ABSTRACT
PURPOSE: The RAS/MEK signaling pathway is essential in carcinogenesis and frequently altered in non-small-cell lung cancer (NSCLC), notably by KRAS mutations (KRASm) that affect 25%-30% of non-squamous NSCLC. This study aims to explore the impact of KRASm subtypes on disease phenotype and survival outcomes. PATIENTS AND METHODS: We conducted a retrospective analysis of the French Epidemiological Strategy and Medical Economics database for advanced or metastatic lung cancer from 2011 to 2021. Patient demographics, histology, KRASm status, treatment strategies, and outcomes were assessed. RESULTS: Of 10 177 assessable patients for KRAS status, 17.6% had KRAS p.G12C mutation, 22.6% had KRAS non-p.G12C mutation, and 59.8% were KRASwt. KRASm patients were more often smokers (96.3%) compared with KRASwt (85.8%). A higher proportion of programmed death-ligand 1 ≥50% was found for KRASm patients: 43.5% versus 38.0% (P < 0.01). KRASm correlated with poorer outcomes. First-line median progression-free survival was shorter in the KRASm than the KRASwt cohort: 4.0 months [95% confidence interval (CI) 3.7-4.3 months] versus 5.1 months (95% CI 4.8-5.3 months), P < 0.001. First-line overall survival was shorter for KRASm than KRASwt patients: 12.6 months (95% CI 11.6-13.6 months) versus 15.4 months (95% CI 14.6-16.2 months), P = 0.012. First-line chemoimmunotherapy offered better overall survival in KRAS p.G12C (48.8 months) compared with KRAS non-p.G12C (24.0 months) and KRASwt (22.5 months) patients. Second-line overall survival with immunotherapy was superior in the KRAS p.G12C subgroup: 12.6 months (95% CI 8.1-18.6 months) compared with 9.4 months (95% CI 8.0-11.4 months) for KRAS non-p.G12C and 9.6 months (8.4-11.0 months) for KRASwt patients. CONCLUSION: We highlighted distinct clinical profiles and survival outcomes according to KRASm subtypes. Notably KRAS p.G12C mutations may provide increased sensitivity to immunotherapy, suggesting potential therapeutic implications for sequencing or combination of therapies. Further research on the impact of emerging KRAS specific inhibitors are warranted in real-world cohorts.
Subject(s)
Carcinoma, Non-Small-Cell Lung , Lung Neoplasms , Mutation , Proto-Oncogene Proteins p21(ras) , Humans , Carcinoma, Non-Small-Cell Lung/genetics , Carcinoma, Non-Small-Cell Lung/mortality , Carcinoma, Non-Small-Cell Lung/pathology , Carcinoma, Non-Small-Cell Lung/drug therapy , Male , Female , Proto-Oncogene Proteins p21(ras)/genetics , Lung Neoplasms/genetics , Lung Neoplasms/mortality , Lung Neoplasms/pathology , Lung Neoplasms/drug therapy , Retrospective Studies , Aged , Middle Aged , France/epidemiologyABSTRACT
In this case series, we present four unique cases of Riga-Fede disease (RFD), a rare disorder characterized by mucosal trauma as a result of repetitive tongue protrusion against the incisors, leading to the development of a large oral mass/ulceration. Due to the rapid development and growth of these lesions mimicking malignancy, it is important for the general and pediatric otolaryngologist to correctly diagnose and treat this benign disorder. This series highlights the variable clinical presentations, along with comorbidities of RFD, as well as the importance of interdisciplinary care between the pediatric otolaryngologist and pediatric dentist in its management. Laryngoscope, 2024.
ABSTRACT
Pulmonary alveolar proteinosis (PAP) is a life-threatening, rare lung syndrome for which there is no cure and no approved therapies. PAP is a disease of lipid accumulation characterized by alveolar macrophage foam cell formation. While much is known about the clinical presentation, there is a paucity of information regarding temporal changes in lipids throughout the course of disease. Our objectives were to define the detailed lipid composition of alveolar macrophages in PAP patients at the time of diagnosis and during treatment. We performed comprehensive mass spectrometry to profile the lipid signature of alveolar macrophages obtained from three independent mouse models of PAP and from PAP and non-PAP patients. Additionally, we quantified changes in macrophage-associated lipids during clinical treatment of PAP patients. We found remarkable variations in lipid composition in PAP patients, which were consistent with data from three independent mouse models. Detailed lipidomic analysis revealed that the overall alveolar macrophage lipid burden inversely correlated with clinical improvement and response to therapy in PAP patients. Specifically, as PAP patients experienced clinical improvement, there was a notable decrease in the total lipid content of alveolar macrophages. This crucial observation suggests that the levels of these macrophage-associated lipids can be utilized to assess the efficacy of treatment. These findings provide valuable insights into the dysregulated lipid metabolism associated with PAP, offering the potential for lipid profiling to serve as a means of monitoring therapeutic interventions in PAP patients.
