Disruption of HSD17B12 in mouse hepatocytes leads to reduced body weight and defect in the lipid droplet expansion associated with microvesicular steatosis.

The function of hydroxysteroid dehydrogenase 12 (HSD17B12) in lipid metabolism is poorly understood. To study this further, we created mice with hepatocyte-specific knockout of HSD17B12 (LiB12cKO). From 2 months on, these mice showed significant fat accumulation in their liver. As they aged, they also had a reduced whole-body fat percentage. Interestingly, the liver fat accumulation did not result in the typical formation of large lipid droplets (LD); instead, small droplets were more prevalent. Thus, LiB12KO liver did not show increased macrovesicular steatosis with the increasing fat content, while microvesicular steatosis was the predominant feature in the liver. This indicates a failure in the LD expansion. This was associated with liver damage, presumably due to lipotoxicity. Notably, the lipidomics data did not support an essential role of HSD17B12 in fatty acid (FA) elongation. However, we did observe a decrease in the quantity of specific lipid species that contain FAs with carbon chain lengths of 18 and 20 atoms, including oleic acid. Of these, phosphatidylcholine and phosphatidylethanolamine have been shown to play a key role in LD formation, and a limited amount of these lipids could be part of the mechanism leading to the dysfunction in LD expansion. The increase in the Cidec expression further supported the deficiency in LD expansion in the LiB12cKO liver. This protein is crucial for the fusion and growth of LDs, along with the downregulation of several members of the major urinary protein family of proteins, which have recently been shown to be altered during endoplasmic reticulum stress.

Gene Regulatory Network Analysis of Decidual Stromal Cells and Natural Killer Cells.

Human reproductive success relies on the proper differentiation of the uterine endometrium to facilitate implantation, formation of the placenta, and pregnancy. This process involves two critical types of decidual uterine cells: endometrial/decidual stromal cells (dS) and uterine/decidual natural killer (dNK) cells. To better understand the transcription factors governing the in vivo functions of these cells, we analyzed single-cell transcriptomics data from first-trimester terminations of pregnancy, and for the first time conducted gene regulatory network analysis of dS and dNK cell subpopulations. Our analysis revealed stromal cell populations that corresponded to previously described in vitro decidualized cells and senescent decidual cells. We discovered new decidualization driving transcription factors of stromal cells for early pregnancy, including DDIT3 and BRF2, which regulate oxidative stress protection. For dNK cells, we identified transcription factors involved in the immunotolerant (dNK1) subpopulation, including IRX3 and RELB, which repress the NFKB pathway. In contrast, for the less immunotolerant (dNK3) population we predicted TBX21 (T-bet) and IRF2-mediated upregulation of the interferon pathway. To determine the clinical relevance of our findings, we tested the overrepresentation of the predicted transcription factors target genes among cell type-specific regulated genes from pregnancy disorders, such as recurrent pregnancy loss and preeclampsia. We observed that the predicted decidualized stromal and dNK1-specific transcription factor target genes were enriched with the genes downregulated in pregnancy disorders, whereas the predicted dNK3-specific targets were enriched with genes upregulated in pregnancy disorders. Our findings emphasize the importance of stress tolerance pathways in stromal cell decidualization and immunotolerance promoting regulators in dNK differentiation.

Serum hydroxysteroid (17beta) dehydrogenase 1 concentration in pregnant women correlates with pregnancy-associated plasma protein A but does not serve as an independent marker for preeclampsia†.

