bluuebunny/crossref_metadata_2025 huggingface dataset:
Refinement of the water‐in‐oil reverse microemulsion process for the encapsulation of proteins within silica nanoparticles
Authors: Strong Elijah W , Tarnai Brian D , Anderson C. William
Published: June 2021

Over the past decade, interest in nanoparticles as a therapeutic tool has skyrocketed as technological advances have made them cheaper and easier to use. Nanoparticles in themselves act as vehicles for proteins, enzymes, drugs, etc. and carry them throughout a biological system to a specific target. However, with proteins being simply bound to the nanoparticles, problems arise with these proteins being susceptible to denaturation. Encapsulation of proteins within nanoparticles provides an advantage over simple binding and gives many benefits through its protection of the proteins. Through encapsulation, proteins are protected by, in this case, a silica shell. This shell prevents the protein from denaturing from heat, acidity, or metabolism. All the while, the drug or enzyme that is encapsulated should still maintain its function and interact with a target site because the silica shell contains small pores that allow for such reactions to proceed.

To establish and refine the encapsulation protocol in the context of our laboratory, we attempted a water‐in‐oil reverse microemulsion process on myoglobin, green fluorescent protein (GFP), and chymotrypsin. Since silica nanoparticles have been shown to be biocompatible in humans and have a great deal of biological significance, we chose to use a silica‐based approach for nanoparticle synthesis. The silica nanoparticles that we synthesized were mesoporous, which allowed the nanoparticles to be biocompatible due to their low hemolytic activity. Due to such biocompatibility, these nanoparticles can be used for things such as drug delivery, biosensors, imaging, and more.

Results showed that encapsulation was successful through spectrophotometry absorbance and fluorescent microscopy measurements for myoglobin and GFP encapsulation, respectively. The catalytic function of the encapsulated proteins was then shown through absorbance experiments run on encapsulated chymotrypsin. Current work is focusing on using these techniques for the encapsulation of several clinically relevant protein targets of similar size to the examples presented here.

Support or Funding Information

National Science Foundation Award #1626602

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Extracellular MIF and Wnt and eph/ephrin signaling are involved in WWOX‐regulated cell‐cell recognition and migration
Authors: Chang Nan‐Shan , Chou Pei‐Yi , Kuo Hsiang‐Ling , Sie Yong‐Da
Published: June 2021

Loss of tumor suppressor WWOX enhances cancer cell metastasis. Here, we determined that WWOX‐deficient cells fail to recognize the parental wild type cells or any WWOX‐expressing cells of the same or different species. WWOX‐negative cells have loose cell‐cell contacts and migrate individually. WWOX‐positive cells group tightly and migrate collectively. Transiently overexpressed WWOX suppresses cancer cell migration. Knockdown of WWOX by small interfering RNA, or ectopic dominant‐negative WWOX, increases cell migration. WWOX/p53/TIAF1 triad is a known axis of tumor suppression. Ectopic expression of the protein triad suppresses cell migration, anchorage‐independent growth, and induces apoptosis. Mutant p53 at the Ser46 phosphorylation site causes functional loss of the triad. Among all the p53 isoforms, ectopic Δ133p53γ is most potent in inducing apoptosis due, in part, to its activation of the SMAD‐responsive promoter. Time‐lapse microscopy revealed that upon encountering, WWOX‐negative human breast MDA‐MB‐231 cells suppress the migration WWOX‐positive mouse L929 fibroblasts. Few L929 cells undergo apoptosis. Indeed, MDA‐MB‐231 accelerate their migration, contact L929 with protrusions, and then migrate in a retrograde manner. The accelerated migration of MDA‐MB‐231 is associated with rapid activation of the Wnt signaling‐mediated TCF/LEF‐responsive promoter in less than 2 hr. However, L929 exhibit the activation of CRE and NF‐kB promoters in 16 hr and later. Suppression of the Wnt signaling by XAV939 abolishes the retrograde migration of MDA‐MB‐231 and their physical merge with L929. Similarly, inhibition of cell surface ephrin A1, B1 and B2 and extracellular MIF with specific antibodies leads to anterograde migration of MDA‐MB‐231 and restoration of their merge with L929. Together, WWOX‐regulated cell‐to‐cell recognition is associated with cell surface ephrins, extracellular TGF‐β and MIF, and Wnt‐dependent TCF/LEF promoter activation, so as to to avoid unwanted surrounding microenvironment by retrograde migration.

Support or Funding Information

Research was supported by the Department of Defense USA (W81XWH‐08–1‐0682), the Ministry of Science and Education, Taiwan, ROC (NSC99‐2320‐B‐006‐012‐MY3, 102‐2320‐B‐006‐018‐, 102‐2320‐B‐006‐030‐, and 102‐3011‐P‐006‐005‐), and the National Health Research Institute, Taiwan, ROC (NHRI‐EX99–9704BI; NHRI‐EX107‐10734NI) to NS Chang.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Effect of adenosine A1 receptor agonist induced‐hibernation on the arousal pathway in the Arctic Ground Squirrel
Authors: Jenkins Mackenzie , Frare Carla , Drew Kelly
Published: June 2021

Hibernation is a seasonal phenomenon and in the arctic ground squirrel (AGS), the effect of seasonal changes is very evident. In late fall AGS decrease their metabolic demand followed by the entry into hibernation in winter. Previously in the lab it has been shown that N6‐cyclohexyladenosine (CHA), an A1 adenosine receptor agonist, induces hibernation in AGS in a seasonally dependent manner. In winter AGS treated with CHA experience a decrease in metabolic demand and temperature as seen during natural hibernation. However, in summer CHA triggers a brief and transient metabolic suppression that does not lead to hibernation onset.

