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Recent studies have shown that repressive chromatin machinery, including DNA methyltransferases and polycomb repressor complexes, binds to chromosomes throughout mitosis and their depletion results in increased chromosome size. In the present study, we show that enzymes that catalyze H3K9 methylation, such as Suv39h1, Suv39h2, G9a and Glp, are also retained on mitotic chromosomes. Surprisingly, however, mutants lacking histone 3 lysine 9 trimethylation (H3K9me3) have unusually small and compact mitotic chromosomes associated with increased histone H3 phospho Ser10 (H3S10ph) and H3K27me3 levels. Chromosome size and centromere compaction in these mutants were rescued by providing exogenous first protein lysine methyltransferase Suv39h1 or inhibiting Ezh2 activity. Quantitative proteomic comparisons of native mitotic chromosomes isolated from wild-type versus Suv39h1/Suv39h2 double-null mouse embryonic stem cells revealed that H3K9me3 was essential for the efficient retention of bookmarking factors such as Esrrb. These results highlight an unexpected role for repressive heterochromatin domains in preserving transcription factor binding through mitosis and underscore the importance of H3K9me3 for sustaining chromosome architecture and epigenetic memory during cell division.

The Ferric uptake regulator (Fur) is crucial to both pathogenic and non-pathogenic bacteria for the maintenance of iron homeostasis as well as the defence against reactive oxygen species. Based on datasets from the genome-wide mapping of transcriptional start sites and transcriptome data, we identified a high confidence regulon controlled by Fur for the model cyanobacterium Synechocystis sp. PCC 6803 and its close relative, strain 6714, based on the conserved strong iron starvation response and Fur-binding site occurrence. This regulon comprises 33 protein-coding genes and the sRNA IsaR1 that are under the control of 16 or 14 individual promoters in strains 6803 and 6714, respectively. The associated gene functions are mostly restricted to transporters and enzymes involved in the uptake and storage of iron ions, with few exceptions or unknown functional relevance. Within the isiABC operon, we identified a previously neglected gene encoding a small cysteine-rich protein, which we suggest calling, IsiE. The regulation of iron uptake, storage, and utilization ultimately results from the interplay between the Fur regulon, several other transcription factors, the FtsH3 protease, and the sRNA IsaR1.

In vitro differentiation of embryonic stem cells (ESCs) provides a convenient basis for the study of microRNA-based gene regulation that is relevant for early cardiogenic processes. However, to which degree insights gained from in vitro differentiation models can be readily transferred to the in vivo system remains unclear. In this study, we profiled simultaneous genome-wide measurements of mRNAs and microRNAs (miRNAs) of differentiating murine ESCs (mESCs) and integrated putative miRNA-gene interactions to assess miRNA-driven gene regulation. To identify interactions conserved between in vivo and in vitro, we combined our analysis with a recent transcriptomic study of early murine heart development in vivo. We detected over 200 putative miRNA–mRNA interactions with conserved expression patterns that were indicative of gene regulation across the in vitro and in vivo studies. A substantial proportion of candidate interactions have been already linked to cardiogenesis, supporting the validity of our approach. Notably, we also detected miRNAs with expression patterns that closely resembled those of key developmental transcription factors. The approach taken in this study enabled the identification of miRNA interactions in in vitro models with potential relevance for early cardiogenic development. Such comparative approaches will be important for the faithful application of stem cells in cardiovascular research. SA 568/17-2 info:eu-repo/semantics/publishedVersion

Aortic dimensions and distensibility are key risk factors for aortic aneurysms and dissections, as well as for other cardiovascular and cerebrovascular diseases. We tested genome-wide associations of ascending and descending aortic distensibility and area derived from cardiac magnetic resonance imaging (MRI) data of up to 32,590 Caucasian individuals in UK Biobank. We identified 102 loci (including 31 novel associations) tagging genes related to cardiovascular development, extracellular matrix production, smooth muscle cell contraction and heritable aortic diseases. Functional analyses highlighted four signalling pathways associated with aortic distensibility (TGF-β, IGF, VEGF and PDGF). We identified distinct sex-specific associations with aortic traits. We developed co-expression networks associated with aortic traits and applied phenome-wide Mendelian randomization (MR-PheWAS), generating evidence for a causal role for aortic distensibility in development of aortic aneurysms. Multivariable MR suggested a causal relationship between aortic distensibility and cerebral white matter hyperintensities, mechanistically linking aortic traits and brain small vessel disease.

