bluuebunny/crossref_metadata_2025_split huggingface dataset:
The neurobiology of predation: sensory adaptations to carnivory (89.3)
Authors: Sarko Diana , Leitch Duncan , Catania Ken 
Published: June 2021

Study objective: We investigated the central and peripheral nervous systems of northern grasshopper mice (Onychomys leucogaster), the most highly carnivorous rodents in North America, in order to explore potential neural correlates of the transition to carnivory.Methods: Multiunit electrophysiological recordings combined with analysis of flattened sections of neocortex processed for cytochrome oxidase were used to determine the topography of primary somatosensory cortex (S1) and the location and size of both the visual and auditory cortex. These findings were then related to flattened cortical sections of juvenile grasshopper mice labeled with the serotonin transporter (SERT) antibody. The number of myelinated axons in trigeminal, cochlear, and optic nerves were also quantified.Results: An enlarged forepaw representation was discovered within S1. Primary visual cortex (V1) also appeared to be larger in flattened cortex preparations compared to other rodents. Whereas the trigeminal and cochlear nerves were comparable to other rodents, the optic nerve contained a large number of axons.Conclusions: Grasshopper mice utilize a more specialized repertoire of attacks that rely on the forelimb, which perhaps corresponds to a magnification of this representation in S1 relative to other rodents. Enlargements of the optic nerve and V1 may also optimize processing of visual cues to enhance predatory success.Grant Funding Source: Supported by NIH grant # DE016061 and NSF grant # 0844743 to K.C.C.
Adaptive evolution of cis‐regulatory regions among genes dynamically expressed in the juvenile human cortex (89.8)
Authors: Nevell Lisa , Caravas Jason , Uddin Monica , Chugani Harry , Sterner Kirstin , Lipovich Leonard , Grossman Lawrence , Sherwood Chet , Kuzawa Chris , Wildman Derek 
Published: June 2021

The human brain excels in its capacity for phenotypic plasticity and response to experience. The objective of this study is to investigate the evolutionary history of human brain plasticity. We previously argued that genes that play a role in phenotypic plasticity should respond with a dynamic expression pattern during development, and we reported a set of 304 genes more variably expressed in the juvenile human cortex than adult (Sterner et al., 2012). Changes in how these genes are regulated among humans compared with non‐human primates may provide clues as to the genetic underpinnings of enhanced phenotypic plasticity in the developing human brain. Using publically available genomic data, our approach identified single nucleotides that are differ in humans (e.g. 1000 genomes project) compared to non‐human primates in regions that contain regulatory sequence (i.e., 2kb upstream, first intron, 5’‐ and 3’‐UTR). Thirty‐one out of 304 genes are enriched for regulatory sites that are derived and fixed in humans and also show dynamic expression in the juvenile human cortex (e.g., WAS, KLF4, C1QA, CTSH, PRODH, SERTAD1). Changes in the regulatory responses of these genes may contribute to the evolution of unique aspects of human brain development and plasticity.Grant Funding Source: Supported by NSF [BCS0827546], AAA Post‐doctoral Research Fellowship, and WSU Office VP Research
Development of a fluorescent bar‐coding system for cell‐based proteomic libraries (912.13)
Authors: Ritter Seth , Berges Stefanie , Colby David 
Published: June 2021

A major objective for biological scientists is to understand the complex intricacies of cellular maintenance, and environmental response. Proteins constitute a significant fraction of the molecules in biological systems under study; however the tools for proteomic analysis fall short of those that have enabled the thorough cataloging of genes and the quantification of gene transcripts. It is therefore beneficial to produce a technology for high‐throughput, quantitative analysis of the proteome. To this aim we are developing a novel heritable surface protein construct. Such surface proteins will protrude out and operate as scaffolds for fluorescently labeled antibodies, or other high‐affinity reagents. By varying the number of repeating epitope elements in the scaffold a series of fluorescent signals through multiple channels and levels of intensity are generated. These Unique Identifying Proteins (UIP), allow for identification of multiple cell populations simultaneously using multidimensional flow cytometry. We found that eight unique barcodes were possible utilizing three total epitopes, where in any channel at least 60% of cells of a population could be taken while error rates in population identification remained below 5%. This system will enable the coupling of population identification and various fluorescent readouts of interests, offering insight into population statistics as well as single cell analysis.Grant Funding Source: Supported by a grant from the National Science Foundation
Determining the role(s) of prime‐side residues in macromolecular inhibition of trypsin‐fold serine proteases (933.2)
Authors: St Germain Commodore , Batt Anna , Baird Teaster 
Published: June 2021

