bluuebunny/crossref_metadata_2025_split huggingface dataset:
Behavioral Economic Demand for Cocaine During Phendimetrazine Maintenance in Humans
Authors: Strickland Justin Charles , Stoops William Walton 
Published: June 2021

ObjectivesPhendimetrazine is a prodrug for the monoamine releaser phenmetrazine that has been investigated as a pharmacotherapy for cocaine use disorder. Preclinical studies indicate that phendimetrazine reduces cocaine self‐administration. Human laboratory research suggests that phendimetrazine may have limited abuse potential and has a favorable safety profile. However, the efficacy of phendimetrazine for reducing cocaine use in humans is unknown. Behavioral economic demand provides a fine‐grained measure of the relative reinforcing effects of drugs by accounting for the multi‐dimensional nature of reinforcement. The purpose of the present study was to use to evaluate behavioral economic demand for cocaine during phendimetrazine maintenance.MethodTwenty‐nine subjects completed a within‐subjects, placebo‐controlled, inpatient study. Subjects were maintained on oral phendimetrazine (210 mg/day) and placebo. After at least seven maintenance days on each dose, subjects completed a cocaine purchase task in which they were asked to indicate the hypothetical number of cocaine “hits” (i.e., 0.1 g cocaine) they would purchase at various prices/hit. Primary outcomes were two demand indices: 1) intensity (consumption at unconstrained price) and 2) elasticity (change in consumption with increased price). Intensity and elasticity were derived using the exponentiated demand equation. Demand curves were analyzed for nonsystematic data and then evaluated for phendimetrazine effects using linear mixed‐effects models. Demographic and substance use variables were also tested as moderators of treatment efficacy.ResultsThree‐criterion analysis indicated that 93% of demand curves were systematic. The exponentiated demand equation provided an excellent fit to curves under placebo (median R2 = .96) and active dosing (median R2 = .96), with no differences between dose conditions. Phendimetrazine did not alter cocaine demand elasticity (p = .91) or intensity (p = .12). However, BMI moderated the effects of phendimetrazine on intensity, with greater reductions in intensity observed for individuals with higher BMI.ConclusionsThese findings suggest that phendimetrazine maintenance does not universally alter cocaine demand, but may produce reductions in demand intensity for individuals with higher BMI. Future human laboratory studies and clinical trials that evaluate phendimetrazine for cocaine use disorder will be important for establishing the predictive validity of the cocaine purchase task for assessing therapeutic efficacy.Support or Funding InformationNational Institute on Drug Abuse (R01DA036553), National Center for Advancing Translational Sciences (UL1TR000117), and National Science Foundation (1247392).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Metabolomic and Lipidomic Signatures of Cold Tolerance in Bumble Bees
Authors: Keaveny Ellen , Helling Mitchell , Goodenough Andrew , Basile Franco , Rule Daniel C. , Dillon Michael E. 
Published: June 2021

Thermal tolerance is a key physiological trait that often varies among populations and species, and can be a key determinant of current and future geographic distributions. The cellular basis for organismal differences in thermal tolerance is an active area of research, with chemical constituents of the cytosol and cell membrane likely playing important roles in maintaining cell function at low temperatures. For freeze‐avoiding organisms, the abundance and makeup of sugars and related molecules depress the freezing point, reducing the risk of ice formation. At less extreme temperatures, remodeling of membrane lipids can help maintain fluidity (and function) at cold temperatures. We have found striking differences in critical thermal minima (CTmin) of worker bumble bees reared in common‐garden conditions from queens collected at low and high latitudes. We compared metabolomic and lipidomic signatures of these bees to identify key molecules that likely underlie differences in cold tolerance. PCA analyses of GC‐MS data for aqueous abdominal extracts revealed clear separation of low and high latitude populations and reveal higher levels of several key molecules, including L‐proline, in the high latitude population. Lipid extractions and subsequent GC‐MS reveals variation in fatty acid composition of nutrients (triacylglycerols) and of cellular membranes, for which phospholipid composition is also critical for function at low temperatures. This broad spectrum approach to metabolomics and lipidomics allows us to compare a wide range of metabolites between bees differing in cold tolerance, facilitating ongoing research on the mechanistic basis for this physiological trait linked to the ecology of diverse organisms.Support or Funding InformationThis material is based upon work supported by the National Science Foundation under Grant Nos. DEB‐1457659 and RII Track 2‐1826834. Any opinions, findings, and conclusions or recommendations expressed in this material are those of the author(s) and do not necessarily reflect the views of the National Science Foundation.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Creation of a Novel Inbred Mouse Model for High Activity with a Small Muscle Phenotype
Authors: Middleton Kevin M , Smolinsky Amanda N , Garland Theodore 
Published: June 2021

