ABSTRACT
Research over the past decade has suggested important roles for pseudogenes in physiology and disease. In vitro experiments demonstrated that pseudogenes contribute to cell transformation through several mechanisms. However, in vivo evidence for a causal role of pseudogenes in cancer development is lacking. Here, we report that mice engineered to overexpress either the full-length murine B-Raf pseudogene Braf-rs1 or its pseudo "CDS" or "3' UTR" develop an aggressive malignancy resembling human diffuse large B cell lymphoma. We show that Braf-rs1 and its human ortholog, BRAFP1, elicit their oncogenic activity, at least in part, as competitive endogenous RNAs (ceRNAs) that elevate BRAF expression and MAPK activation in vitro and in vivo. Notably, we find that transcriptional or genomic aberrations of BRAFP1 occur frequently in multiple human cancers, including B cell lymphomas. Our engineered mouse models demonstrate the oncogenic potential of pseudogenes and indicate that ceRNA-mediated microRNA sequestration may contribute to the development of cancer.
Subject(s)
Lymphoma, Large B-Cell, Diffuse/genetics , Proto-Oncogene Proteins B-raf/genetics , Pseudogenes , RNA/metabolism , Animals , Base Sequence , Humans , Lymphoma, Large B-Cell, Diffuse/metabolism , Mice , Molecular Sequence Data , Proto-Oncogene Proteins B-raf/metabolismABSTRACT
PTEN dysfunction plays a crucial role in the pathogenesis of hereditary and sporadic cancers. Here, we show that PTEN homodimerizes and, in this active conformation, exerts lipid phosphatase activity on PtdIns(3,4,5)P3. We demonstrate that catalytically inactive cancer-associated PTEN mutants heterodimerize with wild-type PTEN and constrain its phosphatase activity in a dominant-negative manner. To study the consequences of homo- and heterodimerization of wild-type and mutant PTEN in vivo, we generated Pten knockin mice harboring two cancer-associated PTEN mutations (PtenC124S and PtenG129E). Heterozygous Pten(C124S/+) and Pten(G129E/+) cells and tissues exhibit increased sensitivity to PI3-K/Akt activation compared to wild-type and Pten(+/-) counterparts, whereas this difference is no longer apparent between Pten(C124S/-) and Pten(-/-) cells. Notably, Pten KI mice are more tumor prone and display features reminiscent of complete Pten loss. Our findings reveal that PTEN loss and PTEN mutations are not synonymous and define a working model for the function and regulation of PTEN.
Subject(s)
PTEN Phosphohydrolase/genetics , PTEN Phosphohydrolase/metabolism , Signal Transduction , Animals , Embryo, Mammalian/cytology , Female , Humans , Loss of Heterozygosity , Male , Mice , Mutation , Protein Multimerization , Proto-Oncogene Proteins c-akt/metabolismABSTRACT
Tumor cells metastasize to distant organs through genetic and epigenetic alterations, including changes in microRNA (miR) expression. Here we find miR-22 triggers epithelial-mesenchymal transition (EMT), enhances invasiveness and promotes metastasis in mouse xenografts. In a conditional mammary gland-specific transgenic (TG) mouse model, we show that miR-22 enhances mammary gland side-branching, expands the stem cell compartment, and promotes tumor development. Critically, miR-22 promotes aggressive metastatic disease in MMTV-miR-22 TG mice, as well as compound MMTV-neu or -PyVT-miR-22 TG mice. We demonstrate that miR-22 exerts its metastatic potential by silencing antimetastatic miR-200 through direct targeting of the TET (Ten eleven translocation) family of methylcytosine dioxygenases, thereby inhibiting demethylation of the mir-200 promoter. Finally, we show that miR-22 overexpression correlates with poor clinical outcomes and silencing of the TET-miR-200 axis in patients. Taken together, our findings implicate miR-22 as a crucial epigenetic modifier and promoter of EMT and breast cancer stemness toward metastasis.
