Fluorescence Polarization Assay Research Articles

Abstract 3051: JunAP, a peptide antagonist against the activator protein 1 transcription factor complex, demonstrates cancer cell cytotoxicity and reduced invasion in vitro and tumor regression in vivo in TNBC models

Abstract The activator protein 1 (AP-1) transcription factor is a dimeric complex consisting of proteins from the JUN, FOS, MAF, and ATF families. AP-1 complexes have been identified as novel therapeutic targets in cancer due to their role in tumor growth, invasion, metastasis, angiogenesis and chemoresistance. Dimerization of components of the AP-1 complex via leucine zipper domain interactions is required for DNA binding and subsequent transcriptional activity. We designed a peptide (Jun antagonizing peptide, JunAP) targeting the basic leucine zipper motif of AP-1 to antagonize AP-1 complex formation and prevent associated activity. Fluorescence polarization and DNA ELISA assays demonstrate target binding and selectivity of JunAP towards AP-1 family members, co-immunoprecipitation experiments reveal that JunAP blocks protein-protein interactions between cJun and its key binding partners, and reporter assays confirm inhibition of AP-1 transcriptional activity in vitro. Pathway analysis of RNA sequencing data confirmed by qPCR analysis shows that JunAP disrupts key pathways required for cell survival and migration. Corresponding functional in vitro assays reveal that JunAP exhibits an inhibitory effect on invasion in Boyden chamber assays and demonstrates potent cytotoxicity in Jun-dependent cells. Further, in a BT549 triple negative breast cancer subcutaneous xenograft model, administration of JunAP results in tumor regression, demonstrating the anti-cancer potential of targeting the AP-1 complex. In summary, these data support JunAP as a potent peptide antagonist of the AP-1 transcription factor family that warrants further development as a potential therapeutic option for AP-1 driven tumors. Citation Format: Karen Mendelson, Zachary F. Mattes, Siok Leong, Ricardo Ramirez, Mark Koester, Claudio Scuoppo, Julia Diehl, Erin Gallagher, Jerel Gonzales, Franco Abbate, Lila Ghamsari, Gene Merutka, Barry J. Kappel, Abi Vainstein-Haras, Jim A. Rotolo. JunAP, a peptide antagonist against the activator protein 1 transcription factor complex, demonstrates cancer cell cytotoxicity and reduced invasion in vitro and tumor regression in vivo in TNBC models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3051.

A structural basis for the activation of peroxisome proliferator-activated receptor gamma (PPARγ) by perfluorooctanoic acid (PFOA)

Perfluorooctanoic acid (PFOA) is a widespread environmental pollutant of the perfluoroalkyl substance (PFAS) class that is extremely resistant to environmental and metabolic degradation, leading to bioaccumulation. PFOA exposure has been linked to many health effects including endocrine disruption and metabolic dysregulation, but our understanding of the molecular mechanisms resulting in these outcomes remains incomplete. One target affected by PFOA is the ligand regulated nuclear receptor peroxisome proliferator-activated receptor gamma (PPARγ) which plays a critical role in controlling metabolic homeostasis through regulating processes such as adipogenesis, glucose homeostasis, inflammation and osteogenesis. It has been previously established that PFOA activates PPARγ through binding to the PPARγ ligand binding domain (PPARγ LBD) leading to increased expression of PPARγ controlled target genes. However, the mechanism by which PFOA achieves this has remained elusive. Here, we employed a combination of X-ray crystallography and fluorescence polarization assays to provide a structural basis for PFOA mediated activation of PPARγ via binding to the PPARγ LBD. Using X-ray crystallography, the cocrystal structure of the PPARγ LBD:PFOA complex was solved. This revealed that PFOA occupies three distinct sites, two within the PPARγ LBD and one within the activation function 2 (AF2) on the protein surface. Structural comparison of PFOA binding with previously reported PPARγ:ligand complexes supports that PFOA activates PPARγ by a partial agonist mechanism at micromolar concentrations. Fluorescence polarization assays also revealed that PFOA binding to the AF2 is unlikely to occur in a cellular context and confirmed that PFOA behaves as a partial agonist in vitro, weakly recruiting a coactivator peptide to the AF2 of the PPARγ LBD. This discovery provides an advancement in understanding PFOA mediated regulation of PPARγ, giving new insight regarding regulation of PPARγ by PFAS and PFAS substitutes in general and can be applied to the design and assessment of safer PFAS.

