Endogenous Interaction Research Articles

Thrombocytopenia Independently Leads to Changes in Monocyte Immune Function.

While platelets have well-studied hemostatic functions, platelets are immune cells that circulate at the interface between the vascular wall and white blood cells. The physiological implications of these constant transient interactions are poorly understood. Activated platelets induce and amplify immune responses, but platelets may also maintain immune homeostasis in healthy conditions, including maintaining vascular integrity and T helper cell differentiation, meaning that platelets are central to both immune responses and immune quiescence. Clinical data have shown an association between low platelet counts (thrombocytopenia) and immune dysfunction in patients with sepsis and extracorporeal membrane oxygenation, further implicating platelets as more holistic immune regulators, but studies of platelet immune functions in nondisease contexts have had limited study. We used in vivo models of thrombocytopenia and in vitro models of platelet and monocyte interactions, as well as RNA-seq and ATAC-seq (assay for transposase-accessible chromatin with sequencing), to mechanistically determine how resting platelet and monocyte interactions immune program monocytes. Circulating platelets and monocytes interact in a CD47-dependent manner to regulate monocyte metabolism, histone methylation, and gene expression. Resting platelet-monocyte interactions limit TLR (toll-like receptor) signaling responses in healthy conditions in an innate immune training-like manner. In both human patients with sepsis and mouse sepsis models, thrombocytopenia exacerbated monocyte immune dysfunction, including increased cytokine production. Thrombocytopenia immune programs monocytes in a manner that may lead to immune dysfunction in the context of sepsis. This is the first demonstration that sterile, endogenous cell interactions between resting platelets and monocytes regulate monocyte metabolism and pathogen responses, demonstrating platelets to be immune rheostats in both health and disease.

TREX reveals proteins that bind to specific RNA regions in living cells

Different regions of RNA molecules can often engage in specific interactions with distinct RNA-binding proteins (RBPs), giving rise to diverse modalities of RNA regulation and function. However, there are currently no methods for unbiased identification of RBPs that interact with specific RNA regions in living cells and under endogenous settings. Here we introduce TREX (targeted RNase H-mediated extraction of crosslinked RBPs)—a highly sensitive approach for identifying proteins that directly bind to specific RNA regions in living cells. We demonstrate that TREX outperforms existing methods in identifying known interactors of U1 snRNA, and reveals endogenous region-specific interactors of NORAD long noncoding RNA. Using TREX, we generated a comprehensive region-by-region interactome for 45S rRNA, uncovering both established and previously unknown interactions that regulate ribosome biogenesis. With its applicability to different cell types, TREX is an RNA-centric tool for unbiased positional mapping of endogenous RNA–protein interactions in living cells.

The Traces of Dysregulated lncRNAs-Associated ceRNA Axes in Retinoblastoma: A Systematic Scope Review

Purpose Long non-coding RNAs are an essential component of competing endogenous RNA regulatory axes and play their role by sponging microRNAs and interfering with the regulation of gene expression. Because of the broadness of competing endogenous RNA interaction networks, they may help investigate treatment targets in complicated disorders. Methods This study performed a systematic scoping review to assess verified loops of competing endogenous RNAs in retinoblastoma, emphasizing the competing endogenous RNAs axis related to long non-coding RNAs. We used a six-stage approach framework and the PRISMA guidelines. A systematic search of seven databases was done to locate suitable papers published before February 2022. Two reviewers worked independently to screen articles and collect data. Results Out of 363 records, fifty-one articles met the inclusion criteria, and sixty-three axes were identified in desired articles. The majority of the research reported several long non-coding RNAs that were experimentally verified to act as competing endogenous RNAs in retinoblastoma: XIST/NEAT1/MALAT1/SNHG16/KCNQ1OT1, respectively. At the same time, around half of the studies investigated unique long non-coding RNAs. Conclusions Understanding the many features of this regulatory system may aid in elucidating the unknown etiology of Retinoblastoma and providing novel molecular targets for therapeutic and clinical applications.

Detecting Native Protein-Protein Interactions by APEX2 Proximity Labeling in Drosophila Tissues.

