bluuebunny/crossref_metadata_2025 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
Prostate Cancer Aggressiveness is Positively Associated with Oxidative Stress and Inversely Associated with Plasma Total Antioxidant Capacity (LB371)
Authors: Vance Terrence M. , Lee Sang Gil , Azabdaftari Gissou , Su L. Joseph , Fontham Elizabeth T. H. , Bensen Jeannette T. , Mohler James L. , Chen Ming‐Hui , Koo Sung I. , Chun Ock K.
Published: June 2021

Prostate cancer (PCa) is the most common non‐cutaneous cancer in men in the US. Although the causes for PCa have yet to be determined, oxidative stress (OS) may be involved. In this study, urinary 8‐isoprostane and plasma total antioxidant capacity (TAC) were measured in a subsample of research subjects (n = 261 plasma, 279 urine) in the North Carolina‐Louisiana Prostate Cancer Project to determine the association between OS, TAC, and PCa aggressiveness. Compared with low and intermediate aggressive cases of PCa, high aggressive cases were found to have lower plasma TAC (mean: 366 vs 610 mM Trolox, n = 22 vs. 239; Wilcoxon rank test, P = 0.05) and higher urinary 8‐isoprostane levels (mean: 177 vs. 147 ng/mmol creatinine, n = 27 vs. 252; log transformed, t‐test, P < 0.05). Plasma TAC was negatively correlated with urinary 8‐isoprostane levels (ρ = ‐0.15; P < 0.05). In simple logistic regression models of high vs. low and intermediate aggressive cases, PCa aggressiveness was negatively associated with TAC, T3 vs. T1 OR=0.19 (95% CI, 0.04, 0.88; P < 0.05), and positively associated with urinary 8‐isoprostanes, T3 vs. T1 OR=3.16 (95% CI, 0.97, 10.30; P = 0.06). These findings suggest that the body’s compromised antioxidant defense and oxidative stress may contribute to PCa aggressiveness.

Acknowledgement: The authors thank the staff, advisory committees and research subjects participating in the North Carolina‐Louisiana Prostate Cancer Project (PCaP) study for their important contributions.

Grant Funding Source: Funded by NIH Cancer Epidemiology Small Grant #1R03CA159421‐01A1 and Department of Defense contract DAMD 17‐03‐2‐0052
Obesity is associated with increased red blood cell folate despite lower intake and serum levels of this vitamin among adults in the United States (LB411)
Authors: Bird Julia , Liu Zhenhua
Published: June 2021

Background: Folates are essential cofactors in various metabolic pathways. Limited evidence has investigated how obesity affects folate metabolism. We investigated the association between body mass index (BMI) and obesity‐related metabolic factors with blood folate status.

Methods: A representative study population of 3829 US adults was derived from the National Health and Nutrition Examination Survey (2003‐2006). Regression analyses were performed to examine the association between BMI and obesity‐related metabolic factors with serum folate and red blood cell (RBC) folate.

Results: Serum folate levels were lowest in both the underweight and obese groups, closely paralleling with the lower dietary folate intakes in these two groups. In contrast, RBC folate incrementally increased with BMI. Regression analyses demonstrated a negative relationship between BMI and serum folate, but a positive relationship for RBC folate. These relationships were significant for 19‐50 year old individuals, but not for elders (蠅 51 yrs).

Conclusion: Obesity is associated with decreased serum folate, and concurs with lower total intake among the obese. In contrast, obesity is positively associated with increased RBC folate. These relationships are significant for young and middle‐aged adults, but not for elders. RBC folate appears to be a better biomarker of cellular folate status in obese individuals.

