scholarly journals Exogenous GbHMGS1 Overexpression Improves the Contents of Three Terpenoids in Transgenic Populus

Forests ◽  
2021 ◽  
Vol 12 (5) ◽  
pp. 595
Author(s):  
Ya-Qiong Wu ◽  
Tong-Li Wang ◽  
Yue Xin ◽  
Shu-Jing Huang ◽  
Gui-Bin Wang ◽  
...  

Ginkgo biloba L. has attracted much attention due to its medicinal properties, particularly those of its terpenoid and flavonoid contents. However, the content and utilization efficiency of terpenoids remain low. The enzyme 3-hydroxy-3-methylglutaryl CoA synthase (HMGS) is a major rate-limiting factor, and RNA-seq has revealed that the mRNA expression of this enzyme is differentially expressed during terpenoid biosynthesis. Here, we investigated the function of the GbHMGS1 gene and its overexpression in Populus. We compared the metabolite contents of nontransgenic (CK) Populus with those of transgenic Populus lines through metabolomics analysis. Our results indicate that the GbHMGS1 protein is localized in the cytoplasm. Significant differences in chemical characteristics were found between the transgenic and CK plants, and a total of 31 differentially expressed metabolites were upregulated in the transgenic plants. We also found higher contents of lanosterol (triterpenoid), dehydroabietic acid (diterpenoid), and phytol (diterpenoid) in the transgenic Populus plants than in their CK counterparts. We thus speculate that GbHMGS1 might regulate plant-related product formation and increase metabolite contents. This study revealed the molecular mechanism governing metabolite synthesis and suggested that one triterpenoid and two diterpenoids with significant upregulation can be used as markers for the breeding of plants with specific terpenoid metabolism-related characteristics.

Plants ◽  
2020 ◽  
Vol 9 (7) ◽  
pp. 891 ◽  
Author(s):  
Mobina Ulfat ◽  
Habib-ur-Rehman Athar ◽  
Zaheerud-din Khan ◽  
Hazem M. Kalaji

Salinity is one of the major abiotic stresses prevailing throughout the world that severely limits crop establishment and production. Every crop has an intra-specific genetic variation that enables it to cope with variable environmental conditions. Hence, this genetic variability is a good tool to exploit germplasms in salt-affected areas. Further, the selected cultivars can be effectively used by plant breeders and molecular biologists for the improvement of salinity tolerance. In the present study, it was planned to identify differential expression of genes associated with selective uptake of different ions under salt stress in selected salt-tolerant canola (Brassica napus L.) cultivar. For the purpose, an experiment was carried out to evaluate the growth response of different salt-sensitive and salt-tolerant canola cultivars. Plants were subjected to 200 mM NaCl stress. Canola cultivars—Faisal Canola, DGL, Dunkled, and CON-II—had higher growth than in cvs Cyclone, Ac-EXcel, Legend, and Oscar. Salt-tolerant cultivars were better able to maintain plant water status probably through osmotic adjustment as compared to salt-sensitive cultivars. Although salt stress increased shoot Na+ and shoot Cl− contents in all canola cultivars, salt-tolerant cultivars had a lower accumulation of these toxic nutrients. Similarly, salt stress reduced shoot K+ and Ca2+ contents in all canola cultivars, while salt-tolerant cultivars had a higher accumulation of K+ and Ca2+ in leaves, thereby having greater shoot K+/Na+ and Ca2+/Na+ ratios. Nutrient utilization efficiency decreased significantly in all canola cultivars due to the imposition of salt stress; however, it was greater in salt-tolerant cultivars—Faisal Canola, DGL, and Dunkled. Among four salt-tolerant canola cultivars, cv Dunkled was maximal in physiological attributes, and thus differentially expressed genes (DEGs) were assessed in it by RNA-seq analysis using next-generation sequencing (NGS) techniques. The differentially expressed genes (DEG) in cv Dunkled under salt stress were found to be involved in the regulation of ionic concentration, photosynthesis, antioxidants, and hormonal metabolism. However, the most prominent upregulated DEGs included Na/K transporter, HKT1, potassium transporter, potassium channel, chloride channel, cation exchanger, Ca channel. The RNA-seq data were validated through qRT-PCR. It was thus concluded that genes related to the regulation of ionic concentrate are significantly upregulated and expressed under salt stress, in the cultivar Dunkled.


2021 ◽  
Vol 80 (Suppl 1) ◽  
pp. 12.2-12
Author(s):  
I. Muller ◽  
M. Verhoeven ◽  
H. Gosselt ◽  
M. Lin ◽  
T. De Jong ◽  
...  

