'Synthesis, antiviral activity, molecular docking, and molecular dynamics studies of ethoxy phthalimide pyrazole derivatives against Cytomegalovirus and Varicella-Zoster virus: potential consequences and strategies for developing new antiviral treatments'.

Substituted ethoxy phthalimide pyrazole derivatives (6a-e) have been produced using a one-pot synthesis technique. Spectral analysis was used to establish the molecular structure of the synthesized compounds, and they were examined in silico and in vitro for their ability to bind to and inhibit replication of the AD-169 strain, the Davis strain of CMV, the OKA strain and the 07/1 strain of Varicella-Zoster virus (VZV). Molecular Docking was used to estimate the binding mechanism and energy of compounds 4, 6a-e to their respective target proteins, thymidine kinase (TK), Varicella-Zoster protease (VZP) of VZV and tegument protein pp71 (TPpp71) of Cytomegalovirus (CMV). The MIC50 and EC50 were utilized to evaluate the antiviral and cytotoxic activities of test compounds in human embryonic lung (HEL) cells against the two reference medicines, Ganciclovir and Acyclovir. The chemicals studied showed a high affinity for binding sites and near binding sites of target proteins by generating H-bonds, carbon-hydrogen bonds, π-anion, π-sulfur, π-sigma, alkyl and π-alkyl interactions. All of the test compounds (6a-e) had higher binding energy than the standard medications. The ADME/T data suggests that these potential inhibitors are less toxic. Drug-protein complexes are structurally compact and demonstrate minimal conformational change in molecular dynamics (MDs) simulations, indicating stability and stiffness. MM-PBSA and post-simulation analysis can predict lead compound active cavity binding stability. By inhibiting multitargeted proteins, these synthetic compounds may improve antiviral therapy. Our research suggests that these unique synthesized chemicals may be useful and accessible adjuvant antiviral therapy for Varicella Zoster and CMV. HighlightsTwo components synthesis of substituted ethoxy phthalimide pyrazole derivatives (6a-e).Tested compounds (6a-e) have antiviral and cytotoxicity activity against CMV and Varicella-Zoster virus (VZV) in HEL cells.Compounds bind to TK, Varicella-Zoster protease (VZP) of VZV, and modeled TPpp71 of Cytomegalovirus (CMV).In comparison to reference drugs, compounds have strong binding free energy and interactions with VZV and CMV protein complexes.The RMSD, RMSF, Rg, residual correlative motion (RCM), No. of hydrogen bonds, protein secondary structure content, per-residue protein secondary structure and MM/PBSA energy calculated for the selected compound with thymidine kinase (TK), VZP of VZV, and modeled tegument protein pp71 (TPpp71) of CMV through MD simulation studies for 50 ns.In comparison to the two reference drugs, ligands/compounds were found to meet the Lipinski rule of five and to have strong biological activity.Communicated by Ramaswamy H. Sarma.

New drug discovery of cardiac anti-arrhythmic drugs: insights in animal models.

Cardiac rhythm regulated by micro-macroscopic structures of heart. Pacemaker abnormalities or disruptions in electrical conduction, lead to arrhythmic disorders may be benign, typical, threatening, ultimately fatal, occurs in clinical practice, patients on digitalis, anaesthesia or acute myocardial infarction. Both traditional and genetic animal models are: In-vitro: Isolated ventricular Myocytes, Guinea pig papillary muscles, Patch-Clamp Experiments, Porcine Atrial Myocytes, Guinea pig ventricular myocytes, Guinea pig papillary muscle: action potential and refractory period, Langendorff technique, Arrhythmia by acetylcholine or potassium. Acquired arrhythmia disorders: Transverse Aortic Constriction, Myocardial Ischemia, Complete Heart Block and AV Node Ablation, Chronic Tachypacing, Inflammation, Metabolic and Drug-Induced Arrhythmia. In-Vivo: Chemically induced arrhythmia: Aconitine antagonism, Digoxin-induced arrhythmia, Strophanthin/ouabain-induced arrhythmia, Adrenaline-induced arrhythmia, and Calcium-induced arrhythmia. Electrically induced arrhythmia: Ventricular fibrillation electrical threshold, Arrhythmia through programmed electrical stimulation, sudden coronary death in dogs, Exercise ventricular fibrillation. Genetic Arrhythmia: Channelopathies, Calcium Release Deficiency Syndrome, Long QT Syndrome, Short QT Syndrome, Brugada Syndrome. Genetic with Structural Heart Disease: Arrhythmogenic Right Ventricular Cardiomyopathy/Dysplasia, Dilated Cardiomyopathy, Hypertrophic Cardiomyopathy, Atrial Fibrillation, Sick Sinus Syndrome, Atrioventricular Block, Preexcitation Syndrome. Arrhythmia in Pluripotent Stem Cell Cardiomyocytes. Conclusion: Both traditional and genetic, experimental models of cardiac arrhythmias' characteristics and significance help in development of new antiarrhythmic drugs.

