Systematic assessment of long-read RNA-seq methods for transcript identification and quantification.

The Long-read RNA-Seq Genome Annotation Assessment Project Consortium was formed to evaluate the effectiveness of long-read approaches for transcriptome analysis. Using different protocols and sequencing platforms, the consortium generated over 427 million long-read sequences from complementary DNA and direct RNA datasets, encompassing human, mouse and manatee species. Developers utilized these data to address challenges in transcript isoform detection, quantification and de novo transcript detection. The study revealed that libraries with longer, more accurate sequences produce more accurate transcripts than those with increased read depth, whereas greater read depth improved quantification accuracy. In well-annotated genomes, tools based on reference sequences demonstrated the best performance. Incorporating additional orthogonal data and replicate samples is advised when aiming to detect rare and novel transcripts or using reference-free approaches. This collaborative study offers a benchmark for current practices and provides direction for future method development in transcriptome analysis.

Cholesterol promotes Cytolysin A activity by stabilizing the intermediates during pore formation.

Pore-forming toxins (PFTs) form nanoscale pores across target membranes causing cell death. Cytolysin A (ClyA) from Escherichia coli is a prototypical α-helical toxin that contributes to cytolytic phenotype of several pathogenic strains. It is produced as a monomer and, upon membrane exposure, undergoes conformational changes and finally oligomerizes to form a dodecameric pore, thereby causing ion imbalance and finally cell death. However, our current understanding of this assembly process is limited to studies in detergents, which do not capture the physicochemical properties of biological membranes. Here, using single-molecule imaging and molecular dynamics simulations, we study the ClyA assembly pathway on phospholipid bilayers. We report that cholesterol stimulates pore formation, not by enhancing initial ClyA binding to the membrane but by selectively stabilizing a protomer-like conformation. This was mediated by specific interactions by cholesterol-interacting residues in the N-terminal helix. Additionally, cholesterol stabilized the oligomeric structure using bridging interactions in the protomer-protomer interfaces, thereby resulting in enhanced ClyA oligomerization. This dual stabilization of distinct intermediates by cholesterol suggests a possible molecular mechanism by which ClyA achieves selective membrane rupture of eukaryotic cell membranes. Topological similarity to eukaryotic membrane proteins suggests evolution of a bacterial α-toxin to adopt eukaryotic motifs for its activation. Broad mechanistic correspondence between pore-forming toxins hints at a wider prevalence of similar protein membrane insertion mechanisms.

Garcinol and Its Role in Chronic Diseases.

The various bioactive compounds isolated from leaves and fruits of Garcinia sps plants, have been characterized and experimentally demonstrated to be anti-oxidant, anti-inflammatory and anti-cancer in nature. Garcinol, a polyisoprenylated benzophenone, obtained from plant Garcinia indica has been found to be an effective inhibitor of several key regulatory pathways (e.g., NF-kB, STAT3 etc.) in cancer cells, thereby being able to control malignant growth of solid tumours in vivo. Despite its high potential as an anti-neoplastic modulator of several cancer types such as head and neck cancer, breast cancer, hepatocellular carcinoma, prostate cancer, colon cancer etc., it is still in preclinical stage due to lack of systematic and conclusive evaluation of pharmacological parameters. While it is promising anti-cancer effects are being positively ascertained for therapeutic development, studies on its effectiveness in ameliorating other chronic diseases such as cardiovascular diseases, diabetes, allergy, neurodegenerative diseases etc., though seem favourable, are very recent and require in depth scientific investigation.

Inhibition of STAT3 dimerization and acetylation by garcinol suppresses the growth of human hepatocellular carcinoma in vitro and in vivo.