The membrane-embedded FtsH proteases found in bacteria, chloroplasts, and mitochondria are involved in diverse cellular processes including protein quality control and regulation. The genome of the model cyanobacterium Synechocystis sp PCC 6803 encodes four FtsH homologs designated FtsH1 to FtsH4. The FtsH3 homolog is present in two hetero-oligomeric complexes: FtsH2/3, which is responsible for photosystem II quality control, and the essential FtsH1/3 complex, which helps maintain Fe homeostasis by regulating the level of the transcription factor Fur. To gain a more comprehensive insight into the physiological roles of FtsH hetero-complexes, we performed genome-wide expression profiling and global proteomic analyses of Synechocystis mutants conditionally depleted of FtsH3 or FtsH1 grown under various nutrient conditions. We show that the lack of FtsH1/3 leads to a drastic reduction in the transcriptional response to nutrient stress of not only Fur but also the Pho, NdhR, and NtcA regulons. In addition, this effect is accompanied by the accumulation of the respective transcription factors. Thus, the FtsH1/3 complex is of critical importance for acclimation to iron, phosphate, carbon, and nitrogen starvation in Synechocystis. Germany Federal Ministry of Education and Research [031L0106B] Grant Agency of the Czech RepublicGrant Agency of the Czech Republic [P501-12-G055] Czech Ministry of Education Ministry of Education, Youth & Sports - Czech Republic [LO1416] Portuguese Fundacao para a Ciencia e a Tecnologia (Foundation for Science and Technology) [PTDC/BIA-MIC/4418/2012, IF/00881/2013, UID/Multi/04326/2013] United Kingdom Biotechnology and Biological Sciences Research Council (BBSRC)Biotechnology and Biological Sciences Research Council (BBSRC) [BB/M012166/1, BB/M000265/1] European Research CouncilEuropean Research Council (ERC) [338895]

Background: Meningioma is the most frequent primary intracranial tumour. Surgical resection remains the main therapeutic option as pharmacological intervention is hampered by poor knowledge of their proteomic signature. There is an urgent need to identify new therapeutic targets and biomarkers of meningioma. Methods: We performed proteomic profiling of grade I, II and III frozen meningioma specimens and three normal healthy human meninges using LC-MS/MS to analyse global proteins, enriched phosphoproteins and phosphopeptides. Differential expression and functional annotation of proteins was completed using Perseus, IPA® and DAVID. We validated differential expression of proteins and phosphoproteins by Western blot on a meningioma validation set and by immunohistochemistry. Findings: We quantified 3888 proteins and 3074 phosphoproteins across all meningioma grades and normal meninges. Bioinformatics analysis revealed commonly upregulated proteins and phosphoproteins to be enriched in Gene Ontology terms associated with RNA metabolism. Validation studies confirmed significant overexpression of proteins such as EGFR and CKAP4 across all grades, as well as the aberrant activation of the downstream PI3K/AKT pathway, which seems differential between grades. Further, we validated upregulation of the total and activated phosphorylated form of the NIMA-related kinase, NEK9, involved in mitotic progression. Novel proteins identified and validated in meningioma included the nuclear proto-oncogene SET, the splicing factor SF2/ASF and the higher-grade specific protein, HK2, involved in cellular metabolism. Interpretation: Overall, we generated a proteomic thesaurus of meningiomas for the identification of potential biomarkers and therapeutic targets. Fund: This study was supported by Brain Tumour Research. Keywords: Meningioma, Proteomics, Phosphoproteins, Differential expression, Grade-specific, RNA metabolism