The Role of Acetyl Zingerone and Its Derivatives in Inhibiting UV-Induced, Incident, and Delayed Cyclobutane Pyrimidine Dimers.

Cyclobutane pyrimidine dimers (CPDs) are ultraviolet radiation (UV)-induced carcinogenic DNA photoproducts that lead to UV signature mutations in melanoma. Previously, we discovered that, in addition to their incident formation (iCPDs), UV exposure induces melanin chemiexcitation (MeCh), where UV generates peroxynitrite (ONOO-), which oxidizes melanin into melanin-carbonyls (MCs) in their excited triplet state. Chronic MeCh and energy transfer by MCs to DNA generates CPDs for several hours after UV exposure ends (dark CPD, dCPDs). We hypothesized that MeCh and the resulting dCPDs can be inhibited using MeCh inhibitors, and MC and ONOO- scavengers. Here, we investigated the efficacy of Acetyl Zingerone (AZ), a plant-based phenolic alkanone, and its chemical analogs in inhibiting iCPDs and dCPDs in skin fibroblasts, keratinocytes, and isogenic pigmented and albino melanocytes. While AZ and its methoxy analog, 3-(4-Methoxy-benzyl)-Pentane-2,4-dione (MBPD) completely inhibited the dCPDs, MBPD also inhibited ~50% of iCPDs. This suggests the inhibition of ~80% of total CPDs at any time point post UV exposure by MBPD, which is markedly significant. MBPD downregulated melanin synthesis, which is indispensable for dCPD generation, but this did not occur with AZ. Meanwhile, AZ and MBPD both upregulated the expression of nucleotide excision repair (NER) pathways genes including Xpa, Xpc, and Mitf. AZ and its analogs were non-toxic to the skin cells and did not act as photosensitizers. We propose that AZ and MBPD represent "next-generation skin care additives" that are safe and effective for use not only in sunscreens but also in other specialized clinical applications owing to their extremely high efficacy in blocking both iCPDs and dCPDs.

Metabolomic analysis shows dysregulation in amino acid and NAD+ metabolism in palmitate treated hepatocytes and plasma of non-alcoholic fatty liver disease spectrum.

There is an alarming increase in incidence of fatty liver disease worldwide. The fatty liver disease spectrum disease ranges from simple steatosis (NAFL) to steatohepatitis (NASH) which culminates in cirrhosis and cancer. Altered metabolism is a hallmark feature associated with fatty liver disease and palmitic acid is the most abundant saturated fatty acid, therefore, the aim of this study was to compare metabolic profiles altered in hepatocytes treated with palmitic acid and also the differentially expressed plasma metabolites in spectrum of nonalcoholic fatty liver. The metabolites were analyzed by liquid chromatography-mass spectrometry (LC-MS) platform. Hepatocyte cell lines PH5CH8 and HepG2 cells when treated with 400 µM dose of palmitic acid showed typical features of steatosis. Metabolomic analysis of lipid treated hepatocyte cell lines showed differential changes in phenylalanine and tyrosine pathways, fatty acid metabolism and bile acids. The key metabolites tryptophan, kynurenine and carnitine differed significantly between subjects with NAFL, NASH and those with cirrhosis. As the tryptophan-kynurenine axis is also involved in denovo synthesis of NAD+, we found significant alterations in the NAD+ related metabolites in both palmitic acid treated and also fatty liver disease with cirrhosis. The study underscores the importance of amino acid and NAD+supplementation as promising strategies in fatty liver disorder.

