Multiple Activation Mechanisms Research Articles

Purified CDT toxins and a clean deletion within the CDT locus provide novel insights into the contribution of binary toxin in cellular inflammation and Clostridioides difficile infection.

Clostridioides difficile is a spore-forming pathogen and the most common cause of healthcare-associated diarrhea and colitis in the United States. Besides producing the main virulence factors, toxin A (TcdA) and toxin B (TcdB), many of the common clinical strains encode the C. difficile transferase (CDT) binary toxin. The role of CDT in the context of C. difficile infection (CDI) is poorly understood. Inflammation is a hallmark of CDI and multiple mechanisms of inflammasome activation have been reported for TcdA, TcdB, and the organism. Some studies have suggested that CDT contributes to this inflammation through a TLR2-dependent priming mechanism that leads to the suppression of protective eosinophils. Here, we show that CDT does not prime but instead activates the inflammasome in bone marrow-derived dendritic cells (BMDCs). In bone marrow-derived macrophages (BMDMs), the cell binding and pore-forming component of the toxin, CDTb, alone activates the inflammasome and is dependent on K+ efflux. The activation is not observed in the presence of CDTa and is not observed in BMDMs derived from Nlrp3-/- mice suggesting the involvement of the NLRP3 inflammasome. However, we did not observe evidence of CDT-dependent inflammasome priming or activation in vivo. Mice were infected with R20291 and an isogenic CRISPR/Cas9-generated R20291 ΔcdtB strain of C. difficile. While CDT contributes to increased weight loss and cecal edema at 2 days post infection, the relative levels of inflammasome-associated cytokines, IL-1β and IL-18, in the cecum and distal colon are unchanged. We also saw CDT-dependent weightloss in Nlrp3-/- mice, suggesting that the increased weightloss associated with the presence of CDT is not a result of NLRP3-dependent inflammasome activation. This study highlights the importance of studying gene deletions in the context of otherwise fully isogenic strains and the challenge of translating toxin-specific cellular responses into a physiological context, especially when multiple toxins are acting at the same time.

Cu single atoms mediated multiple active site reconfiguration to trigger Dual-pathway nonradical peroxymonosulfate activation process

Developing atomically dispersed metal sites is vital to enhance the activation of peroxymonosulfate (PMS). However, for catalyst systems where single atoms and nanoclusters coexist, how to identify the independent and synergistic effects of atomic sites and investigate the possible coexistence of multiple activation mechanisms still remains a great challenge. Herein, CuSA/CoOx-CeO2 catalysts with Cu single atoms and CoOx nanoclusters were fabricated for PMS activation. The introduction of Cu single atoms led to the reconstruction and diversification of catalytic active sites of CoOx-CeO2. The synergy between CoOx nanoclusters and neighboring Cu single atoms in CuSA/CoOx-CeO2 catalyst led to the 1O2 pathway of PMS activation, while the isolated Cu atom induced the Cu(III) oxidation pathway. The introducing of adjacent Cu single atoms caused the redistribution of the site charges in CoOx nanoclusters, which enhanced the charge transfer and promoted the adsorption and activation behavior of PMS molecules. During the evolution of 1O2, the synergistic effect of Cu single atoms and CoOx nanoclusters increased the stability of the reaction intermediate state (*OO*SO3), reducing the total Gibbs free energy change (ΔG) required for 1O2 formation. Compared with the cooperative site, the isolated Cu-O4 sites exhibited stronger charge transfer capacity to PMS molecules and were more conducive to the activation of the local substrate electronic structure, facilitating the formation and stabilization of Cu(III)–OH metastable intermediates. Overall, this study demonstrated the importance about the construction of multi-site induced multipath coexisting PMS activation system and the accurate identification of active sites.

Molecular Mechanism of Yangshen Maidong Decoction in the Treatment of Chronic Heart Failure based on Network Pharmacology, Molecular Docking, and Molecular Dynamics Simulations.

