Genome wide nucleosome landscape shapes 3D chromatin organization.

The hierarchical chromatin organization begins with formation of nucleosomes, which fold into chromatin domains punctuated by boundaries and ultimately chromosomes. In a hierarchal organization, lower levels shape higher levels. However, the dependence of higher-order 3D chromatin organization on the nucleosome-level organization has not been studied in cells. We investigated the relationship between nucleosome-level organization and higher-order chromatin organization by perturbing nucleosomes across the genome by deleting Imitation SWItch (ISWI) and Chromodomain Helicase DNA-binding (CHD1) chromatin remodeling factors in budding yeast. We find that changes in nucleosome-level properties are accompanied by changes in 3D chromatin organization. Short-range chromatin contacts up to a few kilo-base pairs decrease, chromatin domains weaken, and boundary strength decreases. Boundary strength scales with accessibility and moderately with width of nucleosome-depleted region. Change in nucleosome positioning seems to alter the stiffness of chromatin, which can affect formation of chromatin contacts. Our results suggest a biomechanical "bottom-up" mechanism by which nucleosome distribution across genome shapes 3D chromatin organization.

Exploring the mechanism of interaction of glipizide with DNA: Combined in vitro and bioinformatics approach.

DNA, vital for biological processes, encodes hereditary data for protein synthesis, shaping cell structure and function. Since revealing its structure, DNA has become a target for various therapeutically vital molecules, spanning antidiabetic to anticancer drugs. These agents engage with DNA-associated proteins, DNA-RNA hybrids, or bind directly to the DNA helix, triggering diverse downstream effects. These interactions disrupt vital enzymes and proteins essential for maintaining cell structure and function. Analysing drug-DNA interactions has significantly advanced our understanding of drug mechanisms. Glipizide, an antidiabetic drug, is known to cause DNA damage in adipocytes. However, its extract mechanism of DNA interaction is unknown. This study delves into the interaction between glipizide and DNA utilizing various biophysical tools and computational technique to gain insights into the interaction mechanism. Analysis of UV-visible and fluorescence data reveals the formation of complex between DNA and glipizide. The binding affinity of glipizide to DNA was of moderate strength. Examination of thermodynamic parameters at different temperatures suggests that the binding was entropically spontaneous and energetically favourable. Various experiments such as thermal melting assays, viscosity measurement, and dye displacement assays confirmed the minor grove nature of binding of glipizide with DNA. Molecular dynamics studies confirmed the glipizide forms stable complex with DNA when simulated by mimicking the physiological conditions. The binding was mainly favoured by hydrogen bonds and glipizide slightly reduced nucleotide fluctuations of DNA. The study deciphers the mechanism of interaction of glipizide with DNA at molecular levels.

Multifaceted functions of RNA-binding protein vigilin in gene silencing, genome stability, and autism-related disorders.

RNA-binding proteins (RBPs) are emerging as important players in regulating eukaryotic gene expression and genome stability. Specific RBPs have been shown to mediate various chromatin-associated processes ranging from transcription to gene silencing and DNA repair. One of the prominent classes of RBPs is the KH domain-containing proteins. Vigilin, an evolutionarily conserved KH domain-containing RBP has been shown to be associated with diverse biological processes like RNA transport and metabolism, sterol metabolism, chromosome segregation, and carcinogenesis. We have previously reported that vigilin is essential for heterochromatin-mediated gene silencing in fission yeast. More recently, we have identified that vigilin in humans plays a critical role in efficient repair of DNA double-stranded breaks and functions in homology-directed DNA repair. In this review, we highlight the multifaceted functions of vigilin and discuss the findings in the context of gene expression, genome organization, cancer, and autism-related disorders.

The mTORC1-G9a-H3K9me2 axis negatively regulates autophagy in fatty acid-induced hepatocellular lipotoxicity.

