bluuebunny/crossref_metadata_2025 huggingface dataset:
Mucus‐inspired low‐fouling barriers based on self‐assembled glycopeptide nanofibers
Authors: Hudalla Gregory , Restuccia Antonietta
Published: June 2021

Mucus is a glycoprotein‐rich gel that protects epithelial tissue surfaces from non‐specific interactions with micro‐organisms and macromolecules. Synthetic mucus‐like barriers could enable new opportunities to confer implantable materials with non‐fouling properties that are essential for biocompatibility and long‐term efficacy. However, mucus glycoproteins (i.e., “mucins”) derived from natural sources are highly variable and recombinant mucin production is impractical, which has led to increasing interest in synthetic mucin analogs. Here we report synthetic carbohydrate‐modified peptides (i.e., “glycopeptides”) that self‐assemble into beta‐sheet nanofibers in water. The dense carbohydrate coating on the surface of glycopeptide nanofibers is similar to that of naturally derived mucins. Carbohydrates decorating the surface of glycopeptide nanofibers are specifically recognized by their cognate lectins. Additionally, the type of carbohydrate appended onto the nanofiber can be efficiently varied via glycosyltransferase enzymes. Using this approach, we synthesized nanofibers decorated with n‐acetylglucosamine (GlcNAc) and n‐acetyllactosamine (LacNAc) that selectively bind to wheat germ agglutinin (WGA) and galectins, respectively. At millimolar concentrations in water, glycopeptide nanofibers entangled into viscoelastic hydrated gels (i.e., “hydrogels”) (Figure 1a–b). GlcNAc‐decorated glycopeptide hydrogels bound and retained WGA, but were permeable to various non‐lectin proteins, including albumin and green fluorescent protein, as well as the mannose‐binding lectin, concanavalin A. GlcNAc‐decorated glycopeptide hydrogels coated onto a glass surface reduced E. coli adhesion by ~90%, whereas non‐glycosylated peptide hydrogels only reduced bacterial adhesion by 50%. Additionally, glycopeptide hydrogels coated on glass robustly inhibited adhesion of macrophages (Figure 1c) and fibroblasts (Figure 1d) in the presence of serum, whereas non‐glycosylated peptide hydrogels were permissive to non‐specific cell adhesion. Together, these data demonstrate that self‐assembling glycopeptides can provide synthetic barriers with selective macromolecule permeability and resistance to non‐specific cell interactions similar to natural mucinous gels. Moving forward, we anticipate that glycopeptide hydrogels will be broadly useful as mucus‐inspired surface coatings to improve the biocompatibility and efficacy of implantable devices, drug delivery vehicles, and biosensors.

Support or Funding Information

NSF Career award, DMR‐1455201

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Comprehensive High Throughput One‐Pot Analysis of Glycoproteins through Tool for Rapid Analysis of Glycopeptide by Permethylation (TRAP) Method.
Authors: Shajahan Asif , Supekar Nitin T. , Heiss Christian , Azadi Parastoo
Published: June 2021

Glycoproteins plays key roles in various cellular functions such as protein folding, protein targeting, receptor function, cell–cell recognition, protein stability, signaling, and other cellular functions. Currently a comprehensive and universal tool for the automated analysis of glycopeptides through their tandem mass spectrometry and data processing is a tedious process. We recently developed a method, Tool for Rapid Analysis of glycopeptide by Permethylation (TRAP), that simplifies glycoprotein analysis by integrating glycan sequencing and glycopeptide analysis in a single experiment. We employed this approach for the detailed analysis of glycosidic linkages, composition, glycan sequencing, glycosylation site mapping and sequencing of peptide moiety of glycopeptides of glycoproteins from both prokaryotes and eukaryotes. Here we are presenting an easy to use, robust, suitable for large as well as small sample sets and also cost‐effective glycoprotein analysis tool through TRAP approach. A permethylation protocol which can be automated and suitable for reliable permethylation of glycopeptides from the protease digest of glycoproteins was developed. Initial studies on glycopeptides from a model glycoprotein bovine RNAse B indicates that improved sensitivity and throughput of permethylation reactions can be achieved in conducting the reaction in microscale. This approach would enable convenient, rapid, and reliable high‐throughput screening of protein glycosylation and can be employed as a cost‐effective kit for glycosylated biomarker discoveries. In order to improvise the comprehensive glycosylation analysis and throughput, we have also developed an LC‐MS/MS platform for the analysis of glycopeptides processed through TRAP. We have conducted the analysis of standard glycoproteins such as bovine RNase B, human transferrin, and bovine fetuin and our preliminary results indicate that TRAP hyphened with LC‐MS/MS enables characterization of isomers of each glycoform at each glycosylation sites of complex glycoprotein mixtures.

