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Integrating multi-omics technologies to decipher microbiome functions.

Multi-omics approaches have revolutionized our understanding of microbial communities by enabling simultaneous interrogation of genomic, transcriptomic, proteomic, and metabolomic data. The systematic integration and analysis of these deep datasets help decipher the functional roles of microbiomes, providing critical insights into microbial activities, interactions, and dynamics across diverse environments. Biological complexity makes multi-omics analysis of a single, isolated organism demanding but highly informative, yet this complexity increases further when samples comprise hundreds to thousands of individual species. As microbiome research continues to expand into clinical, environmental, and engineered systems, standardized workflows, benchmarked datasets, and community-driven initiatives are essential to ensure reproducibility, standardization and interpretability. Establishing and disseminating best practices for experimental design, data processing, and integrative analyses will be critical for maximizing comparability and scientific rigor across studies. This perspective highlights recent advances in multi-omics microbiome research, outlines key obstacles in data integration and metadata harmonization, and proposes a collaborative roadmap for scalable, FAIR-compliant multi-omics investigations and potentially disruptive Artificial Intelligence (AI) advances comparable to those of AlphaFold in the field of microbiome science.

Multiomics↗

Age-specific hormonal decline is accompanied by transcriptional changes in human sebocytes in vitro.

The importance of hormones in endogenous aging has been displayed by recent studies performed on animal models and humans. To decipher the molecular mechanisms involved in aging we maintained human sebocytes at defined hormone-substituted conditions that corresponded to average serum levels of females from 20 (f20) to 60 (f60) years of age. The corresponding hormone receptor expression was demonstrated by reverse transcription-polymerase chain reaction (RT-PCR), Western blotting and immunocytochemistry. Cells at f60 produced significantly lower lipids than at f20. Increased mRNA and protein levels of c-Myc and increased protein levels of FN1, which have been associated with aging, were detected in SZ95 sebocytes at f60 compared to those detected at f20 after 5 days of treatment. Expression profiling employing a cDNA microarray composed of 15 529 cDNAs identified 899 genes with altered expression levels at f20 vs. f60. Confirmation of gene regulation was performed by real-time RT-PCR. The functional annotation of these genes according to the Gene Ontology identified pathways related to mitochondrial function, oxidative stress, ubiquitin-mediated proteolysis, cell cycle, immune responses, steroid biosynthesis and phospholipid degradation - all hallmarks of aging. Twenty-five genes in common with those identified in aging kidneys and several genes involved in neurodegenerative diseases were also detected. This is the first report describing the transcriptome of human sebocytes and its modification by a cocktail of hormones administered in age-specific levels and provides an in vitro model system, which approximates some of the hormone-dependent changes in gene transcription that occur during aging in humans.

Aging↗

Gene expression variation between mouse inbred strains.

BACKGROUND: In this study, we investigated the effect of genetic background on expression profiles. We analysed the transcriptome of mouse hindlimb muscle of five frequently used mouse inbred strains using spotted oligonucleotide microarrays. RESULTS: Through ANOVA analysis with a false discovery rate of 10%, we show that 1.4% of the analysed genes is significantly differentially expressed between these mouse strains. Differential expression of several of these genes has been confirmed by quantitative RT-PCR. The number of genes affected by genetic background is approximately ten-fold lower than the number of differentially expressed genes caused by a dystrophic genetic defect. CONCLUSIONS: We conclude that evaluation of the effect of background on gene expression profiles in the tissue under study is an effective and sensible approach when comparing expression patterns in animal models with heterogeneous genetic backgrounds. Genes affected by the genetic background can be excluded in subsequent analyses of the disease-related changes in expression profiles. This is often a more effective strategy than backcrossing and inbreeding to obtain isogenic backgrounds.

Animals↗

Microarray based comparison of two Escherichia coli O157:H7 lineages.

