Social bacteria use chemical communication to coordinate and synchronize gene expression via the quorum-sensing (QS) regulatory pathway. In Pectobacterium, a causative agent of the blackleg and soft-rot diseases on potato plants and tubers, expression of the virulence factors is collectively controlled by the QS-signals N-acylhomoserine lactones (NAHLs). Several soil bacteria, such as the actinobacterium Rhodococcus erythropolis, are able to degrade NAHLs, hence quench the chemical communication and virulence of Pectobacterium. Here, next-generation sequencing was used to investigate structural and functional genomics of the NAHL-degrading R. erythropolis strain R138. The R. erythropolis R138 genome (6.7 Mbp) contained a single circular chromosome, one linear (250 kbp) and one circular (84 kbp) plasmid. Growth of R. erythropolis and P. atrosepticum was not altered in mixed-cultures as compared with monocultures on potato tuber slices. HiSeq-transcriptomics revealed that no R. erythropolis genes were differentially expressed when R. erythropolis was cultivated in the presence vs absence of the avirulent P. atrosepticum mutant expI, which is defective for QS-signal synthesis. By contrast 50 genes (<1% of the R. erythropolis genome) were differentially expressed when R. erythropolis was cultivated in the presence vs absence of the NAHL-producing virulent P. atrosepticum. Among them, quantitative real-time reverse-transcriptase-PCR confirmed that the expression of some alkyl-sulfatase genes decreased in the presence of a virulent P. atrosepticum, as well as deprivation of organic sulfur such as methionine, which is a key precursor in the synthesis of NAHL by P. atrosepticum.
Depression risk is exacerbated by genetic factors and stress exposure; however, the biological mechanisms through which these factors interact to confer depression risk are poorly understood. One putative biological mechanism implicates variability in the ability of cortisol, released in response to stress, to trigger a cascade of adaptive genomic and non-genomic processes through glucocorticoid receptor (GR) activation. Here, we demonstrate that common genetic variants in long-range enhancer elements modulate the immediate transcriptional response to GR activation in human blood cells. These functional genetic variants increase risk for depression and co-heritable psychiatric disorders. Moreover, these risk variants are associated with inappropriate amygdala reactivity, a transdiagnostic psychiatric endophenotype and an important stress hormone response trigger. Network modeling and animal experiments suggest that these genetic differences in GR-induced transcriptional activation may mediate the risk for depression and other psychiatric disorders by altering a network of functionally related stress-sensitive genes in blood and brain.
Invasive phytoplasmas wreak havoc on coconut palms worldwide, leading to high loss of income, food insecurity and extreme poverty of farmers in producing countries. Phytoplasmas as strictly biotrophic insect-transmitted bacterial pathogens instigate distinct changes in developmental processes and defence responses of the infected plants and manipulate plants to their own advantage; however, little is known about the cellular and molecular mechanisms underlying host-phytoplasma interactions. Further, phytoplasma-mediated transcriptional alterations in coconut palm genes have not yet been identified. This study evaluated the whole transcriptome profiles of naturally infected leaves of Cocos nucifera ecotype Malayan Red Dwarf in response to yellow decline phytoplasma from group 16SrXIV, using RNA-Seq technique. Transcriptomics-based analysis reported here identified genes involved in coconut innate immunity. The number of down-regulated genes in response to phytoplasma infection exceeded the number of genes up-regulated. Of the 39,873 differentially expressed unigenes, 21,860 unigenes were suppressed and 18,013 were induced following infection. Comparative analysis revealed that genes associated with defence signalling against biotic stimuli were significantly overexpressed in phytoplasma-infected leaves versus healthy coconut leaves. Genes involving cell rescue and defence, cellular transport, oxidative stress, hormone stimulus and metabolism, photosynthesis reduction, transcription and biosynthesis of secondary metabolites were differentially represented. Our transcriptome analysis unveiled a core set of genes associated with defence of coconut in response to phytoplasma attack, although several novel defence response candidate genes with unknown function have also been identified. This study constitutes valuable sequence resource for uncovering the resistance genes and/or susceptibility genes which can be used as genetic tools in disease resistance breeding.
