BEST4+ cells in the intestinal epithelium.

The recent development of single-cell transcriptomics highlighted the existence of a new lineage of mature absorptive cells in the human intestinal epithelium. This subpopulation is characterized by the specific expression of Bestrophin 4 (BEST4) and of other marker genes including OTOP2, CA7, GUCA2A, GUCA2B, and SPIB. BEST4+ cells appear early in development and are present in all regions of the small and large intestine at a low abundance (<5% of all epithelial cells). Location-specific gene expression profiles in BEST4+ cells suggest their functional specialization in each gut region, as exemplified by the small intestine-specific expression of the ion channel CFTR. The putative roles of BEST4+ cells include sensing and regulation of luminal pH, tuning of guanylyl cyclase-C signaling, transport of electrolytes, hydration of mucus, and secretion of antimicrobial peptides. However, most of these hypotheses lack functional validation, notably because BEST4+ cells are absent in mice. The presence of BEST4+ cells in human intestinal organoids indicates that this in vitro model should be suitable to study their role. Recent studies showed that BEST4+ cells are also present in the intestinal epithelium of macaque, pig, and zebrafish and, here, we report their presence in rabbits, which suggests that these species could be appropriate animal models to study BEST4+ cells during the development of diseases and their interactions with environmental factors such as diet or the microbiota. In this review, we summarize the existing literature regarding BEST4+ cells and emphasize the description of their predicted roles in the intestinal epithelium in health and disease.NEW & NOTEWORTHY BEST4+ cells are a novel subtype of mature absorptive cells in the human intestinal epithelium highlighted by single-cell transcriptomics. The gene expression profile of BEST4+ cells suggests their role in pH regulation, electrolyte secretion, mucus hydration, and innate immune defense. The absence of BEST4+ cells in mice requires the use of alternative animal models or organoids to decipher the role of this novel type of intestinal epithelial cells.

Should we really use graph neural networks for transcriptomic prediction?

The recent development of deep learning methods have undoubtedly led to great improvement in various machine learning tasks, especially in prediction tasks. This type of methods have also been adapted to answer various problems in bioinformatics, including automatic genome annotation, artificial genome generation or phenotype prediction. In particular, a specific type of deep learning method, called graph neural network (GNN) has repeatedly been reported as a good candidate to predict phenotypes from gene expression because its ability to embed information on gene regulation or co-expression through the use of a gene network. However, up to date, no complete and reproducible benchmark has ever been performed to analyze the trade-off between cost and benefit of this approach compared to more standard (and simpler) machine learning methods. In this article, we provide such a benchmark, based on clear and comparable policies to evaluate the different methods on several datasets. Our conclusion is that GNN rarely provides a real improvement in prediction performance, especially when compared to the computation effort required by the methods. Our findings on a limited but controlled simulated dataset shows that this could be explained by the limited quality or predictive power of the input biological gene network itself.

Asterics: a simple tool for the ExploRation and Integration of omiCS data.

BACKGROUND: The rapid development of omics acquisition techniques has induced the production of a large volume of heterogeneous and multi-level omics datasets, which require specific and sometimes complex analyses to obtain relevant biological information. Here, we present ASTERICS (version 2.5), a publicly available web interface for the analyses of omics datasets. RESULTS: ASTERICS is designed to make both standard and complex exploratory and integration analysis workflows easily available to biologists and to provide high quality interactive plots. Special care has been taken to provide a comprehensive documentation of the implemented analyses and to guide users toward sound analysis choices regarding some specific omics data. Data and analyses are organized in a comprehensive graphical workflow within ASTERICS workspace to facilitate the understanding of successive data editions and analyses leading to a given result. CONCLUSION: ASTERICS provides an easy to use platform for omics data exploration and integration. The modular organization of its open source code makes it easy to incorporate new workflows and analyses by external contributors. ASTERICS is available at https://asterics.miat.inrae.fr and can also be deployed using provided docker images.

Plasma 1H-NMR metabolic and amino acid profiles of newborn piglets from two lines divergently selected for residual feed intake.