Subject(s)
Breast Neoplasms/pathology , Chromatin Assembly and Disassembly , Epithelial-Mesenchymal Transition , Gene Expression Regulation, Neoplastic , MicroRNAs/metabolism , Neoplasm Metastasis , Neoplastic Stem Cells/metabolism , 5-Methylcytosine/analogs & derivatives , Animals , Breast Neoplasms/metabolism , Cytosine/analogs & derivatives , Cytosine/metabolism , Humans , Mice , Mice, Transgenic , Neoplasm Transplantation , Proto-Oncogene Proteins/metabolism , RNA Interference , Transplantation, HeterologousABSTRACT
von Willebrand factor (VWF) plays a key role in normal hemostasis, and deficiencies of VWF lead to clinically significant bleeding. We sought to identify novel modifiers of VWF levels in endothelial colony-forming cells (ECFCs) using single-cell RNA sequencing (scRNA-seq). ECFCs were isolated from patients with low VWF levels (plasma VWF antigen levels between 30 and 50 IU/dL) and from healthy controls. Human umbilical vein endothelial cells were used as an additional control cell line. Cells were characterized for their Weibel Palade body (WPB) content and VWF release. scRNA-seq of all cell lines was performed to evaluate for gene expression heterogeneity and for candidate modifiers of VWF regulation. Candidate modifiers identified by scRNA-seq were further characterized with small-interfering RNA (siRNA) experiments to evaluate for effects on VWF. We observed that ECFCs derived from patients with low VWF demonstrated alterations in baseline WPB metrics and exhibit impaired VWF release. scRNA-seq analyses of these endothelial cells revealed overall decreased VWF transcription, mosaicism of VWF expression, and genes that are differentially expressed in low VWF ECFCs and control endothelial cells (control ECs). An siRNA screen of potential VWF modifiers provided further evidence of regulatory candidates, and 1 such candidate, FLI1, alters the transcriptional activity of VWF. In conclusion, ECFCs from individuals with low VWF demonstrate alterations in their baseline VWF packaging and release compared with control ECs. scRNA-seq revealed alterations in VWF transcription, and siRNA screening identified multiple candidate regulators of VWF.
Subject(s)
von Willebrand Diseases , von Willebrand Factor , Human Umbilical Vein Endothelial Cells/metabolism , Humans , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism , Single-Cell Analysis , Weibel-Palade Bodies/metabolism , von Willebrand Diseases/metabolism , von Willebrand Factor/metabolismABSTRACT
During embryonic gonad coalescence, primordial germ cells (PGCs) follow a carefully choreographed migratory route circumscribed by guidance signals towards somatic gonadal precursor cells (SGPs). In Drosophila melanogaster, SGP-derived Hedgehog (Hh), which serves as a guidance cue for the PGCs, is potentiated by mesodermally restricted HMGCoA-reductase (Hmgcr) and the ABC transporter Multi-drug-resistant-49 (Mdr49). Given the importance of cholesterol modification in the processing and long-distance transmission of the Hh ligand, we have analyzed the involvement of the Niemann-Pick disease type C-1a (NPC1a) protein, a cholesterol transporter, in germ cell migration and Hedgehog signaling. We show that mesoderm-specific inactivation of Npc1a results in germ cell migration defects. Similar to Mdr49, PGC migration defects in the Npc1a embryos are ameliorated by a cholesterol-rich diet. Consistently, reduction in Npc1a weakens the ability of ectopic HMG Coenzyme A reductase (Hmgcr) to induce germ cell migration defects. Moreover, compromising Npc1a levels influences Hh signaling adversely during wing development, a process that relies upon long-range Hh signaling. Last, doubly heterozygous embryos (Mdr49/Npc1a) display enhanced germ cell migration defects when compared with single mutants (Npc1a/+ or Mdr49/+), supporting cooperative interaction between the two.
Subject(s)
Drosophila Proteins/metabolism , Membrane Proteins/metabolism , Animals , Cell Movement/genetics , Cell Movement/physiology , Drosophila Proteins/genetics , Drosophila melanogaster , Gene Expression Regulation, Developmental/genetics , Gene Expression Regulation, Developmental/physiology , Germ Cells/metabolism , Heterozygote , Membrane Proteins/genetics , Neurons/metabolism , Niemann-Pick C1 Protein , Signal Transduction/genetics , Signal Transduction/physiologyABSTRACT
Huntington's disease (HD) is a chronic neurodegenerative disorder characterized by a late clinical onset despite ubiquitous expression of the mutant Huntingtin gene (HTT) from birth. Transcriptional dysregulation is a pivotal feature of HD. Yet, the genes that are altered in the prodromal period and their regulators, which present opportunities for therapeutic intervention, remain to be elucidated. Using transcriptional and chromatin profiling, we found aberrant transcription and changes in histone H3K27acetylation in the striatum of R6/1 mice during the presymptomatic disease stages. Integrating these data, we identified the Elk-1 transcription factor as a candidate regulator of prodromal changes in HD. Exogenous expression of Elk-1 exerted beneficial effects in a primary striatal cell culture model of HD, and adeno-associated virus-mediated Elk-1 overexpression alleviated transcriptional dysregulation in R6/1 mice. Collectively, our work demonstrates that aberrant gene expression precedes overt disease onset in HD, identifies the Elk-1 transcription factor as a key regulator linked to early epigenetic and transcriptional changes in HD, and presents evidence for Elk-1 as a target for alleviating molecular pathology in HD.
