ABSTRACT
The Population Assessment of Tobacco and Health (PATH) Study is a nationally representative, longitudinal study of the US population on tobacco use and its effects on health, collecting data annually since 2013. The COVID-19 pandemic interrupted in-person survey data collections around the world. In the USA, this included a PATH Study data collection focused on youth (13-17) and young adults (18-19) as well as other US surveys on tobacco use. Given that it was necessary to pause data collection and considering that tobacco-use behaviours could be expected to change along with pandemic-related changes in the social environment, the original design for the 2020 PATH Study data collection for youth and young adults was modified. Also, the PATH Study Adult Telephone Survey was developed to address the need for adult tobacco use monitoring in this unprecedented time. This article describes the modifications made to the 2020 PATH Study design and protocol to provide nationally representative data for youth and adults after the onset of the COVID-19 pandemic as well as the implications of these modifications for researchers.
ABSTRACT
Expression of G6PD is controlled by changes in the degree of splicing of the G6PD mRNA in response to nutrients in the diet. This regulation involves an exonic splicing enhancer (ESE) in exon 12 of the mRNA. Using the G6PD model, we demonstrate that nutrients and hormones control the activity of serine-arginine-rich (SR) proteins, a family of splicing co-activators, and thereby regulate the splicing of G6PD mRNA. In primary rat hepatocyte cultures, insulin increased the amount of phosphorylated SR proteins, and this effect was counteracted by arachidonic acid. The results of RNA affinity analysis with nuclear extracts from intact liver demonstrated that the SR splicing factor proteins SRSF3 and SRSF4 bound to the G6PD ESE. Consequently, siRNA-mediated depletion of SRSF3, but not SRSF4, in liver cells inhibited accumulation of both mRNA expressed from a minigene containing exon 12 and the endogenous G6PD mRNA. Consistent with the functional role of SRSF3 in regulating splicing, SRSF3 was observed to bind to the ESE in both intact cells and in animals using RNA immunoprecipitation analysis. Furthermore, refeeding significantly increased the binding of SRSF3 coincident with increased splicing and expression of G6PD. Together, these data establish that nutritional regulation of SRSF3 activity is involved in the differential splicing of the G6PD transcript in response to nutrients. Nutritional regulation of other SR proteins presents a regulatory mechanism that could cause widespread changes in mRNA splicing. Nutrients are therefore novel regulators of mRNA splicing.
Subject(s)
Gene Expression Regulation , Glucosephosphate Dehydrogenase/metabolism , Liver/metabolism , RNA-Binding Proteins/physiology , RNA/metabolism , Animals , Arachidonic Acid/chemistry , Hep G2 Cells , Hepatocytes/cytology , Hepatocytes/metabolism , Humans , Male , Mice , Mice, Inbred C57BL , Protein Binding , RNA Splicing , RNA, Messenger/metabolism , RNA-Binding Proteins/genetics , Rats , Rats, Sprague-Dawley , Serine-Arginine Splicing Factors , Transcription, GeneticABSTRACT
Nutrient regulation of glucose-6-phosphate dehydrogenase (G6PD) expression occurs through changes in the rate of splicing of G6PD pre-mRNA. This posttranscriptional mechanism accounts for the 12- to 15-fold increase in G6PD expression in livers of mice that were starved and then refed a high-carbohydrate diet. Regulation of G6PD pre-mRNA splicing requires a cis-acting element in exon 12 of the pre-mRNA. Using RNA probes to exon 12 and nuclear extracts from livers of mice that were starved or refed, proteins of 60 kDa and 37 kDa were detected bound to nucleotides 65-79 of exon 12 and this binding was decreased by 50% with nuclear extracts from refed mice. The proteins were identified as hnRNPs K, L, and A2/B1 by LC-MS/MS. The decrease in binding of these proteins to exon 12 during refeeding was not accompanied by a decrease in the total amount of these proteins in total nuclear extract. HnRNPs K, L and A2/B1 have known roles in the regulation of mRNA splicing. The decrease in binding of these proteins during treatments that increase G6PD expression is consistent with a role for these proteins in the inhibition of G6PD mRNA splicing.
