A Pliable Mediator Acts as a Functional Rather Than an Architectural Bridge between Promoters and Enhancers.

While Mediator plays a key role in eukaryotic transcription, little is known about its mechanism of action. This study combines CRISPR-Cas9 genetic screens, degron assays, Hi-C, and cryoelectron microscopy (cryo-EM) to dissect the function and structure of mammalian Mediator (mMED). Deletion analyses in B, T, and embryonic stem cells (ESC) identified a core of essential subunits required for Pol II recruitment genome-wide. Conversely, loss of non-essential subunits mostly affects promoters linked to multiple enhancers. Contrary to current models, however, mMED and Pol II are dispensable to physically tether regulatory DNA, a topological activity requiring architectural proteins. Cryo-EM analysis revealed a conserved core, with non-essential subunits increasing structural complexity of the tail module, a primary transcription factor target. Changes in tail structure markedly increase Pol II and kinase module interactions. We propose that Mediator's structural pliability enables it to integrate and transmit regulatory signals and act as a functional, rather than an architectural bridge, between promoters and enhancers.

A proximal enhancer regulates RORA expression during early human Th17 cell differentiation.

Gene regulatory elements, such as enhancers, greatly influence cell identity by tuning the transcriptional activity of specific cell types. Dynamics of enhancer landscape during early human Th17 cell differentiation remains incompletely understood. Leveraging ATAC-seq-based profiling of chromatin accessibility and comprehensive analysis of key histone marks, we identified a repertoire of enhancers that potentially exert control over the fate specification of Th17 cells. We found 23 SNPs associated with autoimmune diseases within Th17-enhancers that precisely overlapped with the binding sites of transcription factors actively engaged in T-cell functions. Among the Th17-specific enhancers, we identified an enhancer in the intron of RORA and demonstrated that this enhancer positively regulates RORA transcription. Moreover, CRISPR-Cas9-mediated deletion of a transcription factor binding site-rich region within the identified RORA enhancer confirmed its role in regulating RORA transcription. These findings provide insights into the potential mechanism by which the RORA enhancer orchestrates Th17 differentiation.

A systematic comparison of FOSL1, FOSL2 and BATF-mediated transcriptional regulation during early human Th17 differentiation.

Th17 cells are essential for protection against extracellular pathogens, but their aberrant activity can cause autoimmunity. Molecular mechanisms that dictate Th17 cell-differentiation have been extensively studied using mouse models. However, species-specific differences underscore the need to validate these findings in human. Here, we characterized the human-specific roles of three AP-1 transcription factors, FOSL1, FOSL2 and BATF, during early stages of Th17 differentiation. Our results demonstrate that FOSL1 and FOSL2 co-repress Th17 fate-specification, whereas BATF promotes the Th17 lineage. Strikingly, FOSL1 was found to play different roles in human and mouse. Genome-wide binding analysis indicated that FOSL1, FOSL2 and BATF share occupancy over regulatory regions of genes involved in Th17 lineage commitment. These AP-1 factors also share their protein interacting partners, which suggests mechanisms for their functional interplay. Our study further reveals that the genomic binding sites of FOSL1, FOSL2 and BATF harbour hundreds of autoimmune disease-linked SNPs. We show that many of these SNPs alter the ability of these transcription factors to bind DNA. Our findings thus provide critical insights into AP-1-mediated regulation of human Th17-fate and associated pathologies.

Advances in two-dimensional transition metal dichalcogenides-based sensors for environmental, food, and biomedical analysis: A review.