Funding: This work was supported by the Fundação para a Ciência e a Tecnologia (PTDC/BIM-MED/3363/2014) and Scientific Employment Stimulus to JI (Norma Transitória 8189/2018), predoctoral fellowship to JG (FCT; PD/BD/136919/2018) and postdoctoral fellowship to FC (DAI/ 2019/08/SAICTPAC/0047/2015), and iNOVA4Health-UID/Multi/04462/2013, a program financially supported by Fundação para a Ciência e a Tecnologia/Ministério da Educação e Ciência, through national funds and co-funded by FEDER under the PT2020 Partnership Agreement. Deciphering the clues of a regenerative mechanism for the mammalian adult heart would save millions of lives in the near future. Heart failure due to cardiomyocyte loss is still one of the significant health burdens worldwide. Here, we show the potential of a single molecule, DAND5, in mouse pluripotent stem cell-derived cardiomyocytes specification and proliferation. Dand5 loss-of-function generated the double of cardiac beating foci compared to the wild-type cells. The early formation of cardiac progenitor cells and the increased proliferative capacity of Dand5 KO mESC-derived cardiomyocytes contribute to the observed higher number of derived cardiac cells. Transcriptional profiling sequencing and quantitative RT-PCR assays showed an upregulation of early cardiac gene networks governing cardiomyocyte differentiation, cell cycling, and cardiac regenerative pathways but reduced levels of genes involved in cardiomyocyte maturation. These findings prompt DAND5 as a key driver for the generation and expansion of pluripotent stem cell-derived cardiomyocytes systems with further clinical application purposes. publishersversion published

Meningioma are the most frequent primary intracranial tumour. Management of aggressive meningioma is complex, and development of effective biomarkers or pharmacological interventions is hampered by an incomplete knowledge of molecular landscape. Here, we present an integrated analysis of two complementary omics studies to investigate alterations in the &ldquo, transcriptome&ndash, proteome&rdquo, profile of high-grade (III) compared to low-grade (I) meningiomas. We identified 3598 common transcripts/proteins and revealed concordant up- and downregulation in grade III vs. grade I meningiomas. Concordantly upregulated genes included FABP7, a fatty acid binding protein and the monoamine oxidase MAOB, the latter of which we validated at the protein level and established an association with Food and Drug Administration (FDA)-approved drugs. Notably, we derived a plasma signature of 21 discordantly expressed genes showing positive changes in protein but negative in transcript levels of high-grade meningiomas, including the validated genes CST3, LAMP2, PACS1 and HTRA1, suggesting the acquisition of these proteins by tumour from plasma. Aggressive meningiomas were enriched in processes such as oxidative phosphorylation and RNA metabolism, whilst concordantly downregulated genes were related to reduced cellular adhesion. Overall, our study provides the first transcriptome&ndash, proteome characterisation of meningioma, identifying several novel and previously described transcripts/proteins with potential grade III biomarker and therapeutic significance.

T-cell acute lymphoblastic leukemia (T-ALL) and T-lymphoblastic lymphomas (T-LBL) are aggressive malignancies of thymocytes. The role of thymic microenvironmental cells and stromal factors in thymocyte malignant transformation and T-ALL development remains little explored. Here, using the TEL-JAK2 transgenic (TJ2-Tg) mouse model of T-ALL/LBL, which is driven by constitutive JAK/STAT signaling and characterized by the acquisition of Notch1 mutations, we sought to identify stromal cell alterations associated with thymic leukemogenesis. Immunofluorescence analyses showed that thymic lymphomas presented epithelial areas characterized by keratin (Krt) 5 and Krt8 expression, adjacently to epithelial-free areas negative for Krt expression. Both areas contained abundant laminin (extracellular matrix) and ER-TR7+ (fibroblasts) CD31+ (endothelial) and CD11c+ (dendritic) cells. Besides Krt5, Krt-positive areas harbored medullary thymic epithelial cells (TECs) labeled by Ulex europaeus agglutinin-1. By performing flow cytometry and RNA sequencing analyses of thymic lymphomas, we observed an enrichment in medullary TEC markers in detriment of cortical TEC markers. To assess whether TECs are important for T-ALL/LBL development, we generated TJ2-Tg mice heterozygous for the FoxN1 transcription factor nude null mutation (Foxn1+/nu). Strikingly, in TJ2-Tg;Foxn1+/nu compound mice, both emergence of malignant cells in preleukemic thymi and overt T-ALL onset were significantly delayed. Moreover, in transplantation assays, leukemic cell expansion within the thymus of recipient Foxn1+/nu mice was reduced as compared with control littermates. Since thymopoesis is largely normal in Foxn1+/nu mice, these results indicate that FoxN1 haploinsufficiency in TECs has a more profound impact in thymic leukemogenesis.

