Although the occurrence of both types of metabolic evolution are different, the neighboring genes are generally regulated by the same or related regulation factors. Therefore, the translation-based approaches associated with genomics, and transcriptomics are able to be employed for functional genomics focusing on plant secondary metabolism. Here, we present a survey of the current understanding of neighboring genes involved in plant secondary metabolism. Additionally, a genomic overview of neighboring genes of four model plants and transcriptional co-expression network neighboring genes to detect metabolic gene clusters in Arabidopsis is provided. Finally, the insights functional genomics have provided concerning the evolution and mechanistic regulation of both the formation and operation of metabolic neighboring clusters is discussed.

Transcriptome profiles reveal that gibberellin-related genes regulate weeping traits in crape myrtle

Plant architecture includes vital traits that influence and benefit crops, and economically important trees. Different plant architectures provide natural beauty. Weeping ornamental plants are aesthetically appealing to people. The regulatory mechanism controlling the weeping trait is poorly understood in crape myrtle. To investigate the weeping trait mechanism, transcriptional profiling of different organs in weeping and upright crape myrtle was performed based on phenotype. Phenotypic and histological analyses demonstrated that endodermal cells were absent, and that new shoot phenotypes could be rescued by the GA3 treatment of weeping plants. The transcriptional analysis and coexpression network analysis (WGCNA) of differentially expressed genes indicated that GA synthesis and signal transduction pathways play a role in weeping traits. When the expression level of a negative element of GA signaling, LfiGRAS1, was reduced by virus-induced gene silencing (VIGS), new branches grew in infected plants in a negatively geotropic manner. An integrated analysis implied that GA had a strong influence on weeping crape myrtle by interacting with other factors. This study helps to elucidate the mechanism governing the weeping trait and can improve the efficiency of breeding in Lagerstroemia.

Functional Heterogeneity within the Developing Zebrafish Epicardium

SummaryThe epicardium is essential during cardiac development, homeostasis, and repair, and yet fundamental insights into its underlying cell biology, notably epicardium formation, lineage heterogeneity, and functional cross-talk with other cell types in the heart, are currently lacking. In this study, we investigated epicardial heterogeneity and the functional diversity of discrete epicardial subpopulations in the developing zebrafish heart. Single-cell RNA sequencing uncovered three epicardial subpopulations with specific genetic programs and distinctive spatial distribution. Perturbation of unique gene signatures uncovered specific functions associated with each subpopulation and established epicardial roles in cell adhesion, migration, and chemotaxis as a mechanism for recruitment of leukocytes into the heart. Understanding which mechanisms epicardial cells employ to establish a functional epicardium and how they communicate with other cardiovascular cell types during development will bring us closer to repairing cellular relationships that are disrupted during cardiovascular disease.

A transcriptional co-expression network-based approach to identify prognostic biomarkers in gastric carcinoma.

BackgroundGastric carcinoma is a very diverse disease. The progression of gastric carcinoma is influenced by complicated gene networks. This study aims to investigate the actual and potential prognostic biomarkers related to survival in gastric carcinoma patients to further our understanding of tumor biology.MethodsA weighted gene co-expression network analysis was performed with a transcriptome dataset to identify networks and hub genes relevant to gastric carcinoma prognosis. Data was obtained from 300 primary gastric carcinomas (GSE62254). A validation dataset (GSE34942 and GSE15459) and TCGA dataset confirmed the results. Gene ontology, the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis, and gene set enrichment analysis (GSEA) were performed to identify the clusters responsible for the biological processes and pathways of this disease.ResultsA brown transcriptional module enriched in the organizational process of the extracellular matrix was significantly correlated with overall survival (HR = 1.586, p = 0.005, 95% CI [1.149–2.189]) and disease-free survival (HR = 1.544, p = 0.008, 95% CI [1.119–2.131]). These observations were confirmed in the validation dataset (HR = 1.664, p = 0.006, 95% CI [1.155–2.398] in overall survival). Ten hub genes were identified and confirmed in the validation dataset from this brown module; five key biomarkers (COL8A1, FRMD6, TIMP2, CNRIP1 and GPR124 (ADGRA2)) were identified for further research in microsatellite instability (MSI) and epithelial-tomesenchymal transition (MSS/EMT) gastric carcinoma molecular subtypes. A high expression of these genes indicated a poor prognosis.ConclusionA transcriptional co-expression network-based approach was used to identify prognostic biomarkers in gastric carcinoma. This method may have potential for use in personalized therapies, however, large-scale randomized controlled clinical trials and replication experiments are needed before these key biomarkers can be applied clinically.

