Variation In Gene Expression Levels Research Articles

Linking genomic prediction of fillet fat content in Atlantic salmon to underlying changes in lipid metabolism regulation

Fillet fat content is an important production trait in Atlantic salmon (Salmo salar) because it influences the flavor, texture, and nutritional properties of the product. Selection can be applied to alter muscle fat content, however how such selection changes the underlying molecular physiology of these animals is unknown. Here, we examine the link between genomic predicted breeding value for fillet fat of 184 fish and gene expression in the liver with an emphasis on lipid metabolism genes. We found that salmon with higher genomic breeding values for fillet fat had higher expression of genes in lipid metabolism pathways. This included key lipid metabolism genes hmgcrab, fasn-b, fads2d5, and fads2d6, and lipid transporters fatp2f, fabp7b, and apobc. We also found several regulators of lipid metabolism with negative correlation to genomic breeding values, including pparg-b, fxr-a, and fxr-b. A quantitative trait loci analysis for variation in gene expression levels (eQTLs) for 167 fillet fat associated genes found that 71 genes had at least one eQTL, and that most were trans eQTLs. Closer examination revealed distinct eQTL clustering on chromosomes 3 and 6, indicating the presence of putative common regulator in these regions. Taken together, these results suggest that salmon with high genomic breeding value for fillet fat have elevated lipid synthesis, elevated lipid transport, and reduced glycerolipid breakdown; and that this is at least partly due to genetic variants that impact the function of top-level transcription factors involved in liver metabolism. Our study sheds light on how genomic selection for reduced or increased fillet fat content in Atlantic salmon alters gene regulation in the liver of the fish. These results could be used to prioritize SNPs with an association to phenotype related gene expression to improve the efficiency and reduce the cost of genomic selection in the future.

Integration of ATAC-Seq and RNA-Seq Analysis to Identify Key Genes in the Longissimus Dorsi Muscle Development of the Tianzhu White Yak.

During the postnatal stages, skeletal muscle development undergoes a series of meticulously regulated alterations in gene expression. However, limited studies have employed chromatin accessibility to unravel the underlying molecular mechanisms governing muscle development in yak species. Therefore, we conducted an analysis of both gene expression levels and chromatin accessibility to comprehensively characterize the dynamic genome-wide chromatin accessibility during muscle growth and development in the Tianzhu white yak, thereby elucidating the features of accessible chromatin regions throughout this process. Initially, we compared the differences in chromatin accessibility between two groups and observed that calves exhibited higher levels of chromatin accessibility compared to adult cattle, particularly within ±2 kb of the transcription start site (TSS). In order to investigate the correlation between alterations in chromatin accessible regions and variations in gene expression levels, we employed a combination of ATAC-seq and RNA-seq techniques, leading to the identification of 18 central transcriptional factors (TFs) and 110 key genes with significant effects. Through further analysis, we successfully identified several TFs, including Sp1, YY1, MyoG, MEF2A and MEF2C, as well as a number of candidate genes (ANKRD2, ANKRD1, BTG2 and LMOD3) which may be closely associated with muscle growth and development. Moreover, we constructed an interactive network program encompassing hub TFs and key genes related to muscle growth and development. This innovative approach provided valuable insights into the molecular mechanism underlying skeletal muscle development in the postnatal stages of Tianzhu white yaks while also establishing a solid theoretical foundation for future research on yak muscle development.

The effect of developmental variation on expression QTLs in a multi parental Caenorhabditis elegans population.

Regulation of gene expression plays a crucial role in developmental processes and adaptation to changing environments. expression quantitative trait locus (eQTL) mapping is a technique used to study the genetic regulation of gene expression using the transcriptomes of recombinant inbred lines (RILs). Typically, the age of the inbred lines at the time of RNA sampling is carefully controlled. This is necessary because the developmental process causes changes in gene expression, complicating the interpretation of eQTL mapping experiments. However, due to genetics and variation in ambient micro-environments, organisms can differ in their "developmental age," even if they are of the same chronological age. As a result, eQTL patterns are affected by developmental variation in gene expression. The model organism Caenorhabditis elegans is particularly suited for studying the effect of developmental variation on eQTL mapping patterns. In a span of days, C. elegans transitions from embryo through 4 larval stages to adult while undergoing massive changes to its transcriptome. Here, we use C. elegans to investigate the effect of developmental age variation on eQTL patterns and present a normalization procedure. We used dynamical eQTL mapping, which includes the developmental age as a cofactor, to separate the variation in development from genotypic variation and explain variation in gene expression levels. We compare classical single marker eQTL mapping and dynamical eQTL mapping using RNA-seq data of ∼200 multi-parental RILs of C. elegans. The results show that (1) many eQTLs are caused by developmental variation, (2) most trans-bands are developmental QTLs, and (3) dynamical eQTL mapping detects additional eQTLs not found with classical eQTL mapping. We recommend that correction for variation in developmental age should be strongly considered in eQTL mapping studies given the large impact of processes like development on the transcriptome.

