Dissection Of Mechanisms Research Articles

The Fim and FhaB adhesins play a crucial role in nasal cavity infection and Bordetella pertussis transmission in a novel mouse catarrhal infection model.

Pulmonary infections caused by Bordetella pertussis used to be the prime cause of infant mortality in the pre-vaccine era and mouse models of pertussis pneumonia served in characterization of B. pertussis virulence mechanisms. However, the biologically most relevant catarrhal disease stage and B. pertussis transmission has not been adequately reproduced in adult mice due to limited proliferation of the human-adapted pathogen on murine nasopharyngeal mucosa. We used immunodeficient C57BL/6J MyD88 KO mice to achieve B. pertussis proliferation to human-like high counts of 108 viable bacteria per nasal cavity to elicit rhinosinusitis accompanied by robust shedding and transmission of B. pertussis bacteria to adult co-housed MyD88 KO mice. Experiments with a comprehensive set of B. pertussis mutants revealed that pertussis toxin, adenylate cyclase toxin-hemolysin, the T3SS effector BteA/BopC and several other known virulence factors were dispensable for nasal cavity infection and B. pertussis transmission in the immunocompromised MyD88 KO mice. In contrast, mutants lacking the filamentous hemagglutinin (FhaB) or fimbriae (Fim) adhesins infected the nasal cavity poorly, shed at low levels and failed to productively infect co-housed MyD88 KO or C57BL/6J mice. FhaB and fimbriae thus appear to play a critical role in B. pertussis transmission. The here-described novel murine model of B. pertussis-induced nasal catarrh opens the way to genetic dissection of host mechanisms involved in B. pertussis shedding and to validation of key bacterial transmission factors that ought to be targeted by future pertussis vaccines.

Lipidomics in Understanding Pathophysiology and Pharmacologic Effects in Inflammatory Diseases: Considerations for Drug Development.

The lipidome has a broad range of biological and signaling functions, including serving as a structural scaffold for membranes and initiating and resolving inflammation. To investigate the biological activity of phospholipids and their bioactive metabolites, precise analytical techniques are necessary to identify specific lipids and quantify their levels. Simultaneous quantification of a set of lipids can be achieved using high sensitivity mass spectrometry (MS) techniques, whose technological advancements have significantly improved over the last decade. This has unlocked the power of metabolomics/lipidomics allowing the dynamic characterization of metabolic systems. Lipidomics is a subset of metabolomics for multianalyte identification and quantification of endogenous lipids and their metabolites. Lipidomics-based technology has the potential to drive novel biomarker discovery and therapeutic development programs; however, appropriate standards have not been established for the field. Standardization would improve lipidomic analyses and accelerate the development of innovative therapies. This review aims to summarize considerations for lipidomic study designs including instrumentation, sample stabilization, data validation, and data analysis. In addition, this review highlights how lipidomics can be applied to biomarker discovery and drug mechanism dissection in various inflammatory diseases including cardiovascular disease, neurodegeneration, lung disease, and autoimmune disease.

Small RNA Expression Profiling Reveals hsa-miR-181d-5p Downregulation Associated With TNF-α Overexpression in Sjögren's Syndrome Patients.