BACKGROUND: Constitutive activation of signal transducer and activator of transcription 3 (STAT3) has been linked with proliferation, survival, invasion and angiogenesis of a variety of human cancer cells, including hepatocellular carcinoma (HCC). Thus, novel agents that can suppress STAT3 activation have potential for both prevention and treatment of HCC. Here we report, garcinol, a polyisoprenylated benzophenone, could suppress STAT3 activation in HCC cell lines and in xenografted tumor of HCC in nude mice model. EXPERIMENTAL DESIGN: Different HCC cell lines have been treated with garcinol and the inhibition of STAT3 activation, dimerization and acetylation have been checked by immunoblotting, immuno-fluorescence, and DNA binding assays. Xenografted tumor model has been generated in nude mice using HCC cell line and effect of garcinol in the inhibition of tumor growth has been investigated. RESULTS: Garcinol could inhibit both constitutive and interleukin (IL-6) inducible STAT3 activation in HCC cells. Computational modeling showed that garcinol could bind to the SH2 domain of STAT3 and suppress its dimerization in vitro. Being an acetyltransferase inhibitor, garcinol also inhibits STAT3 acetylation and thus impairs its DNA binding ability. The inhibition of STAT3 activation by garcinol led to the suppression of expression of various genes involved in proliferation, survival, and angiogenesis. It also suppressed proliferation and induced substantial apoptosis in HCC cells. Remarkably, garcinol inhibited the growth of human HCC xenograft tumors in athymic nu/nu mice, through the inhibition of STAT3 activation. CONCLUSION: Overall, our results suggest that garcinol exerts its anti-proliferative and pro-apoptotic effects through suppression of STAT3 signaling in HCC both in vitro and in vivo.

Efficient removal of Rhodamine B dye using biochar as an adsorbent: Study the performance, kinetics, thermodynamics, adsorption isotherms and its reusability.

Removal of toxic dyes such as Rhodamine B is essential as it pollutes aqueous and soil streams as well. This comprehensive study explores the potential of Calophyllum inophyllum seed char as an efficient bio-adsorbent based on their characteristic properties and a comparative study between various carbon-based adsorbents on the adsorption capacity of Rhodamine B dye. In this study, the char was prepared from Calophyllum inophyllum seed using a slow pyrolysis process (298 K/min) at an optimum temperature of 823 K and used as an adsorbent for the removal of Rhodamine B from water. The resulting char was mesoporous and had 155.389 m2/g surface areas (BET) and 0.628 cc/g pore volume. The formation of pores was observed from the SEM analysis. The adsorption studies were tested and optimized through various parameters such as solution pH, adsorbent dosage, initial dye concentration, stirring speed, contact time, and solution temperature. Maximum 95.5 % removal of Rhodamine B was possible at the pH: 2, stirring speed: 100 rpm, time: 25 min, temperature 308 K, and dose: 1.2 g/L. The highest adsorption capacity at equilibrium was determined to be 169.5 (mg/g) through Langmuir adsorption isotherm studies and followed pseudo 2nd order kinetics. The thermodynamics study confirmed the adsorption processes were spontaneous (ΔG°=-0.735 kJ/mol) and endothermic (ΔH° = 4.1 kJ/mol) processes. The reusability study confirmed that the mesoporous char can be reused as an efficient adsorbent for up to 3 cycles for environmental remediation.

DISPEL: database for ascertaining the best medicinal plants to cure human diseases.

Medicinal plants are anticipated to be one of the most valuable resources for the remedial usage in the treatment of various ailments. The data on key medicinal plants and their therapeutic efficacy against various ailments are quite scattered and not available on a single platform. Moreover, currently there is no means/mechanism of finding the best medicinal plant(s) from numerous plants known to cure any disease. DISPEL (Diseases Plants Eliminate) is a compendium of medicinal plants available across the world that are used to cure infectious as well as non-infectious diseases in humans. The association of a medicinal plant with a disease it cures is hereby referred to as 'medicinal plant-disease cured' linkage. The DISPEL database hosts ∼60 000 'medicinal plant-disease cured' linkages encompassing ∼5500 medicinal plants and ∼1000 diseases. This platform provides interactive and detailed visualization of medicinal plants, diseases and their relations using comprehensible network graph representation. The user has the freedom to search the database by specifying the name of disease(s) as well as the scientific/common name(s) of plant. Each 'medicinal plant-disease cured' relation is scored based on the availability of any medicine/product derived from that medicinal plant, information about active compound(s), knowledge regarding the part of plant that is effective and number of distinct articles/books/websites confirming the effectiveness of the medicinal plant. The user can find the best plant(s) that can be used to cure any desired disease(s). The DISPEL database is the first step towards generating the 'most-effective' combination of plants to cure a disease since it delineates as well as ranks all the therapeutic medicinal plants for that disease. The combination of best medicinal plants can then be used to conduct clinical trials and thus pave the way for their use in clinics for treatment of diseases. Database URL https://compbio.iitr.ac.in/dispel.

Systematic assessment of long-read RNA-seq methods for transcript identification and quantification.

