Conserved long noncoding RNA TILAM promotes liver fibrosis through interaction with PML in HSCs.

BACKGROUND AND AIMS: Fibrosis is the common end point for all forms of chronic liver injury, and the progression of fibrosis leads to the development of end-stage liver disease. Activation of HSCs and their transdifferentiation into myofibroblasts results in the accumulation of extracellular matrix proteins that form the fibrotic scar. Long noncoding RNAs regulate the activity of HSCs and provide targets for fibrotic therapies. APPROACH AND RESULTS: We identified long noncoding RNA TILAM located near COL1A1 , expressed in HSCs, and induced with liver fibrosis in humans and mice. Loss-of-function studies in human HSCs and human liver organoids revealed that TILAM regulates the expression of COL1A1 and other extracellular matrix genes. To determine the role of TILAM in vivo, we annotated the mouse ortholog ( Tilam ), generated Tilam- deficient green fluorescent protein-reporter mice, and challenged these mice in 2 different models of liver fibrosis. Single-cell data and analysis of single-data and analysis of Tilam-deficient reporter mice revealed that Tilam is induced in murine HSCs with the development of fibrosis in vivo. Tilam -deficient reporter mice revealed that Tilam is induced in murine HSCs with the development of fibrosis in vivo. Furthermore, loss of Tilam expression attenuated the development of fibrosis in the setting of in vivo liver injury. Finally, we found that TILAM interacts with promyelocytic leukemia nuclear body scaffold protein to regulate a feedback loop by which TGF-ß2 reinforces TILAM expression and nuclear localization of promyelocytic leukemia nuclear body scaffold protein to promote the fibrotic activity of HSCs. CONCLUSIONS: TILAM is activated in HSCs with liver injury and interacts with promyelocytic leukemia nuclear body scaffold protein to drive the development of fibrosis. Depletion of TILAM may serve as a therapeutic approach to combat the development of end-stage liver disease.

Single-cell transcriptomics stratifies organoid models of metabolic dysfunction-associated steatotic liver disease.

Metabolic dysfunction-associated steatotic liver disease (MASLD) is a growing cause of morbidity with limited treatment options. Thus, accurate in vitro systems to test new therapies are indispensable. While recently, human liver organoid models have emerged to assess steatotic liver disease, a systematic evaluation of their translational potential is still missing. Here, we evaluated human liver organoid models of MASLD, comparatively testing disease induction in three conditions: oleic acid, palmitic acid, and TGF-ß1. Through single-cell analyses, we find that all three models induce inflammatory signatures, but only TGF-ß1 promotes collagen production, fibrosis, and hepatic stellate cell expansion. In striking contrast, oleic acid ameliorates fibrotic signatures and reduces the hepatic stellate cell population. Linking data from each model to gene expression signatures associated with MASLD disease progression further demonstrates that palmitic acid and TGF-ß1 more robustly model inflammation and fibrosis. Our findings highlight the importance of stratifying MASLD organoid models by signatures of clinical disease progression, provide a single-cell reference to benchmark future organoid injury models, and allow us to study evolving steatohepatitis, fibrosis, and HSC susceptibility to injury in a dynamic, multi-lineage human in vitro system.

Conserved long noncoding RNA TILAM promotes liver fibrosis through interaction with PML in hepatic stellate cells.

Background & Aims: Fibrosis is the common endpoint for all forms of chronic liver injury, and progression of fibrosis leads to the development of end-stage liver disease. Activation of hepatic stellate cells (HSCs) and their transdifferentiation to myofibroblasts results in the accumulation of extracellular matrix (ECM) proteins that form the fibrotic scar. Long noncoding (lnc) RNAs regulate the activity of HSCs and may provide targets for fibrotic therapies. Methods: We identified lncRNA TILAM as expressed near COL1A1 in human HSCs and performed loss-of-function studies in human HSCs and liver organoids. Transcriptomic analyses of HSCs isolated from mice defined the murine ortholog of TILAM . We then generated Tilam -deficient GFP reporter mice and quantified fibrotic responses to carbon tetrachloride (CCl 4 ) and choline-deficient L-amino acid defined high fat diet (CDA-HFD). Co-precipitation studies, mass spectrometry, and gene expression analyses identified protein partners of TILAM . Results: TILAM is conserved between human and mouse HSCs and regulates expression of ECM proteins, including collagen. Tilam is selectively induced in HSCs during the development of fibrosis in vivo . In both male and female mice, loss of Tilam results in reduced fibrosis in the setting of CCl 4 and CDA-HFD injury models. TILAM interacts with promyelocytic leukemia protein (PML) to stabilize PML protein levels and promote the fibrotic activity of HSCs. Conclusion: TILAM is activated in HSCs and interacts with PML to drive the development of liver fibrosis. Depletion of TILAM may serve as a therapeutic approach to combat the development of end stage liver disease.

