Seasonal Stability Assessment of Reference Genes for Quantitative Real-Time Polymerase Chain Reaction Normalization in Bombus terrestris.

Bumblebees (B. terrestris) play a crucial role as highly efficient biological agents in commercial pollination. Understanding the molecular mechanisms governing their adaptation to diverse seasonal environments may pave the way for effective management strategies in the future. With the burgeoning advancement in post-genetic studies focusing on B. terrestris, there is a critical need to normalize quantitative real-time PCR (qRT-PCR) data using suitable reference genes. To address this necessity, we employed RefFinder, a software-based tool, to assess the suitability of several candidate endogenous control genes, including actin (ACT), arginine kinase (AK), elongation factor 1 alpha (EF1), glyceraldehyde-3-phosphate (GAPDH), phospholipase (PLA2), and ribosomal proteins (S18, S28). These genes were evaluated for their efficacy as biological endogenous controls by examining their expression patterns across various environmental conditions corresponding to different seasons (Spring, Summer, Autumn, Winter) and tissues (ovary, fat body, thorax, head) in bumblebees. Moreover, the study investigated the significance of selecting appropriate reference genes for three key genes involved in the juvenile hormone (JH) signaling pathways: Krüppel homolog 1 (Kr-h1), methyl farnesoate epoxidase (MFE), and Vitellogenin (Vg). Our research identifies specific genes suitable for normalization in B. terrestris, thereby offering valuable insights into gene expression and functional metabolic genetics under varying seasonal conditions. This catalog of reference genes will serve as a valuable resource for future research endeavors.

Genetics as a key to improving crop photosynthesis.

Since the basic biochemical mechanisms of photosynthesis are remarkably conserved among plant species, genetic modification approaches have so far been the main route to improve the photosynthetic performance of crops. Yet, phenotypic variation observed in wild species and between varieties of crop species implies there is standing natural genetic variation for photosynthesis, offering a largely unexplored resource to use for breeding crops with improved photosynthesis and higher yields. The reason this has not yet been explored is that the variation probably involves thousands of genes, each contributing only a little to photosynthesis, making them hard to identify without proper phenotyping and genetic tools. This is changing, though, and increasingly studies report on quantitative trait loci for photosynthetic phenotypes. So far, hardly any of these quantitative trait loci have been used in marker assisted breeding or genomic selection approaches to improve crop photosynthesis and yield, and hardly ever have the underlying causal genes been identified. We propose to take the genetics of photosynthesis to a higher level, and identify the genes and alleles nature has used for millions of years to tune photosynthesis to be in line with local environmental conditions. We will need to determine the physiological function of the genes and alleles, and design novel strategies to use this knowledge to improve crop photosynthesis through conventional plant breeding, based on readily available crop plant germplasm. In this work, we present and discuss the genetic methods needed to reveal natural genetic variation, and elaborate on how to apply this to improve crop photosynthesis.

Selection and Validation of Reference Genes for RT-qPCR Analysis in Aegilops tauschii (Coss.) under Different Abiotic Stresses.

Aegilops tauschii (Coss.) is an aggressive and serious annual grass weed in China. Its DD genome is a rich source of genetic material and performs better under different abiotic stress conditions (salinity, drought, temperature, etc.). Reverse-transcribed quantitative polymerase chain reaction (RT-qPCR) is a reliable technique for reference gene selection and validation. This work aimed to evaluate the stability of reference gene expression in Ae. tauschii under different abiotic stresses (salinity, drought, hot, and cold) and developmental stages (seedling and development). The results show that the ubiquitin-conjugating enzyme E2 36-like (UBC36) and protein microrchidia 2-like (HSP) are the most stable genes under control and salinity conditions, respectively. Under drought stress conditions, UBC36 is more stable as compared with others. Glyceraldehyde-3-phosphate dehydrogenase (GADPH) is the most stable reference gene during heat stress conditions and thioredoxin-like protein (YLS) under cold stress condition. Phosphate2A serine/threonine-protein phosphatase 2A (PP2A) and eukaryotic translation initiation factor 3 (ETIF3) are the most stable genes at seedling and developmental stages. Intracellular transport protein (CAC) is recommended as the most stable gene under different abiotic stresses and at developmental stages. Furthermore, the relative expression levels of NHX1 and DREB under different levels of salinity and drought stress conditions varied with the most (HSP and UBC36) and least (YLS and ACT) stable genes. This study provides reliable reference genes for understanding the tolerance mechanisms in Ae. tauschii under different abiotic stress conditions.

