bluuebunny/crossref_metadata_2025 huggingface dataset:
Starting and Sustaining Undergraduate Research
Authors: Bell Ellis
Published: June 2021

Research is accepted as a necessary part of the education of undergraduates and this is particularly true in the molecular life sciences. The phrase “undergraduate research” implies that there is something distinct about “undergraduate research” and is often interpreted to mean research that is less challenging [ie more suitable for undergraduates] or less demanding of facilities and time, which translate into not the type of research that you see at a research intensive institution. Successful research by undergraduates has all the characteristics of successful research by anyone: it is challenging and addresses important problems, using the most appropriate techniques. Initiating and sustaining such research in a primarily undergraduate institution is the focus of this workshop.

Participants will develop a plan to initiate and sustain such research in their own laboratory or institution and will focus on “best practices” for developing a project, recruiting and retaining students, managing a research group from proposal to publication, integrating teaching and research, making appropriate collaborations, and obtaining funding. The workshop will be led by experienced researchers from undergraduate institutions who will be available after the workshop to mentor participants throughout the coming year.

The work is supported by National Science Foundation Grant MCB 0448905 to Ellis Bell
Vascular Structure and Elastic Anisotropy in Cortical Bone
Authors: Dechow Paul C.
Published: June 2021

Elastic properties define the relationship in cortical bone between loading and deformation. Our investigations show the considerable variations in 3D elastic properties in the craniofacial skeleton, in particular, the variations in anisotropy as indicated by the ratio of elastic modulus between the axes of minimum and maximum stiffness within the cortical plane. These variations are an important but generally overlooked aspect of bone quality. Continuing studies are testing whether variations in cortical elastic anisotropy correlate with variations in 3D osteonal structure. A positive correlation would lend support to the claim that 3D osteonal structure is influenced by the orientation of mechanical loads in cortical bone. In the majority of midfacial cortical bone specimens, average osteon orientation weighted by osteon length is within 15 degrees of the direction of maximum stiffness. The anisotropy ratios are moderately correlated with the standard deviation of osteon orientation with R values of about 0.50. Results suggest that osteon orientation is a structural correlate of cortical anisotropy. However, the moderate correlations suggest that other factors, such as collagen orientation, or the quantity and structure of primary bone, are likely also important in determining cortical bone anisotropy at a supraosteonal or bulk level of organization. Supported by NSF BCS‐0240865.
Are Two Legs Better than Four? Comparative Biomechanics & the Evolution of Human Walking & Running
Authors: Raichlen David , Pontzer Herman , Sockol Michael
Published: June 2021

The evolution of bipedalism is considered a defining event of early hominin evolution, however the selection pressures that led to upright walking remain unclear. One popular hypothesis is that bipedalism evolved to reduce energy costs in early hominins. To test this hypothesis, we used chimpanzees as a model of our pre‐bipedal ancestors. We measured bipedal and quadrupedal biomechanics and energetics in a sample of adult chimpanzees and humans. Chimpanzees use approximately 75% more energy than humans during walking, suggesting bipedalism may have been an attractive option for early hominins if selection were acting to reduce energy expenditure. Using inverse dynamics to estimate the amount of muscle volume activated per step, we found that chimpanzees’ flexed limb postures led to large amounts of active muscle volume at the hip and knee, which fully explain their high costs compared to humans. Using a modeling approach, we then assessed how changes in morphology and posture impact estimated energy costs in early hominins. We found that minor changes to either anatomy, posture, or both would have reduced walking costs compared to a chimp‐like ancestor. Therefore, it is increasingly likely that energetics played a major role in the evolution of bipedalism. Research supported by the Leakey Foundation and NSF.
Locomotor hand postures and the functional anatomy of the hominid wrist
Authors: Orr Caley Michael
Published: June 2021

The African apes have stable wrists with limited mobility—probably for energetic efficiency while using the hand as a rigid, propulsive lever during knuckle‐walking (KW). In contrast, the Asian apes have mobile wrists to facilitate a variety of arboreal hand postures. Testing the KW ancestor hypothesis for hominins requires understanding mechanisms of wrist stability in African apes and subsequent identification of related anatomy in the fossil record.

A CT‐based method to track 3D carpal kinematics was used to study midcarpal joint mechanics in Pan troglodytes and Pongo pygmaeus. Midcarpal extension in Pan is distinguished by scaphoid supination on the capitate head, which rapidly close‐packs the midcarpus. Coupled with scaphoid‐centrale fusion and a centrale‐capitate ligament, this limits extension and ensures that the scaphoid and capitate act as a rigid unit. Scaphoid‐centrale fusion and a Pan‐like scaphocapitate joint in early hominins suggest a similar midcarpal mechanism, which may indicate a KW ancestor; alternatively, the mechanism may be a more generalized quadrupedal adaptation. Studying carpal kinematics in quadrupedal monkeys will test this alternative hypothesis. Modifications to the scaphocapitate joint and loss of the centrale‐capitate ligament in later hominins probably resulted in the more mobile modern human wrist.