Grant Funding Source: Supported in part by the USDA/NIFA grant (2014‐67017‐21762, ZL), the USDA Hatch grant (MAS00454, ZL) and the funding from Rays of Hope Center of Breast Cancer Research, Baystate Medical Center (ZL)
Household food security and consumption of a traditional medicinal tincture, sraa tenam, among Cambodian women in Massachusetts (LB480)
Authors: Cordeiro Lorraine , Peterman Jerusha
Published: June 2021

Background: Use of alcohol‐based tinctures is common during pregnancy and the postpartum period among Cambodians and other Southeast Asian populations. The consumption of alcohol‐based tinctures among Cambodians living in the U.S. has not been widely examined. This study investigates the association between household food security status and the consumption of a specific alcohol‐based medicinal tincture, sraa tenam, among Cambodian young women in Massachusetts.

Method: Cambodian women aged 15‐30 years (n=127) were surveyed in this cross‐sectional study. Household food security was assessed using the U.S. Household Food Security 6‐item module. Participants provided data on their knowledge, attitudes, and practices of alcohol‐based medicinal tinctures. Multivariate logistic regression tested the association between the consumption of sraa tenam, an alcohol‐based medicinal tincture, and household food security scores, after adjusting for age, country of birth, and insurance coverage.

Results: Approximately one‐fourth (25.6%) of the sample lived in low/very low food secure households. Forty‐two percent (42%) reported that they had consumed sraa tenam. Women with higher household food security scores were more likely to report consuming this alcohol‐based medicinal tincture compared to those with lower scores (β 0.423; 95% CI: 1.00, 2.33; p<0.05), after controlling for covariates.

Conclusion: Consumption of an alcohol‐based medicinal tincture, sraa tenam, was associated with household food security status among Cambodian women in Massachusetts. Household food insecurity status may be an indicator of low utilization of conventional health care, leading to greater use of traditional medicine as a coping mechanism. Alcohol‐based tinctures have both risks and benefits: the use of alcohol in tinctures may contribute to nutritional deficiencies, and yet, alcohol‐based fermented tinctures may also have potential health benefits with levels of alcohol that may be too low to pose a risk to maternal and infant health. The overall risks and benefits of alcohol‐based medicinal tinctures need to be carefully investigated to support optimal maternal and child health. Raising awareness of traditional practices among health practitioners that serve SE Asian populations may also be important.

Grant Funding Source: USDA
Targeting chemotherapy induced adaptive resistance using hybrid nanoparticles (LB577)
Authors: Kulkarni Ashish , Rao Poornima , Goldman Aaron , Sengupta Shiladitya
Published: June 2021

Chemotherapy is still a standard of care in adjuvant treatment of breast cancer, but it often leads to incomplete tumor response resulting in acquisition of chemoresistance and relapse, primarily due to upregulation of survival signals in tumor cells. To overcome this, chemotherapeutic agent and signaling inhibitor need to be delivered to the same cell sequentially with a precise spatial and temporal control. We designed a hybrid nanoparticle platform that synergistically integrates a cytotoxic agent with an ‘Oncogenic Signaling Inhibitor’ into a single nanovector thereby overcomes adaptive resistance to cytotoxics, increase efficacy & minimize significant off‐target toxicities. As traditional approaches for nanoformulation were incompatible with cytotoxic molecules such as Paclitaxel (PTX) and PI3K inhibitors (PI103), we rationally re‐engineered these molecules by conjugating it to cholesterol to enable supramolecular nanoassembly. Hybrid supramolecular nanoparticles were synthesized by lipid film hydration method. In vitro cytotoxicity studies in multiple cell lines including Breast Cancer (4T1, MDA‐MB‐231), Ovarian Cancer (4306) and Lung Cancer (A549, LLC) showed that hybrid nanoparticles were as effective as free drug combinations and more potent that individual drug nanoparticles. In both 4T1 breast cancer and in a K‐RasLSL/+/Ptenfl/fl ovarian cancer model in vivo, hybrid nanoparticles exerted superior antitumor efficacy as compared with free drugs & individual drug combination (p<0.01, one way ANOVA). Immunohistochemistry staining of tumor tissues for p‐AKT showed that the hybrid nanoparticles had significantly higher inhibition of p‐AKT as compared to individual drug nanoparticles (p<0.01, Student t‐test). These results posit supramolecular nanochemistry as a powerful approach that can potentially impact clinical translation of cytotoxic agents and PI3K inhibitors, and could be extended to enhance the efficacy of molecularly targeted therapeutics. Additionally, rational optimization of the chemical structures to facilitate supramolecular assembly can overcome existing limitations associated with parent molecules, thereby opening up possibility of resuscitating promising drug candidates that had failed to translate to clinics, rejuvenating drug pipelines.