Heart development is a complex process, tightly regulated by numerous molecular mechanisms. Key components of the regulatory network underlying heart development are transcription factors (TFs) and microRNAs (miRNAs), yet limited investigation of the role of miRNAs in heart development has taken place. Here, we report the first parallel genome-wide profiling of polyadenylated RNAs and miRNAs in a developing murine heart. These data enable us to identify dynamic activation or repression of numerous biological processes and signaling pathways. More than 200 miRNAs and 25 long non-coding RNAs were differentially expressed during embryonic heart development compared to the mature heart; most of these had not been previously associated with cardiogenesis. Integrative analysis of expression data and potential regulatory interactions suggested 28 miRNAs as novel regulators of embryonic heart development, representing a considerable expansion of the current repertoire of known cardiac miRNAs. To facilitate follow-up investigations, we constructed HeartMiR (http://heartmir.sysbiolab.eu), an open access database and interactive visualization tool for the study of gene regulation by miRNAs during heart development., Graphical Abstract

Transcriptomic data have become a fundamental resource for stem cell (SC) biologists as well as for a wider research audience studying SC-related processes such as aging, embryonic development and prevalent diseases including cancer, diabetes and neurodegenerative diseases. Access and analysis of the growing amount of freely available transcriptomics datasets for SCs, however, are not trivial tasks. Here, we present StemMapper, a manually curated gene expression database and comprehensive resource for SC research, built on integrated data for different lineages of human and mouse SCs. It is based on careful selection, standardized processing and stringent quality control of relevant transcriptomics datasets to minimize artefacts, and includes currently over 960 transcriptomes covering a broad range of SC types. Each of the integrated datasets was individually inspected andmanually curated. StemMapper's user-friendly interface enables fast querying, comparison, and interactive visualization of quality-controlled SC gene expression data in a comprehensive manner. A proof-of-principle analysis discovering novel putative astrocyte/neural SC lineage markers exemplifies the utility of the integrated data resource. We believe that StemMapper can open the way for new insights and advances in SC research by greatly simplifying the access and analysis of SC transcriptomic data. Portuguese Fundacao para a Ciencia e Tecnologia (FCT) [SFRH/BPD/96890/2013, PTDC/BEX-BID/5410/2014, IF/00881/2013, UID/BIM/04773/2013, UID/Multi/04326/2013]; Programa Doutoral ProRegeM-Mecanismos de Doenca e Medicina Regenerativa [PD/00117/2012]; Plymouth University Peninsula Schools of Medicine and Dentistry, Plymouth, Devon, UK info:eu-repo/semantics/publishedVersion

Background The function of the prion protein, involved in the so-called prion diseases, remains a subject of intense debate and the possibility that it works as a pleiotropic protein through the interaction with multiple membrane proteins is somehow supported by recent reports. Therefore, the use of proteomic and bioinformatics combined to uncover cellular processes occurring together with changes in the expression of the prion protein may provide further insight into the putative pleiotropic role of the prion protein. Results This study assessed the membrane-enriched proteome changes accompanying alterations in the expression of the prion protein. A 2D-DIGE approach was applied to two cell lines after prefractionation towards the membrane protein subset: an embryonic stem cell line and the PK1 subline of neuroblastoma cells which efficiently propagates prion infection. Several proteins were differentially abundant with the increased expression of the prion protein during neural differentiation of embryonic stem cells and with the knockdown of the prion protein in PK1 cells. The identity of around 20% of the differentially abundant proteins was obtained by tandem MS. The catalytic subunit A of succinate dehydrogenase, a key enzyme for the aerobic energy metabolism and redox homeostasis, showed a similar abundance trend as the prion protein in both proteomic experiments. A gene ontology analysis revealed “myelin sheath”, “organelle membrane” and “focal adhesion” associated proteins as the main cellular components, and “protein folding” and “ATPase activity” as the biological processes enriched in the first set of differentially abundant proteins. The known interactome of these differentially abundant proteins was customized to reveal four interactors with the prion protein, including two heat shock proteins and a protein disulfide isomerase. Conclusions Overall, our study shows that expression of the prion protein occurs concomitantly with changes in chaperone activity and cell-redox homeostasis, emphasizing the functional link between these cellular processes and the prion protein. Electronic supplementary material The online version of this article (doi:10.1186/s12864-017-3694-6) contains supplementary material, which is available to authorized users.