Subject(s)
Pulmonary Alveolar Proteinosis , Animals , Mice , Humans , Pulmonary Alveolar Proteinosis/drug therapy , Pulmonary Alveolar Proteinosis/diagnosis , Pulmonary Alveolar Proteinosis/metabolism , Macrophages, Alveolar , Lung/metabolism , Macrophages/metabolism , LipidsABSTRACT
BACKGROUND & AIMS: Metabolic dysfunction-associated steatohepatitis (MASH) is linked to insulin resistance and type 2 diabetes and marked by hepatic inflammation, microvascular dysfunction, and fibrosis, impairing liver function and aggravating metabolic derangements. The liver homeostatic interactions disrupted in MASH are still poorly understood. We aimed to elucidate the plasticity and changing interactions of non-parenchymal cells associated with advanced MASH. METHODS: We characterized a diet-induced mouse model of advanced MASH at single-cell resolution and validated findings by assaying chromatin accessibility, bioimaging murine and human livers, and via functional experiments in vivo and in vitro. RESULTS: The fibrogenic activation of hepatic stellate cells (HSCs) led to deterioration of a signaling module consisting of the bile acid receptor NR1H4/FXR and HSC-specific GS-protein-coupled receptors (GSPCRs) capable of preserving stellate cell quiescence. Accompanying HSC activation, we further observed the attenuation of HSC Gdf2 expression, and a MASH-associated expansion of a CD207-positive macrophage population likely derived from both incoming monocytes and Kupffer cells. CONCLUSION: We conclude that HSC-expressed NR1H4 and GSPCRs of the healthy liver integrate postprandial cues, which sustain HSC quiescence and, through paracrine signals, overall sinusoidal health. Hence HSC activation in MASH not only drives fibrogenesis but may desensitize the hepatic sinusoid to liver homeostatic signals. IMPACT AND IMPLICATIONS: Homeostatic interactions between hepatic cell types and their deterioration in metabolic dysfunction-associated steatohepatitis are poorly characterized. In our current single cell-resolved study of advanced murine metabolic dysfunction-associated steatohepatitis, we identified a quiescence-associated hepatic stellate cell-signaling module with potential to preserve normal sinusoid function. As expression levels of its constituents are conserved in the human liver, stimulation of the identified signaling module is a promising therapeutic strategy to restore sinusoid function in chronic liver disease.
Subject(s)
Diabetes Mellitus, Type 2 , Fatty Liver , Mice , Humans , Animals , Pericytes/metabolism , Diabetes Mellitus, Type 2/metabolism , Liver/pathology , Signal Transduction , Hepatic Stellate Cells/metabolism , Fatty Liver/metabolism , Liver Cirrhosis/pathology , Growth Differentiation Factor 2/metabolismABSTRACT
Adipose tissue is the site of long-term energy storage. During the fasting state, exercise, and cold exposure, the white adipose tissue mobilizes energy for peripheral tissues through lipolysis. The mobilization of lipids from white adipose tissue to the liver can lead to excess triglyceride accumulation and fatty liver disease. Although the white adipose tissue is known to release free fatty acids, a comprehensive analysis of lipids mobilized from white adipocytes in vivo has not been completed. In these studies, we provide a comprehensive quantitative analysis of the adipocyte-secreted lipidome and show that there is interorgan crosstalk with liver. Our analysis identifies multiple lipid classes released by adipocytes in response to activation of lipolysis. Time-dependent analysis of the serum lipidome showed that free fatty acids increase within 30 min of ß3-adrenergic receptor activation and subsequently decrease, followed by a rise in serum triglycerides, liver triglycerides, and several ceramide species. The triglyceride composition of liver is enriched for linoleic acid despite higher concentrations of palmitate in the blood. To further validate that these findings were a specific consequence of lipolysis, we generated mice with conditional deletion of adipose tissue triglyceride lipase exclusively in adipocytes. This loss of in vivo adipocyte lipolysis prevented the rise in serum free fatty acids and hepatic triglycerides. Furthermore, conditioned media from adipocytes promotes lipid remodeling in hepatocytes with concomitant changes in genes/pathways mediating lipid utilization. Together, these data highlight critical role of adipocyte lipolysis in interorgan crosstalk between adipocytes and liver.
