Single-cell resolution of longitudinal blood transcriptome profiles in rheumatoid arthritis, systemic lupus erythematosus and healthy control pregnancies.

OBJECTIVES: Comparative longitudinal analyses of cellular composition and peripheral blood gene expression in Rheumatoid arthritis (RA), systemic lupus erythematosus (SLE) and healthy pregnancies. METHODS: In total, 335 whole blood samples from 84 RA, SLE and healthy controls before pregnancy, at each trimester, 6 weeks, 6 months and 12 months post partum were analysed. We combined bulk and single cell RNA analyses for cell-type estimation, validated by flow cytometry, before combining this in a cell-type adjusted analysis for an improved resolution of unrecognised gene expression changes associated with RA and SLE pregnancies. RESULTS: Patients were well regulated throughout pregnancy, and few had pregnancy complications. In SLE, the interferon signature was augmented during pregnancy, and the pregnancy signature was continued post partum. An altered cell type composition strongly influences the profile. In the pregnancy signature, transcripts involved in galactosylation potentially altering the effector functions of autoantibodies became more evident. Several genes in the adjusted RA signature are expressed in mucosal associated invariant T cells. CONCLUSION: We found distinct RA, SLE and pregnancy signatures, and no expression patterns could be attributed to medication or disease activity. Our results support the need for close postpartum follow-up of patients with SLE. Gene expression patterns in RA were closer to healthy controls than to SLE, and primarily became evident after cell-type adjustment. Adjusting for cell abundance unravelled gene expression signatures less associated with variation in cell-composition and highlighted genes with expression profiles associated with changes in specialised cell populations.

RPA guides UNG to uracil in ssDNA to facilitate antibody class switching and repair of mutagenic uracil at the replication fork.

Activation-induced cytidine deaminase (AID) interacts with replication protein A (RPA), the major ssDNA-binding protein, to promote deamination of cytosine to uracil in transcribed immunoglobulin (Ig) genes. Uracil-DNA glycosylase (UNG) acts in concert with AID during Ig diversification. In addition, UNG preserves genome integrity by base-excision repair (BER) in the overall genome. How UNG is regulated to support both mutagenic processing and error-free repair remains unknown. UNG is expressed as two isoforms, UNG1 and UNG2, which both contain an RPA-binding helix that facilitates uracil excision from RPA-coated ssDNA. However, the impact of this interaction in antibody diversification and genome maintenance has not been investigated. Here, we generated B-cell clones with targeted mutations in the UNG RPA-binding motif, and analysed class switch recombination (CSR), mutation frequency (5' Ig Sµ), and genomic uracil in clones representing seven Ung genotypes. We show that the UNG:RPA interaction plays a crucial role in both CSR and repair of AID-induced uracil at the Ig loci. By contrast, the interaction had no significant impact on total genomic uracil levels. Thus, RPA coordinates UNG during CSR and pre-replicative repair of mutagenic uracil in ssDNA but is not essential in post-replicative and canonical BER of uracil in dsDNA.

TFCheckpoint database update, a cross-referencing system for transcription factors from human, mouse and rat.

Prior knowledge about DNA-binding transcription factors (dbTFs), transcription co-regulators (coTFs) and general transcriptional factors (GTFs) is crucial for the study and understanding of the regulation of transcription. This is reflected by the many publications and database resources describing knowledge about TFs. We previously launched the TFCheckpoint database, an integrated resource focused on human, mouse and rat dbTFs, providing users access to a comprehensive overview of these proteins. Here, we describe TFCheckpoint 2.0 (https://www.tfcheckpoint.org/index.php), comprising 13 collections of dbTFs, coTFs and GTFs. TFCheckpoint 2.0 provides an easy and versatile cross-referencing system for users to view and download collections that may otherwise be cumbersome to find, compare and retrieve.

MicroRNA profiling of psoriatic skin identifies 11 miRNAs associated with disease severity.