Grant Funding Source: National Institute of Health, Department of Defense, BCRP Collaborative Innovator award
Sodium and Proton effects On Inward Proton Transport through Na/K Pumps (LB848)
Authors: Artigas Pablo , Mitchell Travis , Zugaramurdi Camila , Gatto Craig
Published: June 2021

The Na/K pump (NKA) exports 3Na in exchange for 2K, across animal plasma membranes. Ion binding sites I&II can bind Na or K but site‐III exclusively binds Na in a voltage (V)‐dependent fashion. Without external Na or K the NKA imports H generating a current (IH). To crack IH relationship to ion interaction at their sites we investigated the characteristics of IH in ventricular myocytes and in Xenopus oocytes expressing ouabain‐resistant pumps. Lowering pH enlarged IH in both systems. In oocytes at pH 蠄5.6 a rapid increase in IH was followed by a slower inhibition (80% at pH 5). H‐activation was V‐dependent whilst H‐inhibition was V‐independent. In myocytes at pH 6 Nao stimulated IH at [Na] 蠄5 mM and inhibited at higher [Na]. In oocytes, only V‐independent Nao‐dependent inhibition was observed at pH 6 or 7.6 (K0.5~7 mM), but at pH 5 Na induced a nearly identical biphasic response than in myocytes at pH 6. Affinity for Ko was reduced (~10 fold) by pH 5 compared to pH 7.6. Our results suggest that H+ leak through site‐III, that binding of 2Na or 2H to sites‐I&II inhibit permeation and that NKA with mixed Na/H occupancy of sites‐I&II are also permeable to H. Supported by NSF MCB‐1243842.

Grant Funding Source: NSF MCB‐1243842
Therapeutic Role of Ambrisentan for the Treatment of Hypoxic Pulmonary Vasoconstriction: Impact of Intrapulmonary Delivery
Authors: Ferguson Scott , Loomis Zoe , Harral Jules , Redinius Katherine , Dixon Kandice , Anderson Malcolm , Irwin David
Published: June 2021

Hypoxic pulmonary vasoconstriction (HPV) results in robust increases in pulmonary artery pressures (PAP), which underlie the development of pulmonary hypertension (PH), and potentially edema, in those that travel to or work at altitude. Endothelin‐1 receptor (ET‐1) antagonists are currently given orally to treat PH, however intrapulmonary delivery of this therapeutic may provide acute relief of the HPV response at a lower dose and thus could ameliorate the functional impairments caused by PH whilst minimizing the amount of drug required. Despite this potential, the efficacy of intrapulmonary ET‐1 antagonist therapy to treat hypoxia induced PH has yet to be determined. We tested the hypothesis that intrapulmonary delivery of the ET‐1 receptor antagonist Ambrisentan would attenuate an HPV mediated increase in PAP when compared to a saline control. Adult male Sprague‐Dawley rats were randomized into three groups to receive aerosolized saline (Saline, n=8), a low dose (ALD, 1 mg/kg, n=8) or high dose (AHD, 5 mg/kg, n=8) of Ambrisentan (administered via a microinhaler) following the induction of HPV (via 13% inspired O2, simulating ~4000m). PAP were monitored continuously throughout the procedure via indwelling catheters. Pre‐hypoxia PAP values were not different between groups (Saline: 18 ± 1, ALD 19 ± 1, AHD: 17 ± 1 mmHg, P>0.05). Following an initial hypoxia induced increase in PAP (Saline: 37 ± 1, ALD: 30 ± 2, AHD: 33 ± 2 mmHg, P<0.05 vs. pre‐hypoxia for all) intrapulmonary Ambrisentan reduced significantly PAP when compared to Saline (Saline: 34 ± 1, ALD: 23 ± 1, AHD, 23 ±1 mmHg, P<0.05) with no significant differences between ALD and AHD (P>0.05). Intrapulmonary delivery of the ET‐1 receptor antagonist Ambrisentan reduced significantly the increase in PAP following exposure to hypoxia with no significant differences between low and high doses. These results suggest that intrapulmonary delivery of Ambrisentan, at a dose approximating 1/5th of that given orally, may be an effective therapeutic for the treatment of hypoxia induced PH. Given the life threatening complications and impaired work capacity associated with PH and high altitude pulmonary edema (HAPE), individuals traveling to or working at altitude (i.e. military and mining personnel, mountain athletes) as well as those suffering from pathologically induced PH may benefit from acute intrapulmonary delivery of Ambrisentan.