Subject(s)
Fatty Acids, Nonesterified , Lipolysis , Mice , Animals , Lipolysis/physiology , Fatty Acids, Nonesterified/metabolism , Lipidomics , Adipocytes/metabolism , Adipose Tissue/metabolism , Liver/metabolism , Triglycerides/metabolismABSTRACT
The reductive coupling of dienol ethers with N-tosylimines catalyzed by Ni(0) in the presence of a VAPOL-derived phosphoramidite ligand follows an unprecedented regiochemical course; it furnishes syn-configured 1,2-aminoalcohol derivatives in good chemical yields with up to 94% ee.
ABSTRACT
Inguinal hernias are one of the most common surgical pathologies faced by the general surgeon in modern medicine. The cumulative incidence of an inguinal hernia is around 25% in men and 3% in women. The majority of inguinal hernias can be repaired minimally invasively, utilizing either robotic or laparoscopic approaches.
Subject(s)
Hernia, Inguinal , Robotics , Surgeons , Male , Humans , Female , Hernia, Inguinal/diagnosis , Hernia, Inguinal/surgeryABSTRACT
BACKGROUND: Frailty is a measure of physiologic reserve and correlates with surgical outcomes in the elderly. Patients who present with giant paraesophageal hernias (PEH) are typically older than 65. We defined 'giant' as a PEH with 50% or more of the stomach in the chest. We hypothesized that frailty correlates with 30-day complications, length of stay, and discharge destination following laparoscopic giant PEH repair. METHODS: Patients older than 65 to undergo primary laparoscopic repair of a giant PEH at a single academic medical center between 2015 and 2022 were included. Hernia size was determined by preoperative imaging. Frailty was assessed clinically prior to surgery using the modified Frailty Index (mFI), an 11-item instrument that counts clinical deficits associated with frailty. A score ≥ 3 was considered frail. A major complication was a Clavien grade IIIB or higher. RESULTS: Of the 162 patients included in the study, mean age was 74.4 ± 7.2, and 66% of patients were female (n = 128). The mFI was ≥ 3 in 37 patients (22.8%). Frail patients were older (78 ± 7.9 vs. 73 ± 6.6 years, p = 0.02). There was no difference in overall complication rate (40.5% vs. 29.6%, p = 0.22) or major complication rate (8.1% vs. 4.8%, p = 0.20) between frail and non-frail patients. Functionally impaired patients (METS < 4) were more likely to develop a major complication (17.9% vs. 3.0%, p < 0.01). Average length of stay was 2.4 days, and frail patients experienced a longer mean hospital stay (2.5 ± 0.2 vs. 2.3 ± 1.8, p = 0.03). Frail patients were more likely to be discharged to a destination other than home. CONCLUSION: Increased frailty as assessed by the mFI is correlated with length of stay and discharge destination following laparoscopic repair of giant PEH in patients > 65. Complication rates were comparable for both frail and non-frail cohorts.
Subject(s)
Frailty , Hernia, Hiatal , Laparoscopy , Humans , Female , Aged , Aged, 80 and over , Male , Frailty/complications , Hernia, Hiatal/surgery , Hernia, Hiatal/complications , Postoperative Complications/epidemiology , Postoperative Complications/etiology , Postoperative Complications/surgery , Retrospective Studies , Laparoscopy/adverse effects , Laparoscopy/methodsABSTRACT
Diabetes mellitus is one of the major epidemics in the United States. It is heavily associated with obesity and multiple metabolic derangements that lead to long term morbidity, mortality as well as financial burden. Although medical therapy has been the mainstay in the management of diabetes mellitus, there remains a large portion of this patient population which struggles to obtain adequate glycemic control and long-term weight control with medical management alone. Bariatric surgery is a powerful tool in combating diabetes mellitus and affects glucose homeostasis through a variety of pathways. While it does provide a durable pathway for weight loss, improvement in glucose homeostasis is not only affected by the weight loss seen after bariatric surgery. Changes in gut hormone secretion, insulin regulation, and gut microbial composition also affect how these operations improve glucose homeostasis. Through improvement in the management of diabetes mellitus, comorbidities including cardiovascular disease, in turn demonstrate improvement. In this article, we will discuss the role of bariatric (metabolic) surgery as it relates to long term weight loss and the impact that weight loss has on improvement in diabetes mellitus.