BACKGROUND: Previous research has identified the potential for the existence of two separate lineages of Escherichia coli O157:H7. Clinical isolates tended to cluster primarily within one of these two lineages. To determine if there are virulence related genes differentially expressed between the two lineages we chose to utilize microarray technology to perform an initial screening. RESULTS: Using a 610 gene microarray, designed against the E. coli O157 EDL 933 transcriptome, targeting primarily virulence systems, we chose 3 representative Lineage I isolates (LI groups mostly clinical isolates) and 3 representative Lineage II isolates (LII groups mostly bovine isolates). Using standard dye swap experimental designs, statistically different expression (P < 0.05) of 73 genes between the two lineages was revealed. Result highlights indicate that under in vitro anaerobic growth conditions, there is up-regulation of stx2b, ureD, curli (csgAFEG), and stress related genes (hslJ, cspG, ibpB, ibpA) in Lineage I, which may contribute to enhanced virulence or transmission potential. Lineage II exhibits significant up-regulation of type III secretion apparatus, LPS, and flagella related transcripts. CONCLUSION: These results give insight into comparative regulation of virulence genes as well as providing directions for future research. Ultimately, evaluating the expression of key virulence factors among different E. coli O157 isolates has inherent value and the interpretation of such expression data will continue to evolve as our understanding of virulence, pathogenesis and transmission improves.

Escherichia coli O157↗

ForestTreeDB: a database dedicated to the mining of tree transcriptomes.

ForestTreeDB is intended as a resource that centralizes large-scale expressed sequence tag (EST) sequencing results from several tree species (http://foresttree.org/ftdb). It currently encompasses 344,878 quality sequences from 68 libraries, from diverse organs of conifer and hybrid poplar trees. It utilizes the Nimbus data model to provide a hosting system for multiple projects, and uses object-relational mapping APIs in Java and Perl for data accesses within an Oracle database designed to be scalable, maintainable and extendable. Transcriptome builds or unigene sets occupy the focal point of the system. Several of the five current species-specific unigenes were used to design microarrays and SNP resources. The ForestTreeDB web application provides the means for multiple combination database queries. It presents the user with a list of discrete queries to retrieve and download large EST datasets or sequences from precompiled unigene assemblies. Functional annotation assignment is not trivial in conifers which are distantly related to angiosperm model plants. Optimal annotations are achieved through database queries that integrate results from several procedures based open-source tools. ForestTreeDB aims to facilitate sequence mining of coherent annotations in multiple species to support comparative genomic approaches. We plan to continuously enrich ForestTreeDB with other resources through collaborations with other genomic projects.

Databases, Nucleic Acid↗

Role of pituitary hormones on 17alpha-ethinylestradiol-induced cholestasis in rat.

Estrogens cause intrahepatic cholestasis in susceptible women during pregnancy, after administration of oral contraceptives, or during postmenopausal hormone replacement therapy. 17alpha-Ethinylestradiol (EE) is a synthetic estrogen widely used to cause experimental cholestasis in rodents with the aim of examining molecular mechanisms involved in this disease. EE actions on the liver are thought to be mediated by estrogen receptor alpha (ERalpha) and pituitary hormones. We tested this hypothesis by analyzing metabolic changes induced by EE in livers from hypophysectomized (HYPOX) and hypothyroid rats. Microarray studies revealed that the number of genes regulated by EE was increased almost 4-fold in HYPOX rat livers compared with intact males. Little overlap was apparent between the effects of EE in intact and HYPOX rats, demonstrating that pituitary hormones play a critical role in the hepatic effects of EE. Consistently, hypophysectomy protects the liver against induction by EE of serum bilirubin and alkaline phosphatase, two markers of cholestasis and hepatotoxicity and modulates the effects of EE on several genes involved in bile acid homeostasis (e.g., FXR, SHP, BSEP, and Cyp8b1). Finally, we demonstrate a novel mechanism of action of EE through binding and negative regulation of glucocorticoid receptor-mediated transcription. In summary, pituitary- and ERalpha-independent mechanisms contribute to development of EE-induced changes in liver transcriptome. Such mechanisms may be relevant when this model of EE-induced cholestasis is evaluated. The observation that the pharmacological effects of estrogen in liver differ in the absence or presence of the pituitary could be clinically relevant, because different drugs that block actions of pituitary hormones are now available.

Animals↗

Silencing FAF2 mitigates alcohol-induced hepatic steatosis by modulating lipolysis and PCSK9 pathway.