Support or Funding Information

This research was supported by the National Institutes of Health (NIH) funded research, Grant R21GM122633; resource for biomedical glycomics, Grant P41GM103490; instrument grant, Grant S10OD018530; and U.S. Department of Energy grant, Grant DE‐SC0015662, to the Complex Carbohydrate Research Center.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Concentration‐Dependent Actions of Piperidine Alkaloids on the Inhibition of Fetal Movement in Day 40 Pregnant Goats and Comparison to Cell‐Based Models.
Authors: Green Benedict , Welch Kevin D. , Lee Steve T. , Kem William R.
Published: June 2021

Anabasine and anabaseine are potent and effective agonists at nicotinic acetylcholine receptors (nAChR). Anabasine in livestock species is teratogenic and has been shown to cause developmental defects that include arthrogyrposis, kyposis, lordosis, scoliosis, and torticollis. We have postulated that these fetal defects are due to the complete and sustained inhibition of fetal movement. The purpose of this research was to determine if cell‐based predictions of teratogenicity from TE‐671 cell experiments translate to inhibition of fetal movement in a day 40 pregnant goat model. We hypothesized that anabasine, anabaseine, epibatidine, and DMPP which are potent and effective agonists atfetal muscle‐type nAChR expressed by TE‐671 cells, would be effective at completely inhibiting fetal movement in a goat model the putative mechanism of teratogenicity in livestock species. Pregnant goats were I.V. dosed with anabasine, anabaseine, epibatidine, DMPP, or saline control on gestation day 40 and the number of fetal movements were measured by ultrasound periodically for 8 hours. The experiment was designed to only briefly inhibit fetal movement(< 3 h) and as a result, no terata were observed in any of the kids from the pregnant does dosed with any of the compounds in this study. Anabasine, at a dose of 0.8 mg/kg, completely inhibited fetal movement for 1.5 hours after dosing, and its actions were dose‐dependent with an IC50 value of 0.1 mg/kg. Anabaseine, epibatidine, and DMPP did not completely inhibit fetal movement in day 40 pregnant goats. The results from this experiment suggest that while experiments with TE‐671 cells provide valuable information and predictions of the actions of plant alkaloids on fetal movement, in vivo experiments are still required in order to determine the ability of an alkaloid to inhibit fetal movement in livestock species and to predict teratogenic potential.

Support or Funding Information

Funding provided by USDA ARS

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Buprenophine functions as a weak dopamine releaser in the absence of heroin but blocks the dopamine releasing effects of heroin in its presence
Authors: Issacs Dominic , Krystinziak Greg , Schelp Scott , Pultorak Katherine , Oleson Erik
Published: June 2021

With the rise in opioid use and opioid‐related death in modern society, it is imperative to understand how these drugs affect neural circuits, particularly those implicated in drug‐seeking and motivated behavior. Generally, all drugs of abuse are thought to increase mesolimbic dopamine concentrations through various mechanisms, and it is widely believed these fluctuations in dopamine concentrations (and subsequent hypodopaminergic states) are implicated in the acquisition, persistence, and maintenance of addiction. Here we sought to identify increases in phasic dopamine in the nucleus accumbens of rats after intravenous diacetylmorphine (heroin) administration. Then, we sought to identify the effects on phasic dopamine after buprenorphine administration, followed by subsequent heroin administration. Utilizing fast‐scan cyclic voltammetry in awake behaving animals, we administered cumulative animal‐equivalent doses based on clinical investigations that correlated subjective measures of the drug's effect to dosage in human subjects. Either heroin or buprenorphine administered alone causes large increases in dopamine transient amplitude and frequency, whereas heroin administration after buprenorphine causes a decrease in the amplitude and frequency of transients. These data suggest a modulatory effect of buprenorphine on opioid receptors in the presence of exogenous opiates.

Support or Funding Information

Funding for the project was provided by NSF grant IOS‐1557755, NIH grant R03DA038734, Boettcher Young Investigator Award and NARSAD Young Investigator Award to EBO and an institutional UROP to KJP.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Correlation between Toluene Exposure and Toxicity Effects of Toluene on Drosophila Melanogaster Fecundity and Offspring Survivability
Authors: MacGregor Michael Charles , Trivino Vanessa , Luu Brenda , Moreno Angela , Kovar Erin , Arredondo Dalia , Rosell Rosemarie , Ledesma Elmer
Published: June 2021

Toluene is a volatile organic compound that is a common indoor contaminant. According to the U.S. Department of Health and Human Services, chronic toluene exposure negatively affects organ systems, such as nervous and respiratory, and acts as a fetotoxic agent in the development of humans. Toluene exposure causes damage to human myelin sheaths, thus we propose that damage to the fly nervous system will be similar to that seen in humans exposed to toluene. Our ultimate goal is to determine the effects of toluene exposure on developmental and neuronal processes in D. melanogaster. In this first phase, we hypothesize that toluene‐exposed adults, at the interpolated Lethal Exposure (LE50), will demonstrate decreased fecundity and that offspring from toluene‐exposed females will have decreased survivability from the egg to adult stage. We developed a continuous flow system that delivers known concentrations of gaseous toluene to populations of D. melanogaster. Ten to twenty adult female flies per trial were exposed for 24 hours to known concentrations of toluene. Surviving females were collected, counted and mated to non‐exposed adult males. The toluene‐exposed females were placed on a colored agar plate to lay eggs for 24 hours. Eggs were counted to determine fecundity (number of eggs produced by a single female fly). Survivability of eggs to later life stages, was measured by counting number of larvae, pupae, and adults divided by number eggs, larvae, and pupae respectively. The interpolated LE50 of gaseous toluene was between 1000 and 1500 ppm after 24‐hour exposure. Fecundity of toluene‐exposed females decreased by approximately 50% when compared to airexposed females. The survivability of offspring of air‐exposed flies from egg to larval stages was 84% higher than published studies. We observed that larval eclosure from eggs laid by toluene‐exposed females was 35–50% higher on days 1 and 2 when compared to larval eclosure from eggs of air‐exposed exposed females. On days 3–6 the eclosure rate was relatively similar. These data showed toluene's effects on fecundity and survivability were measurable, however, additional replicates are in progress to fully assess the life table characteristics of offspring of toluene‐exposed female flies. Further studies will focus on toluene's effect on nervous and reproductive organs of fruit flies, along with a study on the mutagenic effects of toluene on fly DNA.