Subject(s)
Bariatric Surgery , Weight Loss , Diabetes Mellitus, Type 2/complications , Diabetes Mellitus, Type 2/surgery , Humans , Male , Female , Adult , Obesity , Treatment OutcomeABSTRACT
Cellular metabolism is tightly regulated by growth factor signaling, which promotes metabolic rewiring to support growth and proliferation. While growth factor-induced transcriptional and post-translational modes of metabolic regulation have been well defined, whether post-transcriptional mechanisms impacting mRNA stability regulate this process is less clear. Here, we present the ZFP36/L1/L2 family of RNA-binding proteins and mRNA decay factors as key drivers of metabolic regulation downstream of acute growth factor signaling. We quantitatively catalog metabolic enzyme and nutrient transporter mRNAs directly bound by ZFP36 following growth factor stimulation-many of which encode rate-limiting steps in metabolic pathways. Further, we show that ZFP36 directly promotes the mRNA decay of Enolase 2 (Eno2), altering Eno2 protein expression and enzymatic activity, and provide evidence of a ZFP36/Eno2 axis during VEGF-stimulated developmental retinal angiogenesis. Thus, ZFP36-mediated mRNA decay serves as an important mode of metabolic regulation downstream of growth factor signaling within dynamic cell and tissue states.
Subject(s)
RNA-Binding Proteins , Signal Transduction , RNA-Binding Proteins/genetics , RNA-Binding Proteins/metabolism , Intercellular Signaling Peptides and Proteins/metabolism , RNA Stability/genetics , Tristetraprolin/genetics , Tristetraprolin/metabolismABSTRACT
BACKGROUND: Removal of circulating plasma low-density lipoprotein cholesterol (LDL-C) by the liver relies on efficient endocytosis and intracellular vesicle trafficking. Increasing the availability of hepatic LDL receptors (LDLRs) remains a major clinical target for reducing LDL-C levels. Here, we describe a novel role for RNF130 (ring finger containing protein 130) in regulating plasma membrane availability of LDLR. METHODS: We performed a combination of gain-of-function and loss-of-function experiments to determine the effect of RNF130 on LDL-C and LDLR recycling. We overexpressed RNF130 and a nonfunctional mutant RNF130 in vivo and measured plasma LDL-C and hepatic LDLR protein levels. We performed in vitro ubiquitination assays and immunohistochemical staining to measure levels and cellular distribution of LDLR. We supplement these experiments with 3 separate in vivo models of RNF130 loss-of-function where we disrupted Rnf130 using either ASO (antisense oligonucleotides), germline deletion, or AAV CRISPR (adeno-associated virus clustered regularly interspaced short palindromic repeats) and measured hepatic LDLR and plasma LDL-C. RESULTS: We demonstrate that RNF130 is an E3 ubiquitin ligase that ubiquitinates LDLR resulting in redistribution of the receptor away from the plasma membrane. Overexpression of RNF130 decreases hepatic LDLR and increases plasma LDL-C levels. Further, in vitro ubiquitination assays demonstrate RNF130-dependent regulation of LDLR abundance at the plasma membrane. Finally, in vivo disruption of Rnf130 using ASO, germline deletion, or AAV CRISPR results in increased hepatic LDLR abundance and availability and decreased plasma LDL-C levels. CONCLUSIONS: Our studies identify RNF130 as a novel posttranslational regulator of LDL-C levels via modulation of LDLR availability, thus providing important insight into the complex regulation of hepatic LDLR protein levels.