Chronic heart failure (CHF) is a complex multifactorial clinical syndrome leading to abnormal cardiac structure and function. The severe form of this ailment is characterized by high disability, high mortality, and morbidity. Worldwide, 2-17% of patients die at first admission, of which 17-45% die within 1 year of admission and >50% within 5 years. Yangshen Maidong Decoction (YSMDD) is frequently used to treat the deficiency and pain of the heart. The specific mechanism of action of YSMDD in treating CHF, however, remains unclear. Therefore, a network pharmacology-based strategy combined with molecular docking and molecular dynamics simulations was employed to investigate the potential molecular mechanism of YSMDD against CHF. The effective components and their targets of YSMDD and related targets of CHF were predicted and screened based on the public database. The network pharmacology was used to explore the potential targets and possible pathways that involved in YSMDD treated CHF. Molecular docking and molecular dynamics simulations were performed to elucidate the binding affinity between the YSMDD and CHF targets. Screen results, 10 main active ingredients, and 6 key targets were acquired through network pharmacology analysis. Pathway enrichment analysis showed that intersectional targets associated pathways were enriched in the Prostate cancer pathway, Hepatitis B pathway, and C-type lectin receptor signaling pathways. Molecular docking and molecular dynamics simulations analysis suggested 5 critical active ingredients have high binding affinity to the 5 key targets. This research shows the multiple active components and molecular mechanisms of YSMDD in the treatment of CHF and offers resources and suggestions for future studies.

Synergistic Incorporation of Two ssDNA Activators Enhances the Trans-Cleavage of CRISPR/Cas12a.

CRISPR/Cas12a has been believed to be powerful in molecular detection and diagnostics due to its amplified trans-cleavage feature. However, the activating specificity and multiple activation mechanisms of the Cas12a system are yet to be elucidated fully. Herein, a "synergistic activator effect" is discovered, which supports an activation mechanism that a synergistic incorporation of two short ssDNA activators can promote the trans-cleavage of CRISPR/Cas12a, while either of them is too short to work independently. As a proof-of-concept example, the synergistic activator-triggered CRISPR/Cas12a system has been successfully harnessed in the AND logic operation and the discrimination of single-nucleotide variants, requiring no signal conversion elements or other amplified enzymes. Moreover, a single-nucleotide specificity has been achieved for the detection of single-nucleotide variants by pre-introducing a synthetic mismatch between crRNA and the "helper" activator. The finding of "synergistic activator effect" not only provides deeper insight into CRISPR/Cas12a but also may facilitate its expanded application and power the exploration of the undiscovered properties of other CRISPR/Cas systems.

Comprehensive Genome Characterization of Childhood T-ALL Links Oncogene Activation Mechanism and Subtypes to Prognosis