BACKGROUND: Chronic alcohol consumption leads to lipid accumulation, oxidative stress, cellular damage, and inflammation in the liver, collectively referred to as alcohol-associated liver disease (ALD). FAF2/UBXD8/ETEA (Fas-associated factor 2) is a ubiquitin ligase adaptor protein that plays a crucial role in the ubiquitin-mediated degradation of misfolded proteins in the endoplasmic reticulum. A recent genome-wide association study indicated an association between FAF2 and ALD; however, the exact contribution of FAF2 to ALD pathogenesis remains unclear. METHODS: FAF2 was knocked down using AAV-delivered shRNA in C57/BL6 mice. Mice were subjected to a chronic-plus-single binge ethanol feeding (NIAAA) model. Nine hours after gavage, liver, blood, and other organs of interest were collected for gene expression and biochemical analyses. RESULTS: We first observed a significant elevation in hepatic FAF2 protein expression in individuals with ALD and in mice subjected to an ethanol-binge model. Interestingly, knocking down FAF2 in the liver using adeno-associated virus serotype 8-delivered short hairpin RNA conferred a protective effect against alcohol-induced liver steatosis in ethanol-binged mice. Transcriptomic analysis revealed that differentially expressed genes were enriched in multiple lipid metabolism regulation pathways. Further analysis of transcription factors regulating these differentially expressed genes suggested potential regulation by SREBP1. Several SREBP1 target genes, including Fasn, Scd1, Lpin1, and Pcsk9 (proprotein convertase subtilisin/kexin type 9), were dysregulated in the livers of ethanol-fed FAF2 knockdown mice. Additionally, Pcsk9 could be regulated through the FOXO3-SIRT6 pathway in the livers of ethanol-fed FAF2 knockdown mice, leading to increased liver low-density lipoprotein receptor expression and reduced plasma LDL cholesterol levels. Furthermore, FAF2 knockdown in mouse liver enhanced adipose triglyceride lipase lipolytic activity by upregulating the adipose triglyceride lipase activator, comparative gene identification-58, and downregulating the adipose triglyceridelipase transport inhibitor, Elmod2, contributing to the alleviation of liver steatosis. CONCLUSIONS: Our study uncovers a novel mechanism involving FAF2 in the pathogenesis of ALD.

Animals↗

Transcriptome and proteome expression in activated human CD4 and CD8 T-lymphocytes.

T-lymphocytes (T-cells) are unique in that unlike monocytes, they have no insulin receptors, and are insulin insensitive, but upon activation with antigens develop insulin, IGF-1, and IL-2 receptors, and become insulin sensitive tissues. In vivo activation of these cells has now been demonstrated in patients with diabetic ketoacidosis. We analyzed the genomics and proteomics of activated and non-activated CD4+ and CD8+ T-cells of normal subjects using Affymetrix microarray gene chips and proteomes by SELDI-TOF mass spectrometry analysis. Genes for IL-2, insulin, and IGF-1 receptors were increased at least 2-fold in activated vs non-activated T-cells. Using an expression array containing the entire human genome of 39,500 genes, we evaluated approximately 27,000 genes relevant in physiologic and cellular ontologies. Of these, approximately 10,500 genes were increased in activated cells, compared to about 7,000, which were decreased, and approximately 9500, which were unchanged. Among activated ontologies were signal transduction pathways such as IRS-1, IRS-2, Akt, and glycolytic pathways. To our knowledge this is the first report of an hitherto unreported event. Possible implications of these processes are discussed in the light of their physiological significance.

Adult↗

Transcript analysis reveals an extended regulon and the importance of protein-protein co-operativity for the Escherichia coli methionine repressor.

