bluuebunny/crossref_metadata_2025 huggingface dataset:
Valproate Prevents a Cytosolic vH+ATPase Subunit Insertion on Insulin Granule Membrane and Compromises Insulin Release in Min6 Cells
Authors: Yedulla Nikhil , Naik Akshata R. , Kokotovich Keith M. , Yu Wenxi , Greenberg Miriam L. , Jena Bhanu P.
Published: June 2021

The vacuolar proton pump, vH+ATPase transfers protons (H+) across cell membranes which helps maintain pH gradient within cells necessary for various cellular functions. It is a two‐part pump containing a membrane (V0) and a soluble cytosolic (V1) domain with each domain consisting of multiple subunits. In secretory cells, such as neurons, vH+ATPase acidifies synaptic vesicles, which is essential for neurotransmitter uptake, storage and exocytosis. Valproate, an FDA approved, anti‐ epileptic drug has been shown to disturb vH+ATPase function in yeasts, by inhibiting myo‐inositol phosphatase (MIPS), the first and the rate‐limiting enzyme in inositol biosynthesis leading to inositol depletion. Additionally, vH+ATPase is present on the membrane of insulin secreting granules (ISG) within pancreatic β cells. Several studies have suggested vH+ATPase requirement for insulin maturation within ISGs. Further, pancreatic islets containing a mutant vH+ATPase isoform exhibit compromised insulin release function, however, the mechanism is unknown. Here, we demonstrate that clinical dose of valproate (1mM) significantly reduced glucose‐ stimulated insulin secretion in mouse pancreatic insulinoma (Min6) cells. Moreover, valproate caused maximal inositol depletion in Min6 cells after 5 hours of incubation with the drug. Dual immunofluorescence staining of valproate‐treated Min6 cells demonstrates almost no localization of one of the cytosolic subunits of vH+ATPase (subunit C) on ISGs, unlike in control. These results conclude that inositol depletion in Min6 cells caused by valproate precludes insertion of vH+ATPase subunit C on ISG membrane, rendering the ISGs less competent for glucose‐stimulated insulin secretion.

Support or Funding Information

Work presented in this article was supported in part by the National Science Foundation grants EB00303, CBET1066661 (BPJ); the WSU Interdisciplinary Biomedical Systems Fellow‐ ship and Graduate Research Assistantship (ARN). All authors critically analyzed results and proof read the manuscript. The authors declare no competing financial interests or conflicts.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Ergogenic effects and potential synergism of combined doxapram and acetazolamide in hypoxic rats
Authors: Schroeder Thies , Hamilton Karyn , Irwin David
Published: June 2021

Hypoxemia is a potentially life‐threatening condition that can arise from various conditions, but there is a general lack of effective acute treatments. Voluntary increase of breathing frequency can increase blood oxgen concentrations, however, this does not work during sleep or unconsciousness. Pharmaceutical enhancement of breathing rates is available, but confounded by severe side effects related to respiratory alkalosis. We hypothesized that the addition of a mildly acidifying drug, such as acetazolamide, would be feasible and beneficial, if combined with a respiratory stimulant, such as doxapram. To test the hypothesis, we measured the ability of rats to run in a hypobaric athmosphere of approx. 4,267m/10% O2, after drug injection. Both high (20 mg/kg) and low (10 mg/kg) concentrations of doxapram significantly increased exercising endurance in rats (Log rank test, using a forced exercise wheel model, with or without acetazolamide (5/20 mg/kg) present. Neither low or high acetazolamide improved hypoxic exercise capacity. In an awake hemodynamic rat model, we found that acetazolamide decreased blood pH by approx. 0.05, and doxapram increased it by 0.03 pH points, however, subsequent injection of the respectively other drug normalized blood pH to a common level within 90 minutes. Although both drugs increased blood pO2 independent of each other, addition of the respective other drug did not induce any further increase in pO2. None of the drugs significantly changed pCO2. We conclude that the combination of doxapram and acetazolamide are potentially safe and efficacious in counteracting hypoxemia in rats, caused by hypoxic environments or other factors. Because they cancel out each others effects on blood pH, they might synergize in their ergogenicity of counteracting hypoxemia.

Support or Funding Information

DARPA, Prime Award Number N66001‐10‐C‐2134

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Investigating Peptide Assembly in a Membrane Environment
Authors: Gessel Megan , Kober Megan , Maxwell Zoe , Swain Samantha
Published: June 2021

Objective

This study seeks to determine how the properties of a lipid membrane influence protein folding and assembly in model systems using hydrogen‐detuerium exchange mass spectrometry (HDX‐MS).