As adenosine is known to promote sleep, we decided to investigate if the sleep‐arousal pathways were affecting the seasonal difference in response to CHA. The tuberomammillary nucleus (TMN) is a nucleus of the ascending arousal pathway characterized by fast firing rates to promote wakefulness. Previously we showed a decrease in neuronal activation in the TMN in AGS treated with CHA in both summer and winter seasons.

Here we test the hypothesis that seasonal response to CHA is associated with difference in activation in another nucleus of the ascending arousal pathway, the locus coeruleus (LC).

AGS implanted with body temperature (Tb) transmitter, were treated with CHA or vehicle (0.5mg/kg, IP). AGS were perfused with 4% paraformaldehyde and brains were dissected and cryoprotected in 5%, 10%, 15%, 20%, 30% sucrose. We performed free‐floating immunohistochemistry on 40μm brain slices in four experimental groups: winter CHA, winter control, summer CHA and summer control. We sectioned brains from eight AGS per group for a total of 32 animals and identified active nuclei with cFos immunoreactivity (mouse anti‐cFos 1:20,000, Santa Cruz) and active tyrosine hydroxylase neurons (rabbit anti‐TH). Blinded analysis of cFos immunoreactive neurons was done using bright field microscopy and Metamorph software. Statistical analysis was performed on R.

CHA produced a hibernation‐like response measured by a decrease in Tb in winter AGS but not in the summer. We did not observe any significant difference in neuronal activation in the LC, thus CHA is associated with a decreased neuronal activity in the TMN, resulting in the TMN being the major wakefulness center affected by CHA but not by seasons.

Support or Funding Information

Research was supported by NIH R03 NS081637, NSF IOS‐1258179, RL5GM118990 and Alaska INBRE P20GM103395. The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The gamma‐aminobutyric acid (GABA)A receptor modulates the cytokine response to rhinovirus exposure in human monocytic cells
Authors: Hall David J , Hader Shelby N , Kondiles Bethany R
Published: June 2021

Rhinoviral infections are a major cause of asthma exacerbations. Though productive rhinovirus infections occur predominantly in the bronchial epithelial cells of the upper airway, monocytic‐lineage cells, the most prominent immune cell in the lung, are important in establishing the inflammatory microenvironment observed during rhinoviral asthma exacerbations. Emerging evidence indicates that the gamma‐aminobutyric acid type A receptor (GABAAR) signaling system on leukocytes, airway epithelial cells and airway smooth muscle cells modulates the inflammation in the lung. We sought to determine if GABAAR signaling alters the release of inflammatory mediators from human monocyte‐derived macrophages (MDM) exposed to human rhinovirus (HRV). Results demonstrate that GABAARs on human MDMs are functional and stimulation with GABA or HRV caused GABAARs on the cell surface undergo protein kinase C (PKC)‐mediated endocytic removal and recycling, similar to that found in neurons. GABA treatment of MDMs during HRV exposure attenuated the production of HRV stimulated CCL2 and CXCL10 production by inhibiting the stress kinase June N‐terminal kinase (JNK) and increasing phosphorylation of extracellular regulated kinase (ERK). Interestingly, GABA stimulated the production of interferon‐b through the classical innate immune signaling molecules TANK binding kinase (TBK) and interferon regulated factor 3 (IRF3). Phosphorylation TBK occurred via the lysine deficient kinase (WNK) pathway after the influx of chloride ions when GABAARs are stimulated. Our findings suggest that GABA treatment of MDMs lead to a novel activation of the WNK‐TBK‐IRF3 axis implying the potential for GABA‐mediated treatment of HRV‐induced asthma exacerbations.

Support or Funding Information

Lawrence University, NIH grant R15 AI065505‐01A1 and NSF grant 0521112.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Drosophila CG1275 Expression, Antibody Production, and Western Blotting
Authors: Garcia Brandon Michael , Gorman Maureen J. , Ragan Emily J.
Published: June 2021

Little is known about iron absorption in insects. One potential mechanism involves the action of a ferric reductase to reduce Fe(III) to Fe (II), which can enter the cell through a divalent metal transporter. CG1275 is a potential ferric reductase that is found in Drosophila melanogaster. By replicating, cloning, and expressing the N‐terminus of CG1275 we can develop a reagent to allow us to observe a protein knockdown after RNA interference (RNAi) experiments. We cloned a cDNA encoding the N‐terminal 107 amino acids of CG1275‐PA, the region before the first transmembrane helix, into pET‐32a. This was confirmed by restriction digestion. Expression of the N‐terminus of CG1275 along with a thioredoxin (Trx) tag and 6xHis tag from the vector was performed in Bl21 (DE3) E. coli. We then purified the protein using Nickel‐column affinity chromatography. The purified protein was separated by SDS‐PAGE and the protein bands of interest were cut out and sent for antibody production. The newly developed antibodies were then separated to remove antibodies that recognize the Trx tag. The antibodies to CG1275 were then used for western blots of S2 cell lysates to determine if they recognize CG1275 and are able to verify successful CG1275 knockdown after RNAi experiments.