Discovery of a Potent and Cell-Active Inhibitor of DNA 6mA Demethylase ALKBH1.

N6-Methyladenine (6mA) of DNA has emerged as a novel epigenetic mark in eukaryotes, and several 6mA effector proteins have been identified. However, efforts to selectively inhibit the biological functions of these effector proteins with small molecules are unsuccessful to date. Here we report the first potent and selective small molecule inhibitor (13h) of AlkB homologue 1 (ALKBH1), the only validated 6mA demethylase. 13h showed an IC50 of 0.026 ± 0.013 μM and 1.39 ± 0.13 μM in the fluorescence polarization (FP) and enzyme activity assay, respectively, and a KD of 0.112 ± 0.017 μM in the isothermal titration calorimetry (ITC) assay. The potency of 13h was well explained by the cocrystal structure of the 13h-ALKBH1 complex. Furthermore, 13h displayed excellent selectivity for ALKBH1. In cells, compound 13h and its derivative 16 were able to engage ALKBH1 and modulate the 6mA levels. Collectively, our study identified the first potent, isoform selective, and cell-active ALKBH1 inhibitor, providing a tool compound for exploring the biological functions of ALKBH1 and DNA 6mA.

Elucidating the Signal Transduction Mechanism of the Blue-Light-Regulated Photoreceptor YtvA: From Photoactivation to Downstream Regulation.

The blue-light photoreceptor YtvA from Bacillus subtilis has an N-terminal flavin mononucleotide (FMN)-binding light-oxygen-voltage (LOV) domain that is fused to a C-terminal sulfate transporter and anti-σ factor antagonist (STAS) output domain. To interrogate the signal transduction pathway that leads to photoactivation, the STAS domain was replaced with a histidine kinase, so that photoexcitation of the flavin could be directly correlated with biological activity. N94, a conserved Asn that is hydrogen bonded to the FMN C2═O group, was replaced with Ala, Asp, and Ser residues to explore the role of this residue in triggering the structural dynamics that activate the output domain. Femtosecond to millisecond time-resolved multiple probe spectroscopy coupled with a fluorescence polarization assay revealed that the loss of the hydrogen bond between N94 and the C2═O group decoupled changes in the protein structure from photoexcitation. In addition, alterations in N94 also decreased the stability of the Cys-FMN adduct formed in the light-activated state by up to a factor of ∼25. Collectively, these studies shed light on the role of the hydrogen bonding network in the LOV β-scaffold in signal transduction.

Involvement of the Keap1-Nrf2-ARE pathway in the antioxidant activity of sinomenine

Sinomenine is a pure alkaloid isolated from Sinomenium acutum. This study is aimed to investigate the critical role of the nuclear factor erythroid 2-related factor 2 (Nrf2)-kelch-like ECH-associated protein-1(Keap1)-antioxidant response element (ARE) antioxidative signaling pathway in protecting sinomenine against H2O2-induced oxidative injury. Cytotoxicity and antioxidant experiments to initially determine the protective effects of sinomenine show that sinomenine has no effect on the decreased cell viability and presents similar potency in scavenging all three free radicals. The binding affinity between sinomenine and Keap1 was determined via fluorescence polarization assay, with IC50 of 13.52 μM. Quantum chemical calculation and theoretical simulation illustrated that sinomenine located into the Nrf2-binding site of Keap1 via hydrophobic and hydrogen interactions, showing high stability and binding affinity. On the basis of the stable binding of sinomenine with Keap1, sinomenine efficiently induced nuclear translocation of Nrf2, and increased in ARE activity in a concentration-dependent manner. Quantitative polymerase chain reaction provided further evidences that sinomenine-induced protection upregulated ARE-dependent genes, such as NAD(P)H quinone oxidoreductase 1, hemeoxygenase-1, and glutamate-cysteine ligase modifier subunit. Western blot confirmed that sinomenine increased the expressions of these antioxidative enzymes. Taken together, in vitro and in silico evaluations demonstrate that sinomenine inhibits the binding of Keap1 to Nrf2, promotes the nuclear accumulation of Nrf2 and thus leads to the upregulated expressions of Nrf2-dependent antioxidative genes. Our findings also highlight the use of sinomenine for pharmacological or therapeutic regulation of the Nrf2-Keap1-ARE system, which is a novel strategy to prevent the progression of oxidative injury.