Trypsin‐fold serine proteases are among the most abundant of proteases. By identifying residues that are important for enzyme‐inhibitor interactions in trypsin, we may advance the development of protease‐based therapies in general. Lysine‐60 (K60) is a highly conserved prime‐side residue in trypsin‐fold serine proteases. In trypsin‐inhibitor co‐crystals, K60 hydrogen bonds to tyrosine‐39 (Y39) and is positioned to restrict conformational mobility of phenylalanine‐41 (F41), possibly optimizing the observed hydrogen bond interactions between inhibitors, Y39 and F41. Substitution of K60 with other residues may disrupt these interactions and provide insight to their significance with respect to inhibitor binding. To test this hypothesis, we created trypsin variants K60G, K60A, K60V, K60I, K60M, K60R, Y39S, and Y39F and characterized them with respect to their activities and sensitivities to macromolecular inhibitors soybean trypsin inhibitor (SBTI), bovine pancreatic trypsin inhibitor (BPTI) and ecotin, an inhibitor expressed in E. coli. Our initial results show that, compared to wild‐type trypsin, all variants maintain similar catalytic activity, but‐K60A, K60V, K60I, K60M, and Y39F are more sensitive to inhibition while variants K60R and Y39S are less sensitive to inhibition. Collectively, these results suggest that K60 and Y39 do indeed play significant roles in inhibitor binding.Grant Funding Source: This work was supported by NSF
The forward translocation position of Escherichia coli RNA polymerase is stabilized by KCl and Mg2+ and is strongly dependent on a bridge helix N‐terminal hinge beta’ 778‐GARKGL‐783 (944.2)
Authors: Nedialkov Yuri , Assaf Fadi , Opron Kristopher , Burton Zachary 
Published: June 2021

Exonuclease (exo) III was used to probe the Escherichia coli RNA polymerase (RNAP) ternary elongation complex (TEC) downstream and upstream borders. In the absence of NTPs, RNAP appears to stall primarily in a post‐translocated state and to return slowly to a pre‐translocated state. Exo III mapping, therefore, appears inconsistent with an unrestrained thermal ratchet model for translocation. The forward translocation state is made more stable by elevating the KCl and Mg2+ concentrations into the physiological range (150 versus 40 mM KCl; 5 versus 0.5 mM Mg2+), indicating that, under natural conditions, translocation tends to stick in the post‐translocated position. RNAP mutants in the N‐terminal bridge helix hinge beta’ 778‐GARKGL‐783 strongly affect TEC translocation, NTP binding and stability. Mutations that stiffen the hinge (i.e. GARKGL‐>AARKAL) are strongly defective for holding the forward translocation register and for stable NTP binding but do not destabilize the TEC. Reminiscent of a ball and socket joint, the bulky R780 is surrounded by the beta fork, when the bridge helix is in a relatively straight conformation. An overly flexible hinge (i.e. K781A), however, may result in misaligning R780 within the fork resulting in low transcriptional activity. Molecular dynamics simulation indicates that the GARKGL hinge can completely unwind, but only if the beta’ trigger loop is in an open conformation.Grant Funding Source: NSF and NIH
Defining interaction interfaces important for the establishment of cell polarity in Drosophila neuroblasts (948.8)
Authors: Novak Alexander , Mauser Jonathon , Prehoda Kenneth 
Published: June 2021