MicroRNAs (miRNAs) are small non-coding RNAs (sRNA), that alter gene expression by binding to target messenger RNAs (mRNAs) and repressing translation. Dysregulated miRNA expression has been implicated in the pathogenesis of autoimmune diseases such as Sjögren’s syndrome (SS). The aim of this study was to characterize the global profile of sRNAs in labial salivary glands (LSG) from SS-patients and to validate potential miRNA candidates implicated in glandular inflammation. LSG from 21 SS-patients and 9 sicca controls were analyzed. A global next generation sequencing (NGS)-based sRNA profiling approach was employed to identify direct targets whereby differentially expressed miRNAs were predicted using bioinformatics tools. miRNA levels were validated by TaqMan and target mRNA levels were determined by quantitative real-time PCR. We also performed in vitro assays using recombinant TNF-α. NGS shows that ~30% of sRNAs were miRNAs. In comparison with samples from sicca controls, four miRNAs were found differentially expressed in LSG from SS-patients with low focus score (LFS) and 18 from SS-patients with high focus score (HFS). The miRNA with the most significant changes identified by NGS was hsa-miR-181d-5p and downregulation was confirmed by TaqMan analysis. Levels of TNF-α mRNA, a direct target of hsa-miR-181d-5p, were significantly increased and negatively correlated with hsa-miR-181d-5p presence. Moreover, positive correlations between TNF-α transcript levels, focus score, ESSDAI, and autoantibody levels were also detected. Furthermore, TNF-α stimulation decreased hsa-miR-181d-5p levels in vitro. Downregulation of hsa-miR-181d-5p in LSG from SS-patients could contribute to the glandular pro-inflammatory environment by deregulation of its direct target TNF-α. Further dissection of the pathophysiological mechanisms underlying the hsa-miR-181d-5p-mediated action in inflammatory conditions could be useful to evaluate the benefits of increasing hsa-miR-181d-5p levels for restoration of salivary gland epithelial cell architecture and function.

Identification of substrates of palmitoyl protein thioesterase 1highlights roles of depalmitoylation in disulfide bond formation and synaptic function.

Loss-of-function mutations in the depalmitoylating enzyme palmitoyl protein thioesterase 1 (PPT1) cause neuronal ceroid lipofuscinosis (NCL), a devastating neurodegenerative disease. The substrates of PPT1 are largely undescribed, posing a limitation on molecular dissection of disease mechanisms and therapeutic development. Here, we provide a resource identifying >100 novel PPT1 substrates. We utilized Acyl Resin-Assisted Capture (Acyl RAC) and mass spectrometry to identify proteins with increased in vivo palmitoylation in PPT1 knockout (KO) mouse brains. We then validated putative substrates through direct depalmitoylation with recombinant PPT1. This stringent screen elucidated diverse PPT1 substrates at the synapse, including channels and transporters, G-protein-associated molecules, endo/exocytic components, synaptic adhesion molecules, and mitochondrial proteins. Cysteine depalmitoylation sites in transmembrane PPT1 substrates frequently participate in disulfide bonds in the mature protein. We confirmed that depalmitoylation plays a role in disulfide bond formation in a tertiary screen analyzing posttranslational modifications (PTMs). Collectively, these data highlight the role of PPT1 in mediating synapse functions, implicate molecular pathways in the etiology of NCL and other neurodegenerative diseases, and advance our basic understanding of the purpose of depalmitoylation.

Pangenomics in Microbial and Crop Research: Progress, Applications, and Perspectives.

Advances in sequencing technologies and bioinformatics tools have fueled a renewed interest in whole genome sequencing efforts in many organisms. The growing availability of multiple genome sequences has advanced our understanding of the within-species diversity, in the form of a pangenome. Pangenomics has opened new avenues for future research such as allowing dissection of complex molecular mechanisms and increased confidence in genome mapping. To comprehensively capture the genetic diversity for improving plant performance, the pangenome concept is further extended from species to genus level by the inclusion of wild species, constituting a super-pangenome. Characterization of pangenome has implications for both basic and applied research. The concept of pangenome has transformed the way biological questions are addressed. From understanding evolution and adaptation to elucidating host–pathogen interactions, finding novel genes or breeding targets to aid crop improvement to design effective vaccines for human prophylaxis, the increasing availability of the pangenome has revolutionized several aspects of biological research. The future availability of high-resolution pangenomes based on reference-level near-complete genome assemblies would greatly improve our ability to address complex biological problems.

Assessment of chimeric antigen receptor T cytotoxicity by droplet microfluidics in vitro.

