Regulatory Signals Research Articles

Fibro-adipogenic progenitor cells in skeletal muscle unloading: metabolic and functional impairments

BackgroundSkeletal muscle resident fibro-adipogenic progenitor cells (FAPs) control skeletal muscle regeneration providing a supportive role for muscle stem cells. Altered FAPs characteristics have been shown for a number of pathological conditions, but the influence of temporary functional unloading of healthy skeletal muscle on FAPs remains poorly studied. This work is aimed to investigate how skeletal muscle disuse affects the functionality and metabolism of FAPs.MethodsHindlimb suspension (HS) rat model employed to investigate muscle response to decreased usage. FAPs were purified from m. soleus functioning muscle (Contr) and after functional unloading for 7 and 14 days (HS7 and HS14). FAPs were expanded in vitro, and tested for: immunophenotype; in vitro expansion rate, and migration activity; ability to differentiate into adipocytes in vitro; metabolic changes. Crosstalk between FAPs and muscle stem cells was estimated by influence of medium conditioned by FAP’s on migration and myogenesis of C2C12 myoblasts. To reveal the molecular mechanisms behind unloading-induced alterations in FAP’s functionality transcriptome analysis was performed.ResultsFAPs isolated from Contr and HS muscles exhibited phenotype of MSC cells. FAPs in vitro expansion rate and migration were altered by functional unloading conditions. All samples of FAPs demonstrated the ability to adipogenic differentiation in vitro, however, HS FAPs formed fat droplets of smaller volume and transcriptome analysis showed fatty acids metabolism and PPAR signaling suppression. Skeletal muscle unloading resulted in metabolic reprogramming of FAPs: decreased spare respiratory capacity, decreased OCR/ECAR ratio detected in both HS7 and HS14 samples point to reduced oxygen consumption, decreased potential for substrate oxidation and a shift to glycolytic metabolism. Furthermore, C2C12 cultures treated with medium conditioned by FAPs showed diverse alterations: while the HS7 FAPs-derived paracrine factors supported the myoblasts fusion, the HS14-derived medium stimulated proliferation of C2C12 myoblasts; these observations were supported by increased expression of cytokines detected by transcriptome analysis.Conclusionthe results obtained in this work show that the skeletal muscle functional unloading affects properties of FAPs in time-dependent manner: in atrophying skeletal muscle FAPs act as the sensors for the regulatory signals that may stimulate the metabolic and transcriptional reprogramming to preserve FAPs properties associated with maintenance of skeletal muscle homeostasis during unloading and in course of rehabilitation.

RNA-seq analysis of the biological process and regulatory signal of TGF-β1-mediated changes in ovarian granulosa cells in small-tail Han sheep

Transforming growth factor beta-1 (TGF-β1) regulates the proliferation of ovarian granulosa cells and participates in follicular development in small-tail Han sheep via the SMAD pathway. However, which additional biological processes and regulatory mechanisms are involved in TGF-β1-mediated regulation of granulosa cell changes remains unknown. In this study, TGF-β1-treated (10 ng/mL) ovarian granulosa cells of small-tail Han sheep were used as the model, RNA-Seq was employed to screen differentially expressed genes (DEGs), and rescue experiments were used to verify selected key pathways. In total, 1179 upregulated and 873 downregulated DEGs were screened using RNA-Seq. Gene Ontology (GO) enrichment analysis showed that the DEGs were mainly involved in the biological processes of cell adhesion, cell migration, cell cycle, cell proliferation and apoptosis, and endocrine regulation. Meanwhile, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis indicated that the DEGs were primarily associated with pathways relating to ECM-receptor interaction, PI3K-AKT, focal adhesion, MAPK, TNF, and FOXO signaling, among others. The addition of doramapimod confirmed that the p38 pathway participates in the TGF-β1-induced proliferation and apoptosis of ovarian granulosa cells as well as the regulation of steroid hormone secretion. These results revealed a novel TGF-β1/p38 pathway-mediated mechanism that induces both the proliferation and apoptosis of ovarian granulosa cells. Our findings provide a basis for better understanding the genetic mechanism of TGF-β1 action in follicle development.

