bluuebunny/crossref_metadata_2025 huggingface dataset:
Nutraceutical nanoemulsions: influence of physical states of β‐carotene (crystalized versus dissolved) on bioaccessibility (645.13)
Authors: Xia Ziyuan , McClements David , Xiao Hang
Published: June 2021

β‐Carotene has a poor oral bioaccessibility in the gastrointestinal tract (GIT) because of its high hydrophobicity and melting point. We investigated the bioaccessibilities of solubilized and crystalline forms of β‐carotene using a simulated GIT model. Three systems were compared: (1) soluble β‐carotene in digestible nanoemulsion (d< 200nm); (2) crystalline β‐carotene added to digestible nanoemulsion (d< 200nm); (3) crystalline β‐carotene added to phosphate buffer saline (pH=7.0). Oil‐in‐water nanoemulsions were formed by high‐pressure homogenization using Tween 20 as emulsifier and corn oil as digestible carrier oil. They were then subject to simulated mouth, stomach and small intestine digestion. The rate and extent of free fatty acid production in the small intestine decreased in the order (2)>(1)>(3); whereas the β‐carotene bioaccessibility decreased in the order (1)>>(2)>(3). The low bioaccessibility (1.3%) of system (3) was attributed to the fact that there were few free fatty acids produced to form mixed micelles to solubilize β‐carotene. The bioaccessibility of system (2) was also relatively low (3.6%) compared to system (1) (42%), which suggests that crystalline β‐carotene is less bioaccessible than soluble β‐carotene. This study provides important information for choosing a better delivery condition for lipophilic bioactive components to achieve higher bioaccessibility.

Grant Funding Source: Supported by USDA
Carotenoid content and characteristic profile of Zimbabwean Brassica oleracea green vegetables (645.3)
Authors: Muzhingi Tawanda , Tang Guangwen
Published: June 2021

Green vegetables of the brassica oleracea family are an important part of Zimbabwean diet, consumed daily as casseroles together with the maize based staple food. These green vegetables are rich in carotenoids such as lutein and β‐carotene which are important for macula health and as precursors of vitamin A. In this study five different brassica oleracea vegetable commonly grown and consumed in Zimbabwe where identified as chou mollier, covo, rugare, viscose and drum head cabbage. The green leafy vegetables were cut into pieces and ground into a puree; the carotenoids were extracted with methanol and tetrahydrofuran (THF). The carotenoid contents and profile were compared to kale grown and consumed in the US. Statistical Analysis was conducted with SAS (SAS Inc., NC) using descriptive statistics and ANOVA. All brassica oleracea vegetables analyzed contained lutein, trans β‐carotene and 9 cis β‐carotene with the contents being significantly different among the 6 varieties. Kale grown in the USA had the highest content of lutein (12.5 ±1.3) and β‐carotene (8.5 ±0.8) mg/100g fresh weight respectively. Among the Zimbabwean green vegetables covo variety had the highest contents of lutein (10.4±0.5) and β‐carotene (7.8±0.4) mg/100g fresh weight. rugare (5.2 ±0.3) and cabbage (6.0 ±0.2) mg/100g fresh weight had the lowest trans β‐carotene content. Zimbabwe green vegetable had a 13 cis β‐carotene isomer as opposed to the US kale. Zimbabwe Viscose variety also had phytene and phytophlene carotenoid compounds. The Zimbabwean brassica oleracia vegetable are a very good source of provitamin A carotenoids and lutein. The provitamin A carotenoids content is sufficient enough to justify the promotion of these vegetables as part of a diet to combat vitamin A deficiency which is a public health problem in Zimbabwe. Funding support from International Atomic Agency and USDA Financial support USDA contract #58‐1950‐0‐014

Grant Funding Source: Supported by USDA #58‐1950‐0‐014: IAEA
Cranberry extract promotes anti‐inflammatory activity of Lactobacillus plantarum on HT‐29 human colon cancer cells (647.35)
Authors: Nguyen Thuy , Kimble Lindsey , Mathison Bridget , Chew Boon
Published: June 2021

Probiotics modulate gastrointestinal inflammation by converting non‐digestible dietary nutrients into metabolites for colon epithelial health. Cranberry proanthocyanidins (PACs) can prevent urinary tract infection; however, the role of PACs as a prebiotic in the gastrointestinal tract is unclear. We studied the prebiotic effect of cranberry extract in promoting the anti‐inflammatory activity of Lactobacillus plantarum (Lp) on HT‐29 human colon cancer cells. Lp was incubated overnight with HT‐29 cells at a 10:1 ratio in the presence of 0, 0.01, or 0.1 mg PAC/mL cranberry extract. The presence of Lp decreased (P<0.05) IL‐1β, IL‐2, IL‐6, and TNF‐α production by HT‐29 cells, indicating anti‐inflammatory activity. Addition of cranberry extract to HT‐29+Lp co‐cultures decreased (P<0.05) NF‐κB and IL‐1β gene expression in HT‐29 cells in a dose‐dependent manner when compared to Lp alone. Cranberry extract (0.1 mg PAC/mL) downregulated (P<0.05) COX‐2, and upregulated (P<0.05) IL‐10 expression and superoxide dismutase activity in HT‐29 cells compared to Lp alone, suggesting enhanced antioxidant activity. Cranberry extract at 0.01 mg PAC/mL decreased (P<0.05) while at 0.1 mg PAC/mL increased (P<0.05) IL‐4 and IL‐8 production compared to Lp alone. Therefore, cranberry extract has a potential anti‐inflammatory and anti‐oxidant role in promoting Lp probiotic activity on HT‐29 cells.