Fluorescence Polarization Assays for Organic Compounds in Food Safety.

Elevated concentrations of toxic organic compounds observed in food products pose serious dangers to human health. Both natural and artificial pollutants can cause food contamination. The stages of food production, packaging, transportation, and storage can also largely cause the appearance of undesirable substances in food products. The health consequences of ingesting food containing toxic contaminants range from mild gastroenteritis to deaths resulting from dysfunctional internal organs and neurological syndromes. The World Health Organization (WHO) sets recommendations for the content of such chemicals in food, including a minimum allowable concentration considered safe for human consumption. However, the control of food products from chemical pollutants is necessary. Moreover, fast, sensitive, and inexpensive methods are needed to detect them at the point of need. Currently, immune analysis methods are most widely used to determine pollutants in food. The development of fluorescence polarization immunoassay (FPIA) methods in a competitive format is a powerful and modern tool for detecting organic molecules in various matrices, thereby making FPIA methods useful for food safety applications. Due to the availability of portable devices for measuring the fluorescence polarization signal, FPIA methods can be used at the point of need. The variety of fluorescent labels and recognizing elements (receptors, monoclonal and polyclonal antibodies, and nanobodies) permits fluorescence polarization (FP) assays to detect significantly lower limits of organic substances. The FP assay is a homogeneous, fast, and quantitative method. The development of various formats of FP assays makes them promising in determining food pollutants. This review summarizes publications on FP analyses for detecting organic contaminants (pesticides, hormones, toxins, antibiotics, and other pharmaceuticals) in food products during 2018-2023. Further, it demonstrates the prospects for using this method to determine pollutants at the point of need and for detecting high molecular weight substances, fungi, and bacterial infections during food safety inspections.

Structural and functional characterization of siadenovirus core protein VII nuclear localization demonstrates the existence of multiple nuclear transport pathways.

Adenovirus protein VII (pVII) plays a crucial role in the nuclear localization of genomic DNA following viral infection and contains nuclear localization signal (NLS) sequences for the importin (IMP)-mediated nuclear import pathway. However, functional analysis of pVII in adenoviruses to date has failed to fully determine the underlying mechanisms responsible for nuclear import of pVII. Therefore, in the present study, we extended our analysis by examining the nuclear trafficking of adenovirus pVII from a non-human species, psittacine siadenovirus F (PsSiAdV). We identified a putative classical (c)NLS at pVII residues 120-128 (120PGGFKRRRL128). Fluorescence polarization and electrophoretic mobility shift assays demonstrated direct, high-affinity interaction with both IMPα2 and IMPα3 but not IMPβ. Structural analysis of the pVII-NLS/IMPα2 complex confirmed a classical interaction, with the major binding site of IMPα occupied by K124 of pVII-NLS. Quantitative confocal laser scanning microscopy showed that PsSiAdV pVII-NLS can confer IMPα/β-dependent nuclear localization to GFP. PsSiAdV pVII also localized in the nucleus when expressed in the absence of other viral proteins. Importantly, in contrast to what has been reported for HAdV pVII, PsSiAdV pVII does not localize to the nucleolus. In addition, our study demonstrated that inhibition of the IMPα/β nuclear import pathway did not prevent PsSiAdV pVII nuclear targeting, indicating the existence of alternative pathways for nuclear localization, similar to what has been previously shown for human adenovirus pVII. Further examination of other potential NLS signals, characterization of alternative nuclear import pathways, and investigation of pVII nuclear targeting across different adenovirus species is recommended to fully elucidate the role of varying nuclear import pathways in the nuclear localization of pVII.