Correction: Tagger-A Swiss army knife for multiomics to dissect cell type-specific mechanisms of gene expression in mice.

[This corrects the article DOI: 10.1371/journal.pbio.3000374.].

Immune chromatin reader SP140 regulates microbiota and risk for inflammatory bowel disease.

Inflammatory bowel disease (IBD) is driven by host genetics and environmental factors, including commensal microorganisms. Speckled Protein 140 (SP140) is an immune-restricted chromatin "reader" that is associated with Crohn's disease (CD), multiple sclerosis (MS), and chronic lymphocytic leukemia (CLL). However, the disease-causing mechanisms of SP140 remain undefined. Here, we identify an immune-intrinsic role for SP140 in regulating phagocytic defense responses to prevent the expansion of inflammatory bacteria. Mice harboring altered microbiota due to hematopoietic Sp140 deficiency exhibited severe colitis that was transmissible upon cohousing and ameliorated with antibiotics. Loss of SP140 results in blooms of Proteobacteria, including Helicobacter in Sp140-/- mice and Enterobacteriaceae in humans bearing the CD-associated SP140 loss-of-function variant. Phagocytes from patients with the SP140 loss-of-function variant and Sp140-/- mice exhibited altered antimicrobial defense programs required for control of pathobionts. Thus, mutations within this epigenetic reader may constitute a predisposing event in human diseases provoked by microbiota.

Nanchangmycin regulates FYN, PTK2, and MAPK1/3 to control the fibrotic activity of human hepatic stellate cells.

Chronic liver injury causes fibrosis, characterized by the formation of scar tissue resulting from excessive accumulation of extracellular matrix (ECM) proteins. Hepatic stellate cell (HSC) myofibroblasts are the primary cell type responsible for liver fibrosis, yet there are currently no therapies directed at inhibiting the activity of HSC myofibroblasts. To search for potential anti-fibrotic compounds, we performed a high-throughput compound screen in primary human HSC myofibroblasts and identified 19 small molecules that induce HSC inactivation, including the polyether ionophore nanchangmycin (NCMC). NCMC induces lipid re-accumulation while reducing collagen expression, deposition of collagen in the extracellular matrix, cell proliferation, and migration. We find that NCMC increases cytosolic Ca2+ and reduces the phosphorylated protein levels of FYN, PTK2 (FAK), MAPK1/3 (ERK2/1), HSPB1 (HSP27), and STAT5B. Further, depletion of each of these kinases suppress COL1A1 expression. These studies reveal a signaling network triggered by NCMC to inactivate HSC myofibroblasts and reduce expression of proteins that compose the fibrotic scar. Identification of the antifibrotic effects of NCMC and the elucidation of pathways by which NCMC inhibits fibrosis provide new tools and therapeutic targets that could potentially be utilized to combat the development and progression of liver fibrosis.

Human enteric viruses autonomously shape inflammatory bowel disease phenotype through divergent innate immunomodulation.