Chimeric antigen receptor T (CAR-T) cells are cytotoxic T cells engineered to specifically kill cancer cells expressing specific target receptor(s). Prior CAR-T efficacy tests include CAR expression analysis by qPCR or ELISA, in vitro measurement of interferon-γ (IFNγ) or interleukin-2 (IL-2), and xenograft models. However, the in vitro measurements did not reflect CAR-T cytotoxicity, whereas xenograft models are low throughput and costly. Here, we presented a robust in vitro droplet microfluidic assay for CAR-T cytotoxicity assessment. This method not only enabled assessment of CAR-T cytotoxic activity under different fluid viscosity conditions, but also facilitated measurement of CAR-T expansion and dissection of mechanism of action via phenotype analysis in vitro. Furthermore, our data suggested that label-free cytotoxicity analysis is feasible by acquiring data before and after treatment. Hence, this study presented a novel in vitro method for assessment of cellular cytotoxicity that could potentially be applied to any cytotoxicity experiment with varying solvent composition.

Targeting circDGKD Intercepts TKI's Effects on Up-Regulation of Estrogen Receptor β and Vasculogenic Mimicry in Renal Cell Carcinoma.

Simple SummaryOur aim was to elucidate the molecular mechanisms of how tyrosine kinase inhibitors, including sunitinib, contribute to vasculogenic mimicry (VM) formation and progression in renal cell carcinoma. We demonstrated that sunitinib and axitinib could induce ERβ expression in RCC cell lines and ERβ transcriptionally up-regulated the circular RNA of DGKD (circDGKD, hsa_circ_0058763) expression. The circDGKD could sponge tumor suppressor miR-125-5p family members and consequently led to increased VE-cadherin, the key adhesion molecule in the VM formation process, which is targeted by miR-125-5p at the 3′ UTR, providing novel targets for combination therapy in clinical metastatic RCC patients.Vasculogenic mimicry (VM) has been reported as an alternative channel to increase tumor nutrient supplies and accelerate tumor progression, and is associated with poor survival prognosis in multiple cancers, including renal cell carcinoma (RCC). The currently used anti-angiogenic treatment for metastatic RCC, sunitinib, a tyrosine kinase inhibitor (TKI), has been reported to induce VM formation. Previously we identified that the estrogen receptor β (ERβ) functions as an oncogenic factor to promote RCC progression, supported by the analytic results from The Cancer Genome Atlas (TCGA) database. We have also found evidence that sunitinib induces RCC VM formation by up-regulating ERβ expression. In this study, we further demonstrated that treatment with sunitinib, as well as axitinib, another TKI, could induce ERβ expression in RCC cell lines. Clinical clear cell RCC (ccRCC) patients with higher ERβ expression are more likely to be found VE-cadherin positive and VM positive. Mechanism dissection showed that TKI- induced ERβ transcriptionally up-regulates the circular RNA of DGKD (circDGKD, hsa_circ_0058763), which enhances VE-cadherin expression by sponging the microRNA miR-125-5p family. Targeting circDGKD intercepts sunitinib-pretreatment-induced RCC VM formation, reduces metastases and improves survival in an experimental orthotopic animal model. Targeting ERβ/circDGKD signals may improve the TKI efficacy and provide novel combination therapies for metastatic RCC.

High-dose-androgen-induced autophagic cell death to suppress the Enzalutamide-resistant prostate cancer growth via altering the circRNA-BCL2/miRNA-198/AMBRA1 signaling