We have used DNA arrays to investigate the effects of knocking out the methionine repressor gene, metJ, on the Escherichia coli transcriptome. We assayed the effects in the knockout strain of supplying wild-type or mutant MetJ repressors from an expression plasmid, thus establishing a rapid assay for in vivo effects of mutations characterized previously in vitro. Repression is largely restricted to known genes involved in the biosynthesis and uptake of methionine. However, we identified a number of additional genes that are significantly up-regulated in the absence of repressor. Sequence analysis of the 5' promoter regions of these genes identified plausible matches to met-box sequences for three of these, and subsequent electrophoretic mobility-shift assay analysis showed that for two such loci their repressor affinity is higher than or comparable with the known metB operator, suggesting that they are directly regulated. This can be rationalized for one of the loci, folE, by the metabolic role of its encoded enzyme; however, the links to the other regulated loci are unclear, suggesting both an extension to the known met regulon and additional complexity to the role of the repressor. The plasmid gene replacement system has been used to examine the importance of protein-protein co-operativity in operator saturation using the structurally characterized mutant repressor, Q44K. In vivo, there are detectable reductions in the levels of regulation observed, demonstrating the importance of balancing protein-protein and protein-DNA affinity.

Base Sequence↗

A dual-genome Symbiosis Chip for coordinate study of signal exchange and development in a prokaryote-host interaction.

The soil-dwelling alpha-proteobacterium Sinorhizobium meliloti engages in a symbiosis with legumes: S. meliloti elicits the formation of plant root nodules where it converts dinitrogen to ammonia for use by the plant in exchange for plant photosynthate. To study the coordinate differentiation of S. meliloti and its legume partner during nodule development, we designed a custom Affymetrix GeneChip with the complete S. meliloti genome and approximately 10,000 probe sets for the plant host, Medicago truncatula. Expression profiling of free-living S. meliloti grown with the plant signal molecule luteolin in defined minimal and rich media or of strains altered in the expression of key regulatory proteins (NodD1, NodD3, and RpoN) confirms previous data and identifies previously undescribed regulatory targets. Analyses of root nodules show that this Symbiosis Chip allows the study of gene expression in both partners simultaneously. Our studies detail nearly 5,000 transcriptome changes in symbiosis and document complex transcriptional profiles of S. meliloti in different environments.

Bacterial Proteins↗

Analysis of the transcriptome of the protozoan Theileria parva using MPSS reveals that the majority of genes are transcriptionally active in the schizont stage.

Massively parallel signature sequencing (MPSS) was used to analyze the transcriptome of the intracellular protozoan Theileria parva. In total 1,095,000, 20 bp sequences representing 4371 different signatures were generated from T.parva schizonts. Reproducible signatures were identified within 73% of potentially detectable predicted genes and 83% had signatures in at least one MPSS cycle. A predicted leader peptide was detected on 405 expressed genes. The quantitative range of signatures was 4-52,256 transcripts per million (t.p.m.). Rare transcripts (<50 t.p.m.) were detected from 36% of genes. Sequence signatures approximated a lognormal distribution, as in microarray. Transcripts were widely distributed throughout the genome, although only 47% of 138 telomere-associated open reading frames exhibited signatures. Antisense signatures comprised 13.8% of the total, comparable with Plasmodium. Eighty five predicted genes with antisense signatures lacked a sense signature. Antisense transcripts were independently amplified from schizont cDNA and verified by sequencing. The MPSS transcripts per million for seven genes encoding schizont antigens recognized by bovine CD8 T cells varied 1000-fold. There was concordance between transcription and protein expression for heat shock proteins that were very highly expressed according to MPSS and proteomics. The data suggests a low level of baseline transcription from the majority of protein-coding genes.

Animals↗

ASPIC: a web resource for alternative splicing prediction and transcript isoforms characterization.

Alternative splicing (AS) is now emerging as a major mechanism contributing to the expansion of the transcriptome and proteome complexity of multicellular organisms. The fact that a single gene locus may give rise to multiple mRNAs and protein isoforms, showing both major and subtle structural variations, is an exceptionally versatile tool in the optimization of the coding capacity of the eukaryotic genome. The huge and continuously increasing number of genome and transcript sequences provides an essential information source for the computational detection of genes AS pattern. However, much of this information is not optimally or comprehensively used in gene annotation by current genome annotation pipelines. We present here a web resource implementing the ASPIC algorithm which we developed previously for the investigation of AS of user submitted genes, based on comparative analysis of available transcript and genome data from a variety of species. The ASPIC web resource provides graphical and tabular views of the splicing patterns of all full-length mRNA isoforms compatible with the detected splice sites of genes under investigation as well as relevant structural and functional annotation. The ASPIC web resource-available at http://www.caspur.it/ASPIC/--is dynamically interconnected with the Ensembl and Unigene databases and also implements an upload facility.