Funding: NSF BCS‐622515, Wenner‐Gren Foundation, Sigma Xi, ASU
25‐Hydroxyvitamin D is associated with cognitive function in an elderly population receiving homecare services
Authors: Buell Jennifer S , Scott Tammy , Dawson‐Hughes Bess , Dallal Gerard , Rosenberg Irwin , Folstein Marshal , Tucker Katherine L
Published: June 2021

Vitamin D status has been associated with neurological disorders but little research has explored vitamin D and cognitive function. In this study, we evaluated the cross‐sectional association between 25‐hydroxyvitamin D (25OHD) and cognitive function in 1080 homebound elders (>65y) in Boston, MA. Subjects completed cognitive tests and demographic questionnaires. Blood was drawn and analyzed for 25OHD. After adjusting for age, sex, race, BMI, education, center, renal function, seasonality, and alcohol use, 25OHD was associated with better performance on trails A (β=−0.26; P<0.01), trails B (β=−0.31; P<0.01), digit symbol (β=0.08; P<0.001), matrix reasoning (β=0.02; P<0.03), and block design (β=0.03; P<0.03). 25OHD concentrations >50 nmol/l were associated with better scores on tests of executive function, including trails A (80.5 vs 96; P<0.01), trails B (199s vs. 228s; P<0.01), digit symbol test (31.5 vs. 37; P<0.01), and MMSE (24 vs 26; P<0.001). There were no associations between 25OHD and memory tests. Factor analysis revealed three cognitive factors: memory, executive function, and attention/processing. After covariate adjustment, 25OHD was associated with the executive function factor (β=0.004; P<0.01). 25OHD concentration was positively associated with cognitive performance, particularly with measures of executive function. Funded by NIH AG21790‐01, USDA ARS 58‐1950‐7‐707
Extracellular control of muscle regeneration
Authors: Simon Hans‐Georg , Calve Sarah
Published: June 2021

In urodele amphibian limb regeneration, the muscle provides a reservoir for uncommitted stem‐like blastema cells that will participate in the replacement of the lost structure. However, it is currently not clear what signals activate muscle‐associated satellite cells and/or dedifferentiation of multinucleate myofibers. Cultures of newt muscle fibers allow us to follow these physiological changes at the cellular level using time‐lapse video microscopy.

Our studies on extracellular matrix (ECM) distribution during blastema formation demonstrate upregulation of hyaluronic acid (HA), fibronectin (FN), and Tenascin C, suggesting dynamic changes in the ECM composition. Contemporaneous with ECM reorganization, histological analyses of regenerating blastemas reveal increased satellite cell numbers and migration along with myofiber fragmentation. In order to gain a better understanding of the signaling properties of the ECM in regeneration, we are investigating various ECMs and their effects on cell physiology and morphology. Our data indicate that HA induces fragmentation of myotubes and limits adhesion whereas FN or laminin‐containing matrigel stimulates formation of myotubes.

The establishment of a physiologically relevant environment and its manipulation in vitro will provide new insights into the control of the differentiated state of the cell by its ECM.

Supported by DARPA, NIH, Searle Funds
Dissecting protein complexes with small molecules
Authors: Mapp Anna K
Published: June 2021

Altered transcriptional patterns are associated with all human diseases, either as a cause of the disease or as an effect. For this reason, molecules that interfere with or promote protein‐protein interactions within the transcriptional regulatory network are attractive targets for therapeutic development and as mechanistic probes. Despite the complexity of the transcriptional regulatory system, the design principles for the identification of small molecules that stimulate assembly of the transcriptional machinery complex are evidently straightforward. We find that several structurally distinct amphipathic molecular scaffolds can function in vitro and in human cells as transcriptional activation domains. In addition, structural studies of these molecules in complex with transcriptional machinery proteins reveal that they target the binding surfaces used by natural transcriptional activators and do so with similar affinity and kinetic profile. Finally, straightforward structural modifications of the molecules can be used to convert them into potent inhibitors of transcriptional machinery assembly.

Financial support was provided by the American Cancer Society (RSG 05‐195‐01), the NSF, GSK and Novartis.
Nutritional and Immunological Status and Burden of Infection in Elderly Ecuadorians
Authors: Hamer Davidson H , Sempértegui Fernando , Estrella Bertha , Tucker Katherine L , Rodríguez Alicia , Egas Josefina , Dallal Gerard E , Selhub Jacob , Meydani Simin N
Published: June 2021

The number of the elderly in Latin America is expected to rise substantially. To define the prevalence of infections and micronutrient deficiencies, and immunological status, and to evaluate associations between nutritional status and infection, we performed a cross‐sectional study of elderly Ecuadorians in a low‐income peri‐urban community in Quito, Ecuador. Anthropometric measurements, delayed type hypersensitivity (DTH) skin testing, and blood micronutrient and immunological assays were performed in 352 randomly selected Ecuadorians, aged≥65 y. Relationships between micronutrients, immune function, and history of infection was assessed by multiple linear and logistic regression models. Participants recalled recent episodes of colds/influenza‐like syndromes (63%), cough (61%), urinary tract infection (38%), diarrhea (32%), fever (24%), and pneumonia (3.5%). Anemia and micronutrient deficiencies, especially for vitamins C, D, B6, and B12, folic acid, and zinc were common. Plasma vitamin C was associated with IFN‐γ (P<0.01); and zinc with IFN‐γ and IL‐2 (each P<0.0001). History of respiratory infection was associated with micronutrient deficiency (P<0.0001). The burden of infection, micronutrient deficiencies, and anemia was substantial in elderly Ecuadorians and place them at risk for infections through their negative impact on immune function. Supported by NIH Fogarty International Center (1 R03 TW005779‐01A1), NIA (2R01 AG009140‐10A1), and USDA (#58‐1950‐7‐707).
Assessment of the effects of dietary supplement use (DSU) on Estimated Average Requirement (EAR) and Adequate Intake (AI) in older adults
Authors: Bailey Regan Lucas , Miller Paige E , Hartman Terryl J , Mitchell Diane C , Smiciklas‐Wright Helen
Published: June 2021

