Aromatic essential oils extracted from fresh fruits of Litsea cubeba (Lour.) Pers., have diverse medical and economic values. The dominant components in these essential oils are monoterpenes and sesquiterpenes. Understanding the molecular mechanisms of terpenoid biosynthesis is essential for improving the yield and quality of terpenes. However, the 40 available L. cubeba nucleotide sequences in the public databases are insufficient for studying the molecular mechanisms. Thus, high-throughput transcriptome sequencing of L. cubeba is necessary to generate large quantities of transcript sequences for the purpose of gene discovery, especially terpenoid biosynthesis related genes.
Using Illumina paired-end sequencing, approximately 23.5 million high-quality reads were generated. De
novo assembly yielded 68,648 unigenes with an average length of 834 bp. A total of 38,439 (56%) unigenes were annotated for their functions, and 35,732 and 25,806 unigenes could be aligned to the GO and COG database, respectively. By searching against the Kyoto Encyclopedia of Genes and Genomes Pathway database (KEGG), 16,130 unigenes were assigned to 297 KEGG pathways, and 61 unigenes, which contained the mevalonate and 2-C-methyl-D-erythritol 4-phosphate pathways, could be related to terpenoid backbone biosynthesis. Of the 12,963 unigenes, 285 were annotated to the terpenoid pathways using the PlantCyc database. Additionally, 14 terpene synthase genes were identified from the transcriptome. The expression patterns of the 16 genes related to terpenoid biosynthesis were analyzed by RT-qPCR to explore their putative functions.
RNA sequencing was effective in identifying a large quantity of sequence information. To our knowledge, this study is the first exploration of the L. cubeba transcriptome, and the substantial amount of transcripts obtained will accelerate the understanding of the molecular mechanisms of essential oils biosynthesis. The results may help improve future genetic and genomics studies on the molecular mechanisms behind the chemical composition of essential oils in L. cubeba fruits.
Advanced button mushroom cultivars that are less sensitive to mechanical bruising are required by the mushroom industry, where automated harvesting still cannot be used for the fresh mushroom market. The genetic variation in bruising sensitivity (BS) of Agaricus bisporus was studied through an incomplete set of diallel crosses to get insight in the heritability of BS and the combining ability of the parental lines used and, in this way, to estimate their breeding value. To this end nineteen homokaryotic lines recovered from wild strains and cultivars were inter-crossed in a diallel scheme. Fifty-one successful hybrids were grown under controlled conditions, and the BS of these hybrids was assessed. BS was shown to be a trait with a very high heritability. The results also showed that brown hybrids were generally less sensitive to bruising than white hybrids. The diallel scheme allowed to estimate the general combining ability (GCA) for each homokaryotic parental line and to estimate the specific combining ability (SCA) of each hybrid. The line with the lowest GCA is seen as the most attractive donor for improving resistance to bruising. The line gave rise to hybrids sensitive to bruising having the highest GCA value. The highest negative SCA possibly indicates heterosis effects for resistance to bruising. This study provides a foundation for estimating breeding value of parental lines to further study the genetic factors underlying bruising sensitivity and other quality-related traits, and to select potential parental lines for further heterosis breeding. The approach of studying combining ability in a diallel scheme was used for the first time in button mushroom breeding.
Athetis lepigone Möschler (Lepidoptera: Noctuidae) has recently become an important insect pest of maize (Zea mays) crops in China. In order to understand the characteristics of the different developmental stages of this pest, we used Illumina short-read sequences to perform de novo transcriptome assembly and gene expression analysis for egg, larva, pupa and adult developmental stages. We obtained 10.08 Gb of raw data from Illumina sequencing and recovered 81,356 unigenes longer than 100 bp through a de novo assembly. The total sequence length reached 49.75 Mb with 858 bp of N50 and an average unigene length of 612 bp. Annotation analysis of predicted proteins indicate that 33,736 unigenes (41.47% of total unigenes) are matches to genes in the Genbank Nr database. The unigene sequences were subjected to GO, COG and KEGG functional classification. A large number of differentially expressed genes were recovered by pairwise comparison of the four developmental stages. The most dramatic differences in gene expression were found in the transitions from one stage to another stage. Some of these differentially expressed genes are related to cuticle and wing formation as well as the growth and development. We identified more than 2,500 microsatellite markers that may be used for population studies of A.
lepigone. This study lays the foundation for further research on population genetics and gene function analysis in A. lepigone.
novo transcriptome sequencing is a robust method of predicting miRNA target genes, especially samples without reference genomes. Differentially expressed miRNAs have been previously identified in hemocytes collected from healthy skin and from skin affected by skin ulceration syndrome (SUS) in Apostichopusjaponicus. Target identification for these differentially expressed miRNAs is a major challenge for this non-model organism.