Grant Funding Source: Supported by ARC, Washington State University
Preserved mitochondrial calcium retention capacity in summer‐active Arctic ground squirrels undergoing normothermic myocardial ischemia/reperfusion injury (663.9)
Authors: Dulai Gurpreet , Quinones Quintin , Podgoreanu Mihai , Barnes Brian , Riess Matthias
Published: June 2021

Arctic Ground Squirrels (AGS) exhibit a high degree of endogenous resistance against myocardial ischemia/reperfusion (IR) injury compared to the best protected rat strain (Brown Norway, BN). We tested if this species‐specific myocardial protection is associated with increased mitochondrial calcium retention capacity (CRC). Isolated hearts from BN (n=15) and summer‐active AGS (n=11) underwent 45 min global cardioplegic ischemia and 60 min reperfusion with Krebs solution containing 7.5 mM glucose or 1% Intralipid in addition. Left ventricular pressure was measured isovolumetrically. Cardiac mitochondria were isolated and CRC was measured by spectrofluorometry of calcium green at 503/532 nm excitation/emission for complex I and II substrates in response to intermittent calcium boluses. While multiple regression analysis revealed intralipid, female gender and species (AGS) as predictors of better myocardial function following IR, predictors of improved CRC were AGS and mitochondrial substrate only. These results add to our previous reports of species‐dependent mitochondrial coupling of oxidative phosphorylation as the most important predictor of cardioprotection in our model and underline the importance of broadly preserved mitochondrial function as a key element of attenuated IR injury. Supported by Department of Veterans Affairs (CARA‐026‐10F), NSF, NIH, USAMRMC, FAER and institutional funds.

Grant Funding Source: Supported by Department of Veterans Affairs, NSF, NIH, USAMRMC, and FAER.
Ubiquinol reduces leukocyte‐endothelium interactions in the mesenteric microcirculation following hemorrhagic shock and fluid resuscitation (667.1)
Authors: Shen Qiuhua , Holloway Naomi , Thimmesch Amanda , Wood John , Clancy Richard , Pierce Janet
Published: June 2021

Hemorrhagic shock (HS) and fluid resuscitation (FR) generate reactive oxygen species (ROS) which is a major factor in post‐hemorrhage inflammation and tissue injury. The hypoxia created by HS/reperfusion injury (RI) alters the microvascular blood flow and microvascular oxygen. The objective of the study was to determine if ubiquinol (reduced form of coenzyme Q10) reduces leukocyte‐endothelium interaction following HS/FR. Intravital microscopy was used to measure leukocyte‐endothelium interactions in the rat mesentery following 1‐hour HS and 2‐hour post FR with or without ubiquinol. HS was induced by removing ~40% of the rats’ blood volume resulting in a mean arterial blood pressure (MABP) being ~44 mmHg for 1‐hour. Immediately afterwards ubiquinol (1 mg/100 g body weight) was infused intra‐arterially. RI was produced by replacing the shed blood and Lactate Ringer’s. Below are the findings (n=6, *p < 0.05):

Group
Leukocyte Adherence
Mast Cell Degranulation
Reactive Oxygen Species
Vascular Permeability

Control
10.8 ± 2.3
1.36 ± 0.03
427 ± 82
0.54 ± 0.05

Ubiquinol
2.3 ± 2.0 *
1.02 ± 0.01*
159 ± 23*
0.34 ± 0.02*

These results indicate that ubiquinol protects endothelial function following HS/RI through its antioxidant and its anti‐inflammatory properties.

Grant Funding Source: Supported by DOD, TSNRP, Uniformed Services University of the Health Sciences (HU0001‐11‐1‐TS09).
Cardiomyocyte sialylation and complex N‐glycosylation protect against dilated cardiomyopathy and heart failure (697.2)
Authors: Deng Wei , Ednie Andrew , Bennett Eric
Published: June 2021

Human congenital disorders of glycosylation are characterized by variably reduced glycoprotein glycosylation resulting in multi‐system effects, including cardiac dysfunction. We seek to understand whether and how reduced glycosylation contributes to cardiac symptoms and reported previously that the reduced glycoprotein sialylation that results from the global gene deletion of a cardiac expressing sialyltransferase, ST3Gal4, contributed to pressure‐induced heart failure (FASEB J, 2013, 27:595.2). Here we questioned whether cardiomyocyte‐specific (αMHC‐Cre) deletion of mannosyl (alpha‐1,3‐)‐glycoprotein beta‐1,2‐N‐acetylglucosaminyltransferase, Mgat1, responsible for initiating synthesis of complex and hybrid N‐glycans, contributes to the onset of dilated cardiomyopathy. At age 16 weeks (earliest time point tested), homozygous floxed Mgat1 and αMHC‐Cre positive mice, Mgat1F/FαMHC‐Cre, showed significantly reduced ejection fraction (EF) and fractional shortening (FS) compared to littermate controls (in %, for controls (n=8), EF = 73.8 ± 2.1, FS = 42.5 ± 1.8; for Mgat1F/FαMHC‐Cre mice (n=7), EF = 61.4 ± 2.3; FS = 32.8 ± 1.6; p<0.002). Systolic function of Mgat1F/FαMHC‐Cre mice, but not of control mice, continued to deteriorate with age (at 30 weeks, Mgat1F/FαMHC‐Cre mice (n=5) in %: EF = 38.2 ± 4.5; FS = 18.8 ± 2.5; p<0.0005). Mgat1F/FαMHC‐Cre mice presented with apparent dilated cardiomyopathy indicated by reduced systolic function, a thinner wall and larger internal diameter of the left ventricle (p<0.0005) with no apparent increase in left ventricular mass index, and marked right and left ventricular dilatation observed during necropsy. Together, our data suggest sialylation and complex N‐glycosylation are necessary for normal cardiac function, and reduced cardiomyocyte N‐glycosylation contributes to the onset of significant ventricular systolic dysfunction consistent with dilated cardiomyopathy leading to heart failure.

