Hox Temporal Collinearity: Misleading Fallacy or Essential Developmental Mechanism?

Mapping Intimacies ◽

10.20944/preprints201906.0082.v1 ◽

2019 ◽

Cited By ~ 2

Author(s):

A.J. Durston

Keyword(s):

Careful Study ◽

Rna Seq ◽

Active Chromatin ◽

Hox Gene ◽

Developmental Mechanism ◽

Initiation Phase ◽

Qrt Pcr ◽

Initial Acquisition ◽

Hoxd Cluster ◽

Vertebrate Embryos

Kondo and collaborators recently reported the absence of Hox temporal collinearity in Xenopus tropicalis. They found none in the initiation of accumulation of  Hox transcripts (detected via RNA seq). And none in the initial expression sequence of primary unprorocessed transcripts (Identified by using qRT-PCR against introns or intron-exon boundaries).  Nor in the initial acquisition by Hox gene DNA of a mark for active chromatin.  These findings are in conflict with the idea that temporal collinearity has to do with the initiation of Hox gene transcription or with the opening of and a progression from repressed to active states in Hox chromatin.  But collinear acquisition of the same active chromatin mark has been shown by others in murine 5’ Hoxd cluster genes.The reason for this difference is unknown . This careful study thus indicated that the initiation phase of Hox expression shows no temporal collinearity in X. tropicalis. A previous study in X. laevis from the same group also showed   that the sequence of times for reaching (normalised) half maximal Hox expression showed no temporal collinearity. These conclusions are likely to be correct. These authors do however also conclude that “experimental evidence for the temporal collinearity hypothesis is not strong” There is however strong evidence that Hox temporal collinearity does occur in early vertebrate embryos.   Below. I present and discuss 3 lines of evidence to resolve the present conflict   I argue that Hox temporal collinearity actually does exist and that it is part of a central mechanism in early development.

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Comprehensive Analysis of Transcriptomics and Metabolomics between the Heads and Tails of Angelica Sinensis: Genes Related to Phenylpropanoid Biosynthesis Pathway

Combinatorial Chemistry & High Throughput Screening ◽

10.2174/1386207323999201103221952 ◽

2020 ◽

Vol 23 ◽

Author(s):

Jie Yang ◽

Chi Zhang ◽

Wei-Hong Li ◽

Tian-Er Zhang ◽

Guang-Zhong Fan ◽

...

Keyword(s):

Ferulic Acid ◽

Metabolic Regulation ◽

Comprehensive Analysis ◽

Angelica Sinensis ◽

Rna Seq ◽

Targeted Metabolomics ◽

Kegg Pathways ◽

Qrt Pcr ◽

Phenylpropanoid Biosynthesis ◽

Combined Strategy

Background:: In Traditional Chinese Medicine (TCM), the heads and tails of Angelica sinensis (Oliv.) Diels (AS) is used in treating different diseases due to their different pharmaceutical efficacies. The underline mechanisms, however, have not been fully explored. Objective:: Novel mechanisms responsible for the discrepant activities between AS heads and tails were explored by a combined strategy of transcriptomes and metabolomics. Method:: Six pairs of the heads and tails of AS roots were collected in Min County, China. Total RNA and metabolites, which were used for RNA-seq and untargeted metabolomics analysis, were respectively isolated from each AS sample (0.1 g) by Trizol and methanol reagent. Subsequently, differentially expressed genes (DEGs) and discrepant pharmaceutical metabolites were identified for comparing AS heads and tails. Key DEGs and metabolites were quantified by qRT-PCR and targeted metabolomics experiment. Results:: Comprehensive analysis of transcriptomes and metabolomics results suggested that five KEGG pathways with significant differences included 57 DEGs. Especially, fourteen DEGs and six key metabolites were relation to the metabolic regulation of Phenylpropanoid biosynthesis (PB) pathway. Results of qRT-PCR and targeted metabolomics indicated that higher levels of expression of crucial genes in PB pathway, such as PAL, CAD, COMT and peroxidase in the tail of AS were positively correlated with levels of ferulic acid-related metabolites. The average content of ferulic acid in tails (569.58162.39 nmol/g) was higher than those in the heads (168.73  67.30 nmol/g) (P˂0.01); Caffeic acid in tails (3.82  0.88 nmol/g) vs heads (1.37  0.41 nmol/g) (P˂0.01), and Cinnamic acid in tails (0.24  0.09 nmol/g) vs heads (0.14  0.02 nmol/g) (P˂0.05). Conclusion:: Our work demonstrated that overexpressed genes and accumulated metabolites derived from PB pathway might be responsible for the discrepant pharmaceutical efficacies between AS heads and tails.