Background: Children with relapsed T-cell acute lymphoblastic leukemia (T-ALL) have poor prognosis, and identification of patients at risk for recurrence is required to prevent relapse. Prior genomic studies of T-ALL have failed to identify genetic alterations that are prognostic independent of minimal residual disease (MRD), in part due to limited cohort size, exclusion of patients with refractory disease, and lack of comprehensive, integrated genome and transcriptome sequencing, which is important as many T-ALL drivers occur in non-coding regions of the genome. Aim: To identify all coding and non-coding alterations and identify genomic predictors of relapse or refractory disease. Methods: We performed whole genome, exome, and transcriptome sequencing of 1,313 cases enrolled on the Children's Oncology Group AALL0434 trial of childhood T-ALL. ATAC-seq and HiChIP were used to investigate the consequences of non-coding mutations and structural variants (SV). We used transcriptome profiling by Uniform Manifold Approximation and Projection (UMAP), Leiden algorithm clustering, hierarchical clustering of oncogene expression, and analysis of sequence and structural DNA variants to identify subtypes of T-ALL cases, their driver and associated genomic alterations. Results: Using integrative genomic analysis, we identified the clonal subtype-defining putative drivers in 94% of samples, 60% of which were non-coding regions and required WGS for identification in 28% of cases. We identified 16 T-ALL subtypes, several of which were previously unrecognized and involved partitioning of known subgroups into groups with distinct gene expression and driver alterations. We identified 4 subtypes with deregulation of the TAL1/LMO1/LMO2 core transcriptional circuitry: TAL1-RA, TAL1-RB, TAL1-RB/RPL10, LMO2-refractory (15.5%, 20.4%, 2.8%, 0.9% of all cases). These subtypes were separated by the lower expression of T cell maturation genes CD4/CD8 and RAG in TAL1-RA and more mature double positive TAL1-RB. TAL1-RA was characterized by multiple activation mechanisms for TAL1, LMO1 and/or LMO2; and TAL1-RB with alterations of TAL1, LMO1, LMO2,LYL1 and, TAL2 including rearrangements of these genes to TCR enhancers, chimeric fusion oncoproteins and a diverse range of enhancer hijacking, amplification and SNV events. Activation mechanisms differed between the groups; TAL1-RA commonly had STIL::TAL1 fusion with frequently co-occurring LMO2/LMO1 SNV/Indels or LMO2 enhancer gains, whereas TAL1-RB had frequent TAL1/LMO2/TAL2/LYL1 TCR rearrangements, TAL1 enhancer gains or various other TAL1 rearrangements. Two additional subgroups included 39 cases with TAL1/LMO2 activation and RPL10 mutations (TAL1-RB/RPL10) and the other characterized by refractory disease (LMO2-refractory, day 29 MRD >5%, 8 cases) with deregulation of LMO2 by hijacking of the BCL11B enhancer. We observed striking association between early T-cell precursor phenotype and driver lesions, with several groups highly enriched for ETP-ALL cases. One group (11.5% of all cases, 41.9% of ETP ALL cases, Fig. 1A) had distinct gene expression and deregulation of HOXA13 by SVs, ZFP36L2 or ETV6 by chimeric fusions and MED12 by SNVs. A second group (7.3% cases, 30.4% ETP) had HOXA9 deregulation by HOXA9 SVs or NUP98/NUP214/KMT2/MLLT10 chimeric fusions. Selective HOXA13 deregulation could be explained by genomic breakpoints occurring in different chromatin compartments than rearrangements deregulating other HOXA genes such as HOXA9. Novel HOXA13 SVs include rearrangements to MIR181A1HG and MED13 loci. Event-free survival (EFS) varied by subtype (Fig1B. log rank P<0.0001). Adverse risk subtypes were SPI1-R (P<0.0001, subtype vs. rest of samples), HOXA9 (P<0.0001), LMO2-refractory (P<0.0001) and favorable risk TAL1-RB/RPL10 (P=0.046), NKX2-1 (P=0.0099), TLX1 (P=0.013), TLX3 (P=0.028) and KMT2A-R (P=0.003) subtypes (Fig. 1B). Outcome was associated with key oncogenic pathways: for example, absence of Notch pathway alterations was associated with poor outcome if D29 MRD≥0.01% (5-year EFS <75%), whereas, patients with Notch alterations had good outcome regardless of Day 29 MRD. Conclusion: Comprehensive definition of the genomic landscape of T-ALL requires integrated WGS and RNAseq, and identifies multiple subgroups and driver lesions associated with immunophenotype and outcome. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Multiple activation mechanisms of CaO-based sorbents promoted by pre-sintering-hydration

Highly reactive CaO sorbents based on natural limestone with enhanced stability and re-usability were obtained via a pre-sintering-hydration method. It was found that several active mechanisms including generation of oxygen vacancies, promotion of ion replacement and improvement in pore structure were involved during the process of pre-sintering-hydration. The limestone-based sorbents obtained at a pre-sintering temperature of 1100 °C showed the highest CO2 capture capacity of 0.71 g-CO2 g−1 in the first cycle with a specific surface area of 28.65 m2 g−1. Electron spin-resonance analysis and high-resolution transmission electron microscopy demonstrated the existence of stress-induced oxygen vacancies and the promoting effect of hydration on ion replacement. The CO2 capture capacity of sample Ca-1100Mg-1:7 can still maintain at 0.47 g-CO2 g−1 at the 30th cycle, which was 2.8 times that of Ca-lime. After every 30 cycles, the recovered CO2 capture capacity of sample Ca-1100Mg-1:7 can achieve about 96.9 % of its initial valve. The improved stability and re-usability of the prepared sorbents can be attributed to the combined action of multiple active mechanisms introduced by pre-sintering-hydration treatment.

Multiple Mechanisms of NOTCH1 Activation in Chronic Lymphocytic Leukemia: NOTCH1 Mutations and Beyond.

