Integration of genetic and chromatin modification data pinpoints autoimmune-specific remodeling of enhancer landscape in CD4+ T cells.

CD4+ T cells play a crucial role in adaptive immune responses and have been implicated in the pathogenesis of autoimmune diseases (ADs). Despite numerous studies, the molecular mechanisms underlying T cell dysregulation in ADs remain incompletely understood. Here, we used chromatin immunoprecipitation (ChIP)-sequencing of active chromatin and transcriptomic data from CD4+ T cells of healthy donors and patients with systemic lupus erythematosus (SLE), psoriasis, juvenile idiopathic arthritis (JIA), and Graves' disease to investigate the role of enhancers in AD pathogenesis. By generating enhancer-based gene regulatory networks (eGRNs), we identified disease-specific dysregulated pathways and potential downstream target genes of enhancers harboring AD-associated single-nucleotide polymorphisms (SNPs), which we also validated using chromatin-capture (HiC) data and CRISPR interference (CRISPRi) in primary CD4+ T cells. Our results suggest that alterations in the regulatory landscapes of CD4+ T cells, including enhancers, contribute to the development of ADs and provide a basis for developing new therapeutic approaches.

The molecular design of novel phospholipid-inspired ionic liquid transdermal penetration enhancers: Innovative insights on the action mode and mechanism.

Ionic liquid transdermal penetration enhancers (IL@TPEs) as new enhancement methods have significant advantages in the transdermal drug delivery system. However, the scientific frameworks for the design of efficient IL@TPEs and their applications in transdermal formulations were still lack. So, a series of novel biomimetic phospholipid-inspired IL@TPEs (PIL@TPEs) were designed and synthesized. The developed QSARs proved that enhancement efficacy of PIL@TPEs depended on pKa of drugs and M.W., Polar., and pKa of cations. Surprisingly, the PIL@TPEs dissociated during transdermal process, and skin penetration amounts of acidic drugs was inversely proportional to skin retention amounts of cations, which showed that action modes of PIL@TPEs were different from conventional enhancers. The novel mechanisms of PIL@TPEs were elucidated by quantitative determination of dynamic interaction among cations, anions, drugs, and skins. The PIL@TPEs with high enhancement efficiency owned strong interactions with drugs determined by ATR-FTIR, Raman and NOESY. Moreover, the PIL@TPEs owning better stability in skin ensured the production of strong interactions with lipids and keratins characterized by ATR-FTIR, 1H NMR and CLSM. The good safety of optimized PIL@TPEs was proved by determining cytotoxicity, apoptosis, inflammatory cells, and cytokines. In conclusion, this project will make an important contribution to the design and application of IL@TPEs.

HOXDeRNA activates a cancerous transcription program and super enhancers via genome-wide binding.

The role of long non-coding RNAs (lncRNAs) in malignant cell transformation remains elusive. We previously identified an enhancer-associated lncRNA, LINC01116 (named HOXDeRNA), as a transformative factor converting human astrocytes into glioma-like cells. Employing a combination of CRISPR editing, chromatin isolation by RNA purification coupled with sequencing (ChIRP-seq), in situ mapping RNA-genome interactions (iMARGI), chromatin immunoprecipitation sequencing (ChIP-seq), HiC, and RNA/DNA FISH, we found that HOXDeRNA directly binds to CpG islands within the promoters of 35 glioma-specific transcription factors (TFs) distributed throughout the genome, including key stem cell TFs SOX2, OLIG2, POU3F2, and ASCL1, liberating them from PRC2 repression. This process requires a distinct RNA quadruplex structure and other segments of HOXDeRNA, interacting with EZH2 and CpGs, respectively. Subsequent transformation activates multiple oncogenes (e.g., EGFR, miR-21, and WEE1), driven by the SOX2- and OLIG2-dependent glioma-specific super enhancers. These results help reconstruct the sequence of events underlying the process of astrocyte transformation, highlighting HOXDeRNA's central genome-wide activity and suggesting a shared RNA-dependent mechanism in otherwise heterogeneous and multifactorial gliomagenesis.