Algorithms↗

Molecular characterization of the developmental gene in eyes: through data-mining on integrated transcriptome databases.

OBJECTIVES: Our aim was to utilize publicly available and proprietary sources to discover candidate genes important for ocular development. DESIGN AND METHODS: The collated information on our 5092 non-redundant clusters was grouped and functional annotation was conducted using gene ontology (FatiGO) for categorizing them with respect to molecular function. The web-based viewer technological platform (H-InvDB) was employed for transcription analyses of in-house high quality fetal eye Expressed Sequence Tags (ESTs). Eye-specific ESTs were also analyzed across species by using EMBEST. RESULTS: According to adult eye cDNA libraries, nucleic acid binding and cell structure/cytoskeletal protein genes were the most abundant among the ESTs of fetal eyes. Using cDNA assembly in H-InvDB, 20 (80%) of the 25 most commonly expressed genes in the human eye are also expressed in extraocular tissues. The crystalline gamma S gene is highly expressed in the eye, but not in other tissues. We used EMBEST to compare human fetal eye and octopus eye ESTs and the expression similarity was low (1.6%). This indicated that our fetal eye library contains genes necessary for the developmental process and biological function of the eye, which may not be expressed in the fully developed octopus eyes. The human fetal eye cDNA library also contained highly abundant eye tissue genes, including alphaA-crystallin, eukaryotic translation elongation factor 1 alpha 1 (EEF1A1), bestrophin (VMD2), cystatin C, and transforming growth factor, beta-induced (BIGH3). CONCLUSIONS: Our annotated EST set provides a valuable resource for gene discovery and functional genomic analysis. This display will help to appreciate the strengths and weaknesses of the different technological platforms, so that in future studies the maximum amount of beneficial information can be derived from the appropriate use of each method.

Animals↗

Effector loss and gain drives host range at a fitness cost.

Epidemic preparedness depends on tracking microbial evolution that drives shifts in ecological behaviors such as disease emergence. However, the genetic constraints mediating microbial emergence for generalist and specialist behaviors remain poorly described. Here, we addressed this question by combining comparative and functional genomics with phylogeny-based evolutionary analyses of the cereal pathogen Xanthomonas translucens. We show that a generalist X. translucens subgroup arose from a specialist ancestor, and the loss of a single effector gene, xopAL1, contributed to the generalist host expansion by promoting host jump from barley to wheat. Deleting barley-specialist X. translucens xopAL1 recapitulated the host jump to wheat and demonstrates risk across each globally distributed genetic lineage. However, this niche expansion via XopAL1 loss incurs a significant fitness cost to colonize barley. Moreover, the specialist lineage gained an additional effector gene, xopAJ, which enhanced virulence on barley while restricting oat infection, thereby reinforcing niche specialization. We further conducted transcriptomic analysis of wheat and determined that XopAL1 triggers a defense response that involves the reduction of photosynthetic processes. Our work provides an experimentally validated evolutionary framework to understand mechanisms of intergenera host jump. Overall, we demonstrate that single events of gene loss and gain shape ecological behaviors by creating a dynamic trade-off between niche breadth and specialization.

Triticum↗

Biomphalaria glabrata transcriptome: identification of cell-signalling, transcriptional control and immune-related genes from open reading frame expressed sequence tags (ORESTES).

Biomphalaria glabrata is the major intermediate snail host for Schistosoma mansoni, one of the important schistosomes infecting man. Much remains to be discovered concerning specific molecules mediating the defence events in these intermediate hosts, triggered by invading schistosomes. An expressed sequence tag (EST) gene discovery strategy known as ORESTES has been employed to identify transcripts that might be involved in snail-schistosome interactions in order to examine gene expression patterns in infected B. glabrata. Over 3930 ESTs were sequenced from cDNA libraries made from both schistosome-exposed and unexposed snails using different tissue types, producing a database of 1843 non-redundant clones. The non-redundant set has been assessed for gene ontology and KEGG pathway assignments. This approach has revealed a number of signalling, antioxidant and immune-related gene homologues that, based on current understanding of molluscan and other comparative systems, might play an important role in the molluscan defence response towards infection.