Altered enteric microorganisms in concert with host genetics shape inflammatory bowel disease (IBD) phenotypes. However, insight is limited to bacteria and fungi. We found that eukaryotic viruses and bacteriophages (collectively, the virome), enriched from non-IBD, noninflamed human colon resections, actively elicited atypical anti-inflammatory innate immune programs. Conversely, ulcerative colitis or Crohn's disease colon resection viromes provoked inflammation, which was successfully dampened by non-IBD viromes. The IBD colon tissue virome was perturbed, including an increase in the enterovirus B species of eukaryotic picornaviruses, not previously detected in fecal virome studies. Mice humanized with non-IBD colon tissue viromes were protected from intestinal inflammation, whereas IBD virome mice exhibited exacerbated inflammation in a nucleic acid sensing-dependent fashion. Furthermore, there were detrimental consequences for IBD patient-derived intestinal epithelial cells bearing loss-of-function mutations within virus sensor MDA5 when exposed to viromes. Our results demonstrate that innate recognition of IBD or non-IBD human viromes autonomously influences intestinal homeostasis and disease phenotypes. Thus, perturbations in the intestinal virome, or an altered ability to sense the virome due to genetic variation, contribute to the induction of IBD. Harnessing the virome may offer therapeutic and biomarker potential.

Factors affecting outcomes of embryo transfer in dromedary camels: A retrospective study.

The present study aimed to use a comprehensive approach for evaluating 12 factors related to embryo quality (shape, size and transparency), donors (age, ET type, number of recovered embryos and day of uterine flushing), recipients (age), males (age and individual variations) and environment (season and year) which could affect the outcome of ET in terms of pregnancy (PR) and pregnancy losses in dromedary camels. During three breeding seasons, 116 donor females were mated repeatedly at 12- to 14-day intervals by fertile male camels (n = 33) without stimulation of the ovaries (WSPO). Superovulation (SPO ET) regimen was applied for each donor female twice or thrice per season. In the occasions of applying superovulation regimen, donor females having an ovulatory follicle were mated instead of GnRH administration and superovulation regimen was applied 4 days post-mating (MIX ET). The uteri of all donor females were flushed at Day 8 or 9 post-mating, and a total of 2,095 embryos were recovered and transferred individually to 924 recipient females. Pregnancy diagnoses were conducted at Day 10 after ET (Days 18-19 of gestation) by using progesterone assay and by transrectal ultrasonography (TRU) at Days 30 and 60 of gestation. By using logistic regression analysis, transparency of embryos and age of recipient females had significant effects on PR at Days 18-19 (p < .01), 30 (p < .01) and 60 (p < .01; p < .05, respectively) of gestation. The shape of embryos had significant effects on the PR at Days 30 (p < .05) and 60 (p < .01) of gestation. Type of ET and the breeding season (year) had significant (p < .05) effects on the PR at Day 30, while day of flushing had the same effect on PR at Day 60. Regarding the pregnancy losses, transparency and shape of the embryo, type of ET, breeding season had significant (p < .05) effect on the late embryonic mortalities (LEM) and shape and season of year had significant (p < .01 and p < .05, respectively) effect on LEM/early foetal mortalities (EFM). Regarding male individual factor, there was a tendency for a significant (p = .055) effect of male camels on the PR at Days 18-19 and rate of LEM. In conclusion, transferring a spherical, transparent or a large-sized embryo (>750 µm) into recipient females ageing between 8 and 11 y could greatly improve the PR from Days 18 to 60 of gestation. Also, embryo recovered from donor females with Mix ET type or embryos sired by certain male camel or at Day 8 post-mating of the donor could improve the 2-month PR. In addition, transferring a transparent or spherical-shaped embryo or embryos recovered from donor females with SPO or Mix ET could reduce the pregnancy losses during the first 2 months of pregnancy.

SEAweb: the small RNA Expression Atlas web application.

We present the Small RNA Expression Atlas (SEAweb), a web application that allows for the interactive querying, visualization and analysis of known and novel small RNAs across 10 organisms. It contains sRNA and pathogen expression information for over 4200 published samples with standardized search terms and ontologies. In addition, SEAweb allows for the interactive visualization and re-analysis of 879 differential expression and 514 classification comparisons. SEAweb's user model enables sRNA researchers to compare and re-analyze user-specific and published datasets, highlighting common and distinct sRNA expression patterns. We provide evidence for SEAweb's fidelity by (i) generating a set of 591 tissue specific miRNAs across 29 tissues, (ii) finding known and novel bacterial and viral infections across diseases and (iii) determining a Parkinson's disease-specific blood biomarker signature using novel data. We believe that SEAweb's simple semantic search interface, the flexible interactive reports and the user model with rich analysis capabilities will enable researchers to better understand the potential function and diagnostic value of sRNAs or pathogens across tissues, diseases and organisms.

