Losartan ameliorates renal fibrosis by inhibiting tumor necrosis factor signal pathway.

Losartan is widely used in the treatment of chronic kidney disease (CKD) and has achieved good clinical efficacy, but its exact mechanism is not clear. We performed high-throughput sequencing (HTS) technology to screen the potential target of losartan in treating CKD. According to the HTS results, we found that the tumor necrosis factor (TNF) signal pathway was enriched. Therefore, we conducted in vivo and in vitro experiments to verify it. We found that TNF signal pathway was activated in both unilateral ureteral obstruction (UUO) rats and human proximal renal tubular epithelial cells (HK-2) treated with transforming growth factor-ß1 (TGF-ß1), while losartan can significantly inhibit TNF signal pathway as well as the expression of fibrosis related genes (such as COL-1, α-SMA and Vimentin). These data suggest that losartan may ameliorate renal fibrosis through modulating the TNF pathway.

Transcriptome-Wide Identification and Expression Analysis of Genes Encoding Defense-Related Peptides of Filipendula ulmaria in Response to Bipolaris sorokiniana Infection.

Peptides play an essential role in plant development and immunity. Filipendula ulmaria, belonging to the Rosaceae family, is a medicinal plant which exhibits valuable pharmacological properties. F. ulmaria extracts in vitro inhibit the growth of a variety of plant and human pathogens. The role of peptides in defense against pathogens in F. ulmaria remains unknown. The objective of this study was to explore the repertoire of antimicrobial (AMPs) and defense-related signaling peptide genes expressed by F. ulmaria in response to infection with Bipolaris sorokiniana using RNA-seq. Transcriptomes of healthy and infected plants at two time points were sequenced on the Illumina HiSeq500 platform and de novo assembled. A total of 84 peptide genes encoding novel putative AMPs and signaling peptides were predicted in F. ulmaria transcriptomes. They belong to known, as well as new, peptide families. Transcriptional profiling in response to infection disclosed complex expression patterns of peptide genes and identified both up- and down-regulated genes in each family. Among the differentially expressed genes, the vast majority were down-regulated, suggesting suppression of the immune response by the fungus. The expression of 13 peptide genes was up-regulated, indicating their possible involvement in triggering defense response. After functional studies, the encoded peptides can be used in the development of novel biofungicides and resistance inducers.

Losartan ameliorates renal fibrosis by inhibiting tumor necrosis factor signal pathway / Losartán mejora la fibrosis renal al inhibir la vía de señalización del factor de necrosis tumoral

Losartan is widely used in the treatment of chronic kidney disease (CKD) and has achieved good clinical efficacy, but its exact mechanism is not clear. We performed high-throughput sequencing (HTS) technology to screen the potential target of losartan in treating CKD. According to the HTS results, we found that the tumor necrosis factor (TNF) signal pathway was enriched. Therefore, we conducted in vivo and in vitro experiments to verify it. We found that TNF signal pathway was activated in both unilateral ureteral obstruction (UUO) rats and human proximal renal tubular epithelial cells (HK-2) treated with transforming growth factor-β1 (TGF-β1), while losartan can significantly inhibit TNF signal pathway as well as the expression of fibrosis related genes (such as COL-1, α-SMA and Vimentin). These data suggest that losartan may ameliorate renal fibrosis through modulating the TNF pathway.(AU)

El Losartán es ampliamente utilizado en el tratamiento de la enfermedad renal crónica (CKD) y ha logrado buenos resultados clínicos, pero su mecanismo exacto aún no está claro. Utilizamos la técnica de secuenciación de alto rendimiento (HTS) para detectar posibles dianas de losartán para el tratamiento de la CKD. Según los resultados de HTS, encontramos un enriquecimiento de la vía de señalización del factor de necrosis tumoral (TNF). Así, realizamos experimentos in vivo e in vitro para verificar esto. Encontramos que, tanto en ratas con obstrucción ureteral unilateral (uuo) como en células epiteliales tubulares renales proximal humanas (HK-2) tratadas con factor de crecimiento transformador β1 (TGF-β1), se activó la vía de señalización del TNF. El losartán inhibe significativamente la expresión de las vías de señalización del TNF y genes relacionados con la fibrosis, como COL-1, α-SMA y vicentin. Estos datos sugieren que el losartán puede mejorar la fibrosis renal regulando la vía del TNF.(AU)

Inorganic ions activate lineage-specific gene regulatory networks.