Together with environmental factors, physiological maturity at birth is a major determinant for neonatal survival and postnatal development in mammalian species. Maturity at birth is the outcome of complex mechanisms of intra-uterine development and maturation during the end of gestation. In pig production, piglet preweaning mortality averages 20% of the litter and thus, maturity is a major welfare and economic concern. Here, we used both targeted and untargeted metabolomic approaches to provide a deeper understanding of the maturity in a model of lines of pigs divergently selected on residual feed intake (RFI), previously shown to have contrasted signs of maturity at birth. Analyses were conducted on plasma metabolome of piglets at birth and integrated with other phenotypic characteristics associated to maturity. We confirmed proline and myo-inositol, previously described for their association with delayed growth, as potential markers of maturity. Urea cycle and energy metabolism were found more regulated in piglets from high and low RFI lines, respectively, suggesting a better thermoregulation ability for the low RFI (with higher feed efficiency) piglets.

Feature selection for kernel methods in systems biology.

The substantial development of high-throughput biotechnologies has rendered large-scale multi-omics datasets increasingly available. New challenges have emerged to process and integrate this large volume of information, often obtained from widely heterogeneous sources. Kernel methods have proven successful to handle the analysis of different types of datasets obtained on the same individuals. However, they usually suffer from a lack of interpretability since the original description of the individuals is lost due to the kernel embedding. We propose novel feature selection methods that are adapted to the kernel framework and go beyond the well-established work in supervised learning by addressing the more difficult tasks of unsupervised learning and kernel output learning. The method is expressed under the form of a non-convex optimization problem with a ℓ1 penalty, which is solved with a proximal gradient descent approach. It is tested on several systems biology datasets and shows good performances in selecting relevant and less redundant features compared to existing alternatives. It also proved relevant for identifying important governmental measures best explaining the time series of Covid-19 reproducing number evolution during the first months of 2020. The proposed feature selection method is embedded in the R package mixKernel version 0.8, published on CRAN. Installation instructions are available at http://mixkernel.clementine.wf/.

Network Analyses Reveal Negative Link Between Changes in Adipose Tissue GDF15 and BMI During Dietary-induced Weight Loss.

CONTEXT: Adipose tissue (AT) transcriptome studies provide holistic pictures of adaptation to weight and related bioclinical settings changes. OBJECTIVE: To implement AT gene expression profiling and investigate the link between changes in bioclinical parameters and AT gene expression during 3 steps of a 2-phase dietary intervention (DI). METHODS: AT transcriptome profiling was obtained from sequencing 1051 samples, corresponding to 556 distinct individuals enrolled in a weight loss intervention (8-week low-calorie diet (LCD) at 800 kcal/day) followed with a 6-month ad libitum randomized DI. Transcriptome profiles obtained with QuantSeq sequencing were benchmarked against Illumina RNAseq. Reverse transcription quantitative polymerase chain reaction was used to further confirm associations. Cell specificity was assessed using freshly isolated cells and THP-1 cell line. RESULTS: During LCD, 5 modules were found, of which 3 included at least 1 bioclinical variable. Change in body mass index (BMI) connected with changes in mRNA level of genes with inflammatory response signature. In this module, change in BMI was negatively associated with changes in expression of genes encoding secreted protein (GDF15, CCL3, and SPP1). Through all phases of the DI, change in GDF15 was connected to changes in SPP1, CCL3, LIPA and CD68. Further characterization showed that these genes were specific to macrophages (with LIPA, CD68 and GDF15 expressed in anti-inflammatory macrophages) and GDF15 also expressed in preadipocytes. CONCLUSION: Network analyses identified a novel AT feature with GDF15 upregulated with calorie restriction induced weight loss, concomitantly to macrophage markers. In AT, GDF15 was expressed in preadipocytes and macrophages where it was a hallmark of anti-inflammatory cells.

Major Reorganization of Chromosome Conformation During Muscle Development in Pig.

The spatial organization of the genome in the nucleus plays a crucial role in eukaryotic cell functions, yet little is known about chromatin structure variations during late fetal development in mammals. We performed in situ high-throughput chromosome conformation capture (Hi-C) sequencing of DNA from muscle samples of pig fetuses at two late stages of gestation. Comparative analysis of the resulting Hi-C interaction matrices between both groups showed widespread differences of different types. First, we discovered a complex landscape of stable and group-specific Topologically Associating Domains (TADs). Investigating the nuclear partition of the chromatin into transcriptionally active and inactive compartments, we observed a genome-wide fragmentation of these compartments between 90 and 110 days of gestation. Also, we identified and characterized the distribution of differential cis- and trans-pairwise interactions. In particular, trans-interactions at chromosome extremities revealed a mechanism of telomere clustering further confirmed by 3D Fluorescence in situ Hybridization (FISH). Altogether, we report major variations of the three-dimensional genome conformation during muscle development in pig, involving several levels of chromatin remodeling and structural regulation.