Background:Tocilizumab (TCZ) is a monoclonal antibody that binds to the interleukin 6 receptor (IL-6R), inhibiting IL-6R signal transduction to downstream inflammatory mediators. TCZ has shown to be effective as monotherapy in early rheumatoid arthritis (RA) patients (1). However, approximately one third of patients inadequately respond to therapy and the biological mechanisms underlying lack of efficacy for TCZ remain elusive (1). Here we report gene expression differences, in both whole blood and peripheral blood mononuclear cells (PBMC) RNA samples between early RA patients, categorized by clinical TCZ response (reaching DAS28 < 3.2 at 6 months). These findings could lead to identification of predictive biomarkers for TCZ response and improve RA treatment strategies.Objectives:To identify potential baseline gene expression markers for TCZ response in early RA patients using an RNA-sequencing approach.Methods:Two cohorts of RA patients were included and blood was collected at baseline, before initiating TCZ treatment (8 mg/kg every 4 weeks, intravenously). DAS28-ESR scores were calculated at baseline and clinical response to TCZ was defined as DAS28 < 3.2 at 6 months of treatment. In the first cohort (n=21 patients, previously treated with DMARDs), RNA-sequencing (RNA-seq) was performed on baseline whole blood PAXgene RNA (Illumina TruSeq mRNA Stranded) and differential gene expression (DGE) profiles were measured between responders (n=14) and non-responders (n=7). For external replication, in a second cohort (n=95 therapy-naïve patients receiving TCZ monotherapy), RNA-seq was conducted on baseline PBMC RNA (SMARTer Stranded Total RNA-Seq Kit, Takara Bio) from the 2-year, multicenter, double-blind, placebo-controlled, randomized U-Act-Early trial (ClinicalTrials.gov identifier: NCT01034137) and DGE was analyzed between 84 responders and 11 non-responders.Results:Whole blood DGE analysis showed two significantly higher expressed genes in TCZ non-responders (False Discovery Rate, FDR < 0.05): urotensin 2 (UTS2) and caveolin-1 (CAV1). Subsequent analysis of U-Act-Early PBMC DGE showed nine differentially expressed genes (FDR < 0.05) of which expression in clinical TCZ non-responders was significantly higher for eight genes (MTCOP12, ZNF774, UTS2, SLC4A1, FECH, IFIT1B, AHSP, and SPTB) and significantly lower for one gene (TND2P28M). Both analyses were corrected for baseline DAS28-ESR, age and gender. Expression of UTS2, with a proposed function in regulatory T-cells (2), was significantly higher in TCZ non-responders in both cohorts. Furthermore, gene ontology enrichment analysis revealed no distinct gene ontology or IL-6 related pathway(s) that were significantly different between TCZ-responders and non-responders.Conclusion:Several genes are differentially expressed at baseline between responders and non-responders to TCZ therapy at 6 months. Most notably, UTS2 expression is significantly higher in TCZ non-responders in both whole blood as well as PBMC cohorts. UTS2 could be a promising target for further analyses as a potential predictive biomarker for TCZ response in RA patients in combination with clinical parameters (3).References:[1]Bijlsma JWJ, Welsing PMJ, Woodworth TG, et al. Early rheumatoid arthritis treated with tocilizumab, methotrexate, or their combination (U-Act-Early): a multicentre, randomised, double-blind, double-dummy, strategy trial. Lancet. 2016;388(10042):343-55.[2]Bhairavabhotla R, Kim YC, Glass DD, et al. Transcriptome profiling of human FoxP3+ regulatory T cells. Human Immunology. 2016;77(2):201-13.[3]Gosselt HR, Verhoeven MMA, Bulatovic-Calasan M, et al. Complex machine-learning algorithms and multivariable logistic regression on par in the prediction of insufficient clinical response to methotrexate in rheumatoid arthritis. Journal of Personalized Medicine. 2021;11(1).Disclosure of Interests:None declared


2020 ◽  
Vol 79 (Suppl 1) ◽  
pp. 178-179
Author(s):  
S. Alehashemi ◽  
M. Garg ◽  
B. Sellers ◽  
A. De Jesus ◽  
A. Biancotto ◽  
...  

