We performed silencing and overexpression studies of flavin containing monooxygenase 3 (FMO3) in hyperlipidemic mouse models to examine its effects on trimethylamine N-oxide (TMAO) levels and atherosclerosis. Knockdown of hepatic FMO3 in LDL receptor null (LDLRKO) mice using an antisense oligonucleotide resulted in decreased circulating TMAO levels and atherosclerosis. Surprisingly, we also observed significant decreases in hepatic lipids and in levels of plasma lipids, ketone bodies, glucose and insulin. FMO3 over-expression in transgenic mice, on the other hand, increased hepatic and plasma lipids. Global gene expression analyses suggested that these effects of FMO3 on lipogenesis and gluconeogenesis may be mediated through the PPAR? and KLF15 pathways. In vivo and in vitro results were consistent with the concept that the effects were mediated directly by FMO3 rather than TMA/TMAO; in particular, over-expression of FMO3 in the human hepatoma cell line, Hep3B, resulted in significantly increased glucose secretion and lipogenesis. Our results indicate a major role for FMO3 in modulating glucose and lipid homeostasis in vivo, and they suggest that pharmacologic inhibition of FMO3 to reduce TMAO levels would be confounded by metabolic interactions.
Abnormalities in hepatic lipid metabolism and insulin action are believed to play a critical role in the etiology of nonalcoholic steatohepatitis. Monoacylglycerol acyltransferase (MGAT) enzymes convert monoacylglycerol to diacylglycerol, which is the penultimate step in one pathway for triacylglycerol synthesis. Hepatic expression of Mogat1, which encodes an MGAT enzyme, is increased in the livers of mice with hepatic steatosis, and knocking down Mogat1 improves glucose metabolism and hepatic insulin signaling, but whether increased MGAT activity plays a role in the etiology of nonalcoholic steatohepatitis is unclear. To examine this issue, mice were placed on a diet containing high levels of trans fatty acids, fructose, and cholesterol (HTF-C diet) or a low fat control diet for 4 weeks. Mice were injected with antisense oligonucleotides (ASOs) to knockdown Mogat1 or a scrambled ASO control for 12 weeks while remaining on diet. The HTF-C diet caused glucose intolerance, hepatic steatosis, and induced hepatic gene expression markers of inflammation, macrophage infiltration, and stellate cell activation. Mogat1 ASO treatment, which suppressed Mogat1 expression in liver and adipose tissue, attenuated weight gain, improved glucose tolerance, improved hepatic insulin signaling, and decreased hepatic triacylglycerol content compared with control ASO-treated mice on HTF-C chow. However, Mogat1 ASO treatment did not reduce hepatic diacylglycerol, cholesterol, or free fatty acid content; improve histologic measures of liver injury; or reduce expression of markers of stellate cell activation, liver inflammation, and injury. In conclusion, inhibition of hepatic Mogat1 in HTF-C diet-fed mice improves hepatic metabolic abnormalities without attenuating liver inflammation and injury.
Pericytes, the vascular cells that constitute the outer layer of capillaries, have been shown to have a crucial role in vascular development and stability. Loss of pericytes precedes endothelial cell dysfunction and vascular degeneration in small-vessel diseases, including diabetic retinopathy. Despite their clinical relevance, the cellular pathways controlling survival of retinal pericytes remain largely uncharacterized. Therefore, we investigated the role of Notch signaling, a master regulator of cell fate decisions, in retinal pericyte survival.
C-reactive protein (CRP) binds to damaged cells, activates the classical complement pathway, is elevated in multiple inflammatory conditions, and provides prognostic information on risk of future atherosclerotic events. It is controversial, however, as to whether inhibiting CRP synthesis would have any direct anti-inflammatory effects in humans.
Triantennary N-acetyl galactosamine (GalNAc, GN3: ), a high-affinity ligand for the hepatocyte-specific asialoglycoprotein receptor (ASGPR), enhances the potency of second-generation gapmer antisense oligonucleotides (ASOs) 6-10-fold in mouse liver. When combined with next-generation ASO designs comprised of short S-cEt (S-2'-O-Et-2',4'-bridged nucleic acid) gapmer ASOs, ? 60-fold enhancement in potency relative to the parent MOE (2'-O-methoxyethyl RNA) ASO was observed. GN3: -conjugated ASOs showed high affinity for mouse ASGPR, which results in enhanced ASO delivery to hepatocytes versus non-parenchymal cells. After internalization into cells, the GN3: -ASO conjugate is metabolized to liberate the parent ASO in the liver. No metabolism of the GN3: -ASO conjugate was detected in plasma suggesting that GN3: acts as a hepatocyte targeting prodrug that is detached from the ASO by metabolism after internalization into the liver. GalNAc conjugation also enhanced potency and duration of the effect of two ASOs targeting human apolipoprotein C-III and human transthyretin (TTR) in transgenic mice. The unconjugated ASOs are currently in late stage clinical trials for the treatment of familial chylomicronemia and TTR-mediated polyneuropathy. The ability to translate these observations in humans offers the potential to improve therapeutic index, reduce cost of therapy and support a monthly dosing schedule for therapeutic suppression of gene expression in the liver using ASOs.
Clathrin-mediated endocytosis (CME) is a fundamental process for the regulated internalization of transmembrane cargo and ligands via the formation of vesicles using a clathrin coat. A vesicle coat is initially created at the plasma membrane by clathrin assembly into a lattice, while a specific cargo sorting process selects and concentrates proteins for inclusion in the new vesicle. Vesicles formed via CME traffic to different parts of the cell and fuse with target membranes to deliver cargo. Both clathrin assembly and cargo sorting functions are features of the two gene family consisting of assembly protein 180 kDa (AP180) and clathrin assembly lymphoid myeloid leukemia protein (CALM). In this review, we compare the primary structure and domain organization of CALM and AP180 and relate these properties to known functions and roles in CME and disease.
Nonalcoholic fatty liver disease (NAFLD) spectrum disorders affect approximately 1 billion individuals worldwide. However, the drivers of progressive steatohepatitis remain incompletely defined. Ketogenesis can dispose of much of the fat that enters the liver, and dysfunction in this pathway could promote the development of NAFLD. Here, we evaluated mice lacking mitochondrial 3-hydroxymethylglutaryl CoA synthase (HMGCS2) to determine the role of ketogenesis in preventing diet-induced steatohepatitis. Antisense oligonucleotide-induced loss of HMGCS2 in chow-fed adult mice caused mild hyperglycemia, increased hepatic gluconeogenesis from pyruvate, and augmented production of hundreds of hepatic metabolites, a suite of which indicated activation of the de novo lipogenesis pathway. High-fat diet feeding of mice with insufficient ketogenesis resulted in extensive hepatocyte injury and inflammation, decreased glycemia, deranged hepatic TCA cycle intermediate concentrations, and impaired hepatic gluconeogenesis due to sequestration of free coenzyme A (CoASH). Supplementation of the CoASH precursors pantothenic acid and cysteine normalized TCA intermediates and gluconeogenesis in the livers of ketogenesis-insufficient animals. Together, these findings indicate that ketogenesis is a critical regulator of hepatic acyl-CoA metabolism, glucose metabolism, and TCA cycle function in the absorptive state and suggest that ketogenesis may modulate fatty liver disease.
The Course-Based Undergraduate Research Experiences Network (CUREnet) was initiated in 2012 with funding from the National Science Foundation program for Research Coordination Networks in Undergraduate Biology Education. CUREnet aims to address topics, problems, and opportunities inherent to integrating research experiences into undergraduate courses. During CUREnet meetings and discussions, it became apparent that there is need for a clear definition of what constitutes a CURE and systematic exploration of what makes CUREs meaningful in terms of student learning. Thus, we assembled a small working group of people with expertise in CURE instruction and assessment to: 1) draft an operational definition of a CURE, with the aim of defining what makes a laboratory course or project a "research experience"; 2) summarize research on CUREs, as well as findings from studies of undergraduate research internships that would be useful for thinking about how students are influenced by participating in CUREs; and 3) identify areas of greatest need with respect to CURE assessment, and directions for future research on and evaluation of CUREs. This report summarizes the outcomes and recommendations of this meeting.