To thoroughly understand the function of miRNAs, a normalized cDNA library was sequenced with the Illumina Hiseq2000 technology. A total of 91,098,474 clean reads corresponding to 251,148 unigenes, each with an average length of 494bp, were obtained. Blastx analysis against a nonredundant (nr) NCBI protein database revealed that in this set, 52,680 unigenes coded for 3,893 annotated proteins. Two digital gene expression (DGE) libraries from healthy and SUS samples showed that 4,858 of the unigenes were expressed at significantly different levels; 2,163 were significantly up-regulated, while 2,695 were significantly down-regulated. The computational prediction of miRNA targets from these differentially expressed genes identified 732 unigenes as the targets of 57 conserved and 8 putative novel miRNA families, including spu-miRNA-31 and spu-miRNA-2008.
This study demonstrates the feasibility of identifying miRNA targets by transcriptome analysis. The DGE assembly data represent a substantial increase in the genomic resources available for this species and will provide insights into the gene expression profile analysis and the miRNAs function annotations of further studies.
Hawthorn (Crataegus spp.) is an important pome with a long history as a fruit, an ornamental, and a source of medicine. Fruits of hawthorn are marked by hard stony endocarps, but a hawthorn germplasm with soft and thin endocarp was found in Liaoning province of China. To elucidate the molecular mechanism underlying the soft endocarp of hawthorn, we conducted a de novo assembly of the fruit transcriptome of Crataegus pinnatifida and compared gene expression profiles between the soft-endocarp and the hard-endocarp hawthorn varieties. De novo assembly yielded 52,673 putative unigenes, 20.4% of which are longer than 1,000 bp. Among the high-quality unique sequences, 35,979 (68.3%) had at least one significant match to an existing gene model. A total of 1,218 genes, represented 2.31% total putative unigenes, were differentially expressed between the soft-endocarp hawthorn and the hard-endocarp hawthorn. Among these differentially expressed genes, a number of lignin biosynthetic pathway genes were down-regulated while almost all the flavonoid biosynthetic pathway genes were strongly up-regulated, concomitant with the formation of soft endocarp. In addition, we have identified some MYB and NAC transcription factors that could potentially control lignin and flavonoid biosynthesis. The altered expression levels of the genes encoding lignin biosynthetic enzymes, MYB and NAC transcription factors were confirmed by quantitative RT-PCR. This is the first transcriptome analysis of Crataegus genus. The high quality ESTs generated in this study will aid future gene cloning from hawthorn. Our study provides important insights into the molecular mechanisms underlying soft endocarp formation in hawthorn.
The major histocompatibility complex (MHC) is an excellent molecular marker for the studies of evolutionary ecology and conservation genetics because it is a family of highly polymorphic genes that play a key role in vertebrate immune response. In this study, the functional genes of MHC Class II B (DAB) were isolated for the first time in a vulnerable species, the Chinese egret (Egrettaeulophotes). Using a full length DNA and cDNA produced by PCR and RACE methods, four potential MHC DAB loci were characterized in the genome of this egret and all four were expressed in liver and blood. At least four copies of the MHC gene complex were similar to two copies of the minimal essential MHC complex of chicken, but are less complex than the multiple copies expressed in passerine species. In MHC polymorphism, 19 alleles of exon 2 were isolated from 48 individuals using PCR. No stop codons or frameshift mutations were found in any of the coding regions. The signatures of positive selection detected in potential peptide-binding regions by Bayesian analysis, suggesting that all of these genes were functional. These data will provide the fundamental basis for further studies to elucidate the mechanisms and significance of MHC molecular adaptation in vulnerable Chinese egret and other ardeids.