Poor nutrient intakes are an adaptable risk factor associated with numerous public health outcomes. As older adults represent a vulnerable population for low nutrient intakes they may be ideal candidates for DSU. Usual nutrient intakes were estimated with and without DSU using the mean nutrient intake from 4, 24‐hour recalls in the Geisinger Rural Aging Study (114 m, 158 f; mean age 78.5 ± 6). We examined the impact of DSU on the proportion of our sample who met the EAR and AI for selected nutrients, as well as on the Mean Adequacy Ratio (MAR), an index of nutrient intake (range of 0–1). The nutrients used to calculate the MAR included calcium, folate, pantothenic acid, magnesium, zinc, potassium and Vitamins A, C, D, E, K, B6, and B12. The majority of the sample (74%) reported DSU. The MAR for our sample without accounting for DSU was 0.68 but increased to 0.82 with DSU. Using the EAR, the prevalence of inadequate intakes for nutrients was higher if DSU wasn't accounted for, and a similar trend was observed for those nutrients with an AI. Notably, only 1% of the sample met the AI for Vitamin D through diet alone; however, when DSU was factored in 68% were meeting the AI. Given the widespread prevalence of DSU in older adults and the large contribution of DSU to total nutrient intakes, it is imperative to account for DSU when estimating nutrient adequacies within groups. Supported in part by NIH R21AG023179‐01A1, USDA #58‐1950‐4‐401.
Human hepatic stellate cells and hepatocyte co‐cultures maintain differentiation in vitro and in vivo
Authors: Harding Martha J , Barnaeva Elena , Bustamante Marcia , Lepus Christin M , Rahner Christoph , Pober Jordan S , Sjogren Maria H , Rojkind Marcos
Published: June 2021

Lack of long‐term cultures of mature human hepatocytes (Hep)

that maintain liver‐specific functions have hampered investigations of the effects of chronic drug toxicity and liver pathogens. We investigated if matrix‐producing hepatic stellate cells (HSC) sustained Hep survival and differentiation in vitro and in vivo. Co‐cultures (CC) survived 2 months in vitro, maintained expression of albumin, CYP2E1, CD81, and CK19 mRNAs, began to express α‐fetoprotein and secreted α1‐antitrypsin and fibrinogen. CC proliferated and required passaging every 3–4 weeks. CC prepared with Hep from newborn subjects expressed and sustained the hepatoblast markers Thy1 and Sca 1 in vitro. Transplanted Hep survived in CC for at least 8 weeks when engrafted in vascularized protein gels on the abdominal wall of SCID/bg mice. EM revealed that Hep ultrastructure and polarization was preserved and groups of epithelial cells formed bile duct‐like structures in vivo. Our findings suggest that Hep‐HSC CC sustain normal Hep morphology and differentiation. Accordingly, these CC could be used in studies of liver repopulation, hepatitis infections and long‐term toxicity of drugs and alcohol. Supported by AGA RSA (MJH), NIH‐R01HL085416 (JSP), USUHS MDA905‐03‐1‐0006 (MHS) and NIH‐RO1AA10541(MR) grants.
Protein Network Comparative Genomics
Authors: Ideker Trey
Published: June 2021

With the appearance of large networks of protein‐protein and protein‐DNA interactions as a new type of biological measurement, methods are needed for constructing cellular pathway models using interaction data as the central framework. The key idea is that, by comparing the molecular interaction network with other biological data sets, it will be possible to organize the network into modules representing the repertoire of distinct functional processes in the cell. Three distinct types of network comparisons will be discussed, including those to identify: (1) Protein interaction networks that are conserved across species (2) Networks in control of gene expression changes (3) Networks correlating with systematic phenotypes and synthetic lethals Using these computational modeling and query tools, we are constructing network models to explain the physiological response of yeast to DNA damaging agents.
Alcohol Dehydrogenase Dynamics and Catalysis
Authors: Plapp Bryce V.
Published: June 2021

The catalytic mechanism of horse liver alcohol dehydrogenase has been extensively studied. The enzyme changes conformation when coenzyme and substrates bind. Release of a proton from the catalytic zinc‐water via the proton relay system controls the conformational change and suggest that changes in the coordination of the zinc are involved in substrate binding. Substitutions of various amino acid residues in the active site activate the enzyme by increasing the rate‐limiting release of coenzyme product and affect the conformational change, but the rates of hydride transfer decrease modestly. The hydride ion is transferred with quantum mechanical tunneling, implying that the reacting substrates move closer together at the transition state. The contribution of protein motions is being assessed with studies of various mutated forms of the enzyme. High resolution X‐ray crystallography shows that the reduced nicotinamide ring is puckered in ternary complexes with NADH. X‐ray data can also provide information on protein motions that are correlated with the hydride transfer. Protein dynamics are important for catalysis by liver alcohol dehydrogenase.

(Supported by grants from NSF and NIH)
Exploring the Energy Barriers Involved in Allosteric Transitions in Glutamate Dehydrogenase
Authors: Jarman Erika , Bell Ellis
Published: June 2021

Glutamate Dehydrogenase exhibits negative cooperativity, allosteric activation [by ADP], inhibition [by GTP] and substrate inhibition by glutamate. Each is thought to involve subunit interactions. Ligand induced changes in conformation occur with an energy barrier that must be overcome by energy from ligand binding. In negative cooperativity induced by NAD(P)+ binding, activity goes up but affinity for ligand goes down. We hypothesized that if the increased activity results from enhanced flexibility, raising the thermal energy of the system should overcome the energy barrier resulting in a single form of the enzyme with high activity and low affinity rather than the negative cooperativity. The temperature dependence of the non‐linear Lineweaver Burke plots resulting from negative cooperativity shows that the cooperativity disappears at temperatures above 40°C giving a form with high catalytic efficiency and low affinity, consistent with our hypothesis. At low temperature [15°C or below] the cooperativity also disappears suggesting the energy barrier to trigger the negative cooperativity is too high for ligand derived energy input to overcome. This approach has been extended to examination of both substrate inhibition and heterotropic allosteric regulation in this system to give information about associated energy barriers.