Conjugation of visual enhancers in lateral flow immunoassay for rapid forensic analysis: A critical review.

During crime scene investigations, numerous traces are secured and may be used as evidence for the evaluation of source and/or activity level propositions. The rapid chemical analysis of a biological trace enables the identification of body fluids and can provide significant donor profiling information, including age, sex, drug abuse, and lifestyle. Such information can be used to provide new leads, exclude from, or restrict the list of possible suspects during the investigative phase. This paper reviews the state-of-the-art labelling techniques to identify the most suitable visual enhancer to be implemented in a lateral flow immunoassay setup for the purpose of trace identification and/or donor profiling. Upon comparison, and with reference to the strengths and limitations of each label, the simplistic one-step analysis of noncompetitive lateral flow immunoassay (LFA) together with the implementation of carbon nanoparticles (CNPs) as visual enhancers is proposed for a sensitive, accurate, and reproducible in situ trace analysis. This approach is versatile and stable over different environmental conditions and external stimuli. The findings of the present comparative analysis may have important implications for future forensic practice. The selection of an appropriate enhancer is crucial for a well-designed LFA that can be implemented at the crime scene for a time- and cost-efficient investigation.

Regulatory genome annotation of 33 insect species.

Annotation of newly sequenced genomes frequently includes genes, but rarely covers important non-coding genomic features such as the cis-regulatory modules-e.g., enhancers and silencers-that regulate gene expression. Here, we begin to remedy this situation by developing a workflow for rapid initial annotation of insect regulatory sequences, and provide a searchable database resource with enhancer predictions for 33 genomes. Using our previously developed SCRMshaw computational enhancer prediction method, we predict over 2.8 million regulatory sequences along with the tissues where they are expected to be active, in a set of insect species ranging over 360 million years of evolution. Extensive analysis and validation of the data provides several lines of evidence suggesting that we achieve a high true-positive rate for enhancer prediction. One, we show that our predictions target specific loci, rather than random genomic locations. Two, we predict enhancers in orthologous loci across a diverged set of species to a significantly higher degree than random expectation would allow. Three, we demonstrate that our predictions are highly enriched for regions of accessible chromatin. Four, we achieve a validation rate in excess of 70% using in vivo reporter gene assays. As we continue to annotate both new tissues and new species, our regulatory annotation resource will provide a rich source of data for the research community and will have utility for both small-scale (single gene, single species) and large-scale (many genes, many species) studies of gene regulation. In particular, the ability to search for functionally related regulatory elements in orthologous loci should greatly facilitate studies of enhancer evolution even among distantly related species.

Modelling and calibration of pair-rule protein patterns in Drosophila embryo: From Even-skipped and Fushi-tarazu to Wingless expression networks.

We modelled and calibrated the distributions of the seven-stripe patterns of Even-skipped (Eve) and Fushi-tarazu (Ftz) pair-rule proteins along the anteroposterior axis of the Drosphila embryo, established during early development. We have identified the putative repressive combinations for five Eve enhancers, and we have explored the relationship between Eve and Ftz for complementary patterns. The regulators of Eve and Ftz are stripe-specific DNA enhancers with embryo position-dependent activation rates and are regulated by the gap family of proteins. We achieved remarkable data matching of the Eve stripe pattern, and the calibrated model reproduces gap gene mutation experiments. Extended work inferring the Wingless (Wg) fourteen stripe pattern from Eve and Ftz enhancers have been proposed, clarifying the hierarchical structure of Drosphila's genetic expression network during early development.

Emerging Circulating Biomarkers for Enhanced Cardiovascular Risk Prediction.