Asymmetric cell division leads to the differentiation of daughter cells by aligning the mitotic spindle to polarized fate determinant cortical domains during mitosis. In Drosophila neuroblasts, cell polarity is established through the Par complex protein Bazooka (Baz aka Par‐3) binding to Inscuteable (Insc), while proper mitotic spindle orientation is established through Partner of Inscuteable (Pins) and the G protein, Gai. Loss of any component leads to loss of polarity and subsequent lethality in the developing fly. Although these interactions are fundamentally important, the physical nature of Insc’s interactions with its binding partners is poorly understood. To explore this idea further, we asked: What are the interaction interfaces between Insc and its binding partners? We identified and characterized the minimal interaction interface between Insc, Baz, Pins, and Gαi by MBP affinity chromatography using different constructs of Insc and assaying for binding of the partners. Our study of Insc has revealed that Pins/Insc binding is conserved with their mammalian orthologs using a motif within the ankyrin repeat 1 ‐ 2 region. Baz/Insc binding requires folded Insc ankyrin repeats 2 ‐ 3 plus one adjacent ankyrin repeat. Gαi binds to Insc in a GoLoco‐like manner in the ankyrin 3 ‐ 4 linker. We find that Insc competes with GoLocos for Gai binding. We also find Insc/Gai binding to be nucleotide specific. Using this data we will generate point mutants, which disrupt the individual pathways and look for phenotypes in the developing fly.Grant Funding Source: Supported by NSF REU Site Program in Molecular Biosciences ‐ BIO/DBI 1063144
Oxidized metabolites of linoleic acid mediated hepatotoxicity as a potential mechanism of dietary unsaturated fat and alcohol induced liver injury: in vivo and in vitro studies (959.20)
Authors: Liu Huilin , Ramsden Christopher , Feldstein Ariel , McClain Craig , Kirpich Irina 
Published: June 2021

Introduction: Dietary fat and alcohol both play an important role in the development and progression of alcoholic liver disease. The aim of the present study was to evaluate the role of linoleic acid (LA, a major unsaturated fatty acid in the Western diet), and its bioactive oxidized metabolites (OXLAMs) in alcohol‐induced liver injury. Materials and Methods: In‐vivo study: C57BL/6 mice were fed unsaturated (USF, corn oil enriched) fat diet containing 5% ethanol for 10 days plus a single binge ethanol administration. Liver injury and steatosis as well as 12/15 lipoxygenase and plasma OXLAM levels were evaluated. In‐vitro study: HepG2 cells were exposed to LA and multiple OXLAMs. Seahorse and Cellomics analysis were performed to evaluate effects of treatments on mitochondria function, ER stress, and cell survival. Results: Significant liver injury and steatosis associated with elevated plasma OXLAM levels and hepatic 12/15 lipoxygenase, an enzyme involved in OXLAM production, were observed in response to USF+EtOH. A dose‐dependent decrease in mitochondria oxygen consumption was observed in HepG2 cells in response to multiple OXLAMs. 9‐ and 13‐ HODEs increased cell membrane permeability and ER stress in a dose‐dependent manner, although cell survival was not affected. Conclusion: OXLAM mediated mitochondria dysfunction and ER stress may be a potential mechanism underlying USF+EtOH mediated liver injury.Grant Funding Source: Supported by NIH, DOD and Veterans Administration
Characterizing the effect of porcupine on neural tube closure (979.3)
Authors: Feeney Shea , Galli Lisa , Pay Gina , Burrus Laura 
Published: June 2021

Incomplete closure of the embryonic neural tube is responsible for common birth defects such as spina bifida. Wnts, a group of secreted lipid‐modified signaling proteins, are known to play a critical role in embryonic spinal cord development. Wnt proteins are palymitoylated by the enzyme Porcupine (Porcn), a transmembrane protein localized to the endoplasmic reticulum and Golgi apparatus. The proposed function of palmitoyation is to facilitate the secretion of Wnt proteins. Mutations in human Porcn can result in Focal Dermal Hypoplasia that can manifest multiple abnormalities including neural tube defects. Thus it is hypothesized that inhibition of Porcn in early stage chick embryos will cause abnormal neural tube closure. To characterize Porcn's role in neural tube closure, we developed an in vitro culture method that permitted addition of small molecule inhibitors of Porcn IWP‐1, IWP‐12 and C‐59. Surprisingly, although all of the compounds tested inhibited human Porcn, only IWP‐12 appeared to inhibit chick Porcn. Treatment with IWP‐12 revealed neural tube defects and decreased cell elongation which is consistent with a model in which IWP‐12 inhibits Wnt signaling via the planar cell polarity pathway. To test whether IWP‐1 and C‐59 are species specific inhibitors, we are currently comparing the ability of all three compounds to inhibit human and chick Porcn using an assay that measures Wnt palmitoylation.Grant Funding Source: Supported by NSF RUI MCB‐1244602, NIH‐RIMI P20MD000262, Area Grant NIH #1R15HD070206‐01A1
ABC transporters and uptake in Haemophilus influenzae (997.6)
Authors: Pinkett Heather 
Published: June 2021