Detection of BCR-ABL fusion gene and its transcript variants in chronic myeloid leukaemia patients - a multi-comparison study.

To compare blood cells and plasma for BCR-ABL quantification and to find out the frequency of b2a2, b3a2 and e1a2 transcripts in chronic myeloid leukaemia patients, and to assess the correlation of BCR-ABL transcripts with haematological counts, age and gender. The study was conducted in April 2018 at King Edward Medical University, Lahore, Pakistan, and comprised chronic myeloid leukaemia patients from Mayo Hospital, Lahore. Ribonucleic acid was extracted using commercial extraction kits and detection of BCR-ABL messenger ribonucleic acid and its transcript variants was done by real time polymerase chain reaction. Data was analysed using SPSS 11.5. Of the 48 patients, fusion of b3a2 was detected in 32(66.66%) and b2a2 in 10(32.10%), while fusion e1a2 was not detected at all. No co-expression of transcripts was seen in any patient. No significant correlation was found between transcript type and any of haematological parameters (p>0.05). No significant correlation of transcript type with gender and age was found (p>0.05). BCR-ABL/G6PD ratios in peripheral blood cells were higher than that of plasma (p<0.05). Plasma can be used as an alternative to blood cells for BCR-ABL quantification, and transcript types cannot be easily explained by clinical factors.

TaD27‐B gene controls the tiller number in hexaploid wheat

SummaryTillering is a significant agronomic trait in wheat which shapes plant architecture and yield. Strigolactones (SLs) function in inhibiting axillary bud outgrowth. The roles of SLs in the regulation of bud outgrowth have been described in model plant species, including rice and Arabidopsis. However, the role of SLs genes in wheat remains elusive due to the size and complexity of the wheat genomes. In this study, TaD27 genes in wheat, orthologs of rice D27 encoding an enzyme involved in SLs biosynthesis, were identified. TaD27‐RNAi wheat plants had more tillers, and TaD27‐B‐OE wheat plants had fewer tillers. Germination bioassay of Orobanche confirmed the SLs was deficient in TaD27‐RNAi and excessive in TaD27‐B‐OE wheat plants. Moreover, application of exogenous GR24 or TIS108 could mediate the axillary bud outgrowth of TaD27‐RNAi and TaD27‐B‐OE in the hydroponic culture, suggesting that TaD27‐B plays critical roles in regulating wheat tiller number by participating in SLs biosynthesis. Unlike rice D27, plant height was not affected in the transgenic wheat plants. Transcription and gene coexpression network analysis showed that a number of genes are involved in the SLs signalling pathway and axillary bud development. Our results indicate that TaD27‐B is a key factor in the regulation of tiller number in wheat.

A simple approach to identify functional antibody variable genes in murine hybridoma cells that coexpress aberrant kappa light transcripts by restriction enzyme digestion.

Specific binding to target protein epitopes by a mouse monoclonal antibody (mAb) relies on its variable domains. However, the isolation of functional variable gene transcripts is sometimes hindered by co-expression of aberrant transcripts in hybridoma cells. To develop general strategies for identifying the functional variable transcripts of both heavy (VH) and kappa light (Vκ) chains from mouse hybridomas. VH and Vκ genes of anti-dengue hybridoma clones were PCR-amplified using set of degenerate primers covering all mouse immunoglobulin families. Vκ amplicons were additionally digested with BciVI to eliminate aberrant Vκ transcripts. The productive VH and Vκ sequences were identified by Immunogenetics (IMGT) database analysis and cloned into a dual human IgG expression vector to generate chimeric antibodies (chAbs) in mammalian cells. The reactivity of chAbs was tested by immunoblot and immunofluorescent assays. Among 17 tested hybridoma clones, 400 bp Vκ amplicons were obtained using eight different Vκ primers. Amplicons from productive Vκ transcripts are resistant to BciVI digestion, whereas BciVI-digested amplicons indicated aberrant Vκ transcripts. 500-bp productive VH amplicons could be obtained from all clones using a set of five VH primers. The productive VH/Vκ genes of three anti-dengue NS1 mAbs (m2G6, m1F11 and m1A4) were cloned and mouse-human chAbs were generated. The binding reactivities of the chAbs to dengue NS1 were similar to the original mAbs. A general protocol to identify productive/functional VH and Vκ genes was demonstrated. The method is useful for producing chAbs and genetic archiving of valuable hybridoma cell lines.