MicroRNAs (miRNAs) are small non-coding RNAs that have emerged as central regulators of gene expression and powerful biomarkers of disease. Much is yet unknown about their role in psoriasis pathology. To globally characterize the miRNAome of psoriatic skin, skin biopsies were collected from psoriatic cases (n = 75) and non-psoriatic controls (n = 46) and RNA sequenced. Count data were meta-analysed with a previously published dataset (cases, n = 24, controls, n = 20), increasing the number of psoriatic cases fourfold from previously published studies. Differential gene expression analyses were performed comparing lesional psoriatic (PP), non-lesional psoriatic (PN) and control (NN) skin. Further, functional enrichment and cell-specific analyses were performed. Across all contrasts, we identified 439 significantly differentially expressed miRNAs (DEMs), of which 85 were novel for psoriasis and 11 were related to disease severity. Meta-analyses identified 20 DEMs between PN and NN, suggesting an inherent change in the constitution of all skin in psoriasis. By integrating the miRNA transcriptome with mRNA target interactions, we identified several functionally enriched terms, including "thyroid hormone signalling," "insulin resistance" and various infectious diseases. Cell-specific expression analyses revealed that the upregulated DEMs were enriched in epithelial and immune cells. This study provides the most comprehensive overview of the miRNAome in psoriatic skin to date and identifies a miRNA signature related to psoriasis severity. Our results may represent molecular links between psoriasis and related comorbidities and have outlined potential directions for future functional studies to identify biomarkers and treatment targets.

Non-Coding RNAs in Human Breast Milk: A Systematic Review.

Breast milk is the primary source of nutrition and hydration for the newborn infant but also plays an important role in the child's first immune defense. Additionally, several breast milk factors have been implicated in immune-related health outcomes later in life, including immunoglobulins, cytokines, chemokines, growth factors and, more recently, non-coding RNA (ncRNA) species. In this systematic review, we provide a comprehensive summary of the current literature on endogenous ncRNAs found in human breast milk. Thirty (30) relevant studies were identified and, whilst the majority studies focused on microRNAs (miRNAs), there is evidence that breast milk contains high quantities of RNA which also include long-coding RNAs, circular RNAs, as well as other short RNAs and fragmented tRNA and rRNAs. Among studies investigating miRNAs, miR-148a-3p, miR-30a/d-5p, miR-22-3p, miR-146b-5p, miR-200a/c-3p, and the 5p end of the let-7 miRNAs were commonly reported among the top 10 miRNAs in the cell, lipid, and skim milk fractions of breast milk. Methodological difference and small sample sizes limit the possibility of conclusively identifying which maternal and infant characteristics affect the miRNA profile. The highly expressed miRNAs were generally reported to be similar across lactational stage, milk fraction, maternal and infant characteristics, or infant growth and health. All the same, individual studies identify potential differences in miRNA expression levels which should be confirmed by future studies. Stability, uptake, and physiological functions of miRNAs were also considered in several studies. Breast milk miRNAs are relatively resistant to a range of harsh conditions and uptake experiments suggest that extracellular vesicles containing miRNAs and circular RNAs can be taken up by intestinal epithelial cells. Although the evidence regarding the functional effect of breast milk miRNAs is limited, the predicted functions range from metabolic and biosynthetic processes to signaling pathways, cellular adhesion, communication, growth, and differentiation. Finally, this systematic review highlights some of the methodological challenges and knowledge gaps which can help direct future research in this field. In particular, it is important to further investigate the bioavailability of miRNAs in different milk fractions, and to characterize other ncRNAs which are largely unstudied. Systematic Review Registration: PROSPERO https://www.crd.york.ac.uk/prospero/display_record.php?RecordID=138989, identifier CRD42020138989.

Joint changes in RNA, RNA polymerase II, and promoter activity through the cell cycle identify non-coding RNAs involved in proliferation.

