bluuebunny/crossref_metadata_2025 huggingface dataset:
β‐Hairpins as stability tags for recombinant expression of recalcitrant small helical proteins (951.1)
Authors: Lokensgard Melissa , Love John
Published: June 2021

We have empirically established different expression levels in E. coli for two recombinant proteins with high similarity in sequence, size, and native function. Streptococcal Protein G (Gβ1) and many mutants can be easily expressed in high yield in BL21 (DE3) E. coli. Staphylococcal Protein A (SpAB) can only be expressed with amino‐terminal tags or in periplasmic expression vectors. The primary difference between Gβ1 and SpAB is secondary structure composition‐‐Gβ1 contains two short beta‐hairpins and an alpha helix, and SpAB is a three‐helix bundle. Reasoning that the beta‐hairpins may impart unique stability, several chimeras of SpAB and the Gβ1 beta‐hairpins were created and induced expression levels analyzed by SDS‐PAGE. While the addition the very stable C‐terminal hairpin to the N‐terminus of SpAB (CH‐PA) results in higher yield, this same tag at the C‐terminus of SpAB (PA‐CH) does not. However, addition of 12 amino acids of the N‐terminal hairpin of Gβ1 to either terminus of SpAB (NH‐PA and PA‐NH) enhances recombinant expression significantly. Interestingly, both CH‐PA and NH‐PA sequences begin with hydrophobic residues, but do not appear to be significantly degraded despite well‐established correlations between N‐terminal hydrophobics and proteolysis. An expression yield increase relative to the untagged protein by addition of NH to either terminus is also observed for two entirely unrelated proteins of similar size and secondary structure to SpAB. All of the mutants containing the beta‐hairpin sequence are soluble at neutral pH. Our result may be due to decreased degradation of the chimeric host E. coli proteolytic machinery. Current and future work will explore what changes in structure and physical properties account for the observed yield increase.

Grant Funding Source: NSF
Vitamin D status and metabolism in pregnant, lactating, and nonpregnant women consuming controlled intakes of vitamin D (959.7)
Authors: Park Hey Jun , Brannon Patsy , West Allyson , Yan Jian , Jiang Xinyin , Perry Cydne , Malysheva Olga , Mehta Saurabh , Caudill Marie
Published: June 2021

As part of a 10‐wk controlled feeding study, we investigated the impact of reproductive state on circulating levels of vitamin D metabolites among pregnant (n=26, 27wk gestation), lactating (n=28, 5wk postpartum), and nonpregnant (n=21) women consuming a daily intake of 300 IU vitamin D derived from food (90IU/d) and a prenatal supplement (200IU/d). At study‐end, serum 25‐hydroxyvitamin D (25[OH]D, mean±SD nmol/L) was higher in pregnant women (98±32) than lactating (81±19, P=0.044) and nonpregnant (78±25, P=0.032) women. Similar results were achieved after controlling for confounding factors such as season in a multivariate model. Notably, 300 IU (half of the current RDA) achieved 25(OH)D levels above the RDA target value of 50nmol/L in most of study participants (92%). Study‐end plasma 1,25‐dihydroxyvitamin D (mean [95% CI] pmol/L) was ~2 times higher in pregnant women (303 [252‐364]) than lactating (139 [116‐167], P<.0001) and nonpregnant (163 [139‐191], P<.0001) women. Similarly, study‐end vitamin D binding protein levels (mean [95% CI] mcg/mL) were ~1.8 times higher in pregnant women (370 [291‐470]) than lactating (161 [131‐198], P<.0001) and nonpregnant (205[166‐255], P=0.001) women. In sum, pregnancy dramatically influenced circulating vitamin D metabolites under controlled intakes of vitamin D and related nutrients (e.g., calcium and phosphorus).