Support or Funding Information

NSF (IOS‐1656407)

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Investigating a cytochrome b561 family member in insect cellular iron uptake
Authors: Holst Jessica , Arismendiz Omar Alexis Rodriguez , Ochoa Alma , Gorman Maureen J. , Ragan Emily J.
Published: June 2021

Iron is an essential element to many functions of an animal's life, yet it is also toxic. Because iron is both essential and harmful, tight regulation of its concentration, location and ionization state is vital to animal livelihood. While we understand much about iron uptake in mammals, the mechanism for its transport is poorly understood in insects. We know iron is significant to many aspects of an insect's life, including: energy metabolism, detoxification of pesticides and as a nutrient for symbionts and commensals. We have identified a cytochrome b561 family member in Drosophila melanogaster, CG1275, that is similar to mammalian duodenal cytochrome b, a known ferric reductase involved in iron uptake. CG1275 is well expressed across all life stages of Drosophila, and we hypothesize that it may be involved in the uptake of iron to non‐intestinal insect cells. To test this hypothesis, we are performing RNA interference (RNAi) experiments to attempt knockdown in the expression of the gene coding for this protein. Previously we relied upon traditional polymerase chain reaction (PCR) to qualitatively observe whether knockdown had occurred. Now, by using real‐time PCR, we have quantified the CG1275 knockdown observed in our RNAi experiments. Next we will measure the iron concentration levels of our S2 cells to determine whether CG1275 plays a role in iron uptake to non‐intestinal insect cells.

Support or Funding Information

NSF IOS‐1656407

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Quantitative Comparison of GM1 Gangliosidosis Afflicted Ovine Tissue using Thin‐Layer Chromatography, Mass Spectrometry and Nuclear Magnetic Resonance
Authors: Mata Cindy Venegas , Mitchell Jon C.
Published: June 2021

GM1 ganglioside is a naturally occurring glycosphingolipid that cannot be synthesized in significant quantities; so it needs to be isolated as a natural product. Researchers at Glycoscience Research, Inc. (GRI – Toronto, SD) have developed a novel, verified, ovine source for producing GM1 ganglioside at levels needed for pharmaceutical applications. GM1 gangliosides have been shown to be an effective therapeutic against Huntington's disease as well as a potential promising lead against Parkinson's disease, Alzheimer's, and dementia (Holler, et al., 2016). Research presented here attempts to address the biochemical quantification of purified GM1 ganglioside isolated from afflicted/non‐afflicted lamb muscle to definitively show no significant differences in GM1 levels between the two meat sources. Chloroform:methanol extraction, anion exchange chromatography (AEC) and High Performance Thin‐Layer Chromatography (HPTLC) followed by Nuclear Magnetic Resonance (NMR) and MS/MS Spectrophotometry analysis compares similarities and differences in GM1 concentration levels between muscle tissue from lambs afflicted with the genetic disease of overproducing GM1 ganglioside and normal, wildtype lamb muscle.

Support or Funding Information

This material is based upon work supported by the National Science Foundation/EPSCoR Cooperative Agreement #IIA‐1355423 and by the State of South Dakota, SD‐GOED, NSF I‐Corps Team Program and Dr. Larry and Sue Holler, GlycoScience Research Incorporated

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Synthesis and Development of peptidoglycan fragment microarray and probes to investigate innate immune signaling
Authors: Zhou Junhui , Lazor Klare , Grimes Catherine
Published: June 2021

The innate immune system's interaction with bacteria plays a pivotal role in a variety of human diseases, such as Crohn's disease, asthma, and rheumatoid arthritis. In order to develop better therapies, small molecule probes derived from bacterial cell wall peptidoglycan (PG) are needed. Consequently, we are synthesizing new chemical probes based on PG to better understand their interactions with innate immune receptors. The objective here is to develop novel techniques for the production of cell wall derived libraries and then use those molecules as tools for the investigation of innate immune signaling. It has long been known that a small piece of the bacterial cell wall, muramyl‐dipeptide (MDP), has immune stimulatory properties. More specifically, MDP has been found to bind to Nucleotide Oligomerization Domain‐containing protein 2 (NOD2) to initiate a Nod2‐dependent immune response. The power of microarrays lays in their unprecedented capacity to simultaneously interrogate tens to hundreds of thousands of immobilized probes. Here we developed a synthesis for amine functionalized PG derivatives with varying linker length and position, and applied them to NHS‐functionalized glass slides to investigate their interactions with Nod2, and other nod‐like proteins. N‐acetyl muramic acid (NAM) serves as a key building blocks PG fragments. We report a synthetic strategy to access biorthogonal 3‐lactic acid NAM derivatives in large quantities. By utilizing metabolic cell wall recycling and biosynthetic machineries reported previously in our lab, the NAM derivatives were installed into the backbone of Gram‐positive and Gram‐negative bacteria. The whole cells are labelled via click chemistry and visualized using super‐resolution microscopy.