Abstract PR012: A novel plasma-based epigenomic assay for comprehensive profiling of cancerous and inflammatory disease states

Abstract Background: We developed a method for comprehensive epigenomic profiling from 1 ml of patient (pt) plasma, targeting histone modifications and DNA methylation. This approach can be applied to plasma from pts with cancer and inflammatory conditions, allowing us to infer expression levels of diagnostic markers and drug targets, measure activity of therapeutically targetable transcription factors, and detect non-genomic mechanisms of resistance. By examining the transcriptional status of therapeutic gene targets and pathways, our method addresses the need for new approaches in liquid biopsy (LBx) assays, providing clinically actionable information that enhances the understanding of cancer and inflammatory biology and therapeutic response. Methods: We developed an innovative multimodal assay capable of revealing genome-wide transcriptional activity of promoters and enhancers from chromatin, as well as surveying the DNA methylome. Utilizing this assay on plasma samples from pts with 5 non- cancer conditions and 15 different tumor types, we generated comprehensive genome-wide transcriptional profiles across 21 conditions. Results: We identified transcriptional activation in cancer pt plasma of cancer-associated biomarkers (e.g., HER2, DLL3) in a tumor-specific manner and in autoimmune disorders such as rheumatoid arthritis and systemic lupus erythematosus/lupus nephritis. Epigenomic profiling simultaneously inferred the expression of multiple pertinent ADC and radio-immune targets (e.g., MET, HER2, HER3, TROP2, B7H4 in breast cancer and PSMA in prostate cancer), and SLFN11, a biomarker for sensitivity to these agents. The multimodal platform assesses transcriptional regulation genome-wide, enabling analysis of select genes as well as related pathways. For example, a multimodal classifier robustly predicted HER2 status across multiple HER2-expressing tumors. HER2 classification of breast cancer pts by epigenomic LBx was concordant with standard tissue-based IHC for 64/72 (89%) breast cancer samples (AUC 0.9). Accurate classification was achieved for 11/14 (79%) gastroesophageal adenocarcinoma and 4/4 (100%) ovarian cancer pts. A multi-modal predictive model trained on FOLH1 plasma epigenomic regulatory signals demonstrated significant accuracy in predicting quantitative readouts from PSMA PET scans (total SUVmean) in metastatic castration resistance prostate cancer pts and was associated with response to pluvicto. Epigenomic signatures also delineated activation of the endocrine signaling pathway in breast cancer and multiple mechanisms of resistance to androgen receptor signaling inhibition in prostate cancer, including reactivation of the androgen receptor, glucocorticoid signaling bypass, and histological transformation to a neuroendocrine subtype. Conclusions: This comprehensive epigenomic LBx assay offers a platform for simultaneous genome-wide transcriptional assessment of disease and therapy-relevant genes and pathways, marking a paradigm shift in our ability to assess the underlying transcriptional states of cancer and inflammatory diseases. Citation Format: Travis Clark, Anthony D'Ippolito, Aparna Gorthi, Jonathon Beagan, Jamey Guess, Amy Donahue, Mandy Greene, Alyssa Lau, Fernando Ramirez, Kristian Cibulskis, Jenna Wurster, Michael Coyne, Mary McGillicuddy, Baovy Tran, Tyrone Tamakloe, Hathairat Sawaengsri, Corrie Painter, Juliann Chmielecki, Geoff Otto, Matthew Eaton, Carl Barrett. A novel plasma-based epigenomic assay for comprehensive profiling of cancerous and inflammatory disease states [abstract]. In: Proceedings of the AACR Special Conference: Liquid Biopsy: From Discovery to Clinical Implementation; 2024 Nov 13-16; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(21_Suppl):Abstract nr PR012.