Methods

The folding and assembly of small model peptides in large unilamellar vesicles (LUVs) have been investigated using HDX‐MS. We currently have results for the 13‐residue, cationic peptide indolicidin in LUVs prepared with 1,2‐Dioleoyl‐sn‐glycero‐3‐phosphocholine (DOPC) or 1,2‐Dioleoyl‐sn‐glycero‐3‐phosphoglycerl (DOPG).

Results

Our HDX‐MS results indicate that free indolicidin is fully deuterated at all measured time points, including the shortest (30s). Results with LUVs show differences between LUVs made of DOPC (a zwitterionic phospholipid) and DOPG (an anionic phospholipid). Indolicidin mixed with DOPC LUVs has slightly less deuterium incorporation (~3 fewer deuterium), compared to the free peptide in the absence of LUVs. Indolicidin with DOPG LUVs shows additional protection from hydrogen‐deuterium exchange (~5 fewer deuterium), compared to free indolicidin. Preliminary MS/MS experiments suggest that HDX protection occurs at the C‐terminal end of indolicidin.

Conclusions

Evidence from several studies shows that MPs fold into their “correct” structures even in certain lipid‐free environments, suggesting that the membrane may not be required for proper MP folding. However, many researchers go to great lengths to mimic the physical and chemical environment of the membrane and claim that characteristics including curvature, fluidity, and symmetry are significant. Overall, we plan to investigate three model peptides known to insert into the membrane and oligomerize to form membrane‐active structures. The model peptides represent different structural classes and their individual structures have already been well‐characterized by traditional biophysical methods.

Our results with free indolicidin are consistent with the fact that, in solution, indolicidin remains in a monomeric form and adopts an extended, linear structure. The results with LUVs indicate that the peptide does interact with both types of LUVs. More protection from HDX occurs with the DOPG LUVs, suggesting that the protein may be embedding itself deeper into the DOPG (anionic) LUVs, compared to DOPC (zwitterionic) LUVs. The surface charge of the model membrane may affect the peptide's ability to associate with the membrane. The MS/MS results suggest that the peptide's C‐terminus may be responsible for insertion into both LUV types. We are currently varying the model membranes to examine the effects of fluidity and curvature, as well.

Support or Funding Information

This work was supported by a Major Research Instrumentation grant (MRI1531435) from the National Science Foundation and a grant from the Murdock Charitable Trust.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Organophosphate Hydrolase (OPH) Designed as a Functional Monomer
Authors: Baker Cetara , Garcia Jaffet Santiago , Sweeney Richard , Kirby Stephen
Published: June 2021

Organophosphate hydrolase (OPH) mutants haveshown potential use as a medical countermeasure against a range of differentorganophosphorus compounds (OPs). OPH is typicallyexpressed in bacteria as a homodimer. Two separate monomers (~37kDa each) self‐assemble throughnon‐covalent bonding at the enzyme face close to the putative active site, andenzyme activity is dependent upon dimer formation. OPH homodimers do notsecrete expediently from mammalian cells and are often misfolded. This causespotential problems when trying to express the protein from a heterologous plasmidor viral delivery system. To enhance secretion of OPH from mammalian cells, wesought to increase protein solubility without catastrophic detriment toactivity and without addition of fusion proteins. To this end, we designed OPH tobe expressed as a functional monomer by joining two OPH subunits with a poly‐glycinelinker and expressing it as a single polypeptide in bacteria to evaluateactivity. We created the single polypeptide OPH with a tether 10 or 35 aminoacids in length between the two halves, and named them T10 and T35, respectively. Western blot analysis and paraoxon hydrolysis assays revealedthat T10 was being produced and there was a 71% loss in specific activity comparedto an untethered homodimer positive control. This was a surprise as we expectedT10 to have no enzymatic activity since the tether was too short to allowproper dimer formation. The T35 functional monomer (~76 kDa) was produced showing 29% loss of specific activity. Both T10 and T35 OPH showed high molecular weight aggregate formation greater than 250 kDa in dynamic light scattering and native polyacrylamide gels. Construction of a functional OPH monomer with activity against OPs is instrumental in designing secretable OPHs for adeno‐associatedviral expression. It may also prove useful in the development of chimeric OPHs for maintaining control over the composition of each OPH half, allowing blended activities against a combination of organophosphorus substrates. This is the first attempt known for producing OPH as a functional monomer.