Hydroxysteroid (17beta) dehydrogenase 1 (HSD17B1) is a steroid synthetic enzyme expressed in ovarian granulosa cells and placental syncytiotrophoblasts. Here, HSD17B1 serum concentration was measured with a validated immunoassay during pregnancy at three time points (12-14, 18-20 and 26-28 weeks of gestation). The concentration increased 2.5-fold (P < 0.0001) and 1.7-fold (P = 0.0019) during the follow-up period for control women and women who later developed preeclampsia (PE), respectively, and a significant difference was observed at weeks 26-28 (P = 0.0266). HSD17B1 concentration at all the three time points positively correlated with serum PAPPA measured at the first time point (first time point r = 0.38, P = 1.1 × 10-10; second time point r = 0.27, P = 5.9 × 10-6 and third timepoint r = 0.26, P = 2.3 × 10-5). No correlation was observed between HSD17B1 and placental growth factor (PLGF). Serum HSD17B1 negatively correlated with the mother's weight and body mass index (BMI), mirroring the pattern observed for PAPPA. The univariable logistic regression identified a weak association between HSD17B1 at 26-28 weeks and later development of PE (P = 0.04). The best multivariable model obtained using penalized logistic regression with stable iterative variable selection at 26-28 weeks included HSD17B1, together with PLGF, PAPPA and mother's BMI. While the area under the receiver operating characteristic curve of the model was higher than that of the adjusted PLGF, the difference was not statistically significant. In summary, the serum concentration of HSD17B1 correlated with PAPPA, another protein expressed in syncytiotrophoblasts, and with mother's weight and BMI but could not be considered as an independent marker for PE.

Glycolysis and Heavy Menstrual Bleeding.

Menstrual cycle is a major determinant in female reproductive health. In a recent report, Mao et al. (2022) associated deficient glycolysis with heavy menstrual bleeding. This commentary summarizes these recent findings and the importance of glycolysis and decidualization in endometrial function. It will also discuss if in the light of the recent findings menstrual bleeding is better conceived as a primary endometrial disorder inherent to endometrium or as a secondary endometrial disorder caused by other endometrial conditions.

Cell type markers indicate distinct contributions of decidual stromal cells and natural killer cells in preeclampsia.

In brief: Preeclampsia is a common serious disorder that can occur during pregnancy. This study uses integrative analysis of preeclampsia transcriptomes and single-cell transcriptomes to predict cell type-specific contributions to preeclampsia. Abstract: Preeclampsia is a devastating pregnancy disorder and a major cause of maternal and perinatal mortality. By combining previous transcriptomic results on preeclampsia with single-cell sequencing data, we here predict distinct and partly unanticipated contributions of decidual stromal cells and uterine natural killer cells in early- and late-onset preeclampsia.

COVID-19-specific transcriptomic signature detectable in blood across multiple cohorts.

The coronavirus disease 2019 (COVID-19) caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is spreading across the world despite vast global vaccination efforts. Consequently, many studies have looked for potential human host factors and immune mechanisms associated with the disease. However, most studies have focused on comparing COVID-19 patients to healthy controls, while fewer have elucidated the specific host factors distinguishing COVID-19 from other infections. To discover genes specifically related to COVID-19, we reanalyzed transcriptome data from nine independent cohort studies, covering multiple infections, including COVID-19, influenza, seasonal coronaviruses, and bacterial pneumonia. The identified COVID-19-specific signature consisted of 149 genes, involving many signals previously associated with the disease, such as induction of a strong immunoglobulin response and hemostasis, as well as dysregulation of cell cycle-related processes. Additionally, potential new gene candidates related to COVID-19 were discovered. To facilitate exploration of the signature with respect to disease severity, disease progression, and different cell types, we also offer an online tool for easy visualization of the selected genes across multiple datasets at both bulk and single-cell levels.

Histone H3K4me3 breadth in hypoxia reveals endometrial core functions and stress adaptation linked to endometriosis.