Support or Funding Information

U.S. DOE, Hispanic‐Serving Institutions, STEM Articulation Grant P031C110128

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Peripheral Adipose Arteriole Dilator Response to Amyloid Proteins and Palmitic Acid Recapitulates Central Leptomeningeal Arteriole Response: Validation of a Novel Human Tissue Model
Authors: Truran Seth , Karamanova Nina , Serrano Geidy , Madine Jillian , Davies Hannah , Beach Thomas , Migrino Raymond Q.
Published: June 2021

Background

Cerebrovascular dysfunction is critical in the pathophysiology of Alzheimer's disease and related dementia disorders (ADRD). The relationship between cardiovascular risk factors (e.g. saturated fatty acid palmitic acid PA) and amyloid proteins (Aβ or medin) in ADRD is unknown. Treatments that worked on animal models have failed to work in humans, pointing to the need to develop human tissue‐based models that recapitulate human condition. We showed feasibility of using cadaver leptomeningeal arterioles (LMA) following rapid autopsy to study central arteriole physiology but availability is extremely limited to few centers. Abdominal adipose arterioles (AA), on the other hand, are relatively easy/safe to obtain but require validation to confirm whether physiologic responses parallel cerebrovascular responses.

Aim

Test the hypothesis that AA endothelium‐dependent dilator responses to Aβ, amyloidogenic medin and PA (agents that impair cerebrovascular function)parallel the responses of LMA.

Methods

AA were isolated fromsubcutaneous fat from living volunteers undergoing routine abdominalsurgery. In brain and body donorsundergoing rapid autopsy, LMA ± abdominal AA were isolated. Arterioles were cannulated, pressurized and preconstricted with endothelin‐1. Baseline (control) dilator response to acetylcholine (10−9–10−4M)was obtained. Following washout, arterioles were exposed for 1 hour to physiologic doses of Aβ 2 μM, medin 5 μMor PA 150 μM and post‐treatment dilator response was measured and compared tobaseline; responses between AA and LMA were also compared.

Results

See fig. Responses were not significantly different between AA and LMA.

Conclusions

Aβ, medin and PA caused endothelial dysfunction to a similar degree in both LMA and AA. The findings suggest that peripheral AA areacceptable surrogates to study functional response of central cerebrovasculararterioles and may be a valuable novel human tissue model.

Support or Funding Information

NIH (NIAR21AG044723/NINDSU24NS072026/NIAP30AG19610/NIARO1AG019795), VAMerit (BX‐003767/BX007080), British Heart Foundation (FS/12/61/29877), Arizona Department of Health Services, Arizona Biomedical Research Commission(4001/0011/05‐901/1001), Fox Foundation for Parkinson's Research and US Department of Defense (AZ160056).

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Cardioprotective Effects by a Novel Opioid Peptide in Myocardial Ischemia/Reperfusion Injury
Authors: Kim Hanna , McIntyre Anahi , Woodley John , Lopez Alexandra , Dittakavi Tejaswi , Finnegan Matthew , Amuquandoh Kevin , Ambrosino Maxwell , Walker Kiana , Patel Harsh , Chen Qian , Barsotti Robert , Young Lindon H.
Published: June 2021

Preliminary studies in mice have shown that a novel tri‐peptide (Phe‐D‐Arg‐Phe‐Amide, MW=468 g/mol) attenuates ventilation induced diaphragm dysfunction when given as a pretreatment. Tri‐peptide is structurally similar to the SS‐20 peptide (Phe‐D‐Arg‐Phe‐Lys‐Amide), which has been shown to be cardioprotective when given before ischemia, but thought not to have opioid properties (Cho et al., 2007). Our study aims to determine whether pre‐ or posttreatment with tri‐peptide is more efficacious in attenuating the deleterious effects of myocardial ischemia/reperfusion (I/R) injury and whether this cardioprotection is mediated by opioid receptor activation. This was tested by observing whether tri‐peptide‐induced cardioprotection was abolished when administered in conjunction with naloxone, a nonselective opioid receptor antagonist. Tri‐peptide (50μM) was administered prior to I (pretreatment, n=8) or during R (posttreatment, n=8) in isolated perfused rat hearts subjected to I(30 min)/R(45 min) and compared to untreated control hearts (n=7) and hearts pre‐treated with tri‐peptide (50 μM) + naloxone (Nlx, 10μM, n=8). Tri‐peptide pretreatment hearts significantly recovered post‐reperfused left ventricular developed pressure (LVDP) to 59±6% of initial baseline values compared to untreated control, posttreatment tri‐peptide, and pretreatment tri‐peptide + Nlx hearts in which LVDP recovered to only 33±7%, 34±8%, and 26±4% of initial baseline values respectively (p<0.01). Furthermore, tri‐peptide pretreatment hearts exhibited significantly reduced infarct sizes of 26±1% compared to untreated control and posttreatment tri‐peptide hearts, in which infarct sizes were 38±4% and 37±2% respectively (p<0.05) assessed using 1% triphenyltetrazolium chloride staining. Pretreated tripeptide + Nlx hearts showed infarct sizes of 32±3% which was not significantly different from untreated controls. These data suggest that pretreatment and not posttreatment with this novel tri‐peptide was cardioprotective and that this protection was mediated via opioid receptor activation. Future studies will attempt to determine the specific opioid receptor subtype involved in tri‐peptide cardioprotection by testing whether delta or kappa opioid receptor antagonists block the cardioprotective effects of tri‐peptide pretreatment.