Grant Funding Source: USDA, AEB‐ENC and the Beef Checkoff
Thermodynamics and kinetics of calmodulin/roponic chimeras (969.1)
Authors: Wei Chin‐Chuan , Jensen Drake , Bennett Katherine
Published: June 2021

Calmodulin (CaM) and Troponic C (TnC) belong to an EF‐hand protein family which contains 4 EF‐hands and share very similar primary sequences and three dimension structures. However, their specificities to target proteins are quite different. For example, enzymes activated or recognized by CaM are generally not activated by TnC. In the past, chimera proteins containing EF‐hands from CaM and TnC have been used to understand the function of individual EF‐hands of CaM, assuming that the sequence difference between CaM and TnC plays the major role in target recognition. However, it is unclear whether such EF‐hand switching affects the structure and function. Here we generated four chimera proteins (1TnC, 2TnC, 3TnC, and 4TnC), in which the particular EF‐hand of CaM is replaced with the corresponding EF‐hand of TnC, and investigated their structural change upon Ca2+ binding using isothermal titration calorimetry, fluorescence, different scanning calorimetry, and circular dichroism. Furthermore, the interaction of these chimera proteins to the peptides corresponding to the CaM binding sequence of CaM dependent kinase II, Nitric Oxide Synthase, Orai, and Fas receptor, was performed in a similar manner and the resulting data were used to compare to structures built from molecular modeling.

Grant Funding Source: This work is supported in part by National Science Foundation.
Fluorescent biosensors for probing CDK4/cyclin D activity and developing non‐ATP pocket Inhibitors for melanoma, lung cancer and lymphoma (972.2)
Authors: Morris May , Prevel Camille , Pellerano Morgan , Van Thi Nhu Ngoc
Published: June 2021

CDK4/cyclin D kinase constitutes an attractive pharmacological target in lung cancer, melanoma and lymphoma, associated with mutation or amplification of CDK4, cyclin D or p16INK4a, but efforts to develop tools for detection of this kinase in its native environment, as well as selective inhibitors for therapeutic purposes have remained limited.

To this aim we have engineered a fluorescent polypeptide biosensor that reports on CDK4/cyclin D activity in a sensitive and continuous fashion in vitro, in living cells and in biopsies and which allows to monitor response to therapeutics in animal tumour models. We have further designed two biosensors which have been applied to identify competitors of essential protein/protein interfaces between CDK4 and Cyclin D, and allosteric inhibitors that perturb the conformational dynamics of CDK4, respectively, by high throughput screening. These studies highlight the importance of fluorescent biosensors for fundamental research, biomedical developments and drug discovery programmes, providing novel and sensitive approaches to monitor cancer‐associated alterations in protein kinase activities and develop non‐ATP pocket inhibitors.

Grant Funding Source: Grants to MCM “Chercheuse d’Avenir” Région Languedoc‐Roussillon, ARC and INCA
Factors affecting manganese accumulations in gill mitochondria of Crassostrea virginica (996.1)
Authors: Shoneye Temitope , Nuhar Ahmed , Catapane Edward , Carroll Margaret
Published: June 2021

Manganese (Mn) is a neurotoxin causing Manganism in people exposed to high environmental levels. Oxidative stress is implicated as a factor of Mn toxicity. The mechanisms is attributed to toxic levels of free radicals inducing mitochondrial dysfunction. Reports indicate Mn accumulates in mitochondria, however controversy exists to the extent of Mn accumulation in mitochondria and whether this contributes to the cause of Manganism. We have been using the oyster, Crassostrea virginica, to study Mn neurotoxicty. We found Mn disrupts the animals’ dopaminergic system and mitochondrial respiration. Mn is believed to use the mitochondrial Ca2+ uniporter and H+ or Na+/Ca2+ exchanger for movements in and out of mitochondria. We hypothesize Ca2+ channel blockers will alter mitochondrial Mn accumulations. We prepared mitochondrial fractions from gills. Mitochondria were treated with Ca2+ channel blockers prior to Mn exposure, pelleted and washed, then digested in HNO3 and analyzed in an AA. Mitochondria treated with Mn (1 ‐ 5 mM) exhibited a dose dependent accumulation of up to 1000% compared to controls. The channels blockers, LaCl3, diltiazem, verapamil and ruthenium red (0.25 ‐ 2 mM) reduced accumulations. LaCl3 was the most effective. The study shows mitochondria accumulate Mn and the mechanism of influx and efflux can be affected by drugs that interact with mitochondrial Ca2+ uniporter and H+ or Na+/Ca2+ exchanger.