Bacterial ATP‐Binding Cassette (ABC) importers typically select and transport a single substrate or a small group of similar compounds. The current mechanistic model for how substrates are shuttled across the membrane is based on a small group of well‐characterized bacterial transport systems. While there are many overall similarities among transporters, recent research has indicated that there are fundamental differences the two major types of bacterial transporters. Key structural and mechanistic differences amongst ABC transporters suggest that the two classes of transporters initiate the transport cycle utilizing a different mechanism. Since an organism can have both a type I and type II transporter transport the same substrate, understanding the transport process of type II transporters will allow us to decipher how both transport systems work in concert to transport necessary substrates, fundamental to understanding the organism’s physiology. Structural and biochemical studies of type II transporter, MolAB2C2 from H. influenzae have revealed duplicate transport system in a single organism. The results of our work will close critical gaps in the understanding the fundamentals of the transport mechanism in bacterial pathogens, and will yield insights into how type II transporters utilize ATP hydrolysis to coordinate transport across all organisms.Grant Funding Source: NSF
Thermodynamic examination of the ion effect and cellular crowding on nucleic acids (LB210)
Authors: Comfort Hallie , Grover Neena 
Published: June 2021

Ions play essential roles in forming functional RNA structures and can act as nucleophiles in RNA catalytic reactions. Under physiological conditions, folding of RNA requires potassium and magnesium ions. Nearly 30% of the cellular volume is occupied by ions and macromolecules. Cellular crowding affects the stability of nucleic acids by changing the availability of ions and water (excluded volume effect). To mimic cellular conditions, polyethylene glycol (PEG) is used as a “crowder.” RNA folding experiments on Group I introns show formation of more compact RNA structures at lower concentrations of magnesium ions in the presence of PEG. In this study, we are using the pyrophosphate sensor helix of the thiamine pyrophosphate (TPP)‐ binding riboswitch to examine the ion and crowding effects on small RNA. The stability of multiple bulge and helix constructs was studied using thermal denaturation experiments in varying ion and PEG conditions. Different cations, anions, and PEG concentrations influence small DNA and RNA stability. Comparative thermodynamic data from these experiments will be presented.Grant Funding Source: This work was supported by NSF grant, MCB‐0950582 to NG.
Cellular geometry regulates traction stresses (LB228)
Authors: Oakes Patrick , Banerjee Shiladitya , Marchetti M. Cristina , Gardel Margaret 
Published: June 2021

Cells generate mechanical stresses via the action of myosin motors on the actin cytoskeleton. While the molecular origin of force generation is well understood, we currently lack an understanding of the regulation of force transmission at cellular length scales. Here we experimentally decouple the effects of substrate stiffness, focal adhesion density and cell morphology to show that the total amount of work a cell does against the substrate to which it is adhered is regulated by the cell spread area alone. Surprisingly, the number of focal adhesions and the substrate stiffness have little effect on regulating the work done on the substrate by the cell. For a given spread area, the local curvature along the cell edge regulates the distribution and magnitude of traction stresses to maintain a constant strain energy. A physical model of the adherent cell as a contractile gel under a uniform boundary tension and mechanically coupled to an elastic substrate quantitatively captures the spatial distribution and magnitude of traction stresses. With a single choice of parameters, this model accurately predicts the cell's mechanical output as a function of cell geometry.Grant Funding Source: Supported by the NSF, NIH and the Burroughs Wellcome Fund.
Concerted Loop Motion Triggers Induced Fit of FepA to Ferric Enterobactin (LB231)
Authors: Smallwood Chuck , Jordan Lorne , Trinh Vy , Schuerch Daniel , Gala Amparo , Hanson Mathew , Shipelskiy Yan , Majumbdar Aritri , Newton Salete , Klebba Phillip 
Published: June 2021