MicroRNAs (miRNAs) are a class of small, endogenous non-coding RNAs that negatively regulate gene expression, resulting in the silencing of target mRNA transcripts through mRNA cleavage or translational inhibition. MiRNAs play significant roles in various biological and physiological processes in plants. However, the miRNA-mediated gene regulatory network in pineapple, the model tropical non-climacteric fruit, remains largely unexplored. Here, we report a complete list of pineapple mature miRNAs obtained from high-throughput small RNA sequencing and precursor miRNAs (pre-miRNAs) obtained from ESTs. Two small RNA libraries were constructed from pineapple fruits and leaves, respectively, using Illumina's Solexa technology. Sequence similarity analysis using miRBase revealed 579,179 reads homologous to 153 miRNAs from 41 miRNA families. In addition, a pineapple fruit transcriptome library consisting of approximately 30,000 EST contigs constructed using Solexa sequencing was used for the discovery of pre-miRNAs. In all, four pre-miRNAs were identified (MIR156, MIR399, MIR444 and MIR2673). Furthermore, the same pineapple transcriptome was used to dissect the function of the miRNAs in pineapple by predicting their putative targets in conjunction with their regulatory networks. In total, 23 metabolic pathways were found to be regulated by miRNAs in pineapple. The use of high-throughput sequencing in pineapples to unveil the presence of miRNAs and their regulatory pathways provides insight into the repertoire of miRNA regulation used exclusively in this non-climacteric model plant.
The discovery of mesenchymal stem cells (MSCs) from a myriad of tissues has triggered the initiative of establishing tailor-made stem cells for disease-specific therapy. Nevertheless, lack of understanding on the inherent differential propensities of these cells may restrict their clinical outcome. Therefore, a comprehensive study was done to compare the proliferation, differentiation, expression of cell surface markers and gene profiling of stem cells isolated from different sources, viz. bone marrow, Wharton's jelly, adipose tissue and dental pulp. We found that although all MSCs were phenotypically similar to each other, Wharton's jelly (WJ) MSCs and dental pulp stem cells (DPSCs) were highly proliferative as compared to bone marrow (BM) MSCs and adipose tissue (AD) MSCs. Moreover, indistinguishable cell surface characteristics and differentiation capacity were confirmed to be similar among all cell types. Based on gene expression profiling, we postulate that BM-MSCs constitutively expressed genes related to inflammation and immunodulation, whereas genes implicated in tissue development were highly expressed in AD-MSCs. Furthermore, the transcriptome profiling of WJ-MSCs and DPSCs revealed an inherent bias towards the neuro-ectoderm lineage. Based on our findings, we believe that there is no unique master mesenchymal stem cell that is appropriate to treat all target diseases. More precisely, MSCs from different sources exhibit distinct and unique gene expression signatures that make them competent to give rise to specific lineages rather than others. Therefore, stem cells should be subjected to rigorous characterization and utmost vigilance needs to be adopted in order to choose the best cellular source for a particular disease.
Lignosus rhinocerotis (Cooke) Ryvarden (tiger milk mushroom) has long been known for its nutritional and medicinal benefits among the local communities in Southeast Asia. However, the molecular and genetic basis of its medicinal and nutraceutical properties at transcriptional level have not been investigated. In this study, the transcriptome of L. rhinocerotis sclerotium, the part with medicinal value, was analyzed using high-throughput Illumina HiSeqTM platform with good sequencing quality and alignment results. A total of 3,673, 117, and 59,649 events of alternative splicing, novel transcripts, and SNP variation were found to enrich its current genome database. A large number of transcripts were expressed and involved in the processing of gene information and carbohydrate metabolism. A few highly expressed genes encoding the cysteine-rich cerato-platanin, hydrophobins, and sugar-binding lectins were identified and their possible roles in L. rhinocerotis were discussed. Genes encoding enzymes involved in the biosynthesis of glucans, six gene clusters encoding four terpene synthases and one each of non-ribosomal peptide synthetase and polyketide synthase, and 109 transcribed cytochrome P450 sequences were also identified in the transcriptome. The data from this study forms a valuable foundation for future research in the exploitation of this mushroom in pharmacological and industrial applications.
Feline infectious peritonitis (FIP) is a lethal systemic disease caused by FIP virus (FIPV). There are no effective vaccines or treatment available, and the virus virulence determinants and pathogenesis are not fully understood. Here, we describe the sequencing of RNA extracted from Crandell Rees Feline Kidney (CRFK) cells infected with FIPV using the Illumina next-generation sequencing approach. Bioinformatics analysis, based on Felis catus 2X annotated shotgun reference genome, using CLC bio Genome Workbench is used to map both control and infected cells. Kal's Z test statistical analysis is used to analyze the differentially expressed genes from the infected CRFK cells. In addition, RT-qPCR analysis is used for further transcriptional profiling of selected genes in infected CRFK cells and Peripheral Blood Mononuclear Cells (PBMCs) from healthy and FIP-diagnosed cats.