The African lungfish, Protopterus annectens, can undergo aestivation during drought. Aestivation has three phases: induction, maintenance and arousal. The objective of this study was to examine the differential gene expression in the brain of P. annectens during the induction (6 days) and maintenance (6 months) phases of aestivation as compared with the freshwater control using suppression subtractive hybridization. During the induction phase of aestivation, the mRNA expression of prolactin (prl) and growth hormone were up-regulated in the brain of P. annectens, which indicate for the first time the possible induction role of these two hormones in aestivation. Also, the up-regulation of mRNA expression of tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein γ polypeptide and the down-regulation of phosphatidylethanolamine binding protein, suggest that there could be a reduction in biological and neuronal activities in the brain. The mRNA expression of cold inducible RNA-binding protein and glucose regulated protein 58 were also up-regulated in the brain, probably to enhance their cytoprotective effects. Furthermore, the down-regulation of prothymosin α expression suggests that there could be a suppression of transcription and cell proliferation in preparation for the maintenance phase. In general, the induction phase appeared to be characterized by reduction in glycolytic capacity and metabolic activity, suppression of protein synthesis and degradation, and an increase in defense against ammonia toxicity. In contrast, there was a down-regulation in the mRNA expression of prl in the brain of P. annectens during the maintenance phase of aestivation. In addition, there could be an increase in oxidative defense capacity, and up-regulation of transcription, translation, and glycolytic capacities in preparation for arousal. Overall, our results signify the importance of reconstruction of protein structures and regulation of energy expenditure during the induction phase, and the needs to suppress protein degradation and conserve metabolic fuel stores during the maintenance phase of aestivation.
Individuals who rapidly develop hyperthermia during heat exposure (heat-intolerant) are vulnerable to heat associated illness and injury. We recently reported that heat intolerant mice exhibit complex alterations in stress proteins in response to heat exposure. In the present study, we further explored the role of genes and molecular networks associated with heat tolerance in mice.
Heat-induced physiological and biochemical changes were assessed to determine heat tolerance levels in mice. We performed RNA and microRNA expression profiling on mouse gastrocnemius muscle tissue samples to determine novel biological pathways associated with heat tolerance.
Mice (n = 18) were assigned to heat-tolerant (TOL) and heat-intolerant (INT) groups based on peak core temperatures during heat exposures. This was followed by biochemical assessments (Hsp40, Hsp72, Hsp90 and Hsf1 protein levels). Microarray analysis identified a total of 3,081 mRNA transcripts that were significantly misregulated in INT compared to TOL mice (p<0.05). Among them, Hspa1a, Dnajb1 and Hspb7 were differentially expressed by more than two-fold under these conditions. Furthermore, we identified 61 distinct microRNA (miRNA) sequences significantly associated with TOL compared to INT mice; eight miRNAs corresponded to target sites in seven genes identified as being associated with heat tolerance pathways (Hspa1a, Dnajb1, Dnajb4, Dnajb6, Hspa2, Hspb3 and Hspb7).
The combination of mRNA and miRNA data from the skeletal muscle of adult mice following heat stress provides new insights into the pathophysiology of thermoregulatory disturbances of heat intolerance.
RING finger E3 ligases are components of the ubiquitin proteasome system (UPS) that mediate the transfer of ubiquitin to substrates. Single-subunit RING finger E3s binds the E2 ubiquitin-conjugating enzyme and contains recognition sequences for the substrate within the same polypeptide. Here we describe the characterization of a class of RING finger E3 ligases that is conserved among eukaryotes. This class encodes a RING-H2 domain related in sequence to the ATL RING-H2 domain, another class of E3 ligases, and a C2/C2 zing finger at the amino-terminus, formerly described as BZF. In viridiplantae (green algae and land plants), we designed this family as BTL for BZF ATLs. BTLs are putative orthologs of the mammalian Rabring7/BCA2 RING-H2 E3s that have expanded in angiosperms. They are found in numbers ranging from three to thirty-one, which is in contrast to the one to three members normally found in animals, fungi, and protists. Furthermore, the number of sequence LOGOs generated in angiosperms is four times greater than that in other eukaryotes. In contrast to ATLs, which show expansion by tandem duplication, tandemly duplicated BTLs are scarce. The mode of action of Rabring7/BCA2 and BTLs may be similar since both the Rabring7/BCA2 BZF and the ath|BTL4 BZF are likely to mediate the binding of ubiquitin. This study introduces valuable information on the evolution and domain structure of the Rabring7/BCA2/BTL class of E3 ligases which may be important for core eukaryotic genes.
Wax gourd is a widely used vegetable of Cucuribtaceae, and also has important medicinal and health values. However, the genomic resources of wax gourd were scarcity, and only a few nucleotide sequences could be obtained in public databases.
In this study, we examined transcriptome in wax gourd. More than 44 million of high quality reads were generated from five different tissues of wax gourd using Illumina paired-end sequencing technology. Approximately 4 Gbp data were generated, and de novo assembled into 65,059 unigenes, with an N50 of 1,132 bp. Based on sequence similarity search with known protein database, 36,070 (55.4%) showed significant similarity to known proteins in Nr database, and 24,969 (38.4%) had BLAST hits in Swiss-Prot database. Among the annotated unigenes, 14,994 of wax gourd unigenes were assigned to GO term annotation, and 23,977 were found to have COG classifications. In addition, a total of 18,713 unigenes were assigned to 281 KEGG pathways. Furthermore, 6,242 microsatellites (simple sequence repeats) were detected as potential molecular markers in wax gourd. Two hundred primer pairs for SSRs were designed for validation of the amplification and polymorphism. The result showed that 170 of the 200 primer pairs were successfully amplified and 49 (28.8%) of them exhibited polymorphisms.