Simple SummaryMutations of the NOTCH1 gene are a validated prognostic marker in chronic lymphocytic leukemia and a potential predictive marker for anti-CD20-based therapies. At present, the most frequent pathological alteration of the NOTCH1 gene is due to somatic genetic mutations, which have a multifaceted functional impact. However, beside NOTCH1 mutations, other factors may lead to activation of the NOTCH1 pathway, and these include mutations of FBXW7, MED12, SPEN, SF3B1 as well as other B-cell pathways. Understanding the preferential strategies though which CLL cells hijack NOTCH1 signaling may present important clues for designing targeted treatment strategies for the management of CLL.The Notch signaling pathway plays a fundamental role for the terminal differentiation of multiple cell types, including B and T lymphocytes. The Notch receptors are transmembrane proteins that, upon ligand engagement, undergo multiple processing steps that ultimately release their intracytoplasmic portion. The activated protein ultimately operates as a nuclear transcriptional co-factor, whose stability is finely regulated. The Notch pathway has gained growing attention in chronic lymphocytic leukemia (CLL) because of the high rate of somatic mutations of the NOTCH1 gene. In CLL, NOTCH1 mutations represent a validated prognostic marker and a potential predictive marker for anti-CD20-based therapies, as pathological alterations of the Notch pathway can provide significant growth and survival advantage to neoplastic clone. However, beside NOTCH1 mutation, other events have been demonstrated to perturb the Notch pathway, namely somatic mutations of upstream, or even apparently unrelated, proteins such as FBXW7, MED12, SPEN, SF3B1, as well as physiological signals from other pathways such as the B-cell receptor. Here we review these mechanisms of activation of the NOTCH1 pathway in the context of CLL; the resulting picture highlights how multiple different mechanisms, that might occur under specific genomic, phenotypic and microenvironmental contexts, ultimately result in the same search for proliferative and survival advantages (through activation of MYC), as well as immune escape and therapy evasion (from anti-CD20 biological therapies). Understanding the preferential strategies through which CLL cells hijack NOTCH1 signaling may present important clues for designing targeted treatment strategies for the management of CLL.

TERT Expression in Wilms Tumor Is Regulated by Promoter Mutation or Hypermethylation, WT1, and N-MYC.

Simple SummaryThe telomerase enzyme adds repetitive genetic sequences to the ends of chromosomes called telomeres to prevent cellular senescence. Gain of telomerase function is one of the hallmarks of human cancer. The telomerase protein is coded by the gene TERT and increased TERT RNA levels have been associated with disease relapse in Wilms tumor, the most common kidney cancer of childhood. This study aimed to determine the mechanisms of increased TERT expression in Wilms tumor. This study found mutations in the TERT promoter, increased methylation of the TERT promoter, and genomic copy number amplifications of TERT as potential mechanisms of TERT activation. Conversely, this study found that inactivating WT1 mutation was associated with low TERT RNA levels and telomerase activity. N-MYC overexpression in Wilms tumor cells resulted in increased TERT promoter activity and TERT transcription. TERT transcription is associated with molecular and histologic subgroups in Wilms tumor and telomere-targeted therapies warrant future investigation.Increased TERT mRNA is associated with disease relapse in favorable histology Wilms tumor (WT). This study sought to understand the mechanism of increased TERT expression by determining the association between TERT and WT1 and N-MYC, two proteins important in Wilms tumor pathogenesis that have been shown to regulate TERT expression. Three out of 45 (6.7%) WTs and the corresponding patient-derived xenografts harbored canonical gain-of-function mutations in the TERT promoter. This study identified near ubiquitous hypermethylation of the TERT promoter region in WT compared to normal kidney. WTs with biallelic inactivating mutations in WT1 (7/45, 15.6%) were found to have lower TERT expression by RNA-seq and qRT-PCR and lower telomerase activity determined by the telomerase repeat amplification protocol. Anaplastic histology and increased percentage of blastema were positively correlated with higher TERT expression and telomerase activity. In vitro shRNA knockdown of WT1 resulted in decreased expression of TERT, reduced colony formation, and decreased proliferation of WiT49, an anaplastic WT cell line with wild-type WT1. CRISPR-Cas9-mediated knockout of WT1 resulted in decreased expression of telomere-related gene pathways. However, an inducible Wt1-knockout mouse model showed no relationship between Wt1 knockout and Tert expression in normal murine nephrogenesis, suggesting that WT1 and TERT are coupled in transformed cells but not in normal kidney tissues. N-MYC overexpression resulted in increased TERT promoter activity and TERT transcription. Thus, multiple mechanisms of TERT activation are involved in WT and are associated with anaplastic histology and increased blastema. This study is novel because it identifies potential mechanisms of TERT activation in Wilms tumor that could be of therapeutic interests.