Background:Systemic Autoinflammatory diseases present with sterile inflammation. NOMID (Neonatal-Onset Multisystem Inflammatory Disease) is caused by gain-of-function mutations inNLRP3and excess IL-1 production, presents with fever, neutrophilic dermatosis, aseptic meningitis, hearing loss and eye inflammation; CANDLE (Chronic Atypical Neutrophilic Dermatosis, Lipodystrophy and Elevated Temperature) is caused by loss-of-function mutations in proteasome genes that lead to type-1 interferon signaling, characterized by fever, panniculitis, lipodystrophy, cytopenia, systemic and pulmonary hypertension and basal ganglia calcification. IL-1 blockers are approved for NOMID and JAK-inhibitors show efficacy in CANDLE treatment.Objectives:We used proteomic analysis to compare differentially expressed proteins in active NOMID and CANDLE compared to healthy controls before and after treatment, and whole blood bulk RNA seq to identify the immune cell signatures.Methods:Serum samples from active NOMID (n=12) and CANDLE (n=7) before and after treatment (table 1) and age matched healthy controls (HC) (n=7) were profiled using the SomaLogic platform (n=1125 proteins). Differentially expressed proteins in NOMID and CANDLE were ranked after non-parametric tests for unpaired (NOMIDp<0.05, CANDLE,p<0.1) and paired (p<0.05) analysis and assessed by enriched Gene Ontology pathways and network visualization. Whole blood RNA seq was performed (NOMID=7, CANDLE=7, Controls =5) and RPKM values were used to assess immune cells signatures.Table 1.Patient’s characteristicsNOMIDN=12, Male =6CANDLEN=7, Male =6AgeMedian (range)12 (2, 28)16 (3, 20)Ethnicity%White (Hispanic)80 (20)100 (30)GeneticsNLRP3mutation(2 Somatic, 10 Germline)mutations in proteasome component genes(1 digenic, 6 Homozygous/compound Heterozygous)Before treatmentAfter treatmentBefore treatmentAfter treatmentCRPMedian (range) mg/L52 (16-110)5 (0-23)5 (0-101)1 (0-4)IFN scoremedian (range)0NA328 (211-1135)3 (0-548)Results:Compared to control, 205 proteins (127 upregulated, 78 downregulated) were significantly different at baseline in NOMID, compared to 163 proteins (101 upregulated, and 62 downregulated) in CANDLE. 134 dysregulated proteins (85 upregulated, 49 downregulated) overlapped in NOMID and CANDLE (Figure 1). Pathway analysis identified neutrophil and monocyte chemotaxis signature in both NOMID and CANDLE. NOMID patients had neutrophilia and active neutrophils. CANDLE patients exhibited active neutrophils in whole blood RNA. Endothelial cell activation was the most prominent non-hematopoietic signature and suggest distinct endothelial cell dysregulation in NOMID and CANDLE. In NOMID, the signature included neutrophil transmigration (SELE) endothelial cell motility in response to angiogenesis (HGF, VEGF), while in CANDLE the endothelial signatures included extracellular matrix protein deposition (COL8A) suggesting increased vascular stiffness. CANDLE patients had higher expression of Renin, 4 out of 7 had hypertension, NOMID patients did not have hypertension. Treatment with anakinra and baricitinib normalized 143 and 142 of dysregulated proteins in NOMID and CANDLE respectively.Conclusion:Differentially expressed proteins in NOMID and CANDLE are consistent with innate immune cell activation. Distinct endothelial cell signatures in NOMID and CANDLE may provide mechanistic insight into differences in vascular phenotypes. Treatment with anakinra and Baricitinib in NOMID and CANDLE leaves 30% and 13% of the dysregulated proteins unchanged.Acknowledgments:This work was supported by Intramural Research atNational Institute of Allergy Immunology and Infectious Diseases of National Institutes of Health, Bethesda, Maryland, the Center of Human Immunology and was approved by the IRB.Disclosure of Interests:None declared


2021 ◽  
Vol 15 (1) ◽  
Author(s):  
Weitong Cui ◽  
Huaru Xue ◽  
Lei Wei ◽  
Jinghua Jin ◽  
Xuewen Tian ◽  
...  

Abstract Background RNA sequencing (RNA-Seq) has been widely applied in oncology for monitoring transcriptome changes. However, the emerging problem that high variation of gene expression levels caused by tumor heterogeneity may affect the reproducibility of differential expression (DE) results has rarely been studied. Here, we investigated the reproducibility of DE results for any given number of biological replicates between 3 and 24 and explored why a great many differentially expressed genes (DEGs) were not reproducible. Results Our findings demonstrate that poor reproducibility of DE results exists not only for small sample sizes, but also for relatively large sample sizes. Quite a few of the DEGs detected are specific to the samples in use, rather than genuinely differentially expressed under different conditions. Poor reproducibility of DE results is mainly caused by high variation of gene expression levels for the same gene in different samples. Even though biological variation may account for much of the high variation of gene expression levels, the effect of outlier count data also needs to be treated seriously, as outlier data severely interfere with DE analysis. Conclusions High heterogeneity exists not only in tumor tissue samples of each cancer type studied, but also in normal samples. High heterogeneity leads to poor reproducibility of DEGs, undermining generalization of differential expression results. Therefore, it is necessary to use large sample sizes (at least 10 if possible) in RNA-Seq experimental designs to reduce the impact of biological variability and DE results should be interpreted cautiously unless soundly validated.