Phosphorylation of disordered proteins tunes local and global intramolecular interactions

Protein post-translational modifications, such as phosphorylation, are important regulatory signals for diverse cellular functions. In particular, intrinsically disordered protein regions (IDRs) are subject to phosphorylation as a means to modulate their interactions and functions. Toward understanding the relationship between phosphorylation in IDRs and specific functional outcomes, we must consider how phosphorylation affects the IDR conformational ensemble. Various experimental techniques are suited to interrogate the features of IDR ensembles; molecular simulations can provide complementary insights and even illuminate ensemble features that may be experimentally inaccessible. Therefore, we sought to expand the tools available to study phosphorylated IDRs by all-atom Monte Carlo simulations. To this end, we implemented parameters for phosphoserine (pSer) and phosphothreonine (pThr) into the OPLS version of the continuum solvent model, ABSINTH, and assessed their performance in all-atom simulations compared with published findings. We simulated short (<20 residues) and long (>80 residues) phospho-IDRs that, collectively, survey both local and global phosphorylation-induced changes to the ensemble. Our simulations of four well-studied phospho-IDRs show near-quantitative agreement with published findings for these systems via metrics including changes to radius of gyration, transient helicity, and persistence length. We also leveraged the inherent advantage of sequence control in molecular simulations to explore the conformational effects of diverse combinations of phospho-sites in two multiphosphorylated IDRs. Our results support and expand on previous observations that connect phosphorylation to changes in the IDR conformational ensemble. Herein, we describe phosphorylation as a means to alter sequence chemistry, net charge and charge patterning, and intramolecular interactions, which can collectively modulate the local and global IDR ensemble features.

IMMU-72. TYPE I INTERFERON SIGNALING WITHIN GLIOBLASTOMA CELLS PROMOTES T-CELL EXHAUSTION AND RESISTANCE TO CHECKPOINT INHIBITORS

Abstract Glioblastoma (GBM) remains among the most fatal cancers with few treatment modalities and resistance to modern immunotherapeutic treatment paradigms including immune checkpoint inhibitors (ICI). ICIs neutralize the regulatory signals that disable T cells and have demonstrated remarkable efficacy against a variety of cancers, though not in GBM. The impotence of ICI in GBM has been linked to the tumor’s remarkable capacity to elicit T cell exhaustion, though the mechanism remains poorly understood. Chronic interferon (IFN) cytokine signaling is known to promote immunosuppression and Tex in several cancer types as well as resistance to ICI. However, the role of IFN in GBM has not been investigated despite evidence that IFN signatures exist within clinical samples of GBM. Syngeneic murine GBM cell lines CT2A and SB28 were utilized for in vivo and in vitro studies. Anti-mouse PD-1 and CD8 antibodies or PBS control were injected intraperitoneally. CRISPR editing was used to create validated knockouts (KO) of the type I IFN receptor (IFNAR1) in CT2A. Mice bearing IFNAR1 KO intracranial tumors have improved median survival (p &amp;lt; 0.05) and are exquisitely sensitive to checkpoint anti-PD-1 blockade (p &amp;lt; 0.05) with the majority of mice surviving. Furthermore, surviving mice reject the subsequent intracranial re-challenge with parental CT2A tumors compared to controls (p &amp;lt;0.05) suggesting central memory responses mediating rejection. We conclude that abrogation of Type I IFN signaling is sufficient to extend survival which is further enhanced by ICI. Work is ongoing to further elucidate the precise mechanism that mediates this response and to identify a therapeutic intervention that enables tumor-specific inhibition of the IFN pathway with preliminary work suggesting a CD8 T cell mediated response and reversal of the T cell exhaustion phenotype.