Monoacylglycerol acyltransferase (MGAT) enzymes convert monoacylglycerol to diacylglycerol (DAG), a lipid that has been linked to the development of hepatic insulin resistance through activation of protein kinase C (PKC). The expression of genes that encode MGAT enzymes is induced in the livers of insulin-resistant human subjects with nonalcoholic fatty liver disease, but whether MGAT activation is causal of hepatic steatosis or insulin resistance is unknown. We show that the expression of Mogat1, which encodes MGAT1, and MGAT activity are also increased in diet-induced obese (DIO) and ob/obmice. To probe the metabolic effects of MGAT1 in the livers of obese mice, we administered antisense oligonucleotides (ASOs) against Mogat1 to DIO and ob/ob mice for 3 weeks. Knockdown of Mogat1 in liver, which reduced hepatic MGAT activity, did not affect hepatic triacylglycerol content and unexpectedly increased total DAG content. Mogat1 inhibition also increased both membrane and cytosolic compartment DAG levels. However, Mogat1 ASO treatment significantly improved glucose tolerance and hepatic insulin signaling in obese mice. In summary, inactivation of hepatic MGAT activity, which is markedly increased in obese mice, improved glucose tolerance and hepatic insulin signaling independent of changes in body weight, intrahepatic DAG and TAG content, and PKC signaling.
Increased plasma levels of C-reactive protein (CRP) are closely associated with cardiovascular diseases, but whether CRP is directly involved in the pathogenesis of atherosclerosis is still under debate. Many controversial and contradictory results using transgenic mice and rabbits have been published but it is also unclear whether CRP lowering can be used for the treatment of atherosclerosis. In the current study, we examined the effects of the rabbit CRP antisense oligonucleotides (ASO) on the development of atherosclerosis in WHHL rabbits. CRP ASO treatment led to a significant reduction of plasma CRP levels; however, both aortic and coronary atherosclerotic lesions were not significantly changed compared to those of control WHHL rabbits. These results suggest that inhibition of plasma CRP does not affect the development of atherosclerosis in WHHL rabbits.
Previous studies demonstrated that L-Fabp KO mice are more susceptible to lithogenic diet (LD)-induced gallstones because of altered hepatic cholesterol metabolism and increased canalicular cholesterol secretion. Other studies demonstrated that liver-specific deletion of microsomal triglyceride transfer protein (Mttp-LKO) reduced LD-induced gallstone formation by increasing biliary phospholipid secretion. Here we show that mice with combined deletion (i.e., DKO mice) are protected from LD-induced gallstone formation. Following 2 weeks of LD feeding, 73% of WT and 100% of L-Fabp KO mice developed gallstones versus 18% of Mttp-LKO and 23% of DKO mice. This phenotype was recapitulated in both WT and L-Fabp KO mice treated with an Mttp antisense oligonucleotide (M-ASO). Biliary cholesterol secretion was increased in LD-fed L-Fabp KO mice and decreased in DKO mice. However, phospholipid secretion was unchanged in LD-fed Mttp-LKO and DKO mice as well as in M-ASO-treated mice. Expression of the canalicular export pump ABCG5/G8 was reduced in LD-fed DKO mice and in M-ASO-treated L-Fabp KO mice. We conclude that liver-specific Mttp deletion not only eliminates apical lipoprotein secretion from hepatocytes but also attenuates canalicular cholesterol secretion, which in turn decreases LD-induced gallstone susceptibility.
Raised blood C-reactive protein (CRP) level is a predictor of cardiovascular events, but whether blood CRP is causal in the disease process is unknown. The latter would best be defined by pharmacological inhibition of the protein in the context of a randomized case-control study. However, no CRP specific drug is currently available so such a prospective study cannot be performed. Blood CRP is synthesized primarily in the liver and the liver is an organ where antisense oligonucleotide (ASO) drugs accumulate. Taking advantage of this we evaluated the efficacy of CRP specific ASOs in rodents with experimentally induced cardiovascular damage. Treating rats for 4 weeks with a rat CRP-specific ASO achieved >60% reduction of blood CRP. Notably, this effect was associated with improved heart function and pathology following myocardial infarction (induced by ligation of the left anterior descending artery). Likewise in human CRP transgenic mice treated for 2 weeks with a human CRP-specific ASO, blood human CRP was reduced by >70% and carotid artery patency was improved (2 weeks after surgical ligation). CRP specific ASOs might pave the way towards a placebo-controlled trial that could clarify the role of CRP in cardiovascular disease.
Brain-specific AP180 is present in clathrin coats at equal concentration to the adapter complex, AP2, and assembles clathrin faster than any other protein in vitro. Both AP180 and its ubiquitously expressed homolog clathrin assembly lymphoid myeloid leukemia protein (CALM) control vesicle size and shape in clathrin mediated endocytosis. The clathrin assembly role of AP180 is mediated by a long disordered C-terminal assembly domain. Within this assembly domain, a central acidic clathrin and adapter binding (CLAP) sub-domain contains all of the known short binding motifs for clathrin and AP2. The role of the remaining ?16 kDa C-terminal sequence has not been clear. We show that this sequence has a separate function in ensuring efficient binding of clathrin, based on in vitro binding and ex vivo transferrin uptake assays. Sequence alignment suggests the C-terminal sub-domain is conserved in CALM.
The primary risk factor for atherosclerotic cardiovascular disease is LDL cholesterol, which can be reduced by increasing cholesterol excretion from the body. Fecal cholesterol excretion can be driven by a hepatobiliary as well as a non-biliary pathway known as transintestinal cholesterol efflux (TICE). We previously showed that chronic knockdown of the hepatic cholesterol esterifying enzyme sterol O-acyltransferase 2 (SOAT2) increased fecal cholesterol loss via TICE. To elucidate the initial events that stimulate TICE, C57Bl/6 mice were fed a high cholesterol diet to induce hepatic cholesterol accumulation and were then treated for 1 or 2 weeks with an antisense oligonucleotide targeting SOAT2. Within 2 weeks of hepatic SOAT2 knockdown (SOAT2HKD), the concentration of cholesteryl ester in the liver was reduced by 70% without a reciprocal increase in hepatic free cholesterol. The rapid mobilization of hepatic cholesterol stores resulted in a ? 2-fold increase in fecal neutral sterol loss but no change in biliary cholesterol concentration. Acute SOAT2HKD increased plasma cholesterol carried primarily in lipoproteins enriched in apoB and apoE. Collectively, our data suggest that acutely reducing SOAT2 causes hepatic cholesterol to be swiftly mobilized and packaged onto nascent lipoproteins that feed cholesterol into the TICE pathway for fecal excretion.
ACAT2 is the exclusive cholesterol-esterifying enzyme in hepatocytes and enterocytes. Hepatic ABCA1 transfers unesterified cholesterol (UC) to apoAI, thus generating HDL. By changing the hepatic UC pool available for ABCA1, ACAT2 may affect HDL metabolism. The aim of this study was to reveal whether hepatic ACAT2 influences HDL metabolism.
An effective way to reduce LDL cholesterol, the primary risk factor of atherosclerotic cardiovascular disease, is to increase cholesterol excretion from the body. Our group and others have recently found that cholesterol excretion can be facilitated by both hepatobiliary and transintestinal pathways. However, the lipoprotein that moves cholesterol through the plasma to the small intestine for transintestinal cholesterol efflux (TICE) is unknown. To test the hypothesis that hepatic very low-density lipoproteins (VLDL) support TICE, antisense oligonucleotides (ASO) were used to knockdown hepatic expression of microsomal triglyceride transfer protein (MTP), which is necessary for VLDL assembly. While maintained on a high cholesterol diet, Niemann-Pick C1-like 1 hepatic transgenic (L1Tg) mice, which predominantly excrete cholesterol via TICE, and wild type (WT) littermates were treated with control ASO or MTP ASO. In both WT and L1Tg mice, MTP ASO decreased VLDL triglyceride (TG) and cholesterol secretion. Regardless of treatment, L1Tg mice had reduced biliary cholesterol compared to WT mice. However, only L1Tg mice treated with MTP ASO had reduced fecal cholesterol excretion. Based upon these findings, we conclude that VLDL or a byproduct such as LDL can move cholesterol from the liver to the small intestine for TICE.