Applying the latest advances in genomics and phenomics for trait discovery in polyploid wheat.

Improving traits in wheat has historically been challenging due to its large and polyploid genome, limited genetic diversity and in-field phenotyping constraints. However, within recent years many of these barriers have been lowered. The availability of a chromosome-level assembly of the wheat genome now facilitates a step-change in wheat genetics and provides a common platform for resources, including variation data, gene expression data and genetic markers. The development of sequenced mutant populations and gene-editing techniques now enables the rapid assessment of gene function in wheat directly. The ability to alter gene function in a targeted manner will unmask the effects of homoeolog redundancy and allow the hidden potential of this polyploid genome to be discovered. New techniques to identify and exploit the genetic diversity within wheat wild relatives now enable wheat breeders to take advantage of these additional sources of variation to address challenges facing food production. Finally, advances in phenomics have unlocked rapid screening of populations for many traits of interest both in greenhouses and in the field. Looking forwards, integrating diverse data types, including genomic, epigenetic and phenomics data, will take advantage of big data approaches including machine learning to understand trait biology in wheat in unprecedented detail.

Biomarkers of metabolic disorders and neurobehavioral diseases in a PCB- exposed population: What we learned and the implications for future research.

Polychlorinated biphenyls (PCBs) are one of the original twelve classes of toxic chemicals covered by the Stockholm Convention on Persistent Organic Pollutants (POP), an international environmental treaty signed in 2001. PCBs are present in the environment as mixtures of multiple isomers at different degree of chlorination. These compounds are manmade and possess useful industrial properties including extreme longevity under harsh conditions, heat absorbance, and the ability to form an oily liquid at room temperature that is useful for electrical utilities and in other industrial applications. They have been widely used for a wide range of industrial purposes over the decades. Despite a ban in production in 1979 in the US and many other countries, they remain persistent and ubiquitous in environment as contaminants due to their improper disposal. Humans, independent of where they live, are therefore exposed to PCBs, which are routinely found in random surveys of human and animal tissues. The prolonged exposures to PCBs have been associated with the development of different diseases and disorders, and they are classified as endocrine disruptors. Due to its ability to interact with thyroid hormone, metabolism and function, they are thought to be implicated in the global rise of obesity diabetes, and their potential toxicity for neurodevelopment and disorders, an example of gene by environmental interaction (GxE). The current review is primarily intended to summarize the evidence for the association of PCB exposures with increased risks for metabolic dysfunctions and neurobehavioral disorders. In particular, we present evidence of gene expression alterations in PCB-exposed populations to construct the underlying pathways that may lead to those diseases and disorders in course of life. We conclude the review with future perspectives on biomarker-based research to identify susceptible individuals and populations.

Identification of suitable internal control genes for transcriptional studies in Eleusine coracana under different abiotic stress conditions.

Finger millet [Eleusine coracana (L.) Gaertn] is an excellent food and forage crop of arid and semiarid areas in Africa and Asia. It is well adapted to drought, heat, high salinity, poor soil fertility and low pH with an efficient C4 carbon fixation mechanism for high yield potential. To normalize the target gene expression data, the identification of suitable reference genes is essential. Ten candidate reference genes were selected and their expression stability was analyzed in various samples treated with different abiotic stress conditions. Five different statistical algorithms: geNorm, NormFinder, BestKeeper, ΔCt, and RefFinder were used to determine the stability of these genes. Our results revealed GAPDH, EEF1a, ACT and CYC as highly stable reference genes and PP2A and eIF4A as least stable reference genes across all the samples and suggesting that these genes could be used for accurate transcript normalization under abiotic stress. To the best of our knowledge, this is the first report on identification of suitable reference genes for accurate transcript normalization using qRT-PCR in finger millet under abiotic stress.

Effect of polymorphisms in the CAMKMT gene on growth traits in Ujumqin sheep.

