Transcriptomic comparative study on mouse liver injury caused by ultra-high dose rate irradiation and conventional irradiation / 中华放射医学与防护杂志

Objective:To study the effects of FLASH irradiation (FLASH-RT) and conventional irradiation (CONV-RT) on gene expression profile in mouse liver, in order to provide theoretical basis of the potential mechanism of FLASH-RT.Methods:A total of 11 C57BL/6J male mice were divided into healthy control group (Ctrl group), CONV-RT group and FLASH-RT group according to random number table method. Mouse abdomen was treated with 12 Gy CONV-RT or FLASH-RT. Then the mice were killed by neck removal, and the liver tissues were collected to extract total RNA for transcriptome sequencing (RNA-Seq) that was then analyzed by bio-informatics analysis to investigate the changes of gene expression profiles. The mRNA expression levels of Stat1, Irf9 and Rela were verified by quantitative real-time PCR assay.Results:1 762 differentially expressed genes (DEGs) were identified in group FLASH-RT vs. CONV-RT. Among them, 660 genes were up-regulated and 1 102 genes were down-regulated. 1 918 DEGs were identified in groups FLASH-RT vs. Ctrl. Among them, 728 genes were up-regulated and 1 190 genes were down-regulated. 1 569 DEGs were identified in group CONV-RT vs. Ctrl. Among them, 1 046 genes were up-regulated and 523 genes were down-regulated. According to Gene Ontology (GO) analysis, these DEGs from groups FLASH-RT vs. CONV-RT were involved in various functions including defense response to virus, other organisms in cell components, adenylyltransferase activity in molecular function activity. These DEGs from group FLASH-RT vs. Ctrl were involved in various functions including defense response to other oranisms, endoplasmic reticulum chaperone complex, double-stranded RNA binding and so on. These DEGs from group FLASH-RT vs. CONV-RT were involved in several Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways including influenza A, Herpes simplex infection and so on. These DEGs from group FLASH-RT vs. Ctrl were involved in several KEGG pathways including influenza A, NOD-like receptor signaling pathway. Stat1 was likely to be activated by FLASH radiation. The quantitative real-time PCR assay showed that FLASH-RT obviously increased the mRNA expressions of Stat1, Irf9 and Rela ( t=6.62, 2.11, 1.67, P<0.05). Conclusions:FLASH-RT and CONV-RT could alter gene expression profiles in mouse liver tissues, and these DEGs are involved in multiple radiobiological functional pathways. In comparison with CONV-RT, FLASH-RT induces a low level of liver injury, which may due to hypoxia radiation resistance.

A paired comparison study of genetic transmission network base on HIV-1 pol gene DNA or RNA sequence / 中国热带医学

@#Abstract: Objective To understand the difference of DNA sequence and RNA sequence of paired pol region in HIV patients in constructing HIV genetic transmission network, and to provide scientific data for constructing molecular transmission networks using DNA sequences. Methods　The whole blood and plasma samples of HIV patients living in 2014 and newly reported in 2015-2018 in Liuzhou, Guangxi, were collected, DNA and RNA sequences were extracted, amplified, sequenced, spliced and aligned, and then genetic transmission networks were constructed, and the connectivity consistency of genetic transmission networks constructed by DNA and RNA sequences was compared. Results In this study, a total 2 983 participants were investigated, which were 2014 baseline and 2015-2018 newly reported HIV patients, of which 2 590 participants were only DNA sequences in 2014 baseline, 196 HIV patients were both DNA and RNA sequences of paired pol region in 2014 baseline, and 197 newly reported HIV patients were both DNA and RNA sequences of paired pol region in 2015-2018. In 393 DNA and RNA sequences of paired pol region, the genotype of DNA sequence and RNA sequence were consistent, and there was no statistically significant difference in genetic distance between paired DNA sequences and RNA sequences (Z=-2.72, P=1.00). The connection consistency rate of genetic transmission networks constructed by DNA and RNA sequences of paired pol region of 2015-2018 newly reported with the baseline DNA sequences was 91.4% (108/197). And the connection consistency rate of genetic transmission networks constructed by DNA and RNA sequences of paired pol region of 2015-2018 newly reported with the baseline RNA sequences was 97.0% (191/197). There was no statistical difference in antiretroviral therapy to reduce the risk of HIV secondary transmission between genetic transmission networks constructed by DNA and RNA sequences of paired of pol gene newly reported from 2015 to 2018 with baseline DNA sequence. Conclusion　DNA sequence and RNA sequence of paired pol region of HIV patients have good consistency in genotype, genetic distance, and genetic transmission network construction, and both DNA and RNA sequences can be used for genetic transmission network analysis.