A novel method for the synthesis of a wide range of 1,5-disubstituted 1,2-dihydro-1,2,4-triazol-3-ones is described. The key step involves a reaction between a dilithiated BOC-hydrazine and a N-alkoxycarbonylcarboximidothioate. A broad range of aryl and alkyl functional groups are tolerated, providing a versatile route for the synthesis of triazolones.
A combined glycomics and glycoproteomics strategy was developed for the site-specific analysis of N-linked glycosylation heterogeneity from a complex mammalian protein mixture. Initially, global characterization of the N-glycome was performed using porous graphitized carbon liquid chromatography-tandem mass spectrometry (PGC-LC-MS/MS) and the data used to create an N-glycan modification database. In the next step, tryptic glycopeptides were enriched using zwitterionic hydrophilic interaction liquid chromatography (Zic-HILIC) and fractionated by reversed-phase liquid chromatography (RPLC; pH 7.9). The resulting fractions were each separated into two equal aliquots. The first set of aliquots were treated with peptide-N-glycosidase F (PNGase F) to remove N-glycans and the former N-glycopeptides analyzed by nano-RPLC-MS/MS (pH 2.7) and identified by Mascot database search. This enabled the creation of a glycopeptide-centric concatenated database for each fraction. The second set of aliquots was analyzed directly by nanoRPLC-MS/MS (pH 2.7), employing fragmentation by CID and HCD. The assignment of glycan compositions to peptide sequences was achieved by searching the N-glycopeptide HCD MS/MS spectra against the glycopeptide-centric concatenated databases employing the N-glycan modification database. CID spectra were used to assign glycan structures identified in the glycomic analysis to peptide sequences. This multidimensional approach allowed confident identification of 863 unique intact N-linked glycopeptides from 161 rat brain glycoproteins.
Antisense oligonucleotides (ASOs) are a new class of specific therapeutic agents that alter the intermediary metabolism of mRNA, resulting in the suppression of disease-associated gene products. ASOs exert their pharmacological effects after hybridizing, via Watson-Crick base pairing, to a specific target RNA. If appropriately designed, this event results in the recruitment of RNase H, the degradation of targeted mRNA or pre-mRNA, and subsequent inhibition of the synthesis of a specific protein. A key advantage of the technology is the ability to selectively inhibit targets that cannot be modulated by traditional therapeutics such as structural proteins, transcription factors, and, of topical interest, lipoproteins. In this chapter, we will first provide an overview of antisense technology, then more specifically describe the status of lipoprotein-related genes that have been studied using the antisense platform, and finally, outline the general methodology required to design and evaluate the in vitro and in vivo efficacy of those drugs.
The serine hydrolase ?/? hydrolase domain 6 (ABHD6) has recently been implicated as a key lipase for the endocannabinoid 2-arachidonylglycerol (2-AG) in the brain. However, the biochemical and physiological function for ABHD6 outside of the central nervous system has not been established. To address this, we utilized targeted antisense oligonucleotides (ASOs) to selectively knock down ABHD6 in peripheral tissues in order to identify in vivo substrates and understand ABHD6s role in energy metabolism. Here, we show that selective knockdown of ABHD6 in metabolic tissues protects mice from high-fat-diet-induced obesity, hepatic steatosis, and systemic insulin resistance. Using combined in vivo lipidomic identification and in vitro enzymology approaches, we show that ABHD6 can hydrolyze several lipid substrates, positioning ABHD6 at the interface of glycerophospholipid metabolism and lipid signal transduction. Collectively, these data suggest that ABHD6 inhibitors may serve as therapeutics for obesity, nonalcoholic fatty liver disease, and type II diabetes.
2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a potent activator of the aryl hydrocarbon receptor (AhR) and causes chloracne in humans. The pathogenesis and role of AhR in chloracne remains incompletely understood.
Due to their ability to promote positive effects across all of the lipoprotein classes, cholesteryl ester transfer protein (CETP) inhibitors are currently being developed as therapeutic agents for cardiovascular disease. In these studies, we compared an antisense oligonucleotide (ASO) inhibitor of CETP to the CETP small molecule inhibitor anacetrapib. In hyperlipidemic CETP transgenic (tg) mice, both drugs provided comparable reductions in total plasma cholesterol, decreases in CETP activity, and increases in HDL cholesterol. However, only mice treated with the antisense inhibitor showed an enhanced effect on macrophage reverse cholesterol transport, presumably due to differences in HDL apolipoprotein composition and decreases in plasma triglyceride. Additionally, the ASO-mediated reductions in CETP mRNA were associated with less accumulation of aortic cholesterol. These preliminary findings suggest that CETP ASOs may represent an alternative means to inhibit that target and to support their continued development as a treatment for cardiovascular disease in man.
Antisense oligonucleotides and small interfering RNAs, which suppress the translation of specific mRNA target proteins, are emerging as important therapeutic modalities for the treatment of cardiovascular disease. Over the last 25 years, the advances in all aspects of antisense technology, as well as a detailed understanding of the mechanism of action of antisense drugs, have enabled their use as therapeutic agents. These advancements culminated in the FDA approval of the first chronically administered cardiovascular antisense therapeutic, mipomersen, which targets hepatic apolipoprotein B mRNA. This review provides a brief history of antisense technology, highlights the progression of mipomersen from preclinical studies to multiple Phase III registration trials, and gives an update on the status of other cardiovascular antisense therapeutics currently in the clinic.
Niemann-Pick disease, type C1 (NPC1), which arises from a mutation in the NPC1 gene, is characterized by abnormal cellular storage and transport of cholesterol and other lipids that leads to hepatic disease and progressive neurological impairment. Oxidative stress has been hypothesized to contribute to the NPC1 disease pathological cascade. To determine whether treatments reducing oxidative stress could alleviate NPC1 disease phenotypes, the in vivo effects of the antioxidant N-acetylcysteine (NAC) on two mouse models for NPC1 disease were studied. NAC was able to partially suppress phenotypes in both antisense-induced (NPC1ASO) and germline (Npc1-/-) knockout genetic mouse models, confirming the presence of an oxidative stress-related mechanism in progression of NPC1 phenotypes and suggesting NAC as a potential molecule for treatment. Gene expression analyses of NAC-treated NPC1ASO mice suggested NAC affects pathways distinct from those initially altered by Npc1 knockdown, data consistent with NAC achieving partial disease phenotype suppression. In a therapeutic trial of short-term NAC administration to NPC1 patients, no significant effects on oxidative stress in these patients were identified other than moderate improvement of the fraction of reduced CoQ10, suggesting limited efficacy of NAC monotherapy. However, the mouse model data suggest that the distinct antioxidant effects of NAC could provide potential treatment of NPC1 disease, possibly in concert with other therapeutic molecules at earlier stages of disease progression. These data also validated the NPC1ASO mouse as an efficient model for candidate NPC1 drug screening, and demonstrated similarities in hepatic phenotypes and genome-wide transcript expression patterns between the NPC1ASO and Npc1-/- models.
Elevated plasma triglyceride levels have been recognized as a risk factor for the development of coronary heart disease. Apolipoprotein C-III (apoC-III) represents both an independent risk factor and a key regulatory factor of plasma triglyceride concentrations. Furthermore, elevated apoC-III levels have been associated with metabolic syndrome and type 2 diabetes mellitus. To date, no selective apoC-III therapeutic agent has been evaluated in the clinic.
Circulating trimethylamine-N-oxide (TMAO) levels are strongly associated with atherosclerosis. We now examine genetic, dietary, and hormonal factors regulating TMAO levels. We demonstrate that two flavin mono-oxygenase family members, FMO1 and FMO3, oxidize trimethylamine (TMA), derived from gut flora metabolism of choline, to TMAO. Further, we show that FMO3 exhibits 10-fold higher specific activity than FMO1. FMO3 overexpression in mice significantly increases plasma TMAO levels while silencing FMO3 decreases TMAO levels. In both humans and mice, hepatic FMO3 expression is reduced in males compared to females. In mice, this reduction in FMO3 expression is due primarily to downregulation by androgens. FMO3 expression is induced by dietary bile acids by a mechanism that involves the farnesoid X receptor (FXR), a bile acid-activated nuclear receptor. Analysis of natural genetic variation among inbred strains of mice indicates that FMO3 and TMAO are significantly correlated, and TMAO levels explain 11% of the variation in atherosclerosis.