Support or Funding Information

ONR# N00014‐14‐1‐0699

DARPA # N66001‐10‐C‐2134
Interactions Among rsm ncRNAs and Rsm RNA‐binding Proteins in the Plant Pathogen Pseudomonas syringae DC3000
Authors: Rous Clarissa J. , Fishman Max , Filiatrault Melanie J.
Published: June 2021

In response to changing environmental stimuli, many bacterial species utilize the Csr/Rsm system of posttranscriptional gene expression regulation to control metabolism, motility, biofilm formation, and quorum sensing. Most Csr/Rsm RNA binding proteins are thought to bind near the 5′ end of mRNA transcripts to prevent translation. Small noncoding RNA molecules (ncRNAs), containing conserved binding sites, can sequester the Csr/Rsm proteins to allow translation of mRNA targets. This system consists of one to two proteins and two to three ncRNAs in most bacteria. However, the genome of Pseudomonas syringae pv. tomato strain DC3000 is predicted to encode five Rsm proteins and seven ncRNAs and it is currently unknown which ncRNAs and mRNAs targets bind to each protein. This project sought to determine the conditions of protein expression and to identify which ncRNAs and mRNAs targets bind to each protein. DC3000 strains were designed to express each of the RNA‐binding proteins with a peptide tag, to facilitate immunoprecipitation and isolation of bound mRNAs and ncRNAs. We found that RsmA1, 4, and 5 were not expressed under the conditions tested. RsmA2 and RsmA3 were both expressed in stationary phase in Mannitol‐glutamate media. RNA immunoprecipitation (RIP) followed by qRT‐PCR showed that RsmA2 binds to (in order of decreasing abundance) ncRNAs rsmX‐1, rsmX‐2, rsmX‐3, and rsmY; RsmA3 binds to rsmX‐1, rsmX‐2, and rsmY. Transcripts for rsmZ and rsmX‐4 were not detected. The ncRNA rsmX‐5 was expressed but not significantly enriched in RIP, indicating that it does not bind to RsmA2 or 3 under these conditions. Additionally, mRNA targets were identified based on homology to computationally predicted targets in Pseudomonas aeruginosa. None of the predicted mRNA targets (PSPTO 1951, 0823, 3382, 5128, 0816, 1994, 4224) showed enrichment. It may be that these are not targets, or that the proteins were sequestered by the ncRNAs and not binding to mRNA under the experimental conditions. These results have shed light on the highly complex Rsm system in P. syringae and provide a foundation for future experimentation on Rsm protein associations with mRNAs and ncRNAs under various conditions in order to delineate the mechanisms of this global system of gene regulation.