Variational mode decomposition and binary grey wolf optimization-based automated epilepsy seizure classification framework.

This work proposes a variational mode decomposition (VMD) and binary grey wolf optimization (BGWO) based seizure classification framework. VMD decomposes the EEG signal into band-limited intrinsic mode function (BL-IMFs) non-recursively. The frequency domain, time domain, and information theory-based features are extracted from the BL-IMFs. Further, an optimal feature subset is selected using BGWO. Finally, the selected features were utilized for classification using six different supervised machine learning algorithms. The proposed framework has been validated experimentally by 58 test cases from the CHB-MIT scalp EEG and the Bonn University database. The proposed framework performance is quantified by average sensitivity, specificity, and accuracy. The selected features, along with Bayesian regularized shallow neural networks (BR-SNNs), resulted in maximum accuracy of 99.53 and 99.64 for 1 and 2 s epochs, respectively, for database 1. The proposed framework has achieved 99.79 and 99.84 accuracy for 1 and 2 s epochs, respectively, for database 2.

HSc70 interactome reveal major role of macroautophagy and minor role of chaperone mediated autophagy in K-Ras G12V cell proliferation and survival.

Constitutively active K-Ras oncogene mutation at G12V changes the proteome of cells and activates macroautophagy for cell advantage. Inhibition of macroautophagy impairs K-Ras mediated tumor progression to a limited extent with increase of spontaneous tumors due to poorly understood mechanisms. Here, we show that inhibition of macroautophagy in K-Ras G12V mouse embryonic fibroblasts (MEFs) hyper activates chaperon mediated autophagy (CMA). Quantitative identification of CMA substrates through co-immunoprecipitation of CMA component heat shock cognate 70 (Hsc70) demonstrates a shift of proteins from macroautophagy to CMA mediated degradation. However, macroautophagy impairment show significant inhibition on proliferation and CMA hyper activation provides a basal support to macroautophagy-inhibited MEFs for survival. On the other hand, K-Ras G12V MEFs impaired of CMA reduces number of Hsc70 clients but activated macroautophagy significantly compensated CMA loss. Nonetheless, co-inhibition of CMA and macroautophagy had a synergistic detrimental effect on both proliferation and survival of MEFs expressing K-Ras G12V mutant. Our results point to K-Ras G12V MEFs dependency on macroautophagy and CMA partly compensates its loss for survival but not hyper-proliferation; implicating that targeting both macroautophagy and CMA as a promising therapeutic target in G12V mutation associated K-Ras cancers. SIGNIFICANCE: The present study provides a framework of Hsc70 interacting proteins, which differentially interact with Hsc70 in response to autophagy alterations. The role of proteins accumulation and induced proteo-toxicity could be underlying factor in macroautophagy and CMA co-inhibited K-Ras G12V MEFs phenotype. Our study provides rational for adaptive mechanisms in K-Ras tumors inhibited with different autophagy pathways and also supports targeting both macroautophagy and CMA simultaneously as therapeutic target. At the same time current study will help in characterizing the underlying cellular processes that may play a role in escaping tutor suppressor role CMA and macroautophagy in cancers harboring K-Ras G12V mutation that may be further utilized to identify molecular targets for K-Ras-driven cancers.

Decellularization of caprine esophagus using fruit pericarp extract of Sapindus mukorossi.