Support or Funding Information

This study was supported by the Center for Chronic Disorders of Aging, the Division of Research and the Department of Bio‐Medical Sciences at Philadelphia College of Osteopathic Medicine.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Comparing the Efficacy of Pharmacological Preconditioning with Myristic Acid‐conjugated, TAT‐ conjugated and Native Protein Kinase C Epsilon Peptide Activator in Myocardial Ischemia/Reperfusion (MI/R) Models
Authors: McIntyre Anahi , Kim Hanna , Finnegan Matthew , Amuquandoh Kevin , Chen Qian , Barsotti Robert , Young Lindon
Published: June 2021

Protein kinase C epsilon (PKCɛ) activation is thought to be central in mediating cardioprotection conferred by myocardial ischemic preconditioning. PKCɛ activation via PKCɛ activator peptide (PKCɛ+, HDAPIGYD, MW= 887 g/mol) as pretreatment (PT) is a pharmacological means to mimic preconditioning. However, unconjugated or native PKCɛ+ requires the use of cell permeabilization methods for efficacious intracellular targeting to improve cardiac function and reduce necrotic cell death. Therefore, conjugating PKCɛ+ to known intracellular delivery moieties may be a method to effectively activate PKCɛ for cardioprotection. To test this hypothesis, we subjected isolated perfused rat hearts to PT with PKCɛ+ conjugated to either myristic acid (Myr‐HDAPIGYD, MW= 1097 g/mol, 10mM, n=5) or transactivating (TAT) carrier peptide (YGRKKRRQRRR‐CC‐HDAPIGYD, MW= 2632 g/mol, 10mM, n=6) to evaluate the efficacy of these intracellularly targeted peptide analogs in attenuating contractile dysfunction and infarct size after MI (30 min)/R (90 min) in comparison with native PKCɛ+ PT (10mM, n=5) and untreated control hearts (n=6). We found that compared to control hearts, PT with Myr‐PKCɛ+ and TAT‐PKCɛ+ significantly reduced infarct size from 41±3% to 29 ±1% and 28±2% (p<0.05) respectively compared to control hearts assessed by 1% triphenyltetrazolium chloride staining of heart tissue. By contrast, hearts PT with native PKCɛ+ showed almost no change in infarct size compared to controls, 41±3% versus 35±2%. Both myristic acid‐ and TAT‐conjugated PKCɛ+ PT hearts restored post‐reperfused left ventricular developed pressure (LVDP) to 52±5% and 50±10% compared to both native PKCɛ+ PT and control hearts which only recovered to 33±9% and 38±4% of initial baseline values respectively, but this improvement was not statistically significant. These preliminary results indicate that increasing cellular permeability of PKCɛ+ via conjugation to either myristic acid or TAT significantly improved its efficaciousness in attenuating infarct size when given before ischemia as a pharmacologic mimic of ischemic preconditioning. These results suggest that Myr‐ or TAT‐conjugated PKCɛ+ may be an effective treatment to attenuate cell death in coronary bypass or organ transplantation settings.

Support or Funding Information

This study was supported by the Center for Chronic Disorders of Aging, the Division of Research and the Department of Bio‐Medical Sciences at Philadelphia College of Osteopathic Medicine.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The Effects of Metformin on Methylglyoxal‐induced cardiomyocytes cell damage
Authors: Sandhu Robinderpal , Lefkovitz Dylan , Kim Alisa , Wieczorek Peter , Barsotti Robert , Young Lindon , Chen Qian
Published: June 2021

Methylglyoxal is a precursor of advanced glycation end products which is closely related to vascular complication in diabetes. However, the direct effects of methylglyoxal on cardiac myocytes still need to be elucidated. This study investigated the dose‐dependent effects of methylglyoxal on H9C2 myoblastic cell. Furthermore, we determined if metformin would reduce methylglyoxal caused cell damage. Cell viability was evaluated by a cell counting kit‐8 assay and intracellular reactive oxygen species (ROS) was evaluated via a dichlorofluorescin diacetate (DCFDA) assay. After incubation of different doses of methylglyoxal (5 μM‐1400 μM, n=12–18) for 24 hours, lower dose range of methylglyoxal (5 μM‐800 μM) slightly increased the cell viability by 15±4% compared to the control (n=12). By contrast, higher dose methylglyoxal (1000 μM, 1200 μM, 1400 μM) significantly reduced cell viability to 74 ± 6%, 63 ± 5%, and 56 ± 7%, respectively (all p<0.05) compared to control cells. Meanwhile, higher dose of methylglyoxal (1000 μM, 1200 μM, 1400 μM) also increased intracellular ROS by 42 ± 27% (n=4), 89 ± 23% (n=4, p<0.05), and 109 ± 11% (n=3, p<0.05), respectively. By contrast, administration of metformin (1–40 mM) with methylglyoxal (1200 μM) (n=4) dose‐dependently increased cell viability. Metformin (40 mM) increased cell viability by 123± 41% (p<0.05) and reduced intracellular ROS by 70% (n=1) when compared to methylglyoxal treated cells. The preliminary data suggests that higher dose of methylglyoxal causes cardiac cells damage possibly by increased oxidative stress. Metformin can reduce oxidative stress and protect cardiac cells under higher methylglyoxal concentration and may reduce vascular complications in diabetic patients.

