Unravelling a novel CTNND1-RAB6A fusion transcript: Implications in colon cancer cell migration.

The interchange of DNA sequences between genes may occur because of chromosomal rearrangements leading to the formation of chimeric genes. These chimeric genes have been linked to various cancers, accumulated significant interest in recent times. We used paired-end RNA-seq. data of four CRC and one normal sample generated from our previous study. The STAR-Fusion pipeline was utilized to identify the fusion genes unique to CRC. The in-silico identified fusion gene(s) were explored for their diagnostic, prognostic and therapeutic biomarker potential using TCGA-datasets, then validated through PCR and DNA sequencing. Further, cell line-based studies were performed to gain functional insights of the novel fusion transcript CTNND1-RAB6A, which was amplified in one sample. Sequencing revealed that there was a total loss of the CTNND1 gene, whereas RAB6A retained its coding sequence. Further, RAB6A was functionally characterized for its oncogenic potential in HCT116 cell line. RAB6A under-expression was found to be significantly associated with increased cell migration and is proposed to be regulated via the RAB6A-ECR1-Liprin-α axis. We conclude that RAB6A gene may play significant role in CRC oncogenesis, and could be used as a potential biomarker and therapeutic target in future for better management of a subset of CRCs harbouring this fusion.

Loss of PERK function promotes ferroptosis by downregulating SLC7A11 (System Xc⁻) in colorectal cancer.

Ferroptosis, a genetically and biochemically distinct form of programmed cell death, is characterised by an iron-dependent accumulation of lipid peroxides. Therapy-resistant tumor cells display vulnerability toward ferroptosis. Endoplasmic Reticulum (ER) stress and Unfolded Protein Response (UPR) play a critical role in cancer cells to become therapy resistant. Tweaking the balance of UPR to make cancer cells susceptible to ferroptotic cell death could be an attractive therapeutic strategy. To decipher the emerging contribution of ER stress in the ferroptotic process, we observe that ferroptosis inducer RSL3 promotes UPR (PERK, ATF6, and IRE1α), along with overexpression of cystine-glutamate transporter SLC7A11 (System Xc-). Exploring the role of a particular UPR arm in modulating SLC7A11 expression and subsequent ferroptosis, we notice that PERK is selectively critical in inducing ferroptosis in colorectal carcinoma. PERK inhibition reduces ATF4 expression and recruitment to the promoter of SLC7A11 and results in its downregulation. Loss of PERK function not only primes cancer cells for increased lipid peroxidation but also limits in vivo colorectal tumor growth, demonstrating active signs of ferroptotic cell death in situ. Further, by performing TCGA data mining and using colorectal cancer patient samples, we demonstrate that the expression of PERK and SLC7A11 is positively correlated. Overall, our experimental data indicate that PERK is a negative regulator of ferroptosis and loss of PERK function sensitizes colorectal cancer cells to ferroptosis. Therefore, small molecule PERK inhibitors hold huge promise as novel therapeutics and their potential can be harnessed against the apoptosis-resistant condition.

Integrated Analysis Identifies Novel Fusion Transcripts in Laterally Spreading Tumors Suggestive of Distinct Etiology Than Colorectal Cancers.