Biological detergents like sodium deoxycholate, sodium dodecyl sulphate and Triton X-100 impairs the collagenous and non-collagenous proteins, glycosaminoglycans and growth factors. Further, certain chemical and enzymes are responsible for residual cytotoxicity in the decellularized extracellular matrix. The main focus of this study was to explore the decellularization property of soap nut pericarp extract (SPE) for development of decellularized tubular esophageal scaffold. For this 2.5, 5.0 and 10% concentrations of SPE were used for decellularization of caprine esophageal tissues. Histological analysis of hematoxylin and eosin and Masson's trichrome stained tissue samples confirmed decellularization with preservation of extracellular matrix microarchitecture. Scanning electron microscopic images of luminal surface of decellularized esophageal matrix showed randomly oriented collagen fibres with large interconnected pores and cells were absent. However, the external surface was more textured with fibrous structures and collagen fibres were well preserved. DAPI stained decellularized tissues revealed complete removal of nuclear components, verified by DNA content measurement and SDS-PAGE. The FTIR spectra of decellularized esophagus shows absorption peaks of amide A, B, I, II and III. Elastic modulus of the decellularized esophagus scaffolds increased (P > 0.05) as compared to native tissues. Histological and scanning electron microscopic evaluation of in vitro seeded scaffolds showed attachment and growth of primary chicken embryo fibroblasts over and within the decellularized scaffolds. It was concluded that 5% SPE is ideal for preparation of cytocompatible decellularized caprine esophageal scaffold with well-preserved extracellular matrix architecture and, may be used as an alternative to biological detergents and other chemicals.

Long noncoding RNAs in intestinal homeostasis, regeneration, and cancer.

Signaling pathways that regulate homeostasis and regeneration are found to be deregulated in various human malignancies. Accordingly, attempts have been made to target them at the protein level with little success. However, studies using high-throughput sequencing technologies suggest that only about 2% of the genome translates into proteins, whereas about 75% of the genome is transcribed into noncoding RNAs. Among noncoding RNAs, long noncoding RNAs (lncRNAs) have received tremendous attention in recent years as a crucial player in the regulation of almost all cellular processes involved in tissue homeostasis as well as in the development of various malignancies, including intestinal cancer. Emerging evidence suggests that lncRNAs play an instrumental role in the regulation of intestinal stem cells, injury-induced regeneration, and initiation and progression of intestinal tumors. Here, we summarize the recently discovered lncRNAs during intestinal homeostasis, regeneration, and tumorigenesis. We further present lncRNAs as diagnostic and therapeutic markers in intestinal pathologies.

Development of decellularized aortic scaffold for regenerative medicine using Sapindus mukorossi fruit pericarp extract.

The aim of this study is to develop a novel decellularization method using aqueous extract of soap nut pericarp (SPE) and its evaluation using hematoxylin-eosin staining, scanning electron microscopy, diamidino-2-phenylindol (DAPI) staining, mechanical testing, sodium dodecyl sulfate polyacrylamide gel electrophoresis and DNA quantification. The presently available decellularization agent raises some concerns due to the potential for presence of residual cytotoxic agents in the extracellular matrix. Histological analysis of hematoxylin and eosin and masson's trichrome stained processed aortic samples shows complete decellularization with preservation of extracellular matrix microarchitecture at 120 h. Further, staining of tissue samples with DAPI demonstrates complete removal of DNA fragments. Quantitative evaluation of DNA in the decellularized aorta tissues demonstrated a significant (P < 0.01) decrease in DNA content as compared to native tissues. Collagen quantification assay indicate no significant (P> 0.05) difference in its content between native and decellularized caprine aorta. Tensile strength of the decellularized scaffolds decreased non-significantly (P > 0.05) when compared to native tissues. There was no significant (P > 0.05) difference in young's modulus of elasticity, stiffness and stretch ratio between native aortic tissues and decellularized aortic scaffolds. Histological and scanning electron microscopic examination of in vitro cultured scaffold demonstrated the cell viability and proliferation of primary chicken embryo fibroblasts. SPE treatment is thus capable of producing cytocompatible decellularized caprine aorta scaffold with preservation of extracellular matrix architecture for vascular tissue engineering and could be applied widely as one of the decellularization agent.