Grant Funding Source: NYSED‐ 0516041171, NSF‐0622197
Human DNA Polymerase β’s endonuclease activity prevents mitotic chromosome instability (LB119)
Authors: Carron Emily , Chen Desheng , Makridakis Nick
Published: June 2021

Most tumors have numerical and structural chromosome aberrations that can provide selective growth advantages. It has been reported that overexpression of DNA Polymerase B (Pol β) contributes to chromosome instability by inducing aneuplody, telomere fusions, and a deficient mitotic checkpoint. However, the mechanism is currently unknown. Here, we report that Pol β’s endonuclease activity plays an important role in maintaining chromosome stability by collaborating with Topoisomerase IIα. Our results indicate that Pol β has endonuclease activity, and that this activity is required to prevent chromosomal instability during mitosis as deletion of Pol β induces aneuploidy, the formation of anaphase bridges and micronuclei. Moreover, cells expressing the endonuclease deficient E216K mutant accumulate more anaphase bridges and micronuclei than those expressing wild‐type Pol β. Consistent with these observations, Pol β localizes near the centromere in metaphase. This data suggests that Pol β’s endonuclease activity plays a role in mitotic chromosome segregation.

Grant Funding Source: DOD grant PC094628, and NIH grant 8 P20 GM103518 (to NM
Comparative metacarpal volume and relative thumb muscle mass in humans and non‐human primates (LB35)
Authors: Perez Adela , Patel Biren
Published: June 2021

Fine precision (pad‐to‐pad) grasping is facilitated by having a relative long thumb and short fingers. Power grasping is aided by having large thumb muscle forces generated by large thumb muscles (e.g., adductor pollicis). Reconstructing precision grasping in fossils is easily done by examining bone lengths. Reconstructing muscle force is more difficult because soft tissue seldom preserves in fossils. The goal of this study was to find a metric that can estimate thumb muscle mass from skeletal elements. Accordingly, we examined the relationship between thumb muscle mass and metacarpal 1 (Mc1) volume in a sample (>220) of human and non‐human primates (>20 species). Relative thumb muscle masses (all thumb muscles, thenar muscles only, adductor pollicis only) were calculated as a percentage of total hand muscles mass. Relative Mc1 volume was calculated as a percentage of Mc2‐5 volume. Metacarpal volumes were obtain from 3D surfaces models derived from CT scanning. Regression analyses demonstrated a significant relationship (p<0.01) between relative Mc1 volume and each measure of relative thumb muscle mass. Relative muscle volumes calculated for the fossil hominin Ardipithecus ramidus are lower than modern humans, further supporting the claims that its hands were more ape‐like in function.

Grant Funding Source: Supported by the National Science Foundation (BCS 1317047) and USC URAP
Therapeutic efficacy of cerium oxide nanoparticles in treatment of sepsis induced renal failure (LB608)
Authors: Manne Nandini , Arvapalli Ravikumar , Nepal Niraj , Nandyala Geeta , Rice Kevin , Shinichi Asano , Blough Eric
Published: June 2021

Severe sepsis is a complex disease of multifactorial etiology that is characterized by systemic inflammation which can rapidly progress to multi‐organ dysfunction and death. Although most of the deaths due to sepsis are related to the release of inflammatory mediators, treatments to date have largely centered on the administration of antibiotics, i.v. fluids, NSAIDS, and vasopressors. Studies have shown that the release of inflammatory mediators is mediated, at least in part, through the generation of reactive oxygen species (ROS). Other work has shown that cerium oxide (CeO2) nanoparticles can function to scavenge ROS and that these particles exhibit anti‐bacterial activity. On the basis of these data, we hypothesized a CeO2 nanoparticle based treatment strategy would diminish sepsis‐induced mortality, sepsis‐associated inflammation and that these improvements would be associated with improved renal function. Male Sprague Dawley rats were subjected to polymicrobial sepsis and treated with 0.5mg/kg of CeO2 nanoparticles intravenously. Our preliminary data suggests that the CeO2 nanoparticle intervention improved animal survivability and promoted the restoration of core body temperature. Changes in survivability and thermoregulation were associated with an attenuation in the sepsis‐induced release of β2 microglobulin, cystatin, gluthatione‐s transferase alpha, gluthatione‐s transferase mu, kidney injury molecule‐1, osteopontin and a reduction in serum total ROS. Histological analysis demonstrated that CeO2 nanoparticles decreased sepsis‐induced proximal tubular dilatation and loss of brush border. Taken together, these data suggest that CeO2 nanoparticle based interventions may exhibit potential for the treatment of sepsis‐induced renal failure and death.