Subject(s)
Epigenomics , Huntington Disease/genetics , ets-Domain Protein Elk-1/genetics , ets-Domain Protein Elk-1/metabolism , Animals , Corpus Striatum/metabolism , Dependovirus , Disease Models, Animal , Histones/metabolism , Huntingtin Protein/genetics , Huntington Disease/drug therapy , Mice , Mice, Transgenic , Neurons/metabolism , Nuclear Proteins/metabolismABSTRACT
OBJECTIVE: The aim of the study was to investigate whether inhibition of Sonic Hedgehog (SHH) pathway would prevent progression of Barrett's Esophagus (BE) to esophageal adenocarcinoma. BACKGROUND: The hedgehog signaling pathway is a leading candidate as a molecular mediator of BE and esophageal adenocarcinoma (EAC). Repurposed use of existing off-patent, safe and tolerable drugs that can inhibit hedgehog, such as itraconazole, could prevent progression of BE to EAC. METHODS: The efficacy of itraconazole was investigated using a surgical rat reflux model of Barrett's Metaplasia (BM). Weekly intraperitoneal injections of saline (control group) or itraconazole (treatment group; 200âmg/kg) were started at 24 weeks postsurgery. Esophageal tissue was harvested at 40 weeks. The role of the Hh pathway was also evaluated clinically. Esophageal tissue was harvested after 40 weeks for pathological examination and evaluation of the SHH pathway by immunohistochemistry. RESULTS: BM was present in control animals 29 of 31 (93%) versus itraconazole 22 of 24 (91%). EAC was significantly lower in itraconazole 2 of 24 (8%) versus control 10 of 31 (32%), respectively (P = 0.033). Esophageal SHH levels were lower in itraconazole vs control (P = 0.12). In esophageal tissue from humans with recurrent or persistent dysplastic BE within 24 months of ablative treatment, strong SHH and Indian Hedgehog expression occurred in distal BE versus proximal squamous epithelium, odds ratio = 6.1 (95% confidence interval: 1.6, 23.4) and odds ratio = 6.4 (95% confidence interval: 1.2, 32.8), respectively. CONCLUSION: Itraconazole significantly decreases EAC development and SHH expression in a preclinical animal model of BM. In humans, BE tissue expresses higher SHH, Indian Hedgehog, and bone morphogenic protein levels than normal squamous esophageal epithelium.
Subject(s)
Adenocarcinoma/drug therapy , Adenocarcinoma/etiology , Barrett Esophagus/complications , Esophageal Neoplasms/drug therapy , Esophageal Neoplasms/etiology , Hedgehog Proteins/antagonists & inhibitors , Itraconazole/pharmacology , Itraconazole/therapeutic use , Adenocarcinoma/pathology , Animals , Disease Models, Animal , Disease Progression , Esophageal Neoplasms/pathology , Male , Neoplasm Invasiveness , Rats , Rats, Sprague-DawleyABSTRACT
INTRODUCTION: In response to the increasing complexity of care for patients with bleeding disorders, we established new clinical teams for our hemophilia treatment center (HTC). AIMS: We undertook a quality improvement project to improve the coordination and communication with our patients by establishing primary assignments of clinical staff to individual patients (primary teams). METHODS: A quality improvement project group was formed that established the goals and assignment of primary teams. Patients were surveyed for their knowledge of their primary teams as well as their ability to schedule and contact their primary providers. As a measure of the effects on clinical staff, a balancing survey was also conducted among providers impacted by the clinical assignment of teams. RESULTS: Our results demonstrate improvements across both coordination and communication as reported by patients. Additionally, the assignment of primary teams was met with high satisfaction and improvement in coordination and communication as reported by the clinical staff members of the HTC. CONCLUSIONS: Initiation of a quality improvement project and the creation of a primary team system were feasible at a large HTC and resulted in improvements in both patient-reported and staff-reported outcomes of coordination and communication of care.