Subject(s)
Heterogeneous-Nuclear Ribonucleoprotein Group A-B/metabolism , Heterogeneous-Nuclear Ribonucleoprotein K/metabolism , Heterogeneous-Nuclear Ribonucleoprotein L/metabolism , RNA Precursors/metabolism , RNA Splicing , Animals , Blotting, Western , Chromatography, Liquid , Dietary Carbohydrates/administration & dosage , Dietary Carbohydrates/metabolism , Electrophoresis, Gel, Two-Dimensional , Exons/genetics , Glucosephosphate Dehydrogenase/genetics , Heterogeneous-Nuclear Ribonucleoprotein Group A-B/chemistry , Heterogeneous-Nuclear Ribonucleoprotein K/chemistry , Heterogeneous-Nuclear Ribonucleoprotein L/chemistry , Male , Mass Spectrometry , Mice , Mice, Inbred C57BL , Molecular Weight , Oligoribonucleotides/genetics , Oligoribonucleotides/metabolism , Protein Binding , RNA Precursors/genetics , RNA Processing, Post-Transcriptional , RNA-Binding Proteins/chemistry , RNA-Binding Proteins/metabolism , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , StarvationABSTRACT
This retrospective study examined spinal-related hospitalizations of U.S. Army soldiers deployed to Afghanistan and Iraq. Spinal cord injuries (SCI) and vertebral column injuries (VCI) were identified using International Classification of Disease, 9th Revision, Clinical Modification diagnosis codes. In our study, spinal hospitalizations represented 8.2% of total injury admissions. Risk factors for SCI and VCI incidences were determined using Poisson regression. Lack of previous deployment experience increased risk of having SCI by 33% and VCI by 24% in Iraq (similar increases, but not statistically significant in Afghanistan). Male soldiers had 4.85 times higher risk for SCI in Iraq and 69% higher risk in Afghanistan than female soldiers. In Afghanistan, almost 60% of spinal episodes included traumatic brain injury (TBI), compared to about 40% in Iraq. In both theaters, mild TBI accounted for more than 50% of all TBI-spinal episodes. Sixteen percent of SCI inpatient episodes in Afghanistan and 13% in Iraq were associated with paralysis, with median bed days of 46 and 33 days compared to a median of 6 days in both theaters for nonparalysis spinal injuries. The mortality rate was 2.5 times lower in Afghanistan than in Iraq.
Subject(s)
Hospitalization/statistics & numerical data , Military Personnel/statistics & numerical data , Spinal Cord Injuries/epidemiology , Adolescent , Adult , Afghan Campaign 2001- , Female , Humans , Incidence , International Classification of Diseases , Iraq War, 2003-2011 , Male , Middle Aged , Retrospective Studies , Risk Factors , United States/epidemiologyABSTRACT
Dental Disease and Non-Battle Injuries (D-DNBI) continue to be a problem among U.S. Army active duty (AD), U.S. Army National Guard (ARNG), and U.S. Army Reserve (USAR) deployed soldiers to Operation Iraqi Freedom/Operation New Dawn in Iraq and Operation Enduring Freedom in Afghanistan. A previous study reported the annual rates to be 136 D-DNBI per 1,000 personnel for AD, 152 for ARNG, and 184 for USAR. The objectives of this study were to describe D-DNBI incidence and to determine risk factors for dental encounters and high severity diagnoses for deployed soldiers. The 78 diagnoses were classified into three categories based on severity. Poisson regression was used to compare D-DNBI rates and logistic regression was used to analyze the risk of high severity D-DNBI. In both campaigns, Reserve had a higher risk of D-DNBI than active duty. For Afghanistan, ARNG and USAR demonstrated over 50% increased risk of D-DNBI compared to AD. In Iraq, USAR had a 17% increased risk over AD. Females had a higher risk of D-DNBI (>50%) compared to males in both campaigns. High severity D-DNBI made up 2.77% of all diagnoses. Within Afghanistan, there was a 4.6% increased risk of high severity D-DNBI for each additional deployment month.