Cardiovascular disease (CVD) continues to be the primary cause of mortality worldwide, underscoring the importance of identifying additional cardiovascular risk factors. The consensus is that lipid levels alone do not fully reflect the status of atherosclerosis, thus necessitating extensive research on cardiovascular biomarkers. This review encompasses a wide spectrum of methodologies for identifying novel risk factors or biomarkers for CVD. Inflammation, oxidative stress, plaque instability, cardiac remodeling, and fibrosis play pivotal roles in CVD pathogenesis. We introduce and discuss several promising biomarkers-namely, osteocalcin, angiogenin, lipoprotein-associated phospholipase A2, growth differentiation factor 15, galectin-3, growth stimulation expressed gene 2, and microRNAs, all of which have potential implications in the assessment and management of cardiovascular risk.

A novel C-4-modified isotetrone acts as a potent bio-enhancer to augment the activities of anti-tuberculosis drugs against Mycobacterium tuberculosis.

Mycobacterium tuberculosis is a deadly pathogen that claims millions of lives every year. Current research focuses on finding new anti-tuberculosis drugs that are safe and effective, with lesser side effects and toxicity. One important approach is to identify bio-enhancers that can improve the effectiveness of anti-tuberculosis drugs, resulting in reduced doses and shortened treatment times. The present study investigates the use of C-4 modified isotetrones as bio-enhancers. A series of studies suggest an isotetrone, labeled as C11, inhibits growth, improves MIC, MBC and enhances the killing of M. tuberculosis H37Rv strain when used in combination with the first line and injectable anti-TB drugs in a dose-dependent manner. The combination of C11 and rifampicin also reduces the generation of spontaneous mutants against rifampicin and reaches a mutation prevention concentration (MPC) with moderate rifampicin concentrations. The identified compounds are effective against the MDR strain of M. tuberculosis and non-cytotoxic in HepG2 cells. We find that C11 induces the generation of reactive oxygen species (ROS) inside macrophages and within bacteria, resulting in better efficacy.

Enhancer landscape of lung neuroendocrine tumors reveals regulatory and developmental signatures with potential theranostic implications.

Well-differentiated low-grade lung neuroendocrine tumors (lung carcinoids or LNETs) are histopathologically classified as typical and atypical LNETs, but each subtype is still heterogeneous at both the molecular level and its clinical manifestation. Here, we report genome-wide profiles of primary LNETs' cis-regulatory elements by H3K27ac ChIP-seq with matching RNA-seq profiles. Analysis of these regulatory landscapes revealed three regulatory subtypes, independent of the typical/atypical classification. We identified unique differentiation signals that delineate each subtype. The "proneuronal" subtype emerges under the influence of ASCL1, SOX4, and TCF4 transcription factors, embodying a pronounced proneuronal signature. The "luminal-like" subtype is characterized by gain of acetylation at markers of luminal cells and GATA2 activation and loss of LRP5 and OTP. The "HNF+" subtype is characterized by a robust enhancer landscape driven by HNF1A, HNF4A, and FOXA3, with notable acetylation and expression of FGF signaling genes, especially FGFR3 and FGFR4, pivotal components of the FGF pathway. Our findings not only deepen the understanding of LNETs' regulatory and developmental diversity but also spotlight the HNF+ subtype's reliance on FGFR signaling. We demonstrate that targeting this pathway with FGF inhibitors curtails tumor growth both in vitro and in xenograft models, unveiling a potential vulnerability and paving the way for targeted therapies. Overall, our work provides an important resource for studying LNETs to reveal regulatory networks, differentiation signals, and therapeutically relevant dependencies.

Insufficient correctness of package inserts for psychotropic drugs in Germany.

Package inserts often cause displeasure among patients as they are perceived as misleading and confusing. The aim of this study was to find out how comprehensible, complete, and truthful package inserts are formulated in Germany. 311 package inserts for antipsychotics (mGPCR antagonists) and antidepressants (NE/5-HT enhancers) from different manufacturers and dosages were analysed. The analysis criteria included the description of the effect, the warning of increased suicide risk, the explanation of interactions with co-medication, food and stimulants, as well as alcohol and the warning of impaired roadworthiness. In addition, the timeliness of the information regarding pregnancy and breastfeeding was checked and the symptoms mentioned under the topic of discontinuation symptoms and adverse drug reactions were compared. For most parameters, inconsistencies among various products, deficiencies and incorrect information were noted. This was particularly true for the items pregnancy and breastfeeding. Thus, package inserts for mGPCR antagonists and NE/5-HT enhancers need to be updated and standardised urgently. Such measures will reduce confusion among patients and increase drug adherence.