Given challenges of studying skeletal form and function in humans, experimental models of skeletal loading are important for understanding both basic and applied questions of the musculoskeletal and associated physiological systems (e.g., energy balance, metabolism, behavior). Experimental approaches have frequently involved controlled loading, often axial loads placed on the ulna or tibia. Although appealing in allowing for precise estimates of stress and strain, these approaches often generate extremely high tissue strains, resulting in potentially pathological overload. Conversely, natural models of locomotor loading result in unknown levels of tissue strain but benefit by representing behaviors that animals will perform in life. Voluntary locomotion via an activity wheel can be used to explore the behavioral and physiological challenges of long‐term voluntary exercise. Furthermore, because activity is a heritable trait, it can be selected upon to gradually increase across generations. From an outbred experimental population of Hsd:ICR mice that had already experienced 51 generations of selective breeding for high levels of voluntary wheel‐running behavior (resulting in an approximately 2.5–3‐fold increase in activity over non‐selected controls), we began inbreeding via sib‐sib mating while simultaneously continuing selection for activity. These mice were also homozygous for an allele that results in an approximately 50% reduction in limb muscle mass. At generation 30 of inbreeding, the Hsd:ICR‐Myh4minimsc mice were found to engage in an average of 487 minutes (females) and 371 minutes (males) of activity, with a mean speed of 13.1 m/min (females) and 14.7 m/min (males). These activity values place Hsd:ICR‐Myh4minimsc at the high end of duration and the mid‐range of speed, when compared with other inbred strains. When housed without wheels and compared with outbred ICR mice, Hsd:ICR‐Myh4minimsc mice have significantly lower body mass (27.9 ± 3.7 g sd vs 40.1 ± 5.9 g) and abdominal adipose tissue (0.18 ± 0.06 g vs. 0.30 ± 0.06 g) at 17 weeks of age. Compared with other inbred, high‐activity strains, Hsd:ICR‐Myh4minimsc has advantages with good overall health, strong breeding success, and large litters (median 10 pups/litter). After 39 generations of brother‐sister mating, we estimate > 99.9% homozygosity, at which point the line was transferred to the Mutant Mouse Resource and Research Center, where it will be available to the community as a model system for studies of high voluntary exercise, energy balance, skeletal phenotypes, and musculoskeletal integration.Support or Funding InformationNSF DEB‐1655362 (to TG); NIH NIAMS EARDA sub‐award from 5G11HD052368‐03 (to KMM); California University San Bernardino Department of Biology (to KMM); University of Missouri School of Medicine (to KMM)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Sex Differences in Androgen Receptor Enhancer Activity and Expression during Mus musculus Embryonic Development
Authors: Schuetz Emily K , Reno Philip L 
Published: June 2021

Study objectiveThe objective of this study is to compare the sex‐specific enhancer function of a cis‐regulatory element during mammary gland development to the expression of the androgen receptor (AR) in mammary tissues.IntroductionConserved noncoding elements regulate gene expression during mammalian development with remarkable precision. AR is critical for the development of male specific phenotypes in many tissues, yet little is known regarding the tissue‐specific regulation of AR expression. We have previously identified a 7.7kb sequence near the AR gene that activates lacZ reporter gene expression in prenatal and postnatal mice. Embryologically, the enhancer drives consistent lacZ expression in developing hair follicles and the genital tubercle. However, expression in the mammary ridge/glands occurs in only half of the transgenic embryos produced. Each of these structures are known sites of expression of AR or are affected by androgens during growth and development. We hypothesize that the variability in this enhancer function during mammary gland development may be sex dependent.MethodsWe utilized a previously established transgenic mouse line containing the 7.7kb AR enhancer mouse sequence linked to a basal promoter driving lacZ. Mice were collected and stained during different stages of embryonic development to visualize localization of AR enhancer activity. The sex of the embryos were identified by PCR amplification of the Sry gene. We compared enhancer driven lacZ expression to endogenous AR expression determined by in situ hybridization (ISH) in mouse embryos.ResultsWe have surveyed lacZ expression at E15.5 (embryonic day) and E16.5. Despite consistent expression in other tissues, mammary gland expression of lacZ occurs only in female embryos. No mammary associated lacZ expression is visible in males. These expression results are consistent with AR gene expression as detected by ISH.ConclusionWe show that there are sex differences in enhancer activity of AR during mouse development. AR is crucial for inhibiting the development of female‐specific traits as well as promoting the male phenotype. The lack of lacZ expression in male embryos suggests the inhibitory effects of androgens have been sufficient to either eliminate the mammary associated cell types that normally express AR or the expression of an upstream trans‐acting factor that activates this AR enhancer. This mouse line will be useful for tracing developmental progression of AR expressing tissues and signalling during mammalian integumentary development as well as the development of sexually dimorphic characteristics.Support or Funding InformationFunding provided by NSF IOS‐1656315 & NSF BCS‐1638812 and PCOM's Center for Chronic Disorders of Aging.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
microRNA 520b Mediates ATF5 Expression under Diverse Cellular Stress in Cancer Cells
Authors: Gaither Kari A , Madarampalli Bhanupriya , Lazarus Philip 
Published: June 2021