Our study enriches the genomic resources of wax gourd and provides powerful information for future studies. The availability of this ample amount of information about the transcriptome and SSRs in wax gourd could serve as valuable basis for studies on the physiology, biochemistry, molecular genetics and molecular breeding of this important vegetable crop.
Beclin-1, a key regulator of autophagy. Microtubule-associated protein 1 light chain 3 (LC3), is involved in autophagsome formation during autophagy. The autophagic genes beclin-1 and LC3 paly an important role in the development and progression of tumor. This study was designed to investigate the expression of beclin-1 and LC3 and to clarify their clinical significance in hypopharyngeal squamous cell carcinoma (HSCC).
Eighty-two surgical hypopharyngeal squamous cell carcinoma specimens and fifty-four adjacent non-cancerous mucosal epithelial tissues were obtained. Beclin-1 and LC3-II expression was examined by immunohistochemistry, real-time RT-PCR and Western blotting assays. Correlations with patient clinical characteristics and overall survival were evaluated.
Beclin-1 was positively expressed in 42.7% (35/82) of HSCC specimens (adjacent non-cancerous tissues, 79.6%, 43/54; P<0.0001). Furthermore, 41.5% (34/82) of HSCC specimens exhibited high LC3 immunoreactivity (adjacent non-cancerous tissues, 74.1%, 40/54; P=0.0002). Beclin-1 and LC3-II mRNA transcript levels were significantly lower in HSCCs than in paired non-cancerous tissues (P<0.0001, P=0.0001, respectively). Similarly, western blotting assays showed that beclin-1 and LC3-II were markedly decreased in HSCCs (P=0.02, P=0.004, respectively). A positive correlation was observed between the mRNA transcript levels of beclin-1 and LC3-II in HSCCs (r=0.51, P<0.0001; 95%CI: 0.273 to 0.689). Beclin-1 and LC3 expression were significantly correlated with T categories, differentiation and lymph node metastasis. Negative beclin-1 immuoreactivity and low LC3 expression were associated with poorer overall survival in HSCC patients (P<0.0001, P=0.0145, respectively). Multivariate analysis revealed that beclin-1 was an independent prognositic factor for overall survival.
Beclin-1 and LC3-II are downregulated in HSCCs and their aberrant expression correlates with poor prognosis of HSCCs.
Fasciclin-like arabinogalactan (FLA) protein is a cell-wall-associated protein playing crucial roles in regulating plant growth and development, and it was characterized in different plants including Upland cotton (Gossypium hirsutum L.). In cDNA-AFLP analysis of 25 DPA (days post anthesis) fiber mRNA, two FLA gene-related transcripts exhibit differential expression between Sea Island cotton (G. barbadense L.) and Upland cotton. Based on the transcript-derived fragment, RACE-PCR and realtime PCR technique, GbFLA5 full-length cDNA was isolated and its expression profiles were characterized in both cotton plant tissues and secondary cell wall (SCW) fibers in this study. The 1154 bp GbFLA5 cDNA contains an ORF of 720 bp, encoding GbFLA5 protein of 239 amino acids residues in length with an estimated molecular mass of 25.41 kDa and isoelectric point of 8.63. The deduced GbFLA5 protein contains an N-terminal signal sequence, two AGP-like domains, a single fasciclin-like domain, and a GPI anchor signal sequence. Phylogenetic analysis shows that GbFLA5 protein is homologous to some known SCW-specific expressed FLAs of plant developing xylem, tension wood and cotton fibers. In the SCW deposition stage from 15 to 45 DPA detected, FLA5 maintains a significantly higher expression level in Sea Island cotton fibers than in Upland cotton fibers. The increasing FLA5 transcript abundance coincided with the SCW deposition process and the expression intensity differences coincided with their fiber strength differences between Sea Island cotton and Upland cotton. These expression profile features of GbFLA5 in cotton fibers revealed its tissue-specific and SCW developmental stage-specific expression characters. Further analysis suggested that GbFLA5 is a crucial SCW-specific protein which may contribute to fiber strength by affecting cellulose synthesis and microfibril deposition orientation.