Biodegradable and conventional mulches inhibit nitrogen fixation by peanut root nodules – potentially related to microplastics in the soil

Mulching has been demonstrated to improve the soil environment and promote plant growth. However, the effects of mulching and mulch-derived microplastics (MPs) on nitrogen fixation by root nodules remain unclear. In this study, we investigated the effects of polyethylene (PE) and polylactic acid-polybutylene adipate-co-terephthalate (PLA-PBAT) film mulching on nitrogen fixation by root nodules after 4 years of continuous mulching using 15N tracer technology. Additionally, we examined the relationship between nitrogen fixation and MPs. We found a reduction in the proportion of nitrogen fixation by nodules (54.3 %−58.7 %) due to mulching. This decrease may be attributed to reduced dinitrogenase activity and flavonoid content at the seedling stage caused by mulching, and mulching with PLA-PBAT films significantly decreased the abundance of Bradyrhizobium at maturity. Furthermore, combined analysis of nitrogen-fixing bacteria (nifH) and metabolomes indicated that N-lauroylethanolamine may act as a regulatory signal influencing the root nodule nitrogen fixation process and that mulching resulted in significant changes in its content. The mantel test and PLS-PM suggest that microplastic from mulching may harm root nodule nitrogen fixation. This study reveals the influence of mulching on plant nitrogen uptake and the potential threat of mulch-derived microplastics, with a special focus on root nodule nitrogen fixation.

A one-day journey to the suburbs: circadian clock in the Drosophila visual system.

Living organisms, which are constantly exposed to cyclical variations in their environment, need a high degree of plasticity in their visual system to respond to daily and seasonal fluctuations in lighting conditions. In Drosophila melanogaster, the visual system is a complex tissue comprising different photoreception structures that exhibit daily rhythms in gene expression, cell morphology, and synaptic plasticity, regulated by both the central and peripheral clocks. In this review, we briefly summarize the structure of the circadian clock and the visual system in Drosophila and comprehensively describe circadian oscillations in visual structures, from molecules to behaviors, which are fundamental for the fine-tuning of visual sensitivity. We also compare some features of the rhythmicity in the visual system with that of the central pacemaker and hypothesize about the differences in the regulatory signals and mechanisms that control these two clocks.

Experimental models of fibrosis

Fibrosis, as a pathological process, is characterized by excessive accumulation of extracellular matrix and can affect various organs and tissues, including the lungs, liver, heart and kidneys, leading to serious morbidity and poor quality of life. The main mechanisms of fibrosis are disturbances in cellular signaling pathways, their regulation, interaction, including disturbances in the exchange of cellular regulatory signals, impaired mechanisms of cell adhesion, and changes in the extracellular matrix. All this makes the search for new agents with antifibrotic activity to be urgent. The review examines the basic mechanisms of the development of fibrosis with an emphasis on experimental models, as well as the potential and limitations of experimental models of fibrosis in the context of further search and study of new drugs with antifibrotic activity.

Beyond Cancer Cells: How the Tumor Microenvironment Drives Cancer Progression.

Liver cancer represents a substantial global health challenge, contributing significantly to worldwide morbidity and mortality. It has long been understood that tumors are not composed solely of cancerous cells, but also include a variety of normal cells within their structure. These tumor-associated normal cells encompass vascular endothelial cells, fibroblasts, and various inflammatory cells, including neutrophils, monocytes, macrophages, mast cells, eosinophils, and lymphocytes. Additionally, tumor cells engage in complex interactions with stromal cells and elements of the extracellular matrix (ECM). Initially, the components of what is now known as the tumor microenvironment (TME) were thought to be passive bystanders in the processes of tumor proliferation and local invasion. However, recent research has significantly advanced our understanding of the TME's active role in tumor growth and metastasis. Tumor progression is now known to be driven by an intricate imbalance of positive and negative regulatory signals, primarily influenced by specific growth factors produced by both inflammatory and neoplastic cells. This review article explores the latest developments and future directions in understanding how the TME modulates liver cancer, with the aim of informing the design of novel therapies that target critical components of the TME.