Grant Funding Source: Supported by NSF grant # IOS‐1146882
Early life nutrition influences physical activity level and the contractile and metabolic phenotype of the mouse plantaris (706.17)
Authors: Ferguson David , Martinez C.J. , Scull Brooks , Fiorotto Marta
Published: June 2021

The objective was to determine the effects of postnatal nutrition on skeletal muscle composition and voluntary physical activity. Newborn FVB female pups (n=18) were fostered by dams fed a control (CON; 20% protein) or protein‐restricted (PR; 8% protein) diet. After 3 wks all pups were weaned to a CON diet and individually housed with locked (SEDentary) or free‐moving (EX) running wheels. After 3 wks the contractile and metabolic properties of the plantaris were characterized by immunohistochemistry for myosin heavy chain (MHC) isoforms and succinate dehydrogenase (SDH) enzyme. Compared to SED CON, SED PR pups had a 60% higher proportion of oxidative fibers (P=0.03), 16% more intermediate fibers (P=0.06), and fewer glycolytic fibers (P=0.01); SED PR mice had 11% fewer MHC IIB fibers (P=0.03), 40% more IIX fibers (P=0.001) and similar IIA fibers. In EX CON pups the proportion of oxidative (P=0.024) and IIA fibers increased by 70% and 75%, respectively (p=0.001), and glycolytic, IIB (p=0.003) and IIX (p=0.005) fibers decreased. There was no effect of EX on the metabolic composition of the fibers in PR mice, whereas the changes in MHC were similar to those of EX CON mice. PR mice ran less than CON pups (55.2±4.8 vs 130.1 ± 5.1 km, respectively; P<0.001).Thus, early postnatal PR appears to result in the dissociation between the metabolic and contractile properties of skeletal muscle that may contribute to impaired wheel running.

Grant Funding Source: Supported by USDA
Co‐administration of Neptazane and aminophylline to men abrogates the decline in endurance exercise performance in simulated high‐altitude (708.6)
Authors: Scalzo Rebecca , Binns Scott , Giordano Gregory , Paris Hunter , Sevits Kyle , Beals Joseph , Biela Laurie , Larson Dennis , Luckasen Gary , Irwin David , Schroeder Thies , Hamilton Karyn , Bell Christopher
Published: June 2021

During a sojourn to high altitude, endurance exercise performance is diminished; pharmacotherapy may abrogate this performance deficit. We hypothesized oral administration of Neptazane (N), Aminophylline (A), and/or N combined with A would attenuate hypoxia‐mediated decrements in endurance exercise performance in humans. Healthy males (26±1 years, body mass index: 24.9±0.4 kg/m2; mean±SE) were randomly assigned to one of four treatments: placebo (n=9), N (250 mg; n=10), A (400 mg; n=9), or N (250 mg) combined with A (400 mg; n=8). On two separate occasions, the first in normoxia (21% O2) and the second in hypoxia (15% O2), participants sat for 4.5 hours before completing a standardized exercise bout (30 min, stationary cycling, 100 W) followed by a time trial (time to cycle 12.5 km). The magnitude of time trial performance decrement in hypoxia did not differ (P>0.09) between placebo (+3.0±1.0 min), N (+1.4±0.5 min) and A (+1.8±0.4 min), however the performance decrement with N combined with A was less than placebo (+0.6±0.5 min; P=0.01). This improvement may have been partially mediated by increased SpO2 in hypoxia with N combined with A, compared with placebo (73±1 vs. 79±2%; P<0.02). In adult men, N combined with A abrogated the hypoxia‐mediated decrement in endurance exercise performance. Co‐administration of N and A may promote endurance exercise performance during a sojourn to high altitude.

Grant Funding Source: Supported by DARPA: N66001‐10‐C‐2134
Dibutyl phthalate and chromium, contaminants found in urban rivers, increase aggressive behavior of the freshwater prawn Macrobrachium rosenbergii (728.26)
Authors: Vicente Laura , Pérez Erick , Hernández Andrea , Sosa María , Rivera Nilsa
Published: June 2021

Freshwater ecosystems are being affected by many sources of pollution related to anthropogenic activities, particularly in highly developed urbanized areas. Dominance and fighting behavior in crustaceans vary depending on a number of circumstances such as food availability, shelter, reproductive partners, environment, health status, and exposure to contaminants. The impact of urban contaminants on crustacean behavior has not been thoroughly studied yet and may serve as a means to monitor potential contamination in rivers in highly populated regions, such as the island of Puerto Rico, We hypothesized that organic compounds, such as phthalates, and heavy metal contaminants present in urban rivers of Puerto Rico can affect interactive behaviors of the freshwater prawn even at relatively low concentrations. Submissive prawns of interacting pairs were injected with dibutyl phthalate (DBP) or chromium (Cr III), at levels allowed by the Environmental Protection Agency for drinking water. Results show that low concentrations of both DBP and Cr III increase levels of aggression of submissive prawns in a reversible and repeatable manner. It is possible that these contaminants may have an effect in the mechanisms and neural circuitry underlying aggression in the freshwater prawn. Additional studies using immunohistochemistry and electrophysiology approaches will be conducted to test this possibility.