BACKGROUND: Laterally spreading tumors (LSTs) of the colon and rectum are a class of abnormality which spreads laterally and appears ulcerated. They are a subclass of colorectal cancer (CRCs) with higher invasive potential than CRCs. Moreover, the etiology of LST still remains obscure. METHODS: This study aimed to identify unique fusion transcript(s) in LSTs and evaluate their role in LST development and progression. RNA-Seq data for LST samples from the EMBL-EBI database were used to identify fusion transcripts. An integrated approach using Gene Ontology, pathway analysis, hub gene, and co-expression network analysis functionally characterized fusion transcripts to shed light upon the etiology of LSTs. RESULT: We identified 48 unique fusion genes in LSTs. GO terms were enriched in mRNA metabolic (p ≤ 2.06E-06), mRNA stabilization (p ≤ 1.60E-05), in cytosol (1.20E-05), RBP (p ≤ 2.30E-04), and RNA binding activity (p ≤ 3.51E-08) processes. Pathway analysis revealed an inflammatory phenotype of LSTs suggesting a distinct etiology than CRCs as pathways were enriched in salmonella infection (p ≤ 4.41 e-03), proteoglycans in cancer (p ≤ 1.18 e-02), and insulin signaling (p ≤ 2.13 e-02). Our exclusion and inclusion criteria and hub gene analysis finally identified 9 hub genes. Co-expression analysis of hub genes identified the most significant transcription factors (NELFE, MYC, TAF1, MAX) and kinases (MAPK14, CSNK2A1, CDK1, MAPK1) which were implicated in various cancer pathways. Furthermore, an overall survival analysis of hub genes was performed. Our predefined criterion resulted in the enrichment of NPM1-PTMA (NPM1: p ≤ 0.005) and HIST1H2BO-YBX1 (YBX1: p ≤ 0.02) fusion transcripts, significantly associated with the patient's overall survival. CONCLUSION: Our systematic analysis resulted in novel fusion genes in LSTs suggesting a different etiology than CRCs. Fusion transcripts were observed more frequently in non-granular LSTs suggestive of genetically more unstable than granular LST. We hypothesize that NPM1-PTMA and HIST1H2BO-YBX1 could be implicated in LST development and progression and may also serve as a prognostic or diagnostic biomarker in future for better management of LSTs.

EZH2-H3K27me3 mediated KRT14 upregulation promotes TNBC peritoneal metastasis.

Triple-Negative Breast Cancer (TNBC) has a poor prognosis and adverse clinical outcomes among all breast cancer subtypes as there is no available targeted therapy. Overexpression of Enhancer of zeste homolog 2 (EZH2) has been shown to correlate with TNBC's poor prognosis, but the contribution of EZH2 catalytic (H3K27me3) versus non-catalytic EZH2 (NC-EZH2) function in TNBC progression remains elusive. We reveal that selective hyper-activation of functional EZH2 (H3K27me3) over NC-EZH2 alters TNBC metastatic landscape and fosters its peritoneal metastasis, particularly splenic. Instead of H3K27me3-mediated repression of gene expression; here, it promotes KRT14 transcription by attenuating binding of repressor SP1 to its promoter. Further, KRT14 loss significantly reduces TNBC migration, invasion, and peritoneal metastasis. Consistently, human TNBC metastasis displays positive correlation between H3K27me3 and KRT14 levels. Finally, EZH2 knockdown or H3K27me3 inhibition by EPZ6438 reduces TNBC peritoneal metastasis. Altogether, our preclinical findings suggest a rationale for targeting TNBC with EZH2 inhibitors.

Transcriptomic landscape of early age onset of colorectal cancer identifies novel genes and pathways in Indian CRC patients.

Past decades of the current millennium have witnessed an unprecedented rise in Early age Onset of Colo Rectal Cancer (EOCRC) cases in India as well as across the globe. Unfortunately, EOCRCs are diagnosed at a more advanced stage of cancer. Moreover, the aetiology of EOCRC is not fully explored and still remains obscure. This study is aimed towards the identification of genes and pathways implicated in the EOCRC. In the present study, we performed high throughput RNA sequencing of colorectal tumor tissues for four EOCRC (median age 43.5 years) samples with adjacent mucosa and performed subsequent bioinformatics analysis to identify novel deregulated pathways and genes. Our integrated analysis identifies 17 hub genes (INSR, TNS1, IL1RAP, CD22, FCRLA, CXCL3, HGF, MS4A1, CD79B, CXCR2, IL1A, PTPN11, IRS1, IL1B, MET, TCL1A, and IL1R1). Pathway analysis of identified genes revealed that they were involved in the MAPK signaling pathway, hematopoietic cell lineage, cytokine-cytokine receptor pathway and PI3K-Akt signaling pathway. Survival and stage plot analysis identified four genes CXCL3, IL1B, MET and TNS1 genes (p = 0.015, 0.038, 0.049 and 0.011 respectively), significantly associated with overall survival. Further, differential expression of TNS1 and MET were confirmed on the validation cohort of the 5 EOCRCs (median age < 50 years and sporadic origin). This is the first approach to find early age onset biomarkers in Indian CRC patients. Among these TNS1 and MET are novel for EOCRC and may serve as potential biomarkers and novel therapeutic targets in future.