Development of levamlodipine long-acting patches based on an ion-pair strategy: Investigation of the mechanism for reducing skin irritation.

The aim of this study was to develop a long-acting transdermal patch of levamlodipine (LAM) using an ion-pair strategy to reduce the skin irritation induced by topical application of LAM and explore the mechanism underlying the improvement of skin irritation. The formulation was optimized through porcine in vitro transdermal experiments and rabbit in vivo skin irritation tests. The obtained formulation consisted of poly (2-Ethylhexyl acrylate-co-N-Vinyl-2-pyrrolidone-co-N-(2-Hydroxyethyl) acrylamide) (PENH) as the adhesive matrix, 13.00 % levamlodipine-sorbic acid ion-pair complex (LAM-SA) (w/w), and 10 % isopropyl myristate (IPM) (w/w), with a patch thickness of 70 µm, achieving an erythema index of 188 for rabbit skin and 117-187 for human skin (264 for rabbit skin and 110-260 for human skin in the absence of sorbic acid (SA)). In vivo rabbit and human skin erythema analysis and H&E staining verified that the optimized ion-pair patch effectively reduced skin irritation. Drug distribution experiments in the skin, ATR-FTIR, and molecular simulation were used to characterize the mechanism by which the ion-pair reduced skin irritation. Excessive accumulation of LAM in the epidermis induced secondary structural changes in keratin, resulting in skin barrier damage and inflammatory response. The formation of the LAM-SA ion pair altered physicochemical properties of LAM, reducing drug retention in the epidermis and, thereby, reducing skin irritation. This study demonstrated the potential of the ion-pair strategy to improve the safety of transdermal drug delivery system (TDDS) and provided a means for reducing skin irritation caused by the active pharmaceutical ingredient (API) itself.

Enhancing topical delivery of ISRIB: Optimizing cream formulations with chemical enhancers and pH adjustment.

Chemical warfare agents, particularly vesicants like lewisite, pose a threat due to their ability to cause skin damage through accidental exposure or deliberate attacks. Lewisite rapidly penetrates the skin, causing inflammation and blistering. This study focuses on developing a cream formulation of a therapeutic agent, called integrated stress response inhibitor (ISRIB), to treat lewisite-induced injuries. Moreover, animal studies demonstrate a molecular target engagement (ISR) and significant efficacy of ISRIB against lewisite-induced cutaneous injury. The goal of this formulation is to enhance the delivery of ISRIB directly to affected skin areas using an oil-in-water cream emulsion system. We investigated various excipients, including oils, surfactants, emollients, and permeation enhancers, to optimize ISRIB's solubility and penetration through the skin. The result of this study indicated that the optimal formulation includes 30 % w/w of N-Methyl-2-pyrrolidone, dimethyl sulfoxide and Azone® at a pH of 5. 5. It delivered the highest amount of ISRIB into the skin, demonstrating highest skin absorption with no detectable systemic exposure. Additionally, characterization of the cream, including texture analysis, emulsion type, and content uniformity, confirmed its' suitability for topical application. These findings suggest that ISRIB cream formulation is a promising approach for the localized treatment of skin injuries caused by lewisite.

CASCADES, a novel SOX2 super-enhancer-associated long noncoding RNA, regulates cancer stem cell specification and differentiation in glioblastoma.