Defective autophagy and lipotoxicity are the hallmarks of nonalcoholic fatty liver disease. However, the precise molecular mechanism for the defective autophagy in lipotoxic conditions is not fully known. In the current study, we elucidated that activation of the mammalian target of rapamycin complex 1 (mTORC1)-G9a-H3K9me2 axis in fatty acid-induced lipotoxicity blocks autophagy by repressing key autophagy genes. The fatty acid-treated cells show mTORC1 activation, increased histone methyltransferase G9a levels, and suppressed autophagy as indicated by increased accumulation of the key autophagic cargo SQSTM1/p62 and decreased levels of autophagy-related proteins LC3II, Beclin1, and Atg7. Our chromatin immunoprecipitation analysis showed that decrease in autophagy was associated with increased levels of the G9a-mediated repressive H3K9me2 mark and decreased RNA polymerase II occupancy at the promoter regions of Beclin1 and Atg7 in fatty acid-treated cells. Inhibition of mTORC1 in fatty acid-treated cells decreased G9a-mediated H3K9me2 occupancy and increased polymerase II occupancy at Beclin1 and Atg7 promoters. Furthermore, mTORC1 inhibition increased the expression of Beclin1 and Atg7 in fatty acid-treated cells and decreased the accumulation of SQSTM1/p62. Interestingly, the pharmacological inhibition of G9a alone in fatty acid-treated cells decreased the H3K9me2 mark at Atg7 and Beclin1 promoters and restored the expression of Atg7 and Beclin1. Taken together, our findings have identified the mTORC1-G9a-H3K9me2 axis as a negative regulator of the autophagy pathway in hepatocellular lipotoxicity and suggest that the G9a-mediated epigenetic repression is mechanistically a key step during the repression of autophagy in lipotoxic conditions.

Canola Oil based Poly(ester-ether-amide-urethane) Nanocomposite and Its Anti-Corrosive Coatings.

The environmental and health hazards associated with petro-based chemicals have motivated the researchers to replace them partially or wholly with renewable resource-based polymers. Vegetable oils serve as an excellent alternative to this end as they are cost effective, eco-friendly, easily available and rich with functional groups amenable to chemical reactions. The aim of the research work is to prepare Canola oil [CANO] derived poly (ester-ether-amide-urethane) (CPEEUA) nanocomposite coating material using N,N-bis (2-hydroxyethyl) fatty amide [CFA] obtained from CANO, Lactic acid [LA], and reinforced with Fumed Silica [FS]. CPEEUA was obtained by esterification, etherification, and urethanation reactions and its structure was confirmed from FTIR and NMR spectral analyses. CPEEUA/FS coatings were found to be scratch resistant, flexible, well-adhered to mild steel panels, and hydrophobic with 2.0-2.5 kg scratch hardness, 150lb/inch impact resistance and >90° contact angle value. They exhibited good corrosion protection in 3.5 wt% NaCl solution as investigated by Potentiodynamic Polarization and Electrochemical Impedance tests. CPEEUA coatings are safe for usage up to 200 °C.

Autism-Associated Vigilin Depletion Impairs DNA Damage Repair.

Vigilin (Vgl1) is essential for heterochromatin formation, chromosome segregation, and mRNA stability and is associated with autism spectrum disorders and cancer: vigilin, for example, can suppress proto-oncogene c-fms expression in breast cancer. Conserved from yeast to humans, vigilin is an RNA-binding protein with 14 tandemly arranged nonidentical hnRNP K-type homology (KH) domains. Here, we report that vigilin depletion increased cell sensitivity to cisplatin- or ionizing radiation (IR)-induced cell death and genomic instability due to defective DNA repair. Vigilin depletion delayed dephosphorylation of IR-induced γ-H2AX and elevated levels of residual 53BP1 and RIF1 foci, while reducing Rad51 and BRCA1 focus formation, DNA end resection, and double-strand break (DSB) repair. We show that vigilin interacts with the DNA damage response (DDR) proteins RAD51 and BRCA1, and vigilin depletion impairs their recruitment to DSB sites. Transient hydroxyurea (HU)-induced replicative stress in vigilin-depleted cells increased replication fork stalling and blocked restart of DNA synthesis. Furthermore, histone acetylation promoted vigilin recruitment to DSBs preferentially in the transcriptionally active genome. These findings uncover a novel vigilin role in DNA damage repair with implications for autism and cancer-related disorders.