Our previous genome-wide association study in sheep revealed that OAR3-84073899.1 (SNP31) in intron 8 of the CAMKMT gene was significantly associated with post-weaning gain at the genomic level. Herein, we performed a replication study to investigate single nucleotide polymorphisms (SNPs) within the CAMKMT gene exons, and 1000 bp of the 5'- and 3'-intranslated regions (UTRs) and their associations with growth traits in Ujumqin sheep. Five SNPs were identified through DNA pool sequencing technology: SNP26 in the 5'-UTR, SNP06 in exon 5, SNP07 in exon 8 and SNP27 and SNP28 in the 3'-UTR. Six SNPs, including SNP31 in intron 8, were genotyped in the validation group of 343 Ujumqin sheep, and each SNP was classified into three genotypes. The chi-square test suggested that all the variations were in Hardy-Weinberg equilibrium (P > 0.05) except for SNP28 and SNP31. Linkage disequilibrium analysis showed that SNP07 and SNP31 were strongly linked. An association analysis suggested that SNP06 was significantly associated with chest girth at 6 months of age (P < 0.05). SNP07 exhibited significant correlation with body weight and chest girth at 4 months of age and with body weight, chest girth and chest width at 6 months of age (P < 0.05). SNP27 was highly associated with body weight and chest girth at 4 months of age (P < 0.05), and SNP28 was extremely significantly associated with body weight and chest girth at 4 months of age and with chest girth at 6 months of age (P < 0.01). SNP31 was significantly associated with body weight and shin circumference at 4 months of age and with post-weaning gain (P < 0.05). Association analysis of the combined effect of SNP07 and SNP31 showed significant correlation with body weight and chest girth at four of months of age (P < 0.05) and with body weight and chest girth at 6 months of age (P < 0.05). These results indicate that the SNPs could be used as meritorious and available genetic markers in growth traits breeding and that the CAMKMT gene may be one of the key candidate genes that affect Ujumqin economic traits.

NAL1 allele from a rice landrace greatly increases yield in modern indica cultivars.

Increasing crop production is essential for securing the future food supply in developing countries in Asia and Africa as economies and populations grow. However, although the Green Revolution led to increased grain production in the 1960s, no major advances have been made in increasing yield potential in rice since then. In this study, we identified a gene, SPIKELET NUMBER (SPIKE), from a tropical japonica rice landrace that enhances the grain productivity of indica cultivars through pleiotropic effects on plant architecture. Map-based cloning revealed that SPIKE was identical to NARROW LEAF1 (NAL1), which has been reported to control vein pattern in leaf. Phenotypic analyses of a near-isogenic line of a popular indica cultivar, IR64, and overexpressor lines revealed increases in spikelet number, leaf size, root system, and the number of vascular bundles, indicating the enhancement of source size and translocation capacity as well as sink size. The near-isogenic line achieved 13-36% yield increase without any negative effect on grain appearance. Expression analysis revealed that the gene was expressed in all cell types: panicles, leaves, roots, and culms supporting the pleiotropic effects on plant architecture. Furthermore, SPIKE increased grain yield by 18% in the recently released indica cultivar IRRI146, and increased spikelet number in the genetic background of other popular indica cultivars. The use of SPIKE in rice breeding could contribute to food security in indica-growing regions such as South and Southeast Asia.

Dynamic metabolite QTL analyses provide novel biochemical insights into kernel development and nutritional quality improvement in common wheat.

Despite recent advances in crop metabolomics, the genetic control and molecular basis of the wheat kernel metabolome at different developmental stages remain largely unknown. Here, we performed widely targeted metabolite profiling of kernels from three developmental stages (grain-filling kernels [FKs], mature kernels [MKs], and germinating kernels [GKs]) using a population of 159 recombinant inbred lines. We detected 625 annotated metabolites and mapped 3173, 3143, and 2644 metabolite quantitative trait loci (mQTLs) in FKs, MKs, and GKs, respectively. Only 52 mQTLs were mapped at all three stages, indicating the high stage specificity of the wheat kernel metabolome. Four candidate genes were functionally validated by in vitro enzymatic reactions and/or transgenic approaches in wheat, three of which mediated the tricin metabolic pathway. Metabolite flux efficiencies within the tricin pathway were evaluated, and superior candidate haplotypes were identified, comprehensively delineating the tricin metabolism pathway in wheat. Finally, additional wheat metabolic pathways were re-constructed by updating them to incorporate the 177 candidate genes identified in this study. Our work provides new information on variations in the wheat kernel metabolome and important molecular resources for improvement of wheat nutritional quality.