Impact of Neurotransmitters on the Fatty Acid Composition and the Pigments of the Green Microalga Scenedesmus quadricauda

Apart from their functions in the nervous system of animals, neurotransmitters operate as regulatory agents and signals in diverse kingdoms of life. Some neurotransmitters have recently been revealed to exert specific effects on microalgae, predominantly functioning as algal growth stimulators. This article presents new data on the effects of such neurotransmitters as serotonin, norepinephrine, dopamine, histamine, and acetylcholine on the fatty acid and pigment composition of the green microalga Scenedesmus quadricauda (Turp.) Breb. K-1149. It was established that acetylcholine and, to a lesser extent, histamine increased the total fatty acid content of S. quadricauda cells, whereas serotonin and dopamine decreased the fatty acid content. Acetylcholine, histamine, and norepinephrine elevated the percentage of polyunsaturated fatty acids; in contrast, serotonin and dopamine increased the share of saturated fatty acids. Acetylcholine and, to a lesser extent, norepinephrine increased the total chlorophyll content per gram of dry weight in S. quadricauda, while histamine decreased the chlorophyll content. Histamine also increased the chlorophyll a/chlorophyll b and carotenoid/chlorophyll ratios, which were decreased by dopamine. The data obtained are of biotechnological and ecological interest. The stimulation of fatty acid accumulation and the increase in the percentage of polyunsaturated species was caused by the neurotransmitters acetylcholine and histamine at low (1–10 μM) concentrations, which potentially enables facilitating the biotechnological production of health-promoting preparations for therapeutic and cosmetic purposes. However, other neurotransmitters (dopamine and serotonin) tested increased the relative content of saturated fatty acids; therefore, they apparently can be used to stimulate biofuel production, since saturated fatty acid-rich lipids are advantageous raw materials for biodiesel production. The impact of neurotransmitters on microalgal fatty acid composition and photosystem components may be considered in terms of ongoing chemical interaction between microalgae and other aquatic ecosystem components that are known to produce neurotransmitters.

Pharyngeal Pumping Assay for Quantifying Feeding Behavior in Caenorhabditis elegans.

C. elegans is a well-established nematode model organism, with 83% of its genes conserved in humans with translation potential. C. elegans is translucent, with clearly defined cellular organization, and robustly identifiable under a microscope, being an excellent model for studying feeding behavior. Its neuromuscular pharyngeal pump undergoes a pumping motion that can be quantified to study feeding behavior at specific treatment conditions and in genetically modified worms. Understanding the evolutionarily conserved feeding behaviors and regulatory signals is vital, as unhealthy eating habits increase the risk of associated diseases. The current protocol was developed to identify and study evolutionary conserved signals regulating feeding behavior. The protocol described here is very robust in calculating the pumping rate (pumping per minute) as it directly counts the pharyngeal pumping for 30 s. This protocol uses basic laboratory instrumentation, such as a stereomicroscope with an attached camera and a computer with a video program that can be used to count manually. The advantages of studying C. elegans feeding include understanding the genetic basis of feeding regulation, dysregulation of feeding behavior in a disease model, the influence of toxic or environmental substances in feeding behavior, and modulation of feeding behavior by pharmacological agents. Key features • Quantifies pharyngeal pumping, which can be used to study up/downstream signaling in feeding regulation. • Uses a phenotype (pharyngeal pumping) that is easy to score. • Requires only a stereomicroscope with a camera to record the pharyngeal pumping, which can be counted manually.

Fluorescent tagging of endogenous IRS2 with an auxin-dependent degron to assess dynamic intracellular localization and function