Proper regulation of the cell cycle is necessary for normal growth and development of all organisms. Conversely, altered cell cycle regulation often underlies proliferative diseases such as cancer. Long non-coding RNAs (lncRNAs) are recognized as important regulators of gene expression and are often found dysregulated in diseases, including cancers. However, identifying lncRNAs with cell cycle functions is challenging due to their often low and cell-type specific expression. We present a highly effective method that analyses changes in promoter activity, transcription, and RNA levels for identifying genes enriched for cell cycle functions. Specifically, by combining RNA sequencing with ChIP sequencing through the cell cycle of synchronized human keratinocytes, we identified 1009 genes with cell cycle-dependent expression and correlated changes in RNA polymerase II occupancy or promoter activity as measured by histone 3 lysine 4 trimethylation (H3K4me3). These genes were highly enriched for genes with known cell cycle functions and included 57 lncRNAs. We selected four of these lncRNAs-SNHG26, EMSLR, ZFAS1, and EPB41L4A-AS1-for further experimental validation and found that knockdown of each of the four lncRNAs affected cell cycle phase distributions and reduced proliferation in multiple cell lines. These results show that many genes with cell cycle functions have concomitant cell-cycle dependent changes in promoter activity, transcription, and RNA levels and support that our multi-omics method is well suited for identifying lncRNAs involved in the cell cycle.

Comprehensive transcriptomic analyses of tissue, serum, and serum exosomes from hepatocellular carcinoma patients.

BACKGROUND: The expression of microRNAs (miRNAs) is a promising prognostic and diagnostic tool in hepatocellular carcinoma (HCC). Here we performed small RNA sequencing (sRNA-seq) of tissue, serum and serum exosomes to investigate changes in miRNA expression between the different sample types and correlated the expression with clinical parameters. We also performed gene expression arrays on tumor and normal tissue. RESULTS: Paired tissue, serum and serum exosomes sequencing revealed consistent positive correlation of miR-21 between serum exosomes and tumor tissue, indicating that miR-21 could be exported from tissue to circulation via exosomes. We found that let-7 miRNAs are generally upregulated in serum exosomes compared to whole serum, indicating that these miRNAs could be preferentially loaded into exosomes. Comparing serum from HCC patients with serum from healthy individuals revealed a global increase of miRNAs in serum from HCC patients, including an almost 4-fold increase of several miRNAs, including the liver-specific miR-122. When correlating miRNA expression with clinical parameters we detected significant association between hepatitis B virus (HBV) infection and miR-122 in serum as well as several serum and tissue-miRNAs that correlated with surgery type. We found that miR-141 and miR-146 correlated with cirrhosis in tumor tissue and normal tissue, respectively. Finally, high expression of miR-21 in tumors were associated with poor survival. Focusing on gene expression we found several significant messenger RNAs (mRNAs) between tumor and normal tissue and a Gene Ontology (GO) analysis revealed that these changes were mainly related to cell cycle and metabolism. Further, we detected mRNAs that correlated with cirrhosis and HBV infection in tissue. Finally, GO analysis of predicted targets for miRNAs down-regulated in tumor found that these were enriched for functions related to collagen synthesis. CONCLUSIONS: Our combined data point to altered miRNA and mRNA expression contributing to both generally impaired lipid metabolism and increased cell proliferation and a miRNA-driven increase in collagen synthesis in HCC. Our results further indicate a correlation in miRNA expression between exosomes, serum, and tissue samples suggesting export from tumors via exosomes. This correlation could provide a basis for a more tumor-specific miRNA profile in serum.

Nephronectin mediates p38 MAPK-induced cell viability via its integrin-binding enhancer motif.

Nephronectin (NPNT) is an extracellular matrix (ECM) protein involved in kidney development. We recently reported intracellular NPNT as a potential prognostic marker in breast cancer and that NPNT promotes metastasis in an integrin-dependent manner. Here, we used reverse-phase protein array (RPPA) to analyze NPNT-triggered intracellular signaling in the 66cl4 mouse breast cancer cell line. The results showed that the integrin-binding enhancer motif is important for the cellular effects upon NPNT interaction with its receptors, including phosphorylation of p38 mitogen-activated protein kinase (MAPK). Furthermore, analysis using prediction tools suggests involvement of NPNT in promoting cell viability. In conclusion, our results indicate that NPNT, via its integrin-binding motifs, promotes cell viability through phosphorylation of p38 MAPK.

Gene regulation knowledge commons: community action takes care of DNA binding transcription factors.