Joint Automatic Metabolite Identification and Quantification of a Set of 1H NMR Spectra.

Metabolomics is a promising approach to characterize phenotypes or to identify biomarkers. It is also easily accessible through NMR, which can provide a comprehensive understanding of the metabolome of any living organisms. However, the analysis of 1H NMR spectrum remains difficult, mainly due to the different problems encountered to perform automatic identification and quantification of metabolites in a reproducible way. In addition, methods that perform automatic identification and quantification of metabolites are often designed to process one given complex mixture spectrum at a time. Hence, when a set of complex mixture spectra coming from the same experiment has to be processed, the approach is simply repeated independently for every spectrum, despite their resemblance. Here, we present new methods that are the first to either align spectra or to identify and quantify metabolites by integrating information coming from several complex spectra of the same experiment. The performances of these new methods are then evaluated on both simulated and real datasets. The results show an improvement in the metabolite identification and in the accuracy of metabolite quantifications, especially when the concentration is low. This joint procedure is available in version 2.0 of ASICS package.

The maturity in fetal pigs using a multi-fluid metabolomic approach.

In mammalian species, the first days after birth are an important period for survival and the mortality rate is high before weaning. In pigs, perinatal deaths average 20% of the litter, with important economic and societal consequences. Maturity is one of the most important factors that influence piglet survival at birth. Maturity can be defined as the outcome of complex mechanisms of intra-uterine development and maturation during the last month of gestation. Here, we provide new insights into maturity obtained by studying the end of gestation at two different stages (3 weeks before term and close to term) in two breeds of pigs that strongly differ in terms of neonatal survival. We used metabolomics to characterize the phenotype, to identify biomarkers, and provide a comprehensive understanding of the metabolome of the fetuses in late gestation in three fluids (plasma, urine, and amniotic fluid). Our results show that the biological processes related to amino acid and carbohydrate metabolisms are critical for piglet maturity. We confirm the involvement of some previously described metabolites associated with delayed growth (e.g., proline and myo-inositol). Altogether, our study proposes new routes for improved characterization of piglet maturity at birth.

Multi-species annotation of transcriptome and chromatin structure in domesticated animals.

BACKGROUND: Comparative genomics studies are central in identifying the coding and non-coding elements associated with complex traits, and the functional annotation of genomes is a critical step to decipher the genotype-to-phenotype relationships in livestock animals. As part of the Functional Annotation of Animal Genomes (FAANG) action, the FR-AgENCODE project aimed to create reference functional maps of domesticated animals by profiling the landscape of transcription (RNA-seq), chromatin accessibility (ATAC-seq) and conformation (Hi-C) in species representing ruminants (cattle, goat), monogastrics (pig) and birds (chicken), using three target samples related to metabolism (liver) and immunity (CD4+ and CD8+ T cells). RESULTS: RNA-seq assays considerably extended the available catalog of annotated transcripts and identified differentially expressed genes with unknown function, including new syntenic lncRNAs. ATAC-seq highlighted an enrichment for transcription factor binding sites in differentially accessible regions of the chromatin. Comparative analyses revealed a core set of conserved regulatory regions across species. Topologically associating domains (TADs) and epigenetic A/B compartments annotated from Hi-C data were consistent with RNA-seq and ATAC-seq data. Multi-species comparisons showed that conserved TAD boundaries had stronger insulation properties than species-specific ones and that the genomic distribution of orthologous genes in A/B compartments was significantly conserved across species. CONCLUSIONS: We report the first multi-species and multi-assay genome annotation results obtained by a FAANG project. Beyond the generation of reference annotations and the confirmation of previous findings on model animals, the integrative analysis of data from multiple assays and species sheds a new light on the multi-scale selective pressure shaping genome organization from birds to mammals. Overall, these results emphasize the value of FAANG for research on domesticated animals and reinforces the importance of future meta-analyses of the reference datasets being generated by this community on different species.

Adjacency-constrained hierarchical clustering of a band similarity matrix with application to genomics.