Substrate Stiffness Modulates TGF-β Activation and ECM-Associated Gene Expression in Fibroblasts.

Transforming growth factor-β (TGF-β) is a multifunctional cytokine that regulates the expression of ECM-associated genes during early injury. Tissue fibrosis development is driven by synergistic cues between the evolving biochemical and mechanical milieu. Few studies have addressed the role of substrate stiffness on TGF-β activity and extracellular matrix (ECM)-associated genes. We used a commercial formulation of polydimethylsiloxane (PDMS) to fabricate substrates of 40 kPa, 300 kPa, and 1.5 MPa stiffness, and cultured the HMF3S fibroblasts on substrates. We quantified TGF-β protein secreted by HMF3S cells on different substrates using a TGF-β responsive promoter reporter assay. We also tested for variations in gene expression levels on the substrates using RT-PCR and Western blotting and determined the MMP-2 and MMP-9 activities with gelatin zymography. The results showed that TGF-β protein activation was significantly compromised at lower stiffnesses. The expression of integrin α5 decreased on lower stiffness substrates and correlated with inefficient TGF-β protein activation. Collagen I, collagen III, and MMP-2 expression levels were lower on softer substrates; there was little MMP-9 activity on all substrates. Cell and nuclear morphologies were more rounded on compliant substrates, correlating with increased tubulin expression. Proliferations were higher on stiffer substrates, whereas cells on softer substrates showed cell cycle arrest. These results demonstrated critical feedback mechanisms between substrate stiffness and ECM regulation by fibroblasts, relevant in fibrosis.

Does the egg capsule protect against chronic UV-B radiation? A study based on encapsulated and decapsulated embryos of cuttlefish Sepia officinalis.

Although the egg capsule plays a crucial role in the embryonic development of cephalopods, its ability to protect embryos from Ultraviolet (UV) radiation is unknown. Our study evaluated the photoprotection mechanisms of S. officinalis to UV-B radiation and estimated the ability of the black capsule to act as a physical shield against it. Embryos with and without capsule and juveniles were exposed to four experimental UVB conditions for 55 days. The effects of different UVB doses were evaluated in terms of morphological abnormalities and differences in gene expression between each group. We observed that the development might be severely impaired in embryos exposed to UVB without capsule protection, and these effects were time- and UVB-dose-dependent. In addition, we found variations in gene expression levels (light-sensitive, stress response and DNA repair) in different tissues as a function of UVB doses. We suggest a relationship between morphological abnormalities and the limit of molecular regulation. These results suggest that the quantitative differences in expression are essential for defining the survivability of the embryo face to UVB. Thus, we demonstrated that the egg capsule could ensure successful embryonic development of the cuttlefish S. officinalis even at high doses of UVB.

ADGR: Admixture-Informed Differential Gene Regulation.

The regulatory elements in proximal and distal regions of genes are involved in the regulation of gene expression. Risk alleles in intronic and intergenic regions may alter gene expression by modifying the binding affinity and stability of diverse DNA-binding proteins implicated in gene expression regulation. By focusing on the local ancestral structure of coding and regulatory regions using the paired whole-genome sequence and tissue-wide transcriptome datasets from the Genotype-Tissue Expression project, we investigated the impact of genetic variants, in aggregate, on tissue-specific gene expression regulation. Local ancestral origins of the coding region, immediate and distant upstream regions, and distal regulatory region were determined using RFMix with the reference panel from the 1000 Genomes Project. For each tissue, inter-individual variation of gene expression levels explained by concordant or discordant local ancestry between coding and regulatory regions was estimated. Compared to European, African descent showed more frequent change in local ancestral structure, with shorter haplotype blocks. The expression level of the Adenosine Deaminase Like (ADAL) gene was significantly associated with admixed ancestral structure in the regulatory region across multiple tissue types. Further validations are required to understand the impact of the local ancestral structure of regulatory regions on gene expression regulation in humans and other species.