A large gap remains between the amount of knowledge in scientific literature and the fraction that gets curated into standardized databases, despite many curation initiatives. Yet the availability of comprehensive knowledge in databases is crucial for exploiting existing background knowledge, both for designing follow-up experiments and for interpreting new experimental data. Structured resources also underpin the computational integration and modeling of regulatory pathways, which further aids our understanding of regulatory dynamics. We argue how cooperation between the scientific community and professional curators can increase the capacity of capturing precise knowledge from literature. We demonstrate this with a project in which we mobilize biological domain experts who curate large amounts of DNA binding transcription factors, and show that they, although new to the field of curation, can make valuable contributions by harvesting reported knowledge from scientific papers. Such community curation can enhance the scientific epistemic process.Database URL: http://www.tfcheckpoint.org.

Genes2GO: A web application for querying gene sets for specific GO terms.

Gene ontology annotations have become an essential resource for biological interpretations of experimental findings. The process of gathering basic annotation information in tables that link gene sets with specific gene ontology terms can be cumbersome, in particular if it requires above average computer skills or bioinformatics expertise. We have therefore developed Genes2GO, an intuitive R-based web application. Genes2GO uses the biomaRt package of Bioconductor in order to retrieve custom sets of gene ontology annotations for any list of genes from organisms covered by the Ensembl database. Genes2GO produces a binary matrix file, indicating for each gene the presence or absence of specific annotations for a gene. It should be noted that other GO tools do not offer this user-friendly access to annotations. AVAILABILITY: Genes2GO is freely available and listed under http://www.semantic-systems-biology.org/tools/externaltools/.

The gastrin and cholecystokinin receptors mediated signaling network: a scaffold for data analysis and new hypotheses on regulatory mechanisms.

BACKGROUND: The gastrointestinal peptide hormones cholecystokinin and gastrin exert their biological functions via cholecystokinin receptors CCK1R and CCK2R respectively. Gastrin, a central regulator of gastric acid secretion, is involved in growth and differentiation of gastric and colonic mucosa, and there is evidence that it is pro-carcinogenic. Cholecystokinin is implicated in digestion, appetite control and body weight regulation, and may play a role in several digestive disorders. RESULTS: We performed a detailed analysis of the literature reporting experimental evidence on signaling pathways triggered by CCK1R and CCK2R, in order to create a comprehensive map of gastrin and cholecystokinin-mediated intracellular signaling cascades. The resulting signaling map captures 413 reactions involving 530 molecular species, and incorporates the currently available knowledge into one integrated signaling network. The decomposition of the signaling map into sub-networks revealed 18 modules that represent higher-level structures of the signaling map. These modules allow a more compact mapping of intracellular signaling reactions to known cell behavioral outcomes such as proliferation, migration and apoptosis. The integration of large-scale protein-protein interaction data to this literature-based signaling map in combination with topological analyses allowed us to identify 70 proteins able to increase the compactness of the map. These proteins represent experimentally testable hypotheses for gaining new knowledge on gastrin- and cholecystokinin receptor signaling. The CCKR map is freely available both in a downloadable, machine-readable SBML-compatible format and as a web resource through PAYAO ( http://sblab.celldesigner.org:18080/Payao11/bin/). CONCLUSION: We have demonstrated how a literature-based CCKR signaling map together with its protein interaction extensions can be analyzed to generate new hypotheses on molecular mechanisms involved in gastrin- and cholecystokinin-mediated regulation of cellular processes.

EssOilDB: a database of essential oils reflecting terpene composition and variability in the plant kingdom.