Functional and transcriptional connectivity of communities in breast cancer co-expression networks

Transcriptional co-expression networks represent the concerted gene regulation programs by means of statistical inference of co-expression patterns. The rich phenomenology of transcriptional processes behind complex phenotypes such as cancer, is often captured (at least partially) in the connectivity structure of transcriptional co-expression networks. By analyzing the community structure of these networks, we may develop a deeper understanding of that phenomenology. We identified the modular structure of a transcriptional co-expression network obtained from breast cancer gene expression as well as a non-cancer adjacent breast tissue network as a control. We then analyzed the biological functions associated to the resulting communities by means of enrichment analysis. We also generated two projected networks for both, tumor and control networks: The first one is a projection to a network in which nodes are communities and edges represent topologically adjacent communities, indicating co-expression patterns between them. For the second projection, a bipartite network was generated containing a layer of modules and a layer of biological processes, with links between modules and the functions in which they are enriched; from this bipartite network, a projection to the community layer was obtained. From the analysis of the communities and projections, we were able to discern distinctive patterns of regulation between tumors and controls. Even though the connectivity structure of transcriptional co-expression networks is quite different, the topology of the projected networks is somehow similar, indicating functional compartmentalization, in both tumor and control conditions. However, the biological functions represented in the corresponding modules resulted notably different, with the tumor network comprising functional modules enriched for well-known hallmarks of cancer.

Genome-wide RNAseq study of the molecular mechanisms underlying microglia activation in response to pathological tau perturbation in the rTg4510 tau transgenic animal model

BackgroundActivation of microglia, the resident immune cells of the central nervous system, is a prominent pathological hallmark of Alzheimer’s disease (AD). However, the gene expression changes underlying microglia activation in response to tau pathology remain elusive. Furthermore, it is not clear how murine gene expression changes relate to human gene expression networks.MethodsMicroglia cells were isolated from rTg4510 tau transgenic mice and gene expression was profiled using RNA sequencing. Four age groups of mice (2-, 4-, 6-, and 8-months) were analyzed to capture longitudinal gene expression changes that correspond to varying levels of pathology, from minimal tau accumulation to massive neuronal loss. Statistical and system biology approaches were used to analyze the genes and pathways that underlie microglia activation. Differentially expressed genes were compared to human brain co-expression networks.ResultsStatistical analysis of RNAseq data indicated that more than 4000 genes were differentially expressed in rTg4510 microglia compared to wild type microglia, with the majority of gene expression changes occurring between 2- and 4-months of age. These genes belong to four major clusters based on their temporal expression pattern. Genes involved in innate immunity were continuously up-regulated, whereas genes involved in the glutamatergic synapse were down-regulated. Up-regulated innate inflammatory pathways included NF-κB signaling, cytokine-cytokine receptor interaction, lysosome, oxidative phosphorylation, and phagosome. NF-κB and cytokine signaling were among the earliest pathways activated, likely driven by the RELA, STAT1 and STAT6 transcription factors. The expression of many AD associated genes such as APOE and TREM2 was also altered in rTg4510 microglia cells. Differentially expressed genes in rTg4510 microglia were enriched in human neurodegenerative disease associated pathways, including Alzheimer’s, Parkinson’s, and Huntington’s diseases, and highly overlapped with the microglia and endothelial modules of human brain transcriptional co-expression networks.ConclusionThis study revealed temporal transcriptome alterations in microglia cells in response to pathological tau perturbation and provides insight into the molecular changes underlying microglia activation during tau mediated neurodegeneration.