Insulin Receptor Substrate 2 (IRS2) is a signaling adaptor protein for the insulin (IR) and Insulin-like Growth Factor-1 (IGF-1R) receptors. In breast cancer, IRS2 contributes to both initiation of primary tumor growth and establishment of secondary metastases through regulation of cancer stem cell (CSC) function and invasion. However, how IRS2 mediates its diverse functions is not well understood. We used CRISPR/Cas9-mediated gene editing to modify endogenous IRS2 to study the expression, localization, and function of this adaptor protein. A cassette containing an auxin inducible degradation (AID) sequence, 3X-FLAG tag and mNeon-green was introduced at the N-terminus of the IRS2 gene to provide rapid and reversible control of IRS2 protein degradation and analysis of endogenous IRS2 expression and localization. Live fluorescence imaging of these cells revealed that IRS2 shuttles between the cytoplasm and nucleus in response to growth regulatory signals in a PI3K-dependent manner. Inhibition of nuclear export or deletion of a putative nuclear export sequence in the C-terminal tail promotes nuclear retention of IRS2, implicating nuclear export in the mechanism by which IRS2 intracellular localization is regulated. Moreover, the acute induction of IRS2 degradation reduces tumor cell invasion, demonstrating the potential for therapeutic targeting of this adaptor protein. Our data highlight the value of our model of endogenously tagged IRS2 as a tool to study IRS2 localization and function.

Bridging the Knowledge Gap: Utilization of Mediator Subunits for Crop Improvement.

The Mediator complex is a multisubunit transcription coregulator that transfers regulatory signals from different transcription factors to RNA polymerase II (Pol II) to control Pol II-dependent transcription in eukaryotes. Studies on Arabidopsis Mediator subunits have revealed their unique or overlapping functions in various aspects of plant growth, stress adaptation and metabolite homeostasis. Therefore, the utilization of the plant Mediator complex for crop improvement has been of great interest. Advances in genome editing and sequencing techniques have expedited the characterization of Mediator subunits in economically important crops such as tomato, rice, wheat, soybean, sugarcane, pea, chickpea, rapeseed and hop. In this review, we summarize recent progress in understanding the molecular mechanisms of how the Mediator complex regulates crop growth, development and adaptation to environmental stress. We also discuss the conserved and diverse functions of the Mediator complex in different plant species. In addition, we propose several future research directions to deepen our understanding of the important roles of Mediator subunits and their interacting proteins, which would provide promising targets for genetic modification to develop new cultivars with desirable agronomic traits.

Investigation of the relationship between COVID-19 and pancreatic cancer using bioinformatics and systems biology approaches.

The coronavirus disease 2019 (COVID-19) pandemic, caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus, poses a huge threat to human health. Pancreatic cancer (PC) is a malignant tumor with high mortality. Research suggests that infection with SARS-CoV-2 may increase disease severity and risk of death in patients with pancreatic cancer, while pancreatic cancer may also increase the likelihood of contracting SARS-CoV-2, but the link is unclear. This study investigated the transcriptional profiles of COVID-19 and PC patients, along with their respective healthy controls, using bioinformatics and systems biology approaches to uncover the molecular mechanisms linking the 2 diseases. Specifically, gene expression data for COVID-19 and PC patients were obtained from the Gene Expression Omnibus datasets, and common differentially expressed genes (DEGs) were identified. Gene ontology and pathway enrichment analyses were performed on the common DEGs to elucidate the regulatory relationships between the diseases. Additionally, hub genes were identified by constructing a protein-protein interaction network from the shared DEGs. Using these hub genes, we conducted regulatory network analyses of microRNA/transcription factors-genes relationships, and predicted potential drugs for treating COVID-19 and PC. A total of 1722 and 2979 DEGs were identified from the transcriptome data of PC (GSE119794) and COVID-19 (GSE196822), respectively. Among these, 236 common DEGs were found between COVID-19 and PC based on protein-protein interaction analysis. Functional enrichment analysis indicated that these shared DEGs were involved in pathways related to viral genome replication and tumorigenesis. Additionally, 10 hub genes, including extra spindle pole bodies like 1, holliday junction recognition protein, marker of proliferation Ki-67, kinesin family member 4A, cyclin-dependent kinase 1, topoisomerase II alpha, cyclin B2, ubiquitin-conjugating enzyme E2 C, aurora kinase B, and targeting protein for Xklp2, were identified. Regulatory network analysis revealed 42 transcription factors and 23 microRNAs as transcriptional regulatory signals. Importantly, lucanthone, etoposide, troglitazone, resveratrol, calcitriol, ciclopirox, dasatinib, enterolactone, methotrexate, and irinotecan emerged as potential therapeutic agents against both COVID-19 and PC. This study unveils potential shared pathogenic mechanisms between PC and COVID-19, offering novel insights for future research and therapeutic strategies for the treatment of PC and SARS-CoV-2 infection.