Inorganic biomaterials have been shown to direct cellular responses, including cell-cell and cell-matrix interactions. Notably, ions released from these inorganic biomaterials play a vital role in defining cell identity, and promoting tissue-specific functions. However, the effect of inorganic ions on cellular functions have yet to be investigated at the transcriptomic level, representing a critical knowledge gap in the development of next-generation bioactive materials. To address this gap, we investigated the impact of various inorganic ions including silver, copper, titanium, and platinum on human mesenchymal stem cells (hMSCs). Our finding showed that silver and copper induce osteogenic and chondrogenic differentiation respectively, through enrichment of lineage-specific gene expression program. In particular, silver effectively induced Wingless/Integrated (Wnt) and mitogen-activated protein kinase (MAPK) signaling, which are vital for osteogenesis. On the other hand, copper specifically stimulated Transforming growth factor beta (TGFß) signaling, while suppressing Janus kinase/signal transducers and activators of transcription (JAK-STAT) signaling, thereby promoting chondrogenesis. In contrast, platinum, and tantalum, ions didn't stimulate regenerative responses. Together, our findings highlight the potential of inorganic biomaterials in tissue regeneration strategies, which currently rely largely on growth factors and small molecule therapeutics. STATEMENT OF SIGNIFICANCE: This research emphasizes the critical role of bioactive inorganic ions in controlling lineage-specific gene expression patterns in mesenchymal stem cells, effectively modulating the transcriptome landscape and directing cell fate. The study lays the foundation for a systematic database of biomaterial candidates and their effects on cellular functions, which will ultimately streamline the translation of new biomaterials into clinical applications.

Comparison of differentially expressed genes in longissimus dorsi muscle of Diannan small ears, Wujin and landrace pigs using RNA-seq.

Introduction: Pig growth is an important economic trait that involves the co-regulation of multiple genes and related signaling pathways. High-throughput sequencing has become a powerful technology for establishing the transcriptome profiles and can be used to screen genome-wide differentially expressed genes (DEGs). In order to elucidate the molecular mechanism underlying muscle growth, this study adopted RNA sequencing (RNA-seq) to identify and compare DEGs at the genetic level in the longissimus dorsi muscle (LDM) between two indigenous Chinese pig breeds (Diannan small ears [DSE] pig and Wujin pig [WJ]) and one introduced pig breed (Landrace pig [LP]). Methods: Animals under study were from two Chinese indigenous pig breeds (DSE pig, n = 3; WJ pig, n = 3) and one introduced pig breed (LP, n = 3) were used for RNA sequencing (RNA-seq) to identify and compare the expression levels of DEGs in the LDM. Then, functional annotation, Gene Ontology (GO) enrichment analysis, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis, and Protein-Protein Interaction (PPI) network analysis were performed on these DEGs. Then, functional annotation, Gene Ontology (GO) enrichment analysis, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis, and Protein-Protein Interaction (PPI) network analysis were performed on these DEGs. Results: The results revealed that for the DSE, WJ, and LP libraries, more than 66, 65, and 71 million clean reads were generated by transcriptome sequencing, respectively. A total of 11,213 genes were identified in the LDM tissue of these pig breeds, of which 7,127 were co-expressed in the muscle tissue of the three samples. In total, 441 and 339 DEGs were identified between DSE vs. WJ and LP vs. DSE in the study, with 254, 193 up-regulated genes and 187, 193 down-regulated genes in DSE compared to WJ and LP. GO analysis and KEGG signaling pathway analysis showed that DEGs are significantly related to contractile fiber, sarcolemma, and dystrophin-associated glycoprotein complex, myofibril, sarcolemma, and myosin II complex, Glycolysis/Gluconeogenesis, Propanoate metabolism, and Pyruvate metabolism, etc. In combination with functional annotation of DEGs, key genes such as ENO3 and JUN were identified by PPI network analysis. Discussion: In conclusion, the present study revealed key genes including DES, FLNC, PSMD1, PSMD6, PSME4, PSMB4, RPL11, RPL13A, ROS23, RPS29, MYH1, MYL9, MYL12B, TPM1, TPM4, ENO3, PGK1, PKM2, GPI, and the unannotated new gene ENSSSCG00000020769 and related signaling pathways that influence the difference in muscle growth and could provide a theoretical basis for improving pig muscle growth traits in the future.