A novel Bayesian Latent Class Model (BLCM) evaluates multiple continuous and binary tests: A case study for Brucella abortus in dairy cattle

Bovine brucellosis, primarily caused by Brucella abortus, severely affects both animal health and human well-being. Accurate diagnosis is crucial for designing informed control and prevention measures. Lacking a gold standard test makes it challenging to determine optimal cut-off values and evaluate the diagnostic performance of tests. In this study, we developed a novel Bayesian Latent Class Model that integrates both binary and continuous testing outcomes, incorporating additional fixed (parity) and random (farm) effects, to calibrate optimal cut-off values by maximizing Youden Index. We tested 651 serum samples collected from six dairy farms in two regions of Henan Province, China with four serological tests: Rose Bengal Test, Serum Agglutination Test, Fluorescence Polarization Assay, and Competitive Enzyme-Linked Immunosorbent Assay. Our analysis revealed that the optimal cut-off values for FPA and C-ELISA were 94.2 mP and 0.403 PI, respectively. Sensitivity estimates for the four tests ranged from 69.7% to 89.9%, while specificity estimates varied between 97.1% and 99.6%. The true prevalences in the two study regions in Henan province were 4.7% and 30.3%. Parity-specific odds ratios for positive serological status ranged from 1.2 to 2.2 for different parity groups compared to primiparous cows. This approach provides a robust framework for validating diagnostic tests for both continuous and discrete tests in the absence of a gold standard test. Our findings can enhance our ability to design targeted disease detection strategies and implement effective control measures for brucellosis in Chinese dairy farms.

Comparison of the Glucocorticoid Receptor Binding and Agonist Activities of Typical Glucocorticoids: Insights into Their Endocrine Disrupting Effects.

Over the past decades, the synthetic glucocorticoids (GCs) have been widely used in clinical practice and animal husbandry. Given the health hazard of these toxic residues in food, it is necessary to explore the detailed interaction mechanisms of typical GCs and their main target glucocorticoid receptor (GR). Hence, this work compared the GR binding and agonist activities of typical GCs. Fluorescence polarization assay showed that these GCs were potent ligands of GR. Their GR binding affinities were in the order of methylprednisolone>betamethasone≈prednisolone>dexamethasone, with IC50 values of 1.67, 2.94, 2.95, and 5.58 nM. Additionally, the limits of detection of dexamethasone, betamethasone, prednisolone, and methylprednisolone were 0.32, 0.14, 0.19, and 0.09 μg/kg in fluorescence polarization assay. Reporter gene assay showed that these GCs induced GR transactivation in a dose-dependent manner, confirming their GR agonist activities. Among which, dexamethasone at the concentration of 100 nM produced a maximal induction of more than 11-fold over the blank control. Molecular docking and molecular dynamics simulations suggested that hydrogen-bonding and hydrophobic interactions played an important role in stabilizing the GC-GR-LBD complexes. In summary, this work might help to understand the GR-mediated endocrine disrupting effects of typical GCs.

Evaluation of natural products from virtual screenings as SARS-CoV-2 main protease inhibitors using combinational experiments

Recently, andrographolide, kaempferol, maslinic acid, rutin, and schaftoside have been identified as potent SARS-CoV-2 main protease (Mpro) inhibitors via molecular docking studies. However, no comprehensive in vitro testing of these compounds against Mpro has been conducted. In this study, we rigorously evaluated the in vitro inhibition of Mpro by these compounds using combinational experiments, including fluorescence resonance energy transfer (FRET), fluorescence polarization (FP), and dimerization-dependent red fluorescent protein (ddRFP) assays. Our data revealed that these compounds are not Mpro inhibitors based on the results from a set of in vitro assays. These results suggest that an efficient combination of a molecular docking approach and an experimental assay is essential for the discovery of Mpro inhibitors in the future.