Unusual presentation of jejunal hemangioma on Tc-99m pertechnetate scan with single-photon emission computerized tomography-computed tomography.

Small bowel hemangioma is a rare benign tumor in the pediatric population. The usual presentation of these tumors is melena, anemia, or hematochezia. Our case demonstrates the usefulness of Meckel's/Tc-99m pertechnetate scan with single-photon emission computerized tomography/computerized tomography in diagnosing a vascular lesion in the small bowel in a child presenting with melena, unresponsive to medical management. We present a case of incidentally detected jejunal hemangioma during Tc-99m pertechnetate scintigraphy which would help the nuclear medicine physician and surgeon, to be cognizant of this atypical presentation in their clinical practice.

Detection of UV-Induced Thymine Dimers.

Ultraviolet rays induce interstrand and intrastrand DNA cross-links, usually thymine-thymine cyclobutane dimer (T-T) and thymine-thymine pyrimidine-pyrimidone (6-4) photoproduct (T (6-4) T). These DNA cross-links, if left unrepaired, increase the risk of these mutation being incorporated in the genetic material (i.e., DNA). Numerous studies have reported the mutagenic potential of above mentioned DNA adducts in prokaryotes, yeast and mammalian cells. Different techniques have been developed to identify such DNA adducts such as immuno-Southern blotting. This is a routinely used quantitative method to determine especially the amount of thymine dimers formed, following irradiation. In this chapter, the detailed methodology to identify thymine dimers formation is provided, using specific antibody against these adducts.

Oncogenic BRAF Deletions That Function as Homodimers and Are Sensitive to Inhibition by RAF Dimer Inhibitor LY3009120.

UNLABELLED: We have identified previously undiscovered BRAF in-frame deletions near the αC-helix region of the kinase domain in pancreatic, lung, ovarian, and thyroid cancers. These deletions are mutually exclusive with KRAS mutations and occur in 4.21% of KRAS wild-type pancreatic cancer. siRNA knockdown in cells harboring BRAF deletions showed that the MAPK activity and cell growth are BRAF dependent. Structurally, the BRAF deletions are predicted to shorten the ß3/αC-helix loop and hinder its flexibility by locking the helix in the active αC-helix-in conformation that favors dimer formation. Expression of L485-P490-deleted BRAF is able to transform NIH/3T3 cells in a BRAF dimer-dependent manner. BRAF homodimer is confirmed to be the dominant RAF dimer by proximity ligation assays in BRAF deletion cells, which are resistant to the BRAF inhibitor vemurafenib and sensitive to LY3009120, a RAF dimer inhibitor. In tumor models with BRAF deletions, LY3009120 has shown tumor growth regression, whereas vemurafenib is inactive. SIGNIFICANCE: This study discovered oncogenic BRAF deletions with a distinct activation mechanism dependent on the BRAF dimer formation in tumor cells. LY3009120 is active against these cells and represents a potential treatment option for patients with cancer with these BRAF deletions, or other atypical BRAF mutations where BRAF functions as a dimer.

Inhibition of RAF Isoforms and Active Dimers by LY3009120 Leads to Anti-tumor Activities in RAS or BRAF Mutant Cancers.

LY3009120 is a pan-RAF and RAF dimer inhibitor that inhibits all RAF isoforms and occupies both protomers in RAF dimers. Biochemical and cellular analyses revealed that LY3009120 inhibits ARAF, BRAF, and CRAF isoforms with similar affinity, while vemurafenib or dabrafenib have little or modest CRAF activity compared to their BRAF activities. LY3009120 induces BRAF-CRAF dimerization but inhibits the phosphorylation of downstream MEK and ERK, suggesting that it effectively inhibits the kinase activity of BRAF-CRAF heterodimers. Further analyses demonstrated that LY3009120 also inhibits various forms of RAF dimers including BRAF or CRAF homodimers. Due to these unique properties, LY3009120 demonstrates minimal paradoxical activation, inhibits MEK1/2 phosphorylation, and exhibits anti-tumor activities across multiple models carrying KRAS, NRAS, or BRAF mutation.