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Transcriptome Analysis of Responses to Dengue Virus 2 Infection in Aedes albopictus (Skuse) C6/36 Cells

Viruses ◽

10.3390/v13020343 ◽

2021 ◽

Vol 13 (2) ◽

pp. 343

Author(s):

Manjin Li ◽

Dan Xing ◽

Duo Su ◽

Di Wang ◽

Heting Gao ◽

...

Keyword(s):

Dengue Virus ◽

Aedes Albopictus ◽

Bioinformatics Analysis ◽

Interaction Mechanism ◽

Transcriptional Analysis ◽

Functional Verification ◽

Mosquito Vector ◽

Rna Seq ◽

Sequencing Data ◽

Qrt Pcr

Dengue virus (DENV), a member of the Flavivirus genus of the Flaviviridae family, can cause dengue fever (DF) and more serious diseases and thus imposes a heavy burden worldwide. As the main vector of DENV, mosquitoes are a serious hazard. After infection, they induce a complex host–pathogen interaction mechanism. Our goal is to further study the interaction mechanism of viruses in homologous, sensitive, and repeatable C6/36 cell vectors. Transcriptome sequencing (RNA-Seq) technology was applied to the host transcript profiles of C6/36 cells infected with DENV2. Then, bioinformatics analysis was used to identify significant differentially expressed genes and the associated biological processes. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was performed to verify the sequencing data. A total of 1239 DEGs were found by transcriptional analysis of Aedes albopictus C6/36 cells that were infected and uninfected with dengue virus, among which 1133 were upregulated and 106 were downregulated. Further bioinformatics analysis showed that the upregulated DEGs were significantly enriched in signaling pathways such as the MAPK, Hippo, FoxO, Wnt, mTOR, and Notch; metabolic pathways and cellular physiological processes such as autophagy, endocytosis, and apoptosis. Downregulated DEGs were mainly enriched in DNA replication, pyrimidine metabolism, and repair pathways, including BER, NER, and MMR. The qRT-PCR results showed that the concordance between the RNA-Seq and RT-qPCR data was very high (92.3%). The results of this study provide more information about DENV2 infection of C6/36 cells at the transcriptome level, laying a foundation for further research on mosquito vector–virus interactions. These data provide candidate antiviral genes that can be used for further functional verification in the future.

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Application of qRT-PCR and RNA-Seq analysis for the identification of housekeeping genes useful for normalization of gene expression values during Striga hermonthica development

Molecular Biology Reports ◽

10.1007/s11033-012-2417-y ◽

2012 ◽

Vol 40 (4) ◽

pp. 3395-3407 ◽

Cited By ~ 22

Author(s):

M. Fernández-Aparicio ◽

K. Huang ◽

E. K. Wafula ◽

L. A. Honaas ◽

N. J. Wickett ◽

...

Keyword(s):

Gene Expression ◽

Housekeeping Genes ◽

Striga Hermonthica ◽

Rna Seq ◽

Qrt Pcr

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Ocena zmian profilu ekspresji genów kandydujących w podkładkach jabłoni o odmiennym stopniu tolerancji mrozowej

Biuletyn Instytutu Hodowli i Aklimatyzacji Roślin ◽

10.37317/biul-2020-pb81 ◽

2020 ◽

pp. 21-32

Author(s):

Sylwia Keller-Przybyłkowicz ◽

Mariusz Lewandowski

Keyword(s):

De Novo ◽

Rna Seq ◽

Qrt Pcr

Celem przeprowadzonych badań była identyfikacja genów sprzężonych z cechą mrozoodporności podkładek jabłoni. Ocenę zmian w poziomie ekspresji wyizolowanych genów przeprowadzono metodami RNAseq i qRT-PCR, dla podkładek zróżnicowanych po względem stopnia tolerancji mrozowej: P 66 (tolerancyjna) i M.9 (wrażliwa).W wyniku przeprowadzonych odczytów sekwencji RNA (sekwencjonowanie de novo w systemie Illumina Solid) dla w/w podkładek zidentyfikowano około 167 milionów odczytów unikatowych sekwencji, z których do wstępnych badań weryfikacyjnych wytypowano 15 o zróżnicowanym profilu ekspresji. Sekwencje poddano adnotacji funkcjonalnej. Wytypowane geny kodują: białka strukturalne i integralne błon komórkowych i wakuoli komórkowych, czynników transkrypcyjnych, białek regulujących transport międzykomórkowy i wewnątrzkomórkowy, białek hydrolizujących wiązania C-O i C-N oraz białek wiążących makro- i mikroelementy. Celem weryfikacji typu regulacji sekwencji transkryptomu uzyskanych z sekwencjonowania nowej generacji (NGS), dla tych samych prób przeprowadzono ilościową analizę transkryptu genów (qRT-PCR). Spośród badanych genów, trzy reprezentowały identyczny typ regulacji w badanych układach eksperymentalnych RNA-seq i qRT-PCR. Wytypowane geny stanowią potencjalne sekwencje kandydujące do sporządzenia markerów funkcjonalnych, umożliwiających wczesną selekcję podkładek jabłoni tolerancyjnych na mróz.