Grant Funding Source: This work was supported in part from DOE grant (DE‐PS02‐09ER‐01 to E.R.B)
Characterization of YML020W in Saccharomyces cerevisiae
Authors: Shoemaker Katherine , Keeney Jill
Published: June 2021

Saccharomyces cerevisiae is a single‐celled, eukaryotic yeast commonly known as baker's or brewer's yeast. As a model organism, genes can be easily inserted and removed allowing researchers to utilize deletion genetics to better understand the function of open reading frames (ORFs) within the S. cerevisiae genome. ORFs are characterized using gene ontology, which includes three categories: biological processes, cellular components, and molecular functions. The characterization of genes within the S. cerevisiae genome is of importance in order to understand the functionality of these uncharacterized genes. Through a better understanding of how genes and their products interact with one another, we can gain insight into the function of S. cerevisiae as a model organism. The purpose of this study is to characterize the S. cerevisiae ORF YML020W, which was first identified in a screen for resistance to the apoptosis‐inducing drug, KP1019. In order to further the understanding of the structure and function of this ORF, a deletion strain of YML020W was constructed. Genetic interactions with DUN1 and YML020W were characterized through the construction of a double deletion mutant. To determine the location of the gene within the cell, the protein product associated with YML020W was fluorescently labeled at the C‐terminus and observed using fluorescence microscopy where clusters of YML020Wp were visible. These strategies will add details to gene ontology and provide information concerning of the importance of YML020W in S. cerevisiae.

Support or Funding Information

This work was supported by Juniata College, Howard Hughes Medical Institute, and the National Science Foundation
A new approach to characterize the molecular interactions between TiO2 nanoparticles and dietary flavonoids using surface‐enhanced Raman spectroscopy
Authors: Cao Xiaoqiong , Gao Zili , Zheng Jinkai , He Lili , Xiao Hang
Published: June 2021

TiO2 nanoparticles are commonly presented in food additives (E171), and have been associated with potential adverse effects on health. TiO2 nanoparticles has a specific surface reactivity and bind bidentate ligands from the environment. Flavonoids are widely found in fruits and vegetables, and can act as electro‐donating agents for TiO2 nanoparticles. Knowledge on the molecular interactions between TiO2 nanoparticles and food components such as flavonoid compounds will facilitate the understanding of the fate of TiO2 nanoparticles in food and in the gastrointestinal tract. In this study, we utilized surface‐enhanced Raman spectroscopy (SERS) to develop a rapid and accurate characterization method for the interactions between TiO2 nanoparticles and flavonoids. TiO2 anatase nanoparticles were mixed with seven different flavonoids (i.e. apigenin, luteolin, fisetin, kaempferol, quercetin, myricetin and baicalin) to form a complex that was subjected to SERS analysis. Our results demonstrated that the complex produced distinctive SERS ‘fingerprints’, which allowed further analysis of the molecular interaction. Phenolic groups in the flavonoids played pivotal roles in the interaction between flavonoids and TiO2 nanoparticles. Difference in the numbers and positions of the phenolic groups contributed to the distinctive SERS spectra of different complex. There is no report has been found on the application of SERS in studying the interactions between TiO2 nanoparticles and dietary flavonoids. We successfully developed a simple and accurate SERS‐based method to characterize the molecular interaction between TiO2 nanoparticles and various dietary flavonoid compounds.

Support or Funding Information

This study was partially support by fund from USDA.
Using Mutagenesis and Synthetic Substrates to Explore Regiochemical Control by Soybean‐Lipoxygenase‐1
Authors: Clapp Charles H. , Kahler Kirsten M. , Plumeri Patricia , Karaisz Fred , Dodson Cassidy R.
Published: June 2021