ATF5 is a widely expressed transcription factor that modulates cell survival, proliferation, and differentiation. It is upregulated during such diverse stresses as endoplasmic reticulum stress, amino acid deprivation, and oxidative stress. We had previously found that ATF5 is elevated and acts as a survival factor in several cancer cell lines compared to non‐transformed cells. Cellular stress pathways are implicated in cancer progression, treatment resistance, and metastasis, and upregulation of ATF5 has been implicated in each of these areas for several cancer types, including breast cancer. While the regulation of ATF5 expression is not fully understood, we hypothesize that microRNA (miRNA) play a role in regulating ATF5 under diverse cellular stress conditions. We used in silico analysis to identify potential miRNA candidates (129‐5p, 433‐3p, and 520b) and verified the ability of these candidates to interact with the ATF5 3′ untranslated region (UTR) by luciferase assay. In addition, miRNA were transfected into HeLa human cervical carcinoma cells and their capacity to regulate ATF5 expression was assessed under a variety of cellular stresses. Expression levels of ATF5 protein, ATF5 mRNA, and candidate miRNA were assessed by either Western blot analysis or RT‐qPCR. Of the three miRNA candidates, only miRNA 520b reversed the upregulation of ATF5 under diverse stress conditions in HeLa cells, resulting in a significant reduction (p<0.05) in ATF5 protein expression (~ 40% decrease) compared to control. Under stress conditions in non‐transfected HeLa cells, a rapid and significant decrease (p<0.05) in miRNA 520b (~70% reduction) was observed. This inversely correlated with a significant increase (p<0.05) in ATF5 mRNA (~2‐fold) and increased levels of protein expression (>2‐fold) in cells exposed to stress. Additionally, preliminary data indicate that the ability of miR‐520b to regulate ATF5 is also observed in other cell lines including MCF7 and MDA‐MD‐231 human breast adenocarcinoma cells. Further studies are warranted to determine the mechanism underlying the depressed levels of miRNA 520b under stress, and whether the upregulation of ATF5 in this context leads to enhanced cell survival. Our findings reveal a novel cellular stress‐activated mechanism of ATF5 regulation in cervical carcinoma and breast adenocarcinoma cells, which may lead to novel therapeutic interventions for breast and other cancers in the future.Support or Funding InformationThis work was supported by grants from the National Science Foundation Graduate Research Fellowship Program (grant DGE‐1347973 to Kari Gaither), and the Health Sciences and Services Authority of Spokane, Washington (grant WSU002292 to College of Pharmacy and Pharmaceutical Sciences, Washington State University).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Structure Guided Functional Studies of a Lipoprotein Involved in Salmonella Copper Homeostasis
Authors: Soens Ross , May John 
Published: June 2021

Cells from aerobic organisms require copper ions to generate biochemical energy and for certain enzyme‐catalyzed redox reactions. However, recent studies suggest that when bacteria are engulfed by phagosomes within macrophages, Cu2+ is used in high concentrations, among other molecules, to kill the bacterium. To infect a host, bacteria such as Salmonella enterica must survive this toxic environment containing high Cu2+ levels. A recently discovered protein of unknown function, DcrB, plays a role in copper homeostasis within Salmonella enterica, a major cause of food‐borne illness. DcrB is a small lipoprotein anchored to the periplasmic face of the cytoplasmic membrane. Currently, Salmonella phenotypic assays have shown DcrB is necessary for growth in high levels of Cu2+. The hypothesis of this project is that DcrB binds copper and possibly other divalent cations. To test this hypothesis, we used thermal stability assays paired with structure‐based functional studies. We examined the effect of divalent cations on the stability of purified DcrB protein using a fluorescence‐based thermal shift assay and circular dichroism. Furthermore, we are using our recent crystallographic structure of DcrB to identify possible metal ion binding sites and to test whether each hypothesized metal ion binding site is required for the ability of DcrB to confer resistance to Cu2+. For each site, we used site‐directed mutagenesis to generate an allele of dcrB that encodes a DcrB variant lacking the functional side‐chains at the site. Then, we determined whether a multicopy plasmid that contains the mutant allele of dcrB can complement the copper sensitivity of a dcrB knockout mutant of Salmonella enterica. Our results have established the specificity of DcrB for binding divalent cations and have led to the identification of potential metal binding sites, which we anticipate will shed light on the role of DcrB in metal homeostasis in pathogenic bacteria.Support or Funding InformationThis research was supported by a UW‐La Crosse Faculty Research Grant (JFM), a UW‐La Crosse Undergraduate Research Grant (RWS), and the Dean's Distinguished Fellowship (RWS). This research used resources of the Advanced Photon Source, a U.S. Department of Energy Office of Science User Facility operated for the DOE Office of Science by Argonne National Laboratory under Contract No. DEAC0206CH11357. Use of the LS‐CAT Sector 21 was supported by the Michigan Economic Development Corporation and the Michigan Technology Tri‐Corridor (Grant 085P1000817).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Biosynthetic mechanism of the nonribosomal peptide AMB in Pseudomonas aeruginosa
Authors: Galambos David , Calderone Christopher T 
Published: June 2021