Support or Funding Information

This study was supported by the Center for Chronic Disorders of Aging, the Division of Research and the Department of Bio‐Medical Sciences at Philadelphia College of Osteopathic Medicine.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Does Treadmill Walking affect Neurovascular Coupling during Cognitive Activation in Healthy Individuals?
Authors: Favre Michelle E. , Douglas Samantha B. , Wood Scott J. , Serrador Jorge M.
Published: June 2021

Treadmill desks have become increasingly popular in the workplace to improve physical health. However, it is unknown whether treadmill walking affects neurovascular coupling, which is the increase in cerebral blood flow during cognitive tasks. Additionally, it is unknown whether the speed of treadmill walking affects neurovascular coupling or if there is a sex difference in the response. A total of 49 subjects (22 male, 27 female) walked on a treadmill at four different walking speeds (0.5 mph, 1.5 mph, 2.5 mph and 3.5 mph) in addition to a standing condition (0 mph). While standing or walking at each speed, participants also performed the Stroop color‐word test. Continuous cerebral flow velocity in the right middle cerebral artery (MCAv), blood pressure, heart rate, and end‐tidal CO2 were measured throughout testing. The percent increase in MCAv from the no tasking condition (standing or walking at each speed) to the Stroop tasking condition (Stroop + standing or walking at each speed) was analyzed to measure neurovascular coupling. Results showed a significant effect of walking speed (p=0.003) as well as a significant interaction between walking speed and sex (p=0.012) on the percent increase in MCAv during the Stroop tasking condition (0 mph: Men = 2.6 ± 5.8%, Women = 1.8 ± 3.7%; 0.5 mph: Men = 2.3 ± 4.2%, Women = 3.6 ± 5.2%; 1.5 mph: Men = 4.1 ± 6.2%, Women = 4.2 ± 4.9%; 2.5 mph: Men = 3.4 ± 7.4%, Women = 4.0 ± 4.3%; 3.5 mph: Men = −1.7 ± 7.8%, Women = 3.4 ± 7.0%). There was also a small, but significant effect of walking speed on the difference in end‐tidal CO2 between the Stroop tasking condition and the no tasking condition within each speed (p=0.005; 0 mph: Men = 0.0 ± 1.1 mmHg, Women = −0.1 ± 1.4 mmHg; 0.5 mph: Men = −0.1 ± 0.8 mmHg, Women = 0.0 ± 0.7 mmHg; 1.5 mph: Men = 0.0 ± 1.3 mmHg, Women = 0.2 ± 0.9 mmHg; 2.5 mph: Men = 0.4 ± 1.7 mmHg, Women = 0.1 ± 0.9 mmHg; 3.5 mph: Men = −1.0 ± 2.0 mmHg, Women = −0.2 ± 1.1 mmHg). There was no interaction between speed and sex in end‐tidal CO2. This data suggests that there is a small, but significant reduction in neurovascular coupling in men during the Stroop test at 3.5 mph, but this reduction may be partially explained by the reduction in end‐tidal CO2. This study highlights that neurovascular coupling is preserved during treadmill walking across the slower walking speeds. However, neurovascular coupling at 3.5 mph needs to be further examined with controlled levels of end‐tidal CO2 to support whether neurovascular coupling is reduced at this faster walking speed in men.

Support or Funding Information

The study was supported by the Department of Defense through CDMRP W81XWH‐14‐1‐0598 and CDMRP W81XWH‐14‐2‐0012.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Isolation Stress During the Adolescence Exacerbates the Locomotor Activity Effects of Cocaine in Rats
Authors: Perez‐Cardona Enrique U. , Rivero Carlos , Freire Jaime , Ortiz Natalia , Sacarello Gabriel , Santiago Ivan , Torres Ricardo , Mendez Andrea , Diaz Abdias , Rodriguez Efrain , Segarra Annabell
Published: June 2021

Adolescence is a complex developmental period characterized by sexual and brain maturation. Stress during adolescence, as abandonment and social isolation, interferes with maturation of higher brain functions such as decision making, learning and memory. Since rats are social animals, we investigate the consequences of housing rats individually during adolescence on the development of addictive behaviors. From postnatal day 23 (weaning), male and female rats were either housed in groups of the same sex or single housed throughout the experiments. At day 35 they were tested for their locomotor response to cocaine (15 mg/kg). Our results indicate that isolation during adolescence increases the locomotor response to cocaine in drug‐naive female rats. Unlike females rats, isolation during the adolescence does not affect the initial locomotor response to cocaine in males. However, these prepubertal males that normally do not show cocaine sensitization at this age, sensitized to cocaine when reared in isolation during adolescence. These results show that rearing rats in isolation exacerbates the behavioral effects of cocaine, and that the effect varies according to the sex of the animal. They also advocate for closer monitoring of neglected and solitary adolescents since they may be at a higher risk to develop drug dependency.

Support or Funding Information

This work supported by NSF under grant #OISE1545803.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Malabsorption of copper and other nutrients as well as intestinal parasite infections cause health problems and lower birthrates in the American Bison
Authors: Rhodes Ryon Sun , Topliff Christina L. , Kelling Clayton L. , Coon Steven
Published: June 2021

Background

Herds of American Bison have been raised on pastures of different Indian Reservations in Montana. It has been discovered that many of these Bison have serious health problems related to malnutrition causing lower birth rates. Plants on these reservations absorb unusually high levels of selenium and molybdenum which the animals feed on. Previous studies indicate metals such as copper are necessary for proper health and birthrates. Copper is necessary for normal conception rates and will cause retarded growth of the offspring. In addition, poor calving rates in apparently healthy bison with adequate protein and energy in diets might simply be from copper deficiency.

