Biology subjects

Deng, J.

Publications and source records attributed to Deng, J..

At least 109 records · Page 6Linked to original sources

bioRxiv2022

Activation of the cGAS-STING innate immune response in cells with deficient mitochondrial topoisomerase TOP1MT

The recognition that cytosolic mtDNA activates cGAS-STING innate immune signaling has unlocked novel disease mechanisms. Here, an uncharacterized variant predicted to affect TOP1MT function, P193L, was discovered in a family with multiple early-onset autoimmune diseases, including Systemic Lupus Erythematosus (SLE). Although there was no previous genetic association between TOP1MT and autoimmune disease, the role of TOP1MT as a regulator of mtDNA led us to investigate whether TOP1MT could mediate the release of mtDNA to the cytosol, where it could then activate the cGAS-STING innate immune pathway known to be activated in SLE and other autoimmune diseases. Through analysis of cells with reduced TOP1MT expression, we show that loss of TOP1MT results in release of mtDNA to the cytosol, which activates the cGAS-STING pathway. We also characterized the P193L variant for its ability to rescue several TOP1MT functions when expressed in TOP1MT knockout cells. We show that the P193L variant is not fully functional, as its re-expression at high levels was unable to rescue mitochondrial respiration deficits, and only showed partial rescue for other functions, including repletion of mtDNA replication following depletion, nucleoid size, steady state mtDNA transcripts levels, and mitochondrial morphology. Additionally, expression of P193L at endogenous levels was unable to rescue mtDNA release-mediated cGAS-STING signaling. Overall, we report a link between TOP1MT and mtDNA release leading to cGAS-STING activation. Moreover, we show that the P193L variant has partial loss of function that may contribute to autoimmune disease susceptibility via cGAS-STING mediated activation of the innate immune system.

cell biology↗

bioRxiv2021

Interactions of host defense and hyper-keratinization in psoriasis

ObjectivesTo understand the crosstalk between the host and microbiota in psoriatic skin, using a systems biology approach based on transcriptomics and microbiome profiling. MethodsWe collected the skin tissue biopsies and swabs in both lesion and non-lesion skin of 13 patients with psoriasis (PsO), 15 patients with psoriatic arthritis (PsA), and healthy skin from 12 patients with ankylosing spondylitis (AS). We performed transcriptome sequencing and metagenomics profiling on the local skin sites to study the similarities and differences in the molecular profiles between the three conditions, and the associations between the host defense and microbiota dynamic. ResultsWe found that lesion and non-lesional samples were remarkably different in terms of their transcriptome profiles. Functional annotation of differentially expressed genes (DEGs) showed a major enrichment in neutrophil activation. By using coexpression gene networks, we identified a gene module that was associated with local psoriasis severity at the site of biopsy. From this module, we extracted a "core" set of genes that were functionally involved in neutrophil activation, epidermal cell differentiation and response to bacteria. Skin microbiome analysis revealed that the abundance of Enhydrobacter, Micrococcus and Leptotrichia were significantly correlated with the "core network" of genes. ConclusionsWe identified a core network that regulates inflammation and hyper-keratinization in psoriatic skin, and is associated with local disease severity and microbiome composition.

systems biology↗

bioRxiv2021

Assessment of AI-based Protein Structure Prediction for the NLRP3 Target

The recent success of AlphaFold and RoseTTAFold has demonstrated the values of AI methods in predicting highly accurate protein structures. Despite the advances, their roles in the context of small-molecule drug discovery need to be thoroughly explored. In this study, we evaluated whether the AI-based models can lead to reliable three-dimensional structures of protein-ligand complexes. The structure we predicted was NLRP3, a challenging protein target in terms of obtaining the three-dimensional model both experimentally and computationally. The conformation of the binding pockets generated by the AI models were carefully characterized and compared with experimental structures. Further molecular docking results indicated that AI-predicted protein structures combined with molecular dynamics simulations can be useful in small-molecule drug discovery.

biochemistry↗

bioRxiv2021

Integrating Task-Based Functional MRI Across Tasks Markedly Boosts Prediction and Reliability of Brain-Cognition Relationship