Molecular subtypes of colorectal cancer: An emerging therapeutic opportunity for personalized medicine.

Molecular subtypes-based therapies offer new potential framework for desired and precise outcome in clinical settings. Current treatment strategies in colorectal cancer are largely 'one drug fit all' model for patients that display same pathological conditions. However, CRC is a very heterogenous set of malignancy that does not support for above criteria. Each subtype displays different pathological and genetic signatures. Based on these features, therapeutic stratification for individual patients may be designed, which may ultimately lead to improved therapeutic outcomes. In this comprehensive review, we have attempted to briefly outline major CRC pathways. A detailed overview of molecular subtypes and their clinical significance has been discussed. Present and future methods, governing CRC subtyping in the era of personalized therapy with a special emphasis on CMS subtypes of CRC has been reviewed. Together, discovery and validation of new CRC patient stratification methods, screening for novel therapeutic targets, and enhanced diagnosis of CRC may improve the treatment outcome.

A comparative screening of abiotic stress tolerance in early flowering rice mutants.

Oryza sativa L. (Rice) a grain crop grown considerably in areas frequented with abiotic stresses has a higher heat and drought tolerance and is deemed a model crop for tolerance studies. Manifold environment testing facilitates recognition of constant genotypes of rice mutants for adverse abiotic stress conditions. Mutants are an imperative genetic reserve which can help as an informative basis of natural disparity. We assessed EMS induced early flowering rice mutants (Control, BM6, BM6´ and BM9) for potential study of stress tolerance under limited water, salinity and high zinc conditions by testing for plant growth (biophysical and biochemical) traits, as well ascytotoxicity and genotoxicity determination assay via MTT and epifluoresence microscopy methods in root tissues of the mutants. Environmental effect was more significant than genotypic contribution on biophysical and biochemical parameters. BM9 mutant was found to be the most stable genotype in tolerance studies among other mutants in artificially created stress environments viz. mannitol (moderated drought), sodium chloride (salinity stress), zinc sulfate (heavy metal stress) and zinc oxide nanoparticles for a time duration. These mutants are a valuable resource for varietal advancements and to determine genes for tolerance to multiple abiotic stresses. To our knowledge this is the first account on validation of manifold abiotic stresses in early flowering rice mutants. This study can additionally assist fastidious detection of stress-tolerance genes in this vital crop.

In silico screening of proteins targeting circulating miRNAs for improved diagnosis of multiple myeloma.

Multiple Myeloma (MM) is a B-cell malignancy, which is characterized by the expansion of clonal plasma cells in the bone marrow, leading to abnormal accumulation of monoclonal antibodies in circulation. Certain circulating miRNAs are deregulated in MM and their differential expression profiles in body fluids can be quantified and used to discriminate between the premalignant and malignant stages of MM. Our study identifies protein which would show affinity for a selected panel of circulating miRNAs deregulated in MM. Human RNA binding proteins were identified based on their unique RNA binding domains and their interacting probabilities with the panel of miRNAs deregulated in MM. miR-26 was used as a negative control for interaction studies. 3-D structure of candidate proteins were determined and molecular docking was performed to confirm the results. Five RNA binding proteins TROVE2, CUGBP2, DHX8, PUM2 and DKC1 were used for molecular docking studies. DKC1 showed significant hydrogen bonding as well as remarkable binding affinity values of -17.4 kcal/mol with miR-720 (2 H-bonds), -16 kcal/mol with miR-1246 (1 H-bond) and -16.9 kcal/mol with miR-1308 (3 H-bonds). Identified protein-miRNA interaction could be used to develop an economical and reliable ELISA based methodology for improved and sensitive diagnosis of MM patients.