An update on the role of ferroptosis in the pathogenesis of osteoporosis.

Ferroptosis is a novel form of programmed cell death, distinguished from apoptosis, autophagy, and programmed necrosis and has received much attention since it was defined in 2012. Ferroptotic cells physiologically exhibit iron metabolism dysregulation, oxidative stress, and lipid peroxidation. Morphologically, they show plasma membrane disruption, cytoplasmic swelling, and mitochondrial condensation. Osteoporosis is taken more and more seriously as the proportion of the aging population continues to increase globally. Interestingly, ferroptosis has been demonstrated to be involved in the development and progression of osteoporosis in many extant studies. The review summarizes iron metabolism, lipid peroxidation, and the different regulatory signals in ferroptosis. Changes in signaling mechanisms within osteoblasts, osteoclasts, and osteocytes after ferroptosis occur are explained here. Studies showed ferroptosis play an important role in different osteoporosis models (diabetes osteoporosis, postmenopausal osteoporosis, glucocorticoid-induced osteoporosis). Inhibitors and EC (Exos) targeting ferroptosis could ameliorate bone loss in osteoporotic mice by protecting cells against lipid peroxidation. Shortly, we hope that more effective and appropriate clinical therapy means will be utilized in the treatment of osteoporosis.

GENet: A Graph-Based Model Leveraging Histone Marks and Transcription Factors for Enhanced Gene Expression Prediction.

Understanding the regulatory mechanisms of gene expression is a crucial objective in genomics. Although the DNA sequence near the transcription start site (TSS) offers valuable insights, recent methods suggest that analyzing only the surrounding DNA may not suffice to accurately predict gene expression levels. We developed GENet (Gene Expression Network from Histone and Transcription Factor Integration), a novel approach that integrates essential regulatory signals from transcription factors and histone modifications into a graph-based model. GENet extends beyond simple DNA sequence analysis by incorporating additional layers of genetic control, which are vital for determining gene expression. Our method markedly enhances the prediction of mRNA levels compared to previous models that depend solely on DNA sequence data. The results underscore the significance of including comprehensive regulatory information in gene expression studies. GENet emerges as a promising tool for researchers, with potential applications extending from fundamental biological research to the development of medical therapies.

The Rbfox1/LASR complex controls alternative pre-mRNA splicing by recognition of multi-part RNA regulatory modules.

The Rbfox proteins regulate alternative pre-mRNA splicing by binding to the RNA element GCAUG. In the nucleus, most of Rbfox is bound to LASR, a complex of RNA-binding proteins that recognize additional RNA motifs. However, it remains unclear how the different subunits of the Rbfox/LASR complex act together to bind RNA and regulate splicing. We used a nuclease-protection assay to map the transcriptome-wide footprints of Rbfox1/LASR on nascent cellular RNA. In addition to GCAUG, Rbfox1/LASR binds RNA containing motifs for LASR subunits hnRNPs M, H/F, C, and Matrin3. These elements are often arranged in tandem, forming multi-part modules of RNA motifs. To distinguish contact sites of Rbfox1 from the LASR subunits, we analyzed a mutant Rbfox1(F125A) that has lost RNA binding but remains associated with LASR. Rbfox1(F125A)/LASR complexes no longer interact with GCAUG but retain binding to RNA elements for LASR. Splicing analyses reveal that in addition to activating exons through adjacent GCAUG elements, Rbfox can also stimulate exons near binding sites for LASR subunits. Mini-gene experiments demonstrate that these diverse elements produce a combined regulatory effect on a target exon. These findings illuminate how a complex of RNA-binding proteins can decode combinatorial splicing regulatory signals by recognizing groups of tandem RNA elements.