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Chorionic Villous Mesenchymal Stem Cells Derived Exosomes Deliver miR-135b-5p to Trophoblasts, Promoting their Proliferation and Invasion by Targeting TXNIP via β-Catenin Pathway

10.21203/rs.3.rs-133394/v1 ◽

2020 ◽

Author(s):

Yijing Chu ◽

Yan Zhang ◽

Guoqiang Gao ◽

Jun Zhou ◽

Yang Lv ◽

...

Keyword(s):

Stem Cells ◽

Mesenchymal Stem Cells ◽

Tissue Injury ◽

Luciferase Reporter ◽

Rna Seq ◽

Qrt Pcr ◽

Reporter Assays ◽

Pcr Assays ◽

Proliferation And Invasion

Abstract Background: Human chorionic villous mesenchymal stem cells (CV-MSCs) are found to be a promising and effective treatment for tissue injury. Trophoblast dysfunction during pregnancies is significantly involved in the pathogenesis of preeclampsia (PE). This work was to understand how CV-MSCs regulated trophoblast function. Methods: In this study, we treated trophoblasts with CV-MSC-derived exosomes and RNA-seq analysis was used to understand the changes in trophoblasts. We examined the levels of TXNIP and β-catenin in trophoblasts by immunohistochemistry, western blot and qRT-PCR assays. Luciferase reporter assays and qRT-PCR assays were used to understand the role of miR135b-5p in the effects of CV-MSC-derived exosomes. The growth and invasion of trophoblasts was evaluated with the CCK-8 and transwell assays. Results: The treatment markedly enhanced the trophoblast proliferation and invasion. Furthermore, a significant decrease of TXNIP expression and inactivation of the β-catenin pathway in CV-MSCs exosomes-treated trophoblasts was observed. Consistent with these findings, TXNIP inhibition exhibited the same effect of promoting trophoblast proliferation and invasion as induced by CV-MSC-derived exosomes, also with the accompaniment of inactivation of β-catenin pathway. In addition, overexpression of TXNIP activated the β-catenin pathway in trophoblasts, and reduced the proliferation and invasion of trophoblasts. Importantly, miR135b-5p was found to be highly expressed in CV-MSC exosomes and interact with TXNIP. The miR-135b-5p overexpression significantly elevated the proliferation and invasion of trophoblasts, which could be attenuated by TXNIP overexpression. Conclusion: Our results suggest that TXNIP-dependent β-catenin pathway inactivation mediated by miR135b-5p which is delivered by CV-MSC-derived exosomes could promote the proliferation and invasion of trophoblasts.

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Identification and validation of miRNA reference genes in poplar under pathogen stress

10.21203/rs.3.rs-225734/v1 ◽

2021 ◽

Author(s):

Lichun Zhang ◽

Xiaoqian Yang ◽

Yiyi Yin ◽

Jinxing Wang ◽

Yanwei Wang

Keyword(s):

Gene Expression ◽

Reference Gene ◽

Small Rna ◽

Reference Genes ◽

Expression Profiles ◽

Small Rna Sequencing ◽

Rna Seq ◽

Internal Standards ◽

Qrt Pcr ◽

Canker Pathogen

Abstract Quantitative real time polymerase chain reaction (qRT-PCR) is a common method to analyze gene expression. Due to differences in RNA quantity, quality, and reverse transcription efficiency between qRT-PCR samples, reference genes are used as internal standards to normalize gene expression. However, few universal genes especially miRNAs have been identified as reference so far. Therefore, it is essential to identify reference genes that can be used across various experimental conditions, stress treatments, or tissues. In this study, 14 microRNAs (miRNAs) and 5.8S rRNA were assessed for expression stability in poplar trees infected with canker pathogen. Using three reference gene analysis programs, we found that miR156g and miR156a exhibited stable expression throughout the infection process. miR156g and miR156a were then tested as internal standards to measure the expression of miR1447 and miR171c, and the results were compared to small RNA sequencing (RNA-seq) data. We found that when miR156a was used as the reference gene, the expression of miR1447 and miR171c were consistent with the small RNA-seq expression profiles. Therefore, miR156a was the most stable miRNAs examined in this study, and could be used as a reference gene in poplar under canker pathogen stress, which should enable comprehensive comparisons of miRNAs expression and avoid the bias caused by different lenth between detected miRNAs and traditional referece genes. The present study has expanded the miRNA reference genes available for gene expression studies in trees under biotic stress.