Capturing individual differences in cognition is central to human neuroscience. Yet our ability to estimate cognitive abilities via brain MRI is still poor in both prediction and reliability. Our study tested if this inability can be improved by integrating MRI signals across the whole brain and across modalities, including task-based functional MRI (tfMRI) of different tasks along with other non-task MRI modalities, such as structural MRI, resting-state functional connectivity. Using the Human Connectome Project (n=873, 473 females, after quality control), we directly compared predictive models comprising different sets of MRI modalities (e.g., seven tasks vs. non-task modalities). We applied two approaches to integrate multimodal MRI, stacked vs. flat models, and implemented 16 combinations of machine-learning algorithms. The stacked model integrating all modalities via stacking Elastic Net provided the best prediction (r=.57), relatively to other models tested, as well as excellent test-retest reliability (ICC=~.85) in capturing general cognitive abilities. Importantly, compared to the stacked model integrating across non-task modalities (r=.27), the stacked model integrating tfMRI across tasks led to significantly higher prediction (r=.56) while still providing excellent test-retest reliability (ICC=~.83). The stacked model integrating tfMRI across tasks was driven by frontal and parietal areas and by tasks that are cognition-related (working-memory, relational processing, and language). This result is consistent with the parieto-frontal integration theory of intelligence. Accordingly, our results contradict the recently popular notion that tfMRI is not reliable enough to capture individual differences in cognition. Instead, our study suggests that tfMRI, when used appropriately (i.e., by drawing information across the whole brain and across tasks and by integrating with other modalities), provides predictive and reliable sources of information for individual differences in cognitive abilities, more so than non-task modalities. HighlightsO_LINon-task MRI (sMRI, rs-fMRI) are often used for the brain-cognition relationship. C_LIO_LITask-based fMRI has been deemed unreliable for capturing individual differences. C_LIO_LIWe tested if drawing task-based fMRI information across regions/tasks improves prediction and reliability of the brain-cognition relationship. C_LIO_LIOur approach boosts prediction of task-based fMRI over non-task MRI. C_LIO_LIOur approach renders task-based fMRI reliable over time. C_LIO_LIOur approach shows the importance of the fronto-parietal areas in cognition. C_LI

neuroscience↗

bioRxiv2021

RUNIMC: An R-based package for imaging mass cytometry data analysis and pipeline validation

We propose a novel pipeline for the analysis of imaging mass cytometry data, comparing an unbiased approach, representing the actual gold standard, with a novel biased method. We made use of both synthetic/ controlled datasets as well as two datasets obtained from FFPE sections of follicular lymphoma, and head and neck patients, stained with a 14 and 29-markers panels respectively. The novel pipeline, denominated RUNIMC, has been completely developed in R and contained in a single package. The novelty resides in the ease with which multi-class random forest classifier can be used to classify image features, making the pathologists and expert classification pivotal, and the use of a random forest regression approach that permits a better detection of cell boundaries, and alleviates the necessity of relying on a perfect nuclear staining.

cancer biology↗

bioRxiv2021

Sex-dependent Lupus Ruminococcus blautia gnavus strain induction of zonulin-mediated intestinal permeability and autoimmunity

Imbalances in the gut microbiome are suspected as contributors to the pathogenesis of Systemic Lupus Erythematosus, and our studies and others have documented that patients with active Lupus nephritis have expansions of the obligate anaerobe, Ruminococcus blautia gnavus (RG). To investigate whether the RG strains in Lupus patients have in vivo pathogenic properties, we colonized C57BL/6 mice with individual RG strains from healthy adults or those from Lupus patients. These strains had a similar capacity for murine intestinal colonization, in antibiotic-preconditioned specific-pathogen-free, as well as germ-free adults, and their neonatally colonized litters. Lupus-derived RG strains induced high levels of intestinal permeability that was significantly greater in female than male mice, whereas the RG species-type strain (ATCC29149/VPI C7-1) from a healthy donor had little or no effects. Lupus RG strain-induced functional alterations were associated dysregulated occluden transcript production in the ileal wall as well as raised serum levels of zonulin, a regulator of tight junction formation between cells that form the gut barrier. Notably, the level of Lupus RG-induced intestinal permeability was significantly correlated with serum IgG anti RG cell-wall lipoglycan antibodies, and to anti-native DNA autoantibodies that are a biomarker for SLE. Strikingly, gut permeability was completely reversed by oral treatment with larazotide acetate, an octapeptide that is a specific molecular antagonist of zonulin. Taken together, these studies document a molecular pathway by which RG strains from Lupus patients induce a leaky gut and autoimmunity that have been implicated in the pathogenesis of flares of clinical Lupus disease.