Support or Funding Information

Cobre (Center of Biomedical Research Excellence at the University of Delaware);

NSF (National Science Foundation);

U01(NIH) grant.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Characterization of the Interaction between ISTL1 and Exo70E2 in Arabidopsis
Authors: Camacho Lizbeth
Published: June 2021

Internal and external stimuli at the plasma membrane are the cause of dynamic changes that affect the composition of the plasma membrane which is responsible for the proper responses, survival, and development of an organism. The Endosomal Sorting Complex Required for Transport, otherwise known as ESCRT, is a highly conserved pathway in all eukaryotes. This pathway allows for endocytosis, and endosomal trafficking which tags proteins for degradation. It is also responsible for the sorting of endocytic vesicles to the vacuole. This mediates the responses of plants. It has also been noted that there is a possible relationship between the ESCRT pathway and the exocyst. The exocyst is an octameric protein complex that directs vesicles after the Golgi complex to specific destinations of the the plasma membrane. It has recently been proposed that Exo70 proteins localize vesicle traffic.

Support or Funding Information

National Science Foundation

NIH Minority Access to Research (MARC) 5 T34 GM 8395‐29 California State University Northridge

Otegui Lab

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Transdermal Detection of Hydrogen Sulfide in Humans
Authors: Wiest Elani Fourie , Brandenburg Jacquelyn , Clark Ross , Friedrichsen Debra , Silaski Grayson , Qeadan Fares , Howdieshell Thomas , Shekarriz Reza , Kanagy Nancy
Published: June 2021

We have developed a novel approach to measure circulating hydrogen sulfide (H2S) non‐invasively as a potential way to diagnose and monitor endothelial dysfunction and peripheral artery disease (PAD). PAD is a life‐threatening condition caused by arterial constriction and obstruction of blood flow leading to limb ischemia. Current methods to diagnose and monitor PAD lack sensitivity, are expensive, and technically difficult. Recent studies indicate that decreased H2S production is an underlying cause of PAD. Reduced plasma H2S also correlates with endothelial dysfunction in individuals with untreated hypertension, diabetes, sleep apnea, and other cardiovascular diseases. The Transdermal Gas Sensor (TAGS) device was designed to measure transdermal H2S to test the hypothesis that the diffusion rate (and therefore gas phase concentration) of H2S is directly proportional to dermal blood flow in individuals without endothelial disease. Healthy volunteers between the ages of 21–65 were recruited. Exclusion criteria included subjects currently treated for hypertension, hyperlipidemia, and diabetes. Smokers and pregnant women were also excluded. We observed a linear association between TAGS readings and Laser Speckle Contrast Imaging (LSCI), a gold standard measure of skin blood flow, when readings were adjusted for age in the model (r = 0.5717, p = 0.0132, n = 18). Therefore the TAGS device is not inferior to LSCI in measuring skin blood flow in healthy individuals using a non‐inferiority margin of 5 (p = 0.0394). The TAGS device has the potential, therefore, to serve as a more sensitive, economical, and easy to use diagnostic tool to use H2S detection as a predictor of dermal blood flow.

Support or Funding Information

NSF: R41 NSF FP00000502. NHLBI: R41HL121871

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Investigating the Evolution of the Allosteric Regulation of Glutamate Dehydrogenase
Authors: Lekova Virzhiniya , Bell Ellis
Published: June 2021

Glutamate dehydrogenase exhibits no allosteric regulation in bacteria and archaea, while in mammals it has complex allosteric regulation. The major activator is ADP, and inhibitor is GTP. The enzyme is a homohexamer in all organisms. Ciliates and animals have an antenna domain, not present in other unicellular eukaryotes, or in bacteria and archaea. Using GDH from E. coli, S. cerevisiae, N. tabacum, the unicellular eukaryotes D. discoideum, T. pyriformis, and P. tetraurelia, D. melanogaster, and Bos taurus sequence alignments show the presence of an antenna domain in GDH enzymes from D. discoideum, T. pyriformis, and P. tetraurelia. Conserved residues in all living organisms have been idntified as well as conserved residues present only in eukaryotes or prokaryotes allowing comparisons to be made between residues involved in structure and catalysis as compared to regulation. Homology models of GDH from these organisms have been used to examine the structural differences of GDH from various evolutionary levels. The antenna domain showed similarity to a methyl transferase of D. aromatica, suggesting that its appearance results from an insertion mutation. These observations suggests complex allosteric regulation is linked to multicellularity and the metabolism of ammonia, as well as usage of glutamate as a neurotransmitter.

Supported by NSF Grant MCB 0448905 to Ellis Bell
Gene Profiles of Dengue Virus Targeted Host Biological Processes and Pathologic Markers
Authors: Muhie Seid , Hammamieh Rasha , Yang David , Jett Marti
Published: June 2021

Dengue is one of the most prevalent arthropod‐borne viral illnesses in humans with neither selective therapy nor an approved vaccine. Understanding its molecular targets and biological processes may contribute towards developing sensitive diagnostic markers and/or effective therapeutics. Leucocytes obtained from 15 US Army Ranger Cadets before and after 9 weeks training were in vitro treated with dengue virus serotype 2. cDNAs of isolated RNAs were profiled using glass microarray chips containing ∼10,000 human gene targets. Statistical analyses gave ∼180 genes specifically regulated by dengue both in the presence and absence of severe physiological stress. Ontological‐enrichment showed that genes forming spliceosome, ribosome, translation initiation and proteosome complexes were up regulated while those involved in translation elongation, protein modification and targeting were down regulated. It seems that dengue virus mainly targets host protein biosynthesis with the net effect of blocking formation of functional host proteins while it gears the host's machineries towards promoting its own replication and pathogenesis. Subsets of 180 genes were also specific to dengue even in the presence of infections with other pathogens. These have the potential to be used as diagnostic markers irrespective of the physiological and pathologic conditions of the host.