Grant Funding Source: NSF HRD‐1137725; NSF DBI‐0932955; NIH NIGMS 5SC3GM084763
The E. coli single stranded DNA binding protein SSB interacts with the UmuD polymerase manager protein (735.7)
Authors: Naniong MarkVic , Silva Michelle , DiBenedetto Arianna , Dang Celeste , Alves Christine , Chang Monyrath , Beuning Penny
Published: June 2021

Escherichia coli single‐stranded DNA binding protein (SSB) binds to and protects the ssDNA present in various DNA processing mechanisms. It has been shown that SSB inhibits replication by binding to the C‐terminal domain of the α subunit of DNA polymerase III, the main replicative polymerase in E. coli. Though this interaction between SSB and α occurs through the globular N‐terminal domain of SSB, it has also been shown that SSB serves as a hub for different proteins involved in DNA processing pathways, through interaction with its C‐terminal tail. One such protein is DNA polymerase V, a Y‐family polymerase encoded by the umuDC genes. The interactions between UmuC, the polymerase subunit of DNA pol V, and SSB have been shown to increase access to the primer termini, and thereby increase the efficiency of pol V. In addition to the previously observed interaction of UmuC with SSB (Arad 2008 JBC 283:8274), we have determined that UmuD also physically interacts with SSB, which is weakened by the SSB‐113 (P177S) mutation. We are currently further characterizing this interaction. Supported by NSF and the American Cancer Society.

Grant Funding Source: NSF and American Cancer Society
N‐terminal conserved cysteines of Env7 are palmitoylated and direct its stability, localization, and vacuolar fusion regulation in S. cerevisiae (739.12)
Authors: Gharakhanian Editte , Manandhar Surya , Calle Erika
Published: June 2021

Palmitoylation at cysteine residues is the only known reversible lipid modification and has been implicated in protein membrane association as well as function. There is growing evidence that many palmitoylated proteins have regulatory roles in dynamic cellular functions including membrane fusion. Recently, we identified Env7 as a palmitoylated vacuolar membrane protein‐kinase involved in negative regulation of vacuolar fusion. Our phylogenetic analyses have established Env7 as an ortholog of human STK16, an endomembrane kinase with no known function. Env7 contains a conserved palmitoylation consensus sequence and substitution of the three consecutive cysteines (Cys13‐15) within that domain renders the protein non‐palmitoylated and cytoplasmic. In this study, we further dissect and define the role of individual cysteines of the consensus sequence in vivo. Our results indicate that all three cysteines serve as palmitoylation substrates, and any pairwise combination is essential and sufficient for near wild type levels of Env7 palmitoylation, membrane association, phosphorylation, and fusion regulatory function. Furthermore, individually, each can serve as a minimum requirement for distinct properties of Env7 in cells. Cys13 is sufficient for membrane association, Cys15 is essential for the fusion/fission regulatory function, and Cys14 or Cys15 is essential for protection of membrane bounded Env7 from proteasomal degradation. A role for Cys14 and Cys15 in correct sorting at the membrane is discussed. Thus, Env7 palmitoylation at the N‐terminal triple cysteines directs not only its membrane association, but also its stability, phosphorylation, and cellular function.

Grant Funding Source: Supported by National Science Foundation
Role of superoxide dismutases and alkyl hydroperoxidases in pathogenesis of Pseudomonas syringae (741.1)
Authors: Bile Ernest , Guo Ming , Becker Will , Alfano Jim
Published: June 2021

Plants, such as Arabidopsis thaliana, release reactive oxygen species (ROS) such as the superoxide anion, hydroxyl radical, and hydroxyl ion as a defense mechanism when they are infected with microorganisms. The production of ROS results in restriction of the growth of pathogens like Pseudomonas syringae. In order to be a successful pathogen, P. syringae must suppress these responses when it infects plants. P. syringae has evolved redox‐related enzymes to detoxify ROS. The sods and Ahps are able to relieve oxidative stresses by detoxifying ROS into less reactive molecules using these redox‐related enzymes during infection. However, it remains unclear whether Sods and Ahp(s) also play a role in pathogenesis.

In this project, we set out to1) Clone and express sodA, sodB and sodC genes in E. coli, 2) Perform pathogenicity assay with the sodC mutant, and 3) Generate deletion mutants lacking both AhpC and AhpF. We successfully cloned the sodA, sodB and sodC genes using plasmid pLN615, and verified their authenticity by immunoblot of the expression extract. Pathogenicity assays revealed that the sodC mutant did not show significant differences in virulence from the wild type strain DC3000 in Arabidopsis plants. This suggests that the sodC gene may be redundant with the sodA and sodB in DC3000. We were also successful in generating P. syringae mutant lacking both AhpC and AhpF using an unmarked mutagenesis approach.

Grant Funding Source: NSF
High‐throughput discovery of post‐transcriptional regulatory elements (741.3)
Authors: Wissink Erin , Fogarty Elizabeth , Grimson Andrew
Published: June 2021

Our objective is to identify novel sequence elements that control mammalian post‐transcriptional gene regulation of messenger RNAs (mRNAs). To do so, we created a high‐throughput, fluorescence‐based screening technology. We used this screen to measure the regulatory potential of thousands of short sequences in the context of a specific position within one human 3′ untranslated region (3′UTR). From this screen, we discovered hundreds of previously unknown regulatory elements and validated dozens of them in an orthogonal reporter assay. To then test the importance of these novel elements in the post‐transcriptional regulation of endogenous human genes, we chose a subset of validated elements and identified human 3′UTRs containing that sequence, and we showed that mutating the identified element disrupts the expression of the tested gene. We are now using our screening technology to examine the importance of local 3′UTR context on the regulatory impact our candidate regulatory elements. Our preliminary data indicate that some elements function in all of the 3′UTRs we have tested, whereas others require specific local context to function. In conclusion, it is clear that many post‐transcriptional regulatory elements remain elusive, and our technology can discover them.