Organochlorine Pesticides Negatively Influenced the Cellular Growth, Morphostructure, Cell Viability, and Biofilm-Formation and Phosphate-Solubilization Activities of Enterobacter cloacae Strain EAM 35.

An in vitro study was conducted to assess the impact of organochlorine pesticides (OCPs) on cellular growth, morphology, cell viability, biofilm-formation activity, and growth-regulating substances of a soil bacterium. Phosphate-solubilizing EAM 35 isolated from rhizosphere soil was molecularly identified as Enterobacter cloacae (accession number MT672578.1). Strain EAM 35 tolerated varying levels of OCPs, viz., benzene hexachloride (BHC), chlorpyrifos (CP), dieldrin (DE), and endosulfan (ES). The toxicity of OCPs to strain EAM 35 was displayed in a concentration-dependent manner. Among the OCPs, ES at a concentration of 200 µM showed a higher toxicity, where it maximally reduced the bacterial synthesis of indole-3-acetic acid (IAA), salicylic acid (SA), and 2,3-dihydroxy-benzoic acid (DHBA) by 73% (p ≤ 0.001), 85% (p ≤ 0.005), and 83% (p ≤ 0.001), respectively, over the control. While comparing the toxicity of OCPs to P-solubilizing activity of E. cloacae after 10 days of growth, the toxicity pattern followed the order ES (mean value = 82.6 µg mL-1) > CP (mean value = 93.2 µg mL-1) > DE (mean value = 113.6 µg mL-1) > BHC (mean value = 127 µg mL-1). Furthermore, OCP-induced surface morphological distortion in E. cloacae EAM 35 was observed as gaps, pits on both cellular facets, and fragmented and disorganized cell structure under a scanning electron microscope (SEM). The membrane-compromised cells increased as the concentrations of OC pesticides increased from 25 to 200 µM. Additionally, microbial counts (log10 CFU/mL) were also affected after pesticide exposure and decreased with increasing concentrations. While assessing the impact of OCPs on inhibition (%) of log10 CFU/mL, 150, 175, and 200 µM concentrations of ES completely reduced the growth of E. cloacae. Similarly, while comparing the toxicity of higher concentrations of OCPs to bacterial growth, sensitivity followed the order ES > DE > CP > BHC. In addition, the biofilm-formation ability of strain EAM 35 was inhibited in a pesticide-dose-dependent manner, and it was statistically (p ≤ 0.05, p ≤ 0.005, and p ≤ 0.001) significant. Conclusively, the present study clearly suggests that before applying pesticides to soil, their recommended dose should carefully be monitored.

Enhanced production of camptothecin by immobilized callus of Ophiorrhiza mungos and a bioinformatic insight into its potential antiviral effect against SARS-CoV-2.

Camptothetin (CPT) is a quinoline alkaloid originally isolated from the Chinese tree, Camptotheca acuminata Decne. CPT was found to have anticancerous and antiviral properties. Derivatives of natural CPT, including topothecan and irinotecan are used clinically to treat a variety of cancers. Apart from Camptotheca acuminata Decne, CPT production was also found in the perennial plant Ophiorrhiza mungos. In this study we attempted the immobilization of the tissue culture grown callus of Ophiorrhiza mungos for the continuous production of a higher concentration of CPT. As evident from previous studies about the antiviral effects of CPT, we wanted to bioinformatically analyze the binding potential of CPT towards two important proteins of SARS-CoV-2, protease (Mpro) and RNA dependent RNA polymerase (RdRp). Further docking analysis of the CPT against the exterior spike glycoprotein of SARS-CoV-2 was also done to determine their potential interaction. The immobilized callus of Ophiorrhiza mungos produced CPT at a concentration of 420 µg/l by the end of 12 days of growth. The HPLC analysis was done to determine the purity of the CPT synthesized by the immobilization technique. The bioinformatic analysis revealed a higher binding efficiency of CPT and its derivatives, toptecan and irinotecan against Mpro and RdRp. The docking analysis of CPT against the spike glycoprotein of SARS-CoV-2 showed hydrogen bonding with the amino acids at K466 with a bond distance of 2.56A° and K355 with a bond distance of 2.40A°. This finding was of particular importance that other compounds including hydroxychloroquine sulphate, lopinavir and ivermectin could bind with the spike protein only by weak Vander wall bonds and no hydrogen bond formation was noticed. Our studies hence evaluate the efficiency of CPT against SARS-CoV-2, by potentially blocking the interaction of the spike glycoprotein with the angiotensin-converting enzyme 2 (ACE2) receptor found on host cells.