Activating the Wnt/ß-Catenin Pathway for the Treatment of Melanoma--Application of LY2090314, a Novel Selective Inhibitor of Glycogen Synthase Kinase-3.

It has previously been observed that a loss of ß-catenin expression occurs with melanoma progression and that nuclear ß-catenin levels are inversely proportional to cellular proliferation, suggesting that activation of the Wnt/ß-catenin pathway may provide benefit for melanoma patients. In order to further probe this concept we tested LY2090314, a potent and selective small-molecule inhibitor with activity against GSK3α and GSK3ß isoforms. In a panel of melanoma cell lines, nM concentrations of LY2090314 stimulated TCF/LEF TOPFlash reporter activity, stabilized ß-catenin and elevated the expression of Axin2, a Wnt responsive gene and marker of pathway activation. Cytotoxicity assays revealed that melanoma cell lines are very sensitive to LY2090314 in vitro (IC50 ~10 nM after 72hr of treatment) in contrast to other solid tumor cell lines (IC50 >10 uM) as evidenced by caspase activation and PARP cleavage. Cell lines harboring mutant B-RAF or N-RAS were equally sensitive to LY2090314 as were those with acquired resistance to the BRAF inhibitor Vemurafenib. shRNA studies demonstrated that ß-catenin stabilization is required for apoptosis following treatment with the GSK3 inhibitor since the sensitivity of melanoma cell lines to LY290314 could be overcome by ß-catenin knockdown. We further demonstrate that in vivo, LY2090314 elevates Axin2 gene expression after a single dose and produces tumor growth delay in A375 melanoma xenografts with repeat dosing. The activity of LY2090314 in preclinical models suggests that the role of Wnt activators for the treatment of melanoma should be further explored.

Co-targeting BRAF and cyclin dependent kinases 4/6 for BRAF mutant cancers.

Selective BRAF inhibitors have demonstrated significant clinical benefit in melanoma patients harboring oncogenic BRAF mutations. However, the majority of such patients either exhibit de novo resistance from the beginning of the treatment or acquire resistance and eventually relapse. Despite tremendous progress in understanding the underlying mechanisms of resistance, overcoming resistance to BRAF inhibitors remains an unmet medical need. Constitutive activation of cyclin-dependent kinases (CDK) 4/6 as a result of genetic aberrations including CDKN2A inactivation and CCND1 amplification is common across many cancer types and frequently co-occurs with oncogenic BRAF mutations. Also, cyclin D1 overexpression is a common feature of resistance to BRAF inhibitors. Here we review CDK4/6 as a therapeutic target in BRAF mutant cancers and discuss emerging evidence supporting a critical role of cyclin D1/CDK4/6 axis in de novo and acquired resistance to BRAF inhibitors. Co-targeting CDK4/6 and BRAF could be a more effective therapy to augment clinical response of BRAF inhibitors and overcome resistance in BRAF mutant cancers.

The CDK4/6 inhibitor LY2835219 overcomes vemurafenib resistance resulting from MAPK reactivation and cyclin D1 upregulation.