microbiology↗

bioRxiv2021

DAB2IP inhibits p53 ubiquitin-mediated degradation by competitively binding to GRP75 and suppresses tumor malignancy in colon cancer

Increasing evidence has shown that DAB2IP acts as a tumor suppressor and plays an inhibition role in many tumors. However, the underlying mechanism is still uncertain. Our study shows that DAB2IP is positively associated with a better prognosis in colon cancer patients with wild-type TP53 expression. In vitro assay shows that DAB2IP elicits potent tumor-suppressive effects on inhibiting cell invasiveness, colony formation and promoting cell apoptosis in wild-type TP53 colon cancer cell lines. Subsequently, DAB2IP is demonstrated to up-regulate the stability of wild-type TP53 by inhibiting its degradation in a ubiquitin-proteasome-dependent manner. Using mass spectrometry profiling, we unveil that DAB2IP and p53 could both interact with the ubiquitin ligase-related protein, GRP75. Mechanistically, DAB2IP could competitively bind with GRP75, thus reducing GRP75-mediated p53 ubiquitination and degradation. Finally, animal experiments also reveal that DAB2IP inhibits the tumor progression in vivo. In conclusion, our study presents a novel function of DAB2IP in GRP75-driven wild-type p53 degradation, which provides a new insight in DAB2IP-induced tumor suppression and provides a novel molecular aspect of the p53 pathway.

cancer biology↗

bioRxiv2021

An exploration of metabolic and transcriptional responses in mouse skeletal muscles responding to acute sedentary hypoxia

Skeletal muscles are involved in responses to acute hypoxia as the largest organ in the body. However, as a hypoxic-tolerant tissue, responses in skeletal muscles caused by acute sedentary hypoxia are much less studied. We measured metabolites in skeletal muscles from mice exposed to 8% O2 for 0 minute, 15 minutes and 2 hours and studied the potential relationship between metabolite levels and mRNA levels by reconstructing genome-based metabolic networks and meta-analyzing differentially expressed genes acquired in skeletal muscles after 2 hours of 8% O2 exposure. The metabolite measurement indicated a significant increase in glutamine metabolism but not lactate metabolism in mouse skeletal muscles after 2 hours of hypoxia, where the metabolic responses as a whole were moderate. The central-dogma based metabolic flux analysis suggested an involvement of glutamine metabolism, though, as a whole, metabolite changes and gene changes didnt show a high correlation. Among metaoblites, glutamine metabolism indicated a significant response and a consistent change which could be interpreted by genome-based network analysis. In summary, though this study suggested a moderate metabolic response which has a weak correlation with gene expression change as a whole, glutamine metabolism indicated rapid responses in skeletal muscles responding to acute sedentary hypoxia.

systems biology↗

bioRxiv2021

A proteomics sample metadata representation for multiomics integration, and big data analysis.