Support or Funding Information

The views expressed in this abstract are those of the author(s) and do not reflect the official policy of the Department of Army, Department of Defense, or the U.S. Government. This research was supported by the Defense Threat Reduction Agency – Joint Science and Technology Office, Medical S&T Division. This research was supported in part by an appointment to the Postgraduate Research Participation Program at the U.S. Army Medical Research Institute of Chemical Defense administered by the Oak Ridge Institute for Science and Education through an interagency agreement between the U.S. Department of Energy and USAMRMC.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Roles of Galectins in Infection: First barrier or Trojan Horse?
Authors: Vasta Gerardo R.
Published: June 2021

Carbohydrate structures on the cell surface encode complex information that through specific recognition by carbohydrate‐binding proteins (lectins), modulates interactions between cells, cells and the extracellular matrix, or mediates recognition of potential microbial pathogens. Galectins are a family of β‐galactoside‐binding lectins characterized by a unique sequence motif in the carbohydrate recognition domain, and evolutionary and structural conservation from fungi to invertebrates and vertebrates, including mammals. Since their discovery in the 1970s, their biological roles, initially understood as limited to recognition of endogenous (“self”) carbohydrate ligands in embryogenesis and early development, dramatically expanded in later years by the discovery of their roles in tissue repair, cancer, adipogenesis, and regulation of immune homeostasis. In recent years, however, evidence has also accumulated to support the notion that galectins can bind (“non‐self”) glycans on the surface of potentially pathogenic microbes, and function as recognition and effector factors in innate immunity. Thus, this evidence has established a new paradigm by which galectins can function not only as pattern recognition receptors but also as effector factors, by binding to the microbial surface and inhibiting adhesion and/or entry into the host cell, directly killing the potential pathogen by disrupting its surface structures, or functioning as opsonins, by promoting phagocytosis and pathogen clearance from circulation. Strikingly, some viruses (HIV, Nipah, IHNV), bacteria (Chlamydia, Streptococcus), and protistan parasites (Trichomonas, Leishmania, Perkinsus) have the capacity to take advantage of the aforementioned recognition roles of the vector/host galectins, for successful attachment or invasion. The recent findings mentioned above have not only revealed an extraordinary functional diversification in this otherwise structurally conserved lectin family, but also suggest unique evolutionary adaptations of the surface glycome of some microorganisms to use host recognition for cell surface adhesion and infection. Thus, it is proposed that galectin‐mediated innate immune recognition and effector mechanisms, which throughout evolution have remained effective for preventing or fighting viral, bacterial and parasitic infection, have been “subverted” by certain pathogens to gain entry into the host.

Support or Funding Information

Supported by grant 5R01GM070589 from the National Institutes of Health, and grants IOS‐0822257 and IOS‐1063729 from 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.
Sustained mentorship promotes the development of active learning strategies in undergraduate biology classrooms: Evidence gained from the Promoting Active Learning and Mentoring (PALM) Network
Authors: Wolyniak Michael J , Wick Susan
Published: June 2021

Here we report on the work of the Promoting Active Learning and Mentoring Network (PALM), a National Science Foundation‐supported project started in 2016 and dedicated to leveraging the expertise found in life science professional societies to provide long‐term mentorship in developing active learning techniques in the classroom. PALM mentees work one‐on‐one with mentors that are seasoned in active learning strategies and travel to their mentor's institution to observe classes, plan modifications for their own classes, and strategize the best active learning techniques that are suited to their class' particular needs. A monthly online journal club and regular opportunities to meet in conjunction with the Society for the Advancement of Biology Education Research (SABER) and ASCB meetings provide a dynamic teaching and learning community for mentees and mentors alike. After their mentorship, mentees are encouraged to work with their colleagues on active learning strategies and to become a PALM mentor to further the spread of effective active learning strategies to an ever‐wider audience.

We have assessed the efficacy of PALM mentorship through the use of the Classroom Observation Protocol for Undergraduate STEM (COPUS) protocol (Smith et al., 2013) on pre‐mentorship and post‐mentorship videos of mentee teaching. The COPUS results have revealed a consistent shift in mentee teaching practices from a strict focus on lecture to a more diffuse combination of classroom activities in which the student is more directly active and engaged in the class and the instructor is more directly engaging students in a more personalized manner. While the specific changes in classroom practice vary between individual mentees due to the distinct needs and environments of each class, there is a consistent picture of more student‐centered classrooms that are more engaging and dynamic for both student and instructor. Overall, the COPUS data suggests that the long‐term mentoring strategies brought forth by PALM are an effective and sustainable means to promote the development of active learning techniques among undergraduate biology instructors.