Transcriptional noise adjusted for expression levels reveals genes with high transcriptional noise that are highly expressed, functionally related, and co-regulated in yeast.

Understanding the relationship between variability in single-cell and non-single-cell gene expression studies will aid in understanding the role of and mechanisms that lead to variability in biological systems. Studies on the variation of gene expression levels in yeast normally focus on single cells and use the coefficient of variance (CV) as a measure of noise. The CV is typically negatively correlated with gene expression levels, so most of the studies using yeast find that genes with high transcriptional noise are lowly expressed. We find adjusting noise for expression levels using linear/natural log polynomial, and local fits and analyzing many non-single-cell RNA-seq sets identifies genes with high median transcriptional noise that are different than those that have high median CVs. Interestingly, these genes are heavily regulated by transcription factors that are related to variability and stochastic processes based on single-cell studies, including Msn2p, Msn4p, Hsf1p, and Crz1p but are not associated with genes with high median CVs based on non-single-cell gene expression data. In addition, adjusting noise for expression levels in a single-cell RNA-seq data set adds value by finding genes that have noisy gene expression levels and their associated transcription factors that are not found to be associated with genes with high CVs in the single-cell expression data or a comparable non-single-cell gene expression data. Lastly, S. cerevisiae genes with noisy expression tend to have orthologs with noisy gene expression in C. albicans, indicating transcriptional noise is evolutionarily conserved.

EQTLs as causal instruments for the reconstruction of hormone linked gene networks.

Hormones act within in highly dynamic systems and much of the phenotypic response to variation in hormone levels is mediated by changes in gene expression. The increase in the number and power of large genetic association studies has led to the identification of hormone linked genetic variants. However, the biological mechanisms underpinning the majority of these loci are poorly understood. The advent of affordable, high throughput next generation sequencing and readily available transcriptomic databases has shown that many of these genetic variants also associate with variation in gene expression levels as expression Quantitative Trait Loci (eQTLs). In addition to further dissecting complex genetic variation, eQTLs have been applied as tools for causal inference. Many hormone networks are driven by transcription factors, and many of these genes can be linked to eQTLs. In this mini-review, we demonstrate how causal inference and gene networks can be used to describe the impact of hormone linked genetic variation upon the transcriptome within an endocrinology context.

Plasticity across levels: Relating epigenomic, transcriptomic, and phenotypic responses to osmotic stress in a halotolerant microalga.

Phenotypic plasticity, the ability of a given genotype to produce alternative phenotypes in response to its environment of development, is an important mechanism for coping with variable environments. While the mechanisms underlying phenotypic plasticity are diverse, their relative contributions need to be investigated quantitatively to better understand the evolvability of plasticity across biological levels. This requires relating plastic responses of the epigenome, transcriptome, and organismal phenotype, and investigating how they vary with the genotype. Here we carried out this approach for responses to osmotic stress in Dunaliella salina, a green microalga that is a model organism for salinity tolerance. We compared two strains that show markedly different demographic responses to osmotic stress, and showed that these phenotypic responses involve strain‐ and environment‐specific variation in gene expression levels, but a relative low—albeit significant—effect of strain × environment interaction. We also found an important genotype effect on the genome‐wide methylation pattern, but little contribution from environmental conditions to the latter. However, we did detect a significant marginal effect of epigenetic variation on gene expression, beyond the influence of genetic differences on epigenetic state, and we showed that hypomethylated regions are correlated with higher gene expression. Our results indicate that epigenetic mechanisms are either not involved in the rapid plastic response to environmental change in this species, or involve only few changes in trans that are sufficient to trigger concerted changes in the expression of many genes, and phenotypic responses by multiple traits.

Development of the binary vector pTACAtg1 for stable gene expression in plant: Reduction of gene silencing in transgenic plants carrying the target gene with long flanking sequences.