Tagger-A Swiss army knife for multiomics to dissect cell type-specific mechanisms of gene expression in mice.

A deep understanding of how regulation of the multiple levels of gene expression in mammalian tissues give rise to complex phenotypes has been impeded by cellular diversity. A handful of techniques were developed to tag-select nucleic acids of interest in specific cell types, thereby enabling their capture. We expanded this strategy by developing the Tagger knock-in mouse line bearing a quad-cistronic transgene combining enrichment tools for nuclei, nascent RNA, translating mRNA, and mature microRNA (miRNA). We demonstrate that Tagger can capture the desired nucleic acids, enabling multiple omics approaches to be applied to specific cell types in vivo using a single transgenic mouse line.

DNA Methyltransferase 1 Controls Nephron Progenitor Cell Renewal and Differentiation.

BACKGROUND: Nephron number is a major determinant of long-term renal function and cardiovascular risk. Observational studies suggest that maternal nutritional and metabolic factors during gestation contribute to the high variability of nephron endowment. However, the underlying molecular mechanisms have been unclear. METHODS: We used mouse models, including DNA methyltransferase (Dnmt1, Dnmt3a, and Dnmt3b) knockout mice, optical projection tomography, three-dimensional reconstructions of the nephrogenic niche, and transcriptome and DNA methylation analysis to characterize the role of DNA methylation for kidney development. RESULTS: We demonstrate that DNA hypomethylation is a key feature of nutritional kidney growth restriction in vitro and in vivo, and that DNA methyltransferases Dnmt1 and Dnmt3a are highly enriched in the nephrogenic zone of the developing kidneys. Deletion of Dnmt1 in nephron progenitor cells (in contrast to deletion of Dnmt3a or Dnm3b) mimics nutritional models of kidney growth restriction and results in a substantial reduction of nephron number as well as renal hypoplasia at birth. In Dnmt1-deficient mice, optical projection tomography and three-dimensional reconstructions uncovered a significant reduction of stem cell niches and progenitor cells. RNA sequencing analysis revealed that global DNA hypomethylation interferes in the progenitor cell regulatory network, leading to downregulation of genes crucial for initiation of nephrogenesis, Wt1 and its target Wnt4. Derepression of germline genes, protocadherins, Rhox genes, and endogenous retroviral elements resulted in the upregulation of IFN targets and inhibitors of cell cycle progression. CONCLUSIONS: These findings establish DNA methylation as a key regulatory event of prenatal renal programming, which possibly represents a fundamental link between maternal nutritional factors during gestation and reduced nephron number.

The codon sequences predict protein lifetimes and other parameters of the protein life cycle in the mouse brain.

The homeostasis of the proteome depends on the tight regulation of the mRNA and protein abundances, of the translation rates, and of the protein lifetimes. Results from several studies on prokaryotes or eukaryotic cell cultures have suggested that protein homeostasis is connected to, and perhaps regulated by, the protein and the codon sequences. However, this has been little investigated for mammals in vivo. Moreover, the link between the coding sequences and one critical parameter, the protein lifetime, has remained largely unexplored, both in vivo and in vitro. We tested this in the mouse brain, and found that the percentages of amino acids and codons in the sequences could predict all of the homeostasis parameters with a precision approaching experimental measurements. A key predictive element was the wobble nucleotide. G-/C-ending codons correlated with higher protein lifetimes, protein abundances, mRNA abundances and translation rates than A-/U-ending codons. Modifying the proportions of G-/C-ending codons could tune these parameters in cell cultures, in a proof-of-principle experiment. We suggest that the coding sequences are strongly linked to protein homeostasis in vivo, albeit it still remains to be determined whether this relation is causal in nature.

Precisely measured protein lifetimes in the mouse brain reveal differences across tissues and subcellular fractions.