The Long-read RNA-Seq Genome Annotation Assessment Project (LRGASP) Consortium was formed to evaluate the effectiveness of long-read approaches for transcriptome analysis. The consortium generated over 427 million long-read sequences from cDNA and direct RNA datasets, encompassing human, mouse, and manatee species, using different protocols and sequencing platforms. These data were utilized by developers to address challenges in transcript isoform detection and quantification, as well as de novo transcript isoform identification. The study revealed that libraries with longer, more accurate sequences produce more accurate transcripts than those with increased read depth, whereas greater read depth improved quantification accuracy. In well-annotated genomes, tools based on reference sequences demonstrated the best performance. When aiming to detect rare and novel transcripts or when using reference-free approaches, incorporating additional orthogonal data and replicate samples are advised. This collaborative study offers a benchmark for current practices and provides direction for future method development in transcriptome analysis.

Synthesis of dye-sensitized TiO2/Ag doped nano-composites using UV photoreduction process for phenol degradation: A comparative study.

This study investigates a comparison between the photocatalytic action of two nanocomposites (TiO2 and TiO2(Ag) doped) on the degradation of phenol from water. The nanocomposites were synthesized by the UV photo-reduction process to get a silver metal loading of 0.25, 0.5, 0.75, and 1% (w/w). In addition to this, Eriochrome Cyanine Red (ECR) and Eosin Yellow (EY) both anionic dyes were used for sensitization of Ag-doped TiO2 photo-catalyst such as TiO2(Ag)ECR and TiO2(Ag)EY. The TiO2(Ag-1.0)EY photo-catalyst indicated higher absorbance compared to the TiO2(Ag-1.0)ECR in the 400-700 nm range (visible range). The degradation of phenol was tested by varying the pH, silver loading and catalyst dosage. The maximum degradation of phenol was 98% in 180 min at pH 7 in presence of 1% (w/w) silver loading with 0.5 gL-1 dosage of photo-catalyst TiO2(Ag-1.0)EY. At this condition, the reduction in the phenol concentration was noticed from 20 mg/L to 0.4 mg/L.

Mutant KRAS regulates transposable element RNA and innate immunity via KRAB zinc-finger genes.

RAS genes are the most frequently mutated oncogenes in cancer, yet the effects of oncogenic RAS signaling on the noncoding transcriptome remain unclear. We analyzed the transcriptomes of human airway and bronchial epithelial cells transformed with mutant KRAS to define the landscape of KRAS-regulated noncoding RNAs. We find that oncogenic KRAS signaling upregulates noncoding transcripts throughout the genome, many of which arise from transposable elements (TEs). These TE RNAs exhibit differential expression, are preferentially released in extracellular vesicles, and are regulated by KRAB zinc-finger (KZNF) genes, which are broadly downregulated in mutant KRAS cells and lung adenocarcinomas in vivo. Moreover, mutant KRAS induces an intrinsic IFN-stimulated gene (ISG) signature that is often seen across many different cancers. Our results indicate that mutant KRAS remodels the repetitive noncoding transcriptome, demonstrating the broad scope of intracellular and extracellular RNAs regulated by this oncogenic signaling pathway.

Using multiscale molecular dynamics simulations to obtain insights into pore forming toxin mechanisms.

Pore forming toxins (PFTs) are virulent proteins released by several species, including many strains of bacteria, to attack and kill host cells. In this article, we focus on the utility of molecular dynamics (MD) simulations and the molecular insights gleaned from these techniques on the pore forming pathways of PFTs. In addition to all-atom simulations which are widely used, coarse-grained MARTINI models and structure-based models have also been used to study PFTs. Here, the emphasis is on methods and techniques involved while setting up, monitoring, and evaluating properties from MD simulations of PFTs in a membrane environment. We draw from several case studies to illustrate how MD simulations have provided molecular insights into protein-protein and protein-lipid interactions, lipid dynamics, conformational transitions and structures of both the oligomeric intermediates and assembled pore structures.

First record of Lobothorax typus from Odisha coast, India, with molecular characterization and observation of its biphasic moulting.

Two distinct morphologies of Cymothoidae isopod, Lobothorax typus were collected from the marine water of Bay of Bengal, Goapalpur-on-Sea as the first record of this parasite from coastal water of Odisha, India. All specimens were found attached to the buccal region of different individuals of the same host fish Trichiurus lepturus. With the aid of COI gene sequencing and morphological analysis, the individuals were found to be conspecific. The most prominent variation among the two morphologies includes the size of 5th pereonite and pleon length to total body length ratio. These variations are as a result of the biphasic moulting process. Maximum Likelihood tree analysis based on COI gene sequences concluded the monophyletic taxonomy of different buccal attaching genera under the family Cymothoidae which is in congruence with their morphological divergence.