Investigating Economic and Climate Signals in Groundwater Data

Hydrologic, climatic, regulatory and economic factors interact in complex ways to influence groundwater conditions. These relationships can be difficult to measure and model but are important for water management and for considering viable futures for regional economies. This paper proposes a strategy for an improved understanding of how groundwater levels reflect signals of regulatory, economic and climate factors. Most existing econometric studies of groundwater use focus on large-scale irrigated agriculture and urbanized areas in first world nations, where groundwater extraction data is available. In much of the world, groundwater extraction data are not available for individual farms and other water users. The goal of this study is to explore the responsiveness of groundwater levels (a more widely available measure of groundwater conditions) to regulatory, economic and climate signals in rural areas, using a growing body of remotely sensed land cover data. Pooled Ordinary Least Squares econometric models examine the effects of regulatory, climatic and economic factors on groundwater levels in rural Arizona. Groundwater regulations, recharge projects, housing units and irrigated acreage have statistically significant relationships to groundwater levels in the study areas. This approach can be valuable for understanding factors that influence groundwater conditions and which may be useful in managing groundwater use in widespread areas where groundwater extraction data is unavailable.

Deep learning and direct sequencing of labeled RNA captures transcriptome dynamics.

In eukaryotes, genes produce a variety of distinct RNA isoforms, each with potentially unique protein products, coding potential or regulatory signals such as poly(A) tail and nucleotide modifications. Assessing the kinetics of RNA isoform metabolism, such as transcription and decay rates, is essential for unraveling gene regulation. However, it is currently impeded by lack of methods that can differentiate between individual isoforms. Here, we introduce RNAkinet, a deep convolutional and recurrent neural network, to detect nascent RNA molecules following metabolic labeling with the nucleoside analog 5-ethynyl uridine and long-read, direct RNA sequencing with nanopores. RNAkinet processes electrical signals from nanopore sequencing directly and distinguishes nascent from pre-existing RNA molecules. Our results show that RNAkinet prediction performance generalizes in various cell types and organisms and can be used to quantify RNA isoform half-lives. RNAkinet is expected to enable the identification of the kinetic parameters of RNA isoforms and to facilitate studies of RNA metabolism and the regulatory elements that influence it.

Developing a transdisciplinary tool for water risk management and decision-support in Ontario, Canada

Extant literature reveals limited examination of risk management strategies and tools to support decision-making for sustainable water management in the private sector in Ontario, Canada. Moreover, a gap persists in understanding how water risks are prioritized and managed in the private sector. Addressing these gaps, this transdisciplinary study applied a novel normative-analytical risk governance theoretical framework to water security risks, which combines analytical risk estimation with normative priorities and insights of practitioners, to examine contextually-attuned water risk management strategies and develop a decision-support tool. Using mixed methods, the study first employed a survey to elicit practitioner priorities for seven water risk indicators and investigated water risk management approaches. Then, interviews were conducted to obtain in-depth understanding about the priorities, strategies, opportunities, and role of trust in water risk management. The study found that a combination of regulatory, voluntary, and multi-stakeholder participatory approaches is needed, contingent on the severity of water risks, sector, location, and context. Moreover, the criteria of flexibility, efficiency, strategic incentives, and economic and regulatory signals, are essential. Finally, using secondary data analysis, the study integrated interdisciplinary risk data with practitioner priorities to develop a first-of-a-kind decision-support tool for water risk management in Ontario, ‘WATR-DST’. WATR-DST is an automated tool that applies the study’s findings and assists multi-sector water-related decisions, practices, and investments by providing contextually-attuned risk information in a user-friendly format. Based on the user inputs (location, sector, and source type), it displays the severity of seven water risks, qualitative themes under public and media attention, and recommends water risk management strategies. Thus, the study contributes to knowledge in sustainability management, risk analysis, and environmental management by demonstrating the novel application of the normative-analytical framework for water risk management in the private sector. WATR-DST is a key contribution envisioned to improve multi-sector water-related decisions in Ontario.