By spectroscopic analyses of extrinsic fluorophore_labeled site‐directed Cys residues in 7 surface loops of Escherichia coli FepA, we characterized the binding and transport of ferric enterobactin (FeEnt). Changes in fluorescence emissions reflected conformational motion of loops that altered the environment of the fluorophore, and we observed these dynamics as quenching phenomena during FeEnt binding and transport in living cells or outer membrane vesicles. Cys residues in each of the 7 surface loops (L2, L3, L4, L5, L7 L8, and L11) behaved individually and characteristically with regard to both fluorophore maleimide reactivity and conformational motion. During FeEnt binding the loops closed around FeEnt in approximately one second, in the order L11 > L3 > L2 > L7 > L8 > L5 > L4. This sequence created a hierarchy that correlated with the structure of FepA: L11, L3 and L2 sit opposite L7, L8, L5 and L4 across the outer vestibule, suggesting that the loops bind the metal complex similar to two hands clasping around an object. Although L3 followed a distinctly biphasic exponential decay during FeEnt binding, fluorescence from the other loops followed simpler, single exponential decay processes. Fluorescence measurements of FeEnt transport, by either microscopic or spectroscopic methodologies, demonstrated that ligand uptake occurs uniformly throughout the cell envelope, and susceptibility of FeEnt uptake to the proton ionophore m‐chlorophenyl hydrazone (CCCP) at concentrations as low as 5 uM. The latter result recapitulates the sensitivity of inner membrane major facilitator transporters to CCCP (Kaback, 1974), providing further evidence of the electrochemical gradient as a driving force for TonB‐dependent metal transport.Grant Funding Source: Supported by National Institutes of Health Grant GM53836 and National Science Foundation Grant MCB09522999
Mapping Critical Residues in the Epithelial Sodium Channel (LB292)
Authors: Hayford Corey , Stewart‐Ebert Lauren , Booth Rachell 
Published: June 2021

The epithelial sodium channel (ENaC) is responsible for sodium re‐absorption in the kidneys, lungs, and other epithelial tissues, which contributes to overall fluid homeostasis. While the intracellular termini of each of the three ENaC subunits (α, β, and γ) are necessary for insertion and retrieval of functional ENaCs, the role of the extracellular loop region is rudimentary. Expression of functional ENaC in yeast cells induces a salt sensitive phenotype, which was used to screen mutant ENaCs for function. In an effort to identify critical residues in the extra‐cellular region, error‐prone polymerase chain reaction (EP‐PCR) was used to generate random mutations within this region. Twelve mutant αENaCs were expressed in yeast and screened in a yeast pronging assay for function. Three αENaC mutants (CH3, CH5, and CH7) demonstrated no salt sensitivity (i.e. loss of functional ENaC). Expression of ENaC mutants in yeast lacking salt sensitivity was verified by western blot and mutant ENaC DNA was sequenced. Further characterization of these three mutant ENaCs is currently underway.Grant Funding Source: Supported by NIH R15 GM86798 (R. Booth & W. David), Welch Foundation Departmental Grant AI‐0045, NSF CHE‐1156579, and Research Corporation CC6531 (R. Booth)
Lipopolysaccharide Binding Protein is Associated with Diet Composition and Metabolic Markers in Obese Adults (LB340)
Authors: Clifford Kerry , Piazza Bryana , Keshavarzian Ali , Tangney Christy , Coltman Anne , Hartney Chris , Shawron Krista , Raeisi Shohreh , Forsyth Christopher , Hutkins Robert , Krumbeck Janina , Rasmussen Heather 
Published: June 2021

Intestinal microbiota is modulated by dietary intake and can have a profound impact on systemic health through initiation of systemic inflammation [via bacterial products like lipopolysaccharide (endotoxin)] and promotion of inflammation associated disorders like metabolic syndrome. Whether specific dietary components are related to markers of gut and systemic health, in particular, lipopolysaccharide binding protein or LBP (low level is a measure of systemic endotoxin exposure) is currently unknown. Bloods acquired from 32 healthy, obese (BMI, mean ±SD; 36.6 ± 6.1 kg/m2) subjects were analyzed for metabolic markers and LBP concentrations; three‐day food records were also completed. Median (IQR) age was 51 (40, 55), 56% were African American, 78 % were female, and 37% had metabolic syndrome. Subjects had a mean waist circumference of 116.5 ± 13.4 cm and systolic/diastolic blood pressures of 132.3 ± 15.6/78.8 ± 13.3 mm Hg. Median (IQR) LBP was 10.3 μg/ml (6.3, 23.3). Mean ± SD daily energy intake was 2,112 ± 636 kcal, saturated fat (g) 29.8 ± 97.3 (12.8 ± 3.3% of energy), mean fiber/1000 kcal (g) 8.4± 3.0, and median sugar intake (IQR) 78g (49,100). There was an inverse correlation between LBP and the following parameters: systolic blood pressure (r=‐0.41, p=0.04), saturated fat (g) (r=‐0.49, p=0.01), and calcium (r=‐0.41, p=0.03). The inverse relationships observed between LBP and both saturated fat and calcium indicates that foods high in these dietary components, such as high fat diary, may impact systemic inflammation through a gut‐mediated mechanism.Grant Funding Source: USDA NIFA AFRI