Sensitivity-improved blocking agent-free fluorescence polarization assay through surface modification using polyethylene glycol.

Fluorescence polarization (FP) assays are widely used to quantify biomolecules, and their combination with microfluidic devices has the potential for application in onsite analysis. However, the hydrophobic surface of polydimethylsiloxane (PDMS)-based microfluidic devices and the amphiphilicity of the blocking agents can cause the nonspecific adsorption of biomolecules, which in turn reduces the sensitivity of the FP assay. To address this, we demonstrated an FP assay with improved sensitivity in microfluidic devices using a polyethylene glycol-based surface modification to avoid the use of blocking agents. We evaluated the effectiveness of the modification in inhibiting nonspecific protein adsorption and demonstrated the improved sensitivity of the FP immunoassay (FPIA). Our study addressed the lack of sensitivity of FP assays in microfluidic devices, particularly for the quantification of low-abundance analytes.

Fluorescence polarization assay for screening FANCM-RMI inhibitors to target the alternative lengthening of telomeres.

Alternative Lengthening of Telomeres (ALT) is a mechanism used by 10-15% of all cancers to achieve replicative immortality, bypassing the DNA damage checkpoint associated with short telomeres that leads to cellular senescence or apoptosis. ALT does not occur in non-cancerous cells, presenting a potential therapeutic window for cancers where this mechanism is active. Disrupting the FANCM-RMI interaction has emerged as a promising therapeutic strategy that induces synthetic ALT lethality in genetic studies on cancer cell lines. There are currently no chemical inhibitors reported in the literature, in part due to the lack of reliable biophysical or biochemical assays to screen for FANCM-RMI disruption. Here we describe the development of a robust competitive fluorescence polarization (FP) assay that quantifies target binding at the FANCM-RMI interface. The assay employs a labeled peptide tracer TMR-RaMM2 derived from the native MM2 binding motif, which binds to recombinant RMI1-RMI2 and can be displaced by competitive inhibitors. We report the methods for recombinant production of RMI1-RMI2, design and evaluation of the tracer TMR-RaMM2, along with unlabeled peptide inhibitor controls to enable ALT-targeted drug discovery.

High-Throughput Screening for LC3/GABARAP Binders Utilizing the Fluorescence Polarization Assay.

The characterization of interactions between autophagy modifiers (Atg8-family proteins) and their natural ligands (peptides and proteins) or small molecules is important for a detailed understanding of selective autophagy mechanisms and for the design of potential Atg8 inhibitors that affect the autophagy processes in cells. The fluorescence polarization (FP) assay is a rapid, cost-effective, and robust method that provides affinity and selectivity information for small molecules and peptide ligands targeting human Atg8 proteins.This chapter introduces the basic principles of FP assays. In addition, a case study on peptide interaction with human Atg8 proteins (LC3/GABARAPs) is described. Finally, data analysis and quality control of FP assaysare discussed for the proper calculation of Ki values for the measured compounds.

Discovery of GPX4 inhibitors through FP-based high-throughput screening

Ferroptosis is a form of non-apoptotic cell death, regulated by phospholipid hydroperoxide glutathione peroxidase 4 (GPX4), a selenoprotein with a selenocysteine residue (sec) in the active site. GPX4 is a promising target for cancer cells in therapy-resistant conditions via ferroptosis, which can reduce the level of lipid reactive oxygen species (ROS). So far, all existing GPX4 inhibitors covalently bind to GPX4 via a reactive alkyl chloride moiety or masked nitrile-oxide electrophiles with poor selectivity and pharmacokinetic properties and most were obtained by cell phenotype-based screening. Lacking of effective high-throughput screening methods for GPX4 protein limits the discovery of GPX4 inhibitors. Here, we report a fluorescence polarization (FP)-based high throughput screening (HTS) assay for GPX4-U46C-C10A-C66A in vitro, and found Metamizole sodium from our in-house compound library inhibits GPX4-U46C-C10A-C66A enzyme activity. Structure-activity relationships (SAR) demonstrated the importance of sulfonyl group on interaction between Metamizole sodium and GPX4-U46C-C10A-C66A. Our FP assay could be an effective tool for discovery of GPX4 inhibitors and Metamizole sodium was a potential inhibitor for GPX4 in vitro.