Subject(s)
Blood Coagulation Disorders/psychology , Quality Improvement , Adolescent , Adult , Blood Coagulation Disorders/diagnosis , Humans , Patient-Centered Care , Quality Improvement/organization & administration , Surveys and Questionnaires , Young AdultABSTRACT
Identifying large expansions of short tandem repeats (STRs), such as those that cause amyotrophic lateral sclerosis (ALS) and fragile X syndrome, is challenging for short-read whole-genome sequencing (WGS) data. A solution to this problem is an important step toward integrating WGS into precision medicine. We developed a software tool called ExpansionHunter that, using PCR-free WGS short-read data, can genotype repeats at the locus of interest, even if the expanded repeat is larger than the read length. We applied our algorithm to WGS data from 3001 ALS patients who have been tested for the presence of the C9orf72 repeat expansion with repeat-primed PCR (RP-PCR). Compared against this truth data, ExpansionHunter correctly classified all (212/212, 95% CI [0.98, 1.00]) of the expanded samples as either expansions (208) or potential expansions (4). Additionally, 99.9% (2786/2789, 95% CI [0.997, 1.00]) of the wild-type samples were correctly classified as wild type by this method with the remaining three samples identified as possible expansions. We further applied our algorithm to a set of 152 samples in which every sample had one of eight different pathogenic repeat expansions, including those associated with fragile X syndrome, Friedreich's ataxia, and Huntington's disease, and correctly flagged all but one of the known repeat expansions. Thus, ExpansionHunter can be used to accurately detect known pathogenic repeat expansions and provides researchers with a tool that can be used to identify new pathogenic repeat expansions.
Subject(s)
Amyotrophic Lateral Sclerosis/genetics , DNA Repeat Expansion , Whole Genome Sequencing/methods , Algorithms , C9orf72 Protein/genetics , Databases, Genetic , Humans , Precision Medicine , Sensitivity and Specificity , SoftwareABSTRACT
BACKGROUND: Exercise prevents falls in older adults. Regular updates of estimated effects of exercise on falls are warranted given the number of new trials, the increasing number of older people globally and the major consequences of falls and fall-related injuries. METHODS: This update of a 2019 Cochrane Review was undertaken to inform the World Health Organization guidelines on physical activity and sedentary behaviour. Searches were conducted in six databases. We included randomised controlled trials evaluating effects of any form of physical activity as a single intervention on falls in people aged 60+ years living in the community. Analyses explored dose-response relationships. The certainty of the evidence was assessed using Grading of Recommendations Assessment, Development and Evaluation (GRADE). RESULTS: This review included 116 studies, involving 25,160 participants; nine new studies since the 2019 Cochrane Review. Exercise reduces the rate of falls by 23% (pooled rate ratio (RaR) 0.77, 95% confidence interval (CI) 0.71 to 0.83, 64 studies, high certainty evidence). Subgroup analysis showed variation in effects of different types of exercise (p < 0.01). Rate of falls compared with control is reduced by 24% from balance and functional exercises (RaR 0.76, 95% CI 0.70 to 0.82, 39 studies, high certainty evidence), 28% from programs involving multiple types of exercise (commonly balance and functional exercises plus resistance exercises, RaR 0.72, 95% CI 0.56 to 0.93, 15 studies, moderate certainty evidence) and 23% from Tai Chi (RaR 0.77, 95% CI 0.61 to 0.97, 9 studies, moderate certainty evidence). The effects of programs that primarily involve resistance training, dance or walking remain uncertain. Interventions with a total weekly dose of 3+ h that included balance and functional exercises were particularly effective with a 42% reduction in rate of falls compared to control (Incidence Rate Ratio (IRR) 0.58, 95% CI 0.45 to 0.76). Subgroup analyses showed no evidence of a difference in the effect on falls on the basis of participant age over 75 years, risk of falls as a trial inclusion criterion, individual versus group exercise, or whether a health professional delivered the intervention. CONCLUSIONS: Given the strength of this evidence, effective exercise programs should now be implemented at scale.