Lipoxygenases catalyze the oxygenation of polyunsaturated fatty acids to produce conjugated diene hydroperoxides. Plant lipoxygenases oxygenate linoleic acid to produce either 13‐hydroperoxy‐9,11‐octadecadienoic acid (13‐HPOD), 9‐hydroperoxy‐10,12‐octadecadienoic acid (9‐HPOD) or a mixture of both. The factors determining the position of oxygen incorporation are not well understood. Hornung et al. (Proc. Natl. Acad. Sci. USA 96 (1999) 4192–4197) identified one critical residue, corresponding to phenylalanine‐557 in soybean lipoxygenase‐1 (SBLO‐1). These workers observed that lipoxygenases such as SBLO‐1 with phenylalanine or histidine in this position generally produce primarily 13‐HPOD, whereas those with valine or threonine produce primarily 9‐HPOD. In the present work, the F557V mutant of SBLO‐1 has been prepared. Consistent with the trend noted by Hornung et al., this mutant produces 30–40% 9‐HPOD from linoleic acid at pH 7.5, compared with less than 10% for the wild‐type enzyme. Hornung et al. suggested that having a smaller residue in position 557 might enable linoleic acid to bind in a head‐first manner with the carboxylate group of the substrate interacting with arginine‐707. Consistent with head‐first binding, the 9‐HPOD formed by the F557V mutant has predominantly (86%) the S‐configuration at carbon‐9. To further test the model, we have investigated the action of SBLO‐1 on linoleoyldiethylamine (LDMA), which has been previously shown to be a good substrate for wild‐type SBLO‐1 at pH 7.0, where the dimethylamino group of LDMA is expected to be protonated and positively charged, which would disfavor interaction with arginine‐707. With this substrate, the amount of oxygenation at carbon‐9 is greater than observed with linoleic acid. This result argues against the hypothesis that oxygenation at carbon‐9 arises from binding of the polar end of the substrate near arginine‐707. Surprisingly, the F557A mutant produces only 9% 9‐HPOD from linoleic acid. This result indicates that the amount of 9‐oxygenation does not always correlate with the size of the residue at position‐557. In sum, the results confirm the importance of residue 557 in determining the regiochemistry of the products, but the structural basis for these effects remains to be elucidated.

Support or Funding Information

NSF CHE‐1213262
The Influence of Conserved Residues and Structural Elements on the Stability and Activity of a Thermophilic Intein
Authors: Minteer Christopher J. , Colelli Katie M. , Reitter Julie N. , Mills Kenneth V.
Published: June 2021

Protein splicing is a self‐catalyzed reaction where an intein is excised from precursor flanking polypeptides (exteins) and the exteins are ligated together to produce a mature protein. We are interested in how specific conserved residues catalyze this process and how structural domains affect intein structure. The structure of an intein from Pyrococcus abyssi (the Pab PolII intein) has a Hyperthermophilic/Thermophilic Hairpin (HTH), only found in the structures of thermophilic inteins. It may add thermal stability by extending the central beta sheet of the typical intein fold. Mutations to destabilize inter‐and intra‐strand salt bridges in the hairpin region may result in global destabilization, and were studied through assays to assess activity and susceptibility to proteolysis. We also are interested in the influence of conserved intein residues on activity. Residues in intein block B may serve to destabilize the scissile peptide bond at the N‐terminal splice junction. We have observed cleavage of that bond at low pH, independent of splicing steps, and are exploring whether block B residues are responsible.

Support or Funding Information

This material is based upon work supported by the National Science Foundation under grants MCB‐1244089 and MCB‐1517138 and by a Henry Dreyfus Teacher‐Scholar Award (KVM).
Characterizing Conformational Ensemble of H‐, K‐, and N‐Ras Using Accelerated Molecular Dynamics and Wide‐Angle X‐ray Solution Scattering (WAXS)
Authors: Guterres Hugo , Zhou Hao , Makowski Lee , Mattos Carla
Published: June 2021

Ras GTPase is involved in cell‐signaling pathways that control cell proliferation, differentiation, and apoptosis. There are three main isoforms of Ras, H‐, K‐, and N‐Ras, which share about 95% sequence identity in their soluble domain. They have very similar three‐dimensional folds, however they have been suggested to display functional specificity. For example, in pancreatic cancer K‐Ras mutations occur at much higher frequency than either H‐ or N‐Ras, whereas in leukemia N‐Ras mutations play the dominant role. These isoforms have also been shown to follow distinct cellular processing pathways and localize to different subcellular compartments. In this study, we combine accelerated Molecular Dynamics (aMD) and wide‐angle X‐ray scattering (WAXS) methods to characterize the structural ensemble of Ras isoforms and measure their dynamic properties. Using aMD, we reduce the energetic barriers to enhance conformational sampling of these proteins. A representative subset of structures from aMD is then experimentally tested using WAXS patterns to quantify their relative population. Each isoform shows at least two major conformations, where one is characterized as a relaxed state and another one as a reactive state, based on their relative potential energy calculated from aMD. These conformations are unique for each of the isoform, where they differ primarily around the active site and regions away from the active site, including helix 3/loop 7. This study will provide significant insights into the conformational features of these small proteins that give rise to their functional specificity.