Transcriptomics Provides Novel Insights into the Regulatory Mechanism of IncRNA HIF1 A-AS1 on Vascular Smooth Muscle Cells

ABSTRACT Introduction: Thoracic aortic aneurysm is a potentially fatal disease with a strong genetic contribution. The dysfunction of vascular smooth muscle cells (VSMCs) contributes to the formation of this aneurysm. Although previous studies suggested that long non-coding ribonucleic acid (RNA) hypoxia inducible factor 1 α-antisense RNA 1 (HIF1A-AS1) exerted a vital role in the progression and pathogenesis of thoracic aortic aneurysm, we managed to find a new regulatory mechanism of HIF1A-AS1 in VSMCs via transcriptomics. Methods: Cell viability was detected by the cell counting kit-8 assay. Cell apoptosis was assessed by Annexin V-fluorescein isothiocyanate/propidium iodide double staining. Transwell migration assay and wound healing assay were performed to check the migration ability of HIF1A-AS1 on VSMCs. The NextSeq XTen system (Illumina) was used to collect RNA sequencing data. Lastly, reverse transcription-quantitative polymerase chain reaction confirmed the veracity and reliability of RNA-sequencing results. Results: We observed that overexpressing HIF1A-AS1 successfully promoted apoptosis, significantly altered cell cycle distribution, and greatly attenuated migration in VSMCs, further highlighting the robust promoting effects of HIF1A-AS1 to thoracic aortic aneurysm. Moreover, transcriptomics was implemented to uncover its underlying mechanism. A total of 175 differently expressed genes were identified, with some of them enriched in apoptosis, migration, and cell cycle-related pathways. Intriguingly, some differently expressed genes were noted in vascular development or coagulation function pathways. Conclusion: We suggest that HIF1A-AS1 mediated the progression of thoracic aortic aneurysm by not only regulating the function of VSMCs, but also altering vascular development or coagulation function.

Transcriptome analysis of mild hypothermia protection against radiation / 中南大学学报(医学版)

OBJECTIVES@#To analyze the differentially expressed genes (DEGs) with radiation-induced rat lung injury, and to reveal the protective mechanism for mild hypothermia in the radiation-induced lung injury in rats at the transcriptome level.@*METHODS@#A total of 10 male SD rats aged 6-8 weeks were randomly divided into 2 groups to establish a rat model of radiation-induced lung injury, and one group was treated with mild hypothermia. RNA was extracted from left lung tissue of each group, and sequenced by BGISEQ-500 platform. Significance analysis of DEGs was carried out by edgeR software. Gene ontology (GO) function enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis were used to analyze the gene function. Then 5 key DEGs were verified by real-time reverse transcription PCR (real-time RT-PCR).@*RESULTS@#There were 2 790 DEGs (false discovery rate<0.001, |log@*CONCLUSIONS@#The DEGs and pathways related to mild hypothermia protection against radiation-induced lung injury in rats are obtained, which provides an experimental basis for the protection of mild hypothermia against radiation-induced lung injury.

Mutations in SARS-CoV-2 viral RNA identified in Eastern India: Possible implications for the ongoing outbreak in India and impact on viral structure and host susceptibility