A multiscale indentation-based technique to correlate acoustic emission with deformation mechanisms in complex alloys

Conventional methodologies to link damage evolution with the activation of deformation mechanisms typically require destructive testing and post-mortem analysis. More recently, mechanical testing combined with acoustic emission analysis has provided a method to fingerprint multiple active deformation mechanisms. However, correlating different acoustic emission signals with active deformation sources remains a non-trivial task due to the simultaneous activation of sources during mechanical testing. Here, we demonstrate a multiscale indentation-based technique to isolate dislocation and martensitic transformation sources in the acoustic emission data of a complex alloy. For this purpose, the acoustic emission signals from macro- and microindentation experiments are compared with microscopy and crystallographic analysis of deformed microstructures. A key feature of this method is the selective activation of the martensitic transformation during microindentation, which enables separate fingerprinting of this deformation mechanism. An unsupervised k mean clustering methodology is leveraged to classify acoustic emission waveforms into clusters, which facilitates the detection of shared deformation mechanisms across different tests. The results of this study provide a rapid non-destructive tool to correlate acoustic emission sources with deformation mechanisms in complex alloys.

A Network Pharmacology-Based Approach for Exploring Key Active Compounds and Pharmacological Mechanisms of Tangshen Formula for Treatment of Diabetic Nephropathy

Diabetic nephropathy (DN) is one of the common and severe microvascular complications of diabetes mellitus (DM). The occurrence and development of DN are related to multiple factors in the human body, which makes DN a complex disease, and the pathogeneses of DN have not yet been fully illustrated. Furthermore, DN lacks effective drugs for treatment nowadays. Chinese herbal medicine (CHM) often shows the feature of multicomponents, multitargets, multipathways, and synergistic effects and shows a promising source of new therapeutic drugs for DN. As a CHM, Tangshen Formula (TSF) was used to treat DN patients in China. However, its bioactive compounds and holistic pharmacological mechanisms on DN are both unclear. A network pharmacology approach was firstly applied to explore multiple active compounds and multiple key pharmacological mechanisms for TSF treating DN by drug-targeted interaction databases, herb-compound-target network, protein-protein interaction network, compound-target-pathway network, and analysis methods. And the results showed that TSF have the characteristic of multicomponents, multitargets, multipathways, and synergistic effects for treating DN. The quercetin, naringenin, kaempferol, and isorhamnetin as key active compounds and the PI3K-Akt signaling pathway, TNF signaling pathway, nonalcoholic fatty liver disease (NAFLD), focal adhesion, rap1 signaling pathway, T cell receptor signaling pathway, MAPK signaling pathway, and insulin resistance as the key molecular mechanisms play important roles in TSF treating DN. Moreover, quercetin, naringenin, kaempferol, and isorhamnetin were successfully detected in TSF by the UHPLC-MS/MS analysis method. And their concentrations were 0.224, 8.295, 0.0564, and 0.0879 mg·kg-1, respectively. The present findings not only provide new insights for a deeper understanding of the constituent basis and pharmacology of TSF but also provide guidance for further pharmacological studies on TSF.

Myofibroblast TGF-β Activation Measurement In Vitro.

Myofibroblasts are critical to processes involved in normal wound healing and during pathological fibrosis. They transdifferentiate from fibroblasts, and in doing so become contractile and capable of secreting large amounts of extracellular matrix proteins. Transforming growth factor-beta (TGFβ) is a key cytokine involved in wound healing and fibrogenesis. TGFβ signaling has long been the subject of experimental therapeutic approaches to inhibit fibrosis in a variety of organ systems. Inhibition of TGFβ can reduce myofibroblast transdifferentiation, contractility, and matrix production. Importantly, TGFβ is released from cells and sequestered in the extracellular matrix in a latent form that requires activation for biological function. There have been multiple mechanisms of TGFβ activation described in a variety of cell types and in cell free systems; however, myofibroblasts have previously been shown to activate TGFβ via cell surface integrins, particularly αvβ5 integrins. This chapter will provide detailed protocols for accurately measuring activation of TGFβ by myofibroblasts in vitro. Levels of active TGFβ usually represent a small proportion of the total amount of latent TGFβ present in the matrix. Methods to measure active TGFβ therefore need to be sensitive and specific to detect the active cytokine only.