Support or Funding Information

NSF MCB‐1244203
HomeStyles Express: Telephone Counseling and a Fuss Free Eating Guide to Promote Positive Feeding Practices
Authors: McKinley Jessica , Martin‐Biggers Jennifer T , Gager Erin , Byrd‐Bredbenner Carol
Published: June 2021

HomeStyles Express uses evidence‐based, pediatric weight management nutrition guidelines to improve lifestyle practices in families with preschool children. Parents (n=50) took the pretest, then each week received via email 1 of 8 4‐page mini‐magazine instructional guides focusing on weight‐related topics and participated in a brief telephone counseling session after reading the guide, then took the posttest. Participants were mostly female (96%), white (84%), and had at least a bachelor's degree (82%). At the pretest, 42% reported, at least sometimes, they encouraged their preschool kids to eat by using food as a reward and 56% used non‐food rewards to get kids to eat certain food. Parents were more likely to pressure children to eat than restrict their food choices (means 2.17±0.82SD and 3.86±0.97SD, on 5‐point scale). Content analysis of individual counseling sessions with parents on the Fuss Free Eating guide, which focuses on positive feeding practices for young children, indicated many had a child who was a “picky eater,” and some bribed children to eat certain foods (e.g., vegetables), offered sweets as rewards, felt children should finish their plates, and/or felt spousal support was requisite to achieving their feeding practices goals. After reading the guide, many parents reported they planned to stop bribing kids to eat and would use non‐food rewards (e.g., reading an extra story before bed) to encourage good behavior. Some reported they planned to offer children a greater variety of foods and/would stop forcing children to finish their plates. When asked why they thought positive feeding practices were important, parents frequently cited health benefits, weight management, and development of good habits. Parents rated the importance of using positive feeding practices highly; mean=9.6±0.79SD on 10‐point scale. Goals set by many parents after reading the guide were to introduce new fruits and vegetables to their kids, eat more meals as a family, communicate more about positive feeding practices with their partners, and avoid bribing kids to eat, and stop using food as rewards. Parents were very confident in their ability to meet these goals, average score=8.9±1.32SD on 10‐point scale. At a subsequent counseling session, most parents reported that they successfully met the positive feeding practices goals they had set. The Fuss Free Eating guide, along with a brief telephone counseling session, has the potential to successfully aid parents in applying positive feeding practices.

Support or Funding Information

USDA NIFA #2011‐68001‐30170 Academy of Nutrition and Dietetics Foundation Ann Hertzler Research Grant
Teacher Characteristics Influence Implementation of a Multi‐Component, School‐Based Nutrition Intervention in Fourth‐Grade Classrooms
Authors: Linnell Jessica D , Smith Martin H , Briggs Marilyn , Brian Kelley M , Scherr Rachel E , Dharmar Madan , Zidenberg‐Cherr Sheri
Published: June 2021

School‐based nutrition interventions are a promising approach to improve children's diets and prevent obesity. Few studies have investigated whether outcomes of these interventions are influenced by teacher characteristics or quality of implementation. This study examines whether teacher characteristics and implementation influenced the effectiveness of a multi‐component nutrition education program to improve dietary behaviors. Schools implemented a multi‐component nutrition education intervention during 2013–2014. Teachers (n=8) facilitated nutrition education using an experiential, garden‐enhanced nutrition curriculum among students in their classrooms (n=173) in two schools. Teacher characteristics of teaching experience, self‐efficacy in teaching nutrition, and knowledge about nutrition were collected at baseline. Implementation factors of fidelity and completion were evaluated using self‐reports and qualitative observations. Student outcomes included body mass index (BMI) percentile‐for‐age, nutrition knowledge, critical thinking skills, and identification of vegetables and were measured pre/post. Relationships were analyzed using multivariate mixed model regression with classroom as a random effect. Pre/post changes were analyzed using t‐tests. Students improved BMI percentile‐for‐age (−1.99 ±7.95), and increased nutrition knowledge (2.28±3.40), critical thinking skills (1.98±3.28), and the ability to identify vegetables (0.87±1.09). Teachers’ characteristics of teaching experience, experience teaching nutrition, attendance in college nutrition course, self‐efficacy, and nutrition knowledge were significantly related to fidelity and completeness of program implementation. This study demonstrated implementation of SHCP by teachers can positively influence student outcomes, though program implementation depends upon teacher characteristics. It may be critical to identify effective professional development models to achieve high quality implementation of nutrition programs in school‐based settings.