Enzyme-catalyzed proximity labeling is a potent technique for the discernment of subtle molecular interactions and subcellular localization, furnishing contextual insights into the protein of interest within cells. Although ascorbate peroxidase2 (APEX2) has proven effective in this approach when overexpressed, its compatibility with endogenous proteins remains untested. We improved this technique for studying native protein-protein interactions in live Drosophila ovary tissue. Through CRISPR/Cas9 genome editing, APEX2 was fused with the endogenous dysfusion gene. By pre-treating the tissue with Triton X-100 to enhance biotin-phenol penetration, we successfully identified multiple proteins interacting with the native Dysfusion proteins that reside on the inner nuclear membrane. Our protocol offers a comprehensive workflow for delineating the interactome networks of ovarian components in Drosophila, aiding future studies on endogenous protein-protein interactions in various tissues of other animals. Key features • Elucidating Protein-protein interactions provides deeper insights into the regulation of gene expression in molecular network and complex signaling pathways, advancing protein engineering and drug design. • This protocol utilizes CRISPR/Cas9 knock-in technology to tag endogenous proteins with the APEX2 to label nearby proteins within a 20 nm radius in Drosophila melanogaster. • We optimize APEX2-proximity labeling by using Triton X-100 pre-treatment to enhance biotin-phenol penetration into Drosophila ovaries, enabling endogenous proteins enrichment under native conditions.

The AD Exposome

Efforts to find human genes for Alzheimer Disease and for longevity have generally neglected environmental influences. Brain aging and ADRDA may be understood in the AD Exposome framework, which includes external and endogenous interactions from embryo to old age (Finch and Kulminski 2019; Finch and Haghani 2021). Environmental factors in brain aging and longevity are as strong as common aging genes illustrated by the 15-year difference in the onset of dementia across SES (Finch 2023). Gene-environment interactions merit further attention.

Abstract B006: High RAS-RAF binding as assessed via proximity ligation assay is associated with sensitivity to KRASG12C inhibitors in NSCLC

Abstract KRASG12C inhibitors such as sotorasib and adagrasib have shown efficacy in patients with non-small cell lung cancer (NSCLC). However, a substantial proportion of patients do not respond. Therefore, there is a clear need for predictive biomarkers to guide treatment. The proximity ligation assay (PLA) is an immunofluorescent-based approach utilized to analyze endogenous protein-protein interactions. The method allows for the assessment of signaling-associated protein complexes, which can serve as potential targets for specific inhibitors. In particular, KRASG12C inhibitors can disrupt these complexes by interfering with the interaction between RAS and its effector RAF. By providing insights into the presence of these protein complexes, PLA offers a predictive readout for inhibitor sensitivity. We thus conducted a preclinical study to evaluate if a RAS-CRAF PLA could inform on the presence of RAS-RAF protein complexes and predict the response to KRASG12C inhibitors. To assess the specificity of the panRAS-CRAF PLA, we employed RNA interference in a KRASG12C-mutant H358 NSCLC cell line, wherein knockdown of either panRAS or CRAF resulted in a reduction in the panRAS-CRAF PLA signal. Pharmacological inhibition of RAS-RAF interaction with sotorasib also reduced the panRAS-CRAF PLA signal. We next performed panRAS-CRAF PLA in a panel of 11 KRASG12C-mutant NSCLC cell lines, revealing a higher number of panRAS-CRAF PLA spots in H358, LU65, and HCC1171. To further investigate the RAS-RAF interaction, we assessed the abundance of CRAF-bound RAS-GTP by quantifying the binding of RAS-GTP to the RAS-binding domain of CRAF. H358, LU65, and HCC1171 exhibited the highest levels of RAS-GTP among the 11 cell lines. We observed a strong correlation between the number of panRAS-CRAF PLA foci and the levels of CRAF-bound RAS-GTP (r = 0.87, P = 0.0002). These results indicated that the enhanced level of RAS-GTP for CRAF proteins facilitates the formation of RAS-CRAF complexes, thereby augmenting the panRAS-CRAF PLA signals. We then evaluated the in vitro antitumor activity of sotorasib across the G12C-mutated cell lines and found that the 50% inhibitory concentration values of sotorasib for H358, LU65, and HCC1171 were below 30 nmol/L. The panRAS-CRAF PLA was strongly associated with the sensitivity to sotorasib (r = 0.81, P = 0.002). We subsequently assessed the association of panRAS-CRAF PLA with efficacy of adagrasib in a patient-derived xenograft (PDX) cohort. PanRAS-CRAF PLA was assessed on sections of formalin-fixed paraffin-embedded tumors derived from vehicle-treated control groups at the end of efficacy studies in 18 NSCLC PDX models. Our findings revealed a significant association between high panRAS-CRAF PLA signal and increased sensitivity to adagrasib (r = 0.61, P = 0.008). Our results suggest that panRAS-CRAF PLA may serve as a predictive marker to identify patients who could derive optimal benefit from KRASG12C inhibitors, and may direct combination therapy approaches to those patients less likely to benefit from single-agent therapy. Citation Format: Ryoji Kato, Hitendra S. Solanki, Denis Imbody, Anurima Majumder, Yaakov Stern, Liznair Bridenstine, Harika Gundlapalli, Ida Aronchik, Joseph Johnson, Eric B. Haura. High RAS-RAF binding as assessed via proximity ligation assay is associated with sensitivity to KRASG12C inhibitors in NSCLC [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr B006.