Grant Funding Source: Supported by NIGMS, NSF, and the American Cancer Society
The balance between protein folding and aggregation in the early stages of a protein’s life: role of the ribosome and molecular chaperones (752.6)
Authors: Liu Yue , Cavagnero Silvia
Published: June 2021

Protein biosynthesis takes place within the ribosomal machinery in the presence of the molecular chaperones trigger factor (TF) and DnaK, which are involved in the early stages of a protein’s life in bacteria. To date, the role of the ribosome, DnaK and TF in preventing co‐ and post‐translational protein aggregation is poorly understood. In this study, we used sperm whale apomyoglobin as a model protein to investigate how the ribosome, trigger factor and DnaK affect the balance between protein folding and aggregation, in the context of an E. coli cell free system. We detected both the soluble and insoluble fractions of newly synthesized apomyoglobin in a cell‐free transcription‐translation environment in the presence and absence of TF and DnaK. We took advantage of in situ labeling the N‐terminal methionine via the unnatural BODIPY‐tRNA fMet. Given that the genes encoding TF and DnaK cannot be both knocked out within the same cell strain, we developed a short peptide that can specifically inhibit DnaK activity, and added it into the cell free extract of a tig knock out strain to explore the translation in the absence of both DnaK and trigger factor. We also studied how these two chaperones and the ribosome affect incomplete nascent protein aggregation by examining the solubility of different truncated apomyoglobin constructs. Our results show that elimination of both TF and DnaK activity leads to accumulation of insoluble truncated polypeptides but does not affect the solubility of the full‐length protein. Our findings demonstrate that molecular chaperones are important for preventing any post‐translational aggregation of incomplete polypeptides but the ribosome alone suffices in promoting the successful folding of the full‐length protein.

Grant Funding Source: Supported by NSF grant
Investigating the GADPH‐Sir2 interaction in S. cerevisiae (763.2)
Authors: Palmer Claire
Published: June 2021

The yeast glyceraldehyde‐phosphate dehydrogenase (GAPDH), Tdh3, has been found to interact with the silencing protein Sir2 in S. cerevisiae. Sir2 is a protein deacetylase that removes acetyl groups from the tails of histones H3 and H4. Deacetylated histones are associated with more densely packed chromatin and thus, transcriptional silencing. The Sir2‐GAPDH interaction is being investigated in order to further understand this connection between metabolism and gene silencing. We are interested in determining what part of Sir2 is necessary for this interaction with Tdh3. To this end, Sir2 mutant strains for use in coimmunoprecipitation experiments have been created. We hope to identify strains in which coimmunoprecipitation of Sir2 with Tdh3 is unsuccessful, indicating that these strains are missing a part of Sir2 that is essential for Tdh3 interaction. We are also investigating the possibility that Sir2 could be interacting with Tdh3 in its capacity as a deacetylase. A previous genetic screen suggests that Sir2 may also interact with two ribosomal proteins, Rpl22a and Rps7a. Tdh3, a similar GAPDH protein, Tdh2, Rpl22a and Rps7a are therefore being tested for acetylation by immunoprecipitatation and probing with an anti‐acetyl lysine antibody. Thus far, no evidence of acetylation on Tdh3 has been observed. However, further experiments are necessary to confirm this result. Finally, to better understand Tdh3’s role in silencing, we are creating a library of Tdh3 mutants defective for promoting silencing.

Grant Funding Source: Supported by National Science Foundation
Protein splicing of a temperature‐dependent intein from an extreme thermophile (768.10)
Authors: Siegart Nicolle , Colelli Kathryn , Williams Jennie , Reitter Julie , Mills Kenneth
Published: June 2021

Inteins are intervening polypeptides that self‐catalyze their own excision from the flanking polypeptides, or exteins, in a process called protein splicing. We are studying an intein that interrupts genes from the archaebacteria Pyrococcus abyssi. The Pyrococcus abyssi (Pab) PolII intein only splices at elevated temperatures. The intein contains an N‐terminal cysteine and is preceded by an asparagine in the N‐extein at the ‐1 position. We have shown that protein cleavage occurs independent of the canonical splicing mechanism when the protein contains an alanine in place of the cysteine and an aspartate in place of the asparagine. In addition, we created a split version of the intein to determine whether when split into two fragments and recombined, the intein is capable of undergoing trans splicing.

Grant Funding Source: Supported by the National Science Foundation, the Dreyfus Foundation, and The BD Corporation
Structural analysis of human carbonic anhydrase II and a thermophillic intein (768.3)
Authors: Haley Daniel , Siegart Nicolle , Reitter Julie , Mills Kenneth
Published: June 2021

We are interested in various structural aspects of human Carbonic Anhydrase II (hCAII) and the Pyrococcus horikoshii PolII intein (Pho PolII). A combination of analyses including x‐ray crystallography and biochemical assays will be used to study each of the two proteins. We will compare the activity and structure of hCAII to hCAII where the active‐site zinc ions have been replaced with rhenium. Additionally, we will assay both zinc and rhenium hCAII for enzymatic functionality using a p‐NPA hydrolysis assay. Thus far, we have optimized expression in E. coli and purification of hCAII and have assayed for functionality. Our goal with the PolII intein is to obtain structures for the unspliced precursor and the excised intein in order to do structural comparisons. Our lab has recently crystallized the precursor and my focus is on the intein. The PolII precursor, which comes from a sea‐dwelling, thermophillic archaea, was mutated so that a stop codon preceded the poly‐His tag at the C‐terminal end of the intein before expression and purification were optimized. In order to isolate the intein from its remaining N‐extein, we used DTT to facilitate N‐terminal cleavage and will use size exclusion chromatography to separate the products.

Grant Funding Source: Supported by the National Science Foundation under grant MCB‐1244089 and by the Dreyfus Foundation.
Protein splicing of an intein from the extreme halophile Halobacterium salinarum (768.9)
Authors: Jaramillo Mario , Nicastri Michael , Reitter Julie , Mills Kenneth
Published: June 2021

Protein splicing is the self‐catalyzed excision of an intervening polypeptide (intein) from flanking polypeptides (exteins), concomitant with the ligation of the extein. An intein in the archaebacterium Halobacterium salinarum (Hsa) interrupts the DNA Polymerase II. We have observed that the Hsa intein splices poorly in Escherichia coli cells, unlike two highly related inteins found in Pyrococcus abyssi and Methanoculleus marisnigri, which are not halophilic. To test if splicing activity depends on salinity, we altered the expression conditions by varying the salinity of the growth media and of in vitro activity assays. Future experiments will involve in vivo techniques by over‐expressing the intein and the flanking exteins into Hsa, and by using antibodies to detect possible splicing of the native protein.