Direct massively parallel sequencing of SARS-CoV-2 genome was undertaken from nasopharyngeal andoropharyngeal swab samples of infected individuals in Eastern India. Seven of the isolates belonged to the A2aclade, while one belonged to the B4 clade. Specific mutations, characteristic of the A2a clade, were alsodetected, which included the P323L in RNA-dependent RNA polymerase and D614G in the Spike glycoprotein. Further, our data revealed emergence of novel subclones harbouring nonsynonymous mutations, viz.G1124V in Spike (S) protein, R203K, and G204R in the nucleocapsid (N) protein. The N protein mutationsreside in the SR-rich region involved in viral capsid formation and the S protein mutation is in the S2 domain,which is involved in triggering viral fusion with the host cell membrane. Interesting correlation was observedbetween these mutations and travel or contact history of COVID-19 positive cases. Consequent alterations ofmiRNA binding and structure were also predicted for these mutations. More importantly, the possibleimplications of mutation D614G (in SD domain) and G1124V (in S2 subunit) on the structural stability of Sprotein have also been discussed. Results report for the first time a bird’s eye view on the accumulation ofmutations in SARS-CoV-2 genome in Eastern India.

Expression of immune-related genes in peripheral blood leukocytes of postmenopausal osteoporosis patients / 中国组织工程研究

BACKGROUND: Bones are currently considered as an immune organ. A variety of immune cells that originate from the bone marrow can interact with the cells of the skeletal system to jointly regulate bone metabolism. Explorations on the pathogenesis of postmenopausal osteoporosis as well as treatment-related molecular targets and signal pathways can help prevention and treatment of the disease. OBJECTIVE: To investigate the expression profiles of immune-related genes in peripheral blood leukocytes of postmenopausal osteoporosis patients using RNA-Seq technology. METHODS: Forty female patients who had experienced menopause for 0 to 20 years and were hospitalized due to fractures were enrolled. They were divided into normal bone mass group (T >-1) and osteoporosis group (T 2), and 131 genes were up-regulated and 56 genes were down-regulated. We identified in total 29 differentially expressed immune-related genes including 25 up-regulated and 4 down-regulated ones. There was significant difference in expression between the osteoporosis and normal bone mass groups for genes, including KIR3DL1, KIR3DL2, KIR2DL4, KLRD1 and HSPA6 (P < 0.05). These differentially expressed genes are potentially important for the natural killer cell-mediated cytotoxicity by the KEGG pathway analysis. KIR3DL1, KIR3DL2, KIR2DL4, KLRD1 and HSPA6 may be closely related to the natural killer cell-mediated cytotoxicity during the occurrence of postmenopausal osteoporosis.

Circular RNA expression pattern and competing endogenous RNA network involved in rotator cuff tendinopathy / 中国修复重建外科杂志

Objective: To detect the differentially expressed circular RNA (circRNA) in rotator cuff tendinopathy and analyze the potential molecular mechanism of these parental genes. Methods: Ten supraspinatus tendons donated from patients who underwent tendon repair surgery between June 2018 and June 2019 were used for RNA-sequence. All rotator cuff tendinopathy and normal tendon samples were confirmed by MRI, histological staining, and observation by arthroscopy. All pathological tendons were matched with tendon samples for patients' age, gender, body mass index, and Bonar score. The bioinformatic analysis was performed based on the differentially expressed circRNA and their parental genes, including gene ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and competing endogenous RNA (ceRNA) network construction. Results: There were 94 differentially expressed circRNAs, including 31 up-regulated and 63 down-regulated, detected between the rotator cuff tendinopathy and normal tendon samples with |log2 fold change (FC)| >2, P<0.05. GO analysis showed that the genes were mostly enriched in response to cyclic adenosine monophosphate (cAMP). KEGG pathway analysis showed that the most genes were enriched in extracellular matrix-receptor interaction, protein digestion and absorption, cell cycle, and nuclear factor κB signaling pathway. ceRNA networks showed the interactions among circRNAs, mRNAs, and miRNAs. And circRNA.8951-has-miR-6089-DNMT3B was the most sum max energy. Conclusion: This bioinformatic study reveals several potential therapeutic targets for rotator cuff tendinopathy, which paves the way to better treatment and prevention of this disorder.