Trimethylation of histone H3 at lysine 4 (H3K4me3) is a marker of active promoters. Broad H3K4me3 promoter domains have been associated with cell type identity, but H3K4me3 dynamics upon cellular stress have not been well characterized. We assessed this by exposing endometrial stromal cells to hypoxia, which is a major cellular stress condition. We observed that hypoxia modifies the existing H3K4me3 marks and that promoter H3K4me3 breadth rather than height correlates with transcription. Broad H3K4me3 domains mark genes for endometrial core functions and are maintained or selectively extended upon hypoxia. Hypoxic extension of H3K4me3 breadth associates with stress adaptation genes relevant for the survival of endometrial cells including transcription factor KLF4, for which we found increased protein expression in the stroma of endometriosis lesions. These results substantiate the view on broad H3K4me3 as a marker of cell identity genes and reveal participation of H3K4me3 extension in cellular stress adaptation.

Overexpression of Human Estrogen Biosynthetic Enzyme Hydroxysteroid (17beta) Dehydrogenase Type 1 Induces Adenomyosis-like Phenotype in Transgenic Mice.

Hydroxysteroid (17beta) dehydrogenase type 1 (HSD17B1) is an enzyme that converts estrone to estradiol, while adenomyosis is an estrogen-dependent disease with poorly understood pathophysiology. In the present study, we show that mice universally over-expressing human estrogen biosynthetic enzyme HSD17B1 (HSD17B1TG mice) present with adenomyosis phenotype, characterized by histological and molecular evaluation. The first adenomyotic changes with endometrial glands partially or fully infiltrated into the myometrium appeared at the age of 5.5 months in HSD17B1TG females and became more prominent with increasing age. Preceding the phenotype, increased myometrial smooth muscle actin positivity and increased amount of glandular myofibroblast cells were observed in HSD17B1TG uteri. This was accompanied by transcriptomic upregulation of inflammatory and estrogen signaling pathways. Further, the genes upregulated in the HSD17B1TG uterus were enriched with genes previously observed to be induced in the human adenomyotic uterus, including several genes of the NFKB pathway. A 6-week-long HSD17B1 inhibitor treatment reduced the occurrence of the adenomyotic changes by 5-fold, whereas no effect was observed in the vehicle-treated HSD17B1TG mice, suggesting that estrogen is the main upstream regulator of adenomyosis-induced uterine signaling pathways. HSD17B1 is considered as a promising drug target to inhibit estrogen-dependent growth of endometrial disorders. The present data indicate that HSD17B1 over-expression in TG mice results in adenomyotic changes reversed by HSD17B1 inhibitor treatment and HSD17B1 is, thus, a potential novel drug target for adenomyosis.

Dirichlet process mixture models for single-cell RNA-seq clustering.

Clustering of cells based on gene expression is one of the major steps in single-cell RNA-sequencing (scRNA-seq) data analysis. One key challenge in cluster analysis is the unknown number of clusters and, for this issue, there is still no comprehensive solution. To enhance the process of defining meaningful cluster resolution, we compare Bayesian latent Dirichlet allocation (LDA) method to its non-parametric counterpart, hierarchical Dirichlet process (HDP) in the context of clustering scRNA-seq data. A potential main advantage of HDP is that it does not require the number of clusters as an input parameter from the user. While LDA has been used in single-cell data analysis, it has not been compared in detail with HDP. Here, we compare the cell clustering performance of LDA and HDP using four scRNA-seq datasets (immune cells, kidney, pancreas and decidua/placenta), with a specific focus on cluster numbers. Using both intrinsic (DB-index) and extrinsic (ARI) cluster quality measures, we show that the performance of LDA and HDP is dataset dependent. We describe a case where HDP produced a more appropriate clustering compared to the best performer from a series of LDA clusterings with different numbers of clusters. However, we also observed cases where the best performing LDA cluster numbers appropriately capture the main biological features while HDP tended to inflate the number of clusters. Overall, our study highlights the importance of carefully assessing the number of clusters when analyzing scRNA-seq data.

Differential ATAC-seq and ChIP-seq peak detection using ROTS.