Subject(s)
Military Personnel/statistics & numerical data , Stomatognathic Diseases/epidemiology , Adult , Afghan Campaign 2001- , Female , Humans , Incidence , Iraq War, 2003-2011 , Male , Middle Aged , Retrospective Studies , Risk Factors , Severity of Illness Index , Sex Factors , United States/epidemiology , Young AdultABSTRACT
BACKGROUND: In the past, the U.S. Army Reserve (USAR) and Army National Guard (ARNG) have exhibited lower levels of medical and dental readiness than active duty (AD) Soldiers when activated for deployment. OBJECTIVE: The objective was to compare dental disease and nonbattle injury (D-DNBI) incidence rates and describe the most common D-DNBI diagnoses in Army AD, ARNG, and USAR Soldiers deployed to Iraq (Operation Iraqi Freedom/Operation New Dawn) and Afghanistan or Kuwait (Operation Enduring Freedom). METHODS: Data from the Center for AMEDD Strategic Studies (CASS) were used to determine D-DNBI encounter rates and diagnoses for deployed Army Soldiers. RESULTS: "Dental Caries" was the leading diagnosis (10.00%) for Soldiers in both theaters. For Operation Iraqi Freedom, D-DNBI rates were highest in 2010 at 144.05 per 1,000 Soldiers per year (AD 135.77, ARNG 151.39 and USAR 183.76). In comparison, D-DNBI rates in Operation Enduring Freedom were highest in 2012 with an overall rate of 85.77 per 1,000 Soldiers per year (AD 72.48, ARNG 129.38 and USAR 129.52). CONCLUSIONS: In both campaigns, the data suggest that ARNG and USAR Soldiers had higher D-DNBI rates when compared to AD Soldiers. Further investigation is needed to decrease D-DNBI rates and to determine risk factors that may influence D-DNBI rates among Army components during deployments.
Subject(s)
Military Personnel/statistics & numerical data , Stomatognathic Diseases/epidemiology , Afghan Campaign 2001- , Humans , Incidence , Iraq War, 2003-2011 , Retrospective Studies , United States/epidemiologyABSTRACT
Repeated exposure to cocaine induces neuroadaptations which contribute to the rewarding properties of cocaine. Using cocaine-induced conditioned place preference (CPP) as an animal model of reward, earlier studies have shown that sigma (σ) receptor ligands can attenuate the acquisition, expression and reactivation of CPP. However, the underlying molecular mechanisms that are associated with these changes are not yet understood. In the present study, CM156, a novel antagonist with high selectivity and affinity for σ receptors was used to attenuate the expression of cocaine-induced CPP in mice. Immediately following the behavioral evaluations, mouse brain tissues were collected and alterations in gene expression in half brain samples were profiled by cDNA microarray analysis. Microarray data was analyzed by three distinct normalization methods and four genes were consistently found to be upregulated by cocaine when compared to saline controls. Each of these gene changes were found by more than one normalization method to be reversed by at least one dose of CM156. Quantitative real time PCR confirmed that a single administration of CM156 was able to reverse the cocaine-induced increases in three of these four genes: metastasis associated lung adenocarcinoma transcript 1 (malat1), tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein (ywhaz), and transthyretin (ttr). These genes are involved in processes related to neuroplasticity and RNA editing. The data presented herein provides evidence that pharmacological intervention with a putative σ receptor antagonist reverses alterations in gene expression that are associated with cocaine-induced reward.
Subject(s)
14-3-3 Proteins/genetics , Brain Chemistry/drug effects , Brain Chemistry/genetics , Cocaine/antagonists & inhibitors , Conditioning, Operant/drug effects , Gene Expression/drug effects , Piperazines/pharmacology , Receptors, sigma/antagonists & inhibitors , Sulfur Compounds/pharmacology , Analysis of Variance , Animals , Behavior, Animal/drug effects , Binding Sites/drug effects , Cocaine/pharmacology , DNA, Complementary/genetics , Male , Mice , Mice, Inbred C57BL , Microarray Analysis , Motor Activity/drug effects , Prealbumin/genetics , RNA/biosynthesis , RNA/isolation & purification , RNA, Long Noncoding , RNA, Untranslated/genetics , Real-Time Polymerase Chain Reaction , Reinforcement Schedule , Reward , Seizures/chemically inducedABSTRACT
The inhibition of glucose-6-phosphate dehydrogenase (G6PD) expression by arachidonic acid occurs by changes in the rate of pre-mRNA splicing. Here, we have identified a cis-acting RNA element required for regulated splicing of G6PD mRNA. Using transfection of G6PD RNA reporter constructs into rat hepatocytes, the cis-acting RNA element involved in this regulation was localized to nucleotides 43-72 of exon 12 in the G6PD mRNA. In in vitro splicing assays, RNA substrates containing exon 12 were not spliced. In contrast, RNA substrates containing other regions (exons 8 and 9 or exons 10 and 11) of the G6PD mRNA were efficiently spliced. Furthermore, exon 12 can inhibit splicing when substituted for other exons in RNA substrates that are readily spliced. This activity of the exon 12 regulatory element suggests that it is an exonic splicing silencer. Consistent with its activity as a splicing silencer, spliceosome assembly was inhibited on RNA substrates containing exon 12 compared with RNAs representing other regions of the G6PD transcript. Elimination of nucleotides 43-72 of exon 12 did not restore splicing of exon 12-containing RNA; thus, the 30-nucleotide element may not be exclusively a silencer. The binding of heterogeneous nuclear ribonucleoproteins K, L, and A2/B1 from both HeLa and hepatocyte nuclear extracts to the element further supports its activity as a silencer. In addition, SR proteins bind to the element, consistent with the presence of enhancer activity within this sequence. Thus, an exonic splicing silencer is involved in the inhibition of splicing of a constitutively spliced exon in the G6PD mRNA.