Evaluation of internal reference genes for quantitative expression analysis by real-time reverse transcription-PCR in somatic cells from goat milk.

Reverse transcription (RT) quantitative real-time PCR (qPCR) is the most accurate and easy-to-perform technique to measure the expression level of a selected gene of interest by quantifying mRNA transcripts. The use of reference genes is commonly accepted as the most reliable approach to normalize RT-qPCR data and reduce possible errors generated in the quantification of gene expression. The optimal number and choice of reference genes are experimentally validated for specific tissues or cell types and experimental designs. To date, data on qPCR normalization in goats are scarce and the most suitable reference genes in this species have been identified for only a limited number of tissues. The aim of this study was to determine an optimal combination of stably expressed reference genes in caprine milk somatic cells (MSC) from healthy and infected mammary glands. For the purpose, we performed RT-qPCR for 10 commonly used reference genes from various functional classes and then determined their expression level in MSC from goats intramammary challenged with Staphylococcus aureus and in MSC from healthy controls, with a view to select genes whose stability would be unaffected under infection conditions. The geNorm and NormFinder algorithms were used for validating the reference genes. Furthermore, to demonstrate the importance of normalization of gene expression with appropriate reference genes, we tested the effect of using a combination of the least stable genes for expression analysis evaluation. On the basis of our evaluation, we recommend the use of a panel of reference genes that should include G6PD, YWHAZ, and ACTB for caprine MSC gene expression profiling. The expression of the 2 genes of interest, pentraxin-related protein (PTX3) and secreted phosphoprotein 1 (SPP1), was evaluated by RT-qPCR in all samples collected pre- and postinfection, and the recommended reference genes were used to normalize the data. Our study provides a validated panel of optimal reference genes for the identification of genes differentially expressed by qRT-PCR in caprine MSC. Moreover, we provided a set of intron-spanning primer sequences that could be suitable for gene expression experiments using SYBR Green chemistry on other caprine tissues and cells.

Validation of a metabolite-GWAS network for Populus trichocarpa family 1 UDP-glycosyltransferases.

Metabolite genome-wide association studies (mGWASs) are increasingly used to discover the genetic basis of target phenotypes in plants such as Populus trichocarpa, a biofuel feedstock and model woody plant species. Despite their growing importance in plant genetics and metabolomics, few mGWASs are experimentally validated. Here, we present a functional genomics workflow for validating mGWAS-predicted enzyme-substrate relationships. We focus on uridine diphosphate-glycosyltransferases (UGTs), a large family of enzymes that catalyze sugar transfer to a variety of plant secondary metabolites involved in defense, signaling, and lignification. Glycosylation influences physiological roles, localization within cells and tissues, and metabolic fates of these metabolites. UGTs have substantially expanded in P. trichocarpa, presenting a challenge for large-scale characterization. Using a high-throughput assay, we produced substrate acceptance profiles for 40 previously uncharacterized candidate enzymes. Assays confirmed 10 of 13 leaf mGWAS associations, and a focused metabolite screen demonstrated varying levels of substrate specificity among UGTs. A substrate binding model case study of UGT-23 rationalized observed enzyme activities and mGWAS associations, including glycosylation of trichocarpinene to produce trichocarpin, a major higher-order salicylate in P. trichocarpa. We identified UGTs putatively involved in lignan, flavonoid, salicylate, and phytohormone metabolism, with potential implications for cell wall biosynthesis, nitrogen uptake, and biotic and abiotic stress response that determine sustainable biomass crop production. Our results provide new support for in silico analyses and evidence-based guidance for in vivo functional characterization.

Validation of Reference Genes for Studying Different Abiotic Stresses in Oat (Avena sativa L.) by RT-qPCR.