Supported by NSF Grant MCB 0448905 to Ellis Bell
The Role of Dynamics in the Activity and Regulation of Oligomeric Dehydrogenases
Authors: Bell Ellis
Published: June 2021

Three oligomeric dehydrogenases, each catalyzing similar reactions have been studied to examine the role of protein dynamics in both catalysis and allosteric regulation. Global stability has been assessed using DSC or Guanidine Hydrochloride unfolding approaches. Limited proteolysis and H/D exchange approaches have been used to examine the effects of ligands on local flexibility. A novel technique, combining guanidine hydrochloride and dynamic light scattering approaches is used to assess the stability of subunit interfaces. These techniques, combined with site directed mutagenesis and the use of alternative substrates and allosteric activators or inhibitors reveal a pattern of flexibility effects which indicate that efficient substrates or efficient enzymes have significantly more flexible local structure and subunit interface interactions than less efficient substrates or enzymes. Similarly allosteric activators increase both global and local flexibility while allosteric inhibitors decrease flexibility. Overall these studies show that flexibility plays a critical role in catalytic efficiency and that allosteric regulation can be achieved by ligand induced modulations of flexibility rather than simple induced switching from one conformation to another.

The work is supported by National Science Foundation Grant MCB 0448905 to Ellis Bell
Ligand Induced Changes in Flexibility & Allosteric Interactions in Glutamate Dehydrogenase
Authors: Scheel Carolyn , Bell Ellis
Published: June 2021

Higher eukaryotic glutamate dehydrogenases exhibit complex homo and hetero tropic regulation involving subunit interactions and ligand induced conformational changes. Ligands either have little effect or significantly increase or decrease global stability of the protein. Correlation of guanidine hydrochloride induced unfolding and dynamic light scattering allow the dissociation of the subunits to be followed and indicates that Norvaline which does not support subunit interactions or efficient catalysis significantly stabilizes the protein also affects subunit dissociation. Limited proteolysis studies using immobilized trypsin is allowing differential effects of some ligands to be localized to specific regions of the polypeptide chain. GDH is digested with immobilized protease and the time dependence of fragmentation followed using MALDI tof mass spectrometry and Protein Prospector to identify cleavage sites from the masses of the resultant fragments. Other studies using H/D exchange coupled with a complete proteolysis using pepsin as the protease will allow a more precise view of the conformational changes. Overall these studies indicate that both activity and allosteric regulation of this complex enzyme are linked to changes in conformational flexibility rather than discrete ligand induced changes in conformation.

Funded by NSF Grant MCB 0448905 to EB
Probing the Active and Allosteric Sites of Thermotoga maritima ADPGlucose Pyrophosphorylase: Mutational Analyses of the glgC1 and glgC2 Proteins
Authors: Matsui Mikiko , Wiig Jared , Lim Shannon , Harake Tala , Lippert Rachel , Gatzke Janice , Reyes David , Panosian Onnig , Tennison Brent , Susoeff Michael , Nguyen Kelly , Axelrod Herb L , Lesley Scott A , Meyer Christopher R
Published: June 2021

The T. maritima ADP‐glucose pyrophosphorylases (ADPG PPases) glgC1 and glgC2 share only 36.9% identity and ~30% identity with other ADPG PPases. The recombinant His‐tagged and native proteins have been purified to homogeneity and found to be functionally identical. The glgC1 protein was inactive while the glgC2 protein displayed ~15 fold lower apparent affinity for substrates and Vmax compared to other ADPG PPases and was insensitive to typical effector molecules. In vitro formation of a glgC1/C2 complex resulted in a ~20‐fold stimulation of activity and 2.2‐fold activation by FBP. Co‐expression, crystallization studies and complete kinetic characterization of the complex are in progress. A comprehensive series of altered proteins have been generated and purified to homogeneity to target putative functional amino acids in glgC1 and glgC2. The kinetics analyses of the I24R and D244N glgC2 proteins revealed changes in allostery and activity, respectively. Characterization of other proteins harboring mutations implicated in catalysis and regulation, separately and in complex, is underway. Supported in part by NSF Grant 0448676.
Molecular Characterization of Thermus Thermophilus ADPGlucose Pyrophosphorylase
Authors: Gonzalez Jacob , Watanabe Go , Orry Andrew , Abagyan Ruben , Meyer Christopher R
Published: June 2021

ADPGlucose Pyrophosphorylase (ADPG PPase) catalyzes a key reaction in glucan synthesis and is regulated by allosteric metabolites. Little is known about thermophilic forms of this enzyme. We have successfully expressed and purified the recombinant Thermus thermophilus HB 27 enzyme and collected kinetic data at both 37°C and 75°C. In addition, three unique prolines in the sequence were identified compared to a related ADPG PPase, which may in part account for heat stability. Unexpectedly, the Vmax at 37°C was ~70% of that measured at 75°C. The S0.5 for ATP and Mg2+ in the absence of activators (G6P, FBP, and F6P) were in fair agreement with the results obtained at 75°C. The S0.5 for G1P in the absence of activators decreased ~5‐fold, which may account for the catalytic efficiency. The specific activity for CTP, GTP, UTP, and TTP were determined to be 0.6%, 0.3%, 0.15%, and 0.001%, respectively, of the activity with ATP as a substrate. Interestingly, the fold‐activation increased dramatically for CTP and UTP, to ~9 and ~30‐fold by FBP, respectively. The P100A, P122A, and P195A mutations were successfully generated by site‐directed mutagenesis. Preliminary studies suggest P122A has some effect on the activity of Th.t ADPG PPase. Detailed heat stability tests as well as probing the effects of double and triple proline substitutions are in progress. Further, molecular modeling and in silico docking experiments have indicated a role for K43 in G6P binding and roles for P362, R79, and R306 in FBP binding. These and other sites are being probed by mutagenesis. Supported in part by NSF grant 0448676 and NIH MARC 2 T34‐GM008612‐09.
Studies on the Role of Subunit d for V‐ATPase Reversible Disassembly
Authors: Ediger Benjamin , Parra Karlett
Published: June 2021