Computational Modeling of NLRP3 Identifies Enhanced ATP Binding and Multimerization in Cryopyrin-Associated Periodic Syndromes

Cyropyrin-associated periodic syndromes (CAPS) are clinically distinct syndromes that encompass a phenotypic spectrum yet are caused by alterations in the same gene, NLRP3. Many CAPS cases and other NLRP3-autoinflammatory diseases (NLRP3-AIDs) are directly attributed to protein-coding alterations in NLRP3 and the subsequent dysregulation of the NLRP3 inflammasome leading to IL-1β-mediated inflammatory states. Here, we used bioinformatics tools, computational modeling, and computational assessments to explore the proteomic consequences of NLRP3 mutations, which potentially drive NLRP3 inflammasome dysregulation. We analyzed 177 mutations derived from familial cold autoinflammatory syndrome (FCAS), Muckle-Wells Syndrome (MWS), and the non-hereditary chronic infantile neurologic cutaneous and articular syndrome, also known as neonatal-onset multisystem inflammatory disease (CINCA/NOMID), as well as other NLRP3-AIDs. We found an inverse relationship between clinical severity and the severity of predicted structure changes resulting from mutations in NLRP3. Bioinformatics tools and computational modeling revealed that NLRP3 mutations that are predicted to be structurally severely-disruptive localize around the ATP binding pocket and that specific proteo-structural changes to the ATP binding pocket lead to enhanced ATP binding affinity by altering hydrogen-bond and charge interactions. Furthermore, we demonstrated that NLRP3 mutations that are predicted to be structurally mildly- or moderately-disruptive affect protein-protein interactions, such as NLRP3-ASC binding and NLRP3-NLRP3 multimerization, enhancing inflammasome formation and complex stability. Taken together, we provide evidence that proteo-structural mechanisms can explain multiple mechanisms of inflammasome activation in NLRP3-AID.

Abstract 15888: Platelet Activation is Associated With All-cause Mortality in Patients With Covid-19

Background: COVID-19 is a global pandemic with patients at increased risk for all-cause mortality. Virus-platelet interactions are linked to viral pathogenesis and increased risk of adverse events. Aim: To investigate the relationship between in vivo platelet activity markers and all-cause mortality in hospitalized patients with COVID-19. Method: Plasma samples were collected from 100 hospitalized patients on the day of PCR-confirmed COVID-19 diagnosis. Thromboxane B 2 (TxB 2 ), P-selectin, and soluble CD40 ligand (sCD40L) were measured in plasma, and mean platelet volume (MPV) assessed. Subjects were followed until discharge or death. Results: Among 100 patients, the median age was 65 years (IQR: 55, 75), 39% were female, and 32 died or experienced a thrombotic event. The baseline platelet activation markers, P-selectin (p=0.02), sCD40L (p=0.03) and MPV (p=0.005) were higher in patients who died. After adjustment for age, sex, race/ethnicity, platelet count, antiplatelet therapy, and chronic obstructive pulmonary disease, TxB 2 (p=0.036), P-selectin (p=0.007), sCD40L (p=0.02) and MPV (p=0.01) were each independently associated with death after multivariable adjustment ( Table 1 ). Conclusions: Biomarkers of platelet activation are significantly associated with death or thrombosis in patients hospitalized with COVID-19. Our findings suggest multiple platelet activation mechanisms may contribute to adverse events. Further investigation into the mechanistic role of platelets in COVID-19 pathogenesis and the potential role of antiplatelet therapy is warranted. Table 1. Multivariable regression models of all-cause mortality. Odds ratios (OR) from logistic regression analysis per SD increase for biomarker levels adjusted for age, sex, race, antiplatelet therapy, platelet count, and chronic obstructive pulmonary disease (COPD).

The Cellular and Molecular Determinants of Naphthoquinone-Dependent Activation of the Aryl Hydrocarbon Receptor.