Oat (Avena sativa L.) is a widely cultivated cereal with high nutritional value and it is grown mainly in temperate regions. The number of studies dealing with gene expression changes in oat continues to increase, and to obtain reliable RT-qPCR results it is essential to establish and use reference genes with the least possible influence caused by experimental conditions. However, no detailed study has been conducted on reference genes in different tissues of oat under diverse abiotic stress conditions. In our work, nine candidate reference genes (ACT, TUB, CYP, GAPD, UBC, EF1, TBP, ADPR, PGD) were chosen and analysed by four statistical methods (GeNorm, Normfinder, BestKeeper, RefFinder). Samples were taken from two tissues (leaves and roots) of 13-day-old oat plants exposed to five abiotic stresses (drought, salt, heavy metal, low and high temperatures). ADPR was the top-rated reference gene for all samples, while different genes proved to be the most stable depending on tissue type and treatment combinations. TUB and EF1 were most affected by the treatments in general. Validation of reference genes was carried out by PAL expression analysis, which further confirmed their reliability. These results can contribute to reliable gene expression studies for future research in cultivated oat.

iRGvalid: A Robust in silico Method for Optimal Reference Gene Validation.

BACKGROUND: Appropriate reference genes are critical to accurately quantifying relative gene expression in research and clinical applications. Numerous efforts have been made to select the most stable reference gene(s), but a consensus has yet to be achieved. In this report, we propose an in silico reference gene validation method, iRGvalid, that can be used as a universal tool to validate the reference genes recommended from different resources so as to identify the best ones without a need for any wet lab validation tests. METHODS: iRGvalid takes advantage of high throughput gene expression data and is built on a double-normalization strategy. First, the expression level of each individual gene is normalized against the total gene expression level of each sample, followed by a target gene normalization to the candidate reference gene(s). Linear regression analysis is then performed between the pre- and post- normalized target gene across the whole sample set to evaluate the stability of the reference gene(s), which is positively associated with the Pearson correlation coefficient, Rt. The higher the Rt value, the more stable the reference gene. We applied iRGvalid to 14 candidate reference genes to validate and identify the most stable reference genes in four cancer types: lung adenocarcinoma, breast cancer, colon adenocarcinoma, and nasopharyngeal cancer. The stability of the reference gene is evaluated both individually and in groups of all possible combinations. RESULTS: Highly stable reference genes resulted in high Rt values regardless of the target gene used. The highest stability was achieved with a specific combination of 3 to 6 reference genes. A few genes were among the best reference genes across the cancer types studied here. CONCLUSION: iRGvalid provides an easy and robust method to validate and identify the most stable reference gene or genes from a pool of candidate reference genes. The inclusivity of large expression data sets as well as the direct comparison of candidate reference genes makes it possible to identify reference genes with universal quality. This method can be used in any other gene expression studies when large cohorts of expression data are available.

Targeted mutation of transcription factor genes alters metaxylem vessel size and number in rice roots.

Root metaxylem vessels are responsible for axial water transport and contribute to hydraulic architecture. Variation in metaxylem vessel size and number can impact drought tolerance in crop plants, including rice, a crop that is particularly sensitive to drought. Identifying and validating candidate genes for metaxylem development would aid breeding efforts for improved varieties for drought tolerance. We identified three transcription factor candidate genes that potentially regulate metaxylem vessel size and number in rice based on orthologous annotations, published expression data, and available root and drought-related QTL data. Single gene knockout mutants were generated for each candidate using CRISPR-Cas9 genome editing. Root metaxylem vessel area and number were analyzed in 6-week-old knockout mutants and wild-type plants under well-watered and drought conditions in the greenhouse. Compared with wild type, LONESOME HIGHWAY (OsLHW) mutants had fewer, smaller metaxylem vessels in shallow roots and more, larger vessels in deep roots in drought conditions, indicating that OsLHW may be a repressor of drought-induced metaxylem plasticity. The AUXIN RESPONSE FACTOR 15 mutants showed fewer but larger metaxylem vessel area in well-watered conditions, but phenotypes were inconsistent under drought treatment. ORYZA SATIVA HOMEBOX 6 (OSH6) mutants had fewer, smaller metaxylem vessels in well-watered conditions with greater effects on xylem number than size. OSH6 mutants had larger shoots and more, deeper roots than the wild type in well-watered conditions, but there were no differences in performance under drought between mutants and wild type. Though these candidate gene mutants did not exhibit large phenotypic effects, the identification and investigation of candidate genes related to metaxylem traits in rice deepen our understanding of metaxylem development and are needed to facilitate incorporation of favorable alleles into breeding populations to improve drought stress tolerance.