Subject(s)
Exons/genetics , Gene Expression Regulation , Glucosephosphate Dehydrogenase/genetics , RNA Splicing , RNA, Messenger/genetics , Silencer Elements, Transcriptional , Animals , Binding Sites , Blotting, Western , Cells, Cultured , Chromatography, Affinity , Chromatography, Liquid , Glucosephosphate Dehydrogenase/metabolism , HeLa Cells , Hepatocytes/cytology , Hepatocytes/metabolism , Heterogeneous-Nuclear Ribonucleoproteins/metabolism , Humans , Introns/genetics , Male , Mass Spectrometry , Plasmids/genetics , RNA, Messenger/metabolism , Rats , Rats, Sprague-Dawley , Ribonucleases/metabolism , Ribonucleoproteins/metabolism , Spliceosomes/metabolism , Transcription, GeneticABSTRACT
Polyunsaturated fatty acids are potent inhibitors of lipogenic gene expression in liver. The lipogenic enzyme glucose-6-phosphate dehydrogenase (G6PD) is unique in this gene family, in that fatty acids inhibit at a post-transcriptional step. In this study, we have provided evidence for a signaling pathway for the arachidonic acid inhibition of G6PD mRNA abundance. Arachidonic acid decreases the insulin induction of G6PD expression; by itself, arachidonic acid does not inhibit basal G6PD mRNA accumulation. The insulin stimulation of G6PD involves the phosphoinositide 3-kinase (PI 3-kinase) pathway (Wagle, A., Jivraj, S., Garlock, G. L., and Stapleton, S. R. (1998) J. Biol. Chem. 273, 14968-14974). Incubation of hepatocytes with arachidonic acid blocks the activation of PI 3-kinase by insulin as observed by a decrease in Ser(473) phosphorylation of Akt, the downstream effector of PI 3-kinase. The decrease in PI 3-kinase activity was associated with an increase in Ser(307) phosphorylation of IRS-1. Western analysis demonstrated increased phosphorylation of p38 mitogen-activated protein kinase (MAPK) in arachidonic acid-treated cells, whereas extracellular signal-regulated kinase and c-Jun NH(2)-terminal kinase activity was not changed. Incubating the hepatocytes with the p38 MAPK inhibitor, SB203580, blocked the arachidonic acid inhibition of G6PD mRNA accumulation. Furthermore, SB203580 decreased the arachidonic acid-mediated Ser(307) phosphorylation of IRS-1 and rescued Akt activation that was otherwise decreased by arachidonic acid. Thus, arachidonic acid inhibits the insulin stimulation of G6PD mRNA accumulation by stimulating the p38 MAPK pathway, thereby inhibiting insulin signal transduction.
Subject(s)
Arachidonic Acid/pharmacology , Glucosephosphate Dehydrogenase/biosynthesis , Insulin/pharmacology , p38 Mitogen-Activated Protein Kinases/metabolism , Animals , Cells, Cultured , Enzyme Activation/drug effects , Enzyme Induction/drug effects , Fatty Acids, Unsaturated/pharmacology , Gene Expression/drug effects , Glucosephosphate Dehydrogenase/antagonists & inhibitors , Glucosephosphate Dehydrogenase/genetics , Hepatocytes/enzymology , Insulin Receptor Substrate Proteins , Male , Phosphatidylinositol 3-Kinases/metabolism , Phosphoproteins/metabolism , Phosphorylation , Protein Kinase Inhibitors/pharmacology , Proto-Oncogene Proteins c-akt/metabolism , RNA, Messenger/analysis , Rats , Rats, Sprague-Dawley , Serine/metabolism , Signal Transduction/drug effects , Tumor Necrosis Factor-alpha/pharmacology , p38 Mitogen-Activated Protein Kinases/antagonists & inhibitorsABSTRACT
Continuing our studies on proctolin (Arg-Tyr-Leu-Pro-Thr) we performed the synthesis and biological evaluation of 52 analogues substituted in position 2, 3, 4, and 5 of the peptide chain. The peptides were bioassayed for cardiotropic activity in vitro on Tenebrio molitor and myotropic activity on foregut of Schistocerca gregaria. Twenty analogues retained 20-80% of proctolin activity.