BACKGROUND: Genomic data analyses such as Genome-Wide Association Studies (GWAS) or Hi-C studies are often faced with the problem of partitioning chromosomes into successive regions based on a similarity matrix of high-resolution, locus-level measurements. An intuitive way of doing this is to perform a modified Hierarchical Agglomerative Clustering (HAC), where only adjacent clusters (according to the ordering of positions within a chromosome) are allowed to be merged. But a major practical drawback of this method is its quadratic time and space complexity in the number of loci, which is typically of the order of 10 4 to 10 5 for each chromosome. RESULTS: By assuming that the similarity between physically distant objects is negligible, we are able to propose an implementation of adjacency-constrained HAC with quasi-linear complexity. This is achieved by pre-calculating specific sums of similarities, and storing candidate fusions in a min-heap. Our illustrations on GWAS and Hi-C datasets demonstrate the relevance of this assumption, and show that this method highlights biologically meaningful signals. Thanks to its small time and memory footprint, the method can be run on a standard laptop in minutes or even seconds. AVAILABILITY AND IMPLEMENTATION: Software and sample data are available as an R package, adjclust, that can be downloaded from the Comprehensive R Archive Network (CRAN).

ASICS: an R package for a whole analysis workflow of 1D 1H NMR spectra.

MOTIVATION: In metabolomics, the detection of new biomarkers from Nuclear Magnetic Resonance (NMR) spectra is a promising approach. However, this analysis remains difficult due to the lack of a whole workflow that handles spectra pre-processing, automatic identification and quantification of metabolites and statistical analyses, in a reproducible way. RESULTS: We present ASICS, an R package that contains a complete workflow to analyse spectra from NMR experiments. It contains an automatic approach to identify and quantify metabolites in a complex mixture spectrum and uses the results of the quantification in untargeted and targeted statistical analyses. ASICS was shown to improve the precision of quantification in comparison to existing methods on two independent datasets. In addition, ASICS successfully recovered most metabolites that were found important to explain a two level condition describing the samples by a manual and expert analysis based on bucketing. It also found new relevant metabolites involved in metabolic pathways related to risk factors associated with the condition. AVAILABILITY AND IMPLEMENTATION: ASICS is distributed as an R package, available on Bioconductor. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Large-Scale Measurement of mRNA Degradation in Escherichia coli: To Delay or Not to Delay.

In this study, we compared different computational methods used for genome-wide determination of mRNA half-lives in Escherichia coli with a special focus on the impact on considering a delay before the onset of mRNA decay after transcription arrest. A wide variety of datasets were analyzed coming from different technical methods for mRNA quantification (microarrays, RNA-seq, and RT-qPCR) and different bacterial growth conditions. The exponential decay of mRNA levels was fitted using both linear and exponential models and with or without a delay. We showed that for all the models, independently of mRNA quantification methods and growth conditions, ignoring the delay resulted in only a modest overestimation of the half-life. For approximately 80% of the mRNAs, differences in mRNA half-life values were less than 34s. The correlation between half-lives estimated with and without a delay was extremely high. However, the slope of the linear regression between the half-lives with and without a delay tended to decrease with the delay. For the few mRNAs for which taking into account the delay influenced the estimated half-life, the impact was dependent on the model and the growth condition. The smallest impact was obtained for the linear model.

Unsupervised multiple kernel learning for heterogeneous data integration.

Motivation: Recent high-throughput sequencing advances have expanded the breadth of available omics datasets and the integrated analysis of multiple datasets obtained on the same samples has allowed to gain important insights in a wide range of applications. However, the integration of various sources of information remains a challenge for systems biology since produced datasets are often of heterogeneous types, with the need of developing generic methods to take their different specificities into account. Results: We propose a multiple kernel framework that allows to integrate multiple datasets of various types into a single exploratory analysis. Several solutions are provided to learn either a consensus meta-kernel or a meta-kernel that preserves the original topology of the datasets. We applied our framework to analyse two public multi-omics datasets. First, the multiple metagenomic datasets, collected during the TARA Oceans expedition, was explored to demonstrate that our method is able to retrieve previous findings in a single kernel PCA as well as to provide a new image of the sample structures when a larger number of datasets are included in the analysis. To perform this analysis, a generic procedure is also proposed to improve the interpretability of the kernel PCA in regards with the original data. Second, the multi-omics breast cancer datasets, provided by The Cancer Genome Atlas, is analysed using a kernel Self-Organizing Maps with both single and multi-omics strategies. The comparison of these two approaches demonstrates the benefit of our integration method to improve the representation of the studied biological system. Availability and implementation: Proposed methods are available in the R package mixKernel, released on CRAN. It is fully compatible with the mixOmics package and a tutorial describing the approach can be found on mixOmics web site http://mixomics.org/mixkernel/. Contact: jerome.mariette@inra.fr or nathalie.villa-vialaneix@inra.fr. Supplementary information: Supplementary data are available at Bioinformatics online.