A Biolistic-Mediated Virus-Induced Gene Silencing in Apocynaceae to Map Biosynthetic Pathways of Alkaloids.

Monoterpene indole alkaloids (MIAs) are specialized metabolites synthesized in many plants of the Apocynaceae family including Catharanthus roseus and Rauvolfia sp. MIAs are part of the chemical arsenal that plants evolved to face pet and herbivore attacks, and their high biological activities also confer pharmaceutical properties exploited in human pharmacopeia. Developing robust and straightforward tools to elucidate each step of MIA biosynthetic pathways thus constitutes a prerequisite to the understanding of Apocynaceae defense mechanisms and to the exploitation of MIA cytotoxicity through their production by metabolic engineering. While protocols of virus-induced gene silencing (VIGS) based on Agrobacterium-based transformation have emerged, the recalcitrance of Apocynaceae to this type of transformation prompted us to develop an universal procedure of VIGS vector inoculation. Such procedure relies on the delivery of the transforming plasmids through a particle bombardment performed using a biolistic device and offers the possibility to overcome host specificity to silence genes in any plant species. Using silencing of geissoschizine oxidase as an example, we described the main steps of this biolistic mediated VIGS in C. roseus and R. tetraphylla.

Handling the Hurdles on the Way to Anti-tuberculosis Drug Development.

The global epidemic of tuberculosis (TB) imposes a sustained epidemiologic vigilance and investments in research by governments. Mycobacterium tuberculosis, the main causative agent of TB in human beings, is a very successful pathogen, being the main cause of death in the population among infectious agents. In 2018, ~10 million individuals were contaminated with this bacillus and became ill with TB, and about 1.2 million succumbed to the disease. Most of the success of the M. tuberculosis to linger in the population comes from its ability to persist in an asymptomatic latent state into the host and, in fact, the majority of the individuals are unaware of being contaminated. Even though TB is a treatable disease and is curable in most cases, the treatment is lengthy and laborious. In addition, the rise of resistance to first-line anti-TB drugs elicits a response from TB research groups to discover new chemical entities, preferably with novel mechanisms of action. The pathway to find a new TB drug, however, is arduous and has many barriers that are difficult to overcome. Fortunately, several approaches are available today to be pursued by scientists interested in anti-TB drug development, which goes from massively testing chemical compounds against mycobacteria, to discovering new molecular targets by genetic manipulation. This review presents some difficulties found along the TB drug development process and illustrates different approaches that might be used to try to identify new molecules or targets that are able to impair M. tuberculosis survival.

Transcriptional Profiling and Biological Pathway(s) Analysis of Type 2 Diabetes Mellitus in a Pakistani Population.

The epidemic of type 2 diabetes mellitus (T2DM) is an important global health concern. Our earlier epidemiological investigation in Pakistan prompted us to conduct a molecular investigation to decipher the differential genetic pathways of this health condition in relation to non-diabetic controls. Our microarray studies of global gene expression were conducted on the Affymetrix platform using Human Genome U133 Plus 2.0 Array along with Ingenuity Pathway Analysis (IPA) to associate the affected genes with their canonical pathways. High-throughput qRT-PCR TaqMan Low Density Array (TLDA) was performed to validate the selected differentially expressed genes of our interest, viz., ARNT, LEPR, MYC, RRAD, CYP2D6, TP53, APOC1, APOC2, CYP1B1, SLC2A13, and SLC33A1 using a small population validation sample (n = 15 cases and their corresponding matched controls). Overall, our small pilot study revealed a discrete gene expression profile in cases compared to controls. The disease pathways included: Insulin Receptor Signaling, Type II Diabetes Mellitus Signaling, Apoptosis Signaling, Aryl Hydrocarbon Receptor Signaling, p53 Signaling, Mitochondrial Dysfunction, Chronic Myeloid Leukemia Signaling, Parkinson's Signaling, Molecular Mechanism of Cancer, and Cell Cycle G1/S Checkpoint Regulation, GABA Receptor Signaling, Neuroinflammation Signaling Pathway, Dopamine Receptor Signaling, Sirtuin Signaling Pathway, Oxidative Phosphorylation, LXR/RXR Activation, and Mitochondrial Dysfunction, strongly consistent with the evidence from epidemiological studies. These gene fingerprints could lead to the development of biomarkers for the identification of subgroups at high risk for future disease well ahead of time, before the actual disease becomes visible.