We are grateful to DTRA for funding.
Conserved hydrophobic ensembles in protein kinases: their integrating and regulatory roles
Authors: Kornev Alexandr P. , Ten Eyck Lynn F. , Taylor Susan S.
Published: June 2021

Protein kinases represent a large protein superfamily which regulates numerous processes in living cells. Structures of several serine‐threonine and tyrosine kinases were investigated by recently developed bioinformatics method: Local Spatial Patterns alignment. Previously this method proved to be an effective tool, which is capable to detect highly conserved spatial patterns of amino acid residues. These residues do not form traditional motifs in terms of sequence or protein fold and can not be detected by traditional methods. An example of that was discovery of “the spine” in protein kinases, an important regulatory element. In this work we report detailed analysis of the protein kinase catalytic core. A set of conserved ensembles comprised mostly by hydrophobic residues was detected. The formations traverse protein kinase molecules and play integrating and regulatory roles. They are anchored to the F‐helix located in the middle of the large lobe, which acts as an organizing “hub” providing exact positioning of the key catalytic and regulatory elements.

This work was supported by National Science Foundation Grant NSF‐DBI 99111196 and National Institute of General Medical Sciences Grants GM70996 (to L.F.T.E.); GM19301 and National Science Foundation Grant DBI0217951 (to S.S.T.).
The role of metal‐thiol conjugates in heavy metal detoxification in invertebrate epithelial lysosomes
Authors: Roggenbeck Barbara , Sterling Kenneth M. , Ahearn Gregory A.
Published: June 2021

Hepatopancreas lysosomal membrane vesicles (LMV) from the American lobster (Homarus americanus) were loaded with 1 mM alpha‐ketoglutarate (AKG) and used to study the effects of metal‐binding compounds on zinc uptake. Transport studies using 65Zn indicated that addition of 1 mM L‐cysteine to outside media caused a 3‐fold increase in zinc uptake. Identical experiments using 1 mM glutathione stimulated a 4‐fold increase in zincuptake. Inhibition studies with 1 mM para‐amino hippuric acid (PAH) in the presence of either 1 mM L‐cysteine or 1 mM glutathione caused a 2‐fold decrease in zinc uptake. These data indicated the presence of an Organic Anion Transporter (e.g., OAT‐1) in lobster lysosomal membranes. Based on our zinc uptake studies Western blot analysis was carried out on the LMV using a rabbit anti‐rat OAT‐1 antibody. The Western blot results showed the presence of an orthologous OAT‐1‐like protein (approximate MW of 80 kD) signal from lobster LMV. A cross‐reacting signal from LMV of approximately 50 kD was also observed which may correspond to a splicing variant or an under glycosylated version of the lobster OAT‐1‐like protein. These data suggest a possible role of the OAT‐1 transport system in long‐term sequestration of metal‐thiol conjugates in invertebrates via lysosomes as a means of heavy metal detoxification. Supported by NSF grant IBN04‐21986.
Effective mechanisms in restoring blood pressure following hemorrhage
Authors: Mathis Keisa W. , Whitaker Annie M. , Molina Patricia E.
Published: June 2021

Acute alcohol intoxication (AAI) prior to injury and hemorrhage (HEM) accentuates hypotension and impairs blood pressure (BP) recovery during fluid resuscitation. The impaired hemodynamic counterregulation to HEM in AAI rodents is associated with attenuation of circulating levels of arginine vasopressin (AVP), epinephrine (Epi) and norepinephrine (NE). We hypothesize that restoration of the neuroendocrine response, either through central activation or systemically through vasopressor administration, will improve BP recovery and outcome from HEM during AAI. Parallel studies determined that intracerebroventricular (ICV) administration of neostigmine (acetylcholinesterase inhibitor) improves recovery of HEM‐ and AAI‐induced hypotension (A6488). The aim of the present study was to determine the efficacy of AVP in restoring the pressor response and to investigate the contribution of AVP in mediating the pressor response elicited by ICV neostigmine. A constant intravenous infusion of AVP (0.01 U/kg/min) was administered to catheterized, conscious male Sprague‐Dawley rats following a fixed‐pressure (40 mmHg) HEM. We observed improved pressor response to HEM in AVP‐treated animals. In a different set of animals, administration of a selective AVP receptor antagonist (V1a; 10μg/kg) blunted the neostigmine (1 μg) induced rise in BP. These results show improved BP recovery following HEM due to increased AVP activity produced by systemic administration and following central cholinergic activation. Furthermore, neostigmine produced a marked elevation in Epi (315±88%; P =0.001) and NE (89±29%, P=0.005) levels, suggesting that its pressor response cannot be solely attributed to enhanced AVP release. Whether similar effectiveness is achieved during AAI remains to be established and is focus of current investigation. Supported by DOD PR‐054196 and NIAAA‐AA7577.
Inhibition of NO, KATP and prostanoids decreases hypoxia‐induced, but not H2S‐induced, contractions in rat aorta
Authors: Predmore Benjamin Lee , Alendy Maikel J. , Olson Kenneth R. , Julian David
Published: June 2021