Organisms are often exposed to environmental pressures that affect homeostasis, so it is important to understand the biological basis of stress-response. Various biological mechanisms have evolved to help cells cope with potentially cytotoxic changes in their environment. miRNAs are small non-coding RNAs which are able to regulate mRNA stability. It has been suggested that miRNAs may tip the balance between continued cytorepair and induction of apoptosis in response to stress. There is a wealth of data in the literature showing the effect of environmental stress on miRNAs, but it is scattered in a large number of disparate publications. Meta-analyses of this data would produce added insight into the molecular mechanisms of stress-response. To facilitate this we created and manually curated the miRStress database, which describes the changes in miRNA levels following an array of stress types in eukaryotic cells. Here we describe this database and validate the miRStress tool for analysing miRNAs that are regulated by stress. To validate the database we performed a cross-species analysis to identify miRNAs that respond to radiation. The analysis tool confirms miR-21 and miR-34a as frequently deregulated in response to radiation, but also identifies novel candidates as potentially important players in this stress response, including miR-15b, miR-19b, and miR-106a. Similarly, we used the miRStress tool to analyse hypoxia-responsive miRNAs. The most frequently deregulated miRNAs were miR-210 and miR-21, as expected. Several other miRNAs were also found to be associated with hypoxia, including miR-181b, miR-26a/b, miR-106a, miR-213 and miR-192. Therefore the miRStress tool has identified miRNAs with hitherto unknown or under-appreciated roles in the response to specific stress types. The miRStress tool, which can be used to uncover new insight into the biological roles of miRNAs, and also has the potential to unearth potential biomarkers for therapeutic response, is freely available at http://mudshark.brookes.ac.uk/MirStress.
The adhesion disc of living remoras (Echeneoidea: Echeneidae) represents one of the most remarkable structural innovations within fishes. Although homology between the spinous dorsal fin of generalized acanthomorph fishes and the remora adhesion disc is widely accepted, the sequence of evolutionary-rather than developmental-transformations leading from one to the other has remained unclear. Here, we show that the early remora †Opisthomyzon (Echeneoidea: †Opisthomyzonidae), from the early Oligocene (Rupelian) of Switzerland, is a stem-group echeneid and provides unique insights into the evolutionary assembly of the unusual body plan characteristic of all living remoras. The adhesion disc of †Opisthomyzon retains ancestral features found in the spiny dorsal fins of remora outgroups, and corroborates developmental interpretations of the homology of individual skeletal components of the disc. †Opisthomyzon indicates that the adhesion disc originated in a postcranial position, and that other specializations (including the origin of pectination, subdivision of median fin spines into paired lamellae, increase in segment count and migration to a supracranial position) took place later in the evolutionary history of remoras. This phylogenetic sequence of transformation finds some parallels in the order of ontogenetic changes to the disc documented for living remoras.
Lipin-1 catalyzes the formation of diacylglycerol from phosphatidic acid. Lipin-1 mutations cause lipodystrophy in mice and acute myopathy in humans. It is heavily phosphorylated, and the yeast ortholog Pah1p becomes membrane-associated and active upon dephosphorylation by the Nem1p-Spo7p membrane complex. A mammalian ortholog of Nem1p is the C-terminal domain nuclear envelope phosphatase 1 (CTDNEP1, formerly "dullard"), but its Spo7p-like partner is unknown, and the need for its existence is debated. Here, we identify the metazoan ortholog of Spo7p, TMEM188, renamed nuclear envelope phosphatase 1-regulatory subunit 1 (NEP1-R1). CTDNEP1 and NEP1-R1 together complement a nem1?spo7? strain to block endoplasmic reticulum proliferation and restore triacylglycerol levels and lipid droplet number. The two human orthologs are in a complex in cells, and the amount of CTDNEP1 is increased in the presence of NEP1-R1. In the Caenorhabditis elegans embryo, expression of nematode CTDNEP1 and NEP1-R1, as well as lipin-1, is required for normal nuclear membrane breakdown after zygote formation. The expression pattern of NEP1-R1 and CTDNEP1 in human and mouse tissues closely mirrors that of lipin-1. CTDNEP1 can dephosphorylate lipins-1a, -1b, and -2 in human cells only in the presence of NEP1-R1. The nuclear fraction of lipin-1b is increased when CTDNEP1 and NEP1-R1 are co-expressed. Therefore, NEP1-R1 is functionally conserved from yeast to humans and functions in the lipin activation pathway.
We recently reported that inhibition of 11?-hydroxysteroid dehydrogenase 1 (11?-HSD1) by antisense oligonucleotide (ASO) improved hepatic lipid metabolism independent of food intake. In that study, 11?-HSD1 ASO-treated mice lost weight compared with food-matched control ASO-treated mice, suggesting treatment-mediated increased energy expenditure. We have now examined the effects of 11?-HSD1 ASO treatment on adipose tissue metabolism, insulin sensitivity, and whole-body energy expenditure. We used an ASO to knock down 11?-HSD1 in C57BL/6J mice consuming a Western-type diet (WTD). The 11?-HSD1 ASO-treated mice consumed less food, so food-matched control ASO-treated mice were also evaluated. We characterized body composition, gene expression of individual adipose depots, and measures of energy metabolism. We also investigated glucose/insulin tolerance as well as acute insulin signaling in several tissues. Knockdown of 11?-HSD1 protected against WTD-induced obesity by reducing epididymal, mesenteric, and subcutaneous white adipose tissue while activating thermogenesis in brown adipose tissue. The latter was confirmed by demonstrating increased energy expenditure in 11?-HSD1 ASO-treated mice. The 11?-HSD1 ASO treatment also protected against WTD-induced glucose intolerance and insulin resistance; this protection was associated with smaller cells and fewer macrophages in epididymal white adipose tissue as well as enhanced in vivo insulin signaling. Our results indicate that ASO-mediated inhibition of 11?-HSD1 can protect against several WTD-induced metabolic abnormalities. These effects are, at least in part, mediated by increases in the oxidative capacity of brown adipose tissue.
Training future doctors to develop an appropriate professional persona is an important goal of medical student education and residency training. Most medical education research paradigms on professionalism have focused largely on lapses (e.g. yelling as an example of communication failure) and tend to emphasise behaviour that should be avoided. The assumption is that, if left unchecked, students will see these negative behaviours exhibited by their role models and possibly emulate them, allowing the potential reinforcement of the inappropriate behaviours.
We examined the feasibility of using CYP2D6 genotyping to determine optimal tamoxifen dose and investigated whether the key active tamoxifen metabolite, endoxifen, could be increased by genotype-guided tamoxifen dosing in patients with intermediate CYP2D6 metabolism.
Depolarization of nerve terminals stimulates rapid dephosphorylation of two isoforms of dynamin I (dynI), mediated by the calcium-dependent phosphatase calcineurin (CaN). Dephosphorylation at the major phosphorylation sites Ser-774/778 promotes a dynI-syndapin I interaction for a specific mode of synaptic vesicle endocytosis called activity-dependent bulk endocytosis (ADBE). DynI has two main splice variants at its extreme C terminus, long or short (dynIxa and dynIxb) varying only by 20 (xa) or 7 (xb) residues. Recombinant GST fusion proteins of dynIxa and dynIxb proline-rich domains (PRDs) were used to pull down interacting proteins from rat brain nerve terminals. Both bound equally to syndapin, but dynIxb PRD exclusively bound to the catalytic subunit of CaNA, which recruited CaNB. Binding of CaN was increased in the presence of calcium and was accompanied by further recruitment of calmodulin. Point mutations showed that the entire C terminus of dynIxb is a CaN docking site related to a conserved CaN docking motif (PXIXI(T/S)). This sequence is unique to dynIxb among all other dynamin variants or genes. Peptide mimetics of the dynIxb tail blocked CaN binding in vitro and selectively inhibited depolarization-evoked dynI dephosphorylation in nerve terminals but not of other dephosphins. Therefore, docking to dynIxb is required for the regulation of both dynI splice variants, yet it does not regulate the phosphorylation cycle of other dephosphins. The peptide blocked ADBE, but not clathrin-mediated endocytosis of synaptic vesicles. Our results indicate that Ca(2+) influx regulates assembly of a fully active CaN-calmodulin complex selectively on the tail of dynIxb and that the complex is recruited to sites of ADBE in nerve terminals.