Platelet CD47 Maintains Circulating Monocyte Immune Homeostasis

In addition to their well-studied hemostatic functions, platelets are immune cells. Platelets circulate at the interface between the vascular wall and white blood cells, and transient platelet-leukocyte complexes are found in both healthy and disease states, positioning platelets to provide physiologic cues of both vascular health and injury. Roles for activated platelets in inducing and amplifying immune responses have received an increasing amount of research attention, but our past studies also showed that normal platelet counts are needed in healthy conditions to maintain immune homeostasis. Circulating monocytes are a part of the first line of host defenses that recognize and clear pathogens in part by releasing inflammatory cytokines, including IL-6, IL-8 and TNFα, that trigger a robust inflammatory reaction. However, this needs to be tightly regulated and in disease conditions like sepsis, dysregulated monocyte responses contribute to a “cytokine storm” that leads to exaggerated inflammation, tissue damage and potentially mortality. The cellular and molecular mechanisms that regulate monocyte immune phenotypes are complex and include roles for platelets. Considering the immune regulatory role of platelets and that thrombocytopenia is associated with worse sepsis outcomes, we hypothesize that resting platelets maintain monocyte immune homeostasis in healthy conditions and thrombocytopenia independently leads to monocyte dysregulation that exacerbates sepsis responses in a manner that is dependent on direct platelet-monocyte CD47 homotypic interactions that regulate monocyte immunometabolism and gene expression. Compared to monocytes from mice with normal platelet counts, circulating monocytes from thrombocytopenic mice had increased toll-like receptor (TLR) induced cytokine responses. Furthermore, ex vivo co-incubation of resting platelets with platelet naïve bone marrow monocytes, induced monocyte metabolic programming skewed toward glycolysis, consistent with our in vitro and in vivo RNA-Seq results. Enhanced glycolysis was mediated by AKT/mTOR pathway activation and resulted in durable changes in TLR agonist responses. Assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-Seq) on monocytes from thrombocytopenic mice showed open chromatin at LPS response genes, and resting platelet interactions with monocytes induced histone H3K9 and H3K27 trimethylation in a CD47 dependent manner. Using mouse models of thrombocytopenia and sepsis, we found that normal platelet numbers limited monocyte immune dysregulation and systemic inflammation. This was dependent on platelet CD47 as only WT platelets, not CD47 -/- platelet transfusion rescued the exaggerated inflammatory responses in thrombocytopenic mice (Fig 1). Both plasma IL-6 and IL-8 and circulating monocyte IL6 and IL8 gene expression from human patients with sepsis and non-septic conditions inversely correlated with patient platelet counts, further suggesting a resting platelet role in maintaining monocyte immune quiescence. Overall, our studies demonstrate that resting platelets maintain monocyte immune homeostasis by limiting TLR responses through regulating monocyte immunometabolic processes that lead to epigenetic changes in monocyte immune response genes (Fig 2). Thrombocytopenia therefore independently leads to immune dysregulation in sepsis, exacerbating systemic inflammatory processes. This is the first demonstration of sterile endogenous cell interactions that regulate innate immune-metabolism and monocyte pathogen responses.