Therapeutic strategies against hDOT1L as a potential drug target in MLL-rearranged leukemias.

Therapeutic intervention of proteins participating in chromatin-mediated signaling with small-molecules is a novel option to reprogram expression networks for restraining disease states. Protein methyltransferases form the prominent family of such proteins regulating gene expression via epigenetic mechanisms thereby representing novel targets for pharmacological intervention. Disruptor of telomeric silencing, hDot1L is the only non-SET domain containing histone methyltransferase that methylates histone H3 at lysine 79. H3K79 methylation mediated by hDot1L plays a crucial role in mixed lineage leukemia (MLL) pathosis. MLL fusion protein mediated mistargeting of DOT1L to aberrant gene locations results in ectopic H3K79 methylation culminating in aberrant expression of leukemogenic genes like HOXA9 and MEIS1. hDOT1L has thus been proposed as a potential target for therapeutic intervention in MLL. This review presents the general overview of hDOT1L and its functional role in distinct biological processes. Furthermore, we discuss various therapeutic strategies against hDOT1L as a promising drug target to vanquish therapeutically challenging MLL.

D-Ring-Modified Analogues of Luotonin A with Reduced Planarity: Design, Synthesis, and Evaluation of Their Topoisomerase Inhibition-Associated Cytotoxicity.

A- and D-ring-modified luotonin-inspired heterocycles have been synthesized and were evaluated for their activity against the viability of four cancer cell lines in vitro, namely, MCF7, HCT116, JURKAT, and NCI-H460. The analysis of results indicated that two of the synthesized derivatives displayed good inhibition against the growth of the human colon cancer HCT116 cell line, with potencies lower than but in the same order of magnitude as camptothecin (CPT). These two luotonin analogues also showed an activity similar to that of the highly potent alkaloid CPT as inhibitors of topoisomerase I and also inhibited topoisomerase II. These results show that complete planarity is not a strict requirement for topoisomerase inhibition by luotonin-related compounds, paving the way to the design of analogues with improved solubility.

Epigenetics: An emerging field in the pathogenesis of nonalcoholic fatty liver disease.

Nonalcoholic fatty liver disease (NAFLD) is a major health concern associated with increased mortality due to cardiovascular disease, type II diabetes, insulin resistance, liver disease, and malignancy. The molecular mechanism underlying these processes is not fully understood but involves hepatic fat accumulation and alteration of energy metabolism and inflammatory signals derived from various cell types including immune cells. During the last two decades, epigenetic mechanisms have emerged as important regulators of chromatin alteration and the reprogramming of gene expression. Recently, epigenetic mechanisms have been implicated in the pathogenesis of NAFLD and nonalcoholic steatohepatitis (NASH) genesis. Epigenetic mechanisms could be used as potential therapeutic targets and as noninvasive diagnostic biomarkers for NAFLD. These mechanisms can determine disease progression and prognosis in NAFLD. In this review, we discuss the role of epigenetic mechanisms in the progression of NAFLD and potential therapeutic targets for the treatment of NAFLD.

In silico approaches for investigating the binding propensity of apigenin and luteolin against class I HDAC isoforms.