Hypothesis

Decreased copper absorption is due to increased absorption of selenium and molybdenum and parasitic infections.

Methods

When the animals are euthanized liver samples and fecal samples are removed for analysis. Body scores were taken to access the states of malnutrition and number of offspring were counted to access birthrates.

Results

Increased toxic levels of selenium (3.7ppm) and Molybdenum (4.2ppm) and low levels of copper (10ppm) have been detected in tissue samples from the livers of the bison. However increased levels of Zinc have also been detected in these animals (125ppm). In addition, the presence or increased levels of intestinal parasites have also been detected in the fecal matter taken these animals. These parasites which include Coccidia and various worm infestations may also be also interfering with the normal absorption of copper and other nutrients in the animals.

Conclusions

Malabsorption of nutrients such as copper may be caused by increased absorption of selenium, molybdenum or zinc as well as infections from parasites in the Bison digestive tract.

Support or Funding Information

USDA 2016‐38424‐25559

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Mass Spectrometry‐Based Proteomics Reveals a Regulatory Role for DYRK1A in DNA Damage Repair
Authors: Guard Steven E. , Poss Zach , Ebmeier Chris , Old William
Published: June 2021

Down syndrome, the most common genetic cause of intellectual disability, results from trisomic chromosome 21. A section of chromosome 21 containing 33 genes, known as the Down syndrome critical region, plays a crucial role in producing the cognitive defects characteristic of Down syndrome. The dual‐specificity tyrosine phosphorylation regulated kinase 1A (DYRK1A), located within the Down syndrome critical region, is necessary for normal brain development from flies to humans. Neurological defects in mice arise from an additional DYRK1A gene copy, while heterozygous loss of function causes microcephaly, facial dysmorphia, impaired motor function and behavioral problems in humans, demonstrating the sensitivity of DYRK1A function to gene dosage. However, the molecular mechanisms underlying these phenotypic changes are not fully understood. Defining the protein interacting partners of DYRK1A and subsequent function of these relationships is crucial for understanding the implication of trisomic chromosome 21 on human health.

Most known functions and substrates of this kinase are cytoplasmic, leaving the smaller subpopulation of DYRK1A in the nucleus relatively unstudied. To systematically investigate the nuclear interacting partners of DYRK1A, immunoprecipitation of the endogenous protein in conjunction with mass‐spectrometry based proteomics was used to generate a deep interactome. This nuclear interactome reveals interactions between DYRK1A and several E3 ubiquitin ligases that act as negative regulators of DNA double stranded break repair. Efficient repair of these lesions is necessary for maintaining genome stability and is dependent on a ubiquitin signaling cascade. CRISPR knockout of DYRK1A and inhibition of this kinase with the selective inhibitor harmine decreases the efficiency of DNA double strand break repair following ionizing radiation. The persistence of DNA double strand break markers increased in DYRK1A knock out cells and in a dose dependent manner with harmine at late time points following the initial damage.

Maintaining genomic stability during expansion of neural progenitors is crucial for brain development. The rapid expansion of progenitors during typical neurogenesis is of particular risk, as early‐born cortical progenitors are hypersensitive to replication‐associated DNA damage. Inaccurate repair of genomic lesions at such a time can result in severe and ill effects on the developing organism. The results from this study strongly implicate DYRK1A gene dosage in the efficient repair of DNA double strand breaks, and in a neural context could contribute to the neurodevelopment defects associated with DYRK1A.

Support or Funding Information

Blumenthal Fund Fellowship; Linda Crnic Institute; DARPA

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Biophysical characterization of interactions between a uridylated histone mRNA degradation intermediate and SLBP
Authors: Harris Sarah Elizabeth , Mihailescu Mihaela‐Rita , Lackey Patrick
Published: June 2021

Histone mRNAs are a tightly cell‐cycled regulated group of mRNAs that are strongly expressed at the beginning of S‐phase and rapidly degraded when DNA replication ends. This cell cycle regulation is controlled by histone mRNA's unique 3′ end; instead of the traditional poly(A) tail, histone mRNA instead ends with a 3′ stem‐loop. This stem‐loop is an important factor in controlling the expression and degradation of histone mRNA during S‐phase, along with two key protein binding partners: the stem‐loop binding protein (SLBP) and the human 3′ exonuclease (3′hEXO). At the beginning of degradation, 3′hEXO trims 2–7 nucleotides from the 3′ end. These nucleotides are then often replaced by a short uridylation that preserves the length of the stem‐loop. The exact function of these uridylated intermediates and whether they affect histone mRNA metabolism is currently unknown. We characterized the structure of a stem‐loop with seven nucleotides trimmed from the 3′ end and replaced with seven uridines using nuclear magnetic resonance (NMR) to show that a stem‐loop still forms, despite this trimming‐and‐replacement mechanism that turns the six‐membered stem into a nine‐membered stem. We also characterized interactions between SLBP, 3′hEXO, and the trimmed and uridylated RNA using electrophoretic shift mobility assays (EMSA) and found that the RNA does, in fact, interact with both proteins. This raises new questions about the nature of the uridylated stem‐loop and its function in the translation and degradation of histone mRNA.

