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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Genome-wide Analysis of STAT3-Mediated Transcription during Early Human Th17 Cell Differentiation.

The development of therapeutic strategies to combat immune-associated diseases requires the molecular mechanisms of human Th17 cell differentiation to be fully identified and understood. To investigate transcriptional control of Th17 cell differentiation, we used primary human CD4+ T cells in small interfering RNA (siRNA)-mediated gene silencing and chromatin immunoprecipitation followed by massive parallel sequencing (ChIP-seq) to identify both the early direct and indirect targets of STAT3. The integrated dataset presented in this study confirms that STAT3 is critical for transcriptional regulation of early human Th17 cell differentiation. Additionally, we found that a number of SNPs from loci associated with immune-mediated disorders were located at sites where STAT3 binds to induce Th17 cell specification. Importantly, introduction of such SNPs alters STAT3 binding in DNA affinity precipitation assays. Overall, our study provides important insights for modulating Th17-mediated pathogenic immune responses in humans.

Comparative analysis of human and mouse transcriptomes of Th17 cell priming.

Uncontrolled Th17 cell activity is associated with cancer and autoimmune and inflammatory diseases. To validate the potential relevance of mouse models of targeting the Th17 pathway in human diseases we used RNA sequencing to compare the expression of coding and non-coding transcripts during the priming of Th17 cell differentiation in both human and mouse. In addition to already known targets, several transcripts not previously linked to Th17 cell polarization were found in both species. Moreover, a considerable number of human-specific long non-coding RNAs were identified that responded to cytokines stimulating Th17 cell differentiation. We integrated our transcriptomics data with known disease-associated polymorphisms and show that conserved regulation pinpoints genes that are relevant to Th17 cell-mediated human diseases and that can be modelled in mouse. Substantial differences observed in non-coding transcriptomes between the two species as well as increased overlap between Th17 cell-specific gene expression and disease-associated polymorphisms underline the need of parallel analysis of human and mouse models. Comprehensive analysis of genes regulated during Th17 cell priming and their classification to conserved and non-conserved between human and mouse facilitates translational research, pointing out which candidate targets identified in human are worth studying by using in vivo mouse models.

Identification of global regulators of T-helper cell lineage specification.

BACKGROUND: Activation and differentiation of T-helper (Th) cells into Th1 and Th2 types is a complex process orchestrated by distinct gene activation programs engaging a number of genes. This process is crucial for a robust immune response and an imbalance might lead to disease states such as autoimmune diseases or allergy. Therefore, identification of genes involved in this process is paramount to further understand the pathogenesis of, and design interventions for, immune-mediated diseases. METHODS: We aimed at identifying protein-coding genes and long non-coding RNAs (lncRNAs) involved in early differentiation of T-helper cells by transcriptome analysis of cord blood-derived naïve precursor, primary and polarized cells. RESULTS: Here, we identified lineage-specific genes involved in early differentiation of Th1 and Th2 subsets by integrating transcriptional profiling data from multiple platforms. We have obtained a high confidence list of genes as well as a list of novel genes by employing more than one profiling platform. We show that the density of lineage-specific epigenetic marks is higher around lineage-specific genes than anywhere else in the genome. Based on next-generation sequencing data we identified lineage-specific lncRNAs involved in early Th1 and Th2 differentiation and predicted their expected functions through Gene Ontology analysis. We show that there is a positive trend in the expression of the closest lineage-specific lncRNA and gene pairs. We also found out that there is an enrichment of disease SNPs around a number of lncRNAs identified, suggesting that these lncRNAs might play a role in the etiology of autoimmune diseases. CONCLUSION: The results presented here show the involvement of several new actors in the early differentiation of T-helper cells and will be a valuable resource for better understanding of autoimmune processes.

A validated gene regulatory network and GWAS identifies early regulators of T cell-associated diseases.

Early regulators of disease may increase understanding of disease mechanisms and serve as markers for presymptomatic diagnosis and treatment. However, early regulators are difficult to identify because patients generally present after they are symptomatic. We hypothesized that early regulators of T cell-associated diseases could be found by identifying upstream transcription factors (TFs) in T cell differentiation and by prioritizing hub TFs that were enriched for disease-associated polymorphisms. A gene regulatory network (GRN) was constructed by time series profiling of the transcriptomes and methylomes of human CD4(+) T cells during in vitro differentiation into four helper T cell lineages, in combination with sequence-based TF binding predictions. The TFs GATA3, MAF, and MYB were identified as early regulators and validated by ChIP-seq (chromatin immunoprecipitation sequencing) and small interfering RNA knockdowns. Differential mRNA expression of the TFs and their targets in T cell-associated diseases supports their clinical relevance. To directly test if the TFs were altered early in disease, T cells from patients with two T cell-mediated diseases, multiple sclerosis and seasonal allergic rhinitis, were analyzed. Strikingly, the TFs were differentially expressed during asymptomatic stages of both diseases, whereas their targets showed altered expression during symptomatic stages. This analytical strategy to identify early regulators of disease by combining GRNs with genome-wide association studies may be generally applicable for functional and clinical studies of early disease development.

Expression profiles of long non-coding RNAs located in autoimmune disease-associated regions reveal immune cell-type specificity.

BACKGROUND: Although genome-wide association studies (GWAS) have identified hundreds of variants associated with a risk for autoimmune and immune-related disorders (AID), our understanding of the disease mechanisms is still limited. In particular, more than 90% of the risk variants lie in non-coding regions, and almost 10% of these map to long non-coding RNA transcripts (lncRNAs). lncRNAs are known to show more cell-type specificity than protein-coding genes. METHODS: We aimed to characterize lncRNAs and protein-coding genes located in loci associated with nine AIDs which have been well-defined by Immunochip analysis and by transcriptome analysis across seven populations of peripheral blood leukocytes (granulocytes, monocytes, natural killer (NK) cells, B cells, memory T cells, naive CD4(+) and naive CD8(+) T cells) and four populations of cord blood-derived T-helper cells (precursor, primary, and polarized (Th1, Th2) T-helper cells). RESULTS: We show that lncRNAs mapping to loci shared between AID are significantly enriched in immune cell types compared to lncRNAs from the whole genome (α <0.005). We were not able to prioritize single cell types relevant for specific diseases, but we observed five different cell types enriched (α <0.005) in five AID (NK cells for inflammatory bowel disease, juvenile idiopathic arthritis, primary biliary cirrhosis, and psoriasis; memory T and CD8(+) T cells in juvenile idiopathic arthritis, primary biliary cirrhosis, psoriasis, and rheumatoid arthritis; Th0 and Th2 cells for inflammatory bowel disease, juvenile idiopathic arthritis, primary biliary cirrhosis, psoriasis, and rheumatoid arthritis). Furthermore, we show that co-expression analyses of lncRNAs and protein-coding genes can predict the signaling pathways in which these AID-associated lncRNAs are involved. CONCLUSIONS: The observed enrichment of lncRNA transcripts in AID loci implies lncRNAs play an important role in AID etiology and suggests that lncRNA genes should be studied in more detail to interpret GWAS findings correctly. The co-expression results strongly support a model in which the lncRNA and protein-coding genes function together in the same pathways.

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.