Grant Funding Source: Supported by the National Science Foundation under grant MCB‐1244089 and by the Dreyfus Foundation
Discovery of a unique starch degradation system in the thermophilic red alga Cyanidioschyzon merolae (769.18)
Authors: Husodo Satrio , Gentry Matthew
Published: June 2021

Starch is a key component in many aspects of daily life, including nutrition, biofuel production, and industrial processing: 50‐80% of daily caloric intake comes from starch; >20% of corn starch produced in the U.S. is converted to ethanol; and starch is a cheap and renewable industrial feedstock for producing paper, textiles, adhesives, plastics, and pharmaceuticals. Starch is comprised of glucose chains that intertwine into double helices that pack together to make starch insoluble and inaccessible to most enzymes, including the amylases that release its energy cache. Starch‐based feedstocks are generated by modifying starch using physical, chemical, and enzymatic methods. Physical modifications include cyclic heating/cooling between 50°C and >100°C. The addition of acids and bases to this process both catalyzes glucan chain hydrolysis and converts insoluble starch to a structure that improves accessibility of glucan chains to amylases. Alternatively, plants utilize an orchestrated reversible phosphorylation system to efficiently solubilize the starch surface, a process that can be mimicked to improve in vitro starch degradation. We recently discovered a unique dikinase‐phosphatase system in the thermophilic red alga Cyanidioschyzon merolae that is integral for starch degradation and different from systems seen in Kingdom Plantae. First, we defined the evolutionary phylogeny of this unique system. We cloned the genes, purified the enzymes from a recombinant expression system, and defined their kinetics and substrate specificities. Lastly, we demonstrated that these enzymes dramatically increase in vitro starch degradation of amylases. Collectively, this study provides a basis for understanding how glucan kinases and phosphatases function at the molecular level.

Grant Funding Source: NSF#1252345
Probing conformational changes with multiwavelength collisional quenching (769.6)
Authors: Bell Ellis , Barkdoll‐Weil Forest , Metz Carlos , Varnes Philip , Bartle Emily , Li Chun
Published: June 2021

Collisional quenching approaches using neutral or charged quencher molecules has long been used to indicate conformational changes in proteins Here we introduce a novel approach to collisional quenching using multiwavelength analysis of spectral data collected at two different excitation wavelengths with a variety of small molecule and protein test fluorophores. The results show that not only can detailed information about the local environment of tryptophans in proteins be obtained (even proteins with multiple tryptophans), but information is derived resulting from tyrosine‐tryptophan resonance energy transfer in proteins… Using Acrylamide, Iodide and Cesium collisional quenchers with both tyrosine and tryptophan derivatives and with model proteins with one or more tryptophans we demonstrate the utility of the approach for defining the local environment and changes induced by ligand binding, Furthermore, during the course of these studies we revealed that pi‐cation effects disrupt resonance energy transfer between tryptophan (acting as either donor or acceptor) and other moieties in the protein.

This work was supported by NSF Grants MCB‐104995 and MCB 0448905 to EB

Grant Funding Source: Supported by NSF Grants MCB‐104995 and MCB 0448905 to EB
Probing the role of the N‐terminus of Thermodesulfovibrio yellowstonii ADPglcuose pyrophosphorylase in allosteric regulation (769.9)
Authors: Yik Eric , Susoeff Michael , Kaur Sandeep , Dua Gautam , Holling Jaqueline , Meyer Christopher
Published: June 2021

ADPGlucose Pyrophosphorylase (ADPG PPase) catalyzes the rate‐limiting step of glucan biosynthesis in plants and bacteria. The successful engineering of ADPG PPase will allow for the production of more renewable and biodegradable carbon. Thermodesulfovibrio yellowstonii (Td.y) ADPG PPase shows only ~30% identity to other bacterial forms of the enzyme; at positions E15, F18, F23, S25, and S28 the Td.y enzyme differs from consensus in the glycine rich (positions 9‐17) and KRAKPAV regions (positions 23‐29), areas known to play roles in activity and allosteric regulation. These positions were targeted for mutagenesis and the enzymes purified using hydroxyapitite and DEAE chromatography. For wild‐type (WT), kinetic studies revealed a Vmax of 5.3 U/mg and S0.5 values of 4.0 mM, 13.9 mM, and 1.2 mM for ATP, Mg, and G1P, respectively. Metabolites PEP, G6P, and 3PGA increased the Vmax by 1.5‐3 fold and decreased the S0.5 values for ATP by 4.2, 10.3, and 3.8 fold, respectively. Initial kinetics for the variant S25A gave a Vmax ~3 fold higher and a 10‐fold lower S0.5 value for ATP compared to WT. S25 in Td.y may play a role in setting the activity level in the absence of effectors. Further experiments are being conducted to fully characterize S25A and other variants, particularly with respect to allosteric regulation. Efforts are also underway to crystallize these enzymes to solve the three dimensional structures.

Grant Funding Source: NSF Grant 0448676 and a CSUPERB Presidential Commission Grant
Effects of alcohol on the outer hair cell protein Prestin (775.2)
Authors: Minten Elizabeth
Published: June 2021

Prestin, a transmembrane motor protein, is a member of the SLC26 family and is highly expressed in mammalian outer hair cells located in the cochlea. Outer hair cells, in response to changes in membrane potentials, adjust in length and rigidity. The production of these mechanical changes is thought to amplify the vibrations in the cochlea, increasing hearing sensitivity in mammals by 100‐fold. Since its identification in 2000, prestin has been found to be the motor protein in outer hair cells and is necessary for electromotility of the outer hair cell and for cochlear amplification.