Inhibitory Mechanism on Combination of Phytic Acid with Methanolic Seed Extract of Syzygium cumini and Sodium Chloride over Bacillus subtilis.

The antibiotic resistance in bacteria responsible for causing community and health care-associated infection displayed a major threat to global health. Use of broad-spectrum antibiotics for the treatment of various ailments poses serious side effects. In the present research, we investigated the combined role of 2% phytic acid with 2% methanolic seed extract of Syzygium cumini and 0.5% sodium chloride for inhibition of Bacillus subtilis and Pseudomonas aeruginosa and found it to be efficient over B. subtilis. The zone of inhibition by present mixture was found to be 2.9 ± 0.0004 and 1.9 ± 0.0006 cm against Bacillus subtilis and P. aeruginosa in comparison to individual component. Mixture was found more potent against B. subtilis and selected for further study. The underlying mechanism involved in inhibitory action of this mixture was determined by Scanning electron microscope, DNA fragmentation and propidium iodide staining. Scanning electron microscopy revealed that inhibition of B. subtilis by this mixture is mainly due to the disruption of bacterial cell membrane, leakage of internal cellular content which ultimately leads to the death of bacterial cells. DNA fragmentation showed apoptotic hallmark through degradation caused by mixture against B. subtilis at various time intervals. Likewise, PI staining also revealed the disruption of bacterial membrane by the mixture as the PI gives fluorescence after binding with DNA. The present study concludes that inhibitory potential of this mixture is mainly due to disruption of bacterial cell membrane, degradation of DNA and creation of pores in the membrane. The mixture could be used for inhibition of food pathogen B. subtilis.

Antimicrobial action of methanolic seed extracts of Syzygium cumini Linn. on Bacillus subtilis.

Phytochemicals of Syzygium cumini are used for the treatment of various diseases as a traditional medicine but the mechanism behind their action is not well reported. Antimicrobial activity of methanolic seed extract of S. cumini was done by agar well diffusion assay on Bacillus subtilis and its zone of inhibition was found to be 20.06 mm in comparison to control having no zone of inhibition. MIC of S. cumini was found to be 0.3 mg/ml. Genomic DNA degradation of B. subtilis reveals apoptosis and FE-scanning electron microscope indicates cell wall cracking on several intervals of time. Results of propidium iodide staining showed few bacterial cells were stained in control; however population of stained cells increased after exposing them for varying period of time. Flow cytometric kinetic data analysis on the membrane permeabilization in bacterial cell showed the significant contribution of antimicrobial potential of the seed extract on antimicrobial-induced permeabilization. In silico analysis revealed two components of S. cumini methanolic extract to be active against four enzymes (PDB ID-1W5D, 4OX3, 3MFD and 5E2F) which are crucial for plasma membrane synthesis in B. subtilis. Moreover lupeol showed highest binding energy for macromolecule 1W5D and 4OX3 forming one hydrogen bond each whereas stigmasterol showed the highest binding energy for macromolecule 3MFD and 5E2F forming four hydrogen bonds and alkyl bonds respectively. It demonstrates that methanolic seed extracts of S. cumini could be used for inhibition of food born infection caused by B. subtilis and also an alternative of prevalent antibiotics.

Genetic and epigenetic markers in colorectal cancer screening: recent advances.

INTRODUCTION: Colorectal cancer (CRC) is a heterogenous disease which develops from benign intraepithelial lesions known as adenomas to malignant carcinomas. Acquired alterations in Wnt signaling, TGFß, MAPK pathway genes and clonal propagation of altered cells are responsible for this transformation. Detection of adenomas or early stage cancer in asymptomatic patients and better prognostic and predictive markers is important for improving the clinical management of CRC. Area covered: In this review, the authors have evaluated the potential of genetic and epigenetic alterations as markers for early detection, prognosis and therapeutic predictive potential in the context of CRC. We have discussed molecular heterogeneity present in CRC and its correlation to prognosis and response to therapy. Expert commentary: Molecular marker based CRC screening methods still fail to gain trust of clinicians. Invasive screening methods, molecular heterogeneity, chemoresistance and low quality test samples are some key challenges which need to be addressed in the present context. New sequencing technologies and integrated omics data analysis of individual or population cohort results in GWAS. MPE studies following a GWAS could be future line of research to establish accurate correlations between CRC and its risk factors. This strategy would identify most reliable biomarkers for CRC screening and management.