RNA-sequencing Profiles of Cell Cycle–Related Genes Upregulated during the G2-Phase in Giardia lamblia

To identify the component(s) involved in cell cycle control in the protozoan Giardia lamblia, cells arrested at the G1/S- or G2-phase by treatment with nocodazole and aphidicolin were prepared from the synchronized cell cultures. RNA-sequencing analysis of the 2 stages of Giardia cell cycle identified several cell cycle genes that were up-regulated at the G2-phase. Transcriptome analysis of cells in 2 distinct cell cycle stages of G. lamblia confirmed previously reported components of cell cycle (PcnA, cyclin B, and CDK) and identified additional cell cycle components (NEKs, Mad2, spindle pole protein, and CDC14A). This result indicates that the cell cycle machinery operates in this protozoan, one of the earliest diverging eukaryotic lineages.

Prediction of itching diagnostic marker through RNA sequencing of contact hypersensitivity and skin scratching stimulation mice models

Pruritus (itching) is classically defined as an unpleasant cutaneous sensation that leads to scratching behavior. Although the scientific criteria of classification for pruritic diseases are not clear, it can be divided as acute or chronic by duration of symptoms. In this study, we investigated whether skin injury caused by chemical (contact hypersensitivity, CHS) or physical (skin-scratching stimulation, SSS) stimuli causes initial pruritus and analyzed gene expression profiles systemically to determine how changes in skin gene expression in the affected area are related to itching. In both CHS and SSS, we ranked the Gene Ontology Biological Process terms that are generally associated with changes. The factors associated with upregulation were keratinization, inflammatory response and neutrophil chemotaxis. The Kyoto Encyclopedia of Genes and Genomes pathway shows the difference of immune system, cell growth and death, signaling molecules and interactions, and signal transduction pathways. Il1a , Il1b and Il22 were upregulated in the CHS, and Tnf, Tnfrsf1b, Il1b, Il1r1 and Il6 were upregulated in the SSS. Trpc1 channel genes were observed in representative itching-related candidate genes. By comparing and analyzing RNA-sequencing data obtained from the skin tissue of each animal model in these characteristic stages, it is possible to find useful diagnostic markers for the treatment of itching, to diagnose itching causes and to apply customized treatment.

The effects of painful procedures during neonatal period on rats' hippocampal gene expression profiles in later life / 中国新生儿科杂志

Objective To study the effects of painful procedures during neonatal period on rats' hippocampal gene expression in later life.Method A total of 28 newborn male rats were randomly assigned into two equal groups.From day1 (P1) to day7(P7) after birth,rats in the pain group received four times of needle acupuncture at the foot everyday,simulating the infants' experience in the NICU.Rats in control group were touched at the same side of the foot with a cotton swab.Transcriptome sequencing of the hippocampus of the two groups were examined on day 8 (P8) and day 21 (P21).The gene expression profiles were established and the differentially expressed genes were screened for functional analysis.Quantitative Real-time polymerase chain reaction (qRT-PCR) was used to confirm these differentially expressed genes (GABRB1,GRIN2A,IL1 RAPL1) related to pain-stimulated response or brain cognition,and one of the key genes was further verified using Western blotting.Result The sequencing results showed that there were only 6 differentially expressed genes in hippocampal tissues of rats on P8 in the two groups.Howerer,the number increased to 53 on P21 and 85.0％ of these genes were down-regulated (45/53).Gene Ontology (GO) function enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis showed that the differentially expressed genes were mainly expressed on the cell membrane,voltage-gated ion channels,synapses,neurotransmitter receptors,immune responses,etc.The qRT-PCR and Western-blot results of key genes were consistent with the transcriptome sequencing.Conclusion Pain stimuli at an early stage after birth may trigger differentially expression of voltage-gated ion channel proteins,neurotransmitter receptors,and some key genes such as GABRB1 on hippocampal synaptic cell membranes in rats.These phenomenon may provide initial explanation for the molecular mechanism of early pain stimuli on neonatal brain development.