Subject(s)
Liver , Receptors, LDL , Cholesterol, LDL/metabolism , Receptors, LDL/genetics , Receptors, LDL/metabolism , Liver/metabolism , Carrier Proteins/metabolism , Ubiquitination , Lipoproteins, LDL/metabolismABSTRACT
In cell models, changes in the 'accessible' pool of plasma membrane (PM) cholesterol are linked with the regulation of endoplasmic reticulum sterol synthesis and metabolism by the Aster family of nonvesicular transporters; however, the relevance of such nonvesicular transport mechanisms for lipid homeostasis in vivo has not been defined. Here we reveal two physiological contexts that generate accessible PM cholesterol and engage the Aster pathway in the liver: fasting and reverse cholesterol transport. During fasting, adipose-tissue-derived fatty acids activate hepatocyte sphingomyelinase to liberate sequestered PM cholesterol. Aster-dependent cholesterol transport during fasting facilitates cholesteryl ester formation, cholesterol movement into bile and very low-density lipoprotein production. During reverse cholesterol transport, high-density lipoprotein delivers excess cholesterol to the hepatocyte PM through scavenger receptor class B member 1. Loss of hepatic Asters impairs cholesterol movement into feces, raises plasma cholesterol levels and causes cholesterol accumulation in peripheral tissues. These results reveal fundamental mechanisms by which Aster cholesterol flux contributes to hepatic and systemic lipid homeostasis.
Subject(s)
Cholesterol , Liver , Cholesterol/metabolism , Biological Transport/physiology , Liver/metabolism , Homeostasis , Fatty Acids/metabolismABSTRACT
Studies have linked bibliometric indices with the academic level of plastic surgeons, but this relationship has not been explored with residency program directors (PDs). As teachers of the next generation, PDs' academic performance is an important component of residency program success. We sought to identify distinguishing characteristics of integrated plastic surgery programs, focusing on their PD bibliometric indices. Methods: We identified plastic surgery programs based on 2021 Doximity reputation and research output rankings, respectively, and then divided them into four quartiles (Q1-Q4). PD academic history and bibliometric indices (h-index, the number of publications, and citations) were collected through Doximity profiles and program websites: PubMed, Scopus, Google Scholar, American Society of Plastic Surgeons, and Accreditation Council for Graduate Medical Education. Results: Eighty-four programs were identified. There was a significant positive relationship between h-index, the number of publications, and type of research with reputation ranking (P < 0.05). After adjusting for years of experience post-training, h-index (OR = 1.24; P < 0.001) and the number of publications (OR = 1.05, P < 0.001) were significantly associated with reputation ranking. There was a statistically significant relationship between PD research fellowship completion and research output ranking (P < 0.01). After adjusting for years of experience post-training, h-index (OR = 1.05; P = 0.047) and the number of publications (OR = 1.01; P = 0.04) were significantly associated with research output ranking. Conclusion: Higher ranked programs tend to have PDs who have a strong record of scholarly activity, as evidenced by certain bibliometric indices.
ABSTRACT
BACKGROUND: To compare opioid prescribing practices of resident physicians across a variety of surgical and nonsurgical specialties; to identify factors which influence prescribing practices; and to examine resident utilization of best practice supplemental resources. METHODS: An anonymous survey which assessed prescribing practices was completed by residents from one of several different subspecialties, including internal medicine, obstetrics and gynecology, general surgery, neurosurgery, orthopedic surgery, and urology. Fisher's exact test assessed differences in prescribing practices between specialties. RESULTS: Only 35% of residents reported receiving formal training in safe opioid prescribing. Overall, the most frequently reported influences on prescribing practices were the use of standardized order sets for specific procedures, attending preference, and patient's history of prescribed opioids. Resident physicians significantly underutilize best practice supplemental resources, such as counseling patients on pain expectations prior to prescribing opioid medication; contacting established pain specialists; screening patients for opioid abuse; referring to the Prescription Monitoring Program; and counseling patients on safe disposal of unused pills (P < .001). DISCUSSION: The incorporation of comprehensive prescribing education into resident training and the utilization of standardized order sets can promote safe opioid prescribing.