Genetic modification in plants helps us to understand molecular mechanisms underlying on plant fitness and to improve profitable crops. However, in transgenic plants, the value of gene expression often varies among plant populations of distinct lines and among generations of identical individuals. This variation is caused by several reasons, such as differences in the chromosome position, repeated sequences, and copy number of the inserted transgene. Developing a state-of-art technology to avoid the variation of gene expression levels including gene silencing has been awaited. Here, we developed a novel binary plasmid (pTACAtg1) that is based on a transformation-competent artificial chromosome (TAC) vector, harboring long genomic DNA fragments on both sides of the cloning sites. As a case study, we cloned the cauliflower mosaic virus 35S promoter:β-glucuronidase (35S:GUS) gene cassettes into the pTACAtg1, and introduced it with long flanking sequences on the pTACAtg1 into the plants. In isolated transgenic plants, the copy number was reduced and the GUS expressions were detected more stably than those in the control plants carrying the insert without flanking regions. In our result, the reduced copy number of a transgene suppressed variation and silencing of its gene expression. The pTACAtg1 vector will be suitable for the production of stable transformants and for expression analyses of a transgene.

Development of a Porcine Model to Assess the Effect of In Situ Knee Joint Loading on Site-Specific Cartilage Gene Expression.

Cyclic mechanical loading of cartilage induces stresses and fluid flow, which are thought to modulate chondrocyte metabolism. The uneven surface, plus the heterogeneity of cartilage within a joint, makes stress and fluid pressure distribution in the tissue nonuniform, and gene expression may vary at different sites as a function of load magnitude, frequency, and time. In previous studies, cartilage explants were used for loading tests to investigate biological responses of the cartilage to mechanical loading. In contrast, we used loading tests on intact knee joints, to better reflect the loading conditions in a joint, and thus provide a more physiologically relevant mechanical environment. Gene expression levels in loaded samples for a selection of relevant genes were compared with those of the corresponding unloaded control samples to characterize potential differences. Furthermore, the effects of load magnitude and duration on gene expression levels were investigated. We observed differences in gene expression levels between samples from different sites in the same joint and between corresponding samples from the same site in loaded and unloaded joints. Consistent with previous findings, our results indicate that there is a critical upper and lower threshold of loading for triggering the expression of certain genes. Variations in gene expression levels may reflect the effect of local loading, topography, and structure of the cartilage in an intact joint on the metabolic activity of the associated cells.

Association between Gene Expression Levels of Candidate Cytarabine Pathway and Treatment Outcome Using Publically Available Data from Target Project

Acute myeloid leukemia (AML) is the second most common form of childhood leukemia and has the worst prognosis of all major childhood cancers. Improving the treatment outcome for patients with AML remains a major clinical challenge. The nucleoside analog, cytarabine (Ara-C), has been the mainstay of AML chemotherapy for more than 40 years. However, extensive inter-patient variation in treatment response, development of resistance, and severe toxicity remain as major hurdles to effective Ara-C chemotherapy. It is a pro-drug that requires activation to Ara-CTP by multiple phosphorylation steps. Thus, inter-patient variation in gene expression levels of candidate genes involved in cytarabine metabolism can impact therapeutic outcome. In last few years' significant efforts have been invested in generating genomic, transcriptomic and epigenetic data in various cancer patients including AML. TCGA (The Cancer Genome Atlas) is one of the most commonly available such resource, recently another resource available to scientific community TARGET (Therapeutically Applicable Research to Generate Effective Treatments), a project initiated by the National Cancer Institute (NCI) in 2006. This databased was created with the aim to take multi-omics integrative approach to characterize molecular alterations associated with poor outcomes in some common children's cancers including AML. In this study, we leveraged TARGET database to extract gene expression levels (generated using Affymetrix Gene ST Array) to investigate whether inter-patient differences in expression levels of 11 key candidate genes in cytarabine metabolic pathway are predictive of minimal residual disease (MRD: positive or negative) and thus outcome. These genes included DCK and CMPK1 encoding for drug activating enzymes, CDA, DCTD, NT5C2, NT5C3A, important for cytarabine inactivation; drug influx transporter SLC29A1; RRM1 and RRM2 as ribonucleotide inhibitors; CTPS1 and NME3 which play a key role in the synthesis of nucleoside tri-phosphates. We also investigated correlation of the expression levels of these genes with each other to get insight into whether these genes could be co-regulated. In order to have consistency we only included data (gene expression and clinical and patient demographic, characteristics) from 105 AML patients where expression levels were available from bone marrow specimens and also restricted our analysis to AAML03P1 and AAML0531 clinical cohorts. Our resultsshow significant correlation in expression levels between cytarabine metabolic pathway genes resulting in three clusters: First cluster included DCK, NT5C2, NT5C3A, RRM1 and CMPK1 with positive correlation with each other's ranging between (r=0.619 to 0.720 p-value Our results from association analysis with MRD showed that cluster 1 genes had higher expression levels in patients with positive MRD. On the other hand, cluster 2 genes have all a higher expression in the MRD negative. To get the combinatorial effect of gene-expression on different clinical endpoints we defined a combination score (CS) using CS = (DCK*NT5C2*NT5C3A*CMPK1*CTPS1*RRM1*RRM2*DCTD)/(CDA*NME3*SLC29A1). Higher CS was associated with positive MRD (P= 0.012) (Figure 2) and lower overall survival rates (r= - 0.232, P-value = 0.017). By generating a combinatorial expression score for cytarabine pathway genes, we have been able to identify features that might predict MRD, overall survival and thus outcome in patients treated with cytarabine. An interesting fact that we observed positive correlations between activating genes - DCK, CMPK with inactivating genes such as NT5C2 or reductases RRM1/RRM2 that negatively impact the drug activation. One possible explanation, when DCK is highly expressed, CMPK1 responsible for the second phosphorylation step increases. At the same time, RRM1 and RRM2 known as DCK inhibitors and NT5C2 become highly expressed to balance high DCK level. This might also be a plausible reason for not observing consistent correlations between expression levels of ara-C pathway genes with outcome. Download : Download high-res image (178KB) Download : Download full-size image Disclosures No relevant conflicts of interest to declare.