We examine the concept of translational research from the perspective of evaluators charged with assessing translational efforts. One of the major tasks for evaluators involved in translational research is to help assess efforts that aim to reduce the time it takes to move research to practice and health impacts. Another is to assess efforts that are intended to increase the rate and volume of translation.
Serum levels of apolipoprotein CIII (apoCIII) are increased in type 1 diabetic patients, and when ? cells are exposed to these diabetic sera, apoptosis occurs, an effect abolished by an antibody against apoCIII. We have investigated the BB rat, an animal model that develops a human-like type 1 diabetes, and found that apoCIII was also increased in sera from prediabetic rats. This increase in apoCIII promoted ?-cell death. The endogenous levels of apoCIII were reduced by treating prediabetic animals with an antisense against this apolipoprotein, resulting in a significantly delayed onset of diabetes. ApoCIII thus serves as a diabetogenic factor, and intervention with this apolipoprotein in the prediabetic state can arrest disease progression. These findings suggest apoCIII as a target for the treatment of type 1 diabetes.
Protein phosphorylation and glycosylation are the most common post-translational modifications observed in biology, frequently on the same protein. Assembly protein AP180 is a synapse-specific phosphoprotein and O-linked beta-N-acetylglucosamine (O-GlcNAc) modified glycoprotein. AP180 is involved in the assembly of clathrin coated vesicles in synaptic vesicle endocytosis. Unlike other types of O-glycosylation, O-GlcNAc is nucleocytoplasmic and reversible. It was thought to be a terminal modification, that is, the O-GlcNAc was not found to be additionally modified in any way. We now show that AP180 purified from rat brain contains a phosphorylated O-GlcNAc (O-GlcNAc-P) within a highly conserved sequence. O-GlcNAc or O-GlcNAc-P, but not phosphorylation alone, was found at Thr-310. Analysis of synthetic GlcNAc-6-P produced identical fragmentation products to GlcNAc-P from AP180. Direct O-linkage of GlcNAc-P to a Thr residue was confirmed by electron transfer dissociation MS. A second AP180 tryptic peptide was also glycosyl phosphorylated, but the site of modification was not assigned. Sequence similarities suggest there may be a common motif within AP180 involving glycosyl phosphorylation and dual flanking phosphorylation sites within 4 amino acid residues. This novel type of protein glycosyl phosphorylation adds a new signaling mechanism to the regulation of neurotransmission and more complexity to the study of O-GlcNAc modification.
Ru(II) complexes of TsDPEN containing two alkyl groups on the non-tosylated nitrogen atom are poor catalysts for asymmetric transfer hydrogenation of ketones and imines; this observation provides direct evidence for the importance of the N-H interaction in the transition state for ketone reduction.
11?-hydroxysteroid dehydrogenase 1 (11?-HSD1) converts inactive 11-keto derivatives to active glucocorticoids within tissues and may play a role in the metabolic syndrome (MS). We used an antisense oligonucleotide (ASO) to knock down 11?-HSD1 in livers of C57BL/6J mice consuming a Western-type diet (WTD). 11?-HSD1 ASO-treated mice consumed less food, so we compared them to ad libitum-fed mice and to food-matched mice receiving control ASO. Knockdown of 11?-HSD1 directly protected mice from WTD-induced steatosis and dyslipidemia by reducing synthesis and secretion of triglyceride (TG) and increasing hepatic fatty acid oxidation. These changes in hepatic and plasma lipids were not associated with reductions in genes involved in de novo lipogenesis. However, protein levels of both sterol regulatory element-binding protein (SREBP) 1 and fatty acid synthase were significantly reduced in mice treated with 11?-HSD1 ASO. There was no change in hepatic secretion of apolipoprotein (apo)B, indicating assembly and secretion of smaller apoB-containing lipoproteins by the liver in the 11?-HSD1-treated mice. Our results indicate that inhibition of 11?-HSD1 by ASO treatment of WTD-fed mice resulted in improved plasma and hepatic lipid levels, reduced lipogenesis by posttranslational regulation, and secretion of similar numbers of apoB-containing lipoproteins containing less TG per particle.
Chronic elevations of plasma apolipoprotein B (apoB) are strongly associated with cardiovascular disease. We have previously demonstrated that inhibition of hepatic apoB mRNA using antisense oligonucleotides (ASO) results in reductions of apoB, VLDL, and LDL in several preclinical animal models and humans. In this study, we evaluated the anti-atherogenic effects of a murine-specific apoB ASO (ISIS 147764) in hypercholesterolemic LDLr deficient (LDLr(-/-)) mice. ISIS 147764 was administered weekly at 25-100 mg/kg for 10-12 weeks and produced dose-dependent reductions of hepatic apoB mRNA and plasma LDL by 60-90%. No effects on these parameters were seen in mice receiving control ASOs. ApoB ASO treatment also produced dose-dependent reductions of aortic en face and sinus atherosclerosis from 50-90%, with high-dose treatment displaying less disease than the saline-treated, chow-fed LDLr(-/-) mice. No changes in intestinal cholesterol absorption were seen with apoB ASO treatment, suggesting that the cholesterol-lowering pharmacology of 147764 was primarily due to inhibition of hepatic apoB synthesis and secretion. In summary, ASO-mediated suppression of apoB mRNA expression profoundly reduced plasma lipids and atherogenesis in LDLr(-/-) mice, leading to the hypothesis that apoB inhibition in humans with impaired LDLr activity may produce similar effects.
The nuclear receptor superfamily of ligand-activated transcription factors plays a central role in the regulation of cellular responses to chemical challenge. Nuclear receptors are activated by a wide range of both endogenous and exogenous chemicals, and their target genes include those involved in the metabolism and transport of the activating chemical. Such target gene activation, thus, acts to remove the stimulating xenobiotic or to maintain homeostatic levels of endogenous chemicals. Given the dual nature of this system it is important to understand how these two roles are balanced, such that xenobiotics are efficiently removed while not impacting negatively on homeostasis of endogenous chemicals. Using DNA microarray technology we have examined the transcriptome response of primary rat hepatocytes to two nuclear receptor ligands: Pregnenalone-16?-carbonitrile (PCN), a xenobiotic PXR agonist, and lithocholic acid, an endogenous mixed PXR/VDR/FXR agonist. We demonstrate that despite differences in the profile of activated nuclear receptors, transcriptome responses for these two ligands are broadly similar at lower concentrations, indicating a conserved general response. However, as concentrations of stimulating ligand rises, the transcriptome responses diverge, reflecting a need for specific responses to the two stimulating chemicals. Finally, we demonstrate a novel feed-back loop for PXR, whereby ligand-activation of PXR suppresses transcription of the PXR gene, acting to attenuate PXR protein expression levels at higher ligand concentrations. Through in silico simulation we demonstrate that this feed-back loop is an important factor to prevent hyperexpression of PXR target genes such as CYP3A and confirm these findings in vitro. This novel insight into the regulation of the PXR-mediated regulatory signal networks provides a potential mechanistic rationale for the robustness in steroid homeostasis within the cell.
As future physicians, questions about when medical students realize they will have to teach remain under-explored. Aim: To understand when students serving in pre-clinical teaching roles make the connection between teaching and being a physician.