AIM: Aberrant activity of class I histone deacetylases (HDACs) has strong implications for various cancers. Targeting these HDACs with synthetic HDAC inhibitors has shown significant side effects such as atrial fibrillation and QT prolongation emphasizing the need of natural inhibitors as substitutes to synthetic ones. RESULTS: The binding propensity of the two plant-derived inhibitors apigenin and luteolin towards class I HDAC isoforms was checked using extra-precision molecular docking and implicit solvation MMGBSA. Apigenin showed a superior binding affinity against these isoforms as compared to luteolin. Both inhibitors docked stable to the binding pocket of these HDACs as determined by molecular dynamics simulation study. CONCLUSION: Apigenin and luteolin may serve as substitutes to synthetic inhibitors for effective HDAC based anticancer therapy.

Functionalized spirooxindole-indolizine hybrids: Stereoselective green synthesis and evaluation of anti-inflammatory effect involving TNF-α and nitrite inhibition.

Stereoselective synthesis of a small library of novel spiroheterocyclic hybrids including indolizine, oxindole, and substituted piperidine units has been accomplished in [bmim]Br using a [3 + 2] cycloaddition strategy in good yield and were tested for their anti-inflammatory activities. The effects of compounds (4a-o) against inflammation were studied using carrageenan-induced hind paw oedema, croton oil-induced ear oedema, and cotton pellet-induced granuloma models. Among the heterocyclic hybrids, compounds 4d, 4g, and 4o showed significant anti-inflammatory activities against acute and chronic inflammatory models. These compounds also showed significant inhibition of PGE2, TNF-α, and nitrite levels in carrageenan-induced hind paw oedema. Thus it is evident from our study that these novel spiroheterocyclic hybrids 4d, 4g, and 4o displayed significant anti-inflammatory effects that involve the reduction of PGE2, TNF-α, and nitrite levels.

Highly functionalized pyrrolidine analogues: stereoselective synthesis and caspase-dependent apoptotic activity.

Novel spiropyrrolidine heterocyclic hybrids were synthesized for the first time in a sustainable fashion employing a 1,3-dipolar cycloaddition strategy to form a new class of azomethine ylides generated from tyrosine and acenaphthenequinone. Following their synthesis and characterization, these heterocyclic hybrids were tested for their anticancer activities by incubation at different concentrations and durations with different cancer and non-cancer cell cultures, and the results indicated a potential therapeutic activity. Further analysis of cancer cell death revealed that it occurred through a caspase-related apoptotic pathway, specifically mediated by caspase-3. These results demonstrated that the obtained spiropyrrolidine heterocyclic hybrids may be good hit compounds for the development of potential therapeutic agents for the treatment of malignant tumors.

Combinatorial In Silico Strategy towards Identifying Potential Hotspots during Inhibition of Structurally Identical HDAC1 and HDAC2 Enzymes for Effective Chemotherapy against Neurological Disorders.

Histone deacetylases (HDACs) regulate epigenetic gene expression programs by modulating chromatin architecture and are required for neuronal development. Dysregulation of HDACs and aberrant chromatin acetylation homeostasis have been implicated in various diseases ranging from cancer to neurodegenerative disorders. Histone deacetylase inhibitors (HDACi), the small molecules interfering HDACs have shown enhanced acetylation of the genome and are gaining great attention as potent drugs for treating cancer and neurodegeneration. HDAC2 overexpression has implications in decreasing dendrite spine density, synaptic plasticity and in triggering neurodegenerative signaling. Pharmacological intervention against HDAC2 though promising also targets neuroprotective HDAC1 due to high sequence identity (94%) with former in catalytic domain, culminating in debilitating off-target effects and creating hindrance in the defined intervention. This emphasizes the need of designing HDAC2-selective inhibitors to overcome these vicious effects and for escalating the therapeutic efficacy. Here we report a top-down combinatorial in silico approach for identifying the structural variants that are substantial for interactions against HDAC1 and HDAC2 enzymes. We used extra-precision (XP)-molecular docking, Molecular Mechanics Generalized Born Surface Area (MMGBSA) for predicting affinity of inhibitors against the HDAC1 and HDAC2 enzymes. Importantly, we employed a novel in silico strategy of coupling the state-of-the-art molecular dynamics simulation (MDS) to energetically-optimized structure based pharmacophores (e-Pharmacophores) method via MDS trajectory clustering for hypothesizing the e-Pharmacophore models. Further, we performed e-Pharmacophores based virtual screening against phase database containing millions of compounds. We validated the data by performing the molecular docking and MM-GBSA studies for the selected hits among the retrieved ones. Our studies attributed inhibitor potency to the ability of forming multiple interactions and infirm potency to least interactions. Moreover, our studies delineated that a single HDAC inhibitor portrays differential features against HDAC1 and HDAC2 enzymes. The high affinity and selective HDAC2 inhibitors retrieved through e-Pharmacophores based virtual screening will play a critical role in ameliorating neurodegenerative signaling without hampering the neuroprotective isoform (HDAC1).