Subject(s)
Accidental Falls/prevention & control , Exercise , Randomized Controlled Trials as Topic , Aged , Female , Guidelines as Topic , Humans , Independent Living , Male , Middle Aged , World Health OrganizationABSTRACT
The Cepheid Xpert® Norovirus kit automates sample processing, nucleic acid extraction, and real-time reverse transcription polymerase chain reactions (RT-PCRs) to detect norovirus genogroups I (GI) and II (GII). Eighty-five stool samples collected between February 2015 and May 2017 were used to compare the performance of a user-modified Xpert assay against a clinically validated laboratory-developed test (LDT). Of the 85 samples, 54 were previously archived in -80°C freezer. The remaining 31 were fresh samples tested concurrently with the LDT. The results of all samples tested using the Xpert kit and LDT were found to be concordant, including 12 GI- and 42 GII-positive samples, 1 GI and GII coinfection, and 30 negative samples. Comparison of the assays showed perfect concordance with a kappa coefficient score of 1.00 (95%CI from 1.00 to 1.00). Of the 30 negative stool samples tested, three samples were positive for rotavirus detected using an immunochromatographic assay, with no cross-reactivity shown in both LDT and Xpert assays. In-run sample processing control of the Xpert assay for all negative samples tested showed no/minor inhibition. Compared to the LDT, the Xpert assay produced similar or better Ct values for detection. It also showed better mitigation of PCR inhibition in stool sample testing.
Subject(s)
Caliciviridae Infections/diagnosis , Norovirus/isolation & purification , Reagent Kits, Diagnostic , Real-Time Polymerase Chain Reaction/methods , Adolescent , Adult , Aged , Aged, 80 and over , Biological Specimen Banks , Child , Child, Preschool , Clinical Laboratory Techniques/methods , Cross Reactions , Feces/virology , Female , Gastroenteritis/virology , Genotype , Humans , Infant , Male , Middle Aged , Norovirus/genetics , RNA, Viral/isolation & purification , Sensitivity and Specificity , Young AdultABSTRACT
Von Willebrand Disease (VWD) is considered the most common inherited bleeding disorder. It has multiple subtypes and a primary symptom of mucocutaneous bleeding. Some researchers in this field speculate that inherited disorders of platelet function may be as common but underdiagnosed due to the difficulty of accessing testing. The diagnostic approach for this disease has evolved as new instruments and diagnostic testing have become available. The ISTH-Bleeding Assessment Tool is a validated instrument that is used to screen patients referred for bleeding symptoms for further laboratory testing. The three main screening tests used in the diagnosis of VWD include von Willebrand Factor (VWF) antigen, platelet-dependent VWF activity, and factor VIII activity. Improvements in laboratory assays discussed include changes in how traditional assays are performed as well as the addition of new laboratory assays. The role of genetic testing and management of patients with borderline low von Willebrand factor are also discussed.
Subject(s)
Blood Coagulation Tests/methods , Diagnostic Tests, Routine/methods , Genetic Testing/methods , Humans , von Willebrand DiseasesSubject(s)
Factor VIII/therapeutic use , Hemophilia A/drug therapy , Hemorrhage/prevention & control , Practice Guidelines as Topic/standards , Premedication/methods , Adolescent , Child , Child, Preschool , Female , Hemophilia A/pathology , Humans , Infant , Male , Primary Prevention , Prognosis , Time-to-TreatmentABSTRACT
The earliest stages of Huntington disease are marked by changes in gene expression that are caused in an indirect and poorly understood manner by polyglutamine expansions in the huntingtin (HTT) protein. To explore the hypothesis that DNA methylation may be altered in cells expressing mutated HTT, we use reduced representation bisulfite sequencing (RRBS) to map sites of DNA methylation in cells carrying either wild-type or mutant HTT. We find that a large fraction of the genes that change in expression in the presence of mutant huntingtin demonstrate significant changes in DNA methylation. Regions with low CpG content, which have previously been shown to undergo methylation changes in response to neuronal activity, are disproportionately affected. On the basis of the sequence of regions that change in methylation, we identify AP-1 and SOX2 as transcriptional regulators associated with DNA methylation changes, and we confirm these hypotheses using genome-wide chromatin immunoprecipitation sequencing (ChIP-Seq). Our findings suggest new mechanisms for the effects of polyglutamine-expanded HTT. These results also raise important questions about the potential effects of changes in DNA methylation on neurogenesis and cognitive decline in patients with Huntington disease.