Multiplexed engineering glycosyltransferase genes in CHO cells via targeted integration for producing antibodies with diverse complex-type N-glycans

Therapeutic antibodies are decorated with complex-type N-glycans that significantly affect their biodistribution and bioactivity. The N-glycan structures on antibodies are incompletely processed in wild-type CHO cells due to their limited glycosylation capacity. To improve N-glycan processing, glycosyltransferase genes have been traditionally overexpressed in CHO cells to engineer the cellular N-glycosylation pathway by using random integration, which is often associated with large clonal variations in gene expression levels. In order to minimize the clonal variations, we used recombinase-mediated-cassette-exchange (RMCE) technology to overexpress a panel of 42 human glycosyltransferase genes to screen their impact on antibody N-linked glycosylation. The bottlenecks in the N-glycosylation pathway were identified and then released by overexpressing single or multiple critical genes. Overexpressing B4GalT1 gene alone in the CHO cells produced antibodies with more than 80% galactosylated bi-antennary N-glycans. Combinatorial overexpression of B4GalT1 and ST6Gal1 produced antibodies containing more than 70% sialylated bi-antennary N-glycans. In addition, antibodies with various tri-antennary N-glycans were obtained for the first time by overexpressing MGAT5 alone or in combination with B4GalT1 and ST6Gal1. The various N-glycan structures and the method for producing them in this work provide opportunities to study the glycan structure-and-function and develop novel recombinant antibodies for addressing different therapeutic applications.

Distinctive gene expression patterns and imprinting signatures revealed in reciprocal crosses between cattle sub-species

BackgroundThere are two genetically distinct subspecies of cattle, Bos taurus taurus and Bos taurus indicus, which arose from independent domestication events. The two types of cattle show substantial phenotypic differences, some of which emerge during fetal development and are reflected in birth outcomes, including birth weight. We explored gene expression profiles in the placenta and four fetal tissues at mid-gestation from one taurine (Bos taurus taurus; Angus) and one indicine (Bos taurus indicus; Brahman) breed and their reciprocal crosses.ResultsIn total 120 samples were analysed from a pure taurine breed, an indicine breed and their reciprocal cross fetuses, which identified 6456 differentially expressed genes (DEGs) between the two pure breeds in at least one fetal tissue of which 110 genes were differentially expressed in all five tissues examined. DEGs shared across tissues were enriched for pathways related to immune and stress response functions. Only the liver had a substantial number of DEGs when reciprocal crossed were compared among which 310 DEGs were found to be in common with DEGs identified between purebred livers; these DEGs were significantly enriched for metabolic process GO terms. Analysis of DEGs across purebred and crossbred tissues suggested an additive expression pattern for most genes, where both paternal and maternal alleles contributed to variation in gene expression levels. However, expression of 5% of DEGs in each tissue was consistent with parent of origin effects, with both paternal and maternal dominance effects identified.ConclusionsThese data identify candidate genes potentially driving the tissue-specific differences between these taurine and indicine breeds and provide a biological insight into parental genome effects underlying phenotypic differences in bovine fetal development.