Effective measures for controlling chronic wasting disease (CWD), a contagious prion disease of cervids, remain elusive. We review theoretic relationships between predation and host-parasite dynamics and describe a mathematical model to evaluate the potential influence of random removal through harvest or culling and selective predation by wolves (Canis lupus) upon CWD dynamics in deer (Odocoileus spp.) populations. Imposing nonselective mortality representing a 15% annual harvest or cull 51 yr after CWD introduction lowered both deer population size and steady state CWD. Selective (4×) mortality at the same 15% predation rate caused a more modest reduction in deer population size accompanied by a relatively rapid decline in CWD prevalence and elimination of the disease from a closed population. The impacts of selective predation on epidemic dynamics were sensitive to assumptions on parameter estimates; however, within expected ranges, the results of selective predation were consistent and robust. We suggest that as CWD distribution and wolf range overlap in the future, wolf predation may suppress disease emergence or limit prevalence.
This study measured and analyzed the outdoor airborne endotoxin concentration, on particulate matter (PM²·? and PM¹?), for two cities in the interior of British Columbia, Canada. Samples were collected throughout one seasonal cycle, from October 2005 to September 2006. It was found that concentrations were generally highest in the summer and fall, and lowest in the winter and spring. Temperature and relative humidity were found to be most influential, with highest endotoxin concentrations recorded during warm periods and moderate relative humidity (35 to 75 percent). No clear association of concentration with wind direction was observed. Results were comparable between the two cities considered in this study, and concentrations were similar to or slightly higher than those reported by other studies considering urban locations. Endotoxin concentration was also found to be positively associated with agricultural dust sources identified by a source apportionment study conducted at one of the sampling locations.
The recognition and signaling of DNA double strand breaks involves the participation of multiple proteins, including the protein kinase ATM (mutated in ataxia-telangiectasia). ATM kinase is activated in the vicinity of the break and is recruited to the break site by the Mre11-Rad50-Nbs1 complex, where it is fully activated. In human cells, the activation process involves autophosphorylation on three sites (Ser(367), Ser(1893), and Ser(1981)) and acetylation on Lys(3016). We now describe the identification of a new ATM phosphorylation site, Thr(P)(1885) and an additional autophosphorylation site, Ser(P)(2996), that is highly DNA damage-inducible. We also confirm that human and murine ATM share five identical phosphorylation sites. We targeted the ATM phosphorylation sites, Ser(367) and Ser(2996), for further study by generating phosphospecific antibodies against these sites and demonstrated that phosphorylation of both was rapidly induced by radiation. These phosphorylations were abolished by a specific inhibitor of ATM and were dependent on ATM and the Mre11-Rad50-Nbs1 complex. As found for Ser(P)(1981), ATM phosphorylated at Ser(367) and Ser(2996) localized to sites of DNA damage induced by radiation, but ATM recruitment was not dependent on phosphorylation at these sites. Phosphorylation at Ser(367) and Ser(2996) was functionally important because mutant forms of ATM were defective in correcting the S phase checkpoint defect and restoring radioresistance in ataxia-telangiectasia cells. These data provide further support for the importance of autophosphorylation in the activation and function of ATM in vivo.
Mutations of Comparative Gene Identification-58 (CGI-58) in humans cause triglyceride (TG) accumulation in multiple tissues. Mice genetically lacking CGI-58 die shortly after birth due to a skin barrier defect. To study the role of CGI-58 in integrated lipid and energy metabolism, we utilized antisense oligonucleotides (ASOs) to inhibit CGI-58 expression in adult mice. Treatment with two distinct CGI-58-targeting ASOs resulted in ?80-95% knockdown of CGI-58 protein expression in both liver and white adipose tissue. In chow-fed mice, ASO-mediated depletion of CGI-58 did not alter weight gain, plasma TG, or plasma glucose, yet raised hepatic TG levels ?4-fold. When challenged with a high-fat diet (HFD), CGI-58 ASO-treated mice were protected against diet-induced obesity, but their hepatic contents of TG, diacylglycerols, and ceramides were all elevated, and intriguingly, their hepatic phosphatidylglycerol content was increased by 10-fold. These hepatic lipid alterations were associated with significant decreases in hepatic TG hydrolase activity, hepatic lipoprotein-TG secretion, and plasma concentrations of ketones, nonesterified fatty acids, and insulin. Additionally, HFD-fed CGI-58 ASO-treated mice were more glucose tolerant and insulin sensitive. Collectively, this work demonstrates that CGI-58 plays a critical role in limiting hepatic steatosis and maintaining hepatic glycerophospholipid homeostasis and has unmasked an unexpected role for CGI-58 in promoting HFD-induced obesity and insulin resistance.
Calcineurin is a phosphatase that is activated at the last known stage of mitosis, abscission. Among its many substrates, it dephosphorylates dynamin II during cytokinesis at the midbody of dividing cells. However, dynamin II has several cellular roles including clathrin-mediated endocytosis, centrosome cohesion and cytokinesis. It is not known whether dynamin II phosphorylation plays a role in any of these functions nor have the phosphosites involved in cytokinesis been directly identified. We now report that dynamin II from rat lung is phosphorylated to a low stoichiometry on a single major site, Ser-764, in the proline-rich domain. Phosphorylation on Ser-764 also occurred in asynchronously growing HeLa cells and was greatly increased upon mitotic entry. Tryptic phospho-peptides isolated by TiO(2) chromatography revealed only a single phosphosite in mitotic cells. Mitotic phosphorylation was abolished by roscovitine, suggesting the mitotic kinase is cyclin-dependent kinase 1. Cyclin-dependent kinase 1 phosphorylated full length dynamin II and Glutathione-S-Transferase-tagged-dynamin II-proline-rich domain in vitro, and mutation of Ser-764 to alanine reduced proline-rich domain phosphorylation by 80%, supporting that there is only a single major phosphosite. Ser-764 phosphorylation did not affect clathrin-mediated endocytosis or bulk endocytosis using penetratin-based phospho-deficient or phospho-mimetic peptides or following siRNA depletion/rescue experiments. Phospho-dynamin II was enriched at the mitotic centrosome, but this targeting was unaffected by the phospho-deficient or phospho-mimetic peptides. In contrast, the phospho-mimetic peptide displaced endogenous dynamin II, but not calcineurin, from the midbody and induced cytokinesis failure. Therefore, phosphorylation of dynamin II primarily occurs on a single site that regulates cytokinesis downstream of calcineurin, rather than regulating endocytosis or centrosome function.
Endoplasmic reticulum (ER) membrane cholesterol is maintained at an optimal concentration of ?5 mol % by the net impact of sterol synthesis, modification, and export. Arv1p was first identified in the yeast Saccharomyces cerevisiae as a key component of this homeostasis due to its probable role in intracellular sterol transport. Mammalian ARV1, which can fully complement the yeast lesion, encodes a ubiquitously expressed, resident ER protein. Repeated dosing of specific antisense oligonucleotides to ARV1 produced a marked reduction of ARV1 transcripts in liver, adipose, and to a lesser extent, intestine. This resulted in marked hypercholesterolemia, elevated serum bile acids, and activation of the hepatic farnesoid X receptor (FXR) regulatory pathway. Knockdown of ARV1 in murine liver and HepG2 cells was associated with accumulation of cholesterol in the ER at the expense of the plasma membrane and suppression of sterol regulatory element-binding proteins and their targets. These studies indicate a critical role of mammalian Arv1p in sterol movement from the ER and in the ensuing regulation of hepatic cholesterol and bile acid metabolism.
The following three articles represent the output of a combined effort initiated by the Scientific Regulatory Policy Committee of the Society of Toxicologic Pathology to provide a unified review of current scientific practices and relevant literature and provide suggestions regarding the recognition, interpretation, and risk assessment of hepatic drug metabolizing enzyme (DME) induction studies. The core objective was to provide a review that the scientific community including pathologists, regulatory scientists, toxicologists, investigative scientists, and others would find valuable for managing, designing, and interpreting toxicity studies supporting regulatory filings. Three working groups composed of scientists from industry, academia, and regulatory agencies were convened to review the available literature on important aspects of the interpretation and risk assessment of hepatic microsomal DME enzyme induction in three publications. The three reviews are as follows: "Effects of Hepatic Drug Metabolizing Enzyme Induction on Clinical Pathology Parameters in Animals and Man," Toxicol Pathol "Hepatic Drug Metabolizing Enzyme Induction: Microscopic and Ultrastructural Appearance," Toxicol Pathol "Hepatic Drug Metabolizing Enzyme Induction and Implications for Preclinical and Clinical Risk Assessment," Toxicol Pathol The purpose of this introduction is not to summarize the articles but rather to frame the series and to provide a common mechanistic introduction.