V1V0‐ATPase proton pumps are multisubunit proteins consisting of integral and peripheral subunits regulated by reversible disassembly in response to the extracellular concentration of glucose; it is hypothesized that disassembly is an energy conserving strategy to preserve ATP due to limited glucose. In the absence of glucose, the peropheral sub‐complex (V1) is released into the cytoplasm and the membrane sub‐complex (Vo) remains integral. The disassembled enzyme is catalytically inactive but addition of glucose to glucose‐deprived cells yields reassembly of a functional holoenzyme. This project studies subunit d, a key subunit coupling ATP hydrolysis to proton transport. Subunit d remains bound to the membrane upon disassembly and interacts with different V1 and Vo subunits depending on the V‐ATPase's state of assembly. We used computational models of subunit d to introduce cysteine residues that will be photo‐chemically cross‐linked to neighboring V‐ATPase subunits using the sulfhydryl reagent MBP. This study will map subunit interactions involving subunit d in the assembled and disassembled states. It will generate new information regarding the roles of subunit d for V‐ATPase function and the mechanism of glucose‐dependent dissociation.

Supported by NSF
Mustard Gas‐Induced Alteration of Retinoid Metabolism/Signaling in Guinea Pig Lung
Authors: Roy Somdutta Sinha , Schäffer Michael W , Mukherjee Shyamali , Das Salil K
Published: June 2021

Mustard gas exposure causes inflammatory lung diseases, including Adult Respiratory Distress Syndrome (ARDS). Retinoids (retinoic acid, retinol, retinyl palmitate) are important for growth and differentiation of various cell types. Retinoids are known for their role in regulation of gene expression and as effective inhibitors of chemical carcinogenesis in epithelial tumors such as those of the skin, lung, breast etc. Various environmental toxicants are known to alter the delicate metabolic balance of these compounds in the body. HPLC method has been used for analysis of retinoids in a single run in guinea pig lung after exposure of CEES (2‐chloroethyl ethyl sulfide), a mustard analogue. Adult guinea pigs were intratracheally injected with CEES in ethanol. Control animals were injected with only ethanol. Animals were sacrificed and lungs removed after perfusion with physiological saline. Lung injury was established by measuring leakage of iodinated‐BSA into lung tissue. HPLC analysis of lungs showed that there was an increase in retinoic acid (RA) and a decrease in retinol and retinol esters due to CEES exposure. Expression of RA receptors (α, ß) and cellular retinoic acid binding protein was also increased. Further studies are being carried out to elucidate the functional consequence of the early increase in the level of RA by CEES exposure (Supported by US Army Grant W81XWH‐06‐2‐0044)
Effects of 2‐Chloroethyl Ethyl Sulfide (CEES), a Mustard Gas Analog, on Serotonin/Dopamine Signaling in Guinea Pig Brain
Authors: Gadsden Jessica L , Mukhopadhyay Sutapa , Schäffer Michael W , Roy Somdutta Sinha , Mukherjee Shyamali , Das Salil K
Published: June 2021

We reported earlier that exposure of guinea pigs to CEES, a mustard gas analog, resulted in body weight gain associated with a down‐regulation of the pro‐opiomelanocortin (POMC) gene. However, its mechanism is not clear. It is known that d‐fenfluramine, a serotonin (5‐HT) 2C receptor agonist and an appetite suppressant, causes activation of c‐fos of POMC neurons in the arcuate nucleus of the hypothalamus. POMC neurons in this area express 5‐HT2C receptor mRNA, suggesting that the 5‐HT2C receptor mediates fenfluramine's actions in this area. Serotonin has also been shown to regulate dopamine neurotransmission. Dopamine directs energy balance by exerting effects on food intake through the mesolimbic, meso‐hypothalamic, and nigro‐striatal brain circuitry. HPLC analysis showed a 50% reduction in the levels of serotonin and a small increase in the levels of dopamine in the midbrain due to CEES exposure. Western blot analysis indicated that CEES exposure had no effect on the protein levels of either D1 or D2 dopamine receptors in the frontal and midbrain area. However, the protein levels of D1 receptors were increased and protein levels of D2 receptors were decreased in the lateral brain. These results indicate that down‐regulation of POMC gene by CEES exposure may be mediated by its effect on serotonin/dopaminergic signaling. This project was supported by DOD grant W81XWH‐06‐2‐0044 and NIH grant 5T32HL007735‐12.
Immunological attempts to detect Panulirus argus virus 1 in spiny lobsters leading to the development of an ELISA for a high school science curriculum
Authors: Faulconer Emily Kaye , Ratzlaff Robert E.
Published: June 2021