Androgen deprivation therapy (ADT) is a gold standard treatment for advanced PCa. However, most patients eventually develop the castration-resistant prostate cancer (CRPC) that progresses rapidly despite ongoing systemic androgen deprivation. While early studies indicated that high physiological doses of androgens might suppress rather than promote PCa cell growth in some selective CRPC patients, the exact mechanism of this opposite effect remains unclear. Here we found that Enzalutamide-resistant (EnzR) CRPC cells can be suppressed by the high-dose-androgen (dihydrotestosterone, DHT). Mechanism dissection suggested that a high-dose-DHT can suppress the circular RNA-BCL2 (circRNA-BCL2) expression via transcriptional regulation of its host gene BCL2. The suppressed circRNA-BCL2 can then alter the expression of miRNA-198 to modulate the AMBRA1 expression via direct binding to the 3′UTR of AMBRA1 mRNA. The consequences of high-dose-DHT suppressed circRNA-BCL2/miRNA-198/AMBRA1 signaling likely result in induction of the autophagic cell death to suppress the EnzR CRPC cell growth. Preclinical studies using in vivo xenograft mouse models also demonstrated that AMBRA1-shRNA to suppress the autophagic cell death can weaken the effect of high-dose-DHT on EnzR CRPC tumors. Together, these in vitro and in vivo data provide new insights for understanding the mechanisms underlying high-dose-DHT suppression of the EnzR CRPC cell growth, supporting a potential therapy using high-dose-androgens to suppress CRPC progression in the future.

Fat mass and obesity associated (FTO)-mediated N6-methyladenosine modification of Krüppel-like factor 3 (KLF3) promotes osteosarcoma progression

ARSTRACT N6-methyladenosine (m6A) methylation is the most common and abundant methylation modification of eukaryotic mRNAs, which is involved in tumor initiation and progression. The study aims to explore the potential role and the regulatory mechanism of fat mass and obesity associated (FTO) in osteosarcoma (OS) progression. In this study, we detected the expressions of Krüppel-like factor 3 (KLF3) in OS cells and tissues and found that the mRNA and protein levels of KLF3 were increased in OS cells and tissues and significantly related to tumor size, metastasis, and TNM stage and poor prognosis of OS patients. FTO promoted the proliferation and invasion and suppressed apoptosis of OS cells through cell experiments in vitro. Further mechanism dissection revealed that FTO and YTHDF2 enforced the decay of KLF3 mRNA and decreased its expression. FTO-mediated mRNA demethylation inhibited KLF3 expression in the YTHDF2-dependent manner. Moreover, KLF3 overexpression abrogated FTO-induced oncogenic effects on the proliferation and invasion of OS cells. Overall, our findings showed that FTO-mediated m6A modification of KLF3 promoted OS progression, which may provide a therapeutic target for OS.

KLF8 is activated by TGF-β1 via Smad2 and contributes to ovarian cancer progression.

Krüppel-like factor 8 (KLF8) is a transcription factor expressed abnormally in various cancer types and promotes oncogenic transformation. However, the role of KLF8 in ovarian cancer (OC) progression remains unclear. This study reports that transforming growth factor-β1(TGF-β1)/Smad2/KLF8 axis regulates epithelial-mesenchymal transition (EMT) and contributes to OC progression. We analyzed the KLF8 expression in OC cells and tissues, wherein a significant overexpression of KLF8 was observed. Increased KLF8 expressions were correlated with higher cell proliferation, EMT, migration, and invasion and conferred poor clinical outcomes in OC patients. Overexpressed KLF8 increases F-actin polymerization and induces cytoskeleton remodeling of OC cells. Furthermore, a dissection of the molecular mechanism defined that TGF-β1 triggers KLF8 through the Smad2 pathway and regulates EMT. Pharmacological and genetic inhibition of Smad2 followed by TGF-β1 treatment failed to activate KLF8 expression and induction of EMT. Using promoter-luciferase reporter assays, we defined that upon TGF-β1 activation, phosphorylated Smad2 binds and promotes the KLF8 promoter activity, and knockdown of Smad2 inhibits KLF8 promoter activation. Together, these results demonstrate that TGF-β1 activates KLF8 expression by the Smad2 pathway, and KLF8 contributes to OC progression and may serve as a potential therapeutic strategy for treating OC patients.

Application of omics in hypertension and resistant hypertension.