Support or Funding Information

Funding was provided by UCANR competitive grant #11‐1018, USDA 2011‐38420‐20082, and USDA NIFA Hatch project 221082.
Diets Higher in Animal and Plant Protein Are Associated with Lower Adiposity and Do Not Impair Kidney Function in US Adults
Authors: Berryman Claire E. , Agarwal Sanjiv , Lieberman Harris R. , Fulgoni Victor L. , Pasiakos Stefan M.
Published: June 2021

High protein diets are associated with lower adiposity and higher HDL‐C. Whether these benefits can be attributed to a specific protein source (i.e. non‐dairy animal, plant, dairy) is unknown and concerns remain regarding the impact of higher protein diets on kidney function. Data from NHANES 2007–10 (n=10,977; 19+ y) were combined to evaluate decile trends of protein type on markers of cardiometabolic risk and kidney function. Variables were adjusted for sex, race/ethnicity, age, physical activity, poverty‐income ratio, smoking habits, individual intake (g/kg) for each of the other 2 protein types, BMI (except for weight related variables) and macronutrient (CHO, fiber, total fat and SFA) intake. Total protein intake was 82.3 ± 0.8 g/d (animal: 37.4 ± 0.5, plant: 24.7 ± 0.3, dairy: 13.4 ± 0.3 g/d). Both BMI and waist circumference were inversely associated with animal (β= −0.20, P <0.0001; β= −0.51, P <0.0001, respectively) and plant (β= −0.35, P <0.0001; β= −0.83, P <0.0001, respectively) protein intake. Blood urea nitrogen concentrations increased across deciles for animal (β= 0.31, P <0.0001, D1–D10: 11.6 ± 0.2 to 14.9 ± 0.3 mg/dL) and dairy (β= 0.20, P <0.0001, D1–D10: 12.7 ± 0.2 to 13.9 ± 0.2 mg/dL) but not plant protein intake. Glomerular filtration rate and blood creatinine were not associated with protein intake. Diets higher in plant and animal protein, independent of other dietary factors, are associated with improved cardiometabolic risk, particularly central adiposity, and do not impair kidney function in US adults.

Support or Funding Information

US Army Medical Research and Materiel Command
Comparative Effects of Glutathione Peroxidase‐1 Overexpression and Cu/Zn‐superoxide Dismutase Knockout on Murine Hepatic Fatty Acid Metabolism
Authors: Tao Ling , Leung Annie Ka May , Chakraborty Debrup , Lei Xin Gen
Published: June 2021

Glutathione peroxidase‐1 (GPX1) and Cu/Zn‐superoxide dismutase (SOD1) are two major antioxidant enzymes, while polyunsaturated fatty acids (PUFA) including eicosapentaenoic acid (20:5n‐3, EPA) and docosahexaenoic acid (22:6n‐3, DHA) are health‐promoting nutrients. This study was conducted to determine the effects of GPX1 overexpression and SOD1 knockout on hepatic synthesis, accumulation, and oxidation of PUFA. GPX1 overexpressing (OE, 6–7 mo), and SOD1 knockout (SKO, 2 mo) mice and their respective wild‐type controls were fed a Torula yeast basal diet supplemented with 0 or 5% defatted microalgae (Nannochloropsis oceanica, 16% EPA) for 8 wk (n = 5 to 7 per group). Overexpression of GPX1 decreased (P < 0.01) linoleic acid (18:2n‐6) concentration and raised (P < 0.05) ω‐3/ω‐6 fatty acid ratio in the liver of mice regardless of diets. In comparison, knockout of SOD1 decreased (P < 0.05) concentrations of PUFA, in particular eicosatrienoic acid (20:3n‐3, precursor of EPA) and DHA, in mice fed both diets. The knockout of SOD1 also resulted in lower (P < 0.01) hepatic ω‐3/ω‐6 fatty acid ratio than the wild‐type mice when both genotypes were fed the algae diet. Inclusion of 5% algae in the diet elevated (P < 0.05) the hepatic DHA concentration in only wild‐type mice of SOD1. Results of qPCR analysis showed that knockout of SOD1 down‐regulated (P < 0.05) hepatic mRNA levels of elongase 2 and 5, the major enzymes involved in long chain PUFA synthesis, compared with the wild‐type controls. Meanwhile, the OE mice had lower (P < 0.05) hepatic mRNA levels of PUFA oxidation‐related genes including cyclooxygenase 1 (COX1), COX2, and arachidonate 5‐lipoxygenase than their wild‐type controls. In addition, feeding the algae diet decreased (P < 0.01) non‐esterified fatty acid concentrations in both plasma and liver of the OE mice, and concentration of triglycerides in the liver of the SOD1 wild‐type mice. In conclusion, overexpression of GPX1 and knockout of SOD1 exerted differential effects on synthesis, accumulation and oxidation of hepatic PUFA in mice. Further study requires to measure oxidized metabolites of PUFA and to reveal molecular mechanisms by which GPX1 and SOD1 regulate PUFA metabolism.