Low density ionogels obtained by rapid gellification of tetraethyl orthosilane assisted by ionic liquids.

A non-hydrolytic one pot sol-gel method has been used to synthesize mesoporous silica ionogels with the confined ionic liquid (IL) 1-ethyl 3-methyl imidazolium tetra fluoro-borate [EMIM][BF(4)]. The precursor for obtaining the SiO(2) matrix was tetraethyl orthosilicate (TEOS) and formic acid was used as a catalyst. These ionogels have been characterized by density measurements, TEM, BET, DSC, TGA and FTIR. The incorporation of the ionic liquid [EMIM][BF(4)] enhances the gellification rate which results in the ionogels having very low density (~0.3 g cm(-3)). The low density has been explained on the basis of the creation of 'blind embedded pores' in the matrix (apart from open pores) due to very rapid gellification (~1 min). Morphological studies provide experimental evidence for the presence of blind pores/voids inside the ionogel ingots. We have also shown that the IL entrapped in nanopores (~7-8 nm pore size) of the SiO(2) matrix has different physical properties than the bulk IL viz. (a) the phase transition temperatures (T(g), T(c) and T(m)) of the IL change upon confinement, (b) the thermal stability reduces upon confinement, and (c) the pore wall interaction with the IL results in changes in the C-H vibrations of the imidazolium ring and alkyl chain (the former increasing) which is also indicated in our DFT-calculation.

Studies on imidazolium-based ionic liquids having a large anion confined in a nanoporous silica gel matrix.

The properties of large molecules confined in nanopores are expected to be different from those of the bulk. This study reports changes in the phase behavior and vibrational frequencies of an imidazolium-based ionic liquid (IL), namely, 1-butyl-3-methyl imidazolium octyl sulfate ([BMIM][OcSO(4)]) in a nanoporous silica gel matrix. Nanoporous silica gel matrixes have been synthesized by a one-step sol-gel technique using tetraethylorthosilicate (TEOS) as a starting precursor. The synthesized gel has been characterized by differential scanning calorimetry (DSC), BET, TEM and FTIR. The FTIR spectra show shifts in many vibrational bands; particularly, the vibrational bands related to the imidazolium ring, aliphatic chain, and SO(3) of the IL are found to shift significantly upon confinement. The DSC results show significant changes in the melting point (ΔT(m) ≈ 52 °C), crystallization temperature (ΔT(C) ≈ 14 °C), and glass transition temperature (ΔT(g) ≈ 2 °C). The IL used in the present study has a large anion ([OcSO(4)]), and ΔT(m) for this is much larger than those reported earlier for many other ILs with relatively smaller anions. A new approach, based on the liquid-drop model, has been suggested to explain this.

Properties of ionic liquid confined in porous silica matrix.

Porous silica matrices of different pore sizes with confined ionic liquid (1-butyl-3-methylimidazolium hexafluorophosphate) [BMIM] [PF(6)] were prepared by sol-gel technique using a tetraethyl orthosilicate (TEOS) precursor with an aim to study the changes in physico-chemical properties of ionic liquid on confinement. It is found that on confinement 1) melting point decreases, 2) fluorescence spectra shows a red shift and 3) the vibrational bands are affected particularly those of imadazolium ring, which interacts more with the walls of the silica matrix. Preliminary theoretical calculations suggest that SiO(2) matrix interact more with the heterocyclic group of [BMIM] cation than the tail alkyl chain end group resulting in significant changes in the aromatic vibrations.