Hypertension is a major modifiable risk factor that affects the global health burden. Despite the availability of multiple antihypertensive drugs, blood pressure is often not optimally controlled. The prevalence of true resistant hypertension in treated hypertensive patients is ~2-20%, and these patients are at higher risk for adverse events and poor clinical outcomes. Therefore, an in-depth dissection of the pathophysiological mechanisms of hypertension and resistant hypertension is needed to identify more effective targets for regulating blood pressure. Omics technologies, such as genomics, transcriptomics, proteomics, metabolomics, and microbiomics, can accurately present the characteristics of organisms at varying molecular levels. Integrative omics can further reveal the network of interactions between molecular levels and provide a complete dynamic view of the organism. In this review, we describe the applications, progress, and challenges of omics technologies in hypertension. Specifically, we discuss the application of omics in resistant hypertension. We believe that omics approaches will produce a better understanding of the pathogenesis of hypertension and resistant hypertension and improve diagnostic and therapeutic strategies, thus increasing rates of blood pressure control and reducing the public health burden of hypertension.

A comprehensive analysis of FOX family in HCC and experimental evidence to support the oncogenic role of FOXH1.

Hepatocellular carcinoma (HCC) remains the second leading cause of cancer related deaths worldwide. Understanding about the molecular biology of HCC and development of targeted therapies are still the main focuses of this type of disease. Here, by connecting the expression levels of FOX proteins with their associated clinical characteristics using TCGA LIHC dataset, we found that 27/40 FOX proteins were highly expressed in HCC tumors compared to normal liver tissues and their expression levels were tightly associated with HCC tumor stage, tumor grade and overall survival. Our experimental results also confirmed that FOXH1 indeed played an oncogenic role in HCC development by promoting cell growth and cell migration/invasion. Mechanistic dissection demonstrated that FOXH1-induced cell growth and cell migration/invasion relied on mTOR signaling because inhibition of mTOR signaling by rapamycin could attenuate FOXH1-mediated phenotypic alterations of HCC cells. The results from orthotopic mouse model also validated that FOXH1 promoted HA22T tumor growth via triggering mTOR activation. Overall, this study not only comprehensively examines the clinical values of FOX proteins in HCC but also provides experimental evidence to support the role of FOXH1 in HCC development, building rationale to develop more effective therapies to treat HCC patients.

Excitability properties of mouse and human skeletal muscle fibres compared by muscle velocity recovery cycles

Mouse models of skeletal muscle channelopathies are not phenocopies of human disease. In some cases (e.g. Myotonia Congenita) the phenotype is much more severe, whilst in others (e.g. Hypokalaemic periodic paralysis) rodent physiology is protective. This suggests a species’ difference in muscle excitability properties. In humans these can be measured indirectly by the post-impulse changes in conduction velocity, using Muscle Velocity Recovery Cycles (MVRCs). We performed MVRCs in mice and compared their muscle excitability properties with humans. Mouse Tibialis Anterior MVRCs (n = 70) have only one phase of supernormality (increased conduction velocity), which is smaller in magnitude (p = 9 × 10−21), and shorter in duration (p = 3 × 10−24) than human (n = 26). This abbreviated supernormality is followed by a period of late subnormality (reduced velocity) in mice, which overlaps in time with the late supernormality seen in human MVRCs. The period of late subnormality suggests increased t-tubule Na+/K+-pump activity. The subnormal phase in mice was converted to supernormality by blocking ClC-1 chloride channels, suggesting relatively higher chloride conductance in skeletal muscle. Our findings help explain discrepancies in phenotype between mice and humans with skeletal muscle channelopathies and potentially other neuromuscular disorders. MVRCs are a valuable new tool to compare in vivo muscle membrane properties between species and will allow further dissection of the molecular mechanisms regulating muscle excitability.

Methods for the Comprehensive in vivo Analysis of Energy Flux, Fluid Homeostasis, Blood Pressure, and Ventilatory Function in Rodents.