At this point, while the structure of prestin remains a mystery, previous experiments have indicated that prestin is sensitive to the levels of cholesterol in the cell membrane. Alcohols, which are also known modulators of lipid bilayer properties, could have the potential to have an impact on prestin function. It has been established that the link between the nonlinear capacitance of prestin and its electromotility is a surrogate measure of prestin function. Therefore, the effects of six alcohols ‐ ethanol, butanol, pentanol, hexanol, nonanol, and decanol, were each assessed at differing concentrations using patch‐clamp electrophysiology on HEK cells expressing prestin. The alcohols were chosen to have chain lengths smaller than 13, as alcohols with chain lengths above this cutoff have been shown to have limited biological activity and low solubility in cell buffers. All six of the alcohols tested showed either a depolarizing or polarizing effect.

Grant Funding Source: Supported by NSF, RICE University
Creation of tPA conjugated dendrimer nanoparticles (780.4)
Authors: Paliwal Vipin
Published: June 2021

A drug delivery system was created using nanoparticles called dendrimers. Tissue plasminogen activator (tPA), a clot busting drug given to stroke patients was covalently bonded to dendrimers using surface active organic functional groups including carboxylic acids, amines, and alcohol. Methods included esterification using a heterobifunctional coupling agent EDC (1‐ethyl‐3[3‐dimethylaminpropyl]carbodiimide hydrochloride. A fourth generation dendrimer (hydroxyl‐terminated poly(amidoamine) PAMAM dendrimers (G4‐OH) with 64 binding sites was chosen to bind the human tPA protein molecule (molecular mass of 72 kDa). Several dilutions of dendrimer were tested to achieve the best drug‐nanoparticle stoichiometric ratio and avoid overcrowding. Upon completion of the synthesis the drug‐dendrimer conjugate was purified using centrifugal filtration. The tPA‐dendrimer complex was tested for clot dissolving activity using an in vitro Thrombin assay. It was concluded that Succinamic acid functionalized dendrimer at a 1:1 drug to nanoparticle ratio was found to have the highest clot dissolving activity when compared to control tPA standard. This tPA‐dendrimer conjugate could be more effective, quicker reacting than tPA drug alone and may find use in treating stroke, pulmonary embolism and heart attack patients.

Grant Funding Source: National Science Foundation
Census tract income levels and outdoor physical activity opportunities for families with preschoolers (806.19)
Authors: Li Cheng , Martin‐Biggers Jennifer , Cheng Carolyn , Byrd‐Bredbenner Carol
Published: June 2021

The near home environment influences the PA and sedentary behaviors of adults and children. A valid, reliable, and easy to use self‐report assessment of the PA environment of homes with preschoolers was used to determine availability and accessibility of PA equipment and space in yards (areas immediately outside the home) and neighborhood PA facilities (e.g., playgrounds, low‐cost recreation centers). Scores for yard and neighborhood PA availability and accessibility were compared to the CTIL of the home’s address using 1‐way ANOVA. Homes (n=44) were categorized in low, middle, moderate, and upper CTIL (9%, 9%, 57%, 25%). Significant (p<.05) group differences were found for yard accessibility score and neighborhood availability score, but not for others. Post hoc tests indicated that the highest CTIL homes had more accessible yard PA equipment and space than lower CTIL homes. Highest CTIL homes also had significantly more neighborhood PA availability than lowest CTIL homes and neared significance for neighborhood accessibility (p=0.08). CTIL, as proxies for income, indicate that income disparities affect the PA availability and accessibility of families with preschoolers, and likely are a contributor to differences in obesity rates by income. Thus, eliminating “playground deserts” could be an important step in reducing obesity rates in CTIL areas.

Grant Funding Source: Supported by USDA, National Institute of Food and Agriculture, Grant Number 2011‐68001‐30170
Feasibility and effectiveness of in‐home behavioral economic strategies to increase vegetable intake among low‐income children (808.18)
Authors: Leak Tashara , Swenson Alison , Vickers Zata , Reicks Marla
Published: June 2021

Poor dietary behaviors put children at risk for obesity. The purpose of this intervention is to determine which behavioral economic strategies increase vegetable consumption among low‐income children (9‐12 years) in the Minneapolis/St. Paul metropolitan area. Caregivers (n=44) are randomly assigned 6 (of 10) in‐home behavioral economic strategies; one new strategy/week to be completed at 3 dinner meals for a total of 6 weeks. An example strategy is to make vegetables more available and visible than other foods during dinner. Intervention and control group (n=20) caregivers record child vegetable intake at 3 dinner meals and report intakes weekly to a trained researcher via telephone or in person. Researchers also inquire about barriers (e.g. lack of availability) and facilitators (e.g. time) for implementing strategies. Child vegetable liking, vegetable home inventory data, and three 24‐dietary recalls are also collected at baseline and immediately after the intervention. Most children reported liking more than 5 vegetable types; often the same vegetables available in the home and prepared by caregivers at dinner meals. Mean vegetable intake is being compared for each strategy across intervention and control groups to determine the effectiveness of each. The 6 most effective will be incorporated into the Cooking Matters for Families nutrition education program to increase vegetable intake among children.