B-RAF selective inhibitors, including vemurafenib, were recently developed as effective therapies for melanoma patients with B-RAF V600E mutation. However, most patients treated with vemurafenib eventually develop resistance largely due to reactivation of MAPK signaling. Inhibitors of MAPK signaling, including MEK1/2 inhibitor trametinib, failed to show significant clinical benefit in patients with acquired resistance to vemurafenib. Here, we describe that cell lines with acquired resistance to vemurafenib show reactivation of MAPK signaling and upregulation of cyclin D1 and are sensitive to inhibition of LY2835219, a selective inhibitor of cyclin-dependent kinase (CDK) 4/6. LY2835219 was demonstrated to inhibit growth of melanoma A375 tumor xenografts and delay tumor recurrence in combination with vemurafenib. Furthermore, we developed an in vivo vemurafenib-resistant model by continuous administration of vemurafenib in A375 xenografts. Consistently, we found that MAPK is reactivated and cyclin D1 is elevated in vemurafenib-resistant tumors, as well as in the resistant cell lines derived from these tumors. Importantly, LY2835219 exhibited tumor growth regression in a vemurafenib-resistant model. Mechanistic analysis revealed that LY2835219 induced apoptotic cell death in a concentration-dependent manner in vemurafenib-resistant cells whereas it primarily mediated cell-cycle G1 arrest in the parental cells. Similarly, RNAi-mediated knockdown of cyclin D1 induced significantly higher rate of apoptosis in the resistant cells than in parental cells, suggesting that elevated cyclin D1 activity is important for the survival of vemurafenib-resistant cells. Altogether, we propose that targeting cyclin D1-CDK4/6 signaling by LY2835219 is an effective strategy to overcome MAPK-mediated resistance to B-RAF inhibitors in B-RAF V600E melanoma.

Identification of druggable cancer driver genes amplified across TCGA datasets.

The Cancer Genome Atlas (TCGA) projects have advanced our understanding of the driver mutations, genetic backgrounds, and key pathways activated across cancer types. Analysis of TCGA datasets have mostly focused on somatic mutations and translocations, with less emphasis placed on gene amplifications. Here we describe a bioinformatics screening strategy to identify putative cancer driver genes amplified across TCGA datasets. We carried out GISTIC2 analysis of TCGA datasets spanning 16 cancer subtypes and identified 486 genes that were amplified in two or more datasets. The list was narrowed to 75 cancer-associated genes with potential "druggable" properties. The majority of the genes were localized to 14 amplicons spread across the genome. To identify potential cancer driver genes, we analyzed gene copy number and mRNA expression data from individual patient samples and identified 42 putative cancer driver genes linked to diverse oncogenic processes. Oncogenic activity was further validated by siRNA/shRNA knockdown and by referencing the Project Achilles datasets. The amplified genes represented a number of gene families, including epigenetic regulators, cell cycle-associated genes, DNA damage response/repair genes, metabolic regulators, and genes linked to the Wnt, Notch, Hedgehog, JAK/STAT, NF-KB and MAPK signaling pathways. Among the 42 putative driver genes were known driver genes, such as EGFR, ERBB2 and PIK3CA. Wild-type KRAS was amplified in several cancer types, and KRAS-amplified cancer cell lines were most sensitive to KRAS shRNA, suggesting that KRAS amplification was an independent oncogenic event. A number of MAP kinase adapters were co-amplified with their receptor tyrosine kinases, such as the FGFR adapter FRS2 and the EGFR family adapters GRB2 and GRB7. The ubiquitin-like ligase DCUN1D1 and the histone methyltransferase NSD3 were also identified as novel putative cancer driver genes. We discuss the patient tailoring implications for existing cancer drug targets and we further discuss potential novel opportunities for drug discovery efforts.

Comparative lipidomics in clinical isolates of Candida albicans reveal crosstalk between mitochondria, cell wall integrity and azole resistance.

Prolonged usage of antifungal azoles which target enzymes involved in lipid biosynthesis invariably leads to the development of multi-drug resistance (MDR) in Candida albicans. We had earlier shown that membrane lipids and their fluidity are closely linked to the MDR phenomenon. In one of our recent studies involving comparative lipidomics between azole susceptible (AS) and azole resistant (AR) matched pair clinical isolates of C. albicans, we could not see consistent differences in the lipid profiles of AS and AR strains because they came from different patients and so in this study, we have used genetically related variant recovered from the same patient collected over a period of 2-years. During this time, the levels of fluconazole (FLC) resistance of the strain increased by over 200-fold. By comparing the lipid profiles of select isolates, we were able to observe gradual and statistically significant changes in several lipid classes, particularly in plasma membrane microdomain specific lipids such as mannosylinositolphosphorylceramides and ergosterol, and in a mitochondrial specific phosphoglyceride, phosphatidyl glycerol. Superimposed with these quantitative and qualitative changes in the lipid profiles, were simultaneous changes at the molecular lipid species levels which again coincided with the development of resistance to FLC. Reverse transcriptase-PCR of the key genes of the lipid metabolism validated lipidomic picture. Taken together, this study illustrates how the gradual corrective changes in Candida lipidome correspond to the development of FLC tolerance. Our study also shows a first instance of the mitochondrial membrane dysfunction and defective cell wall (CW) in clinical AR isolates of C. albicans, and provides evidence of a cross-talk between mitochondrial lipid homeostasis, CW integrity and azole tolerance.