Transition metal dichalcogenides (TMDCs) are versatile two-dimensional (2D) nanomaterials used in biosensing applications due to their excellent physical and chemical properties. Due to biomaterial target properties, biosensors' most significant challenge is improving their sensitivity and stability. In environmental analysis, TMDCs have demonstrated exceptional pollutant detection and removal capabilities. Their high surface area, tunable electronic properties, and chemical reactivity make them ideal for sensors and adsorbents targeting various contaminants, including heavy metals, organic pollutants, and emerging contaminants. Furthermore, their unique electronic and optical properties enable sensitive detection techniques, enhancing our ability to monitor and mitigate environmental pollution. In the food analysis, TMDCs-based nanomaterials have shown remarkable potential in ensuring food safety and quality. These nanomaterials exhibit high specificity and sensitivity for detecting contaminants, pathogens, and adulterants in various food matrices. Their integration into sensor platforms enables rapid and on-site analysis, reducing the reliance on centralized laboratories and facilitating timely interventions in the food supply chain. In biomedical studies, TMDCs-based nanomaterials have demonstrated significant strides in diagnostic and therapeutic applications. Their biocompatibility, surface functionalization versatility, and photothermal properties have paved the way for novel disease detection, drug delivery, and targeted therapy approaches. Moreover, TMDCs-based nanomaterials have shown promise in imaging modalities, providing enhanced contrast and resolution for various medical imaging techniques. This article provides a comprehensive overview of 2D TMDCs-based biosensors, emphasizing the growing demand for advanced sensing technologies in environmental, food, and biomedical analysis.

Design, Development, and Evaluation of SA-F127:TPGS Polymeric Mixed Micelles for Improved Delivery of Glipizide Drug: In-vitro, Ex-vivo, and In-vivo Investigations.

The anti-diabetic glipizide (GLN) drug has notable pharmaceutical advantages, but poor aqueous solubility restricts its wide applications. The present work was to develop a mixed polymeric micelle system composed of SA-F127 and TPGS to improve the water solubility and effective delivery of the GLN. First, we synthesized SA-F127 and confirmed it through FTIR, NMR, and GPC techniques. The GLN-PMM were fabricated with the thin-film technique and optimized with CCD design. The developed GLN-PMM was characterized using DLS, Zeta, TEM, Rheology, FTIR, DSC, and XRD measurements. The GLN-PMM manifested a spherical morphology with 67.86 nm particle size, a -3.85 mV zeta potential, and a 0.582±0.06 PDI value. The polymeric mixed micelles showed excellent compatibility with GLN and were amorphous in nature. NMR studies confirmed the encapsulation of GLN in the core of the mixed micelle. In addition, the GLN-PMM micelles were tested in vitro for cumulative drug release, ex vivo for permeation, and in vivo for anti-diabetic investigations. The GLN-PMM release profile in the various pH environments showed over 90% after 24 h, clearly indicating sustained release. The GLN-PMM micelles gave higher 88.86±3.39% GLN permeation from the goat intestine compared with free GLN. In-vivo anti-diabetic investigation proves the powerful anti-diabetic properties of GLN-PMM in comparison to the marketed formulation. These findings demonstrated that the polymeric mixed micelles of SA-F127 and TPGS could be a promising, effective, and environment-friendly approach for oral delivery of the GLN.

Global chromatin state analysis reveals lineage-specific enhancers during the initiation of human T helper 1 and T helper 2 cell polarization.

Naive CD4⁺ T cells can differentiate into specific helper and regulatory T cell lineages in order to combat infection and disease. The correct response to cytokines and a controlled balance of these populations is critical for the immune system and the avoidance of autoimmune disorders. To investigate how early cell-fate commitment is regulated, we generated the first human genome-wide maps of histone modifications that reveal enhancer elements after 72 hr of in vitro polarization toward T helper 1 (Th1) and T helper 2 (Th2) cell lineages. Our analysis indicated that even at this very early time point, cell-specific gene regulation and enhancers were at work directing lineage commitment. Further examination of lineage-specific enhancers identified transcription factors (TFs) with known and unknown T cell roles as putative drivers of lineage-specific gene expression. Lastly, an integrative analysis of immunopathogenic-associated SNPs suggests a role for distal regulatory elements in disease etiology.