Lymph node metastasis-related lncRNA GAS6-AS1 facilitates the progression of esophageal squamous cell carcinoma.

Background: Lymph node metastasis is the main type of metastasis in esophageal squamous cell carcinoma (ESCC), especially when the primary tumor invasion depth reaches above the adventitia layer (T3 stage), the incidence of lymph node metastasis increases sharply. Abnormal expression of long non-coding RNAs (lncRNAs) has been confirmed in ESCC, but there are still many unknown connections between lncRNAs and lymph node metastasis. Methods: We used transcriptome sequencing (RNA-seq) to analyze 10 pairs of ESCC tissues with primary tumor stage T3 and their paired normal epithelium. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to further verify the sequencing results, and survival curve analysis, logistic regression analysis, and receiver operating characteristic (ROC) curve analysis were used to investigate its clinical application value. We investigated the growth and metastasis effects of lncRNA GAS6-AS1 on ESCC cell lines TE-1 and KYSE410 in vitro and in vivo. Other functional experiments included cell apoptosis and cell cycle experiments. Results: Based on our RNA-seq data, lncRNA GAS6-AS1 is highly expressed in ESCC tissues, especially in cancer tissues with lymph node metastasis. The qRT-PCR experiment analysis showed that high expression of GAS6-AS1 was related to poor tumor differentiation and tumor stage. Logistic regression analysis showed that it was an independent risk factor for lymph node metastasis, and ROC analysis validated that it could predict lymph node metastasis. Further survival analysis suggested that high expression of GAS6-AS1 was associated with patients' poor prognosis. In vitro experiments, knocking down GAS6-AS1 inhibited the growth and metastasis of ESCC cells and inhibited tumor growth in vivo. In addition, knocking down GAS6-AS1 can inhibit cell cycle and promote cell apoptosis. Conclusions: Our results revealed that lncRNA GAS6-AS1 obtained from RNA-seq can be used as an independent risk factor for ESCC lymph node metastasis and an effective biomarker to predict, and that it was related to the growth and metastasis of ESCC. It may represent a new biomarker to aid in the assessment of the lymph node metastasis of ESCC.

Transcriptome analysis revealed potential genes involved in thermogenesis in muscle tissue in cold-exposed lambs.

Cold tolerance is an important trait for sheep raised at high altitudes. Muscle tissue, comprising 30-40% of the total body mass, produces heat during cold exposure. However, little is known about the genetic mechanisms of this tissue and its role in thermogenesis in lambs. We examined genes in skeletal muscle tissue in a cold-adapted sheep breed, Altay, and a cold-intolerant sheep breed, Hu, when exposed to low air temperature. Three ewe-lambs of each breed were maintained at -5°C and three ewe-lambs of each breed were maintained at 20°C. After cold exposure for 25 days, the longissimus dorsi of each lamb was collected, and transcriptome profiles were sequenced and analyzed. The results of RNA-seq showed that the average reads among the four groups were 11.0 Gbase. The genome mapping rate averaged 88.1% and the gene mapping rate averaged 82.5%. The analysis of differentially expressed genes (DEGs) indicated that the peroxisome proliferator-activated receptors (PPAR), cAMP, and calcium signaling pathways and muscle contraction in muscle tissue were linked to thermogenesis in cold-exposed lambs. Furthermore, PCK1 (phosphoenolpyruvate carboxykinase1) increased glyceroneogenesis in cold-exposed Altay lambs, and APOC3 (apolipoprotein C3), LPL (lipoprotein lipase), and FABP4 (fatty acid binding protein 4, adipocyte) were involved in the intake and transport of free fatty acids. In Hu sheep, cAMP biosynthesis from ATP hydrolysis was regulated by ADCY10 (adenylate cyclase) and ADORA2a (adenosine A2a receptor). Skeletal muscle contraction was regulated by MYL2 (myosin light chain 2). In conclusion, cold exposure altered the expression level of genes involved in heat production in muscle tissue. Some potential mechanisms were revealed, including calcium ion transport in the calcium signaling pathway, fatty acid metabolism in the PPAR signaling pathway, and cAMP biosynthesis in the cAMP signaling pathway. This study implied that skeletal muscle plays an important role in thermoregulation in lambs.