Genetic Profiles Associated with Chemoresistance in Patient-Derived Xenograft Models of Ovarian Cancer / Journal of the Korean Cancer Association, 대한암학회지

PURPOSE: Recurrence and chemoresistance (CR) are the leading causes of death in patients with high-grade serous carcinoma (HGSC) of the ovary. The aim of this study was to identify genetic changes associated with CR mechanisms using a patient-derived xenograft (PDX) mouse model and genetic sequencing. MATERIALS AND METHODS: To generate a CR HGSC PDX tumor, mice bearing subcutaneously implanted HGSC PDX tumors were treated with paclitaxel and carboplatin. We compared gene expression and mutations between chemosensitive (CS) and CR PDX tumors with whole exome and RNA sequencing and selected candidate genes. Correlations between candidate gene expression and clinicopathological variables were explored using the Cancer Genome Atlas (TCGA) database and the Human Protein Atlas (THPA). RESULTS: Three CR and four CS HGSC PDX tumor models were successfully established. RNA sequencing analysis of the PDX tumors revealed that 146 genes were significantly up-regulated and 54 genes down-regulated in the CR group compared with the CS group. Whole exome sequencing analysis showed 39 mutation sites were identified which only occurred in CR group. Differential expression of SAP25,HLA-DPA1, AKT3, and PIK3R5 genes and mutation of TMEM205 and POLR2A may have important functions in the progression of ovarian cancer chemoresistance. According to TCGA data analysis, patients with high HLA-DPA1 expression were more resistant to initial chemotherapy (p=0.030; odds ratio, 1.845). CONCLUSION: We successfully established CR ovarian cancer PDX mouse models. PDX-based genetic profiling study could be used to select some candidate genes that could be targeted to overcome chemoresistance of ovarian cancer.

Identification of Epithelial-Mesenchymal Transition-related Target Genes Induced by the Mutation of Smad3 Linker Phosphorylation

BACKGROUND: Smad3 linker phosphorylation plays essential roles in tumor progression and metastasis. We have previously reported that the mutation of Smad3 linker phosphorylation sites (Smad3-Erk/Pro-directed kinase site mutant constructs [EPSM]) markedly reduced the tumor progression while increasing the lung metastasis in breast cancer. METHODS: We performed high-throughput RNA-Sequencing of the human prostate cancer cell lines infected with adenoviral Smad3-EPSM to identify the genes regulated by Smad3-EPSM. RESULTS: In this study, we identified genes which are differentially regulated in the presence of Smad3-EPSM. We first confirmed that Smad3-EPSM strongly enhanced a capability of cell motility and invasiveness as well as the expression of epithelial-mesenchymal transition marker genes, CDH2, SNAI1, and ZEB1 in response to TGF-β1 in human pancreatic and prostate cancer cell lines. We identified GADD45B, CTGF, and JUNB genes in the expression profiles associated with cell motility and invasiveness induced by the Smad3-EPSM. CONCLUSIONS: These results suggested that inhibition of Smad3 linker phosphorylation may enhance cell motility and invasiveness by inducing expression of GADD45B, CTGF, and JUNB genes in various cancers.

GEE: An Informatics Tool for Gene Expression Data Explore / 대한의료정보학회지

OBJECTIVES: Major public high-throughput functional genomic data repositories, including the Gene Expression Omnibus (GEO) and ArrayExpress have rapidly expanded. As a result, a large number of diverse high-throughput functional genomic data retrieval systems have been developed. However, high-throughput functional genomic data retrieval remains challenging. METHODS: We developed Gene Expression data Explore (GEE), the first powerful, flexible web and mobile search application for searching whole-genome epigenetic data and microarray data in public databases, such as GEO and ArrayExpress. RESULTS: GEE provides an elaborate, convenient interface of query generation competences not available via various high-throughput functional genomic data retrieval systems, including GEO, ArrayExpress, and Atlas. In particular, GEE provides a suitable query generator using eVOC, the Experimental Factor Ontology (EFO), which is well represented with a variety of high-throughput functional genomic data experimental conditions. In addition, GEE provides an experimental design query constructor (EDQC), which provides elaborate retrieval filter conditions when the user designs real experiments. CONCLUSIONS: The web version of GEE is available at http://www.snubi.org/software/gee, and its app version is available from the Apple App Store.