Developing drought-tolerant rice varieties with higher yield under water stressed conditions provides a viable solution to serious yield-reduction impact of drought. Understanding the molecular regulation of this polygenic trait is crucial for the eventual success of rice molecular breeding programmes. microRNAs have received tremendous attention recently due to its importance in negative regulation. In plants, apart from regulating developmental and physiological processes, microRNAs have also been associated with different biotic and abiotic stresses. Hence here we chose to analyze the differential expression profiles of microRNAs in three drought treated rice varieties: Vandana (drought-tolerant), Aday Sel (drought-tolerant) and IR64 (drought-susceptible) in greenhouse conditions via high-throughput sequencing.
Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is a rare, autosomal recessive disorder associated with mutations in the thymidine phosphorylase (TYMP) gene. The main objective of this study was to characterize the genetic profiles of the deceased proband's family members (N = 4) using DNA sequencing and to determine miRNA deregulation in MNGIE using miRNA microarray profiling and bioinformatic analysis. We found that the genetic profile of the younger sister showed similar TYMP gene mutations as that of the proband with the exception of a heterozygous mutation in exon 10. The miRNA microarray revealed 55 significantly up-regulated and 65 significantly down-regulated miRNAs. These miRNAs have been implicated in various mitochondrial dynamics such as energy metabolism, Krebs cycle, mitochondria-associated apoptosis, and mitophagy. In conclusion, we demonstrate that blood miRNAs are deregulated in the pathogenesis of MNGIE and these changes may have therapeutic implications. Further experimental studies will be required to elucidate the functional miRNA-mRNA interactions in MNGIE.
Background. A common research goal in transcriptome projects is to find genes that are differentially expressed in different phenotype classes. Biologists might wish to validate such gene candidates experimentally, or use them for downstream systems biology analysis. Producing a coherent differential gene expression analysis from RNA-seq count data requires an understanding of how numerous sources of variation such as the replicate size, the hypothesized biological effect size, and the specific method for making differential expression calls interact. We believe an explicit demonstration of such interactions in real RNA-seq data sets is of practical interest to biologists. Results. Using two large public RNA-seq data sets-one representing strong, and another mild, biological effect size-we simulated different replicate size scenarios, and tested the performance of several commonly-used methods for calling differentially expressed genes in each of them. We found that, when biological effect size was mild, RNA-seq experiments should focus on experimental validation of differentially expressed gene candidates. Importantly, at least triplicates must be used, and the differentially expressed genes should be called using methods with high positive predictive value (PPV), such as NOISeq or GFOLD. In contrast, when biological effect size was strong, differentially expressed genes mined from unreplicated experiments using NOISeq, ASC and GFOLD had between 30 to 50% mean PPV, an increase of more than 30-fold compared to the cases of mild biological effect size. Among methods with good PPV performance, having triplicates or more substantially improved mean PPV to over 90% for GFOLD, 60% for DESeq2, 50% for NOISeq, and 30% for edgeR. At a replicate size of six, we found DESeq2 and edgeR to be reasonable methods for calling differentially expressed genes at systems level analysis, as their PPV and sensitivity trade-off were superior to the other methods'. Conclusion. When biological effect size is weak, systems level investigation is not possible using RNAseq data, and no meaningful result can be obtained in unreplicated experiments. Nonetheless, NOISeq or GFOLD may yield limited numbers of gene candidates with good validation potential, when triplicates or more are available. When biological effect size is strong, NOISeq and GFOLD are effective tools for detecting differentially expressed genes in unreplicated RNA-seq experiments for qPCR validation. When triplicates or more are available, GFOLD is a sharp tool for identifying high confidence differentially expressed genes for targeted qPCR validation; for downstream systems level analysis, combined results from DESeq2 and edgeR are useful.