Subject(s)
DNA Methylation , Mutant Proteins/genetics , Nerve Tissue Proteins/genetics , Nuclear Proteins/genetics , Animals , Cell Line , Chromatin Immunoprecipitation , CpG Islands , Disease Models, Animal , Gene Expression , Humans , Huntingtin Protein , Huntington Disease/genetics , Huntington Disease/metabolism , Mice , SOXB1 Transcription Factors/metabolism , Transcription Factor AP-1/metabolismABSTRACT
SOX2 is a master regulator of both pluripotent embryonic stem cells (ESCs) and multipotent neural progenitor cells (NPCs); however, we currently lack a detailed understanding of how SOX2 controls these distinct stem cell populations. Here we show by genome-wide analysis that, while SOX2 bound to a distinct set of gene promoters in ESCs and NPCs, the majority of regions coincided with unique distal enhancer elements, important cis-acting regulators of tissue-specific gene expression programs. Notably, SOX2 bound the same consensus DNA motif in both cell types, suggesting that additional factors contribute to target specificity. We found that, similar to its association with OCT4 (Pou5f1) in ESCs, the related POU family member BRN2 (Pou3f2) co-occupied a large set of putative distal enhancers with SOX2 in NPCs. Forced expression of BRN2 in ESCs led to functional recruitment of SOX2 to a subset of NPC-specific targets and to precocious differentiation toward a neural-like state. Further analysis of the bound sequences revealed differences in the distances of SOX and POU peaks in the two cell types and identified motifs for additional transcription factors. Together, these data suggest that SOX2 controls a larger network of genes than previously anticipated through binding of distal enhancers and that transitions in POU partner factors may control tissue-specific transcriptional programs. Our findings have important implications for understanding lineage specification and somatic cell reprogramming, where SOX2, OCT4, and BRN2 have been shown to be key factors.
Subject(s)
Embryonic Stem Cells , Enhancer Elements, Genetic , Nerve Tissue Proteins , Octamer Transcription Factor-3 , POU Domain Factors , SOXB1 Transcription Factors , Animals , Cell Differentiation/genetics , Cell Line , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Embryonic Stem Cells/cytology , Embryonic Stem Cells/metabolism , Gene Expression Regulation, Developmental , Mice , Nerve Tissue Proteins/genetics , Nerve Tissue Proteins/metabolism , Nucleotide Motifs , Octamer Transcription Factor-3/genetics , Octamer Transcription Factor-3/metabolism , POU Domain Factors/genetics , POU Domain Factors/metabolism , Pluripotent Stem Cells/cytology , Pluripotent Stem Cells/metabolism , Promoter Regions, Genetic , SOXB1 Transcription Factors/genetics , SOXB1 Transcription Factors/metabolismABSTRACT
Transcriptional dysregulation is an early feature of Huntington disease (HD). We observed gene-specific changes in histone H3 lysine 4 trimethylation (H3K4me3) at transcriptionally repressed promoters in R6/2 mouse and human HD brain. Genome-wide analysis showed a chromatin signature for this mark. Reducing the levels of the H3K4 demethylase SMCX/Jarid1c in primary neurons reversed down-regulation of key neuronal genes caused by mutant Huntingtin expression. Finally, reduction of SMCX/Jarid1c in primary neurons from BACHD mice or the single Jarid1 in a Drosophila HD model was protective. Therefore, targeting this epigenetic signature may be an effective strategy to ameliorate the consequences of HD.