Glioblastoma is the most common primary malignant brain tumor in adults, with a median survival of just over 1 year. The failure of available treatments to achieve remission in patients with glioblastoma (GBM) has been attributed to the presence of cancer stem cells (CSCs), which are thought to play a central role in tumor development and progression and serve as a treatment-resistant cell repository capable of driving tumor recurrence. In fact, the property of "stemness" itself may be responsible for treatment resistance. In this study, we identify a novel long noncoding RNA (lncRNA), cancer stem cell-associated distal enhancer of SOX2 (CASCADES), that functions as an epigenetic regulator in glioma CSCs (GSCs). CASCADES is expressed in isocitrate dehydrogenase (IDH)-wild-type GBM and is significantly enriched in GSCs. Knockdown of CASCADES in GSCs results in differentiation towards a neuronal lineage in a cell- and cancer-specific manner. Bioinformatics analysis reveals that CASCADES functions as a super-enhancer-associated lncRNA epigenetic regulator of SOX2. Our findings identify CASCADES as a critical regulator of stemness in GSCs that represents a novel epigenetic and therapeutic target for disrupting the CSC compartment in glioblastoma.

How pharmacology can aid in the diagnosis of mental disorders.

The precise diagnosis of mental disorders constitutes a formidable problem. Mental disorders are currently diagnosed based on clinical symptoms, which are often subjective. Various drug classes, traditionally referred to as "antidepressants," "antipsychotics" and "mood stabilizers" are then used empirically to treat affected patients. The previous decade has witnessed an increasing extension of the use of drug classes beyond their traditional indications (e.g., "antidepressants" in the treatment of anxiety disorders). Therefore, we would like to initiate a discussion in the pharmacological and psychiatric research communities on an alternative classification of mental disorders: Instead of using the traditional categorical classification of mental disorders physicians should rather diagnose symptoms (e.g., anhedonia) without bias to a traditional categorization (e.g., depression). The appropriate most effective drugs are then selected based on these symptoms. Depending on the responsiveness of the patient towards a given drug X, the disease should be classified, e.g., as drug X-responsive disease. This approach will also help us elucidate the still poorly understood molecular mechanisms underlying mental disorders, i.e., drugs can also be viewed and used as molecular diagnostic tools. In several fields of medicine, drugs are already used as molecular diagnostic tools. Thus, there is already precedence for the concept proposed here for mental disorders.

Ultra-processed foods, allergy outcomes and underlying mechanisms in children: An EAACI task force report.

BACKGROUND: Consumption of ultra-processed foods [UPFs] may be associated with negative health outcomes. Limited data exist regarding the potential role of UPFs in the occurrence of allergic diseases. The underlying mechanisms underpinning any such associations are also poorly elucidated. METHODS: We performed a systematic review and narrative evidence synthesis of the available literature to assess associations between UPF consumption and pediatric allergy outcomes (n = 26 papers), including data on the association seen with the gut microbiome (n = 16 papers) or immune system (n = 3 papers) structure and function following PRISMA guidelines. RESULTS: Dietary exposure to fructose, carbonated soft drinks, and sugar intake was associated with an increased risk of asthma, allergic rhinitis, and food allergies in children. Commercial baby food intake was associated with childhood food allergy. Childhood intake of fructose, fruit juices, sugar-sweetened beverages, high carbohydrate UPFs, monosodium glutamate, UPFs, and advanced glycated end-products (AGEs) was associated with the occurrence of allergic diseases. Exposure to UPFs and common ingredients in UPFs seem to be associated with increased occurrence of allergic diseases such as asthma, wheezing, food allergies, atopic dermatitis, and allergic rhinitis, in many, but not all studies. CONCLUSION: More preclinical and clinical studies are required to better define the link between UPF consumption and the risk of allergies and asthma. These observational studies ideally require supporting data with clearly defined UPF consumption, validated dietary measures, and mechanistic assessments to definitively link UPFs with the risk of allergies and asthma.

Beyond modular enhancers: new questions in cis-regulatory evolution.

Our understanding of how cis-regulatory elements work has advanced rapidly, outpacing our evolutionary models. In this review, we consider the implications of new mechanistic findings for evolutionary developmental biology. We focus on three different debates: whether evolutionary innovation occurs more often via the modification of old cis-regulatory elements or the emergence of new ones; the extent to which individual elements are specific and autonomous or multifunctional and interdependent; and how the robustness of cis-regulatory architectures influences the rate of trait evolution. These discussions lead us to propose new questions for the evo-devo of cis-regulation.