Panulirus argus virus 1 (PaV1) is pathogenic to the Caribbean spiny lobster and is lethal to the host 30–90 days after infection. The Caribbean spiny lobster, Panulirus argus, occurs throughout the Caribbean basin roughly from Brazil to Florida. In Florida, it constitutes the single most valuable fishery in the state. Our first objective was to develop an immunological detection assay for the virus. Western blots of rabbit anti‐sera against partially purified virus suggested the presence of antibodies against the virus. Upon further assessment by ELISA using purified PaV1, we determined that the anti‐sera contained mostly antibodies against host tissue antigens. Our second objective was to re‐design the ELISA to fit a high school science curriculum and capture students’ interest in immunology. We used conjugated protein‐A to detect mammalian blood in food. We hope to incorporate the results from our lobster virus efforts to demonstrate how an ELISA might be applied in the study of a new disease. This assay meets classroom safety concerns, can be read visually, and teaches basic immunologic principles. This unit enhances student understanding of immunology by using strategies such as inquiry‐based learning and writing across the curriculum. Funded by The John H. Wallace 2007–2008 Summer Teacher AAI Research Fellowship and the National Science Foundation, Biological Oceanography Program, Award #0452383.
G proteins associated with CXCR5 expressed by prostate cancer cells
Authors: El Haibi Christelle P , Singh Shailesh , Sharma Praveen K. , Lillard James W.
Published: June 2021

Metastasis to bone remains a major clinical concern and the main cause of prostate cancer (PCa) morbidity and mortality. The reasons for preferential establishment and growth of disseminated PCa cells in bone marrow are incompletely understood and require further investigation. We have recently shown that PCa cell lines differentially express CXCR5, and in vivo CXCR5 expression positively correlates with prostate tumor staging, as determined by histological staining of surgical specimens and tissue microarrays. By virtue of its presence in the bone microenvironment, CXCL13 (the ligand for CXCR5) may play a critical role in PCa cell migration and invasion, when compared to normal prostatic epithelial cells. Like other chemokine receptors, CXCR5 is a cell surface G‐protein coupled receptor. We have delineated some events triggered by G‐protein activation through CXCL13 binding to CXCR5 expressed by PCa cell lines derived from bone, brain, and lymph node metastases. G‐protein isoforms expressed by these PCa cell lines were evaluated by Western Blot, while tandem mass spectrometry analysis was used to elucidate the proteins that associate with CXCR5 before and after CXCL13 interaction. We show that PCa cells differentially express G‐protein isoforms to presumably modulate PCa progression in part through CXCR5 activation. This research is supported by USAMRMC (Award # W81XWH‐06‐01‐9562).
Cellular expression and antimicrobial function of a phylogenetically conserved novel histone 1x‐like protein
Authors: Jaso‐Friedmann Liliana , Connor Meghan A. , Moss Lauren D. , Lackay Sarah N. , Leary John H. , Evans Donald L.
Published: June 2021

Soluble linker and core histone‐like proteins isolated from many different types of mucosal and lymphoreticular tissues have antimicrobial activity. Previous reports by others have shown that granule extracts from RAW 264.7 cells and neutrophil extracellular traps contain H1 that may function in bacterial killing. We have previously identified an H1x‐like protein (i.e. NCC cationic antimicrobial protein‐s/ncamp‐1) from fish NK‐like cells (nonspecific cytotoxic cells/NCC) and prepared polyclonal and monoclonal antibodies to this membrane protein. In the present study we identify the (ICR) mouse ncamp‐1 equivalent using polyclonal as well as an anti‐ncamp‐1 monoclonal antibody (mab 9C9). Polychromatic flow cytometry revealed mab 9C9 expression on PBL (Gr‐1+ 21.1% ncamp‐1; DX‐5+ 12.2%+); mesenteric lymph node (CD11c = 42.3% ncamp‐1, Gr‐1 = 24.6%; CD21 = 33.3%); and splenocytes had approximately the same distribution and percentages of ncamp‐1+cells as the LN. Granule extracts were prepared from RAW 264.7 cells and analyzed by Western blots. A 29‐32 kDa signal was produced. Granule extracts were also prepared for an antimicrobial assay. A significant reduction in E. coli CFU was produced by a RAW 264.7 granule extract. The specificity of bacterial killing by this extract was determined by pretreatment of the extract with polyclonal anti‐ncamp‐1. This produced a significant reduction in killing. Pretreatment with pre‐immune serum did not reduce bacterial cell killing by the extract. These studies provided preliminary data that a potential novel class of pattern recognition receptor is phylogenetically conserved and exists on mouse lymphoreticular cells and in macrophage granules. Research supported by USDA award # 2006‐35204‐16562.
Peer education, exercising and eating right (PEER): determining changes in student self‐efficacy and outcome expectations in a teaching partnership
Authors: Khan Naiman , Nasti Chris , Evans Ellen , Chapman‐Novakofski Karen
Published: June 2021

This undergraduate/faculty teaching partnership was a 1 semester feasibility study to support undergraduate course development. Peers (n=6) led weekly discussions with the class (N=39); faculty/GAs conducted lectures. Student (n=39) nutrition self‐efficacy improved when asked if they could resist eating if they had to say no to others (p=0.026), when physically run down (p=0.024), and during depression (p=0.006). Nutrition outcome expectations improved when asked if eating ≥ 5 servings of vegetables/fruits would increase energy (p=0.036), less likely to not enjoy eating (p=0.008), less likely to get cancer/CHD (p=0.031), and being good examples for others (p=0.011). Students expected to have more energy (p=0.01), a desirable weight (p=0.038), less likely not to enjoy eating (p=0.003). Students improved intake of fruits/day (p=0.037), reduced intake of regular salad dressing (p=0.006), watched less TV (p=0.044); there was an increase in shopping/preparing their own food (p=0.044). There were no significant improvements in the outcome expectations/self‐efficacy in exercise behavior. However, there was improvement in amount of strenuous (p=0.032) and moderate (p=0.003) exercise/week. Results show that a faculty‐peer collaboration is not only feasible but can also result in significant nutrition/exercise changes in undergraduate students. Supported by USDA.
Biotin‐dependent cell signaling networks in mouse embryos
Authors: Melendez Rocio Rodriguez , White Brett , Zempleni Janos
Published: June 2021