Effects of Essential Oil Derived from the Bitter Orange (Citrus aurantium) on Growth Performance, Histology and Gene Expression Levels in Common Carp Juveniles (Cyprinus carpio).

The aim of this study was to detect effects of bitter orange (Citrus aurantium) essential oil, commonly called neroli oil (NO) (0, 0.25, 0.50, 1, and 1.5% referred to as NO0 NO0.25, NO0. 05, NO1 and NO1.5, respectively) on growth performance output and expression levels of some growth-related genes in the muscle tissue and some immune-related genes in the head kidney and pathological differences in digestive system organs of common carp Cyprinus carpio. The NO0.25 group had a large improvement in growth efficiency at the end of the 60-day feeding cycle. Real-time PCR (Bio RAD, USA) system was used to detect variations in gene expression levels. Furthermore, NO supplementation of up to 0.25% in muscle tissue controlled the release of growth hormone (GH) and insulin-like growth factor I (IGF-I). Furthermore, in the NO0.25 treatment category, immune response gene levels TNF-α, IL-8 and IL-1ß increased in head kidney tissue. In the histological examination of the liver and intestine, there were significant differences between fish fed with N1 and N1.5 diets. This study confirms that dietary supplementation of NO up to 0.25% can improve common carp growth efficiency and increase the expression of genes (GH and IGF-I) related to muscle growth, TNF-α, IL-8 and IL-1ß genes related to immune status, and liver and intestine histological status of common carp.

In vitro Evaluation of Programmed Cell Death in the Immune System of Pacific Oyster Crassostrea gigas by the Effect of Marine Toxins.

Programmed cell death (PCD) is an essential process for the immune system's development and homeostasis, enabling the remotion of infected or unnecessary cells. There are several PCD's types, depending on the molecular mechanisms, such as non-inflammatory or pro-inflammatory. Hemocytes are the main component of cellular immunity in bivalve mollusks. Numerous infectious microorganisms produce toxins that impair hemocytes functions, but there is little knowledge on the role of PCD in these cells. This study aims to evaluate in vitro whether marine toxins induce a particular type of PCD in hemocytes of the bivalve mollusk Crassostrea gigas during 4 h at 25°C. Hemocytes were incubated with two types of marine toxins: non-proteinaceous toxins from microalgae (saxitoxin, STX; gonyautoxins 2 and 3, GTX2/3; okadaic acid/dynophysistoxin-1, OA/DTX-1; brevetoxins 2 and 3, PbTx-2,-3; brevetoxin 2, PbTx-2), and proteinaceous extracts from bacteria (Vibrio parahaemolyticus, Vp; V. campbellii, Vc). Also, we used the apoptosis inducers, staurosporine (STP), and camptothecin (CPT). STP, CPT, STX, and GTX 2/3, provoked high hemocyte mortality characterized by apoptosis hallmarks such as phosphatidylserine translocation into the outer leaflet of the cell membrane, exacerbated chromatin condensation, DNA oligonucleosomal fragments, and variation in gene expression levels of apoptotic caspases 2, 3, 7, and 8. The mixture of PbTx-2,-3 also showed many apoptosis features; however, they did not show apoptotic DNA oligonucleosomal fragments. Likewise, PbTx-2, OA/DTX-1, and proteinaceous extracts from bacteria Vp, and Vc, induced a minor degree of cell death with high gene expression of the pro-inflammatory initiator caspase-1, which could indicate a process of pyroptosis-like PCD. Hemocytes could carry out both PCD types simultaneously. Therefore, marine toxins trigger PCD's signaling pathways in C. gigas hemocytes, depending on the toxin's nature, which appears to be highly conserved both structurally and functionally.