CRISPR/Cas9 guided genome and epigenome engineering and its therapeutic applications in immune mediated diseases.

Recent developments in the nucleic acid editing technologies have provided a powerful tool to precisely engineer the genome and epigenome for studying many aspects of immune cell differentiation and development as well as several immune mediated diseases (IMDs) including autoimmunity and cancer. Here, we discuss the recent technological achievements of the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-based RNA-guided genome and epigenome editing toolkit and provide an insight into how CRISPR/Cas9 (CRISPR Associated Protein 9) toolbox could be used to examine genetic and epigenetic mechanisms underlying IMDs. In addition, we will review the progress in CRISPR/Cas9-based genome-wide genome and epigenome screens in various cell types including immune cells. Finally, we will discuss the potential of CRISPR/Cas9 in defining the molecular function of disease associated SNPs overlapping gene regulatory elements.

Transcriptional and epigenetic regulation of T-helper lineage specification.

Combined with TCR stimuli, extracellular cytokine signals initiate the differentiation of naive CD4(+) T cells into specialized effector T-helper (Th) and regulatory T (Treg) cell subsets. The lineage specification and commitment process occurs through the combinatorial action of multiple transcription factors (TFs) and epigenetic mechanisms that drive lineage-specific gene expression programs. In this article, we review recent studies on the transcriptional and epigenetic regulation of distinct Th cell lineages. Moreover, we review current study linking immune disease-associated single-nucleotide polymorphisms with distal regulatory elements and their potential role in the disease etiology.

Methods for time series analysis of RNA-seq data with application to human Th17 cell differentiation.

MOTIVATION: Gene expression profiling using RNA-seq is a powerful technique for screening RNA species' landscapes and their dynamics in an unbiased way. While several advanced methods exist for differential expression analysis of RNA-seq data, proper tools to anal.yze RNA-seq time-course have not been proposed. RESULTS: In this study, we use RNA-seq to measure gene expression during the early human T helper 17 (Th17) cell differentiation and T-: cell activation (Th0). To quantify Th17-: specific gene expression dynamics, we present a novel statistical methodology, DyNB, for analyzing time-course RNA-seq data. We use non-parametric Gaussian processes to model temporal correlation in gene expression and combine that with negative binomial likelihood for the count data. To account for experiment-: specific biases in gene expression dynamics, such as differences in cell differentiation efficiencies, we propose a method to rescale the dynamics between replicated measurements. We develop an MCMC sampling method to make inference of differential expression dynamics between conditions. DyNB identifies several known and novel genes involved in Th17 differentiation. Analysis of differentiation efficiencies revealed consistent patterns in gene expression dynamics between different cultures. We use qRT-PCR to validate differential expression and differentiation efficiencies for selected genes. Comparison of the results with those obtained via traditional timepoint-: wise analysis shows that time-course analysis together with time rescaling between cultures identifies differentially expressed genes which would not otherwise be detected. AVAILABILITY: An implementation of the proposed computational methods will be available at http://research.ics.aalto.fi/csb/software/

Identification of early gene expression changes during human Th17 cell differentiation.

Th17 cells play an essential role in the pathogenesis of autoimmune and inflammatory diseases. Most of our current understanding on Th17 cell differentiation relies on studies carried out in mice, whereas the molecular mechanisms controlling human Th17 cell differentiation are less well defined. In this study, we identified gene expression changes characterizing early stages of human Th17 cell differentiation through genome-wide gene expression profiling. CD4(+) cells isolated from umbilical cord blood were used to determine detailed kinetics of gene expression after initiation of Th17 differentiation with IL1ß, IL6, and TGFß. The differential expression of selected candidate genes was further validated at protein level and analyzed for specificity in initiation of Th17 compared with initiation of other Th subsets, namely Th1, Th2, and iTreg. This first genome-wide profiling of transcriptomics during the induction of human Th17 differentiation provides a starting point for defining gene regulatory networks and identifying new candidates regulating Th17 differentiation in humans.