The amount of public proteomics data is increasing at an extraordinary rate. Hundreds of datasets are submitted each month to ProteomeXchange repositories, representing many types of proteomics studies, focusing on different aspects such as quantitative experiments, post-translational modifications, protein-protein interactions, or subcellular localization, among many others. For every proteomics dataset, two levels of data are captured: the dataset description, and the data files (encoded in different file formats). Whereas the dataset description and data file formats are supported by all ProteomeXchange partner repositories, there is no standardized format to properly describe the sample metadata and their relationship with the dataset files in a way that fully allows their understanding or re-analysis. It is left to the users choice whether to provide or not an ad hoc document containing this information. Therefore, in many cases, understanding the study design and data requires going back to the associated publication. This can be tedious and may be restricted in the case of non-open access publications. In many cases, this problem limits the generalization and reuse of public proteomics data. Here we present a standard representation for sample metadata tailored to proteomics datasets produced by the HUPO Proteomics Standards Initiative and supported by ProteomeXchange resources. We repurposed the existing data format MAGE-TAB used routinely in the transcriptomics field to represent and annotate proteomics datasets. MAGETAB-Proteomics defines a set of annotation rules that the datasets submitted to ProteomeXchange should follow, ranging from sample properties to data analysis protocols. We also introduce a crowdsourcing project that enabled the manual curation of over 200 public datasets using MAGE-TAB-Proteomics. In addition, we describe an ecosystem of tools and libraries that were developed to validate and submit sample metadata-related information to ProteomeXchange. We expect that these tools will improve the reproducibility of published results and facilitate the reanalysis and integration of public proteomics datasets.

bioinformatics↗

bioRxiv2021

Image-based Motion Artifact Reduction on Liver Dynamic Contrast Enhanced MRI

Liver MRI images often suffer degraded quality from ghosting or blurring artifact caused by patient respiratory or bulk motion. In this study, we developed a two-stage deep learning model to reduce motion artifact on dynamic contrast enhanced (DCE) liver MRIs. The stage-I network utilized a deep residual network with a densely connected multi-resolution block (DRN-DCMB) network to remove the majority of motion artifacts. The stage-II network applied the perceptual loss to preserve image structural features by updating the parameters of the stage-I network via backpropagation. The stage-I network was trained using small image patches simulated with five types of motion, i.e., rotational, sinusoidal, random, elastic deformation and through-plane, to mimic actual liver motion patterns. The stage-II network training used full-size images with the same types of motion as the stage-I network. The motion reduction deep learning model was testing using simulated motion images and images with real motion artifacts. The resulted images after two-stage processing demonstrated substantially reduced motion artifacts while preserved anatomic details without image blurriness. This model outperformed existing methods of motion reduction artifact on liver DCE-MRI.

bioinformatics↗

bioRxiv2021

A study of the neuronal injury biomarkers pNF-H and UCHL1 in serum, CSF and urine in a cohort of thoracic endovascular aortic repair (TEVAR) patients.

A collection of longitudinal serum, cerebrospinal fluid (CSF) and urine samples were collected from a cohort of 50 patients undergoing thoracic endovascular aortic repairs (TEVAR). Samples were taken multiple time per day out to 5 days post operation and were probed with novel electrochemiluminescent assays specific for the phosphorylated axonal form of the major neurofilament subunit NF-H (pNF-H) and for ubiquitin C-terminal hydrolase 1 (UCHL1). Control blood samples showed small signals for pNF-H and in some cases rather larger signals for UCHL1. The presence of UCHL1 in these control blood samples was convincingly verified by western blotting with multiple well characterized UCHL1 antibodies and by mass spectroscopy. Elevated levels of both pNF-H and UCHL1 in blood and CSF in recovering TEVAR patients were associated with poorer outcomes. In particular release of UCHL1 into blood over several hours following TEVAR and peaking at any time over 1 ng/ml was a very strong predictor of patient death and was associated with renal failure and spinal cord ischemia (SCI). An unexpected finding was that high levels of UCHL1 were detected in certain urine samples, again in association with SCI, renal failure and poor patient outcome. We also present epitope mapping data on the UCHL1 monoclonal antibodies used including data on the widely used commercially available MCA-BH7. These studies suggest that measurement of the levels of both proteins in the blood, CSF and urine of TEVAR patients may be of clinical utility. However this study also raises questions about the origin and significance of UCHL1 both in control blood, in patient blood samples and in urine.

neuroscience↗

bioRxiv2020

Scalable pooled CRISPR screens with single-cell chromatin accessibility profiling