Marginal biotin deficiency develops in 50% of pregnant women. Studies in mice showed that biotin deficiency may cause fetal malformations, including cleft palate and abnormal skeletal development. Here we used DNA microarrays and computational biology approaches to identify biotin‐dependent cell signaling networks that play roles in causing these malformations. BALB/c mice were fed a biotin‐free diet; controls were fed a normal diet. Hyperovulation was induced by hormonal stimulation; 24 h after stimulation oocytes were collected. RNA was reverse transcribed, PCR amplified, fragmented, labeled and hybridized to the Affymetrix Genome 430 2.0 array. Gene expression was analyzed using the IngenuityTM software. Selected genes were validated by real time PCR. The expression of 394 and 998 genes decreased and increased, respectively, in biotin‐deficient oocytes compared with controls. Biotin‐dependent genes clustered in pathways known to play roles in fetal development, e.g., cell cycle control, gene regulation, and cell death. Two signaling networks have been selected for an in‐depth analysis and are currently investigated by using western blot analysis, reporter‐gene constructs, and ChIP assays: Akt and MYCN. Support: University of Nebraska ARD, Hatch Act; NIH DK063945 and ES015206, USDA 2006‐35200‐17138, and NSF EPSCoR EPS‐0701892.
Major parameters of zinc (Zn) homeostasis in adult women with a wide range of habitual zinc intake
Authors: Sheng Xiao‐Yang , Hambidge K Michael , Miller Leland V , Bailey Karl B , Gibson Rosalind S , Westcott Jamie E , Lei Sian , Grunwald Gary , Krebs Nancy F
Published: June 2021

A clear understanding of optimal public health strategies to prevent human Zn deficiency depends on improved understanding of human Zn homeostasis. Adequate Zn nutrition is of special importance in all women of child‐bearing age. The objective of this study was to measure major parameters of Zn homeostasis in healthy young free‐living adult women with a wide range of total dietary Zn (TDZ, mg Zn/d) and phytate (TDP) based on habitual intake. Methodology included administration of two Zn stable isotopes (67Zn & 68Zn) intravenously at different times to measure fractional absorption of Zn (FAZ) and endogenous fecal Zn (EFZ, mg Zn/d). A third tracer, 70Zn, and dysprosium were administered orally with all meals for 6 consecutive days to 24 healthy women. Zn and phytate were measured in duplicate meals, and Zn & Zn isotope ratios in feces and urine. Mean ± SD results included: TDZ 11.7 ± 7.2 mg Zn/d (5.5 – 29 mg Zn/d); FAZ 0.30 ± 0.10; total absorbed Zn (TAZ) 3.0 ± 1.1 mg Zn/d; EFZ 2.7 ± 0.8 mg Zn/d. The slope of linear regression of endogenous zinc in feces + urine vs TAZ = 0.31, r2 = 0.15, p = 0.081. Fitting TAZ data with a saturation response model gave an Amax = 5.5 mg Zn/d, r2 = 0.60. Calculations from these factorial data were consistent with current EAR for Zn for adult women.

Supported by USDA 2003‐00900; GCRC M01RR00051; MO1 RR00069‐41; Science & Technology Commission, Shanghai Municipality 03JC14042.
Fractional Zn absorption (FZA) and total absorbed Zn (TAZ) in elderly are lower than values used to derive the adult Zn Estimated Average Requirement (EAR)
Authors: Chung Carolyn Sue , Montelius Lynn , Nguyen Tuan Q , Gildengorin Ginny , Dare Doris , King Janet
Published: June 2021

Zn intake and metabolism are altered in elderly individuals, yet dietary requirements have not been studied for this group. We measured FZA in response to mixed food diets containing approximately the Zn EAR and the EAR ± 40% consumed for 2 wks in 17 healthy elderly, 10 women and 7 men. All subjects consumed the 3 diets. 70Zn and 67Zn tracers were given orally and intravenously on d8 and 9 of each 2‐week diet period, respectively. Urine collected 3–5 d after infusion was analyzed by ICPMS for 67:66 Zn ratios and by ICP for total urinary and plasma Zn. FZA was determined using tracer/tracee ratios and averaged from d 3–5. TAZ equaled the total dietary zinc intake multiplied by FZA. FZA differed across all diet groups (p<0.0001, ANCOVA), regardless of age or sex. Conversely, TAZ did not vary by diet group, nor did plasma and urinary Zn. At EAR intakes, mean FZA adjusted for age was 29 ± 6.1 and 37 ± 2.9 % in men and women, respectively, and was 40 and 10% lower than absorption values used to determine EARs (48 and 41%). Mean TAZ was 2.99 ± 0.48 and 2.73 ± 0.19 mg/d in men and women, respectively, and was 21 and 17% lower than absorbed Zn values used for EARs (3.8 and 3.3 mg/d). The CV for FZA and TAZ in the elderly was 31% compared to the assumed 10% used to estimate the RDA. The lower measured FZA for these healthy elderly individuals suggests that the current Zn EAR underestimates the average requirement. Supported by USDA NRI 2004‐01842 and NIH MO1‐RR00083‐43.
Neutrophil motion on P‐selectin/ICAM‐1 in an in vitro model of a lung capillary
Authors: Tees David , Sundd Prithu , Goetz Douglas J
Published: June 2021