Support or Funding Information

This project was funded by National Science Foundation grant DBI‐1263103.
Conformational Flexibility Enables Function of a BECN1 Region Essential for Starvation‐Mediated Autophagy
Authors: Sinha Sangita , Mei Yang , Ramanathan Arvind , Glover Karen , Yang Zhongyu , Colbert Christopher
Published: June 2021

BECN1 is essential for autophagy, a critical eukaryotic cellular homeostasis pathway. Here we delineate a highly conserved BECN1 domain (residues 141–171), located between the previously characterized BH3 and coiled‐coil domains, and elucidate its structure and role in autophagy. The 2.0 Å Sulfur‐Single Wavelength Anomalous Diffraction crystal structure of this domain demonstrates that its N‐terminal half is unstructured, while its C‐terminal half is helical, hence we name it the Flexible Helical Domain (FHD). Circular dichroism, Double‐Electron‐Electron Resonance‐Electron Paramagnetic Resonance and Small Angle X‐ray Scattering (SAXS) analyses confirm that this domain is partially disordered even in the context of adjacent BECN1 domains. Molecular dynamic simulations fitted to SAXS data indicate that the FHD transiently samples more helical conformations in solution. Strikingly, the FHD is predicted to fold upon binding, and we find it becomes more helical in the presence 2,2,2‐trifluoroethanol, indicating it may undergo a disorder‐to‐helix conformational change upon binding to a partner. A completely helical conformation would place highly conserved FHD residues on the same face of the helix, suggesting that these residues may be involved in binding. Lastly, cellular studies show that highly conserved FHD residues are essential for the starvation‐induced up‐regulation of autophagy, the first BECN1 region shown to be critical for starvation‐induced autophagy rather than basal autophagy. Thus, our combined data suggest that the FHD likely undergoes a disorder‐to‐helix transition upon binding to appropriate partners, and conserved residues critical for this interaction are essential for starvation‐induced up‐regulation of autophagy.

Support or Funding Information

This work was supported by National Institutes of Health grants RO3 NS090939 (S.S.) and R15 GM113227 (C.C.); a National Science Foundation grant MCB‐1413525 (S.S.); North Dakota EPSCoR doctoral dissertation award for Y.M. (S.S.). AR was supported by a ORNL Laboratory Director's Research and Development SEED proposal 7278.
Deciphering the Metal Dependency of Novel Nicotinate Hydroxylase from Bacillus niacini
Authors: Meek Jessica A , Sherer Laura A , Snider Mark J
Published: June 2021

N‐Heterocyclic compounds are becoming increasingly common in the environment due to their miscibility in water and pose potential threats to wildlife due to their carcinogenic properties. Nicotinic acid (NA), a nontoxic ‐N‐heterocyclic aromatic compound, serves as a useful model for this class of compounds and is known to be degraded by a number of soil bacteria. Knowledge of the chemistries involved in NA degradation by these bacteria can be applied to bioremediation techniques to remove similar contaminates from the environment. Bacillus niacini is a common soil bacterium that is able to completely degrade and utilize nicotinic acid by a pathway that is largely uncharacterized. A gene cluster hypothesized to code the enzymes responsible for nicotinate degradation in B. niacini suggests that four proteins are involved in the first two steps of the nicotinate degradation pathway. The sequences predict that two Mo‐dependent hydroxylases (NicB1 and NicB2), a flavin‐dependent protein (NicA2) and a protein containing an [2Fe‐2S] cluster (NicA1) are potentially important for the first two oxidation steps of nicotinate degradation. To determine the metal dependency and further understand the mechanism of the NicAB enzymes, heterologous expression and purification of each of these enzymes was achieved from E. coli and Pseudomonas chlororaphis, analyzed for metal content and ability to catalyze the double hydroxylation of nicotinate to 2,6‐dihydroxynicotinate.