Catalogue of stage-specific transcripts in Ixodes ricinus and their potential functions during the tick life-cycle.

BACKGROUND: The castor bean tick Ixodes ricinus is an important vector of several clinically important diseases, whose prevalence increases with accelerating global climate changes. Characterization of a tick life-cycle is thus of great importance. However, researchers mainly focus on specific organs of fed life stages, while early development of this tick species is largely neglected. METHODS: In an attempt to better understand the life-cycle of this widespread arthropod parasite, we sequenced the transcriptomes of four life stages (egg, larva, nymph and adult female), including unfed and partially blood-fed individuals. To enable a more reliable identification of transcripts and their comparison in all five transcriptome libraries, we validated an improved-fit set of five I. ricinus-specific reference genes for internal standard normalization of our transcriptomes. Then, we mapped biological functions to transcripts identified in different life stages (clusters) to elucidate life stage-specific processes. Finally, we drew conclusions from the functional enrichment of these clusters specifically assigned to each transcriptome, also in the context of recently published transcriptomic studies in ticks. RESULTS: We found that reproduction-related transcripts are present in both fed nymphs and fed females, underlining the poorly documented importance of ovaries as moulting regulators in ticks. Additionally, we identified transposase transcripts in tick eggs suggesting elevated transposition during embryogenesis, co-activated with factors driving developmental regulation of gene expression. Our findings also highlight the importance of the regulation of energetic metabolism in tick eggs during embryonic development and glutamate metabolism in nymphs. CONCLUSIONS: Our study presents novel insights into stage-specific transcriptomes of I. ricinus and extends the current knowledge of this medically important pathogen, especially in the early phases of its development.

Choosing the Best Gene Predictions with GeneValidator.

GeneValidator is a tool for determining whether the characteristics of newly predicted protein-coding genes are consistent with those of similar sequences in public databases. For this, it runs up to seven comparisons per gene. Results are shown in an HTML report containing summary statistics and graphical visualizations that aim to be useful for curators. Results are also presented in CSV and JSON formats for automated follow-up analysis.Here, we describe common usage scenarios of GeneValidator that use the JSON output results together with standard UNIX tools. We demonstrate how GeneValidator's textual output can be used to filter and subset large gene sets effectively. First, we explain how low-scoring gene models can be identified and extracted for manual curation-for example, as input for genome browsers or gene annotation tools. Second, we show how GeneValidator's HTML report can be regenerated from a filtered subset of GeneValidator's JSON output. Subsequently, we demonstrate how GeneValidator's GUI can be used to complement manual curation efforts. Additionally, we explain how GeneValidator can be used to merge information from multiple annotations by automatically selecting the higher-scoring gene model at each common gene locus. Finally, we show how GeneValidator analyses can be optimized when using large BLAST databases.

Design, optimization and validation of genes commonly used in expression studies on DMH/AOM rat colon carcinogenesis model.

Colorectal cancer (CRC), also known as colon cancer, is the third most common form of cancer worldwide in men and the second in women and is characterized by several genetic alterations, among them the expression of several genes. 1,2-dimethylhydrazine (DMH) and its metabolite azoxymethane (AOM) are procarcinogens commonly used to induce colon cancer in rats (DMH/AOM rat model). This rat model has been used to study changes in mRNA expression in genes involved in this pathological condition. However, a lack of proper detailed PCR primer design in the literature limits the reproducibility of the published data. The present study aims to design, optimize and validate the qPCR, in accordance with the MIQE (Minimum Information for Publication of Quantitative Real-Time PCR Experiments) guidelines, for seventeen genes commonly used in the DMH/AOM rat model of CRC (Apc, Aurka, Bax, Bcl2, ß-catenin, Ccnd1, Cdkn1a, Cox2, Gsk3beta, IL-33, iNOs, Nrf2, p53, RelA, Smad4, Tnfα and Vegfa) and two reference genes (Actb or ß-actin and B2m). The specificity of all primer pairs was empirically validated on agarose gel, and furthermore, the melting curve inspection was checked as was their efficiency (%) ranging from 90 to 110 with a correlation coefficient of r 2 > 0.980. Finally, a pilot study was performed to compare the robustness of two candidate reference genes.