Characterization of sphere-forming HCT116 clones by whole RNA sequencing

PURPOSE: To determine CD133+ cells defined as cancer stem cells (CSCs) in colon cancer, we examined whether CD133+ clones in HCT116 demonstrate known features of CSCs like sphere-forming ability, chemodrug-resistance, and metastatic potential. METHODS: Magnetic cell isolation and cell separation demonstrated that <1% of HCT116 cells expressed CD133, with the remaining cells being CD133- clones. In colon cancer cells, radioresistance is also considered a CSC characteristic. We performed clonogenic assay using 0.4 Gy γ-irradiation. RESULTS: Interestingly, there were no differences between HCT116 parental and HCT116 CD133+ clones when the cells comprised 0.5% of the total cells, and CD133- clone demonstrated radiosensitive changes compared with parental and CD133+ clones. Comparing gene expression profiles between sphere-forming and nonforming culture conditions of HCT116 subclones by whole RNA sequencing failed to obtain specific genes expressed in CD133+ clones. CONCLUSION: Despite no differences of gene expression profiles in monolayer attached culture conditions of each clone, sphere-forming conditions of whole HCT116 subclones, parental, CD133+, and CD133- increased 1,761 coding genes and downregulated 1,384 genes related to CSCs self-renewal and survival. Thus, spheroid cultures of HCT116 cells could be useful to expand colorectal CSCs rather than clonal expansion depending on CD133 expressions.

Zika virus: Genome sequence analysis and nucleic acid detection methods / 第二军医大学学报

Objective To analyze the genome sequence characteristics of Zika virus and to develop nucleic acid detection methods for Zika virus. Methods The phylogenetic tree of 81 kinds of Flavivirus was constructed. The differences of nucleotide and amino acid sequence among Zika virus, type 4 dengue and Japanese encephalitis virus (JEV) were analyzed and compared. The gene mutated sites of Asian and African Zika virus, especially four Zika virus strains from China, were analyzed. A set of primers and probes of real-time quantitative PCR for Zika virus were designed after comparing the genome sequences of Asian and African Zika virus. Results Spondweni and Kedougou viruses were the closest homologously to Zika virus among 81 kinds of Flavivirus. Comparison of full genomic nucleic acid sequence showed that Zika virus was closer to type 4 dengue virus than JEV, whereas comparison of amino acid yielded an opposite result. Compared with traditional Asian type Zika virus, Guangdong GD01 strains had 5 amino acid mutated sites, Zhejiang ZJ03 strains had 6 mutated sites, and VE Ganxian strains had 33 mutated sites. Detection of designed PCR primers and probes for plasmid RNA was positive, with the lower limit of detection being 100 copies/mL and Zika virus RNA was detected to be positive, type 1-4 dengue virus and Japanese encephalitis virus being negative. Conclusion Zika virus and Spondweni virus are the closest homologously. The high mutation character of VE Ganxian strains indicates that Zika might evolve fast. PCR primers and probes designed in this paper can be used for Asian and African type Zika virus detection, with relatively higher sensitivity and specificity.

Haemophilus parainfluenzae Infective Endocarditis Diagnosed by Direct 16S rRNA Sequencing of Vegetation

The HACEK group of microorganisms is responsible for approximately 3-6% of endocarditis cases and is a major cause of culture-negative endocarditis. Here, we report a case of Haemophilus parainfluenzae infective endocarditis that was diagnosed by direct PCR sequencing of 16S rRNA from resected vegetation. A healthy 26-yr-old man was admitted to the emergency room (ER) on March 27, 2011 because of intermittent high fever. The patient was prescribed cefpodoxime for 5 days at the ER. Six and 11 sets of blood cultures were performed at the ER and in a general ward, respectively, using BACTEC Plus Aerobic/F (Becton-Dickinson, USA) and Lytic Anaerobic/F Plus (BD) together. Echocardiography revealed a large vegetation at the posterior mitral valve leaflet. After performing mitral valvoplasty on hospital day (HD) 11, the vegetation tissue was cultured in thioglycolate broth, blood agar, Brucella agar, and MacConkey agar for 7 days, but no organism was grown. Direct PCR sequencing of 16S rRNA of the tissue revealed the presence of H. parainfluenzae. In the 17 sets of blood cultures, bacterial growth was detected in only 2 aerobic bottles of 5 sets taken at HD 9 after 10-day and 14-day incubation. The organism was identified as H. parainfluenzae by using the VITEK NHI card (bioMerieux, France). Direct PCR sequencing of vegetation could be useful in diagnosing bacterial pathogens in infective endocarditis patients, especially in culture-negative cases.