Support or Funding Information

This work was supported by funding from the College of Wooster Department of Chemistry and the National Science Foundation.
A Biophysical Analysis of Aspartic Acid Cyclization Coupled to Peptide Bond Cleavage
Authors: Koulopoulos Michael , Giaccone Zach , Reitter Julie , Mills Kenneth
Published: June 2021

Protein splicing is the process by which a polypeptide chain, called an intein, catalyzes its excision from two flanking polypeptides, called exteins, concomitant with the exteins ligating together. We are interested in how inteins catalyze the splicing reaction and therefore are studying the third step of the mechanism via a fluorescence‐based assay using a model peptide. Currently, we are analyzing peptide bond cleavage due to cyclization of aspartic acid rather than asparagine, which is promoted at low pH and high temperature. We are currently studying the potential catalytic role of an adjacent histidine residue and carefully determining the influence of pH and temperature on the reaction.

Support or Funding Information

This material is based upon work supported by the National Science Foundation under grants MCB‐1244089 and MCB‐1517138 and by a Henry Dreyfus Teacher‐Scholar Award (KVM).
The Tale of Two Inteins: Similar Inteins with Different C‐termini
Authors: Ramsoomair Christian K , Giaccone Zachary T , Reitter Julie N , Mills Kenneth V
Published: June 2021

Protein splicing is a post‐translational process facilitated by an intervening polypeptide, or intein. The intein catalyzes its excision from the flanking polypeptides, or exteins, as well as ligation of the exteins. We studied inteins that interrupt the DNA Polymerase II from the cyanobacteria Synechococcus sp. PCC 7002 (Ssp) and Trichodesmium erythraeum (Tery). The two inteins differ mostly in the residues that promote the third step of splicing, side chain cyclization of the C‐terminal residue coupled to peptide bond cleavage. While the Ssp intein has a native C‐terminal Asn, the Tery intein has a native Gln. We hypothesized that the replacement of the native Asn with Gln might slow splicing, whereas replacement of the native Gln with Asn may accelerate splicing, as cyclization of Asn residues is usually considerably faster. Preliminary results suggest that our hypothesis is correct for isolated side chain cyclization, but that the overall splicing reaction is less affected by the identity of the C‐terminal residue. This suggests that conformational changes after step one, an amide to thioester rearrangement, might affect the rate of side‐chain cyclization.

Support or Funding Information

This material is based upon work supported by the National Science Foundation under grants MCB‐1244089 and MCB‐1517138, a Henry Dreyfus Teacher‐Scholar Award (KVM), and Mrs. Jeremiah W. O'Connor, Jr.
Statistical Analysis of Kinetic Profiles of Anti‐Clumping Agents
Authors: Zem Gregory , Hakobyan H. , Terteryan S. , Baronian T. , Mikaelian E. , Vozovoy E. , Baum T. , Oppenheimer S. B.
Published: June 2021

This study is a continuation of a multi‐year effort using a yeast (Saccharomyces cerevisiae) model to test the effects of various reagents on unclumping of fixed yeast cells. Anti‐clumping reagents could be useful in unclumping cancer cells in the bloodstream, in anti‐thrombocytic applications, and in blocking biofilms and infectivity. Here 3 reagents (sodium sulfate, EDTA and L‐arginine) were tested at 3 concentrations (0.1M range) over a 60 min time course on their ability to unclump yeast in deionized water droplets. In 330 replicate experiments all 3 reagents unclumped yeast with up to a 33 percent increased number of single cells over controls without reagent. Kinetic profiles of each reagent and controls were generated and statistically evaluated. For example only the low concentration of L‐arginine showed results that were generally not significant in the spread between experimentals and controls, while in all other concentrations of L‐arginine and the other two reagents all but 2 of the 28 p values were less than 0.05. This indicated that the spreads between experimentals and controls were generally significant with p values reaching as low as 0.0000007. This simple assay system can easily evaluate hundreds of reagents for potential anti‐clumping activity (support: Sidney Stern Memorial Trust, Cal State contributions from NSF Presidential Award 0731633 and various NIH and NSF grants).