Altruism, social interactions, and the course of a pandemic

Externalities and social preferences, such as altruism, play a key role in the choice of social interactions, which in turn affect the diffusion of a pandemic. We build a dynamic epidemiological model with endogenous social interactions in a frictional environment, also in a variant with heterogeneous agents and a network structure. Taking into account agents’ endogenous behavior and altruism generates markedly different predictions relative to a naïve epidemiological model with exogenous contact rates. Congestion and commitment inefficiencies arise, even under full altruism, and call for policy intervention. We derive the efficient allocation, and show how the Ramsey planner can mitigate the respective externalities.

Impact of P-Glycoprotein-Mediated Drug-Endogenous Substrate Interactions on Androgen and Blood-Brain Barrier Permeability

This report focuses on pharmacokinetic drug–endogenous substrate interactions (DEIs). We hypothesized that P-glycoprotein (P-gp)-mediated DEI might affect androgen kinetics, especially its blood–brain barrier (BBB) permeability. The intracellular accumulation of the endogenous substrates of P-gp, testosterone (TES) and androstenedione (ADO) was increased by several tested drugs in uptake studies using P-gp overexpressing cells, indicating that these drugs inhibit P-gp-mediated efflux of TES of ADO from the cells. In a transport study using rat BBB kit, we found that the BBB limited the penetration of TES and ADO into the central nervous system. In addition, tested drugs that cause DEI were found to increase BBB permeability of TES and ADO via P-gp inhibition. In short, this study provides new findings regarding the possibility that DEI may affect the kinetics of endogenous substrates of P-gp.

Visualization and Quantification of Endogenous Intra-Organelle Protein Interactions at ER-Mitochondria Contact Sites by Proximity Ligation Assays.

Membrane contact sites (MCSs) are areas of close membrane proximity that allow and regulate the dynamic exchange of diverse biomolecules (i.e., calcium and lipids) between the juxtaposed organelles without involving membrane fusion. MCSs are essential for cellular homeostasis, and their functions are ensured by the resident components, which often exist as multimeric protein complexes. MCSs often involve the endoplasmic reticulum (ER), a major site of lipid synthesis and cellular calcium storage, and are particularly important for organelles, such as the mitochondria, which are excluded from the classical vesicular transport pathways. In the last years, MCSs between the ER and mitochondria have been extensively studied, as their functions strongly impact cellular metabolism/bioenergetics. Several proteins have started to be identified at these contact sites, including membrane tethers, calcium channels, and lipid transfer proteins, thus raising the need for new methodologies and technical approaches to study these MCS components. Here, we describe a protocol consisting of combined technical approaches, that include proximity ligation assay (PLA), mitochondria staining, and 3D imaging segmentation, that allows the detection of proteins that are physically close (>40 nm) to each other and that reside on the same membrane at ER-mitochondria MCSs. For instance, we used two ER-anchored lipid transfer proteins, ORP5 and ORP8, which have previously been shown to interact and localize at ER-mitochondria and ER-plasma membrane MCSs. By associating the ORP5-ORP8 PLA with cell imaging software analysis, it was possible to estimate the distance of the ORP5-ORP8 complex from the mitochondrial surface and determine that about 50% of ORP5-ORP8 PLA interaction occurs at ER subdomains in close proximity to mitochondria.

Can digitalization and low-carbonization progress in harmony? Evidence from Chinese cities.