Temporally discordant chromatin accessibility and DNA demethylation define short and long-term enhancer regulation during cell fate specification.

Epigenetic mechanisms govern the transcriptional activity of lineage-specifying enhancers; but recent work challenges the dogma that joint chromatin accessibility and DNA demethylation are prerequisites for transcription. To understand this paradox, we established a highly-resolved timeline of DNA demethylation, chromatin accessibility, and transcription factor occupancy during neural progenitor cell differentiation. We show thousands of enhancers undergo rapid, transient accessibility changes associated with distinct periods of transcription factor expression. However, most DNA methylation changes are unidirectional and delayed relative to chromatin dynamics, creating transiently discordant epigenetic states. Genome-wide detection of 5-hydroxymethylcytosine further revealed active demethylation begins ahead of chromatin and transcription factor activity, while enhancer hypomethylation persists long after these activities have dissipated. We demonstrate that these timepoint specific methylation states predict past, present and future chromatin accessibility using machine learning models. Thus, chromatin and DNA methylation collaborate on different timescales to mediate short and long-term enhancer regulation during cell fate specification.

Smarcd1 subunit of SWI/SNF chromatin-remodeling complexes collaborates with E2a to promote murine lymphoid specification.

Lymphocyte development from murine hematopoietic stem cells (HSCs) entails a loss of self-renewal capacity and a progressive restriction of developmental potential. Previous research from our laboratory suggests that specialized assemblies of ATP-dependent SWI/SNF chromatin-remodeling complexes play lineage-specific roles during murine hematopoiesis. Here, we demonstrate that the Smarcd1 subunit is essential for specification of lymphoid cell fate from multipotent progenitors. Acute deletion of Smarcd1 in murine adult hematopoiesis leads to lymphopenia, characterized by a near-complete absence of early lymphoid progenitors and mature B and T cells, while the myeloid and erythroid lineages remain unaffected. Mechanistically, we demonstrate that Smarcd1 is essential for the coordinated activation of a lymphoid gene signature in murine multipotent progenitors. This is achieved by interacting with the E2a transcription factor at proximal promoters and by regulating the activity of distal enhancers. Globally, these findings identify Smarcd1 as an essential chromatin remodeler that governs lymphoid cell fate.

Enhlink infers distal and context-specific enhancer-promoter linkages.

Enhlink is a computational tool for scATAC-seq data analysis, facilitating precise interrogation of enhancer function at the single-cell level. It employs an ensemble approach incorporating technical and biological covariates to infer condition-specific regulatory DNA linkages. Enhlink can integrate multi-omic data for enhanced specificity, when available. Evaluation with simulated and real data, including multi-omic datasets from the mouse striatum and novel promoter capture Hi-C data, demonstrate that Enhlink outperfoms alternative methods. Coupled with eQTL analysis, it identified a putative super-enhancer in striatal neurons. Overall, Enhlink offers accuracy, power, and potential for revealing novel biological insights in gene regulation.

Enhancers: A Focus on Synthetic Biology and Correlated Gene Expression.

Enhancers are central for the regulation of metazoan transcription but have proven difficult to study, primarily due to a myriad of interdependent variables shaping their activity. Consequently, synthetic biology has emerged as the main approach for dissecting mechanisms of enhancer function. We start by reviewing simple but highly parallel reporter assays, which have been successful in quantifying the complexity of the activator/coactivator mechanisms at enhancers. We then describe studies that examine how enhancers function in the genomic context and in combination with other enhancers, revealing that they activate genes through a variety of different mechanisms, working together as a system. Here, we primarily focus on synthetic reporter genes that can quantify the dynamics of enhancer biology through time. We end by considering the consequences of having many genes and enhancers within a 'local environment', which we believe leads to correlated gene expression and likely reports on the general principles of enhancer biology.