Rice Straw Waste-Based Biogas Production via Microbial Digestion: A Review.

The development of sustainable and renewable energy production is in high demand, and bioenergy production via microbial digestion of organic wastes is in prime focus. Biogas produced from the microbial digestion of organic waste is the most promising among existing biofuel options. In this context, biogas production from lignocellulosic biomass is one of the most viable and promising technologies for sustainable biofuel production. In the present review, an assessment and feasibility advancement have been presented towards the sustainable production of biogas from rice straw waste. Rice straw (RS) is abundantly available, contains a high composition of cellulose, and is found under the category of lignocellulosic waste, but it may cause severe environmental issues if not treated. Whereas, due to its high cellulose and inorganic content, lower cost, and huge availability, this waste can be effectively valorized into biogas production at a lower cost on a commercial scale. Therefore, the present review provides existing insight in this area by focusing on the operational parameter's improvement and advancement in the research for the expansion of mass-scale production at a lower cost. Thus, the presented review analyzed the processing parameters status, associated challenges, and positive endnote solutions for more sustainable viability for biogas production.

Biologically derived copper oxide-based nanocatalyst using Moringa oleifera leaves and its applications in hydrolytic enzymes and biohydrogen production.

Due to the limited availability of fossil fuels, pollution causing serious environmental issues, and their continuously rising price, the development of low-cost efficient enzymes and their implementation in biomass-based bioenergy industries are highly demanded. In the present work, phytogenic fabrication of copper oxide based nanocatalyst has been performed using moringa leaves and has been characterized using different techniques. Herein, the impact of different dosages of as-prepared nanocatalyst on fungal co-cultured cellulolytic enzyme production under co-substrate fermentation using wheat straw and sugarcane bagasse in 4:2 ratios in solid state fermentation (SSF) has been investigated. An optimal concentration of 25 ppm of nanocatalyst influenced the production of 32 IU/gds of enzyme, which showed thermal stability at 70 °C for 15 h. Additionally, enzymatic bioconversion of rice husk at 70 °C librated 41 g/L of total reducing sugars, which led to the production of 2390 mL/L of cumulative H2 in 120 h.

Rice straw derived graphene-silica based nanocomposite and its application in improved co-fermentative microbial enzyme production and functional stability.

Cellulases are the one of the most highly demanded industrial biocatalysts due to their versatile applications, such as in the biorefinery industry. However, relatively poor efficiency and high production costs are included as the key industrial constraints that hinder enzyme production and utilization at economic scale. Furthermore, the production and functional efficiency of the ß-glucosidase (BGL) enzyme is usually found to be relatively low among the cellulase cocktail produced. Thus, the current study focuses on fungi-mediated improvement of BGL enzyme in the presence of a rice straw-derived graphene-silica-based nanocomposite (GSNCs), which has been characterized using various techniques to analyze its physicochemical properties. Under optimized conditions of solid-state fermentation (SSF), co-fermentation using co-cultured cellulolytic enzyme has been done, and maximum enzyme production of 42 IU/gds FP, 142 IU/gds BGL, and 103 IU/gds EG have been achieved at a 5 mg concentration of GSNCs. Moreover, at a 2.5 mg concentration of nanocatalyst, the BGL enzyme showed its thermal stability at 60°C and 70 °C by holding its half-life relative activity for 7 h, while the same enzyme demonstrated pH stability at pH 8.0 and 9.0 for the 10 h. This thermoalkali BGL enzyme might be useful for the long-term bioconversion of cellulosic biomass into sugar.

Microbial cellulase production and stability investigations via graphene like carbon nanostructure derived from paddy straw.