Changes in cellular chromatin states fine-tune transcriptional output and ultimately lead to phenotypic changes. Here we propose a novel application of our reproducibility-optimized test statistics (ROTS) to detect differential chromatin states (ATAC-seq) or differential chromatin modification states (ChIP-seq) between conditions. We compare the performance of ROTS to existing and widely used methods for ATAC-seq and ChIP-seq data using both synthetic and real datasets. Our results show that ROTS outperformed other commonly used methods when analyzing ATAC-seq data. ROTS also displayed the most accurate detection of small differences when modeling with synthetic data. We observed that two-step methods that require the use of a separate peak caller often more accurately called enrichment borders, whereas one-step methods without a separate peak calling step were more versatile in calling sub-peaks. The top ranked differential regions detected by the methods had marked correlation with transcriptional differences of the closest genes. Overall, our study provides evidence that ROTS is a useful addition to the available differential peak detection methods to study chromatin and performs especially well when applied to study differential chromatin states in ATAC-seq data.

Computational strategies for single-cell multi-omics integration.

Single-cell omics technologies are currently solving biological and medical problems that earlier have remained elusive, such as discovery of new cell types, cellular differentiation trajectories and communication networks across cells and tissues. Current advances especially in single-cell multi-omics hold high potential for breakthroughs by integration of multiple different omics layers. To pair with the recent biotechnological developments, many computational approaches to process and analyze single-cell multi-omics data have been proposed. In this review, we first introduce recent developments in single-cell multi-omics in general and then focus on the available data integration strategies. The integration approaches are divided into three categories: early, intermediate, and late data integration. For each category, we describe the underlying conceptual principles and main characteristics, as well as provide examples of currently available tools and how they have been applied to analyze single-cell multi-omics data. Finally, we explore the challenges and prospective future directions of single-cell multi-omics data integration, including examples of adopting multi-view analysis approaches used in other disciplines to single-cell multi-omics.

A relational database to identify differentially expressed genes in the endometrium and endometriosis lesions.

Endometriosis is a common inflammatory estrogen-dependent gynecological disorder, associated with pelvic pain and reduced fertility in women. Several aspects of this disorder and its cellular and molecular etiology remain unresolved. We have analyzed the global gene expression patterns in the endometrium, peritoneum and in endometriosis lesions of endometriosis patients and in the endometrium and peritoneum of healthy women. In this report, we present the EndometDB, an interactive web-based user interface for browsing the gene expression database of collected samples without the need for computational skills. The EndometDB incorporates the expression data from 115 patients and 53 controls, with over 24000 genes and clinical features, such as their age, disease stages, hormonal medication, menstrual cycle phase, and the different endometriosis lesion types. Using the web-tool, the end-user can easily generate various plot outputs and projections, including boxplots, and heatmaps and the generated outputs can be downloaded in pdf-format.Availability and implementationThe web-based user interface is implemented using HTML5, JavaScript, CSS, Plotly and R. It is freely available from https://endometdb.utu.fi/ .

Transcriptomic responses to hypoxia in endometrial and decidual stromal cells.

Human reproductive success depends on a properly decidualized uterine endometrium that allows implantation and the formation of the placenta. At the core of the decidualization process are endometrial stromal fibroblasts (ESF) that differentiate to decidual stromal cells (DSC). As variations in oxygen levels are functionally relevant in endometrium both upon menstruation and during placentation, we assessed the transcriptomic responses to hypoxia in ESF and DSC. In both cell types, hypoxia-upregulated genes in classical hypoxia pathways such as glycolysis and the epithelial mesenchymal transition. In DSC, hypoxia restored an ESF-like transcriptional state for a subset of transcription factors that are known targets of the progesterone receptor, suggesting that hypoxia partially interferes with progesterone signaling. In both cell types, hypoxia modified transcription of several inflammatory transcription factors that are known regulators of decidualization, including decreased transcription of STATs and increased transcription of CEBPs. We observed that hypoxia-upregulated genes in ESF and DSC had a significant overlap with genes previously detected to be upregulated in endometriotic stromal cells. Promoter analysis of the genes in this overlap suggested the hypoxia-upregulated Jun/Fos and CEBP transcription factors as potential drivers of endometriosis-associated transcription. Using immunohistochemistry, we observed increased expression of JUND and CEBPD in endometriosis lesions compared to healthy endometria. Overall, the findings suggest that hypoxic stress establishes distinct transcriptional states in ESF and DSC and that hypoxia influences the expression of genes that contribute to the core gene regulation of endometriotic stromal cells.