Support or Funding Information

National Science Foundation #1624200

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Functional Relevance of Unstructured Regions of AcrA, the Periplasmic Adaptor of the Major Multidrug Efflux System in E. coli
Authors: Ojo Isoiza , Wei Yinan
Published: June 2021

Efflux pumps, combined with low permeability of the outer membrane of gram negative bacteria play a crucial role in bacteria multi‐drug resistance. There are five classes of efflux pumps but the major and most studied of the efflux system in E. coli is the resistance nodulation cell division (RND) pump. This pump is a complex of three proteins: the inner membrane transporter AcrB, the periplasmic adaptor AcrA, and the outer membrane protein channel TolC. Of the three proteins, the structure and dynamic behavior of AcrA remain to be fully elucidated. In this study the functional roles of the unstructured regions at the N‐ and C‐termini of the protein were examined. These structurally unresolved regions include residues 26–37 at the N‐terminus, and residues 376 to 397 at the C‐terminus. We conducted truncation mutation to systematically remove residues from the C‐terminus of AcrA, and found that residues after Q376 are indeed not functionally important. The truncated AcrA, in which E377 is replaced by a stop codon, is fully active. On the N‐terminus, we created a mutant in which four amino acid residues (Q31QGG34) were deleted. The mutant was nonfunctional. To understand the mechanism of function disruption, we modified the membrane anchoring of AcrA, and determined the role of these first 13 residues at the N‐terminus of the mature AcrA sequence.

Support or Funding Information

NSF grant CHE‐1709381

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Automated Evaluation of Microvascular Blood Flow and Oxygenation following Trauma and Hemorrhage in Rats
Authors: Torres Filho Ivo P , Barraza David , Williams Charnae E , Hildreth Kim , Dubick Michael A
Published: June 2021

Studies evaluating blood flow and oxygen partial pressure (PO2) may not directly measure both parameters and may depend on investigator's selection of measuring sites. In addition, relevant systemic parameters may not be simultaneously recorded. Therefore, we implemented a new automated system for blood flow and PO2 acquisition in large tissue areas while collecting systemic information. In 10 animals anesthetized with isoflurane, cardio‐respiratory parameters were continuously recorded, in experiments lasting up to 4 h. Other data were collected at baseline, and after laparotomy and 30 min hemorrhage (40% of total blood volume). A cremaster muscle was spread over a thermostatically‐controlled pedestal fixed to a motorized stage. Sixteen noninvasive PO2 measurements using oxygen‐dependent phosphorescence quenching and fiber‐optics were performed during a computer‐controlled tissue scan. In the same areas used for PO2 measurements, microvascular blood flow was estimated employing laser speckle contrast imaging. Blood was sampled for extensive biochemistry and coagulation profiles. The system was used successfully by different operators. One set of flow and PO2 measurements was completed in less than 90 s. Changes in muscle flow correlated with local PO2 but several systemic parameters did not correlate with blood flow and PO2, emphasizing the importance of performing both local and systemic evaluations. System advantages include integration between local and multiple systemic parameters, unbiased data collection/analysis, easy implementation, improved performance, no need for customized programming, and simplified training compared to intravital microscopy.

Support or Funding Information

Supported by US Army Medical Research and Materiel Command.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The ERG1a K+ Channel Increases Intracellular Calcium and Calpain Activity in C2C12 Myotubes
Authors: Pond Amber Lynn , Whitmore Clayton L. , Pratt Evan E. , Anderson Luke Brian , Rantz Emily , Salyer Amy , Carraro Ugo , Zampieri Sandra , Weilbaecher Rod , Davie Judy K. , Hockerman Gregory H
Published: June 2021

In skeletal muscle increased intracellular calcium concentration ([Ca2+]i; uM range) is necessary for excitation‐contraction coupling; however, smaller increases in [Ca2+]i (nM range) can modulate other physiological processes in non‐contracting muscle. Indeed, fluctuations in localized calcium concentration can serve as a second messenger. In contrast, inappropriate changes in [Ca2+]i can have detrimental effects on muscle tissue and are associated with numerous skeletal muscle pathologies, for example muscular dystrophy, amyotrophic lateral sclerosis, malignant hyperthermia, cancer cachexia, and atrophy. Therefore, [Ca2+]i must be tightly regulated in terms of time, space and amplitude for cellular processes to occur in a properly coordinated fashion. In earlier reports, we showed that the ERG1a K+ channel is upregulated in atrophying skeletal muscle and contributes to increased ubiquitin proteasome proteolysis. Here, using the ratiometric Ca2+ indicator fura‐2AM, we show that ERG1a expression in C2C12 myotubes produces an increase in basal [Ca2+]i as well as a transient increase in [Ca2+]i as a consequence of depolarization. We explored this transient increase in [Ca2+]i using pharmacological agents. The data demonstrate that the ERG1a‐induced increase is not sensitive to the L‐type calcium channel blocker nifedipine, suggesting that it does not result from modulation of Cav1.1 channels. To further support this data, immunoblots reveal that there is no change in Cav1.1 channel abundance. However, the data do demonstrate that the increase in [Ca2+]i is sensitive to the SERCA blocking agent thapsigargin, suggesting that the source of the calcium is the sarcoplasmic reticulum stores. Additionally, the data reveal that ERG1a expression also increases basal calpain activity. In summary, to date the data show that ERG1a increases [Ca2+]i levels and suggest that this increase could occur, at least in part, as a result of release of calcium from sarcoplasmic reticulum stores. Additionally, the data reveal that ERG1a expression also increases calpain activity, suggesting that the increase in intracellular calcium results in increased calpain activity and possibly contributes to the increased proteolysis that occurs in atrophic skeletal muscle.