Cellulases are among the most in-demand bioprocess enzymes, and the high cost of production, combined with their low enzymatic activity, is the main constraint, particularly in the biofuels industry. As a result, low-cost enzyme production modes with high activity and stability have emerged as the primary focus of research. Here, a method for producing a graphene like carbon nanostructure (GLCNs) has been investigated utilizing paddy straw (Ps), and its physicochemical characteristics have been examined using a variety of techniques including XRD, FT-IR, SEM and TEM. Further, the pretreatment of Ps feedstock for cellulase production was done using diluted waste KOH liquid collected during the preparation of the GLCNs. To increase the production and stability of the enzyme, newly prepared GLCNs is utilized as a nanocatalyst. Using 15 mg of GLCNs, 35 IU/gds FP activity was seen after 72 h, followed by 158 IU/gds EG and 114 IU/gds BGL activity in 96 h. This nanocatalyst supported enzyme was thermally stable at 70 °C up to 15 h and exhibited stability at pH 7.0 for 10 h by holding 66 % of its half-life.

Activating transcription factor 3 is a positive regulator of human IFNG gene expression.

IL-12 and IL-18 are essential for Th1 differentiation, whereas the role of IFN-alpha in Th1 development is less understood. In this microarray-based study, we searched for genes that are regulated by IFN-alpha, IL-12, or the combination of IL-12 plus IL-18 during the early differentiation of human umbilical cord blood CD4(+) Th cells. Twenty-six genes were similarly regulated in response to treatment with IL-12, IFN-alpha, or the combination of IL-12 plus IL-18. These genes could therefore play a role in Th1 lineage decision. Transcription factor activating transcription factor (ATF) 3 was upregulated by these cytokines and selected for further study. Ectopic expression of ATF3 in CD4(+) T cells enhanced the production of IFN-gamma, the hallmark cytokine of Th1 cells, whereas small interfering RNA knockdown of ATF3 reduced IFN-gamma production. Furthermore, ATF3 formed an endogenous complex with JUN in CD4(+) T cells induced to Th1. Chromatin immunoprecipitation and luciferase reporter assays showed that both ATF3 and JUN are recruited to and transactivate the IFNG promoter during early Th1 differentiation. Collectively, these data indicate that ATF3 promotes human Th1 differentiation.

Breast Cancer Transcriptional Regulatory Network Reprogramming by using the CRISPR/Cas9 System: An Oncogenetics Perspective.

Breast cancer (BC) is the second most commonly diagnosed cancer in the world. BC develops due to dysregulation of transcriptional profiles, substantial interpatient variations, genetic mutations, and dysregulation of signaling pathways in breast cells. These events are regulated by many genes such as BRCA1/2, PTEN, TP53, mTOR, TERT, AKT, PI3K and others genes. Treatment options for BC remain a hurdle, which warrants a comprehensive understanding that establishes an interlinking connection between these genes in BC tumorigenesis. Consequently, there is an increasing demand for alternative treatment approaches and the design of more effective treatments. In this regard, it is crucial to build the corresponding transcriptional regulatory networks governing BC by using advanced genetic tools and techniques. In the past, several molecular editing technologies have been used to edit genes with several limitations. Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR Associated Protein 9 (CRISPR/Cas9) recently received wise attention due to its potential in biomedical and therapeutic applications. Here, we review the role of various molecular signalling pathways dysregulated in BC development such as PTEN/PI3K/AKT/mTOR as well as BRCA1/BRCA2/TP53/TERT and their interplay between the related gene networks in BC initiation, progression and development of resistance against available targeted therapeutic agents. Use of CRISPR/Cas9 gene-editing technology to generate BC gene-specific transgenic cell lines and animal models to decipher their role and interactions with other gene products has been employed successfully. Moreover, the significance of using CRISPR/Cas9 technology to develop early BC diagnostic tools and treatments is discussed here.

Interactome Networks of FOSL1 and FOSL2 in Human Th17 Cells.