Design, synthesis, and biological evaluation of novel 8-substituted quercetin derivatives targeting the β‑catenin/B-cell lymphoma 9 interaction

The β-catenin/B-cell lymphoma 9 (BCL9) protein–protein interaction (PPI) is a potential target for aberrantly active Wnt/β-catenin signaling which actively participates in initiating and progressing of many cancers. Herein, we discovered novel 8-substituted quercetin derivatives with potential inhibitory activities targeting β-catenin/BCL9 PPI. Among all the derivatives, compound B4 displayed the most promising PPI inhibitory activity with an IC50 value of 2.25 μM in a competitive fluorescence polarization assay and a KD value of 1.44 μM for the β-catenin protein. Furthermore, B4 selectively inhibited the growth of colorectal cancer (CRC) cells, suppressed the transactivation of Wnt signaling, and downregulated the expression of oncogenic Wnt target gene. Especially, B4 showed potent anti-CRC activity in vivo with the tumor growth inhibition (TGI) of 75.99 % and regulated the tumor immune microenvironment.

Fluorescence Polarization Assay Based on a New Recognition Motif QepA for the One-Step Detection of Fluoroquinolones in Eggs.

A recognition motif is vital in determining the specificity and sensitivity of the fluorescence polarization assay (FPA) for detecting chemical contaminants in food. Four candidates (Gyrase, GyrBA, TopIV, and QepA) were prepared for this study. The applicability of QepA was confirmed through DNA cleavage assay, inhibition effects, and mechanism investigations using molecular docking, compared to other counterparts. Finally, a novel FPA based on QepA and a CIP-FITC tracer for the detection of fluoroquinolones (FQs) in eggs was developed. The limits of detection (LODs) for eight fluoroquinolones ranged from 2.2 to 5.1 ng g-1, with enrofloxacin, danofloxacin, and difloxacin meeting the maximum residue limits (MRLs). The spiked recoveries ranged from 65.8 to 103.6% with coefficients of variation (CVs) of 5.4-12.8%. Therefore, a new recognition motif for FQs that did not belong to conventional antibodies was identified, and QepA-based FPA could be a potential tool for rapid, homogeneous, and sensitive monitoring of the residue of FQs in eggs.