Initiation of transcription is essential for most of the cellular responses to environmental conditions and for cell and tissue specificity. This process is regulated through numerous proteins, their ligands and mutual interactions, as well as interactions with DNA. The key such regulatory proteins are transcription factors (TFs) and transcription co-factors (TcoFs). TcoFs are important since they modulate the transcription initiation process through interaction with TFs. In eukaryotes, transcription requires that TFs form different protein complexes with various nuclear proteins. To better understand transcription regulation, it is important to know the functional class of proteins interacting with TFs during transcription initiation. Such information is not fully available, since not all proteins that act as TFs or TcoFs are yet annotated as such, due to generally partial functional annotation of proteins. In this study we have developed a method to predict, using only sequence composition of the interacting proteins, the functional class of human TF binding partners to be (i) TF, (ii) TcoF, or (iii) other nuclear protein. This allows for complementing the annotation of the currently known pool of nuclear proteins. Since only the knowledge of protein sequences is required in addition to protein interaction, the method should be easily applicable to many species.
Based on experimentally validated interactions between human TFs with different TFs, TcoFs and other nuclear proteins, our two classification systems (implemented as a web-based application) achieve high accuracies in distinguishing TFs and TcoFs from other nuclear proteins, and TFs from TcoFs respectively.
As demonstrated, given the fact that two proteins are capable of forming direct physical interactions and using only information about their sequence composition, we have developed a completely new method for predicting a functional class of TF interacting protein partners with high precision and accuracy.
Reverse transcription quantitative polymerase chain reaction (qRT-PCR) has rapidly become the most sensitive and accurate method for the quantification of gene expression. To facilitate gene expression studies and obtain more accurate qRT-PCR data, normalization relative to stable housekeeping genes is required. These housekeeping genes need to show stable expression under the given experimental conditions for the qRT-PCR results to be accurate. Unfortunately, there are no studies on the stability of housekeeping genes used in Spodoptera litura. In this study, eight candidate reference genes, elongation factor 1 alpha (EF1), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ribosomal protein L10 (RPL10), ribosomal protein S3 (RPS3), beta actin (ACTB), beta FTZ-F1 (FTZF1), ubiquinol-cytochrome c reductase (UCCR), and arginine kinase (AK), were evaluated for their suitability as normalization genes under different experimental conditions using the statistical software programs, BestKeeper, geNorm and Normfinder, and the comparative ΔCt method. We determined the expression levels of the candidate reference genes for three biotic factors (developmental stage, tissue and population), and four abiotic treatments (temperature, insecticide, food and starvation). The results indicated that the best sets of candidates as reference genes were as follows: GAPDH and UCCR for developmental stages; RPL10, AK and EF1 for different tissues; RPL10 and EF1 for different populations in China; GAPDH and EF1 for temperature-stressed larvae; AK and ACTB for larvae treated with different insecticides; RPL10, GAPDH and UCCR for larvae fed different diets; RPS3 and ACTB for starved larvae. We believe that these results make an important contribution to gene analysis studies in S. litura and form the basis of further research on stable reference genes in S. litura and other organisms.
Killer immunoglobulin-like receptors (KIRs) are involved in the regulation of natural killer cell cytotoxicity. Within the human genome seventeen KIR genes are present, which all contain a large number of allelic variants. The high level of homology among KIR genes has hampered KIR genotyping in larger cohorts, and determination of gene copy number variation (CNV) has been difficult. We have designed a multiplex ligation-dependent probe amplification (MLPA) technique for genotyping and CNV determination in one single assay and validated the results by next-generation sequencing and with a KIR gene-specific short tandem repeat assay. In this way, we demonstrate in a cohort of 120 individuals a high level of CNV for all KIR genes except for the framework genes KIR3DL3 and KIR3DL2. Application of our MLPA assay in segregation analyses of families from the Centre d’Etude du Polymorphisme Humaine, previously KIR-genotyped by classical techniques, confirmed an earlier reported duplication and resulted in the identification of a novel duplication event in one of these families. In summary, our KIR MLPA assay allows rapid and accurate KIR genotyping and CNV detection, thus rendering improved transplantation programs and oncology treatment feasible, and enables more detailed studies on the role of KIRs in human (auto)immunity and infectious disease.