Identification of Potential Novel Prognosis-Related Genes Through Transcriptome Sequencing, Bioinformatics Analysis, and Clinical Validation in Acute Myeloid Leukemia.

Background: Acute Myeloid Leukemia (AML) is a complex and heterogeneous hematologic malignancy. However, the function of prognosis-related signature genes in AML remains unclear. Methods: In the current study, transcriptome sequencing was performed on 15 clinical samples, differentially expressed RNAs were identified using R software. The potential interactions network was constructed by using the common genes between target genes of differentially expressed miRNAs with transcriptome sequencing results. Functional and pathway enrichment analysis was performed to identify candidate gene-mediated aberrant signaling pathways. Hub genes were identified by the cytohubba plugin in Cytoscape software, which then expanded the potential interactions regulatory module for hub genes. TCGA-LAML clinical data were used for the prognostic analysis of the hub genes in the regulatory network, and GVSA analysis was used to identify the immune signature of prognosis-related hub genes. qRT-PCR was used to verify the expression of hub genes in independent clinical samples. Results: We obtained 1,610 differentially expressed lncRNAs, 233 differentially expressed miRNAs, and 2,217 differentially expressed mRNAs from transcriptome sequencing. The potential interactions network is constructed by 12 lncRNAs, 25 miRNAs, and 692 mRNAs. Subsequently, a sub-network including 15 miRNAs as well as 12 lncRNAs was created based on the expanded regulatory modules of 25 key genes. The prognostic analysis results show that CCL5 and lncRNA UCA1 was a significant impact on the prognosis of AML. Besides, we found three potential interactions networks such as lncRNA UCA1/hsa-miR-16-5p/COL4A5, lncRNA UCA1/hsa-miR-16-5p/SPARC, and lncRNA SNORA27/hsa-miR-17-5p/CCL5 may play an important role in AML. Furthermore, the evaluation of the immune infiltration shows that CCL5 is positively correlated with various immune signatures, and lncRNA UCA1 is negatively correlated with the immune signatures. Finally, the result of qRT-PCR showed that CCL5 is down-regulated and lncRNA UCA1 is up-regulated in AML samples separately. Conclusions: In conclusion, we propose that CCL5 and lncRNA UCA1 could be recognized biomarkers for predicting survival prognosis based on constructing competing endogenous RNAs in AML, which will provide us novel insight into developing novel prognostic, diagnostic, and therapeutic for AML.

Molecular Insights into the Role of Cysteine-Rich Peptides in Induced Resistance to Fusarium oxysporum Infection in Tomato Based on Transcriptome Profiling.

Cysteine-rich peptides (CRPs) play an important role in plant physiology. However, their role in resistance induced by biogenic elicitors remains poorly understood. Using whole-genome transcriptome sequencing and our CRP search algorithm, we analyzed the repertoire of CRPs in tomato Solanum lycopersicum L. in response to Fusarium oxysporum infection and elicitors from F. sambucinum. We revealed 106 putative CRP transcripts belonging to different families of antimicrobial peptides (AMPs), signaling peptides (RALFs), and peptides with non-defense functions (Major pollen allergen of Olea europaea (Ole e 1 and 6), Maternally Expressed Gene (MEG), Epidermal Patterning Factor (EPF)), as well as pathogenesis-related proteins of families 1 and 4 (PR-1 and 4). We discovered a novel type of 10-Cys-containing hevein-like AMPs named SlHev1, which was up-regulated both by infection and elicitors. Transcript profiling showed that F. oxysporum infection and F. sambucinum elicitors changed the expression levels of different overlapping sets of CRP genes, suggesting the diversification of functions in CRP families. We showed that non-specific lipid transfer proteins (nsLTPs) and snakins mostly contribute to the response of tomato plants to the infection and the elicitors. The involvement of CRPs with non-defense function in stress reactions was also demonstrated. The results obtained shed light on the mode of action of F. sambucinum elicitors and the role of CRP families in the immune response in tomato.