Subject(s)
Brain/metabolism , Histones/metabolism , Huntington Disease/metabolism , Lysine/metabolism , Animals , Animals, Genetically Modified , Blotting, Western , Brain/pathology , Brain-Derived Neurotrophic Factor/genetics , Brain-Derived Neurotrophic Factor/metabolism , Cells, Cultured , Drosophila melanogaster/genetics , Drosophila melanogaster/metabolism , Female , Gene Expression Profiling , Histone Demethylases , Humans , Huntingtin Protein , Huntington Disease/genetics , Male , Methylation , Mice , Mice, Inbred C57BL , Mice, Inbred CBA , Nerve Tissue Proteins/genetics , Nerve Tissue Proteins/metabolism , Neurons/cytology , Neurons/metabolism , Oxidoreductases, N-Demethylating/genetics , Oxidoreductases, N-Demethylating/metabolism , Promoter Regions, Genetic/genetics , RNA Interference , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
Alterations in von Willebrand factor (VWF) have an important role in human health and disease. Deficiency of VWF is associated with symptoms of bleeding and excesses of VWF are associated with thrombotic outcomes. Understanding the mechanisms that drive VWF regulation can lead to a better understanding of modulation of VWF levels in humans. We identified clusterin (CLU) as a potential candidate regulator of VWF based on a single cell RNA sequencing (scRNA-seq) analysis in control endothelial cells (ECs) and von Willebrand disease (VWD) endothelial colony-forming-cells (ECFCs). We found that patients with deficiencies of VWF (von Willebrand disease, VWD) had decreased CLU expression and ECs with low VWF expression also had low CLU expression. Based on these findings, we sought to evaluate the role of CLU in the regulation of VWF, specifically as it relates to VWD. As CLU is primarily thought to be a golgi protein involved in protein chaperoning, we hypothesized that knockdown of CLU would lead to decreases in VWF and alterations in Weibel-Palade bodies (WPBs). We used both siRNA- and CRISPR-Cas9-based approaches to modulate CLU in human umbilical vein endothelial cells (HUVECs) and evaluated VWF protein levels, VWF mRNA copy number, and WPB quantity and size. We demonstrated that siRNA-based knockdown of CLU resulted in decreases in VWF content in cellular lysates and supernatants, but no significant change in WPB quantity or size. A CRISPR-Cas9-based knockdown of CLU demonstrated similar findings of decreases in intracellular VWF content but no significant change in WPB quantity or size. Our data suggests that CLU knockdown is associated with decreases in cellular VWF content but does not affect VWF mRNA levels or WPB quantity or size.
Subject(s)
Clusterin , von Willebrand Factor , Humans , Cells, Cultured , Clusterin/genetics , Human Umbilical Vein Endothelial Cells/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism , von Willebrand Diseases , von Willebrand Factor/genetics , von Willebrand Factor/metabolism , Weibel-Palade Bodies/metabolismABSTRACT
The mechanisms underlying the selective regional vulnerability to neurodegeneration in Huntington's disease (HD) have not been fully defined. To explore the role of astrocytes in this phenomenon, we used single-nucleus and bulk RNAseq, lipidomics, HTT gene CAG repeat-length measurements, and multiplexed immunofluorescence on HD and control post-mortem brains. We identified genes that correlated with CAG repeat length, which were enriched in astrocyte genes, and lipidomic signatures that implicated poly-unsaturated fatty acids in sensitizing neurons to cell death. Because astrocytes play essential roles in lipid metabolism, we explored the heterogeneity of astrocytic states in both protoplasmic and fibrous-like (CD44+) astrocytes. Significantly, one protoplasmic astrocyte state showed high levels of metallothioneins and was correlated with the selective vulnerability of distinct striatal neuronal populations. When modeled in vitro, this state improved the viability of HD-patient-derived spiny projection neurons. Our findings uncover key roles of astrocytic states in protecting against neurodegeneration in HD.
Subject(s)
Astrocytes , Huntington Disease , Neurons , Huntington Disease/metabolism , Huntington Disease/genetics , Huntington Disease/pathology , Astrocytes/metabolism , Astrocytes/pathology , Humans , Neurons/metabolism , Huntingtin Protein/genetics , Huntingtin Protein/metabolism , Male , Female , Lipidomics/methods , Middle Aged , Metallothionein/metabolism , Metallothionein/genetics , Brain/metabolism , Brain/pathology , Lipid Metabolism , Aged , MultiomicsSubject(s)
Delivery, Obstetric , Obstetrical Forceps , Female , Humans , Infant, Newborn , PregnancyABSTRACT
An 11-year-old male with hemophilia A and a known high-titer Factor VIII inhibitor was admitted with retroperitoneal hemorrhage. The patient was receiving infusions of recombinant activated Factor VII (rFVIIa) for a recent elbow hemorrhage when retroperitoneal bleeding commenced. Despite increased dosing of rFVIIa and a dose of activated prothrombin complex concentrate (aPCC), he continued to hemorrhage and required several blood transfusions. Factor XIII was administered 1 hour after rFVIIa and the patient demonstrated cessation of bleeding and normalization of clot strength. Factor XIII may act as an adjuvant in effective clot stabilization in patients with hemophilia and inhibitory antibodies.