The full understanding of the mechanisms underlying transcriptional regulatory networks requires unravelling of complex causal relationships. Genome high-throughput technologies produce a huge amount of information pertaining gene expression and regulation; however, the complexity of the available data is often overwhelming and tools are needed to extract and organize the relevant information. This work starts from the assumption that the observation of co-occurrent events (in particular co-localization, co-expression and co-regulation) may provide a powerful starting point to begin unravelling transcriptional regulatory networks. Co-expressed genes often imply shared functional pathways; co-expressed and functionally related genes are often co-localized, too; moreover, co-expressed and co-localized genes are also potential targets for co-regulation; finally, co-regulation seems more frequent for genes mapped to proximal chromosome regions. Despite the recognized importance of analysing co-occurrent events, no bioinformatics solution allowing the simultaneous analysis of co-expression, co-localization and co-regulation is currently available. Our work resulted in developing and valuating CluGene, a software providing tools to analyze multiple types of co-occurrences within a single interactive environment allowing the interactive investigation of combined co-expression, co-localization and co-regulation of genes. The use of CluGene will enhance the power of testing hypothesis and experimental approaches aimed at unravelling transcriptional regulatory networks. The software is freely available at http://bioinfolab.unipg.it/.
The C-repeat (CRT)/dehydration-responsive element (DRE) binding factor (CBF/DREB1) transcription factors play a key role in cold response. However, the detailed roles of many plant CBFs are far from fully understood. A CBF gene (SsCBF1) was isolated from the cold-hardy plant Solanum lycopersicoides. A subcellular localization study using GFP fusion protein indicated that SsCBF1 is localized in the nucleus. We delimited the SsCBF1 transcriptional activation domain to the C-terminal segment comprising amino acid residues 193–228 (SsCBF1193–228). The expression of SsCBF1 could be dramatically induced by cold, drought and high salinity. Transactivation assays in tobacco leaves revealed that SsCBF1 could specifically bind to the CRT cis-elements in vivo to activate the expression of downstream reporter genes. The ectopic overexpression of SsCBF1 conferred increased freezing and high-salinity tolerance and late flowering phenotype to transgenic Arabidopsis. RNA-sequencing data exhibited that a set of cold and salt stress responsive genes were up-regulated in transgenic Arabidopsis. Our results suggest that SsCBF1 behaves as a typical CBF to contribute to plant freezing tolerance. Increased resistance to high-salinity and late flowering phenotype derived from SsCBF1 OE lines lend more credence to the hypothesis that plant CBFs participate in diverse physiological and biochemical processes related to adverse conditions.
Coccidiosis, caused by various Eimeria species, is a major parasitic disease in chickens. However, our understanding on how chickens respond to coccidian infection is highly limited at both molecular and cellular levels. The present study employed the Affymetrix chicken genome array and performed transcriptome analysis on chicken cecal epithelia in response to infection for 4.5 days in vivo by the cecal-specific species E. tenella. By Significance Analysis of Microarrays (SAM), we have identified 7,099 probe sets with q-values at <0.05, in which 4,033 and 3,066 genes were found to be up- or down-regulated in response to parasite infection. The reliability of the microarray data were validated by real-time qRT-PCR of 20 genes with varied fold changes in expression (i.e., correlation coefficient between microarray and qRT-PCR datasets: R2 = 0.8773, p<0.0001). Gene ontology analysis, KEGG pathway mapping and manual annotations of regulated genes indicated that up-regulated genes were mainly involved in immunity/defense, responses to various stimuli, apoptosis/cell death and differentiation, signal transduction and extracellular matrix (ECM), whereas down-regulated genes were mainly encoding general metabolic enzymes, membrane components, and some transporters. Chickens mustered complex cecal eipthelia molecular and immunological responses in response to E. tenella infection, which included pathways involved in cytokine production and interactions, natural killer cell mediated cytotoxicity, and intestinal IgA production. In response to the pathogenesis and damage caused by infection, chicken cecal epithelia reduced general metabolism, DNA replication and repair, protein degradation, and mitochondrial functions.
Variation in the expression of numerous genes is at the basis of plant response to environmental stresses. Non-target-site-based resistance to herbicides (NTSR), the major threat to grass weed chemical control, is governed by a subset of the genes involved in herbicide stress response. Quantitative PCR assays allowing reliable comparison of gene expression are thus key to identify genes governing NTSR. This work aimed at identifying a set of reference genes with a stable expression to be used as an internal standard for the normalisation of quantitative PCR data in studies investigating NTSR to herbicides inhibiting acetolactate synthase (ALS) in the major grass weed Lolium sp. Gene expression stability was assessed in plants resistant or sensitive to two ALS inhibitors, subjected or not to herbicide stress. Using three complementary approaches implemented in the programs BestKeeper, NormFinder and geNorm, cap-binding protein, glyceraldehyde-3-phosphate-dehydrogenase and ubiquitin were identified as the most suitable reference genes. This reference gene set can probably be used to study herbicide response in other weed species. It was used to compare the expression of the genes encoding two herbicide target enzymes (ALS and acetyl-coenzyme A carboxylase) and five cytochromes P450 (CYP) with potential herbicide-degrading activity between plants resistant or sensitive to ALS inhibitors. Overall, herbicide application enhanced CYP gene expression. Constitutive up-regulation of all CYP genes observed in resistant plants compared to sensitive plants suggested enhanced secondary metabolism in the resistant plants. Comprehensive transcriptome studies associated to gene expression analyses using the reference gene set validated here are required to unravel NTSR genetic determinants.