Animals↗

Mouse proteome analysis.

A general overview of the protein sequence set for the mouse transcriptome produced during the FANTOM2 sequencing project is presented here. We applied different algorithms to characterize protein sequences derived from a nonredundant representative protein set (RPS) and a variant protein set (VPS) of the mouse transcriptome. The functional characterization and assignment of Gene Ontology terms was done by analysis of the proteome using InterPro. The Superfamily database analyses gave a detailed structural classification according to SCOP and provide additional evidence for the functional characterization of the proteome data. The MDS database analysis revealed new domains which are not presented in existing protein domain databases. Thus the transcriptome gives us a unique source of data for the detection of new functional groups. The data obtained for the RPS and VPS sets facilitated the comparison of different patterns of protein expression. A comparison of other existing mouse and human protein sequence sets (e.g., the International Protein Index) demonstrates the common patterns in mammalian proteomes. The analysis of the membrane organization within the transcriptome of multiple eukaryotes provides valuable statistics about the distribution of secretory and transmembrane proteins

Animals↗

Contribution of the Saccharomyces cerevisiae transcriptional regulator Leu3p to physiology and gene expression in nitrogen- and carbon-limited chemostat cultures.

Transcriptional regulation of branched-chain amino-acid metabolism in Saccharomyces cerevisiae involves two key regulator proteins, Leu3p and Gcn4p. Leu3p is a pathway-specific regulator, known to regulate six genes involved in branched-chain amino-acid metabolism and one gene in nitrogen assimilation. Gcn4p is a global regulator, involved in the general response to amino-acid and purine starvation. To investigate the contribution of Leu3p in regulation of gene expression, a leu3Delta strain was compared to an isogenic reference strain using DNA-microarray analysis. This comparison was performed for both glucose-grown/ammonium-limited and ethanol-limited/ammonium-excess chemostat cultures. In ethanol-limited cultures, absence of Leu3p led to reduced transcript levels of six of the seven established Leu3p target genes, but did not affect key physiological parameters. In ammonium-limited cultures, absence of Leu3p caused a drastic decrease in storage carbohydrate content. mRNA levels of genes involved in storage carbohydrate metabolism were also found reduced. Under N-limited conditions, the leu3Delta genotype elicited an amino-acid starvation response, leading to increased transcript levels of many amino-acid biosynthesis genes. By combining the transcriptome data with data from earlier studies that measured DNA binding of Leu3p both in vitro and in vivo, BAT1, GAT1 and OAC1 were identified as additional Leu3p-regulated genes. This study demonstrates that unravelling of transcriptional regulation networks should preferably include several cultivation conditions and requires a combination of experimental approaches.

Culture Media↗

PathMED: an R toolkit for single-sample molecular scoring and machine learning with omics data.

MOTIVATION: Molecular scoring is a popular approach for studying pathway-level functional alterations with omics data. Using molecular scores for tasks such as single-sample molecular characterisation, phenotype prediction or disease stratification has several advantages compared to using omics data directly. Molecular scores provide biological interpretability and are more generalisable across datasets, facilitating data integration and machine learning applications. However, numerous scoring methods are available through different software packages, and currently there is a lack of tools to easily use these scores for model training and prediction. RESULTS: We developed pathMED, an R/Bioconductor package that unifies various scoring methods in a simple framework. Furthermore, pathMED also contains a machine learning module to train and test models that use the calculated molecular scores to predict clinical outcomes. We demonstrate some of its potential applications in three use cases using public omics data. We showed the generalisability of machine learning models trained on transcriptomic scores in predicting clinical outcomes when deploying on proteomic scores. We also demonstrated the application of transcriptomics scores in predicting breast cancer treatment response and identifying pathways strongly associated to tumour biology and treatment response. Finally, we demonstrated the benefit of integrating a novel gene set dissection step into the analysis pipeline to resolve disease heterogeneity at the pathway level. AVAILABILITY: PathMED is freely available in the Bioconductor repository (https://bioconductor.org/packages/release/bioc/html/pathMED.html). Code to reproduce the analyses is publicly available at https://github.com/GENyO-BioInformatics/pathMED_article.

Software↗