The turnover of brain proteins is critical for organism survival, and its perturbations are linked to pathology. Nevertheless, protein lifetimes have been difficult to obtain in vivo. They are readily measured in vitro by feeding cells with isotopically labeled amino acids, followed by mass spectrometry analyses. In vivo proteins are generated from at least two sources: labeled amino acids from the diet, and non-labeled amino acids from the degradation of pre-existing proteins. This renders measurements difficult. Here we solved this problem rigorously with a workflow that combines mouse in vivo isotopic labeling, mass spectrometry, and mathematical modeling. We also established several independent approaches to test and validate the results. This enabled us to measure the accurate lifetimes of ~3500 brain proteins. The high precision of our data provided a large set of biologically significant observations, including pathway-, organelle-, organ-, or cell-specific effects, along with a comprehensive catalog of extremely long-lived proteins (ELLPs).

Oasis 2: improved online analysis of small RNA-seq data.

BACKGROUND: Small RNA molecules play important roles in many biological processes and their dysregulation or dysfunction can cause disease. The current method of choice for genome-wide sRNA expression profiling is deep sequencing. RESULTS: Here we present Oasis 2, which is a new main release of the Oasis web application for the detection, differential expression, and classification of small RNAs in deep sequencing data. Compared to its predecessor Oasis, Oasis 2 features a novel and speed-optimized sRNA detection module that supports the identification of small RNAs in any organism with higher accuracy. Next to the improved detection of small RNAs in a target organism, the software now also recognizes potential cross-species miRNAs and viral and bacterial sRNAs in infected samples. In addition, novel miRNAs can now be queried and visualized interactively, providing essential information for over 700 high-quality miRNA predictions across 14 organisms. Robust biomarker signatures can now be obtained using the novel enhanced classification module. CONCLUSIONS: Oasis 2 enables biologists and medical researchers to rapidly analyze and query small RNA deep sequencing data with improved precision, recall, and speed, in an interactive and user-friendly environment. AVAILABILITY AND IMPLEMENTATION: Oasis 2 is implemented in Java, J2EE, mysql, Python, R, PHP and JavaScript. It is freely available at https://oasis.dzne.de.

The landscape of human mutually exclusive splicing.

Mutually exclusive splicing of exons is a mechanism of functional gene and protein diversification with pivotal roles in organismal development and diseases such as Timothy syndrome, cardiomyopathy and cancer in humans. In order to obtain a first genomewide estimate of the extent and biological role of mutually exclusive splicing in humans, we predicted and subsequently validated mutually exclusive exons (MXEs) using 515 publically available RNA-Seq datasets. Here, we provide evidence for the expression of over 855 MXEs, 42% of which represent novel exons, increasing the annotated human mutually exclusive exome more than fivefold. The data provide strong evidence for the existence of large and multi-cluster MXEs in higher vertebrates and offer new insights into MXE evolution. More than 82% of the MXE clusters are conserved in mammals, and five clusters have homologous clusters in Drosophila Finally, MXEs are significantly enriched in pathogenic mutations and their spatio-temporal expression might predict human disease pathology.

Group psychological intervention for maternal depression: A nested qualitative study from Karachi, Pakistan.

AIM: To understand the experience of maternal depression, the factors implicated in accessing health, and the acceptability of the psychosocial intervention. METHODS: The participants were recruited from the paediatrics outpatient department of Civil Hospital Karachi, Pakistan. The study started in December 2009 and completed in December 2010. Women with maternal depression, aged 18-44 years with children aged 0-30 mo who had received nutritional supplements, and participated in the intervention programme [called Learning through Play (LTP) plus] were included in the study. Qualitative interviews were conducted with 8 participants before the intervention and 7 participants after the intervention. A semi structured topic guide was used to conduct the interviews. RESULTS: Framework analysis procedures were used to analyse the qualitative data. Four themes emerged: (1) the women's contextual environment: Interpersonal conflicts, lack of social support and financial issues being the major barriers in assessing healthcare; (2) women's isolation and powerlessness within the environment: Sense of loneliness was identified as a restricting factor to access healthcare; (3) the impact of the intervention (LTP-Plus): Women felt "listened to" and seemed empowered; and (4) empowered transformed women within the same contextual environment: The facilitator provided a "gardening role" in nurturing the women resulting in a positive transformation within the same environment. The women's homes seemed to be more happy homes and there was a positive change in their behaviour towards their children. CONCLUSION: Findings informed the further development and testing of culturally-appropriate psychosocial intervention (LTP+) for addressing maternal depression.