NF-κB is a ubiquitously expressed transcription factor that regulates a large number of genes in response to diverse physiological and pathological stimuli. The regulation of the transcriptional activity of NF-κB is often dependent on its interaction with IκBα. Proteins that bind to IκBα are critical regulators of NF-κB activity. DDRGK1 is a member of the DDRGK domain-containing protein family with unknown function. In this study, we showed that the depletion of DDRGK1 inhibits cell proliferation and invasion. Microarray analysis indicated that the expression of NF-κB target genes showed the most significant decrease after depleting of DDRGK1, suggesting that DDRGK1 may play an important role in the NF-κB signaling pathway. We further demonstrated that DDRGK1 interacts with IκBα and regulates its stability, thereby regulates the NF-κB transcriptional activity. Our findings identify DDRGK1 as an important regulator of the NF-κB pathway.
The whitefly Bemisa tabaci is a species complex of more than 31 cryptic species which include some of the most destructive invasive pests of crops worldwide. Among them, Middle East-Asia Minor 1 (MEAM1) and Mediterranean have invaded many countries and displaced the native whitefly species. The successful invasion of the two species is largely due to their wide range of host plants, high resistance to insecticides and remarkable tolerance to environmental stresses. However, the molecular differences between invasive and indigenous whiteflies remain largely unknown.
Here the global transcriptional difference between the two invasive whitefly species (MEAM1, MED) and one indigenous whitefly species (Asia II 3) were analyzed using the Illumina sequencing. Our analysis indicated that 2,422 genes between MEAM1 and MED; 3,073 genes between MEAM1 and Asia II 3; and 3,644 genes between MED and Asia II 3 were differentially expressed. Gene Ontology enrichment analysis revealed that the differently expressed genes between the invasive and indigenous whiteflies were significantly enriched in the term of ‘oxidoreductase activity’. Pathway enrichment analysis showed that carbohydrate, amino acid and glycerolipid metabolisms were more active in MEAM1 and MED than in Asia II 3, which may contribute to their differences in biological characteristics. Our analysis also illustrated that the majority of genes involved in ‘drug metabolic pathway’ were expressed at a higher level in MEAM1 and MED than in Asia II 3. Taken together, these results revealed that the genes related to basic metabolism and detoxification were expressed at an elevated level in the invasive whiteflies, which might be responsible for their higher resistance to insecticides and environmental stresses.
The extensive comparison of MEAM1, MED and Asia II 3 gene expression may serve as an invaluable resource for revealing the molecular mechanisms underlying their biological differences and the whitefly invasion.
Triple negative breast cancers (TNBC) are difficult to treat due to a lack of targets and heterogeneity. Inhibition of angiogenesis is a promising therapeutic strategy, but has had limited effectiveness so far in breast cancer. To quantify heterogeneity in angiogenesis-related gene expression in breast cancer, we focused on two families – VEGFs and semaphorins – that compete for neuropilin co-receptors on endothelial cells. We compiled microarray data for over 2,600 patient tumor samples and analyzed the expression of VEGF- and semaphorin-related ligands and receptors. We used principal component analysis to identify patterns of gene expression, and clustering to group samples according to these patterns. We used available survival data to determine whether these clusters had prognostic as well as therapeutic relevance. TNBC was highly associated with dysregulation of VEGF- and semaphorin-related genes; in particular, it appeared that expression of both VEGF and semaphorin genes were altered in a pro-angiogenesis direction. A pattern of high VEGFA expression with low expression of secreted semaphorins was associated with 60% of triple-negative breast tumors. While all TNBC groups demonstrated poor prognosis, this signature also correlated with lower 5-year survival rates in non-TNBC samples. A second TNBC pattern, including high VEGFC expression, was also identified. These pro-angiogenesis signatures may identify cancers that are more susceptible to VEGF inhibition.