A CYC/TB1-type TCP transcription factor controls spikelet meristem identity in barley.

Barley possesses a branchless, spike-shaped inflorescence where determinate spikelets attach directly to the main axis, but the developmental mechanism of spikelet identity remains largely unknown. Here we report the functional analysis of the barley gene BRANCHED AND INDETERMINATE SPIKELET 1 (BDI1), which encodes a TCP transcription factor and plays a crucial role in determining barley inflorescence architecture and spikelet development. The bdi1 mutant exhibited indeterminate spikelet meristems that continued to grow and differentiate after producing a floret meristem; some spikelet meristems at the base of the spike formed two fully developed seeds or converted to branched spikelets, producing a branched inflorescence. Map-based cloning analysis showed that this mutant has a deletion of ~600 kb on chromosome 5H containing three putative genes. Expression analysis and virus-induced gene silencing confirmed that the causative gene, BDI1, encodes a CYC/TB1-type TCP transcription factor and is highly conserved in both wild and cultivated barley. Transcriptome and regulatory network analysis demonstrated that BDI1 may integrate regulation of gene transcription cell wall modification and known trehalose-6-phosphate homeostasis to control spikelet development. Together, our findings reveal that BDI1 represents a key regulator of inflorescence architecture and meristem determinacy in cereal crop plants.

Evaluation of a custom transcriptome sequencing library construction reagent with a small amount of cell input / 上海交通大学学报（医学版）

Objective : To verify the feasibility of replacing the expensive commercial reagent SMART-Seq v4 Ultra Low Input RNA Kit (hereinafter referred to as TaKaRa reagent) with a reagent (hereinafter referred to as DIY reagent) which was made by ourselves based on the SMART (switching mechanism at 5' end of RNA template) technology. Methods ¡¤ Four 8-week-old C57BL/6 female mice were randomly di-vided into two groups. One group did not receive any treatment as a control, and the other group was intraperitoneally injected with 1 mL of 4% thioglycollate broth to induce peritoneal macrophages. After 72 hours, RNA was extracted from the peritoneal macrophages. cDNA library con-struction was performed with DIY reagent and TaKaRa reagent respectively. Finally, bioinformatics analysis was performed to compare the RNA sequencing results after use of different library construction reagents from different aspects, such as data quality, gene differential expression analysis, and KEGG (Kyoto encyclopedia of genes and genomes) pathway analysis. Results ¡¤ The results of bioinformatics analysis showed that the sample processed by the DIY reagent and TaKaRa reagent were both of good data quality, and the two reagents had comparative capabil-ity in transcripts capture. Gene coverage of the sequences both showed consistent uniformity. On top of these, the results of differential gene ex-pression analysis and gene pathway analysis were consistent. Conclusion ¡¤ Considering relatively great reduction in experimental cost for li-brary construction, the DIY reagent can replace expensive commercial reagent for library construction experiments with a small amount of cell input.

Clinical utility of RNA sequencing to resolve unusual GNE myopathy with a novel promoter deletion.

INTRODUCTION: UDP N-acetylglucosamine2-epimerase/N-acetylmannosamine-kinase (GNE) gene mutations can cause mostly autosomal-recessive myopathy with juvenile-onset known as hereditary inclusion-body myopathy (HIBM). METHODS: We describe a family of a patient showing an unusual HIBM with both vacuolar myopathy and myositis without quadriceps-sparing, hindering diagnosis. We show how genetic testing with functional assays, clinical transcriptome sequencing (RNA-seq) in particular, helped facilitate both the diagnosis and a better understanding of the genotype-phenotype relationship. RESULTS: We identified a novel 7.08 kb pathogenic deletion upstream of GNE using array comparative genomic hybridization (aCGH) and a common Val727Met variant. Using RNA-seq, we found only monoallelic (Val727Met-allele) expression, leading to ~50% GNE reduction in muscle. Importantly, α-dystroglycan is hypoglycosylated in the patient muscle, suggesting HIBM could be a "dystroglycanopathy." CONCLUSIONS: Our study shows the importance of considering aCGH for GNE-myopathies, and the potential of RNA-seq for faster, definitive molecular diagnosis of unusual myopathies. Muscle Nerve, 2019.