Multiple hot-deck imputation for network inference from RNA sequencing data.

Motivation: Network inference provides a global view of the relations existing between gene expression in a given transcriptomic experiment (often only for a restricted list of chosen genes). However, it is still a challenging problem: even if the cost of sequencing techniques has decreased over the last years, the number of samples in a given experiment is still (very) small compared to the number of genes. Results: We propose a method to increase the reliability of the inference when RNA-seq expression data have been measured together with an auxiliary dataset that can provide external information on gene expression similarity between samples. Our statistical approach, hd-MI, is based on imputation for samples without available RNA-seq data that are considered as missing data but are observed on the secondary dataset. hd-MI can improve the reliability of the inference for missing rates up to 30% and provides more stable networks with a smaller number of false positive edges. On a biological point of view, hd-MI was also found relevant to infer networks from RNA-seq data acquired in adipose tissue during a nutritional intervention in obese individuals. In these networks, novel links between genes were highlighted, as well as an improved comparability between the two steps of the nutritional intervention. Availability and implementation: Software and sample data are available as an R package, RNAseqNet, that can be downloaded from the Comprehensive R Archive Network (CRAN). Contact: alyssa.imbert@inra.fr or nathalie.villa-vialaneix@inra.fr. Supplementary information: Supplementary data are available at Bioinformatics online.

Time course study of the response to LPS targeting the pig immune gene networks.

BACKGROUND: Stress is a generic term used to describe non-specific responses of the body to all kinds of challenges. A very large variability in the response can be observed across individuals, depending on numerous conditioning factors like genetics, early influences and life history. As a result, there is a wide range of individual vulnerability and resilience to stress, also called robustness. The importance of robustness-related traits in breeding strategies is increasing progressively towards the production of animals with a high level of production under a wide range of climatic conditions and management systems, together with a lower environmental impact and a high level of animal welfare. The present study aims at describing blood transcriptomic, hormonal, and metabolic responses of pigs to a systemic challenge using lipopolysaccharide (LPS). The objective is to analyze the individual variation of the biological responses in relation to the activity of the HPA axis measured by the levels of plasma cortisol after LPS and ACTH in 120 juvenile Large White (LW) pigs. The kinetics of the response was measured with biological variables and whole blood gene expression at 4 time points. A multilevel statistical analysis was used to take into account the longitudinal aspect of the data. RESULTS: Cortisol level reaches its peak 4 h after LPS injection. The characteristic changes of white blood cell count to LPS were observed, with a decrease of total count, maximal at t=+4 h, and the mirror changes in the respective proportions of lymphocytes and granulocytes. The lymphocytes / granulocytes ratio was maximal at t=+1 h. An integrative statistical approach was used and provided a set of candidate genes for kinetic studies and ongoing complementary studies focused on the LPS-stimulated inflammatory response. CONCLUSIONS: The present study demonstrates the specific biomarkers indicative of an inflammation in swine. Furthermore, these stress responses persist for prolonged periods of time and at significant expression levels, making them good candidate markers for evaluating the efficacy of anti-inflammatory drugs.

1HNMR-Based metabolomic profiling method to develop plasma biomarkers for sensitivity to chronic heat stress in growing pigs.

The negative impact of heat stress (HS) on the production performances in pig faming is of particular concern. Novel diagnostic methods are needed to predict the robustness of pigs to HS. Our study aimed to assess the reliability of blood metabolome to predict the sensitivity to chronic HS of 10 F1 (Large White × Creole) sire families (SF) reared in temperate (TEMP) and in tropical (TROP) regions (n = 56±5 offsprings/region/SF). Live body weight (BW) and rectal temperature (RT) were recorded at 23 weeks of age. Average daily feed intake (AFDI) and average daily gain were calculated from weeks 11 to 23 of age, together with feed conversion ratio. Plasma blood metabolome profiles were obtained by Nuclear Magnetic Resonance spectroscopy (1HNMR) from blood samples collected at week 23 in TEMP. The sensitivity to hot climatic conditions of each SF was estimated by computing a composite index of sensitivity (Isens) derived from a linear combination of t statistics applied to familial BW, ADFI and RT in TEMP and TROP climates. A model of prediction of sensitivity was established with sparse Partial Least Square Discriminant Analysis (sPLS-DA) between the two most robust SF (n = 102) and the two most sensitive ones (n = 121) using individual metabolomic profiles measured in TEMP. The sPLS-DA selected 29 buckets that enabled 78% of prediction accuracy by cross-validation. On the basis of this training, we predicted the proportion of sensitive pigs within the 6 remaining families (n = 337). This proportion was defined as the predicted membership of families to the sensitive category. The positive correlation between this proportion and Isens (r = 0.97, P < 0.01) suggests that plasma metabolome can be used to predict the sensitivity of pigs to hot climate.