Cardiovascular disease represents the leading cause of death in the United States, and metabolic diseases such as obesity represent the primary impediment to improving cardiovascular health. Rodent (mouse and rat) models are widely used to model cardiometabolic disease, and as a result, there is increasing interest in the development of accurate and precise methodologies with sufficiently high resolution to dissect mechanisms controlling cardiometabolic physiology in these small organisms. Further, there is great utility in the development of centralized core facilities furnished with high-throughput equipment configurations and staffed with professional content experts to guide investigators and ensure the rigor and reproducibility of experimental endeavors. Here, we outline the array of specialized equipment and approaches that are employed within the Comprehensive Rodent Metabolic Phenotyping Core (CRMPC) and our collaborating laboratories within the Departments of Physiology, Pediatrics, Microbiology & Immunology, and Biomedical Engineering at the Medical College of Wisconsin (MCW), for the detailed mechanistic dissection of cardiometabolic function in mice and rats. We highlight selected methods for the analysis of body composition and fluid compartmentalization, electrolyte accumulation and flux, energy accumulation and flux, physical activity, ingestive behaviors, ventilatory function, blood pressure, heart rate, autonomic function, and assessment and manipulation of the gut microbiota. Further, we include discussion of the advantages and disadvantages of these approaches for their use with rodent models, and considerations for experimental designs using these methods.

SmbHLH60 and SmMYC2 antagonistically regulate phenolic acids and anthocyanins biosynthesis in Salvia miltiorrhiza

IntroductionSalvia miltiorrhiza is a renowned traditional Chinese medicinal plant with extremely high medicinal value, especially for cardiovascular and cerebrovascular diseases. The jasmonic acid (JA) signaling pathway plays an important role in the improved biosynthesis of secondary metabolites, which is mediated by a major transcriptional regulator, MYC2. However, the JA regulatory mechanism of secondary metabolites biosynthesis in S. miltiorrhiza is still largely unknown. ObjectivesOur work focuses on the dissection of the molecular mechanism of transcriptional regulation in MeJA-mediated biosynthesis of medicinal components of S. miltiorrhiza. We examined the role of MeJA-responsive bHLH transcription factors (TFs) in improving bioactive secondary metabolites accumulation in S. miltiorrhiza. MethodsHairy root transformation based on CRISPR/Cas9 technique was used to decipher gene function(s). Changes in the content of phenolic acids were evaluated by HPLC. Y1H, EMSA and dual-LUC assays were employed to analyze the molecular mechanism of SmbHLH60 in the regulation on the biosynthesis of phenolic acids and anthocyanins. Y2H, BiFC and pull-down affinity assays were used to corroborate the interaction between SmbHLH60 and SmMYC2. ResultsBeing one of the most significantly negatively regulated bHLH genes by MeJA, a new transcription factor SmbHLH60 was discovered and characterized. Over-expression of SmbHLH60 resulted in significant inhibition of phenolic acid and anthocyanin biosynthesis in S. miltiorrhiza by transcriptionally repressing of target genes such as SmTAT1 and SmDFR, whereas CRISPR/Cas9-generated knockout of SmbHLH60 resulted in the opposite effect. In addition, SmbHLH60 and SmMYC2 formed a heterodimer to antagonistically regulate phenolic acid and anthocyanin biosynthesis. ConclusionOur results clarified that SmbHLH60 is a negativeregulator on the biosynthesis of phenolic acids and anthocyanins. SmbHLH60 competed with SmMYC2 in an antagonistic manner, providing new insights for the molecular mechanism of MeJA-mediated regulation on the biosynthesis of secondary metabolites in S. miltiorrhiza.

The Histone Acetyltransferase MOF Regulates SIRT1 Expression to Suppress Renal Cell Carcinoma Progression.