Animals ◽  
2021 ◽  
Vol 11 (5) ◽  
pp. 1268
Author(s):  
Shengchao Zhang ◽  
Sibtain Ahmad ◽  
Yuxia Zhang ◽  
Guohua Hua ◽  
Jianming Yi

Enhanced plane of nutrition at pre-weaning stage can promote the development of mammary gland especially heifer calves. Although several genes are involved in this process, long intergenic non-coding RNAs (lincRNAs) are regarded as key regulators in the regulated network and are still largely unknown. We identified and characterized 534 putative lincRNAs based on the published RNA-seq data, including heifer calves in two groups: fed enhanced milk replacer (EH, 1.13 kg/day, including 28% crude protein, 25% fat) group and fed restricted milk replacer (R, 0.45 kg/day, including 20% crude protein, 20% fat) group. Sub-samples from the mammary parenchyma (PAR) and mammary fat pad (MFP) were harvested from heifer calves. According to the information of these lincRNAs’ quantitative trait loci (QTLs), the neighboring and co-expression genes were used to predict their function. By comparing EH vs R, 79 lincRNAs (61 upregulated, 18 downregulated) and 86 lincRNAs (54 upregulated, 32 downregulated) were differentially expressed in MFP and PAR, respectively. In MFP, some differentially expressed lincRNAs (DELs) are involved in lipid metabolism pathways, while, in PAR, among of DELs are involved in cell proliferation pathways. Taken together, this study explored the potential regulatory mechanism of lincRNAs in the mammary gland development of calves under different planes of nutrition.


Author(s):  
Mayukh Banerjee ◽  
Ana Ferragut Cardoso ◽  
Laila Al-Eryani ◽  
Jianmin Pan ◽  
Theodore S. Kalbfleisch ◽  
...  

AbstractChronic arsenic exposure causes skin cancer, although the underlying molecular mechanisms are not well defined. Altered microRNA and mRNA expression likely play a pivotal role in carcinogenesis. Changes in genome-wide differential expression of miRNA and mRNA at 3 strategic time points upon chronic sodium arsenite (As3+) exposure were investigated in a well-validated HaCaT cell line model of arsenic-induced cutaneous squamous cell carcinoma (cSCC). Quadruplicate independent HaCaT cell cultures were exposed to 0 or 100 nM As3+ for up to 28-weeks (wk). Cell growth was monitored throughout the course of exposure and epithelial-mesenchymal transition (EMT) was examined employing immunoblot. Differentially expressed miRNA and mRNA profiles were generated at 7, 19, and 28-wk by RNA-seq, followed by identification of differentially expressed mRNA targets of differentially expressed miRNAs through expression pairing at each time point. Pathway analyses were performed for total differentially expressed mRNAs and for the miRNA targeted mRNAs at each time point. RNA-seq predictions were validated by immunoblot of selected target proteins. While the As3+-exposed cells grew slower initially, growth was equal to that of unexposed cells by 19-wk (transformation initiation), and exposed cells subsequently grew faster than passage-matched unexposed cells. As3+-exposed cells had undergone EMT at 28-wk. Pathway analyses demonstrate dysregulation of carcinogenesis-related pathways and networks in a complex coordinated manner at each time point. Immunoblot data largely corroborate RNA-seq predictions in the endoplasmic reticulum stress (ER stress) pathway. This study provides a detailed molecular picture of changes occurring during the arsenic-induced transformation of human keratinocytes.