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Long non-coding RNA LINC00152 regulates cell proliferation and migration by epigenetically repressing LRIG1 expression in cholangiocarcinoma

10.21203/rs.3.rs-76993/v1 ◽

2020 ◽

Author(s):

Ni Wang ◽

Yang Yu ◽

Boming Xu ◽

Chunmei Zhang ◽

Jie Liu ◽

...

Keyword(s):

Cell Proliferation ◽

Biological Function ◽

Rna Seq ◽

Normal Tissues ◽

Qrt Pcr ◽

Non Coding Rna ◽

And Migration ◽

Long Non Coding Rna ◽

Proliferation And Migration

Abstract Background: Recently, long non-coding RNAs (lncRNAs) have been verified to have significant regulatory roles in multiple human cancer processes. Long non-coding RNA LINC00152, located on chromosome 2p11.2, was identified as an oncogenic lncRNA in various cancers. However, the biological function and molecular mechanism of LINC00152 in cholangiocarcinoma (CCA) are still unknown.Methods: Bioinformatic analysis was performed to determine LINC00152 expression levels in the CCA and normal tissues by using raw microarray data downloaded from Gene Expression Omnibus (GSE76297) and The Cancer Genome Atlas (TCGA). Quantitative reverse transcription PCR (qRT-PCR) was used to validate LINC00152 expression in the CCA tissues compared with that in the paired normal tissues. CCK8, colony formation, Edu assays, transwell assays, flow cytometry, and in vivo tumor formation assays were performed to investigate the biological function of LINC00152 on CCA cell phenotypes. RNA-seq was carried out to identify the downstream target gene which was further examined by qRT-PCR, western bolt and rescue experiments. RNA immunoprecipitation (RIP) and Chromatin immunoprecipitation (ChIP) assays were performed to reveal the factors involved in the mechanism of LINC00152 functions in CCA.Results: LINC00152 is significantly upregulated in cholangiocarcinoma. LINC00152 regulated the proliferation and migration of cholangiocarcinoma cells both in vitro and in vivo. RNA-seq revealed that LINC00152 knockdown preferentially affected genes linked with cell proliferation, cell differentiation and cell adhesion. Furthermore, mechanistic investigation validated that LINC00152 could bind EZH2 and modulate the histone methylation of promoter of leucine rich repeats and immunoglobulin like domains 1 (LRIG1), thereby affecting cholangiocarcinoma cells growth and migration.Conclusion: Taken together, these results demonstrated the significant roles of LINC00152 in cholangiocarcinoma and suggested a new diagnostic and therapeutic direction of cholangiocarcinoma.

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Small RNA sequencing evaluation of renal microRNA biomarkers in dogs with X-linked hereditary nephropathy

Scientific Reports ◽

10.1038/s41598-021-96870-y ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Candice P. Chu ◽

Shiguang Liu ◽

Wenping Song ◽

Ethan Y. Xu ◽

Mary B. Nabity

Keyword(s):

Small Rna ◽

Target Genes ◽

Differentially Expressed ◽

Small Rna Sequencing ◽

Rna Seq ◽

Ckd Progression ◽

Critical Pathways ◽

Qrt Pcr ◽

Hereditary Nephropathy ◽

Differentially Expressed Mirnas

AbstractDogs with X-linked hereditary nephropathy (XLHN) are an animal model for Alport syndrome in humans and progressive chronic kidney disease (CKD). Using mRNA sequencing (mRNA-seq), we have characterized the gene expression profile affecting the progression of XLHN; however, the microRNA (miRNA, miR) expression remains unknown. With small RNA-seq and quantitative RT-PCR (qRT-PCR), we used 3 small RNA-seq analysis tools (QIAGEN OmicSoft Studio, miRDeep2, and CPSS 2.0) to profile differentially expressed renal miRNAs, top-ranked miRNA target genes, and enriched biological processes and pathways in CKD progression. Twenty-three kidney biopsies were collected from 5 dogs with XLHN and 4 age-matched, unaffected littermates at 3 clinical time points (T1: onset of proteinuria, T2: onset of azotemia, and T3: advanced azotemia). We identified up to 23 differentially expressed miRNAs at each clinical time point. Five miRNAs (miR-21, miR-146b, miR-802, miR-142, miR-147) were consistently upregulated in affected dogs. We identified miR-186 and miR-26b as effective reference miRNAs for qRT-PCR. This study applied small RNA-seq to identify differentially expressed miRNAs that might regulate critical pathways contributing to CKD progression in dogs with XLHN.

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