Co-Analysis of Genome-Wide DNA Methylation and Gene Expression Suggests Novel Regulatory Mechanism for RUNX1 in Inv(16) Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is characterized by recurring chromosomal abnormalities that encode oncogenic fusion proteins. Approximately 10% of AML cases are associated with a chromosome 16 inversion [inv(16)(p13q22)] or translocation t(16;16)(p13q22). These chromosomal rearrangements results in the formation of the fusion oncogene CBFB-MYH11 and ultimately the fusion protein CBFβ-SMMHC. Although CBFβ-SMMHC was initially considered a dominant negative repressor of RUNX1, we have recently shown that it works together with RUNX1 to activate gene expression through direct target gene binding. RUNX1 is known to regulate gene expression at both transcriptional and epigenetic levels. Aberrant DNA methylation patterns have been reported across different cancer types including AML. Recent studies have shown that DNA methylation negatively influences RUNX1 DNA binding. We hypothesize that changes to DNA methylation and consequently dysregulated RUNX1 DNA binding contribute to the pathogenesis of inv(16) AML. In this study, we examined the epigenetic and transcriptomic landscapes of CBFB-MYH11 knockin mice and assessed the contribution of the resulting fusion protein CBFβ-SMMHC to RUNX1 DNA target binding. To assess the effects of DNA methylation on RUNX1 DNA binding in vitro, we initially performed fluorescence polarization and electrophoretic mobility shift assays using unmethylated and methylated RUNX1 DNA target sequences. RUNX1, RUNX1/CBFβ and RUNX1/CBFβ-SMMHC showed significantly lower binding affinity for methylated DNA compared to unmethylated DNA probes. To determine genome-wide DNA methylation changes, we performed enzymatic methyl sequencing on sorted Lin -Sca1 -c-Kit + bone marrow cells from control and pre-leukemic inv(16) mice. Inv(16) mice showed a significant genome-wide DNA hypermethylation compared to controls (13,478 hyper- vs 705 hypomethylated regions; q-value < 0.05), with 14% of the differentially methylated regions (DMRs) lying in promoters. Genes associated with these DMRs were enriched for gene ontology (GO) processes related to hematopoiesis, such as myeloid cell homeostasis and erythrocyte differentiation, and DNA replication, such as mitotic DNA replication initiation, among others. In addition, several RUNX1 target genes showed promoter hypermethylation, such as Klf1 or the thrombopoietin receptor Mpl. To understand the impact of aberrant DNA methylation on gene expression, we performed RNA-seq using the same cell population. Gene expression changes were observed in inv(16) mice compared to controls, with 741 genes being upregulated and 278 downregulated from a total of 1019 differentially expressed genes (DEG; absolute fold-change ≥ 2, q-value < 0.05). Enriched GO processes identified from the DEGs were associated with immune responses, such as response to interferon-alpha (and beta) and leukocyte activation involved in inflammatory response. We then intersected the DMR (focusing on promoters) and DEG lists to identify genes whose expression might be directly influenced by DNA methylation changes in inv(16) leukemia. Hypermethylation of promoters was associated with 102 genes, with 67% of them showing downregulation of gene expression in inv(16) mice, whereas hypomethylation was associated with 18 genes with 94% showing upregulation of gene expression. These results show that in inv(16) mice, promoter hypermethylation is associated with downregulation of gene expression, and hypomethylation with increased expression. We also confirmed that several RUNX1 target genes showing promoter hypermethylation also had reduced gene expression in inv(16) mice. The initial analysis suggests that CBFβ-SMMHC expression significantly affects the methylation landscape in mice, with important RUNX1 target genes exhibiting promoter hypermethylation and subsequent downregulation of gene expression. We are currently performing chromatin immunocleavage sequencing using the same cell population to determine RUNX1 binding in vivo. Overall, this study explores a novel regulatory mechanism of RUNX1 function through DNA methylation, and ultimately a new role for RUNX1 in the pathogenesis of inv(16) AML.

Accuracy of serological tests for bovine brucellosis: A systematic review and meta-analysis

The direct methods for diagnosis of bovine brucellosis have several limitations, therefore serological tests are the basis for the diagnosis of the disease. However, a meta-analysis estimating the diagnostic sensitivity (DSe) and diagnostic specificity (DSp) on the main tests used in bovine brucellosis control programs worldwide has not been performed. This systematic review and meta-analysis aimed to estimate the DSe, DSp and thereby accuracy of serological tests individually used in the diagnosis of bovine brucellosis. The databases CABI, Cochrane Library, PubMed/MEDLINE, SciELO, Scopus and Web of Science were used to select articles. The search resulted in 5308 studies, of which 71 were selected for systematic review using quality assessment tools and 65 studies were included in the meta-analysis. For the meta-analysis, 178 assays and 11 different serological tests were considered. To estimate DSe and DSp of the tests, studies were divided according to animal selection for the studies: (1) studies that carried out a random or consecutive selection of participants (noncasecontrol studies) and (2) all studies, including casecontrol studies. Considering only the non-case-control studies to estimate the DSe, the tests that exhibited the best and worst performance were the iELISA test (indirect enzyme immunoassay - bacterial suspension as antigen - BS) (96.5%, 95% CI: 94.1–97.9%) and 2ME (2- mercaptoethanol test) (85.0%, 95% CI: 79.6–89.1%), respectively; while for DSp, the FPA (fluorescence polarization assay) (99, 7%, 95% CI: 99.5–99.8%) and PCFIA tests (protein concentration fluorescence immunoassay) (78.5%, 95% CI: 70.0–85.1%) showed better and worse performance, respectively. Overall, our results showed an overestimation in the DSe and DSp of the eleven serological tests assessed when casecontrol studies were included in the meta-analysis, which is a concern considering its impacts on the time and costs associated with populational diagnosis of the diseases, since several of these tests are routinely used in the control and eradication programs of bovine brucellosis worldwide. Furthermore, the tests that exhibited the best DSe and DSp, iELISA (BS) and FPA, respectively, are relatively easy to perform and interpret and the test which showed the best overall accuracy was FPA.