Hypoxia and H2S elicit similar contractile responses in every vertebrate smooth muscle thus far tested, but the mechanisms are poorly understood. In vascular smooth muscle, blockade of nitric oxide (NO), ATP‐sensitive potassium channels (KATP), and prostanoid production have each been shown to modulate aortic responses to hypoxia or H2S, but no study has tested whether the effect of blockade on the responses to hypoxia and H2S are similar in the same tissue preparation. We tested this with aortic rings from Fisher 344 rats using standard vascular myography techniques. We found that both hypoxia and H2S elicit a tri‐phasic, contract‐relax‐contract response. Hypoxia‐induced contractions were significantly reduced by the NOS inhibitor L‐NAME (P<0.029; RM ANOVA, Fisher LSD), the KATP inhibitor glibenclamide (P<0.020), and by a cocktail of glibenclamide, L‐NAME and the prostanoid inhibitors esculetin, clotrimazole and indomethacin (P<0.002), indicating a role for NO, KATP and prostanoids in modulating hypoxia signaling. In contrast, H2S‐induced contractions were not significantly affected by any of these inhibitors (P>0.134; RM ANOVA). Therefore, the mechanisms by which hypoxia and H2S elicit a contract‐relax‐contract response in rat aortic smooth muscle appear to be different. It remains to be tested whether this is true for other smooth muscle tissues. Research support: NSF IBN‐0422139.
Electrophysiological characterization of mosquito NHE8 in Xenopus oocytes
Authors: Piermarini Peter M , Beyenbach Klaus W
Published: June 2021

Na/H exchangers (NHEs) mediate the antiport of Na+ and/or K+ for H+ across cell membranes. In invertebrates and prokaryotes, some NHEs are electrogenic operating as nNa/H or Na/nH exchangers. In the yellow‐fever mosquito Aedes aegypti, a cDNA encoding an isoform of NHE8 (AeNHE8) was recently cloned and shown to mediate Na/H and K/H exchange when expressed in yeast and fibroblast cells (Kang’ethe et al., 2007, Am J Physiol Renal, 292: 1501). The goal of our study was to characterize the electrophysiology of AeNHE8 when expressed in Xenopus oocytes (AeNHE8 oocytes). Measurements of membrane potential (Vm) and intracellular pH (pHi) indicate that AeNHE8 oocytes have a depolarized Vm and a higher pHi compared to controls. In some batches of AeNHE8 oocytes, reducing extracellular [Na+] rapidly lowers pHi, which is reversed by returning [Na+]. In all batches of AeNHE8 oocytes, reducing extracellular [Na+] consistently and reversibly hyperpolarizes Vm. Two‐electrode voltage clamp studies show that AeNHE8 oocytes have a higher conductance than controls and that the conductance is lowered by decreasing extracellular [Na+]. Taken together, these observations indicate that AeNHE8 induces a significant Na+‐conductance when expressed in Xenopus oocytes. Whether the conductance is due to electrogenic nNa/H exchange remains to be determined. Supported by NSF Grant IOB‐0542797 to KWB.
Biomechanics of the primate craniofacial skeleton and its relevance to human evolution
Authors: Wang Qian
Published: June 2021

Since J.T. Robinson's pioneering dietary interpretations of facial morphology in australopiths, studies on biomechanics of skulls have been reshaping our understanding of human evolution. Hypotheses concerning function and evolution in early human forms can be developed and tested through various theoretical and experimental biomechanical approaches, such as mechanical design modeling, geometric analysis, in vivo and in vitro strain gage experiments, and Finite Element Analysis (FEA). As an engineering technique used to analyze how objects of complex design resist load, FEA is particularly useful in examining biomechanics of craniofacial skeletons. Our recent discovery that elastic properties of cortical bones of craniofacial skeletons in closely related species demonstrate similar patterns of variation has allowed the precise extension of FEA to fossils. With Finite Element skull models from both extant and fossil primate species, we are able to investigate global synchronous stress and strain patterns in craniofacial skeletons, examine how they structurally adapt to resisting stress during mastication and other activities, and draw biologically meaningful information on mechanical advantages and evolutionary processes of various features in facial skeletons, e.g., anterior pillar in robust australopiths.

Supported by National Science Foundation HOMINID grants 0725183 and MEDCEN 23750.
The skeletal muscle transcript profile reflects accommodative responses to inadequate protein intake in younger and older males
Authors: Thalacker‐Mercer Anna Elizabeth , Fleet James , Craig Bruce , Campbell Wayne
Published: June 2021

The effects of dietary protein and age on the skeletal muscle transcript profile were assessed in 12 younger (21–43 y) and 10 older (63–79 y) men who completed three controlled feeding trials with protein intakes of 63% (P63), 94% (P94), and 125% (P125) of the recommended dietary allowance (RDA). On d 12 of each trial, a fasting state sample of the vastus lateralis was obtained and Affymetrix U133A Plus 2.0 genechips were used to assess global changes in mRNA levels. 958 transcripts (TR) were differentially expressed (P<0.05) with diet and 853 with diet‐by‐age (P<0.05) using ANOVA. Independent of age, at P63 TR associated with ubiquitin‐dependent protein catabolism and muscle contraction were up‐regulated (UP). At P63 and P94 TR related to apoptosis were UP and TR related to cell differentiation; muscle and organ development; extracellular space; and responses to stimuli and stress were down‐regulated. The diet‐by‐age effect on protein modification (folding) TR was consistent with the older males being less responsive to anabolic stimuli (lower protein synthesis at P125) and more responsive to a catabolic state (protein breakdown at P63). In summary, changes in skeletal muscle mRNA levels to protein intakes near or below the RDA are indicative of an early accommodative response in both age groups, and especially older males.