Molecular Biomarkers for Weight Control in Obese Individuals Subjected to a Multiphase Dietary Intervention.

Context: Although calorie restriction has proven beneficial for weight loss, long-term weight control is variable between individuals. Objective: To identify biomarkers of successful weight control during a dietary intervention (DI). Design, Setting, and Participants: Adipose tissue (AT) transcriptomes were compared between 21 obese individuals who either maintained weight loss or regained weight during the DI. Results were validated on 310 individuals from the same study using quantitative reverse transcription polymerase chain reaction and protein levels of potential circulating biomarkers measured by enzyme-linked immunosorbent assay. Intervention: Individuals underwent 8 weeks of low-calorie diet, then 6 months of ad libitum diet. Outcome Measure: Weight changes at the end of the DI. Results: We evaluated six genes that had altered expression during DI, encode secreted proteins, and have not previously been implicated in weight control (EGFL6, FSTL3, CRYAB, TNMD, SPARC, IGFBP3), as well as genes for which baseline expression differed between those with good and poor weight control (ASPN, USP53). Changes in plasma concentrations of EGFL6, FSTL3, and CRYAB mirrored AT messenger RNA expression; all decreased during DI in individuals with good weight control. ASPN and USP53 had higher baseline expression in individuals who went on to have good weight control. Expression quantitative trait loci analysis found polymorphisms associated with expression levels of USP53 in AT. A regulatory network was identified in which transforming growth factor ß1 (TGF-ß1) was responsible for downregulation of certain genes during DI in good controllers. Interestingly, ASPN is a TGF-ß1 inhibitor. Conclusions: We found circulating biomarkers associated with weight control that could influence weight management strategies and genes that may be prognostic for successful weight control.

Time course of the response to ACTH in pig: biological and transcriptomic study.

BACKGROUND: HPA axis plays a major role in physiological homeostasis. It is also involved in stress and adaptive response to the environment. In farm animals in general and specifically in pigs, breeding strategies have highly favored production traits such as lean growth rate, feed efficiency and prolificacy at the cost of robustness. On the hypothesis that the HPA axis could contribute to the trade-off between robustness and production traits, we have designed this experiment to explore individual variation in the biological response to the main stress hormone, cortisol, in pigs. We used ACTH injections to trigger production of cortisol in 120 juvenile Large White (LW) pigs from 28 litters and the kinetics of the response was measured with biological variables and whole blood gene expression at 4 time points. A multilevel statistical analysis was used to take into account the longitudinal aspect of the data. RESULTS: Cortisol level reached its peak 1 h after ACTH injection. White blood cell composition was modified with a decrease of lymphocytes and monocytes and an increase of granulocytes (F D R<0.05). Basal level of cortisol was correlated with birth and weaning weights. Microarray analysis identified 65 unique genes of which expression responded to the injection of ACTH (adjusted P<0.05). These genes were classified into 4 clusters with distinctive kinetics in response to ACTH injection. The first cluster identified genes strongly correlated to cortisol and previously reported as being regulated by glucocorticoids. In particular, DDIT4, DUSP1, FKBP5, IL7R, NFKBIA, PER1, RGS2 and RHOB were shown to be connected to each other by the glucocorticoid receptor NR3C1. Most of the differentially expressed genes that encode transcription factors have not been described yet as being important in transcription networks involved in stress response. Their co-expression may mean co-regulation and they could thus provide new patterns of biomarkers of the individual sensitivity to cortisol. CONCLUSIONS: We identified 65 genes as biological markers of HPA axis activation at the gene expression level. These genes might be candidates for a better understanding of the molecular mechanisms of the stress response.