Dysregulated function of Th17 cells has implications in immunodeficiencies and autoimmune disorders. Th17 cell differentiation is orchestrated by a complex network of transcription factors, including several members of the activator protein (AP-1) family. Among the latter, FOSL1 and FOSL2 modulate the effector functions of Th17 cells. However, the molecular mechanisms underlying these effects are unclear, owing to the poorly characterized protein interaction networks of FOSL factors. Here, we establish the first interactomes of FOSL1 and FOSL2 in human Th17 cells, using affinity purification-mass spectrometry analysis. In addition to the known JUN proteins, we identified several novel binding partners of FOSL1 and FOSL2. Gene ontology analysis found a significant fraction of these interactors to be associated with RNA-binding activity, which suggests new mechanistic links. Intriguingly, 29 proteins were found to share interactions with FOSL1 and FOSL2, and these included key regulators of Th17 fate. We further validated the binding partners identified in this study by using parallel reaction monitoring targeted mass spectrometry and other methods. Our study provides key insights into the interaction-based signaling mechanisms of FOSL proteins that potentially govern Th17 cell differentiation and associated pathologies.

Evaluation of inhibitory activities of plant extracts on production of LPS-stimulated pro-inflammatory mediators in J774 murine macrophages.

Whole plant methanolic extracts of 14 traditionally used medicinal herbs were evaluated for their anti-inflammatory activity. Extracts of Grindelia robusta, Salix nigra, Arnica montana, and Quassia amara showed up to 4.5-fold inhibition of nitric oxide (NO) production in the J774 murine macrophage cells challenged with LPS without cytotoxicity. These four selected extracts significantly reduced the protein levels of inducible NO synthase (iNOS) and the cyclooxygenase-2 (COX-2) as observed by Western blot analysis. Culture supernatants from cells treated with these extracts indicated 3-5-fold reduction of tumor necrosis factor-alpha (TNF-alpha). However, only G. robusta and Q. amara extracts significantly inhibited (by 50%) IL-1beta and IL-12 secretions. Furthermore, all these plant extracts were shown to prevent the LPS-mediated nuclear translocation of nuclear factor-kappaB (NF-kappaB). All the above observations indicate the anti-inflammatory potential of these plant extracts.

Quantitative Proteomics Reveals the Dynamic Protein Landscape during Initiation of Human Th17 Cell Polarization.

Th17 cells contribute to the pathogenesis of inflammatory and autoimmune diseases and cancer. To reveal the Th17 cell-specific proteomic signature regulating Th17 cell differentiation and function in humans, we used a label-free mass spectrometry-based approach. Furthermore, a comprehensive analysis of the proteome and transcriptome of cells during human Th17 differentiation revealed a high degree of overlap between the datasets. However, when compared with corresponding published mouse data, we found very limited overlap between the proteins differentially regulated in response to Th17 differentiation. Validations were made for a panel of selected proteins with known and unknown functions. Finally, using RNA interference, we showed that SATB1 negatively regulates human Th17 cell differentiation. Overall, the current study illustrates a comprehensive picture of the global protein landscape during early human Th17 cell differentiation. Poor overlap with mouse data underlines the importance of human studies for translational research.

Transcriptional Repressor HIC1 Contributes to Suppressive Function of Human Induced Regulatory T Cells.

Regulatory T (Treg) cells are critical in regulating the immune response. In vitro induced Treg (iTreg) cells have significant potential in clinical medicine. However, applying iTreg cells as therapeutics is complicated by the poor stability of human iTreg cells and their variable suppressive activity. Therefore, it is important to understand the molecular mechanisms of human iTreg cell specification. We identified hypermethylated in cancer 1 (HIC1) as a transcription factor upregulated early during the differentiation of human iTreg cells. Although FOXP3 expression was unaffected, HIC1 deficiency led to a considerable loss of suppression by iTreg cells with a concomitant increase in the expression of effector T cell associated genes. SNPs linked to several immune-mediated disorders were enriched around HIC1 binding sites, and in vitro binding assays indicated that these SNPs may alter the binding of HIC1. Our results suggest that HIC1 is an important contributor to iTreg cell development and function.