1,2-naphthoquinone (1,2-NQ) and 1,4-naphthoquinone (1,4-NQ) are clinically promising biologically active chemicals that have been shown to stimulate the aryl hydrocarbon receptor (AhR) signaling pathway, but whether they are direct or indirect ligands or activate the AhR in a ligand-independent manner is unknown. Given the structural diversity of AhR ligands, multiple mechanisms of AhR activation of gene expression, and species differences in AhR ligand binding and response, we examined the ability of 1,2-NQ and 1,4-NQ to bind to and activate the mouse and human AhRs using a series of in vitro AhR-specific bioassays and in silico modeling techniques. Both NQs induced AhR-dependent gene expression in mouse and human hepatoma cells, but were more potent and efficacious in human cells. 1,2-NQ and 1,4-NQ stimulated AhR transformation and DNA binding in vitro and was inhibited by AhR antagonists. Ligand binding analysis confirmed the ability of 1,2-NQ and 1,4-NQ to competitively bind to the AhR ligand binding cavity and the molecular determinants for interactions were predicted by molecular modeling methods. NQs were shown to bind distinctly differently from that of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and differences were also observed between species. Mutation of amino acid residues (F289, M334, and M342) involved in critical NQ:AhR binding interactions, decreased NQ- and AhR-dependent gene expression, consistent with a role for these residues in binding and activation of the AhR by NQs. These studies provide insights into the molecular mechanism of action of NQs and contribute to the development of emerging NQ-based therapeutics.

Genomic Profiling of Biliary Tract Cancer Cell Lines Reveals Molecular Subtypes and Actionable Drug Targets.

SummaryBiliary tract cancers (BTCs) currently have no approved targeted therapies. Although genomic profiling of primary BTCs has identified multiple potential drug targets, accurate models are needed for their evaluation. Genomic profiling of 22 BTC cell lines revealed they harbor similar mutational signatures, recurrently mutated genes, and genomic alterations to primary tumors. Transcriptomic profiling identified two major subtypes, enriched for epithelial and mesenchymal genes, which were also evident in patient-derived organoids and primary tumors. Interrogating these models revealed multiple mechanisms of MAPK signaling activation in BTC, including co-occurrence of low-activity BRAF and MEK mutations with receptor tyrosine kinase overexpression. Finally, BTC cell lines with altered ERBB2 or FGFRs were exquisitely sensitive to specific targeted agents, whereas surprisingly, IDH1-mutant lines did not respond to IDH1 inhibitors in vitro. These findings establish BTC cell lines as robust models of primary disease, reveal specific molecular disease subsets, and highlight specific molecular vulnerabilities in these cancers.

An Overview of Factors Affecting Renal Clearance

Biological Renal clearance comprises multiple active and passive mechanisms, in turn leading to the formation of urine. In this article, a brief review of passive filtration in glomerular capillaries, active tubular secretion, and reabsorption is discussed. Factors affecting renal clearance have also been brought into the discussion, encompassing drug physicochemical properties, drug concentration, the volume of distribution, protein binding, blood flow to kidneys, biological factors, and drug interactions. Further, the necessity of dose adjustment in patients suffering renal impairment has been highlighted.

The effect of low- and high-penetration light on localized cancer therapy

The design of a delivery system allowing targeted and controlled drug release has been considered one of the main strategies used to provide individualized cancer therapy, to improve survival statistics, and to enhance quality-of-life. External stimuli including low- and high-penetration light have been shown to have the ability to turn drug delivery on and off in a non-invasive remotely-controlled fashion. The success of this approach has been closely related to the development of a variety of drug delivery systems - from photosensitive liposomes to gold nanocages - and relies on multiple mechanisms of drug release activation. In this review, we make reference to the two extremes of the light spectrum and their potential as triggers for the delivery of antitumor drugs, along with the most recent achievements in preclinical trials and the challenges to an efficient translation of this technology to the clinical setting.

Abstract 1507: Multiple mechanisms of Myb gene activation revealed through transcriptome analysis of salivary gland adenoid cystic carcinoma