Achieving high-quality development of the city requires actively promoting coordinated digitalization and low-carbon development. Previous studies have focused on the unidirectional impact of urban digitalization on low-carbonization and there is a lack of research on their interactions. This study uses the generalized spatial three-stage least squares method and the spatial simultaneous equation to investigate the endogenous interactions between urban digitalization and low-carbonization. The properties of the spatiotemporal evolution are then examined using linked coordination degree models, kernel density, and spatial statistical approaches. Finally, using the spatial panel metering model, this study empirically investigates the motivations behind the synergistic advancement of digitalization and low-carbonization. The results show that: (1) There is an endogenous interaction between urban digitalization and low-carbonization and that this interaction pattern is closely linked to geographical proximity. (2) In general, both urban digitalization and low-carbonization have a positive spatial impact and a negative spatial interaction, and their coordination levels have a significant spatial impact. (3) Throughout the research period, the coordination degree of urban digitalization and low carbonization continued to increase, showing a positive spatial correlation and a balanced development trend. (4) Economic development, industrial structure, and human capital accumulation are vital internal drivers of the synergistic advancement of urban digitalization and low carbonization. Government capacities and technological innovations are key external factors that contribute to the synergistic advancement of urban digitalization and low-carbonization. Overall, the paper is essential not only to deepen understanding of the relationship between urban digitalization and low-carbonization but also to formulate policies for their coordinated development.

An Anterior Second Heart Field Enhancer Regulates the Gene Regulatory Network of the Cardiac Outflow Tract.

Conotruncal defects due to developmental abnormalities of the outflow tract (OFT) are an important cause of cyanotic congenital heart disease. Dysregulation of transcriptional programs tuned by NKX2-5 (NK2 homeobox 5), GATA6 (GATA binding protein 6), and TBX1 (T-box transcription factor 1) have been implicated in abnormal OFT morphogenesis. However, there remains no consensus on how these transcriptional programs function in a unified gene regulatory network within the OFT. We generated mice harboring a 226-nucleotide deletion of a highly conserved cardiac enhancer containing 2 GATA-binding sites located ≈9.4 kb upstream of the transcription start site of Nkx2-5 (Nkx2-5∆enh) using CRISPR-Cas9 gene editing and assessed phenotypes. Cardiac defects in Nkx2-5∆enh/∆enh mice were structurally characterized using histology and scanning electron microscopy, and physiologically assessed using electrocardiography, echocardiography, and optical mapping. Transcriptome analyses were performed using RNA sequencing and single-cell RNA sequencing data sets. Endogenous GATA6 interaction with and activity on the NKX2-5 enhancer was studied using chromatin immunoprecipitation sequencing and transposase-accessible chromatin sequencing in human induced pluripotent stem cell-derived cardiomyocytes. Nkx2-5∆enh/∆enh mice recapitulated cyanotic conotruncal defects seen in patients with NKX2-5, GATA6, and TBX1 mutations. Nkx2-5∆enh/∆enh mice also exhibited defects in right Purkinje fiber network formation, resulting in right bundle-branch block. Enhancer deletion reduced embryonic Nkx2-5 expression selectively in the right ventricle and OFT of mutant hearts, indicating that enhancer activity is localized to the anterior second heart field. Transcriptional profiling of the mutant OFT revealed downregulation of important genes involved in OFT rotation and septation, such as Tbx1, Pitx2, and Sema3c. Endogenous GATA6 interacted with the highly conserved enhancer in human induced pluripotent stem cell-derived cardiomyocytes and in wild-type mouse hearts. We found critical dose dependency of cardiac enhancer accessibility on GATA6 gene dosage in human induced pluripotent stem cell-derived cardiomyocytes. Our results using human and mouse models reveal an essential gene regulatory network of the OFT that requires an anterior second heart field enhancer to link GATA6 with NKX2-5-dependent rotation and septation gene programs.

Endogenous oxytocin and human social interactions: A systematic review and meta-analysis.

While there has been an increase in studies investigating the relationship between endogenous oxytocin (OXT) concentrations and human social interactions over the past decades, these studies still seem far from converging, both in methodological terms and in terms of their results. This systematic review and meta-analysis were aimed at a comprehensive evaluation and synthesis of empirical evidence on the relationship between endogenous OXT concentrations and human social interactions by reviewing studies published between 1970 and July 2020 and addressing various related methodological and analytical limitations. Sixty-three studies were included in the qualitative synthesis, and results from 51 studies were pooled in a meta-analysis (n = 3,741 participants). The results indicated that social interaction did not lead to an expected hormonal response in causal designs, either in a pre-post design (g = 0.079) or when comparing experimental conditions with and without social interaction (g = 0.256). However, in correlational designs, the overall mean effect size (ES) of the correlations between indicators of social interaction and OXT concentrations was significantly different from zero (z = 0.137). In both designs, subgroup analyses revealed that studies involving either parent-child interactions, or the utilization of the enzyme-linked immunosorbent assay method for OXT analysis, or unrestricted eating, drinking, or exercise before biofluid collection showed significantly higher than zero mean ESs. This review exposes the observed inconsistencies and suggests that standardized, replicable, and reliable approaches to assessing social interaction and measuring OXT concentrations need to be developed to study neurochemical mechanisms of sociality in humans. (PsycInfo Database Record (c) 2023 APA, all rights reserved).