The membrane-embedded FtsH proteases found in bacteria, chloroplasts, and mitochondria are involved in diverse cellular processes including protein quality control and regulation. The genome of the model cyanobacterium Synechocystis sp PCC 6803 encodes four FtsH homologs designated FtsH1 to FtsH4. The FtsH3 homolog is present in two hetero-oligomeric complexes: FtsH2/3, which is responsible for photosystem II quality control, and the essential FtsH1/3 complex, which helps maintain Fe homeostasis by regulating the level of the transcription factor Fur. To gain a more comprehensive insight into the physiological roles of FtsH hetero-complexes, we performed genome-wide expression profiling and global proteomic analyses of Synechocystis mutants conditionally depleted of FtsH3 or FtsH1 grown under various nutrient conditions. We show that the lack of FtsH1/3 leads to a drastic reduction in the transcriptional response to nutrient stress of not only Fur but also the Pho, NdhR, and NtcA regulons. In addition, this effect is accompanied by the accumulation of the respective transcription factors. Thus, the FtsH1/3 complex is of critical importance for acclimation to iron, phosphate, carbon, and nitrogen starvation in Synechocystis.

Mutations and inadequate methylation profiles of CITED2 are associated with human congenital heart disease (CHD). In mouse, Cited2 is necessary for embryogenesis, particularly for heart development, and its depletion in embryonic stem cells (ESC) impairs cardiac differentiation. We have now determined that Cited2 depletion in ESC affects the expression of transcription factors and cardiopoietic genes involved in early mesoderm and cardiac specification. Interestingly, the supplementation of the secretome prepared from ESC overexpressing CITED2, during the onset of differentiation, rescued the cardiogenic defects of Cited2-depleted ESC. In addition, we demonstrate that the proteins WNT5A and WNT11 held the potential for rescue. We also validated the zebrafish as a model to investigate cited2 function during development. Indeed, the microinjection of morpholinos targeting cited2 transcripts caused developmental defects recapitulating those of mice knockout models, including the increased propensity for cardiac defects and severe death rate. Importantly, the co-injection of anti-cited2 morpholinos with either CITED2 or WNT5A and WNT11 recombinant proteins corrected the developmental defects of Cited2-morphants. This study argues that defects caused by the dysfunction of Cited2 at early stages of development, including heart anomalies, may be remediable by supplementation of exogenous molecules, offering the opportunity to develop novel therapeutic strategies aiming to prevent CHD. Agência financiadora: Fundação para a Ciência e a Tecnologia (FCT) Comissão de Coordenação e Desenvolvimento Regional do Algarve (CCDR Algarve) ALG-01-0145-FEDER-28044; DFG 568/17-2 Algarve Biomedical Center (ABC) Municipio de Loulé info:eu-repo/semantics/publishedVersion

BACKGROUND: Meningioma is the most frequent primary intracranial tumour. Surgical resection remains the main therapeutic option as pharmacological intervention is still hampered by the poor knowledge of the molecular signature of these tumours MATERIAL AND METHODS: In order to elucidate the proteomic profiling of meningiomas and identify proteins involved in their pathogenesis, we completed a comparative mass spectrometry analysis of meningioma tissue of all WHO grades, analysing global proteins, phosphoproteins and phosphopeptides. We performed differential expression analyses and functional annotation studies to identify commonly upregulated proteins and phosphoprotein in all grades of meningioma compared to meningeal tissue as well as grade-specific candidates relevant for tumour progression. Top candidates werevalidated by Western blotting and immunohistochemistry in an additional sample set RESULTS: We confirmed significant overexpression of proteins including EGFR, STAT2 and CKAP4 across all grades, as well as the aberrant activation of the downstream PI3K/AKT pathway, which seems differential between grades. Further, we validated upregulation in all grades of the total and activated phosphorylated form of the NIMArelated kinase, NEK9, involved in mitotic progression. Novel proteins identified in meningioma and validated as commonly overexpressed in all grades were the nuclear proto-oncogene SET and the splicing factor SF2/ASF, while another newly identified protein that was specific for higher grades was hexokinase-2, involved in cellular metabolism CONCLUSION: Overall, we generated a proteomic thesaurus of meningiomas in order to decipher aberrantly expressed proteins and activated pathways; this body of knowledge will eventually lead to the identification of relevant biomarkers and therapeutic targets.