Subject(s)
Internship and Residency , Physicians , Humans , Analgesics, Opioid/therapeutic use , Pain, Postoperative/drug therapy , Drug Prescriptions , Practice Patterns, Physicians'ABSTRACT
BACKGROUND: In mouse liver hepatocytes, nearly half of the surface area of every mitochondrion is covered by wrappER, a wrapping-type of ER that is rich in fatty acids and synthesizes lipoproteins (VLDL) (Anastasia et al. in Cell Rep 34:108873, 2021; Hurtley in Science (80- ) 372:142-143, 2021; Ilacqua et al. in J Cell Sci 135:1-11, 2021). A disruption of the ultrastructure of the wrappER-mitochondria contact results in altered fatty acid flux, leading to hepatic dyslipidemia (Anastasia et al. 2021). The molecular mechanism that regulates the extent of wrappER-mitochondria contacts is unknown. METHODS: We evaluated the expression level of the mitochondrial protein Synj2bp in the liver of normal and obese (ob/ob) mice. In addition, we silenced its expression in the liver using an AAV8 vector. We coupled quantitative EM morphometric analysis to proteomics and lipid analyses on these livers. RESULTS: The expression level of Synj2bp in the liver positively correlates with the extent of wrappER-mitochondria contacts. A 50% reduction in wrappER-mitochondria contacts causes hepatic dyslipidemia, characterized by a gross accumulation of lipid droplets in the liver, an increased hepatic secretion of VLDL and triglycerides, a curtailed ApoE expression, and an increased capacity of mitochondrial fatty acid respiration. CONCLUSION: Synj2bp regulates the extent of wrappER-mitochondria contacts in the liver, thus contributing to the control of hepatic lipid flux.
Subject(s)
Fatty Acids , Liver , Mitochondria , Animals , Mice , Adaptor Proteins, Signal Transducing/metabolism , Fatty Acids/metabolism , Homeostasis , Liver/metabolism , Membrane Proteins/metabolism , Mitochondria/metabolism , Mitochondria/physiology , ProteomicsABSTRACT
Parkin, an E3 ubiquitin ligase, plays an essential role in mitochondrial quality control. However, the mechanisms by which Parkin connects mitochondrial homeostasis with cellular metabolism in adipose tissue remain unclear. Here, we demonstrate that Park2 gene (encodes Parkin) deletion specifically from adipose tissue protects mice against high-fat diet and aging-induced obesity. Despite a mild reduction in mitophagy, mitochondrial DNA content and mitochondrial function are increased in Park2 deficient white adipocytes. Moreover, Park2 gene deletion elevates mitochondrial biogenesis by increasing Pgc1α protein stability through mitochondrial superoxide-activated NAD(P)H quinone dehydrogenase 1 (Nqo1). Both in vitro and in vivo studies show that Nqo1 overexpression elevates Pgc1α protein level and mitochondrial DNA content and enhances mitochondrial activity in mouse and human adipocytes. Taken together, our findings indicate that Parkin regulates mitochondrial homeostasis by balancing mitophagy and Pgc1α-mediated mitochondrial biogenesis in white adipocytes, suggesting a potential therapeutic target in adipocytes to combat obesity and obesity-associated disorders.
Subject(s)
Mitophagy , Organelle Biogenesis , Mice , Humans , Animals , Mitophagy/genetics , Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-alpha/genetics , Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-alpha/metabolism , Adipocytes, White/metabolism , Adiposity , Ubiquitin-Protein Ligases/metabolism , Obesity/genetics , DNA, Mitochondrial/genetics , DNA, Mitochondrial/metabolismABSTRACT
The nickel catalyzed reductive coupling of aldehydes with sorbate esters and related electron-deficient 1,3-dienes are known in the literature to occur at the π-bond proximal to the ester to afford aldol-type products. In stark contrast to this established path, a VAPOL-derived phosphoramidite ligand in combination with a bench-stable nickel precatalyst brokers a regiocomplementary course in that C-C bond formation proceeds exclusively at the distal alkene site to give deoxypropionate type products carrying an acrylate handle; they can be made in either anti- or syn-configured form. In addition to this enabling reverse pathway, the reaction is distinguished by excellent levels of chemo-, diastereo-, and enantioselectivity; moreover, it can be extended to the catalytic formation of F3C-substituted stereogenic centers. The use of a dienyl pinacolboronate instead of a sorbate ester is also possible, which opens access to valuable chiral borylated building blocks in optically active form.