Abstract Adenoid cystic carcinoma (ACC) of the salivary gland is a rare head and neck malignancy with poor long-term prognosis. Hallmark chromosomal translocations result in MYB to NFIB (t(6;9)) and MYBL1 to NFIB (t(8;9)) gene fusions. Gene rearrangement and aberrant expression of the MYB and MYBL1 genes, along with the encoded transcription factors, are the oncogenic driver in the majority of ACC tumors. Previous RNA-sequencing (RNA-seq) data collected from FFPE fixed ACC tumors illustrated MYB and MYBL1 elicit similar gene expression profiles in ACC, suggesting these transcription factors drive ACC oncogenes via similar mechanisms. Using these RNA-seq data we have identified EN1 and SOX4 as important Myb regulated genes and demonstrated activation of their promoters in reporter gene assays. We hope to utilize these reporters in high-throughput screens to identify new drug candidates for ACC. We have utilized RNA-seq data to identify an additional mechanism leading to MYB overexpression that appears unique to ACC tumors. In these tumors transcription of the MYB gene is aberrantly activated through use of an alternative promoter located within the first intron of the gene. Transcripts originating from this promoter have been confirmed in an ACC tumor via 5'RACE and the resulting Myb protein has a small N-terminal truncation eliminating the epitope recognized by a common Myb antibody. The alternative promoter is active and regulated in a cell type specific manner in reporter gene assays. Previous studies have shown chromosomal translocations juxtapose NFIB enhancers to activate MYB gene transcription. ChIP and 3C assays in fresh frozen ACC tumors will be utilized to test the hypothesis that NFIB enhancers are specifically recruited to the alternative MYB promoter, thereby activating gene transcription. Thus, multiple mechanisms lead to the aberrant over-expression and truncation of MYB and drives ACC oncogenesis. Citation Format: Candace Frerich, Kathryn Brayer, Brandon Painter, Scott Ness. Multiple mechanisms of Myb gene activation revealed through transcriptome analysis of salivary gland adenoid cystic carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1507.

Hedgehog pathway and smoothened inhibitors in cancer therapies.

The hedgehog (Hh) pathway plays an important role in cancer development and maintenance, as ~25% of all cancers have aberrant Hh pathway activation. Targeted therapy for inhibition of the Hh pathway was thought to be promising for achieving clinical response in the Hh-dependent cancers. However, the results of new clinical trials with smoothened (SMO) antagonists do not show much success in cancers other than basal cell carcinoma. The studies suggest that the Hh pathway involves multiple mechanisms of activation or inhibition in primary cilia and interactions between several related pathways in different types of cells, which makes this pathway extremely complex. The SMO-specific antagonists may not stop all relevant pathways that may lead to escape or development of resistance. Therefore, in the Hh-dependent cancers, the inhibition of two or more oncogenic pathways (including the Hh pathway) with use of a single agent of a suitable multitarget profile or a combination of drugs seems promising for achieving clinical response in patients and decrease in resistance development with prolonged use of the specific SMO antagonists. Furthermore, for studying the effect of new treatments, the inclusion criteria should be more specific for selection of patients with aberrant Hh pathway activity confirmed by tests. These considerations will be very helpful for choosing the right patients and the right drugs for the best therapeutic outcome.

Crocodylian nuclear factor kappa B.

We deduced the amino acid (aa) sequence of the nuclear factor kappa B (NFκB) protein from genomic data for the American alligator (Alligator mississippiensis), the estuarine crocodile (Crocodylus porosus), and the Indian gharial (Gavialis gangeticus). A 105kDa protein, NFκB1 exhibits complex post-translational processing, multiple mechanisms of activation, and acts as precursor for a p50, a Rel homology transcription factor which influences the expression of key genes for developmental processes, apoptosis, and immune function. The aa sequences of the crocodylian proteins share very high identity with each other (97.2±0.7%), birds (81.0±1.1%, n=6), mammals (75.3±1.6%, n=4), reptiles (80.3±5.1%, n=2), and less identity with fish (55.5±5.5%, n=4) and one amphibian (66.1±0.8%). The crocodylian protein has a well-conserved Rel homology domain, a nuclear localization signal, and a glycine-rich region which facilitates proteasome-mediated generation of p50. The Rel homology domain contains sequences responsible for dimerization, DNA-binding, and nuclear translocation. In addition, seven ankyrin repeats were located, which putatively allow for inhibition of transcriptional regulation by mediating interaction with Inhibitor kappa B. Other features include a death domain, and conserved serine residues, near the C-terminal end, which act as potential phosphorylation sites for activation of the proteolytic generation of p50. Western blot analysis showed both the 105kDa precursor and the 50kDa mature NFκB were expressed in the alligator liver. Nuclear factor κB exhibited diffuse cytoplasmic distribution in alligator hepatocytes, and almost no cytoplasmic localization in infected animals. In addition, nuclear NFκB exhibited specific binding to the consensus NFκB promoter element.