One of the major challenges in designing a peptide-based vaccine is the identification of antigenic regions in an antigen that can stimulate B-cell’s response, also called B-cell epitopes. In the past, several methods have been developed for the prediction of conformational and linear (or continuous) B-cell epitopes. However, the existing methods for predicting linear B-cell epitopes are far from perfection. In this study, an attempt has been made to develop an improved method for predicting linear B-cell epitopes. We have retrieved experimentally validated B-cell epitopes as well as non B-cell epitopes from Immune Epitope Database and derived two types of datasets called Lbtope_Variable and Lbtope_Fixed length datasets. The Lbtope_Variable dataset contains 14876 B-cell epitope and 23321 non-epitopes of variable length where as Lbtope_Fixed length dataset contains 12063 B-cell epitopes and 20589 non-epitopes of fixed length. We also evaluated the performance of models on above datasets after removing highly identical peptides from the datasets. In addition, we have derived third dataset Lbtope_Confirm having 1042 epitopes and 1795 non-epitopes where each epitope or non-epitope has been experimentally validated in at least two studies. A number of models have been developed to discriminate epitopes and non-epitopes using different machine-learning techniques like Support Vector Machine, and K-Nearest Neighbor. We achieved accuracy from ∼54% to 86% using diverse s features like binary profile, dipeptide composition, AAP (amino acid pair) profile. In this study, for the first time experimentally validated non B-cell epitopes have been used for developing method for predicting linear B-cell epitopes. In previous studies, random peptides have been used as non B-cell epitopes. In order to provide service to scientific community, a web server LBtope has been developed for predicting and designing B-cell epitopes (http://crdd.osdd.net/raghava/lbtope/).
Orosomucoid 1-like 3 (ORMDL3) gene was strongly linked with the development of asthma in genetic association studies, and its expression could be significantly induced by allergen in airway epithelial cells of mice. However, the expression mechanism of ORMDL3 was still unclear. Here we have identified and characterized the mouse ORMDL3 gene promoter. Deletion constructs of the 5′ ﬂanking region were fused to a luciferase reporter gene. After transient transfection in mouse fibroblast cell line NIH3T3, a CRE (−27/−20) binding CREB was identified in the core promoter region. Deletion or mutation of the CRE consensus sequence resulted in a significant loss of the promoter activity. EMSA and ChIP assays demonstrated the binding of CREB to the core promoter. Knocking down endogenous CREB led to a reduction in ORMDL3 expression. Conversely, overexpression of CREB up-regulated ORMDL3 expression. Moreover, forskolin, a PKA activator, could facilitate the phosphorylation of CREB, which in turn heightens ORMDL3 expression. H-89, a PKA-specific inhibitor, could significantly inhibit ORMDL3 expression. This study delineates the characterization of mouse ORMDL3 gene promoter and shows signaling pathway cAMP/PKA/CREB plays an important role in regulating ORMDL3 expression, which will be helpful for future animal model studies regarding the regulation or function of ORMDL3 gene.
Thymosin proteins are well known for their actin-binding activity. Thymosin beta 4 (Tβ4) has been associated with biological activities in tissue repair and cell migration via interaction with ATP-synthase in vertebrates, while the information of similar thymosin functions in invertebrates is limited. We have shown previously that ATP-synthase is present on the surface of crayfish hematopoietic tissue (HPT) cells, and that astakine 1 (Ast1, an invertebrate cytokine) was found to interact with this β-subunit of ATP synthase. Here, we identified five different β-thymosins from Pacifastacus leniusculus, designated Pl-β-thymosin1-5. The two dominant isoforms in brain, HPT and hemocytes, Pl-β-thymosin1 and 2, were chosen for functional studies. Both isoforms could bind to the β-subunit of ATP-synthase, and Pl-β-thymosin1, but not Pl-β-thymosin2, significantly increased extracellular ATP formation. Moreover, Pl-β-thymosin1 stimulated HPT cell migration in vitro and Ast1 blocked this effect. Pl-β-thymosin2 increased the circulating hemocyte number at an early stage after injection. Additionally, in vivo injection of Pl-β-thymosin1 resulted in significant reduction of reactive oxygen species (ROS) production in crayfish HPT whereas Pl-β-thymosin2 had a similar but transient effect. Both Pl-β-thymosins induced the expression of Ast1 and superoxide dismutase (SOD) transcripts, while silencing of endogenous Pl-β-thymosin 1 and 2 by RNAi resulted in significant reduction of the Ast1 and SOD transcripts. The diverse effects exhibited by Pl-β-thymosin1 and Pl-β-thymosin2 indicates that these proteins are involved in a complex interaction that regulates the hematopoietic stem cell proliferation and differentiation.