The infection of Cryptococcus neoformans is acquired through the inhalation of desiccated yeast cells and basidiospores originated from the environment, particularly from bird's droppings and decaying wood. Three environmental strains of C. neoformans originated from bird droppings (H4, S48B and S68B) and C. neoformans reference clinical strain (H99) were used for intranasal infection in C57BL/6 mice. We showed that the H99 strain demonstrated higher virulence compared to H4, S48B and S68B strains. To examine if gene expression contributed to the different degree of virulence among these strains, a genome-wide microarray study was performed to inspect the transcriptomic profiles of all four strains. Our results revealed that out of 7,419 genes (22,257 probes) examined, 65 genes were significantly up-or down-regulated in H99 versus H4, S48B and S68B strains. The up-regulated genes in H99 strain include Hydroxymethylglutaryl-CoA synthase (MVA1), Mitochondrial matrix factor 1 (MMF1), Bud-site-selection protein 8 (BUD8), High affinity glucose transporter 3 (SNF3) and Rho GTPase-activating protein 2 (RGA2). Pathway annotation using DAVID bioinformatics resource showed that metal ion binding and sugar transmembrane transporter activity pathways were highly expressed in the H99 strain. We suggest that the genes and pathways identified may possibly play crucial roles in the fungal pathogenesis.
The king cobra (Ophiophagus hannah) is widely distributed throughout many parts of Asia. This study aims to investigate the complexity of Malaysian Ophiophagus hannah (MOh) venom for a better understanding of king cobra venom variation and its envenoming pathophysiology. The venom gland transcriptome was investigated using the Illumina HiSeq™ platform, while the venom proteome was profiled by 1D-SDS-PAGE-nano-ESI-LCMS/MS.
To date, the molecular signalling mechanisms which regulate growth factors-induced MSCs tenogenic differentiation remain largely unknown. Therefore, a study to determine the global gene expression profile of tenogenic differentiation in human bone marrow stromal cells (hMSCs) using growth differentiation factor 5 (GDF5) was conducted. Microarray analyses were conducted on hMSCs cultures supplemented with 100 ng/ml of GDF5 and compared to undifferentiated hMSCs and adult tenocytes. Results of QuantiGene® Plex assay support the use and interpretation of the inferred gene expression profiles and pathways information. From the 27,216 genes assessed, 873 genes (3.21% of the overall human transcriptome) were significantly altered during the tenogenic differentiation process (corrected p<0.05). The genes identified as potentially associated with tenogenic differentiation were ARHGAP29, CCL2, integrin alpha 8 and neurofilament medium polypeptides. These genes, were mainly associated with cytoskeleton reorganization (stress fibers formation) signaling. Pathway analysis demonstrated the potential molecular pathways involved in tenogenic differentiation were: cytoskeleton reorganization related i.e. keratin filament signaling and activin A signaling; cell adhesion related i.e. chemokine and adhesion signaling; and extracellular matrix related i.e. arachidonic acid production signaling. Further investigation using atomic force microscopy and confocal laser scanning microscopy demonstrated apparent cytoskeleton reorganization in GDF5-induced hMSCs suggesting that cytoskeleton reorganization signaling is an important event involved in tenogenic differentiation. Besides, a reduced nucleostemin expression observed suggested a lower cell proliferation rate in hMSCs undergoing tenogenic differentiation. Understanding and elucidating the tenogenic differentiation signalling pathways are important for future optimization of tenogenic hMSCs for functional tendon cell-based therapy and tissue engineering.
Physiological responses to stress are controlled by expression of a large number of genes, many of which are regulated by microRNAs. Since most banana cultivars are salt-sensitive, improved understanding of genetic regulation of salt induced stress responses in banana can support future crop management and improvement in the face of increasing soil salinity related to irrigation and climate change. In this study we focused on determining miRNA and their targets that respond to NaCl exposure and used transcriptome sequencing of RNA and small RNA from control and NaCl-treated banana roots to assemble a cultivar-specific reference transcriptome and identify orthologous and Musa-specific miRNA responding to salinity. We observed that, banana roots responded to salinity stress with changes in expression for a large number of genes (9.5% of 31,390 expressed unigenes) and reduction in levels of many miRNA, including several novel miRNA and banana-specific miRNA-target pairs. Banana roots expressed a unique set of orthologous and Musa-specific miRNAs of which 59 respond to salt stress in a dose-dependent manner. Gene expression patterns of miRNA compared with those of their predicted mRNA targets indicated that a majority of the differentially expressed miRNAs were down-regulated in response to increased salinity, allowing increased expression of targets involved in diverse biological processes including stress signaling, stress defence, transport, cellular homeostasis, metabolism and other stress-related functions. This study may contribute to the understanding of gene regulation and abiotic stress response of roots and the high-throughput sequencing data sets generated may serve as important resources related to salt tolerance traits for functional genomic studies and genetic improvement in banana.