Support or Funding Information

The National Science Foundation CHE‐1659821 (REU)

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Invariant BECN1 C‐x‐x‐C Motifs Are Essential For Starvation‐Induced Autophagy
Authors: Mukhopadhyay Shreya , Li Yue , Sinha Sangita
Published: June 2021

Autophagy is an essential catabolic cellular homeostasis process conserved in all eukaryotes. BECN1, a protein essential for autophagosome nucleation, contains two invariant C‐x‐x‐C motifs (18CxxC21 and 137CxxC140 in human BECN1) that bookend a large BECN1 intrinsically disordered region (IDR). We use circular dichroism spectroscopy to elucidate differences in secondary structural content of the wild‐type and CysTetrad mutant BECN1 IDR. Additionally, we are using small angle X‐ray scattering analyses to further understand the impact of the CysTetrad mutation on the structure of the BECN1 IDR. We also show that helicity of the BECN1 IDR increases upon addition of 2,2,2‐trifluoroethanol (TFE), suggesting that it may undergo binding‐associated helical transitions. Further, we use ICP‐MS to demonstrate that BECN1 binds Zn2+ in a 1:1 molar ratio under both, reducing and non‐reducing environments. Lastly, we show that both C‐x‐x‐C motifs are required, while the IDR is also important, for starvation‐triggered up‐regulation of autophagy.

Support or Funding Information

NIH grants RO3 NS090939 and R15 GM122035 (S.C.S.)

National Science Foundation grant MCB‐1413525 (S.C.S.)

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Mortalin Caught in Action: Structures of the Nucleotide Binding Domain in the Apo, ADP‐bound and AMP+PPi‐bound States
Authors: Moseng Mitchell , Page Richard
Published: June 2021

The heat shock protein 70, Hsp70, chaperone family plays key roles in cellular homeostasis and stress response. Hsp70s not only regulate the folding of nascent proteins, but also prevent aggregation, promote disaggregation, and refold misfolded proteins. The mitochondrial Hsp70, Mortalin, functions in a variety of processes such as mitochondrial protein import and quality control, Fe‐S cluster biogenesis, mitochondrial homeostasis, and regulation of p53, an important tumor suppressor. Mortalin has been implicated in the regulation of apoptosis, cell stress response, neurodegenerative diseases, and has an anti‐apoptotic role in cancer cells. We have elucidated crystal structures of the nucleotide binding domain of human Mortalin in the apo, ADP‐, AMP and AMP states at 2.8 Å, 2.78 Å, and 1.75 Å resolutions, respectively. The crystal structure of Mortalin‐NBD binding to the modified ADP homolog N6propargyl‐ADP has also been determined at 1.49 Å. The overall structures and active site organizations are similar, with a few key differences in side chain positions. Specific residues in the nucleotide binding pocket, which are not conserved in other Hsp70‐family members, lead to lower nucleotide affinity and slower turnover relative to cytosolic Hsp70. ITC and biolayer interferometry experiments performed on the R126W and Y128C disease variants of Mortalin to determine ADP binding and interactions with an inter‐domain linker peptide elucidate how the mutations change Mortalin's functions and how these interactions differ between Mortalin, Hsp70, and Hsc70. ITC experiments have also been performed to test how the MortalinNBD can accommodate modified ADP nucleotides. This structural and binding data may also contribute to the understanding of disease‐associated Mortalin mutations and to improved Mortalin‐targeting antitumor compounds.

Support or Funding Information

NSF 15‐52113

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
An engineered E. coli ribosome with tunable translation rates enhances recombinant protein expression
Authors: Oza Javin P. , Soye Ben Des , Jewett Michael C.
Published: June 2021

Using E. coli for the expression of recombinant proteins in their soluble active form continues to be a challenge in biotechnology. Issues including low yields of full‐length, soluble, and functional proteins fundamentally limit discovery, development, and manufacturing of commercially, industrially, and medically relevant proteins. In many cases, improvements in protein solubility have been achieved by codon optimization, over‐expression of chaperone proteins, solubility tags, and by varying expression conditions. Optimization of these parameters is often done iteratively and on a case‐to‐case basis, which can be slow and expensive. To overcome limitations in recombinant protein expression, we report a genomically engineered strain of E. coli with a tunable ribosome enabling modulation in translation rates under isothermic conditions. Additionally, we show that cell extracts from this strain of E. coli can support a cell‐free protein synthesis platform capable of on‐demand production of recombinant proteins. This platform technology enables two key advances. First, we provide quantitative evidence that tuning protein synthesis rates improves soluble fractions of traditionally intractable proteins such as the human MEK1 and IRKβ. In each case, soluble fractions increased from <20% to >80% of total protein when compared with expression in BL21 cell extracts. 2) This platform technology is plug‐and‐play, allowing the user to identify the optimal translation rate condition for a desired protein in a high‐throughput manner, which can then be scaled‐up for desired protein yields. The timescale from DNA template to the optimized protein expression condition is 24–48 hours, notably faster than traditional methods involving cell transformations, and optimization of expression times and temperatures. Additionally, our data suggest that this approach obviates the need for solubility tags, enabling the expression of proteins in their wild‐type forms. Our work provides a new tool for studying protein folding in the context of ribosomal kinetics, and a new platform for manufacturing traditionally difficult proteins.