Functional interplay between YY1 and CARM1 promotes oral carcinogenesis.

Coactivator associated arginine methyltransferase 1 (CARM1) has been functionally implicated in maintenance of pluripotency, cellular differentiation and tumorigenesis; where it plays regulatory roles by virtue of its ability to coactivate transcription as well as to modulate protein function as an arginine methyltransferase. Previous studies establish an oncogenic function of CARM1 in the context of colorectal and breast cancer, which correlate to its overexpressed condition. However, the mechanism behind its deregulated expression in the context of cancer has not been addressed before. In the present study we uncover an oncogenic function of CARM1 in the context of oral cancer, where it was found to be overexpressed. We also identify YY1 to be a positive regulator of CARM1 gene promoter, where silencing of YY1 in oral cancer cell line could lead to reduction in expression of CARM1. In this context, YY1 showed concomitant overexpression in oral cancer patient samples compared to adjacent normal tissue. Cell line based experiments as well as xenograft study revealed pro-neoplastic functions of YY1 in oral cancer. Transcriptomics analysis as well as qRT-PCR validation clearly indicated pro-proliferative, pro-angiogenic and pro-metastatic role of YY1 in oral cancer. We also show that YY1 is a substrate of CARM1 mediated arginine methylation, where the latter could coactivate YY1 mediated reporter gene activation in vivo. Taken together, CARM1 and YY1 were found to regulate each other in a positive feedback loop to facilitate oral cancer progression.

p53 mediated regulation of coactivator associated arginine methyltransferase 1 (CARM1) expression is critical for suppression of adipogenesis.

Coactivator-associated arginine methyltransferase 1 (CARM1/PRMT4) is a type I arginine methyltransferase that mediates transcriptional activation via methylation of histone H3 on R17, R26, and R42. CARM1 is also a coactivator of transcription of various transcription factors such as NF-kB, MEF2C, ß-catenin, p53, PPAR-gamma etc. CARM1 has been functionally implicated in maintenance of pluripotency, cellular differentiation, and tumorigenesis; where its expression status plays an important role. Although its expression has been shown to be regulated by a few miRNAs in different contexts at post-transcriptional level, transcriptional regulation of CARM1 gene is still unexplored. In this report we demonstrate that CARM1 is a p53 responsive gene, where p53 could suppress CARM1 promoter-driven luciferase expression. CARM1 gene expression was found to be repressed by p53 in 3T3L1 preadipocytes when activated with Nutlin-3a treatment. Ectopic overexpression of CARM1 could rescue inhibitory effect of p53 on adipogenesis, suggesting a role of p53-CARM1 axis of regulation operational in the context of adipocyte differentiation. p53 and CARM1 showed antagonistic regulatory influence on PPAR-gamma expression; which suggests that p53-mediated suppression of adipogenesis could be partly via repression of CARM1 expression. Taken together these observations provide convincing mechanistic explanation for p53 function in the context of adipocyte differentiation process.

Roles for Transcription Factors Sp1, NF-κB, IRF3, and IRF7 in Expression of the Human IFNL4 Gene.

The expression of a biologically active human IFNλ4 depends on the presence of a frameshift deletion polymorphism within the first exon of the interferon lambda 4 (IFNL4) gene. In this report, we use the lung carcinoma-derived cell line, A549, which is genetically viable to express a functional IFNλ4, to address transcriptional requirements of the IFNL4 gene. We show that the GC-rich DNA-binding transcription factor (TF) specificity protein 1 (Sp1) is recruited to the IFNL4 promoter and has a role in induction of gene expression upon stimulation with viral RNA mimic poly(I:C). By using RNAi and overexpression strategies, we also show key roles in IFNL4 gene expression for the virus-inducible TFs, nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), IFN regulatory factor 3 (IRF3), and IRF7. Interestingly, we also observe that overexpression of IFNλ4 influences IFNL4 promoter activity, which may further be dependent on the retinoic acid-inducible gene-I (RIG-I)-like receptor pathway. Together, our work for the first time reports on the functional characterization of the human IFNL4 promoter.