Magnocellular neurons (MCNs) in the hypothalamo-neurohypophysial system (HNS) are highly specialized to release large amounts of arginine vasopressin (Avp) or oxytocin (Oxt) into the blood stream and play critical roles in the regulation of body fluid homeostasis. The MCNs are osmosensory neurons and are excited by exposure to hypertonic solutions and inhibited by hypotonic solutions. The MCNs respond to systemic hypertonic and hypotonic stimulation with large changes in the expression of their Avp and Oxt genes, and microarray studies have shown that these osmotic perturbations also cause large changes in global gene expression in the HNS. In this paper, we examine gene expression in the rat supraoptic nucleus (SON) under normosmotic and chronic salt-loading SL) conditions by the first time using "new-generation", RNA sequencing (RNA-Seq) methods. We reliably detect 9,709 genes as present in the SON by RNA-Seq, and 552 of these genes were changed in expression as a result of chronic SL. These genes reflect diverse functions, and 42 of these are involved in either transcriptional or translational processes. In addition, we compare the SON transcriptomes resolved by RNA-Seq methods with the SON transcriptomes determined by Affymetrix microarray methods in rats under the same osmotic conditions, and find that there are 6,466 genes present in the SON that are represented in both data sets, although 1,040 of the expressed genes were found only in the microarray data, and 2,762 of the expressed genes are selectively found in the RNA-Seq data and not the microarray data. These data provide the research community a comprehensive view of the transcriptome in the SON under normosmotic conditions and the changes in specific gene expression evoked by salt loading.
Despite significant advances in staging and therapies, lung cancer remains a major cause of cancer-related lethality due to its high incidence and recurrence. Clearly, a novel approach is required to develop new therapies to treat this devastating disease. Recent evidence indicates that tumours contain a small population of cells known as cancer stem cells (CSCs) that are responsible for tumour maintenance, spreading and resistant to chemotherapy. The genetic composition of CSCs so far is not fully understood, but manipulation of the specific genes that maintain their integrity would be beneficial for developing strategies to combat cancer. Therefore, the goal of this study isto identify the transcriptomic composition and biological functions of CSCs from non-small cell lung cancer (NSCLC).
The Malaysian giant freshwater prawn, Macrobrachium rosenbergii, is an economically important crustacean worldwide. However, production of this prawn is facing a serious threat from Vibriosis disease caused by Vibrio species such as Vibrio parahaemolyticus. Unfortunately, the mechanisms involved in the immune response of this species to bacterial infection are not fully understood. We therefore used a high-throughput deep sequencing technology to investigate the transcriptome and comparative expression profiles of the hepatopancreas from this freshwater prawn infected with V. parahaemolyticus to gain an increased understanding of the molecular mechanisms underlying the species' immune response to this pathogenic bacteria.
Paphia textile is an important, aquaculture bivalve clam species distributed mainly in China, Philippines, and Malaysia. Recent studies of P. textile have focused mainly on artificial breeding and nutrition analysis, and the transcriptome and genome of P. textile have rarely been reported. In this work, the transcriptome of P. textile foot tissue was sequenced on an Illumina HiSeq™ 2000 platform. A total of 20,219,795 reads were generated, resulting in 4.08 Gb of raw data. The raw reads were cleaned and assembled into 54,852 unigenes with an N50 length of 829 bp. Of these unigenes, 38.92% were successfully annotated based on their matches to sequences in seven public databases. Among the annotated unigenes, 14,571 were assigned Gene Ontology terms, 5448 were classified to Clusters of Orthologous Groups categories, and 6738 were mapped to 228 pathways in the Kyoto Encyclopedia of Genes and Genomes database. For functional marker development, 5605 candidate simple sequence repeats were identified in the transcriptome and 80 primer pairs were selected randomly and amplified in a wild population of P. textile. A total of 36 loci that exhibited obvious repeat length polymorphisms were detected. The transcriptomic data and microsatellite markers will provide valuable resources for future functional gene analyses, genetic map construction, and quantitative trait loci mapping in P. textile.
Rphq2, a minor gene for partial resistance to Puccinia hordei , was physically mapped in a 188 kbp introgression with suppressed recombination between haplotypes of rphq2 and Rphq2 barley cultivars.