Nonribosomal peptide synthetases (NRPSs) are enzymes found in bacteria and fungi that synthesize a class of natural products called non‐ribosomal peptides. The biosynthesis of the toxin AMB (L‐2‐amino‐4‐trans‐3‐butenoic acid) by Pseudomonas aeruginosa is directed by the amb gene cluster, which encodes two NRPSs (AmbB and AmbE) and two hydroxylases (AmbC and AmbD). Based on sequence homology and previous results, we predicted that the biosynthesis of AMB occurs through the action of the two hydroxylases on an Ala‐Glu‐Ala substrate tethered to AmbE. The modular, linear structure of NRPSs also allows us to predict the function of individual domains in a similar fashion. However, we do not know the regioselectivity or timing of these hydroxylases within the larger synthetic pathway. Sequence homology also indicates that the hydroxylated substrate may be subsequently dehydrated through a novel dehydratase function by the C‐domain. Here, we report evidence that AmbC is a β‐hydroxylase and that AmbD is a ɣ‐hydroxylase. We observed that both enzymes act on a substrate attached to the T1 domain of the AmbE NRPS. Our approach used a mutated AmbE construct containing only the first four (X‐A‐MT‐T) domains of the NRPS. We incubated the truncated construct with a Glu substrate, AmbC, AmbD, and SAM, which is the methyl source for the MT (methylation) domain in vivo. Analysis using HPLC showed a product that is consistent with hydroxylation of the Glu substrate. Confirmation of the predicted activity of AmbC and AmbD combined with a deeper understanding of the timing and regioselectivity of these enzymes will be useful in deciphering whether the C‐domain harbors novel dehydratase activity.Support or Funding InformationRUI: Condensation Domain‐Catalyzed Dehydration, National Science FoundationThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Refinement of the water‐in‐oil reverse microemulsion process for the encapsulation of proteins within silica nanoparticles
Authors: Strong Elijah W , Tarnai Brian D , Anderson C. William 
Published: June 2021

Over the past decade, interest in nanoparticles as a therapeutic tool has skyrocketed as technological advances have made them cheaper and easier to use. Nanoparticles in themselves act as vehicles for proteins, enzymes, drugs, etc. and carry them throughout a biological system to a specific target. However, with proteins being simply bound to the nanoparticles, problems arise with these proteins being susceptible to denaturation. Encapsulation of proteins within nanoparticles provides an advantage over simple binding and gives many benefits through its protection of the proteins. Through encapsulation, proteins are protected by, in this case, a silica shell. This shell prevents the protein from denaturing from heat, acidity, or metabolism. All the while, the drug or enzyme that is encapsulated should still maintain its function and interact with a target site because the silica shell contains small pores that allow for such reactions to proceed.To establish and refine the encapsulation protocol in the context of our laboratory, we attempted a water‐in‐oil reverse microemulsion process on myoglobin, green fluorescent protein (GFP), and chymotrypsin. Since silica nanoparticles have been shown to be biocompatible in humans and have a great deal of biological significance, we chose to use a silica‐based approach for nanoparticle synthesis. The silica nanoparticles that we synthesized were mesoporous, which allowed the nanoparticles to be biocompatible due to their low hemolytic activity. Due to such biocompatibility, these nanoparticles can be used for things such as drug delivery, biosensors, imaging, and more.Results showed that encapsulation was successful through spectrophotometry absorbance and fluorescent microscopy measurements for myoglobin and GFP encapsulation, respectively. The catalytic function of the encapsulated proteins was then shown through absorbance experiments run on encapsulated chymotrypsin. Current work is focusing on using these techniques for the encapsulation of several clinically relevant protein targets of similar size to the examples presented here.Support or Funding InformationNational Science Foundation Award #1626602This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.