Plant essential oils are complex mixtures of volatile organic compounds, which play indispensable roles in the environment, for the plant itself, as well as for humans. The potential biological information stored in essential oil composition data can provide an insight into the silent language of plants, and the roles of these chemical emissions in defense, communication and pollinator attraction. In order to decipher volatile profile patterns from a global perspective, we have developed the ESSential OIL DataBase (EssOilDB), a continually updated, freely available electronic database designed to provide knowledge resource for plant essential oils, that enables one to address a multitude of queries on volatile profiles of native, invasive, normal or stressed plants, across taxonomic clades, geographical locations and several other biotic and abiotic influences. To our knowledge, EssOilDB is the only database in the public domain providing an opportunity for context based scientific research on volatile patterns in plants. EssOilDB presently contains 123 041 essential oil records spanning a century of published reports on volatile profiles, with data from 92 plant taxonomic families, spread across diverse geographical locations all over the globe. We hope that this huge repository of VOCs will facilitate unraveling of the true significance of volatiles in plants, along with creating potential avenues for industrial applications of essential oils. We also illustrate the use of this database in terpene biology and show how EssOilDB can be used to complement data from computational genomics to gain insights into the diversity and variability of terpenoids in the plant kingdom. EssOilDB would serve as a valuable information resource, for students and researchers in plant biology, in the design and discovery of new odor profiles, as well as for entrepreneurs--the potential for generating consumer specific scents being one of the most attractive and interesting topics in the cosmetic industry. Database URL: http://nipgr.res.in/Essoildb/

TFcheckpoint: a curated compendium of specific DNA-binding RNA polymerase II transcription factors.

SUMMARY: Gene regulatory network assembly and analysis requires high-quality knowledge sources that cover functional aspects of the various components of the gene regulatory machinery. A multiplicity of resources exists with information about mammalian transcription factors (TFs); yet, only few of these provide sufficiently accurate classifications of the functional roles of individual TFs, or standardized evidence that would justify the information on which these functional classifications are based. We compiled the list of all putative TFs from nine different resources, ignored factors such as general TFs, mediator complexes and chromatin modifiers, and for the remaining factors checked the available literature for references that support their function as a true sequence-specific DNA-binding RNA polymerase II TF (DbTF). The results are available in the TFcheckpoint database, an exhaustive collection of TFs annotated according to experimental and other evidence on their function as true DbTFs. TFcheckpoint.org provides a high-quality and comprehensive knowledge source for genome-scale regulatory network studies. AVAILABILITY: The TFcheckpoint database is freely available at www.tfcheckpoint.org

ZifBASE: a database of zinc finger proteins and associated resources.

BACKGROUND: Information on the occurrence of zinc finger protein motifs in genomes is crucial to the developing field of molecular genome engineering. The knowledge of their target DNA-binding sequences is vital to develop chimeric proteins for targeted genome engineering and site-specific gene correction. There is a need to develop a computational resource of zinc finger proteins (ZFP) to identify the potential binding sites and its location, which reduce the time of in vivo task, and overcome the difficulties in selecting the specific type of zinc finger protein and the target site in the DNA sequence. DESCRIPTION: ZifBASE provides an extensive collection of various natural and engineered ZFP. It uses standard names and a genetic and structural classification scheme to present data retrieved from UniProtKB, GenBank, Protein Data Bank, ModBase, Protein Model Portal and the literature. It also incorporates specialized features of ZFP including finger sequences and positions, number of fingers, physiochemical properties, classes, framework, PubMed citations with links to experimental structures (PDB, if available) and modeled structures of natural zinc finger proteins. ZifBASE provides information on zinc finger proteins (both natural and engineered ones), the number of finger units in each of the zinc finger proteins (with multiple fingers), the synergy between the adjacent fingers and their positions. Additionally, it gives the individual finger sequence and their target DNA site to which it binds for better and clear understanding on the interactions of adjacent fingers. The current version of ZifBASE contains 139 entries of which 89 are engineered ZFPs, containing 3-7F totaling to 296 fingers. There are 50 natural zinc finger protein entries ranging from 2-13F, totaling to 307 fingers. It has sequences and structures from literature, Protein Data Bank, ModBase and Protein Model Portal. The interface is cross linked to other public databases like UniprotKB, PDB, ModBase and Protein Model Portal and PubMed for making it more informative. CONCLUSION: A database is established to maintain the information of the sequence features, including the class, framework, number of fingers, residues, position, recognition site and physio-chemical properties (molecular weight, isoelectric point) of both natural and engineered zinc finger proteins and dissociation constant of few. ZifBASE can provide more effective and efficient way of accessing the zinc finger protein sequences and their target binding sites with the links to their three-dimensional structures. All the data and functions are available at the advanced web-based search interface http://web.iitd.ac.in/~sundar/zifbase.