RepViz: a replicate-driven R tool for visualizing genomic regions.

OBJECTIVE: Visualization of sequencing data is an integral part of genomic data analysis. Although there are several tools to visualize sequencing data on genomic regions, they do not offer user-friendly ways to view simultaneously different groups of replicates. To address this need, we developed a tool that allows efficient viewing of both intra- and intergroup variation of sequencing counts on a genomic region, as well as their comparison to the output of user selected analysis methods, such as peak calling. RESULTS: We present an R package RepViz for replicate-driven visualization of genomic regions. With ChIP-seq and ATAC-seq data we demonstrate its potential to aid visual inspection involved in the evaluation of normalization, outlier behavior, detected features from differential peak calling analysis, and combined analysis of multiple data types. RepViz is readily available on Bioconductor ( https://www.bioconductor.org/packages/devel/bioc/html/RepViz.html ) and on Github ( https://github.com/elolab/RepViz ).

Decidualization of Human Endometrial Stromal Fibroblasts is a Multiphasic Process Involving Distinct Transcriptional Programs.

Decidual stromal cells differentiate from endometrial stromal fibroblasts (ESFs) under the influence of progesterone and cyclic adenosine monophosphate (cAMP) and are essential for implantation and the maintenance of pregnancy. They evolved in the stem lineage of placental (eutherian) mammals coincidental with the evolution of implantation. Here we use the well-established in vitro decidualization protocol to compare early (3 days) and late (8 days) gene transcription patterns in immortalized human ESF. We document extensive, dynamic changes in the early and late decidual cell transcriptomes. The data suggest the existence of an early signal transducer and activator of transcription (STAT) pathway dominated state and a later nuclear factor κB (NFKB) pathway regulated state. Transcription factor expression in both phases is characterized by putative or known progesterone receptor ( PGR) target genes, suggesting that both phases are under progesterone control. Decidualization leads to proliferative quiescence, which is reversible by progesterone withdrawal after 3 days but to a lesser extent after 8 days of decidualization. In contrast, progesterone withdrawal induces cell death at comparable levels after short or long exposure to progestins and cAMP. We conclude that decidualization is characterized by a biphasic gene expression dynamic that likely corresponds to different phases in the establishment of the fetal-maternal interface.

Evolution: Oxygen and early animals.

The biology of sponges provides clues about how early animals may have dealt with low levels of oxygen.

The evolution of early cellular systems viewed through the lens of biological interactions.

The minimal cell concept represents a pragmatic approach to the question of how few genes are required to run a cell. This is a helpful way to build a parts-list, and has been more successful than attempts to deduce a minimal gene set for life by inferring the gene repertoire of the last universal common ancestor, as few genes trace back to this hypothetical ancestral state. However, the study of minimal cellular systems is the study of biological outliers where, by practical necessity, coevolutionary interactions are minimized or ignored. In this paper, we consider the biological context from which minimal genomes have been removed. For instance, some of the most reduced genomes are from endosymbionts and are the result of coevolutionary interactions with a host; few such organisms are "free-living." As few, if any, biological systems exist in complete isolation, we expect that, as with modern life, early biological systems were part of an ecosystem, replete with organismal interactions. We favor refocusing discussions of the evolution of cellular systems on processes rather than gene counts. We therefore draw a distinction between a pragmatic minimal cell (an interesting engineering problem), a distributed genome (a system resulting from an evolutionary transition involving more than one cell) and the looser coevolutionary interactions that are ubiquitous in ecosystems. Finally, we consider the distributed genome and coevolutionary interactions between genomic entities in the context of early evolution.