BackgroundRenal cell carcinoma (RCC) is one of the most common and lethal human urological malignancies around the world. Although many advancements in diagnostic and therapeutic strategies have been acquired, the prognosis of patients with metastatic RCC was poor. Thus, there is an urgent need to understand the molecular mechanism of RCC.MethodsThe quantitative real-time PCR (qRT-PCR) was used to detect the RNA expression of MOF in human RCC tissues and cell lines. The protein expression of MOF was analyzed with immunohistochemistry (IHC) and Western blot. To understand the regulatory mechanism of MOF in liver cancer, ChIP-qPCR assay and dual-luciferase assay were performed. Moreover, a series of in vivo and in vitro experiments were conducted to evaluate the effect of MOF on renal cell carcinoma progression.ResultsIn the present study, we found that Males absent on the first (MOF), a histone acetyltransferase involved in transcription activation, was significantly decreased in both RCC tissues and RCC cells compared to normal tissues and non-cancer cells. Moreover, MOF downregulation was associated with advanced histological grade, pathologic stage and distant metastasis of RCC patients. Ectopic expression of MOF could significantly attenuate cell proliferation and promote cell apoptosis. Besides, MOF overexpression also suppressed migration of RCC cells through inhibiting epithelial-mesenchymal transition (EMT). Importantly, the inhibition of tumor growth by MOF was further confirmed by in vivo studies. Mechanism dissection revealed that MOF could transcriptionally upregulate the expression of SIRT1, leading to attenuated STAT3 signaling, which was involved in cell proliferation and migration. Moreover, SIRT1 knockdown could restore the biological function induced by MOF overexpression.ConclusionsOur findings indicated that MOF serves as a tumor suppressor via regulation of SIRT1 in the development and progression of RCC, and MOF might be a potent biomarker for diagnosis and prognosis prediction of RCC patients.

Abstract P5-09-01: Using isogenic model systems to determine mechanisms regulating mutant p53 protein stability in breast cancer cells

Abstract Two of the most common events in triple-negative breast cancers (TNBC) are mutation of the tumor suppressor gene TP53 and development of aneuploidy. This is often the case in aggressive and molecularly heterogeneous cancer subtypes such as TNBC, where ~80% of cases harbor a mutation in TP53. In addition to losing wild-type (WT) tumor-suppressive function, mutant p53 proteins are proposed to acquire gain-of-function (GOF) activity, leading to novel oncogenic phenotypes. Mutant p53 proteins can accumulate to high levels in tumors, and this stabilization is thought to be a pre-requisite for mutant p53 to exert its GOF effects. Mechanisms underlying the accumulation of mutant p53 are not well understood. To study mutant p53 GOF mechanisms and phenotypes, we used CRISPR/Cas9-mediated genome editing and developed two isogenic TNBC cell line models (one non-transformed and one tumor-derived) that express the most frequently occurring p53 missense mutations (R175H and R273H), are deficient for functional p53 protein (null), or retain the wild-type (WT) protein. In these engineered models, endogenous p53 expression is regulated by the native p53 promoter, thus providing a controlled system for rigorous functional experimentation across different p53 states. Through functional genomics analyses comparing isogenic TNBC cell lines, which initially differed only by the TP53 genotype, we have evaluated the relationship between mutant p53 and development of aneuploidy and determined that development of aneuploidy and not TP53 genotype contributed to several previously reported mutant p53 GOF phenotypes in vitro and in vivo1. Immunoblotting, immunofluorescence, and immunohistochemistry experiments revealed that mutant p53-expressing cell lines display a range of p53 protein levels, with several maintaining low levels of p53, similar to the cell lines which contain WT p53. In addition, mutant p53 levels correlate with aneuploidy, but not mean nuclear area or whole-genome doubling status, and protein levels increase over time in cell lines that become aneuploid. Using our various clonal cell lines that exhibit differing p53 protein stability, we are currently determining if p53 stability is dysregulated in aneuploid cell lines after copy number gain or loss of genes encoding critical p53 regulators, leading to the accumulation of the mutant protein. Analysis of cancer cell line genetic dependencies and human tumor data from The Cancer Genome Atlas (TCGA) has identified genetic and clinical correlates with increased mutant p53 protein levels. Utilizing biochemical and bioinformatic approaches, we aim to uncover mechanisms for increased mutant p53 stability and identify novel regulators of both WT and mutant p53 proteins. The dissection of mechanisms that contribute to stabilization of p53 protein will not only give insight into the regulation and tumor suppressive function of WT p53, but it has the potential for clinical translation in human cancers that have high-frequency p53 mutation and high levels of mutant p53 protein, as targeting mutant p53 through pharmacologic inhibition or antibody-based therapies is an active area of investigation. 1Article in press in Nature Communications Citation Format: Lindsay Redman-Rivera, Hailing Jin, Brian D Lehmann, Jennifer A Pietenpol. Using isogenic model systems to determine mechanisms regulating mutant p53 protein stability in breast cancer cells [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-09-01.