Nitrogen is an essential nutrient for all living organisms. In cyanobacteria, a group of oxygenic photosynthetic bacteria, nitrogen homeostasis is maintained by an intricate regulatory network around the transcription factor NtcA. Although mechanisms controlling NtcA activity appear to be well understood, the sets of genes under its control (i.e., its regulon) remain poorly defined. In this protocol, we describe the procedure for chromatin immunoprecipitation using NtcA antibodies, followed by DNA sequencing analysis (ChIP-seq) during early acclimation to nitrogen starvation in the cyanobacterium Synechocystis sp. PCC 6803 (hereafter Synechocystis). This protocol can be extended to analyze any DNA-binding protein in cyanobacteria for which suitable antibodies exist.

Glycerol offers several advantages as a substrate for biotechnological applications. An important step toward using the popular production host Saccharomyces cerevisiae for glycerol-based bioprocesses has been the fact that in recent studies commonly used S. cerevisiae strains were engineered to grow in synthetic medium containing glycerol as the sole carbon source. For metabolic engineering projects of S. cerevisiae growing on glycerol, characterized promoters are missing. In the current study, we used transcriptome analysis and a yECitrine-based fluorescence reporter assay to select and characterize 25 useful promoters. The promoters of the genes ALD4 and ADH2 showed 4.2-fold and 3-fold higher activities compared to the well-known strong TEF1 promoter. Moreover, the collection contains promoters with graded activities in synthetic glycerol medium and different degrees of glucose repression. To demonstrate the general applicability of the promoter collection, we successfully used a subset of the characterized promoters with graded activities in order to optimize growth on glycerol in an engineered derivative of CEN.PK, in which glycerol catabolism exclusively occurs via a non-native DHA pathway.

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive malignancy of thymocytes. The role of thymic microenvironmental cells and stromal factors in thymocyte malignant transformation and T-ALL development remains little explored. Here, using the TEL-JAK2 transgenic (TJ2-Tg) mouse model of T-ALL, which is driven by constitutive JAK/STAT signaling and characterized by the acquisition ofNotch1mutations, we sought to identify stromal cell alterations associated with thymic leukemogenesis. Immunofluorescence analyses showed that thymic lymphomas presented epithelial areas characterized by keratin 5 and keratin 8 expression, adjacently to keratin-negative, epithelial-free areas. Both keratin-positive and -negative areas stained conspicuously with ER-TR7 (a fibroblast marker), laminin, and CD31 (an endothelial cell marker). Besides keratin 5, keratin-positive areas were also labeled by theUlex Europaeusagglutinin-1 medullary thymic epithelial cell (TEC) marker. To assess whether TECs are important for T-ALL development, we generated TJ2-Tg mice heterozygous for the FoxN1 transcription factornudenull mutation. In contrast tonudehomozygous mice, which lack thymus and thymocytes, heterozygous mutant mice present only mild thymocyte maturation defects. In TJ2-Tg;Foxn1+/nucompound mice both emergence of malignant cells in pre-leukemic thymi and overt T-ALL onset were significantly delayed. Moreover, in transplantation assays leukemic cell expansion in the thymus of recipientFoxn1+/numice was reduced as compared to control littermates. These results indicate that FoxN1 insufficiency impairs specifically thymic leukemogenesis but not thymocyte development.SummaryIn a mouse model of T-ALL, several cellular alterations were detected in thymic lymphomas, including altered epithelial distribution and increased proportion of fibroblasts or endothelial cells. Reduced dosage of FoxN1, a thymic epithelial transcription factor, delayed leukemogenesis in these mice.

The most fascinating aspect of transcriptomics is that the entire set of RNA molecules or transcripts (messenger RNAs, microRNAs, long noncoding RNAs) produced in a population of cells or in tissues can be analyzed simultaneously. While capturing transcript abundance on genome-wide level has become a routine task thanks to high-throughput technologies, analyzing the transcriptome continues to be a challenging task, since the RNA content of a biological entity is heterogeneous and can vary substantially. The abundance of individual transcripts varies from a few copies to hundreds or thousands of copies per cell. The kind and copy numbers of individual transcripts expressed at a given time depend on the developmental stage, external conditions, and environmental stimuli. Quantitative and qualitative alterations of RNAs can be directly linked to the molecular mechanism of disease, reflect the downstream consequences of these disease processes, or even possess prognostic and predictive value.