Subject(s)
Electrons , Nickel , Nickel/chemistry , Stereoisomerism , Ligands , Aldehydes/chemistry , Catalysis , Alkenes/chemistry , Esters , Polyenes , AcrylatesABSTRACT
The low-density lipoprotein receptor (Ldlr) and apolipoprotein E (Apoe) germline knockout (KO) models have provided fundamental insights in lipid and atherosclerosis research for decades. However, testing new candidate genes in these models requires extensive breeding, which is highly time and resource consuming. In this chapter, we provide methods for rapidly modeling hypercholesterolemia and atherosclerosis as well as testing new genes in adult mice through somatic gene editing. Adeno-associated viral (AAV) vectors are exploited to deliver the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 genome editing system (AAV-CRISPR) to the liver. This tool enables rapid and efficient editing of lipid- and atherosclerosis-related genes in the liver.
Subject(s)
Atherosclerosis , Hypercholesterolemia , Hyperlipidemias , Animals , Atherosclerosis/genetics , CRISPR-Cas Systems/genetics , Gene Editing/methods , Hypercholesterolemia/genetics , MiceABSTRACT
OBJECTIVE: Distinct dominant mutations in the calcium-permeable ion channel TRPV4 (transient receptor potential vanilloid 4) typically cause nonoverlapping diseases of either the neuromuscular or skeletal systems. However, accumulating evidence suggests that some patients develop mixed phenotypes that include elements of both neuromuscular and skeletal disease. We sought to define the genetic and clinical features of these patients. METHODS: We report a 2-year-old with a novel R616G mutation in TRPV4 with a severe neuropathy phenotype and bilateral vocal cord paralysis. Interestingly, a different substitution at the same residue, R616Q, has been reported in families with isolated skeletal dysplasia. To gain insight into clinical features and potential genetic determinants of mixed phenotypes, we perform in-depth analysis of previously reported patients along with functional and structural assessment of selected mutations. RESULTS: We describe a wide range of neuromuscular and skeletal manifestations and highlight specific mutations that are more frequently associated with overlap syndromes. We find that mutations causing severe, mixed phenotypes have an earlier age of onset and result in more marked elevations of intracellular calcium, increased cytotoxicity, and reduced sensitivity to TRPV4 antagonism. Structural analysis of the two mutations with the most dramatic gain of ion channel function suggests that these mutants likely cause constitutive channel opening through disruption of the TRPV4 S5 transmembrane domain. INTERPRETATION: These findings demonstrate that the degree of baseline calcium elevation correlates with development of mixed phenotypes and sensitivity to pharmacologic channel inhibition, observations that will be critical for the design of future clinical trials for TRPV4 channelopathies.
Subject(s)
Peripheral Nervous System Diseases , TRPV Cation Channels , Calcium , Calcium Channels/genetics , Gain of Function Mutation , Humans , Mutation , Peripheral Nervous System Diseases/genetics , Phenotype , TRPV Cation Channels/chemistry , TRPV Cation Channels/geneticsABSTRACT
BACKGROUND: The intestine occupies the critical interface between cholesterol absorption and excretion. Surprisingly little is known about the role of de novo cholesterol synthesis in this organ, and its relationship to whole body cholesterol homeostasis. Here, we investigate the physiological importance of this pathway through genetic deletion of the rate-limiting enzyme. METHODS: Mice lacking 3-hydroxy-3-methylglutaryl-coenzyme A reductase (Hmgcr) in intestinal villus and crypt epithelial cells were generated using a Villin-Cre transgene. Plasma lipids, intestinal morphology, mevalonate pathway metabolites, and gene expression were analyzed. RESULTS: Mice with intestine-specific loss of Hmgcr were markedly smaller at birth, but gain weight at a rate similar to wild-type littermates, and are viable and fertile into adulthood. Intestine lengths and weights were greater relative to body weight in both male and female Hmgcr intestinal knockout mice. Male intestinal knockout had decreased plasma cholesterol levels, whereas fasting triglycerides were lower in both sexes. Lipidomics revealed substantial reductions in numerous nonsterol isoprenoids and sterol intermediates within the epithelial layer, but cholesterol levels were preserved. Hmgcr intestinal knockout mice also showed robust activation of SREBP-2 (sterol-regulatory element binding protein-2) target genes in the epithelium, including the LDLR (low-density lipoprotein receptor). At the cellular level, loss of Hmgcr is compensated for quickly after birth through a dramatic expansion of the stem cell compartment, which persists into adulthood. CONCLUSIONS: Loss of Hmgcr in the intestine is compatible with life through compensatory increases in intestinal absorptive surface area, LDLR expression, and expansion of the resident stem cell compartment.