Inhibition of Hsp110-STAT3 interaction in endothelial cells alleviates vascular remodeling in hypoxic pulmonary arterial Hypertension model

BackgroundPulmonary arterial hypertension (PAH) is a progressive and devastating disease characterized by pulmonary vascular remodeling which is associated with the malignant phenotypes of pulmonary vascular cells. Recently, the effects of heat shock protein 110 (Hsp110) in human arterial smooth muscle cells were reported. However, the underlying roles and mechanisms of Hsp110 in human pulmonary arterial endothelial cells (HPAECs) that was disordered firstly at the early stage of PAH remain unknown.MethodsIn this research, the expression of Hsp110 in PAH human patients and rat models was investigated, and the Hsp110 localization was determined both in vivo and in vitro. The roles and mechanism of elevated Hsp110 in excessive cell proliferation and migration of HPAECs were assessed respectively exposed to hypoxia. Small molecule inhibitors targeting Hsp110-STAT3 interaction were screened via fluorescence polarization, anti-aggregation and western blot assays. Moreover, the effects of compound 6 on HPAECs abnormal phenotypes in vitro and pulmonary vascular remodeling of hypoxia-indued PAH rats in vivo by interrupting Hsp110-STAT3 interaction were evaluated.ResultsOur studies demonstrated that Hsp110 expression was increased in the serum of patients with PAH, as well as in the lungs and pulmonary arteries of PAH rats, when compared to their respective healthy subjects. Moreover, Hsp110 levels were significantly elevated in HPAECs under hypoxia and mediated its aberrant phenotypes. Furthermore, boosted Hsp110-STAT3 interaction resulted in abnormal proliferation and migration via elevating p-STAT3 and c-Myc in HPAECs. Notably, we successfully identified compound 6 as potent Hsp110-STAT3 interaction inhibitor, which effectively inhibited HPAECs proliferation and migration, and significantly ameliorated right heart hypertrophy and vascular remodeling of rats with PAH.ConclusionsOur studies suggest that elevated Hsp110 plays a vital role in HPAECs and inhibition of the Hsp110-STAT3 interaction is a novel strategy for improving vascular remodeling. In addition, compound 6 could serve as a promising lead compound for developing first-in-class drugs against PAH.

SARS-CoV-2 ORF3a-Mediated NF-κB Activation Is Not Dependent on TRAF-Binding Sequence.

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has caused a global pandemic of Coronavirus Disease 2019 (COVID-19). Excessive inflammation is a hallmark of severe COVID-19, and several proteins encoded in the SARS-CoV-2 genome are capable of stimulating inflammatory pathways. Among these, the accessory protein open reading frame 3a (ORF3a) has been implicated in COVID-19 pathology. Here we investigated the roles of ORF3a in binding to TNF receptor-associated factor (TRAF) proteins and inducing nuclear factor kappa B (NF-κB) activation. X-ray crystallography and a fluorescence polarization assay revealed low-affinity binding between an ORF3a N-terminal peptide and TRAFs, and a dual-luciferase assay demonstrated NF-κB activation by ORF3a. Nonetheless, mutation of the N-terminal TRAF-binding sequence PIQAS in ORF3a did not significantly diminish NF-κB activation in our assay. Our results thus suggest that the SARS-CoV-2 protein may activate NF-κB through alternative mechanisms.