Support: USDA 03‐35200‐13779 & 98‐35200‐6151; NIH R01 AG15750, and Purdue Research Foundation.
Rapid, noninvasive phenotyping of the mouse right ventricle using MRI
Authors: Chuang Joyce Shaw‐An , Omens Jeff , McCulloch Andrew , Sheikh Farah , Frank Larry
Published: June 2021

Genetically modified mouse models are powerful tools in cardiovascular disease research. Several techniques have been used to quantify global geometry and function of the mouse left ventricle. However, characterization of the right ventricle (RV) has been more difficult because of its non‐symmetric shape. Magnetic resonance imaging (MRI) is commonly used to obtain in vivo data of human or large animal hearts and is particularly useful in characterizing smaller hearts. We have developed a technique for quickly generating a 3D biventricular model of a mouse heart from gated, high‐resolution MR data. Spatial data in both the short and long‐axis directions are collected to accurately reconstruct both ventricles for volume and mass measurements. Our technique was used to study RV function in cardiac specific desmoplakin knockout (DPKO) mice. DP mutations have been linked to human patients with arrhythmogenic right ventricular dysplasia/cardiomyopathy (ARVD/C), a disease characterized by RV dysfunction. The results show that when compared to wildtype mice, DPKO mice have larger RV end diastolic volumes (90±4 vs. 48±5 ul) and lower ejection fractions (16±3 vs. 39±4%). Funding Source: NSF
Studies on the Regulatory Roles of the Yeast V‐ATPase Subunit C
Authors: Gonzales Kristen , Parra Karlett J.
Published: June 2021

V‐ATPases are highly conserved ATP‐driven pumps. Located at the membrane of acidic intracellular organelles such as lysosomes and vacuoles, V‐ATPases pump protons from the cytosol and acidify the luminal pH. They consist of two domains; a peripheral domain (V1) and a membrane‐bound domain (Vo). V1 is responsible for ATP hydrolysis, and Vo functions as the pore for the transfer of protons. V‐ATPases are dynamic molecules. The V1Vo complex is assembled when glucose concentrations in the growing medium are high. However, as glucose is consumed, the complex disassembles into three components: V1 without subunit C, Vo, and subunit C. Disassembly inactivates the V‐ATPase pump, and readdition of glucose triggers reassociation of the three components, as well as reactivation of the enzyme. Since subunit C dissociates from the rest of the complex upon glucose depletion and reassociates in the presence of glucose, it is proposed that this subunit regulates interactions between the V1 and Vo domains. The purpose of this study is to determine the role of subunit C in V‐ATPase regulation. We tagged the C‐terminus of subunit C with GST in order to perform pull‐down experiments. We will then determine if subunit C interacts with other molecules upon dissociating from V1 Vo. In the long‐term, this study will provide new insights into the mechanisms that control V‐ATPase function, organelle acidification, and glucose‐triggered responses.

Supported by NIH‐IMSD and NSF
SKATE: Recognition, experience, willingness and intake of fruit and vegetable snacks in rural school aged children at risk for obesity
Authors: McCabe‐Sellers Beverly J , Strickland Earline , Lovera Dalia , Bogle Margaret L
Published: June 2021

School Kids Access to Treats to Eat (SKATE) was a fruit and vegetable snack feeding study that assessed food recognition, food experience, and willingness to try specific fruits and vegetables of 186 elementary school children in Grades 4–6 at high risk for obesity. This rural school had a 100% free breakfast and lunch program, averaging 95% daily participation. Fruit and vegetable snacks were offered as an integral part of the school day and were available to all consented, assented children. Study participation was over 84 percent. A food recognition and experience checklist was used at baseline and at week nine to assess changes in a pilot fruit and vegetable snack program that offered pre‐weighed gram portions and post‐weighed refuse of 13 fruits and 5 vegetables snacks. Willingness to try new foods was measured by interviews using flash cards of specific foods. Teachers served as role models by consuming snacks with their classes and by incorporating food facts sheets into lesson plans. Children who initially recognized the foods were more willing to try and also consumed higher percentages of the snacks. While the majority reported being willing to try the snacks, even more actually tried the snacks, demonstrating fruit and vegetable snacks as a feasible approach to promote healthier food choices among children at high risk for obesity. Supported by USDA, ARS #6251‐53000‐004‐00D.
A culturally relevant nutrition education program targeting Hmong immigrants
Authors: Wakimoto Patricia , Donohue Susan , Fujii Mary L , Peterson Lisa , Martin Anna , Sutherland Barbara
Published: June 2021