Pooled CRISPR screens have been used to identify genes responsible for specific phenotypes and diseases, and, more recently, to connect genetic perturbations with multi-dimensional gene expression profiles. Here, we describe a method to link genome-wide chromatin accessibility to genetic perturbations in single cells. This scalable, cost-effective method combines pooled CRISPR perturbations with a single-cell combinatorial indexing assay for transposase-accessible chromatin (CRISPR-sciATAC). Using a human and mouse species-mixing experiment, we show that CRISPR-sciATAC separates single cells with a low doublet rate. Then, in human myelogenous leukemia cells, we apply CRISPR-sciATAC to target 21 chromatin-related genes that are frequently mutated in cancer and 84 subunits and cofactors of chromatin remodeling complexes, generating chromatin accessibility data for ~30,000 single cells. Using this large-scale atlas, we correlate loss of specific chromatin remodelers with changes in accessibility -- globally and at the binding sites of individual transcription factors. For example, we show that loss of the H3K27 methyltransferase EZH2 leads to increased accessibility at heterochromatic regions involved in embryonic development and triggers expression of multiple genes in the HOXA and HOXD clusters. At a subset of regulatory sites, we also analyze dynamic changes in nucleosome spacing upon loss of chromatin remodelers. CRISPR-sciATAC is a high-throughput, low-cost single-cell method that can be applied broadly to study the role of genetic perturbations on chromatin in normal and disease states.

genomics↗

bioRxiv2020

Effect of cutting depth during sugarcane harvest on root characteristics and yield

Ratooning is an important cultivation practice in sugarcane production around the world, with underground buds on the remaining stalk acting as the source for establishment of a subsequent ratoon crop. However, the optimal depth of cutting during harvest in terms of yield and root growth remains unknown. We carried out a two-year field study to determine the effects of three cutting depths (0, 5 and 10 cm below the surface) ratoon cane root and yield. Results showed that cutting to a depth of 5 cm increased the root fresh weight and root volume by 32-40% and 49-85%, respectively, compared to cutting depths of 0 and 10 cm. Remarkably, cutting to a depth of 5 cm also had a significant effect on the development of fine roots, which is closely linked to cane yield. The effect was particularly noticeable in terms of two root traits, root volume and the surface area of roots with a diameter of 1.0-2.0mm, and root length and the number of root tips in roots with a diameter of 0-0.5mm. As a result, a cutting depth of 5 cm below the surface increased cane yield by 35 and 25% compared to depths of 0 and 10 cm below the surface, respectively. Overall, these findings suggest that a cutting depth of 5 cm is optimal in terms of sugarcane yield, largely due to the enhanced effect on root traits, especially the development of fine roots. These findings will help optimize sugarcane ratoon management and improve the ratoon cycle.

plant biology↗

bioRxiv2020

Neonatal LPS exposure results in ATP8A2 down-regulation in the prefrontal cortex and depressive-like behaviors in mice through increasing IFN-γ level

Neonatal lipopolysaccharide (LPS) exposure can cause depressive-like behaviors in rodents involving elevated interferon (IFN)-{gamma}. Studies have linked down-regulation of prefrontal cortex (PFC) ATPase phospholipid transporting 8A2(ATP8A2) expression to depressive-like behaviors. In non-neuronal cells, IFN-{gamma} could reduce ATP8A2 expression. We therefore hypothesized that neonatal LPS exposure might induce PFC ATP8A2 down-regulation by increasing IFN-{gamma} level. Here, C57BL6/J mice of both sexes received 3-dose-injections of LPS (50g/kg bodyweight, i.p.) on postnatal day (PND)5, PND7 and PND9. LPS-treated mice showed a transiently decreased PFC ATP8A2 expression. Moreover, a negative correlation of PFC ATP8A2 expression was found with IFN-{gamma} level. Using neutralizing mAb, IFN-{gamma} was identified as the key mediator of LPS-induced PFC ATP8A2 decrease. Besides, neutralizing IFN-{gamma} during neonatal LPS exposure attenuated the depressive-like behaviors in adulthood. In sum, neonatal LPS exposure reduced ATP8A2 level in PFC in mice via increasing IFN-{gamma} level, maybe associated with mechanism underlying LPS-induced brain and behavior impairments.