Tax Revenues and State Expenditures in the US with Balanced-Budget Requirements Using Panel VAR

We examine Granger causality between state expenditures and tax revenues in the US with Balanced-Budget Requirements (BBRs). BBRs are statutory or constitutional rules to prevent states from spending more than tax revenues. Policymakers may adjust taxes, change expenditures, or do both to achieve BBRs. Panel VAR is introduced to address endogenous interactions between state expenditures and tax revenues. Panel VAR provides a unifying empirical framework and identification strategy which might be lacking in the previous literature. The empirical result supports that tax revenues Granger cause state expenditures. As BBRs are about planning, tax revenues make states plan on how to spend them. Considering BBRs with the empirical results from this study, states expenditures should be adjusted close to (projected) tax revenues. Additionally, this study finds that the business cycle has counter-intuitive effects. Tax revenues did not change when the US experienced the recession during 2008-2009 with the increases in intergovernmental transfer from the federal. State expenditures also increased during the recession.

Zebrafish gonad mutant models reveal neuroendocrine mechanisms of brain sexual dimorphism and male mating behaviors of different brain regions

BackgroundSexually dimorphic mating behaviors differ between sexes and involve gonadal hormones and possibly sexually dimorphic gene expression in the brain. However, the associations among the brain, gonad, and sexual behavior in teleosts are still unclear. Here, we utilized germ cells-free tdrd12 knockout (KO) zebrafish, and steroid synthesis enzyme cyp17a1-deficient zebrafish to investigate the differences and interplays in the brain–gonad–behavior axis, and the molecular control of brain dimorphism and male mating behaviors.MethodsTdrd12+/−; cyp17a1+/− double heterozygous parents were crossed to obtain tdrd12−/−; cyp17a1+/+ (tdrd12 KO), tdrd12+/+; cyp17a1−/− (cyp17a1 KO), and tdrd12−/−; cyp17a1−/− (double KO) homozygous progenies. Comparative analysis of mating behaviors were evaluated using Viewpoint zebrafish tracking software and sexual traits were thoroughly characterized based on anatomical and histological experiments in these KOs and wild types. The steroid hormone levels (testosterone, 11-ketotestosterone and 17β-estradiol) in the brains, gonads, and serum were measured using ELISA kits. To achieve a higher resolution view of the differences in region-specific expression patterns of the brain, the brains of these KOs, and control male and female fish were dissected into three regions: the forebrain, midbrain, and hindbrain for transcriptomic analysis.ResultsQualitative analysis of mating behaviors demonstrated that tdrd12−/− fish behaved in the same manner as wild-type males to trigger oviposition behavior, while cyp17a1−/− and double knockout (KO) fish did not exhibit these behaviors. Based on the observation of sex characteristics, mating behaviors and hormone levels in these mutants, we found that the maintenance of secondary sex characteristics and male mating behavior did not depend on the presence of germ cells; rather, they depended mainly on the 11-ketotestosterone and testosterone levels secreted into the brain–gonad regulatory axis. RNA-seq analysis of different brain regions revealed that the brain transcript profile of tdrd12−/− fish was similar to that of wild-type males, especially in the forebrain and midbrain. However, the brain transcript profiles of cyp17a1−/− and double KO fish were distinct from those of wild-type males and were partially biased towards the expression pattern of the female brain. Our results revealed important candidate genes and signaling pathways, such as synaptic signaling/neurotransmission, MAPK signaling, and steroid hormone pathways, that shape brain dimorphism and modulate male mating behavior in zebrafish.ConclusionsOur results provide comprehensive analyses and new insights regarding the endogenous interactions in the brain–gonad–behavior axis. Moreover, this study revealed the crucial candidate genes and neural signaling pathways of different brain regions that are involved in modulating brain dimorphism and male mating behavior in zebrafish, which would significantly light up the understanding the neuroendocrine and molecular mechanisms modulating brain dimorphism and male mating behavior in zebrafish and other teleost fish.Graphical