Transcriptional divergence of the duplicated hypoxia-inducible factor alpha genes in zebrafish.

Oxygen availability has been a major force in shaping the physiological evolution of animals. Under reduced oxygen availability (hypoxia) major changes in gene expression are mediated by hypoxia-inducible factors (HIF alphas). Tetrapods have three hif alpha genes, whereas zebrafish (Danio rerio) and other cyprinids have six due to a teleost lineage-specific genome duplication. We studied the transcriptional divergence of the six teleost-specific hif alphas by inspecting the tissue-specific transcription patterns in adult zebrafish and by monitoring the early developmental transcription of normoxia- and hypoxia-grown zebrafish embryos. Overall we observed the highest hif alpha mRNA levels in tissues that are important for hypoxic survival, including the brain, gill and heart. Of the paralogs that have not previously received attention (hif alpha-1A, hif alpha-2B and hif alpha-3B) especially the hif alpha-2B transcription levels suggest functional relevance. The hif alpha-1A/B paralogs that have considerable coding sequence divergence displayed more overall transcriptional divergence than the hif alpha-2A/B paralog pair. The hif alpha-2A/B paralogs that are similarly conserved in coding sequence had a divergent transcription pattern during early development. When zebrafish grown in modest hypoxia were compared to normoxia grown fish, only hif alpha-3A transcription was significantly altered. These results suggest that, in zebrafish, the evolutionary retention of each hif alpha paralog pair has involved unique patterns of coding sequence divergence, adult tissue-specific transcriptional divergence or developmental transcriptional divergence.

Revisiting redox-active antioxidant defenses in response to hypoxic challenge in both hypoxia-tolerant and hypoxia-sensitive fish species.

It is not known whether changes in antioxidant levels always occur in fish in response to the oxidative stress that usually accompanies a hypoxic challenge. The studies of antioxidant responses to hypoxia in fish have mostly focused on very anoxia-tolerant species and indicate that there is an enhancement of antioxidant defenses. Here we present new data on redox-active antioxidants from three species, which range in their tolerance to hypoxia: the epaulette shark, threespine stickleback, and rainbow trout, together with a compilation of results from other studies that have measured oxidative stress parameters in hypoxia-exposed fish. The results suggest that in general, fish do not show an increase in redox-active antioxidant defense in response to oxidative stress associated with hypoxia. Rather, the changes in antioxidant defenses during hypoxia are very much species- and tissue-specific and are not linked to the level of hypoxia tolerance of the fish species.

Subfunctionalization of cyprinid hypoxia-inducible factors for roles in development and oxygen sensing.

Among vertebrates, teleost fishes have evolved the most impressive adaptations to variable oxygen tensions in water (Shoubridge and Hochachka 1980; Nilsson and Randall 2010). Under conditions of oxygen deprivation (hypoxia), major changes in gene expression are mediated by hypoxia-inducible factors (HIF alpha). Here we show that hif alpha genes were duplicated in the teleost specific whole-genome duplication. Although one of each paralogous gene pair was lost in most teleosts, both copies were retained in cyprinids. Computational analyses suggest that these duplicates have become subfunctionalized with complementary changes in coding and regulatory sequences within each paralogous gene pair. We tested our predictions with comparisons of hif alpha transcription in zebrafish, a cyprinid, and sturgeon, an outgroup that diverged from teleosts before the duplication event. Our experiments revealed distinct transcriptional profiles in the cyprinid duplicates: while one of each paralogous pair maintained the ancestral developmental response, the other was more sensitive to changes in oxygen tension. These results demonstrate the subfunctionalization of cyprinid hif alpha paralogs for specialized roles in development and the hypoxic stress response.