Design, synthesis and antiproliferative activity of decarbonyl luotonin analogues.

A small library of benzimidazole-fused pyrrolo[3,4-b]quinoline has been synthesized from readily available benzimidazole 2-carbaldehyde and various substituted arylamines in good to excellent yields utilizing an intramolecular Povarov reaction catalyzed by boron trifluoride diethyl etharate as the key final step. The compounds thus synthesized can be considered as decarbonyl analogues of the anticancer alkaloid luotonin A and were evaluated in a DNA relaxation assay for their ability to inhibit human topoisomerase I. Interestingly, two of the compounds showed a remarkable activity that is comparable to that of the standard drug camptothecin. The compounds were also evaluated for their cytotoxic effect in four highly aggressive human cancer cell lines, namely KB, MDA-MB231 (breast), LNCap (prostate), and HT1080 (fibrosarcoma). Some of the compounds obtained showed promising cytotoxicities for these four cell lines.

Plant Derived Inhibitor Sulforaphane in Combinatorial Therapy Against Therapeutically Challenging Pancreatic Cancer.

Pancreatic cancer is one of the most aggressive human cancers and is expected to surpass breast cancer to become the third chief cause of cancer-related deaths in the United States. While conventional treatment approaches such as surgery and classic chemotherapy have slightly improved the relative five year survival rate to 8% yet it is the lowest survival rate for any major cancer. This emphasizes the serious need of more effective and well tolerated therapies to reverse the poor prognosis of the defined neoplasm. Aberrant expression of histone deacetylase (HDAC) enzymes has been implicated in pancreatic cancer signalling. The inhibitors of these enzymes namely HDAC inhibitors (HDACi) are the novel agents which are currently being tested. These inhibitors modulate both histone and nonhistone proteins and have shown multiple biological effects including cell cycle arrest, differentiation and apoptosis in several cancer models. This article focuses on plant-derived HDAC inhibitor Sulforaphane (SFN) as a promising antipancreatic cancer agent. Moreover, we discuss the distinct molecular mechanisms triggered by SFN to exert cytotoxic effect in the predefined cancer models. Finally we describe the combinatorial therapeutic strategy involving SFN with other anticancer agents. This novel approach circumvents herculean cancer chemoresistance and alleviates toxicity, the main drawbacks of monotherapy.

The many faces of histone H3K79 methylation.

Dot1/DOT1L (disruptor of telomeric silencing-1) is an evolutionarily conserved histone methyltransferase that methylates lysine 79 located within the globular domain of histone H3. Dot1 was initially identified by a genetic screen as a disruptor of telomeric silencing in Saccharomyces cerevisiae; further, it is the only known non-SET domain containing histone methyltransferase. Methylation of H3K79 is involved in the regulation of telomeric silencing, cellular development, cell-cycle checkpoint, DNA repair, and regulation of transcription. hDot1L-mediated H3K79 methylation appears to have a crucial role in transformation as well as disease progression in leukemias involving several oncogenic fusion proteins. This review summarizes the multiple functions of Dot1/hDOT1L in a range of cellular processes.