Crosstalk between glucocorticoid and mineralocorticoid receptors boosts glucocorticoid-induced killing of multiple myeloma cells

The glucocorticoid receptor (GR) is a crucial drug target in multiple myeloma as its activation with glucocorticoids effectively triggers myeloma cell death. However, as high-dose glucocorticoids are also associated with deleterious side effects, novel approaches are urgently needed to improve GR action in myeloma. Here, we reveal a functional crosstalk between GR and the mineralocorticoid receptor (MR) that plays a role in improved myeloma cell killing. We show that the GR agonist dexamethasone (Dex) downregulates MR levels in a GR-dependent way in myeloma cells. Co-treatment of Dex with the MR antagonist spironolactone (Spi) enhances Dex-induced cell killing in primary, newly diagnosed GC-sensitive myeloma cells. In a relapsed GC-resistant setting, Spi alone induces distinct myeloma cell killing. On a mechanistic level, we find that a GR–MR crosstalk likely arises from an endogenous interaction between GR and MR in myeloma cells. Quantitative dimerization assays show that Spi reduces Dex-induced GR–MR heterodimerization and completely abolishes Dex-induced MR–MR homodimerization, while leaving GR–GR homodimerization intact. Unbiased transcriptomics analyses reveal that c-myc and many of its target genes are downregulated most by combined Dex-Spi treatment. Proteomics analyses further identify that several metabolic hallmarks are modulated most by this combination treatment. Finally, we identified a subset of Dex-Spi downregulated genes and proteins that may predict prognosis in the CoMMpass myeloma patient cohort. Our study demonstrates that GR–MR crosstalk is therapeutically relevant in myeloma as it provides novel strategies for glucocorticoid-based dose-reduction.

Cathepsin D Attenuates the Proliferation of Vascular Smooth Muscle Cells Induced by the AGE/RAGE Pathway by Suppressing the ERK Signal.

In this study, we aimed to clarify the role and mechanism by which Cathepsin D (CTSD) mediates the advanced glycation end products (AGEs)-induced proliferation of vascular smooth muscle cells (VSMCs). We conducted a Western blotting assay and co-immunoprecipitation assay to detect the expression of target proteins and the interaction between different proteins. Cell Counting Kit-8 (CCK-8) assay and 5- ethynyl-2'-deoxyuridine (EdU) were used to evaluate the proliferation. AGEs significantly promoted phenotypic switching and proliferation of VSMCs in a concentration-dependent manner. This effect of AGEs was accompanied by inhibition of CTSD. Both the proliferation of VSMCs and inhibition of CTSD induced by AGEs could be attenuated by the specific inhibitor of the receptor for advanced glycation end products (RAGE), FPS-ZM1. Overexpression of CTSD significantly alleviated these effects of AGEs on VSMCs. The mechanism of CTSD action in VSMCs was also explored. Overexpression of CTSD reduced the activation of p-ERK caused by AGEs. By contrast, the knockdown of CTSD, elicited using a plasmid containing short hairpin RNA (shRNA) against CTSD, further increased the activation of p-ERK compared to AGEs alone. Additionally, co-immunoprecipitation studies revealed an endogenous interaction between CTSD, a protease, and p-ERK, its potential substrate. It has been demonstrated that CTSD downregulates the level of phosphorylated ERK by degrading its target, and this interaction plays a critical role in the proliferation of VSMCs induced by the AGE/RAGE axis. These results provide a novel insight into the prevention and treatment of vascular complications in diabetes.

Design, Synthesis, and Application of Fluorescent Ligands Targeting the Intracellular Allosteric Binding Site of the CXC Chemokine Receptor 2.