Subject(s)
Neuropeptides/chemistry , Oligopeptides/chemistry , Amino Acid Sequence , Amino Acid Substitution , Animals , Digestive System/drug effects , Grasshoppers/drug effects , Grasshoppers/physiology , Insecta , Neuropeptides/chemical synthesis , Neuropeptides/pharmacology , Oligopeptides/chemical synthesis , Oligopeptides/pharmacology , Structure-Activity Relationship , Tenebrio/drug effects , Tenebrio/physiologyABSTRACT
Polyunsaturated fatty acids inhibit the expression of hepatic glucose-6-phosphate dehydrogenase (G6PD) by changes in the amount of G6PD pre-mRNA in the nucleus in the absence of changes in the transcription rate of the gene. We have compared the nuclear accumulation of partially and fully spliced mRNA for G6PD in the livers of mice fed diets high versus low in polyunsaturated fat. Consumption of a diet high in polyunsaturated fat decreased the accumulation of partially spliced forms of the G6PD pre-mRNA. Examining the fate of multiple introns within the G6PD primary transcript indicated that in mice fed a high fat diet, G6PD pre-mRNA containing intron 11 accumulated within the nucleus, whereas G6PD mature mRNA abundance was inhibited 50% or more within the same livers. Transient transfection of RNA reporters into primary hepatocyte cultures was used to localize the cis-acting RNA element involved in this regulated splicing. Reporter RNA produced from constructs containing exon 12 were decreased in amount by arachidonic acid. The extent of this decrease paralleled that seen in the expression of the endogenous G6PD mRNA. The presence of both exon 12 and a neighboring intron within the G6PD reporter RNA was essential for regulation by polyunsaturated fatty acid. Inhibition was not dependent on the presence of the G6PD polyadenylation signal and the 3'-untranslated region, but substitution with the SV40 poly(A) signal attenuated the inhibition by arachidonic acid. Thus, exon 12 contains a putative splicing regulatory element involved in the inhibition of G6PD expression by polyunsaturated fat.
Subject(s)
Fatty Acids, Unsaturated/pharmacology , Glucosephosphate Dehydrogenase/genetics , RNA Precursors/genetics , RNA Splicing/drug effects , Animals , Arachidonic Acid/pharmacology , Cells, Cultured , Gene Expression Regulation/drug effects , Hepatocytes/metabolism , Introns , Male , Mice , Mice, Inbred C57BL , Promoter Regions, Genetic , Rats , Rats, Sprague-DawleyABSTRACT
Understanding how a cell adapts to dietary energy in the form of carbohydrate versus energy in the form of triacylglycerol requires knowledge of how the activity of the enzymes involved in lipogenesis is regulated. Changes in the activity of these enzymes are largely caused by changes in the rate at which their proteins are synthesized. Nutrients within the diet can signal these changes either via altering hormone concentrations or via their own unique signal transduction pathways. Most of the lipogenic genes are regulated by changes in the rate of their transcription. Glucose-6-phosphate dehydrogenase (G6PD) is unique in this group of enzymes in that nutritional regulation of its synthesis involves steps exclusively at a posttranscriptional level. G6PD activity is enhanced by the consumption of diets high in carbohydrate and is inhibited by the consumption of polyunsaturated fat. In this review, evidence is presented that changes in the rate of synthesis of the mature G6PD mRNA involves regulation of the efficiency of splicing of the nascent G6PD transcript. Furthermore, this regulation involves the activity of a cis-acting sequence in the G6PD primary transcript. This sequence in exon 12 is essential for the inhibition of G6PD mRNA splicing by PUFA. Understanding the mechanisms by which nutrients alter nuclear posttranscriptional events will provide new information on the breadth of mechanisms involved in gene regulation.