neuroscience↗

bioRxiv2020

DDR1-INDUCED NEUTROPHIL EXTRACELLULAR TRAPS DRIVE PANCREATIC CANCER METASTASIS

Pancreatic ductal adenocarcinoma (PDAC) tumors are characterized by a desmoplastic reaction and dense collagen that is known to promote cancer progression. A central mediator of pro-tumorigenic collagen signaling is the receptor tyrosine kinase discoid domain receptor 1 (DDR1). DDR1 is a critical driver of a mesenchymal and invasive cancer cell PDAC phenotype. Previous studies have demonstrated that genetic or pharmacologic inhibition of DDR1 prevents PDAC tumorigenesis and metastasis. Here, we investigated whether DDR1 signaling has cancer cell non-autonomous effects that promote PDAC progression and metastasis. We demonstrate that collagen-induced DDR1 activation in cancer cells is a major stimulus for CXCL5 production, resulting in the recruitment of tumor-associated neutrophils (TANs), the formation of neutrophil extracellular traps (NETs) and subsequent cancer cell invasion and metastasis. Moreover, we have identified that collagen-induced CXCL5 production was mediated by a DDR1-PKC{theta}-SYK-NF{kappa}B signaling cascade. Together, these results highlight the critical contribution of collagen I-DDR1 interaction in the formation of an immune microenvironment that promotes PDAC metastasis. SummaryDeng et al find that collagen signaling via DDR1 on human pancreatic cancer cells drives production and release of the cytokine, CXCL5, into systemic circulation. CXCL5 then triggers infiltration of neutrophils into the tumor where they promote cancer cell progression.

cancer biology↗

bioRxiv2020

Fine-scale Population Structure and Demographic History of Han Chinese Inferred from Haplotype Network of 111,000 Genomes

ABSTRACTHan Chinese is the most populated ethnic group across the globe with a comprehensive substructure that resembles its cultural diversification. Studies have constructed the genetic polymorphism spectrum of Han Chinese, whereas high-resolution investigations are still missing to unveil its fine-scale substructure and trace the genetic imprints for its demographic history. Here we construct a haplotype network consisted of 111,000 genome-wide genotyped Han Chinese individuals from direct-to-consumer genetic testing and over 1.3 billion identity-by-descent (IBD) links. We observed a clear separation of the northern and southern Han Chinese and captured 5 subclusters and 17 sub-subclusters in haplotype network hierarchical clustering, corresponding to geography (especially mountain ranges), immigration waves, and clans with cultural-linguistic segregation. We inferred differentiated split histories and founder effects for population clans Cantonese, Hakka, and Minnan-Chaoshanese in southern China, and also unveiled more recent demographic events within the past few centuries, such as Zou Xikou and Chuang Guandong. The composition shifts of the native and current residents of four major metropolitans (Beijing, Shanghai, Guangzhou, and Shenzhen) imply a rapidly vanished genetic barrier between subpopulations. Our study yields a fine-scale population structure of Han Chinese and provides profound insights into the nation’s genetic and cultural-linguistic multiformity.Competing Interest StatementThe authors AL, KK, ST, XW, LW, TL, HW, JD, QZ, XY, and GC work for WeGene (Shenzhen Zaozhidao Technology Co. Ltd. or Shenzhen WeGene Clinical Laboratory).View Full Text

genomics↗

bioRxiv2020

Calcitriol, the active form of vitamin D, is a promising candidate for COVID-19 prophylaxis

COVID-19, the disease caused by SARS-CoV-2 (1), was declared a pandemic by the World Health Organization (WHO) in March 2020 (2). While awaiting a vaccine, several antivirals are being used to manage the disease with limited success (3, 4). To expand this arsenal, we screened 4 compound libraries: a United States Food and Drug Administration (FDA) approved drug library, an angiotensin converting enzyme-2 (ACE2) targeted compound library, a flavonoid compound library as well as a natural product library. Of the 121 compounds identified with activity against SARS-CoV-2, 7 were shortlisted for validation. We show for the first time that the active form of Vitamin D, calcitriol, exhibits significant potent activity against SARS-CoV-2. This finding paves the way for consideration of host-directed therapies for ring prophylaxis of contacts of SARS-CoV-2 patients.