An effector\u2013reporter system to study cellular signal transduction in strawberry fruit (Fragaria ananassa)

An effector–reporter system is a powerful tool used to study cellular signal transduction, but this technique has been traditionally used in protoplasts. A similar system to study cellular signal transduction in fruits has not yet been established. In this study, we aimed to establish an effector–reporter system for strawberry fruit, a model nonclimacteric fruit. We first investigated the characteristics of transient gene expression in strawberry fruits and found marked variation in gene expression levels among individual fruits, and this variation has complicated the establishment of a technical system. To overcome this difficulty, we investigated a sampling strategy based on a statistical analysis of the activity pattern of four different reporters (GUS, GFP, FLuc, and RLuc) among individual fruits and combinations of pairs of reporters (GUS/GFP and RLuc/FLuc). Based on an optimized sampling strategy, we finally established a step-by step protocol for the effector/reporter assay. Using FaMYB10 and FaWRKY71 as the effectors and GUS driven by the FaCHS promoter as the reporter, we demonstrated that this effector/reporter system was practical and reliable. This effector/reporter technique will contribute to an in-depth exploration of the signaling mechanism for the regulation of strawberry fruit ripening.

High heterogeneity undermines generalization of differential expression results in RNA-Seq analysis

BackgroundRNA 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.ResultsOur 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.ConclusionsHigh 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.

Variations in gene expression levels with severity of synovitis in dogs with naturally occurring stifle osteoarthritis.

Osteoarthritis (OA) is one of the major causes of chronic pain in dogs. However, the pathogenesis of OA has not been fully understood in dogs. The objective of this study was to comprehensively investigate the mRNA expression levels of proinflammatory cytokines, inflammatory mediators, nerve growth factor and its receptor, and matrix metalloproteinases in the synovium of dogs with spontaneous OA as well as to elucidate their relationships with the severity of synovitis. Dogs that were diagnosed with stifle OA on the basis of radiographic findings were included, and the degree of synovitis was observed using stifle arthroscopy. The dogs were assigned to two different groups depending on their synovitis scores: the low-grade group (score of 1 or 2; n = 8) and high-grade group (score of 3 to 5; n = 18). The dogs showing no evidence of orthopedic disease were included in the control group (n = 6). Synovial tissue samples were collected from the sites at which synovitis scores were assessed using arthroscopy. Total RNA was extracted from the collected synovial tissue, and cDNA was synthesized. Subsequently, RT-qPCR were performed using canine-specific primer sets for IL1B, IL6, CXCL8, TNF, TGFB1, PTGS2, PTGES, MMP3, MMP13, NGF, NTRK1, and PTGER4. Expression levels of IL1B, IL6, CXCL8, and MMP13 were significantly higher in the high-grade group than in the control group. In addition, expression levels of IL1B, CXCL8, TNF, and PTGS2 were significantly higher in the high-grade group than in the low-grade group. Expression levels of IL1B, IL6, CXCL8, TNF, PTGS2, and PTGER4 showed significant positive correlation with synovitis score. In conclusion, all mRNA expression levels in the synovial membrane varied according to the degree of synovitis in dogs with spontaneous OA. Thus, this study may partially elucidate the pathogenesis of synovitis in dogs with spontaneous OA.

Single-molecule long-read (SMRT) transcriptome sequencing of Mercenaria mercenaria reveals a powerful anti-apoptotic system critical for air exposure endurance

Mercenaria mercenaria is an economically important clam species and exhibits an outstanding resistance to multiple environmental stressors. However, our understanding of their stress adaptability is limited due to a lack of genomic information, such as transcriptome resources. In this study, single-molecule long-read (SMRT) mRNA sequencing was performed to obtain the full-length gill transcriptome reference sequences of M. mercenaria under air exposure stress. In all, 14.5 G subreads were obtained and assembled into 64,603 unigenes, among which 50,613 were successfully annotated. Additionally, 56,295 SSRs, 1457 transcription factors, and 5924 lncRNAs were identified in M. mercenaria transcriptome. Furthermore, numerous apoptosis-related transcripts were identified according to Swiss-Prot annotation and their numbers were counted. We also found that most apoptosis-related transcripts exhibited typical domains of a certain protein family through conserved domain prediction. Additionally, eight typical sequences related to apoptosis pathway were detected by RT-PCR, with the aim to show the sequential variation of gene expression levels under air exposure. These results implied that the complicated apoptosis system, especially the powerful anti-apoptotic system was critical for M. mercenaria to endure air exposure.