Grant Funding Source: Supported by NIFA 2014‐00403
Hummus (H)/garbanzo bean (GB) consumption is associated with increased nutrient intake, better diet quality, and lower body mass index in adult participants (19+ y) from the NHANES (2003‐2010) (810.15)
Authors: O'Neil Carol , Nicklas Theresa , Fulgoni Victor
Published: June 2021

The association between H/GB consumption with nutrient intake, dietary quality, and weight parameters was examined in adults using data from NHANES (2003‐2010). Consumers (n=264) were defined as having any amount of H/GB during either 24‐hour diet recall. Means and ANOVA (covariate adjusted) were determined using appropriate sample weights; significance was p<0.05. Diet quality was calculated using the Healthy Eating Index‐2005 (HEI). Fiber (24.4±0.7 v 10.1±0.1 g/d), PUFA (19.5±0.4 v 17.3±0.1 g/d), vitamins A (787±42 v 640±6 RAE mcg/d), E (10.1±0.5 v 7.5±1.01 mcg/d), and C (119±8 v 86.4±4.3 mg/d), folate (627±16 v 547±4 mcg/d), magnesium (385±13 v 292±4 mg/d), potassium 3103±59 v 2697±12 mg/d), and iron (17.4±0.5 v 15.8±0.1 mg/d) intake was higher in consumers. Total fat (76.4±4.5 v 80.4±0.3 g/d), SFA (22.4±0.7 v 26.6±0.1 g/d), and cholesterol (227±8 v 288±2 mg/d) were lower in consumers. Consumers had higher HEI scores (62.2±1.3 v 51.9±0.2). BMI (26.4±0.5 v 28.6±0.1) and waist circumference (92.2±1.3 v 97.9±0.3 cm) were lower in consumers.Consumers were 43% less likely to be overweight or obese and had a 48% reduced risk of increased waist size. H/GB consumption was associated with better nutrient intake, diet quality and weight parameters in adults.

Grant Funding Source: Supported by Sabra Dipping Co., LLC., and USDA.
Normality of adult dietary protein requirement distribution (820.11)
Authors: Ross Richard , McCabe George , Campbell Wayne , Johnson Craig
Published: June 2021

Nitrogen balance data consistently suggest that the dietary protein requirement of adult humans follows a lognormal distribution. In contrast, statistical theory and the associated physiology imply that the protein requirement for healthy adults is normally distributed. The objective of this research was to reconcile this apparent conflict. An errors‐in‐variables simulation was conducted with parameters drawn from nitrogen balance data obtained in a study of 42 healthy adults (age 21‐81 years) who consumed controlled diets with protein intakes of 0.50, 0.75, and 1.00 g protein•kg‐1•d‐1 during three randomized 18‐day trials. Simulation results indicate that even when the true distribution of the protein requirement is normal, the observed (estimated) distribution is lognormal. Parameter estimates for the normal distribution obtained from the simulation enable policy decisions that more accurately reflect the distribution of the protein requirement in the adult population.

Grant Funding Source: Supported by NIH R01 AG15750 and USDA 98‐35200‐6151
Characterization of muscle plasticity in a neotropical migrant, Dumetella carolinesis (gray catbird) (878.2)
Authors: DeMoranville Kristen , Corder Keely , Russell Dave , Huss Janice , Schaeffer Paul
Published: June 2021

This study focuses on how avian migration is facilitated by changes in organismal metabolism, muscle structure, and muscle metabolism. These specific migratory changes were examined across the annual cycle on‐an organismal level by investigating whole body oxidative capacity (ability to use energy) and, on a tissue level by characterizing muscle tissue size and oxidative capacity. In order to quantify whole body oxidative capacity, summit metabolism was measured to determine the peak oxygen consumed by each individual. Birds were euthanized and the pectoralis (flight muscle) and gastrocnemius (leg muscle) were removed and weighed. Mitochondrial content of the muscle was quantified by measuring enzyme activity via citrate synthase and cytochrome oxidase assays to determine oxidative capacity of the muscles. Summit metabolism and flight muscle mass were greatest during southward migration compared to temperate breeding and tropical wintering. The Gray Catbird displays phenotypic plasticity during migration compared to non‐migratory periods suggesting that augmentation of the flight muscle and increased metabolism are necessary for migration.

Grant Funding Source: Supported by NSF #IOS‐1257455 to PJS
AltitudeOmics: the effect of high altitude ascent and acclimatisation on cerebral blood flow regulation (885.1)
Authors: Fan Jui‐Lin , Subudhi Andrew , Evero Oghenero , Bourdillon Nicolas , Kayser Bengt , Julian Colleen , Panerai Ronney , Lovering Andrew , Roach Robert
Published: June 2021

Adequate oxygen supply to the brain is critical to maintain brain function. Hypoxia presents a unique challenge in maintaining sufficient cerebral oxygen delivery (DO2). We assessed by ultrasound cerebral blood flow (CBF: internal carotid, vertebral arteries and middle cerebral artery velocity [MCAv]) and arterial blood pressure (index of cerebral autoregulation; CA) during rest and hypercapnic breathing (MCAv‐CO2 slope; index of cerebrovascular function) in 21 healthy subjects at sea‐level (SL) and upon arrival at 5260m (ALT1) and after 16 days of acclimatisation (ALT16). Cerebral DO2 was calculated as the product of arterial oxygen content (CaO2) and flow in each respective artery and summed to estimate global CBF. Global CBF increased ~70% upon arrival at ALT1 (P<0.05) and returned to SL values at ALT16 as a result of changes in cerebral vascular resistance. A reciprocal pattern in CaO2 maintained global cerebral DO2 across acclimatisation. MCAv‐CO2 slope was elevated by ~79% upon arrival at ALT1 and further increased by ~89% at ALT16 (P<0.05). Indexes of CA were reduced upon arrival at ALT1 (P<0.05), but did not change with acclimatisation at ALT16 (P>0.10). Cerebral DO2 was well maintained upon acute exposure and acclimatisation to hypoxia. Cerebrovascular function was enhanced with acclimatisation to high altitude, but these changes did not mitigate the reduction in CA associated with hypoxic exposure.

Grant Funding Source: Supported by U.S. Department of Defense