Genome-wide chromatin and gene expression profiling during memory formation and maintenance in adult mice.

Recent evidence suggests that the formation and maintenance of memory requires epigenetic changes. In an effort to understand the spatio-temporal extent of learning and memory-related epigenetic changes we have charted genome-wide histone and DNA methylation profiles, in two different brain regions, two cell types, and three time-points, before and after learning. In this data descriptor we provide detailed information on data generation, give insights into the rationale of experiments, highlight necessary steps to assess data quality, offer guidelines for future use of the data and supply ready-to-use code to replicate the analysis results. The data provides a blueprint of the gene regulatory network underlying short- and long-term memory formation and maintenance. This 'healthy' gene regulatory network of learning can now be compared to changes in neurological or psychiatric diseases, providing mechanistic insights into brain disorders and highlighting potential therapeutic avenues.

MEPD: medaka expression pattern database, genes and more.

The Medaka Expression Pattern Database (MEPD; http://mepd.cos.uni-heidelberg.de/) is designed as a repository of medaka expression data for the scientific community. In this update we present two main improvements. First, we have changed the previous clone-centric view for in situ data to a gene-centric view. This is possible because now we have linked all the data present in MEPD to the medaka gene annotation in ENSEMBL. In addition, we have also connected the medaka genes in MEPD to their corresponding orthologous gene in zebrafish, again using the ENSEMBL database. Based on this, we provide a link to the Zebrafish Model Organism Database (ZFIN) to allow researches to compare expression data between these two fish model organisms. As a second major improvement, we have modified the design of the database to enable it to host regulatory elements, promoters or enhancers, expression patterns in addition to gene expression. The combination of gene expression, by traditional in situ, and regulatory element expression, typically by fluorescence reporter gene, within the same platform assures consistency in terms of annotation. In our opinion, this will allow researchers to uncover new insights between the expression domain of genes and their regulatory landscape.

DNA methylation changes in plasticity genes accompany the formation and maintenance of memory.

The ability to form memories is a prerequisite for an organism's behavioral adaptation to environmental changes. At the molecular level, the acquisition and maintenance of memory requires changes in chromatin modifications. In an effort to unravel the epigenetic network underlying both short- and long-term memory, we examined chromatin modification changes in two distinct mouse brain regions, two cell types and three time points before and after contextual learning. We found that histone modifications predominantly changed during memory acquisition and correlated surprisingly little with changes in gene expression. Although long-lasting changes were almost exclusive to neurons, learning-related histone modification and DNA methylation changes also occurred in non-neuronal cell types, suggesting a functional role for non-neuronal cells in epigenetic learning. Finally, our data provide evidence for a molecular framework of memory acquisition and maintenance, wherein DNA methylation could alter the expression and splicing of genes involved in functional plasticity and synaptic wiring.

Oasis: online analysis of small RNA deep sequencing data.

UNLABELLED: Oasis is a web application that allows for the fast and flexible online analysis of small-RNA-seq (sRNA-seq) data. It was designed for the end user in the lab, providing an easy-to-use web frontend including video tutorials, demo data and best practice step-by-step guidelines on how to analyze sRNA-seq data. Oasis' exclusive selling points are a differential expression module that allows for the multivariate analysis of samples, a classification module for robust biomarker detection and an advanced programming interface that supports the batch submission of jobs. Both modules include the analysis of novel miRNAs, miRNA targets and functional analyses including GO and pathway enrichment. Oasis generates downloadable interactive web reports for easy visualization, exploration and analysis of data on a local system. Finally, Oasis' modular workflow enables for the rapid (re-) analysis of data. AVAILABILITY AND IMPLEMENTATION: Oasis is implemented in Python, R, Java, PHP, C++ and JavaScript. It is freely available at http://oasis.dzne.de. CONTACT: stefan.bonn@dzne.de SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.