Analysis of sinomenine synthesis pathway based on transcriptome sequencing in Sinomenii Caulis / 中草药

Objective: In order to explore the expression of sinomenine content control genes, synthetic control sites and expression pathway. Methods: In this study, high performance liquid chromatography (HPLC) was used to determine the content of sinomenine in the roots and stems of 49 Sinomenii Caulis in six populations. Two populations with large multiple differences in sinomenine content were selected, namely Shanxi Baoji and Guizhou Zunyi. The most representative of them were selected, and their roots and stems were taken for transcriptome sequencing and named as HR/LR and HS/LS. Results: Sequencing results showed that 355 201 transcripts were obtained by splicing clean reads, including 275 491 Unigene transcripts. There were 23 562 and 37 143 differentially expressed genes in HR/LR and HS/LS, respectively. GO database analysis showed that the functions of these differentially expressed genes were significantly enriched in aspartic-type endopeptidase activity and aspartic-type peptidase activity, it is speculated that these two enzymes might be encoded. The results of KEGG enrichment explained that the differentially expressed genes were involved in carbohydrate metabolism, protein binding to cell membrane and vitamin C synthesis. The results of qRT-PCR verified the expression of upstream key genes of the isoquinoline alkaloid synthesis pathway and found that it was positively correlated with the accumulation of sinomenine. Conclusion: This study provided a preliminary understanding of the molecular mechanism that caused the difference in sinomenine content, and provided a reference for further understanding of the accumulation rules and synthesis pathways of sinomenine.

Comparative genomic hybridization and transcriptome sequencing reveal that two genes, OsI_14279 (LOC_Os03g62620) and OsI_10794 (LOC_Os03g14950) regulate the mutation in the γ-rl rice mutant.

We previously established the genetic locus of the rolled-leaf mutant, γ-rl, to chromosome 3. In this study, we performed a comparative genomic hybridization (CGH) analysis to identify the genes responsible for the γ-rl mutant phenotype. This was combined with RNA transcriptome sequencing (RNA-seq) to analyze differences in the mRNA expression in seeds 12 h after germination. Using the reference genome of the "indica type" rice from GenBank, we created a chip with 386,000 high density DNA probes designed to target chromosome 3. The genomic DNA from γ-rl and Qinghuazhan (the wild-type) was used for hybridization against the chip to compare signal differences. We uncovered 49 regions with significant differences in hybridization signals including deletions and insertions. RNA-seq analysis between γ-rl and QHZ identified 1060 differentially expressed genes, which potentially regulate numerous biological activities. Moreover, we identified 72 annotated genes in the 49 regions discovered in CGH. Among these, 44 genes showed differential expression in RNA-seq. qRT-PCR validation of the candidate genes confirmed that seven of the 44 genes showed a significant change in their expression levels. Among these, four genes [OsI_10125 (LOC_Os03g06654), OsI_14045 (LOC_Os03g62490), OsI_14279 (LOC_Os03g62620) and OsI_14326 (LOC_Os03g63250)] were down regulated and three genes [(OsI_10794 (LOC_Os03g14950), OsI_11412 (LOC_Os03g21250) and OsI_14152 (LOC_Os03g61360)] were up regulated with a fold change ≥2.0 and a P value ≤ 0.01. Finally, we constructed transgenic plants to study the in vivo functions of these genes. RNAi knock down of LOC_Os03g62620 resulted in rolled-leaf, lower seed-setting and decreased seed growth phenotypes. Transgenic plants with LOC_Os03g14950 over-expression showed dwarf plants with a shortened leaf phenotype. Our results, LOC_Os03g62620 and LOC_Os03g14950 as the essential genes responsible for creating the γ-rl mutant phenotypes suggested that these genes may play crucial roles in regulating rice leaf development and seed growth.