Leukocyte sequestration in lung capillaries is a key step in the inflammatory response to lung infection. P‐selectin and ICAM‐1 have well defined roles in leukocyte adhesion in systemic venules but their role in pulmonary capillaries is still unclear. Here, a novel in vitro Micropipette Cell Adhesion Assay (MCAA) used P‐selectin, ICAM‐1 or BSA coated capillary‐sized glass microvessels as an in vitro model for a pulmonary capillary. Leukocytes were aspirated into these in vitro capillaries and velocities were determined under pressures representative of lung capillaries. Neutrophil velocities in MCAA were significantly lower on P‐selectin than BSA and decreased with increasing P‐selectin concentration. Pre‐treating P‐selectin coated microvessels with an anti‐P‐selectin mAb resulted in a small but significant increase in velocity. ICAM‐1, expressed alone or with P‐selectin, does not mediate adhesion of unstimulated neutrophils in pulmonary capillary geometry. These results demonstrate that P‐selectin at low density mediates leukocyte adhesion in the pulmonary capillary geometry and that this adhesion is difficult to block with mAbs in this geometry. This work was supported by grant BES‐0547165 from NSF.
Charge effects of the blood‐brain barrier on the transport of charged molecules
Authors: Fu Bingmei M , Li Guanglei , Yuan Wei
Published: June 2021

The endothelial surface glycocalyx layer and the basement membrane (BM) are two important components of the blood‐brain barrier (BBB). They provide large resistance to solute transport across the BBB in addition to the tight junctions in the cleft between endothelial cells. Due to their glycosaminoglycan compositions, they are believed to carry negative charge. To quantify their charge effects, we used quantitative fluorescence imaging microscopy to measure the permeability (P) of the BBB to two similar sized globular proteins, ribonuclease (net charge=+3) and α‐lactalbumin (net charge=−11). The mean Pribonucleasewas 6.2 × 10−7 cm/s (n=11), which is ~4 times of Pα‐lactalbumin. To determine the charge densities in the glycocalyx layer and in the BM, we developed a mathematical model for the transport of charged molecules across the BBB. In this model, constant charge densities are assumed in the surface glycocalyx layer and in the BM. At their interfaces with non‐charge regions of the BBB, the electrostatic and steric exclusions to charged molecules are considered. Our model predicts that to account for the 4‐folds difference in measured Pribonuclease and Pα‐lactalbumin, the charge density in both the surface glycocalyx and the BM would be ~25 meq/l. This work is supported in part by the Andrew Grove Foundation and NSF.
The effects of alcohol binge on vessel reactivity
Authors: Molina Miguel Federico , McDonough Kathleen , Giamio Betsy , Mathis Keisa
Published: June 2021

Alcohol (ALC) consumption is associated with alterations in blood pressure and changes in vascular function. Chronic and excessive ALC consumption increases the risk for hypertension and stroke, while moderate daily intake has been shown to reduce incidence of coronary artery disease leading to a reduction in the risk of myocardial infarction. The underlying mechanism has been suggested to be altered endothelial function, with moderate ALC consumption enhancing endothelial function, while chronic excessive consumption impairs function. Previous studies have shown that acute ALC intoxication (AAI) decreases blood pressure, enhances hypotension following hemorrhagic shock, and impairs pressor response to fluid resuscitation. This study examined the effects of AAI on blood vessel reactivity to phenylephrine (PE), acetylcholine (Ach), and nitroprusside (NP) ex‐vivo. Chronically instrumented, conscious male Sprague‐Dawley rats (300–350 g) received a primed continuous 15 hour intragastric ALC infusion (1.75g/kg + 300 mg/kg/hr). Thirty minutes after discontinuing ALC, animals were sacrificed for isolation of thoracic aorta and mesenteric arteries. Aortic and mesenteric ring segments (1–2 mm) were suspended in myograph baths containing Krebs‐Henseleit bicarbonate buffer, pH 7.4, gassed with 95% O2: 5% CO2. Arterial rings from AAI rats had decreased PE‐induced tension (aorta: 2.28 ± 0.09 vs. 2.6 ± 0.13 g; mesenteric artery: 1.70 ± 0.06 vs. 1.91 ± 0.10), greater (21%) Ach‐mediated relaxation and similar NP‐mediated relaxation. These results indicate that AAI favors vasodilatation and are consistent with enhanced endothelial dilator function. (Supported by DOD PR‐054196).
Differences in Ito and myofilament protein expression may underlie transmurally‐varying electromechanics in the canine left ventricle
Authors: Campbell Stuart G. , Flaim Sarah N. , Leem Chae Hun , McCulloch Andrew D.
Published: June 2021

Myocytes isolated from the endocardial, midmyocardial, and epicardial regions of the canine left ventricle (LV) possess distinct action potential morphologies, Ca2+ transients (CaTs), and timecourses of unloaded shortening. Some of these differences have been linked to variable expression of Ca2+‐handling and ion channel proteins. We used a computational model of myocyte electromechanics to test the hypothesis that identified variations in protein expression account for observed differences in mechanical function between epicardial (EPI), midmyocardial (MID), and endocardial (ENDO) cells. The ENDO cell model displayed a greater delay to onset of shortening compared with EPI and MID cells, in agreement with experiments. Time to onset of cell shortening was strongly affected by the magnitude of the transient outward K+ current (Ito). Simulated shortening in response to experimentally measured ENDO and MID CaTs agreed well with experiments, while responses to the EPI CaT did not. Agreement between measured and simulated EPI shortening was improved by increasing the rate of crossbridge cycling. These results suggest important roles for Ito and altered crossbridge kinetics in modulating electromechanical function within the canine LV. Supported by NSF grant BES‐0506252 and NIH grant P41 RR‐08605.