Support or Funding Information

This work was supported by DARPA and the the David and Lucille Packard Foundation Fellowship.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Investigating the Mechanism of cis Amide Bond Stabilization in Phosphoserine‐Proline Sequences
Authors: Raniszewski Nicole R. , Ganguly Himal K. , Zondlo Neal
Published: June 2021

Phosphorylation is a reversible covalent modification to biomolecules that is central to protein‐protein interactions, signal transduction, and gene expression. When improperly regulated, phosphorylation can trigger oncogenesis and neurodegenerative diseases such as Alzheimer's disease (AD) and chronic traumatic encephalopathy (CTE). Phosphorylation can also act as a structural switch for cis‐trans isomerization of peptide bonds: through intraresidue phosphate‐amide hydrogen bonds, phosphorylated residues promote compact trans conformations. In other contexts, phosphorylated Serine (pSer) residues can promote cis peptide bonds through unknown mechanisms. To investigate the basis for these structural effects, a series of X‐pSer‐Pro‐Z peptides was synthesized with modifications on the proline, N‐terminal to pSer (X), and C‐terminal to Pro (Z).

Dipeptides remove influences from secondary and tertiary protein structures, allowing elucidation of key molecular interactions that affect the cis conformer. Two 4S‐substituted hydroxyproline derivatives, Fmoc‐ 4S‐iodophenyl‐hydroxyproline and Fmoc‐4S‐fluoroproline, were synthesized and incorporated into the peptide. These substitutions promote the cis‐Pro conformation via intraresidue stereoelectronic effects. The initial peptides were examined with C‐terminal amides, while modifications of the N‐terminus allowed for control of sterics. Formyl, acetyl, pivaloyl, 2‐iodobenzoyl, and 4‐iodobenzoyl moieties were included in peptides to provide varying degrees of steric demand to promote alternate conformations at the N‐terminus.

The peptides were analyzed by 1H NMR to quantify the effects of proline, N‐terminal modifications, and phosphorylation on structure. TOCSY and ROESY 1H NMR spectra were employed to differentiate the cis and trans peaks and characterize the conformational changes. Structural changes as a function of phosphate protonation state were identified by pH‐dependent NMR experiments. The downfield shift in the amide region with increasing pH implied a Hydrogen bonding interaction between the phosphate oxygen and its own amide hydrogen. The equilibrium constant for cis‐trans isomerization, Ktrans/cis, increased with increasing pH for the acetyl and 4‐iodobenzoyl derivatives. Pivaloyl and 2‐iodobenzoyl derivatives showed an opposite trend, increasing the population of cis with increasing pH. N‐terminal 2‐iodobenzoyl and acetyl groups show greater preference towards the cis at various pH with respect to other examined peptides. Stabilization of cis amide bonds upon phosphorylation can be used to design tunable scaffolds for peptide conformations.

Support or Funding Information

David M. Heitzer Award, DOD

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Discovery of novel monomeric L‐threonine 3‐dehydrogenase and elucidation of product release mechanism
Authors: Motoyana Tomoharu , Nakano Shogo , Yamamoto Yuta , Tokiwa Hiroaki , Asano Yasuhisa , Ito Sohei
Published: June 2021

Introduction

L‐Threonine (L‐Thr) metabolism is not well studied, however, plays some part in the lipid synthesis of Trypanosoma brucei and the growth of mouse embryonic stem (ES) cells. L‐Thr is initially catalyzed by L‐threonine dehydrogenase (TDH) which is a NAD+‐dependent enzyme catalyzing dehydrogenation of hydroxyl group of L‐Thr to 2‐amino 3‐ketobutyrate (AKB). TDH belonging to short chain dehydrogenase/reductase family has high specificity toward L‐Thr; the specificity is regulated by switching between open state and closed state by binding of substrate (1). On the other hand, the product release mechanism remains to be elucidated. In this study, we discuss the reaction mechanism associated with structural change at the active site by combinational analysis, such as high resolution X‐ray crystal structure, quantum mechanical (QM) calculation method and kinetic analysis.

Methods

In silico screening was performed using INTMSAlign (2) to obtain TDH (mtTDH) derived from metagenome library. At first, we evaluated enzymatic properties of mtTDH. Four crystal structures of mtTDH were determined at 1.25 – 1.9 Å resolution: apo, binary (NAD+‐binding) and two ternary (l‐Ser‐NAD+‐binding and AKB‐NADH‐binding). For the AKB‐NADH‐binding structure, the Inter‐Fragment Interaction Energy (IFIE) were calculated by the Fragment Molecular Orbital (FMO) method. We also performed site‐directed mutagenesis (S74A, S111A, Y136F, T177A, D179A, and D179N) and kinetic studies to elucidate the roles of active site residues.

Results & Discussion

The crystal structure of mtTDH was obtained as a monomeric form; this is consistent with the result of gel‐filtration chromatography. Based on these results, we concluded that mtTDH is monomeric TDH. Structural comparison of the four structures revealed that ligand (AKB or l‐Thr) and eight molecules (six residues and two molecules of water) appeared to form hydrogen bonding interactions each other. In order to evaluate these interactions quantitatively, IFIEs were calculated by FMO method which is a kind of QM calculation method. As a result, surprisingly, D179 had a repulsive interaction with the AKB. Kinetic analysis of mtTDH variants, D179A and D179N, exhibited the remarkably decrease in activity in spite that D179 works against formation of interaction with the ligand. Taken together, D179 may be a sensor detecting the completion of the reaction and important for switching between open state and closed state (3).

Support or Funding Information

This work was supported by JSPS KAKENHI Grants 16K18688, 17KT0010, and 17K06931 and JST by ERATO Grant JPMJER1102.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.