Dissection of the antibacterial mechanism of zinc oxide nanoparticles with manipulable nanoscale morphologies

Despite the extensive uses of ZnO nanoparticles as promising antimicrobial agents to tackle the severe microbial infections, the systematic antibacterial studies on ZnO nanoparticles with manipulable nanoscale morphologies at the genetic expression level remain ill-defined. In this study, via a controllable thermal decomposition, ZnO nanoparticles of different morphologies were facilely prepared. Additionally, the surface PEGylation of ZnO was conducted to obtain the nanoparticles of low biotoxicity. While all the prepared ZnO nanoparticles exhibited the significantly chemical activities, the pronounced antibacterial effect of obtained ZnO nanoparticles was also identified, in which the ultra-small ones (~5 nm) showed the best performance. Moreover, the antibacterial activities of ZnO nanoparticles were studied by bacterial nucleic acid leakage, alkaline phosphatase, biofilm and reactive oxygen species (ROS) assays. Furthermore, the transcriptome analysis of ZnO nanoparticles with different morphologies against Escherichia coli (E. coli) revealed the underlying antibacterial mechanism involved the signal transduction, material transport, energy metabolism and other biological processes. Therefore, the cost-effective preparation of ZnO nanoparticles with distinct morphological features provides insights for the development of application specific antibacterial agents.

CYLD deficiency enhances metabolic reprogramming and tumor progression in nasopharyngeal carcinoma via PFKFB3

Aberrant cancer metabolism contributes to cell proliferation and tumor progression. However, the contribution of enhanced glycolysis, observed during cancer metabolism, to the pathogenesis and progression of nasopharyngeal carcinoma (NPC) remains unclear. CYLD, an NF-κB inhibitor, is frequently deficient in NPC. Here, we investigated the role of CYLD in the metabolic reprogramming of NPC and found that restoration of CYLD expression suppressed glycolysis in NPC cells. Mechanistic dissection showed that CYLD stabilized p53 and facilitated its nuclear translocation, thereby enhancing p53 activity by removing K63-linked and K48-linked ubiquitin chains of p53, which can bind to the PFKFB3 promoter and inhibit its transcription. Additionally, CYLD interacted with FZR1 to promote APC/C-FZR1 E3 ligase activity, which further ubiquitinated and degraded PFKFB3 via the 26S proteasomal system. Furthermore, clinical tissue array analysis indicated that low expression of CYLD was correlated with high expression of PFKFB3 and poor prognosis among patients with NPC. In conclusion, CYLD suppressed PFKFB3 expression via two factors, namely, p53 and FZR1, to inhibit glycolysis and delay tumor growth and progression in NPC. CYLD is a biomarker indicating poor prognosis of patients with NPC.

Single-molecule dissection of coupling mechanisms between cofactor binding and folding of a complex protein

Dissecting protein folding pathways is challenging, especially for large or multidomain proteins. This challenge is further exacerbated when considering that many proteins harbor one or more cofactors in their structure, which can alter their fold and thermodynamic properties. Here, we use optical tweezers to mechanically unfold and refold drosophila cryptochrome (dCRY), a large, multidomain protein that harbors a FAD cofactor in its structure. By applying force and simultaneously varying the FAD concentration and the refolding and rebinding dwell time, we dissect the mechanisms that coupled dCRY folding and FAD binding.