2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Tiankun Hui ◽  
Hongyang Jing ◽  
Xinsheng Lai

Abstract Background Neuromuscular junctions (NMJs) are chemical synapses formed between motor neurons and skeletal muscle fibers and are essential for controlling muscle contraction. NMJ dysfunction causes motor disorders, muscle wasting, and even breathing difficulties. Increasing evidence suggests that many NMJ disorders are closely related to alterations in specific gene products that are highly concentrated in the synaptic region of the muscle. However, many of these proteins are still undiscovered. Thus, screening for NMJ-specific proteins is essential for studying NMJ and the pathogenesis of NMJ diseases. Results In this study, synaptic regions (SRs) and nonsynaptic regions (NSRs) of diaphragm samples from newborn (P0) and adult (3-month-old) mice were used for RNA-seq. A total of 92 and 182 genes were identified as differentially expressed between the SR and NSR in newborn and adult mice, respectively. Meanwhile, a total of 1563 genes were identified as differentially expressed between the newborn SR and adult SR. Gene Ontology (GO) enrichment analyses, Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis and gene set enrichment analysis (GSEA) of the DEGs were performed. Protein–protein interaction (PPI) networks were constructed using STRING and Cytoscape. Further analysis identified some novel proteins and pathways that may be important for NMJ development, maintenance and maturation. Specifically, Sv2b, Ptgir, Gabrb3, P2rx3, Dlgap1 and Rims1 may play roles in NMJ development. Hcn1 may localize to the muscle membrane to regulate NMJ maintenance. Trim63, Fbxo32 and several Asb family proteins may regulate muscle developmental-related processes. Conclusion Here, we present a complete dataset describing the spatiotemporal transcriptome changes in synaptic genes and important synaptic pathways. The neuronal projection-related pathway, ion channel activity and neuroactive ligand-receptor interaction pathway are important for NMJ development. The myelination and voltage-gated ion channel activity pathway may be important for NMJ maintenance. These data will facilitate the understanding of the molecular mechanisms underlying the development and maintenance of NMJ and the pathogenesis of NMJ disorders.


2019 ◽  
Vol 26 (11) ◽  
pp. 1485-1492
Author(s):  
Xiaochun Yi ◽  
Jie Zhang ◽  
Huixiang Liu ◽  
Tianxia Yi ◽  
Yuhua Ou ◽  
...  

The adverse clinical result and poor treatment outcome in recurrent spontaneous abortion (RSA) make it necessary to understand the pathogenic mechanism. The mating combination CBA/J × DBA/2 has been widely used as an abortion-prone model compared to DBA/2-mated CBA/J mice. Here, we used RNA-seq to get a comprehensive catalogue of genes differentially expressed between survival placenta in abortion-prone model and control. Five hundred twenty-four differentially expressed genes were obtained followed by clustering analysis, Gene Ontology analysis, and pathway analysis. We paid more attention to immune-related genes namely “immune response” and “immune system process” including 33 downregulated genes and 28 upregulated genes. Twenty-one genes contribute to suppressing immune system and 7 are against it. Six genes were validated by reverse transcription-polymerase chain reaction, namely Ccr1l1, Tlr4, Tgf-β1, Tyro3, Gzmb, and Il-1β. Furthermore, Tlr4, Tgf-β1, and Il-1β were analyzed by Western blot. Such immune profile gives us a better understanding of the complicated immune processing in RSA and immunosuppression can rescue pregnancy loss.


Genes ◽  
2020 ◽  
Vol 11 (4) ◽  
pp. 397
Author(s):  
Dadong Deng ◽  
Xihong Tan ◽  
Kun Han ◽  
Ruimin Ren ◽  
Jianhua Cao ◽  
...  

The development of the placental fold, which increases the maternal–fetal interacting surface area, is of primary importance for the growth of the fetus throughout the whole pregnancy. However, the mechanisms involved remain to be fully elucidated. Increasing evidence has revealed that long non-coding RNAs (lncRNAs) are a new class of RNAs with regulatory functions and could be epigenetically regulated by histone modifications. In this study, 141 lncRNAs (including 73 up-regulated and 68 down-regulated lncRNAs) were identified to be differentially expressed in the placentas of pigs during the establishment and expanding stages of placental fold development. The differentially expressed lncRNAs and genes (DElncRNA-DEgene) co-expression network analysis revealed that these differentially expressed lncRNAs (DElncRNAs) were mainly enriched in pathways of cell adhesion, cytoskeleton organization, epithelial cell differentiation and angiogenesis, indicating that the DElncRNAs are related to the major events that occur during placental fold development. In addition, we integrated the RNA-seq (RNA sequencing) data with the ChIP-seq (chromatin immunoprecipitation sequencing) data of H3K4me3/H3K27ac produced from the placental samples of pigs from the two stages (gestational days 50 and 95). The analysis revealed that the changes in H3K4me3 and/or H3K27ac levels were significantly associated with the changes in the expression levels of 37 DElncRNAs. Furthermore, several H3K4me3/H3K27ac-lncRNAs were characterized to be significantly correlated with genes functionally related to placental development. Thus, this study provides new insights into understanding the mechanisms for the placental development of pigs.


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