Support or Funding Information

U.S. Department of Defense, PRMRP Grant# PR170326

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Quadrupole ICPMS Determination of Iron in Drosophila melanogaster S2 Cell Cultures using Collision Cell and Desolvation Strategies
Authors: AL‐Ogaidi Hussein A , Ketterer Michael E , Ragan Emily J
Published: June 2021

Inductively coupled plasma mass spectrometry (ICPMS) has become the preferred technique for trace and ultra‐trace determination of elemental concentrations in a wide variety of sample matrices. In our studies of iron uptake and metabolism in insect cells, we evaluated different ICPMS procedures to measure picogram to microgram quantities of iron in whole insects and Drosophila melanogaster S2 cells. While a well‐known spectrophotometric iron assay with ferrozine suffices for microgram amounts of the elements, ICPMS is imperative for lower level work.

The determination of iron by ICPMS faces several significant challenges: samples must be decomposed to mineralize organic matter; the separation of iron from the matrix may be required in some circumstances; all sample preparation and analytical processes must be conducted under sufficiently “clean”, low‐blank conditions; and the interference of polyatomic ions (ArN+, ArO+, ArOH+) upon iron ions must be addressed. To address these challenges, samples were digested and mineralized by heating with nitric acid at 90°C in fluoropolymer containers. “Trace metal” grades of HNO3 were found to be satisfactory for minimizing reagent contributions to the overall blank. As quadrupole mass analyzers cannot distinguish between ions of the same nominal mass (e.g., 40Ar16O+ vs. 56Fe+), alternative strategies must be used to suppress the formation of polyatomic ions so that Fe+ ions can be observed above background signal. We used two different approaches. The first is de‐solvation of the sample aerosol, where water vapor is removed by a membrane dryer with countercurrent Ar gas flow. The second is passage of the extracted ions through a “collision cell” prior to mass analysis, which selectively attenuates the polyatomic ions via ion kinetic energy discrimination. A combination of both approaches lowered solution detection limits for Fe from ~ 50 – 100 micrograms/liter to << 1 microgram/liter. We present optimized procedures for the routine determination of Fe in digested cell and insect tissue samples.

Support or Funding Information

NSF IOS‐1656407

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Ferrozine‐Based Assay for Determining Iron Content in Insect Cells
Authors: Castaneda Mirella , AL‐Ogaidi Hussein , Gorman Maureen J. , Ragan Emily J.
Published: June 2021

We are optimizing a ferrozine‐based assay to analyze the iron content in Drosophila S2 cells. We hypothesize that putative ferric reductases CG1275 or CG8399 may be involved in reducing iron in the hemolymph and allowing uptake into cells. We have modified a ferrozine‐based assay, in which ferrozine complexes with ferrous ions in solution and forms a stable magenta complex species that can be detected at 562 nm. Using this method with an Ultraspec II spectrophotometer we obtained a limit of detection of 0.30 μg/mL. This method provides a reliable, inexpensive and simple way of analyzing and quantifying the iron content within the S2 cells, allowing us to analyze iron content after RNA interference experiments to knock down CG1275 and CG8399.

Support or Funding Information

NSF IOS‐1656407

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Behavioral Economic Demand for Cocaine During Phendimetrazine Maintenance in Humans
Authors: Strickland Justin Charles , Stoops William Walton
Published: June 2021

Objectives

Phendimetrazine 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.

Method

Twenty‐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.

Results

Three‐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.

Conclusions

These 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 Information

National 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 Information

This 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 Information

NSF 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 objective

The 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.

Introduction

Conserved 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.

Methods

We 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.

Results

We 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.

Conclusion

We 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 Information

Funding 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 Information

This 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 Information

This 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 Information

RUI: Condensation Domain‐Catalyzed Dehydration, 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.