Hepatic drug-metabolizing enzyme (DME) induction is an adaptive response associated with changes in preclinical species; this response can include increases in liver weight, hepatocellular hyperplasia and hypertrophy, and upregulated tissue expression of DMEs. Effects of DME induction on clinical pathology markers of hepatobiliary injury and function in animals as well as humans are not well established. This component of a multipart review of the comparative pathology of xenobiotically mediated induction of hepatic metabolizing enzymes reviews pertinent data from retrospective and prospective preclinical and clinical studies. Particular attention is given to studies with confirmation of DME induction and concurrent evaluation of liver and/or serum hepatobiliary marker enzyme activities and histopathology. These results collectively indicate that in the rat, when histologic findings are limited to hepatocellular hypertrophy, DME induction is not expected to be associated with consistent or substantive changes in serum or plasma activity of hepatobiliary marker enzymes such as alanine aminotransferase, alkaline phosphatase, and gamma glutamyltransferase. In the dog and the monkey, published studies also do not demonstrate a consistent relationship across DME-inducing agents and changes in these clinical pathology parameters. However, increased liver alkaline phosphatase or gamma glutamyltransferase activity in dogs treated with phenobarbital or corticosteroids suggests that direct or indirect induction of select hepatobiliary injury markers can occur both in the absence of liver injury and independently of induction of DME activity. Although correlations between tissue and serum levels of these hepatobiliary markers are limited and inconsistent, increases in serum/plasma activities that are substantial or involve changes in other markers generally reflect hepatobiliary insult rather than DME induction. Extrahepatic effects, including disruption of the hypothalamic-pituitary-thyroid axis, can also occur as a direct outcome of hepatic DME induction in humans and animals. Importantly, hepatic DME induction and associated changes in preclinical species are not necessarily predictive of the occurrence, magnitude, or enzyme induction profile in humans.
Traditionally family planning education is limited for obstetrics and gynecology residents training at faith-based institutions. We describe the first formalized educational program to teach contraception, sterilization, and abortion at a Catholic institution.
Large-scale comparative phosphoproteomics studies have frequently been done on whole cells or organs by conventional bottom-up mass spectrometry approaches, that is, at the phosphopeptide level. Using this approach, there is no way to know which protein isoforms the phosphopeptide signal originated from. Also, as a consequence of the scale of these studies, important information on the localization of phosphorylation sites in subcellular compartments is not surveyed. As a case study, we investigated whether the isoforms of dynamin I (dynI), at the whole brain and subcellular level, had differential phosphorylation. We first established that the dynI isoforms xa, xb, and xd were expressed in nerve terminals. Our investigation revealed that dynI xa was constitutively phosphorylated to a higher extent than the other isoforms despite identical sequences in the phosphorylated subdomains. DynI xa had a 10-fold higher stoichiometry of diphosphorylation at Ser-774 and Ser-778 than dynI xb and xd combined. Diphosphorylation was 2-fold enriched in nerve terminals relative to whole brain and was preferentially targeted for stimulus-dependent dephosphorylation. Phospho-Ser-851 and Ser-857 were depleted from nerve terminals. Our data reveals major differential phosphorylation of dynI phosphosites in different variants and in different neuronal compartments that would be completely imperceptible to a large-scale phosphoproteomics approach.
Niemann-Pick type C (NPC) disease is a lysosomal storage disease characterized by the accumulation of cholesterol and glycosphingolipids. The majority of NPC patients die in their teen years due to progressive neurodegeneration; however, half of NPC patients also suffer from cholestasis, prolonged jaundice, and hepatosplenomegaly. We previously showed that a key mediator of NPC liver disease is tumor necrosis factor (TNF) ?, which is involved in both proinflammatory and apoptotic signaling cascades. In this study, we tested the hypothesis that blocking TNF action with an anti-TNF monoclonal antibody (CNTO5048) will slow the progression of NPC liver disease.
Loss of function of Niemann-Pick C1 (NPC1) leads to lysosomal free cholesterol storage, resulting in the neurodegenerative disease Niemann-Pick disease type C (NPC). Significant numbers of patients with NPC also suffer from liver disease. Currently, no treatments exist that alter patient outcome, and it is unknown if recovery from tissue damage can occur even if a treatment were found. Our laboratory developed a strategy to test whether mice can recover from NPC liver disease. We used antisense oligonucleotides to knock down hepatic expression of NPC1 in BALB/C mice for either 9 or 15 weeks. This recapitulated liver disease with hepatomegaly, cell death, and fibrosis. Then, antisense oligonucleotide treatment was halted for an additional 4, 9, or 15 weeks. We report that significant liver recovery occurred even when NPC1 protein expression only partially returned to normal. Several pathological phenotypes were alleviated, including hepatomegaly, cholesterol storage, and liver cell death. Histological examination revealed that foamy cell accumulation was relieved; however, liver fibrosis increased. Additionally, resolution of cholesterol storage and liver cell death took longer in mice with long-term knockdown. Finally, we found that transcription of cholesterol homeostatic genes was significantly disrupted during the recovery phase after long-term knockdown.
Reflective practice may help physicians identify and connect with what they value and find meaningful in their work. There are many practical obstacles in teaching narrative skills and reflection to residents in surgical subspecialties. We aimed to assess the feasibility of designing and implementing a writing workshop series within an obstetrics and gynecology curriculum.
Fenretinide and tamoxifen have additive antitumor effects preclinically. We performed a randomized, placebo-controlled, double-blind adjuvant trial in breast cancer patients treated for 5 years with tamoxifen, with or without fenretinide. Between October 1995 and October 1999, 426 postmenopausal women with hormone receptor-positive breast cancer were randomized. Patients were monitored for efficacy and toxicity. Four hundred and nineteen patients were evaluable. The study was terminated early due to slow accrual. There were no significant differences between treatment groups in DFS, TTR or survival. More patients stopped treatment early on the fenretinide arm than on placebo (P = 0.02). Grade 3/4 toxicities, including visual problems and musculoskeletal complaints were more common in patients receiving fenretinide (P = 0.007). A Night Blindness Questionnaire was used to monitor nyctalopia, which was slightly, but not significantly, more common on fenretinide. In this underpowered study, no significant difference was observed in efficacy between treatment groups. This trial provides important toxicity information about fenretinide, a retinoid that has been used in the prevention setting, because it is the only placebo-controlled, double-blind randomized study ever performed.
A series of kinetic and structural investigations on ruthenium-based catalysts for asymmetric transfer hydrogenation (ATH) of ketones are reported. A method is reported for monitoring the formation of ruthenium hydride species in real time using (1)H NMR spectroscopy.
The complex competency labeled practice-based learning and improvement (PBLI) by the Accreditation Council for Graduate Medical Education (ACGME) incorporates core knowledge in evidence-based medicine (EBM). The purpose of this study was to operationally define a "PBLI-EBM" domain for assessing resident physician competence.
Early residency is a crucial time in the professional development of physicians. As interns assume primary care for their patients, they take on new responsibilities. The events they find memorable during this time could provide us with insight into their developing professional identities.