Reactivation of mitogen-activated protein kinase (MAPK) pathway by FGF receptor 3 (FGFR3)/Ras mediates resistance to vemurafenib in human B-RAF V600E mutant melanoma.

Oncogenic B-RAF V600E mutation is found in 50% of melanomas and drives MEK/ERK pathway and cancer progression. Recently, a selective B-RAF inhibitor, vemurafenib (PLX4032), received clinical approval for treatment of melanoma with B-RAF V600E mutation. However, patients on vemurafenib eventually develop resistance to the drug and demonstrate tumor progression within an average of 7 months. Recent reports indicated that multiple complex and context-dependent mechanisms may confer resistance to B-RAF inhibition. In the study described herein, we generated B-RAF V600E melanoma cell lines of acquired-resistance to vemurafenib, and investigated the underlying mechanism(s) of resistance. Biochemical analysis revealed that MEK/ERK reactivation through Ras is the key resistance mechanism in these cells. Further analysis of total gene expression by microarray confirmed a significant increase of Ras and RTK gene signatures in the vemurafenib-resistant cells. Mechanistically, we found that the enhanced activation of fibroblast growth factor receptor 3 (FGFR3) is linked to Ras and MAPK activation, therefore conferring vemurafenib resistance. Pharmacological or genetic inhibition of the FGFR3/Ras axis restored the sensitivity of vemurafenib-resistant cells to vemurafenib. Additionally, activation of FGFR3 sufficiently reactivated Ras/MAPK signaling and conferred resistance to vemurafenib in the parental B-RAF V600E melanoma cells. Finally, we demonstrated that vemurafenib-resistant cells maintain their addiction to the MAPK pathway, and inhibition of MEK or pan-RAF activities is an effective therapeutic strategy to overcome acquired-resistance to vemurafenib. Together, we describe a novel FGFR3/Ras mediated mechanism for acquired-resistance to B-RAF inhibition. Our results have implications for the development of new therapeutic strategies to improve the outcome of patients with B-RAF V600E melanoma.

Ncb2 is involved in activated transcription of CDR1 in azole-resistant clinical isolates of Candida albicans.

We recently demonstrated that CDR1 overexpression in azole-resistant isolates of Candida albicans is due to its enhanced transcriptional activation and increased mRNA stability. In this study, we provide the first evidence of transcriptional regulation of CDR1 by Ncb2, the ß subunit of NC2, a heterodimeric regulator of transcription. Conditional NCB2 null mutants displayed decreased susceptibility toward azole and an enhanced transcription of CDR1. Interestingly, Ncb2 associated with the CDR1 promoter under both repression and activation; however, an increase in recruitment was observed under both transient and constitutive activation states. By chromatin immunoprecipitation (ChIP) assay, we showed the preferential recruitment of Ncb2 to the core TATA region under activation (azole-resistant isolate), while under repression (azole-susceptible isolate) it was present at the TATA upstream region. Further, ChIP analysis revealed that Ncb2 binding was not restricted to the CDR1 gene; instead, it was observed on the promoters of genes coregulated with CDR1 by the transcription activator Tac1. The tac1Δ null mutants, which fail to show the drug-induced transient activation of CDR1, also showed no increase in Ncb2 recruitment at the promoter. Taken together, our results show that Ncb2, in conjunction with Tac1, is involved in the transcriptional activation of CDR1, opening up new therapeutic possibilities to combat multidrug resistance (MDR) in C. albicans.