microbiology↗

bioRxiv2020

Heat Shock Protein 27 versus Estrogen Therapy for Post-Menopausal Atherosclerosis: Rethinking Mechanisms of Cholesterol Lowering

Aims The estrogen-inducible protein Heat Shock Protein 27 (HSP27) as well as anti-HSP27 antibodies are elevated in healthy subjects compared to cardiovascular disease patients. Vaccination of ApoE-/- mice with recombinant HSP25 (rHSP25, the murine ortholog), boosts anti-HSP25 levels and attenuates atherogenesis. As estrogens promote HSP27 synthesis, cellular release and blood levels, we hypothesize that menopause will result in loss of HSP27 atheroprotection. Hence, we now compare the efficacy of rHSP25 vaccination vs. estradiol (E2) therapy for the prevention of post-menopausal atherogenesis.Methods and Results ApoE-/- mice subjected to ovariectomy (OVX) showed a 65% increase atherosclerotic burden compared to sham mice after 5 weeks of a high fat diet. Relative to vaccination with rC1, a truncated HSP27 control peptide, atherogenesis was reduced by 5-weekly rHSP25 vaccinations (−43%), a subcutaneous E2 slow release pellet (−52%) or a combination thereof (−82%). Plasma cholesterol levels declined in parallel with the reductions in atherogenesis, but relative to rC1/OVX mice plasma PCSK9 levels were 52% higher in E2/OVX and 41% lower in rHSP25/OVX mice (p<0.0001 for both). Hepatic LDLR mRNA levels did not change with E2 treatment but increased markedly with rHSP25 vaccination. Conversely, hepatic PCSK9 mRNA increased 148% with E2 treatment vs. rC1/OVX but did not change with rHSP25 vaccination. In human HepG2 hepatocytes E2 increased PCSK9 promoter activity 303%, while the combination of [rHSP27 + PAb] decreased PCSK9 promoter activity by 64%.Conclusion The reduction in post-OVX atherogenesis and cholesterol levels with rHSP25 vaccination is associated with increased LDLR but not PCSK9 expression. Surprisingly, E2 therapy attenuates atherogenesis and cholesterol levels post-OVX without altering LDLR but increases PCSK9 expression and promoter activity. This is the first documentation of increased PCSK9 expression with E2 therapy and raises questions about balancing physiological estrogenic / PCSK9 homeostasis and targeting PCSK9 in women – are there effects beyond cholesterol?Competing Interest StatementEOB and YXC are inventors on US patents 8343915B2 and 8343916B2; EOB, CS and YXC are inventors on US patent application PCT/CA2016/051018; EOB is the inventor on US patent application 63/031,6402020; all intellectual property pertains to HSP27 diagnostics / therapeutics. EOB is the Scientific Co-Founder of Pemi31 Therapeutics Inc., a startup company that controls the aforementioned intellectual property. EOB, CS and YXC have equity interests in Pemi31 Therapeutics Inc.AbbreviationsAAbanti-HSP27 (or anti-HSP25) antibodiesApoE-/-apolipoprotein E nulla.u.absorbance unitsE217β estradiolHFDhigh fat dietHPRThypoxanthine-phosphoribosyltransferaseHSP27Heat Shock Protein 27HSP27o/eHSP27 over-expressingLDL-Clow density lipoprotein cholesterolLDLRlow density lipoprotein receptorMPmenopausemRNAmessenger RNANF-κBnuclear factor kappa light chain enhancer of activated B cellsONovernightOVXovariectomyPAbpolyclonal anti-HSP27 IgG antibodyPBSphosphate buffered salinePCSK9Proprotein Convertase Subtilisin/Kexin type 9rC1recombinant C-terminal truncation of HSP27 spanning amino acids 90-205rHSP27recombinant HSP27SDS-PAGEsodium dodecyl sulfate and polyacrylamide gelWBWestern BlotView Full Text

pathology↗