People with mental illness around the world continue to suffer from stigmatization and limited care. Previous studies utilizing self-report questionnaires indicate that many medical students regard clinical work with psychiatric patients as unappealing, while the professionalism literature has documented a general decline in students capacity for empathy over the course of medical school. Through in-depth interviews, this study attempts to better understand the formation of medical students perceptions of psychiatry and the implications of that process for a more general understanding of the impact of emotionally-laden experiences on medical students capacity for empathy. Forty-seven fourth-year medical students who had expressed interest or performed well in psychiatry were asked a series of questions to elicit their perceptions of the field of psychiatry. Interview transcripts were systematically coded using content analysis and principles of grounded theory. Stigma, stereotypes, and stressfully intense emotional reactions seemed to adversely affect the students expected satisfaction from and willingness to care for the mentally ill, despite enjoying psychiatrys intellectual content and the opportunity to develop in-depth relationships with patients. Teaching faculty need to directly address the stigma and stereotypes that surround mental illness and actively help medical students cope with the stress that they report experiencing during their psychiatry clerkship in order to improve the recognition and treatment of psychiatric illness by newly graduating physicians. More generally, the relationships that we identify among stress, stigmatization, and stereotyping along an empathic spectrum suggest that increased attention should be paid to the stress that empathy can entail. This perspective may allow for the creation of similarly targeted interventions throughout the medical school curriculum to counteract the decline in empathy, the so-called "hardening of the heart," associated with physician-training worldwide.
Evidence-based practice (EBP) requires practitioners to identify and formulate questions in response to patient encounters, and to seek, select, and appraise applicable clinical research. A standardized workshop format serves as the model for training of medical educators in these skills. We developed an evaluation exercise to assess the ability to identify and solve a problem requiring the use of targeted skills and administered it to 47 North American junior faculty and residents in various specialties at the close of two short workshops in EBP. Prior to the workshop, subjects reported prior training in EBP and completed a previously validated knowledge test. Our post-workshop exercise differed from the baseline measures and required participants to spontaneously identify a suitable question in response to a simulated clinical encounter, followed by a description of a stepwise approach to answering it. They then responded to successively more explicitly prompted queries relevant to their question. We analyzed responses to identify areas of skill deficiency and potential reasons for these deficiencies. Twelve respondents (26%) initially failed to identify a suitable question in response to the clinical scenario. Ability to choose a suitable question correlated with the ability to connect an original question to an appropriate study design. Prior EBP training correlated with the pretest score but not with performance on our exercise. Overall performance correlated with ability to correctly classify their questions as pertaining to therapy, diagnosis, prognosis, or harm. We conclude that faculty and residents completing standard workshops in EBP may still lack the ability to initiate and investigate original clinical inquiries using EBP skills.
Probing the dynamin binding site: Bis-tyrphostin (1, Bis-T), is a potent inhibitor of the phospholipid-stimulated GTPase activity of dynamin I. Analogues of Bis-T have significant potential as a biological probes for the dissection of endocytic pathways. Bis-T-derived compounds were synthesised and evaluated for their ability to inhibit the GTPase activity of dynamin I. Two analogues (23 and 24) represent the first asymmetrically substituted Bis-T analogues to retain dynamin inhibition.Two azidobenzyl amide (4 and 23) and one 3-trifluoromethyl-3H-diazirin-3-ylphenyl (24) analogues of bis-tyrphostin (1, Bis-T) were synthesised as potential photoaffinity labels for the elucidation of the binding site of compound 1 in dynamin I. Of the two azidobenzyl amide analogues (4 and 23), the terminally substituted 23 retained dynamin I GTPase inhibition (IC(50)=6.4+/-2.8 microM) whilst 4, which was substituted on the central carbon of the amide linker, displayed no activity. Analogue 24 also retained inhibitory activity (IC(50)=36+/-9 microM). Photoaffinity labelling experiments did not unequivocally elucidate the binding pocket of compound 1. However, compounds 23 and 24 represent the first asymmetrically substituted Bis-T analogues to retain dynamin inhibitory activity, providing a new direction for analogue synthesis.
Niemann-Pick type C (NPC) is a fatal autosomal recessive lysosomal storage disease clinically characterized by neurodegeneration and liver disease. Heterogeneous mutations in the NPC1 and NPC2 genes cause impaired egress of free cholesterol from lysosomes, leading to accumulation of cholesterol and glycosphingolipids. Key features of NPC liver disease include hepatic apoptosis, inflammation, and fibrosis. It is unclear what signaling events regulate these disease processes in NPC. We hypothesize that tumor necrosis factor alpha (TNF-alpha), which is involved in both proinflammatory and apoptotic signaling cascades, is a key mediator of inflammation, apoptosis, and fibrosis in NPC liver disease. In this study, we evaluated the role of TNF-alpha signaling in NPC liver disease by utilizing NPC1-specific antisense oligonucleotides to knock down NPC1 expression in control and TNF-alpha knockout mice. In the absence of TNF-alpha, NPC1 knockdown produced liver disease with significantly less inflammation, apoptosis, and fibrosis.
The in vivo pharmacokinetics/pharmacodynamics of 2-O-(2-methoxyethyl) (2-MOE) modified antisense oligonucleotides (ASOs), targeting apolipoprotein B-100 (apoB-100), were characterized in multiple species. The species-specific apoB antisense inhibitors demonstrated target apoB mRNA reduction in a drug concentration and time-dependent fashion in mice, monkeys, and humans. Consistent with the concentration-dependent decreases in liver apoB mRNA, reductions in serum apoB, and LDL-C, and total cholesterol were concurrently observed in animal models and humans. Additionally, the long duration of effect after cessation of dosing correlated well with the elimination half-life of 2-MOE modified apoB ASOs studied in mice (t(1/2) congruent with 20 days) and humans (t(1/2) congruent with 30 days) following parental administrations. The plasma concentrations of ISIS 301012, observed in the terminal elimination phase of both mice and monkeys were in equilibrium with liver. The partition ratios between liver and plasma were similar, approximately 6000:1, across species, and thus provide a surrogate for tissue exposure in humans. Using an inhibitory E(max) model, the ASO liver EC(50s) were 101+/-32, 119+/-15, and 300+/-191 microg/g of ASO in high-fat-fed (HF) mice, transgenic mice containing the human apoB transgene, and monkeys, respectively. The estimated liver EC(50) in man, extrapolated from trough plasma exposure, was 81+/-122 microg/g. Therefore, extraordinary consistency of the exposure-response relationship for the apoB antisense inhibitor was observed across species, including human. The cross-species PK/PD relationships provide confidence in the use of pharmacology animal models to predict human dosing for second-generation ASOs targeting the liver.
Cytokinesis involves two phases: (1) membrane ingression followed by (2) membrane abscission. The ingression phase generates a cleavage furrow and this requires co-operative function of the actin-myosin II contractile ring and septin filaments. We demonstrate that the actin-binding protein, EPLIN, locates to the cleavage furrow during cytokinesis and this is possibly via association with the contractile ring components, myosin II and the septin, Sept2. Depletion of EPLIN results in formation of multinucleated cells and this is associated with inefficient accumulation of active myosin II (MRLC(S19)) and Sept2 and their regulatory small GTPases, RhoA and Cdc42, respectively, to the cleavage furrow during the final stages of cytokinesis. We suggest that EPLIN may function during cytokinesis to maintain local accumulation of key cytokinesis proteins at the furrow.
In many parts of the world the practice of medicine and medical education increasingly focus on providing patient care within context of the larger healthcare system. Our purpose is to solicit perceptions of all professional stakeholders (e.g. nurses) of the system regarding the U.S. ACGME competency Systems Based Practice to uncover the extent to which there is agreement or discrepancy among key system stakeholders. Eighty-eight multidisciplinary personnel (n = 88) from two academic medical centers were invited to participate in one of 14 nominal group process sessions. Participants generated and prioritized resident characteristics that they believed were important for effective System Based Practices. Through content analysis the prioritized attribute statements were coded to identify embedded themes of resident roles and behavior. From the themes, three major resident roles emerged: resident as Self-Manager, Team Collaborator, and Patient Advocate. No one professional group (e.g., nurses, attending physicians, social workers) emphasized all of these roles. Some concepts that are emphasized in the ACGME definition like using cost-benefit analysis were conspicuously absent from the healthcare team generated list. We showed that there are gaps between the key stakeholders prioritizations about the ACGME definition of SBP and, more generally, the behaviors and roles identified by healthcare team stakeholders beyond the U.S. This suggests that within the process of developing a comprehensive working understanding of the Systems Based Practice competency (or other similar competencies, such as in CanMEDS), it is necessary to use multiple stakeholders in the system (perhaps including patients) to more accurately identify key resident roles and observable behaviors.
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