The Expanded Food and Nutrition Education Program (EFNEP) is a unique program funded by the USDA designed to assist limited‐resource audiences in acquiring the knowledge, skills, attitudes, and behavior necessary for improved health. The program is intended to contribute to their personal development and the improvement of the total family diet and nutritional well‐being. California is host to a diverse population and a collaborative effort over several years has resulted in an adaptation of a series of lessons that has overcome linguistic and cultural challenges. The objective of this research project was to deliver a culturally relevant adaptation of the EFNEP curriculum and examine outcomes, using a revised evaluation instrument. The lessons included a learner‐centered approach that was complemented with video clips on meal planning, increasing physical activity, reducing sugar intake and promoting families eating together. The lessons were delivered by bilingual, Hmong paraprofessionals. Seventy‐five participants completed the program. Results included gaining practical skills in food handling and safety and improvement in self‐reported behavior changes, including “planning ahead” for meals and saving money when food shopping. This project serves as a successful model with potential for reaching other underserved diverse populations.
Animal source food intake is lower in households with children who experience food insecurity
Authors: Marquis Grace S. , Colecraft Esi K , Sakyi‐Dawson Owuraku , Lartey Anna , Ahunu Ben , Butler Lorna M , Jensen Helen H , Reddy Manju B , Lonergan Elisabeth , Canacoo Emmanuel , Aryeetey Richmond
Published: June 2021

Animal source food (ASF) intake in young children has been associated with improved dietary quality and growth outcomes. The ENAM project provides technical and financial support for income generation activities of caregivers of young children in selected districts in Ghana, with the goal of improving household food security and ASF utilization in children's diets. Baseline data from the ENAM project was analyzed to compare child dietary intakes and anthropometry between 123 households that experienced child‐level food‐insecurity and 391 households in which children were food secure. Caregiver wealth rank (p<0.001), occupation (p=0.004), weekly income (p=0.011), and locality (p<0.001) were associated with food security among the children in the past month. The likelihood of eating ASF and the frequency of consuming ASF within the past week were positively associated with children's food security (p<0.01). However, child WHZ (95% CI: −0.20, 0.22); WAZ (95% CI:−0.18, 0.25) and HAZ (95% CI:−0.22, 0.35) did not differ by food security status. In this setting, food security was related to ASF intake but not anthropometry in 2‐ to 5‐y‐old children. Support was through GL‐CRSP, funded in part by USAID, Grant # PCE‐G‐00‐98‐00036‐00.
Eating competence change in web‐based non‐dieting curriculum appears gender‐specific
Authors: Lohse Barbara Ann , Patterson Jill , Stotts Jodi L , Do Mi‐Young , Greene Geoff W , White Adrienne A
Published: June 2021

The ecSatter Inventory (ecSI), a validated survey with 4 subscales, assessed change in eating competence (EC) following university student (N=133, mean age 19.6 ± 1.2 y) participation in a 10‐lesson web‐based curriculum that utilized a non‐dieting approach to weight management. 4 of the 10 lessons focused on EC constructs related to eating enjoyment, food acceptance (FA), internal regulation (IR) and food contextual skills (CS). EC responses were gender‐specific. For males (N=45), a decrease in eating enjoyment was the only significant change (P=.04). Females (N=88) significantly increased total ecSI score (P=.04) and FA (P=.001); CS tended to increase (P=.07). At baseline, females scored significantly lower than males for total score (29.3 ± 7 vs 34.6 ± 7.1; P<.001), eating enjoyment (10.0±3.4 vs 12.5±2.5; P<.001), FA (4.9 ±2.5 vs 6.0 ±2.5; P=.025), and IR (6.3±1.8 vs 7.4±1.6; P=.001). Following intervention, only eating enjoyment remained significantly lower for females compared to males (10.1±3.6 vs 11.6±2.9; P=.018). EC changes following the intervention were compared between males and females; male EC deteriorated with trends toward or significant decreases in total ecSI score (−.95 vs +1.5; P=.058) and eating enjoyment (−.84 vs +.24; P=.049). Body mass index remained unchanged and unrelated to ecSI scores or gender. Further research is needed to ensure that gender is not masking impact as a result of intervention‐driven cogitation affecting perceived competence upon transitioning from a pre‐contemplative stage in EC. Supported by NRI, USDA CSREES, grant #2005‐ 35215‐15412, PSU Dept Nutritional Sciences
Biotinylation and methylation of histones play a role in regulation of genes associated with hydrogen peroxide induced oxidative stress
Authors: Wijeratne Subhashinee Samudra , Zempleni Janos
Published: June 2021

The expression of approximately 10,000 genes changed in response to oxidative stress by H2O2 in Caco‐2 cells. Three genes, epidermal growth factor receptor (EGFR), the oncogene homolog KRAS, and guanidinoacetate N‐methyltransferase (GAMT), which showed the highest fold‐change in their expressions, were chosen to evaluate whether chromatin remodeling events play a role in stress‐dependent gene regulation. Relative enrichment of two epigenetic markers, biotinylation of lysine 12 in histone H4 (K12BioH4) and dimethylation of lysine 9 in histone H3 (K9Me2H3) in the promoter regions of the above genes was quantified by ChIP assays. Increased expression of EGFR and KRAS in stressed cells was associated with decreased relative abundance of K9Me2H3, whereas decreased expression of GMAT was associated with increased relative abundance of K9Me2H3 in promoter regions. The relative abundance K12BioH4 decreased in promoter regions of all three genes in stressed cells regardless of their observed expression changes, probably due to low expression of holocarboxylase synthetase (HCS), the mediator of biotinylation. These results suggest that epigenetic events may play a role in the expression of EGFR, KRAS and GMAT genes in oxidatively stressed cells. Support: Univ. of Nebraska ARD, Hatch Act, and NIH DK063945 and ES015206, USDA 2006‐35200‐17138, and NSF EPSCoR EPS‐0701892.