The inhibition of CXC chemokine receptor 2 (CXCR2), a key inflammatory mediator, is a potential strategy in the treatment of several pulmonary diseases and cancers. The complexity of endogenous chemokine interaction with the orthosteric binding site has led to the development of CXCR2 negative allosteric modulators (NAMs) targeting an intracellular pocket near the G protein binding site. Our understanding of NAM binding and mode of action has been limited by the availability of suitable tracer ligands for competition studies, allowing direct ligand binding measurements. Here, we report the rational design, synthesis, and pharmacological evaluation of a series of fluorescent NAMs, based on navarixin (2), which display high affinity and preferential binding for CXCR2 over CXCR1. We demonstrate their application in fluorescence imaging and NanoBRET binding assays, in whole cells or membranes, capable of kinetic and equilibrium analysis of NAM binding, providing a platform to screen for alternative chemophores targeting these receptors.

Do Nanodisc Assembly Conditions Affect Natural Lipid Uptake?

Lipids play critical roles in modulating membrane protein structure, interactions, and activity. Nanodiscs provide a tunable membrane mimetic that can model these endogenous protein-lipid interactions in a nanoscale lipid bilayer. However, most studies of membrane proteins with nanodiscs use simple synthetic lipids that lack the headgroup and fatty acyl diversity of natural extracts. Prior research has successfully used natural lipid extracts in nanodiscs that more accurately mimic natural environments, but it is not clear how nanodisc assembly may bias the incorporated lipid profiles. Here, we applied lipidomics to investigate how nanodisc assembly conditions affect the profile of natural lipids in nanodiscs. Specifically, we tested the effects of assembly temperature, nanodisc size, and lipidome extract complexity. Globally, our analysis demonstrates that the lipids profiles are largely unaffected by nanodisc assembly conditions. However, a few notable changes emerged within individual lipids and lipid classes, such as a differential incorporation of cardiolipin and phosphatidylglycerol lipids from the E. coli polar lipid extract at different temperatures. Conversely, some classes of brain lipids were affected by nanodisc size at higher temperatures. Collectively, these data enable the application of nanodiscs to study protein-lipid interactions in complex lipid environments.

Controlling (bio)chemical conversions of health-related plant-based compounds by processing: The case of Brussels sprouts and leek

Brussels sprouts and leek are suppliers of multiple health-related compounds, yet understudied on how processing impacts their concentration. In this study, the plant tissue structure was altered by processing to direct endogenous enzyme-substrate interactions. For this, both conventional and novel processing techniques were employed. Heating intact vegetables prior to high shear mixing resulted in relatively good preservation of health-related compounds, attributed to a prompt inactivation of endogenous enzymes. Oppositely, high shear mixing prior to heating facilitated over 82% conversion for both vitamin C, S-alk(en)yl-L-cysteine sulfoxides (ACSOs) and glucosinolates. Pulsed electric field (PEF) treatment prior to heating, resulted in ±25% ACSOs conversion, yet no glucosinolates conversion. PEF treatment effectiveness is attributed to substrate-enzyme localization in the matrix and cell size. Vitamin K1 remained relatively stable during all treatments. It was postulated that higher concentrations of vitamin C protect carotenoids against oxidation pointing to interaction effects among health-related compounds. Industrial relevance textThis research paper gives more insight into how (the sequence of) commonly used pretreatments (e.g. heating, mixing) and more innovative pretreatments (e.g. pulsed electric field) can have an impact on the health potential of vegetables. The considered vegetables (Brussels sprouts and leek) are highly industrial relevant and insights may be extrapolated to other vegetables of these large plant families. By understanding which (bio)chemical reactions can occur during certain pretreatments (e.g. degradation or potential formation of health-related compounds), industry can perform pretreatments more thoughtfully and consequently steer the health potential of vegetable products. Pulsed electric fields are currently not commonly used in vegetable processing. However, this processing technique can enable certain enzymatic conversions, of which some could result in formation of health beneficial compounds, without losing the vegetable structure. Therefore, applying pulsed electric fields in the vegetable processing industry could have large potential. The aim of this research is to open the door to more research on pulsed electric fields applied on vegetables, so the vegetable industry becomes acquainted with using this technique.