Support or Funding Information

Supported in part by a NIH grant DK53018 and a USDA/DOE Biomass R&D Initiative Grant
Placental growth factor administration prevents hypertension, increased sFlt‐1 levels and reduced glomerular filtration rate responses to placental ischemia
Authors: Spradley Frank T , Tan Adelen Y , Joo Woo S , Daniels Garrett , Kussie Paul , Karumanchi S Ananth , Granger Joey P
Published: June 2021

Preeclampsia is a pregnancy‐specific disorder noted by new‐onset hypertension in the mother accompanied by a variety of other cardiovascular disturbances. Unfortunately, the most effective treatment is early delivery of the fetus and the ischemic placenta. Progress toward treatment has found that placental ischemia/hypoxia induced in animals by reduced uterine perfusion pressure (RUPP) or in human patients stimulates release of hypertensive placental factors into the maternal circulation. The anti‐angiogenic factor sFlt‐1, which antagonizes and reduces bioavailable vascular endothelial growth factor (VEGF) and placental growth factor (PlGF), is elevated in RUPP rats and preeclamptic patients. Although PlGF and VEGF are both natural ligands for sFlt‐1, VEGF also binds with high‐affinity to Flk1 (VEGFR2) causing side‐effects related to excess Flk1 signaling including edema. In contrast, PlGF is specific for sFlt‐1. Thus, we tested the hypothesis that PlGF treatment would reduce placental ischemia‐induced hypertension without any adverse consequences to the mother or fetus. On gestational day 14, Sprague Dawley rats were randomized in to three groups: normal pregnant (NP, N=9), RUPP (N=11) and RUPP + 180 μg/kg/day PlGF (N=6). The rhPlGF (AG31, a purified‐recombinant human PlGF) was infused via intraperitoneal osmotic minipump. Mean arterial blood pressure (MAP, carotid catheter); glomerular filtration rate (GFR); placental and fetal weights; and plasma sFlt‐1 levels were assessed on day 19. Data is mean ± standard error. MAP (NP: 104±1 vs. RUPP: 123±4 mmHg) and plasma sFlt‐1 levels (NP: 26±12 vs. RUPP: 660±150 ng/mL) were significantly higher (P<0.05) in the RUPP rats. Whereas, total placental weights (NP: 6.1±0.6 vs. RUPP: 2.1±0.3 g); total fetal weights (NP: 26.1±2.5 vs. RUPP: 9.2±1.3 g); and GFR (NP: 4.2±0.4 vs. RUPP: 3.8±0.3 mL/min) were significantly reduced (P<0.05) in the RUPP rats. Treatment of RUPP rats with rhPlGF significantly reduced (P<0.05) MAP (105±3 mmHg) and sFlt‐1 levels (79±17 ng/mL); did not alter placental weights (2.6±0.5 g) or fetal weights (12.6±2.3 g); but significantly elevated (P<0.05) GFR (3.0±0.1) compared to untreated RUPP rats. There were no major adverse maternal consequences such as signs of edema with rhPlGF. These data indicate that the increased sFlt‐1 and reduced PlGF known to occur as a result of placental ischemia contribute to the development of maternal hypertension. In conclusion, our finding that rhPlGF prevents placental ischemia‐induced hypertension and restores renal hemodynamics suggests a strong therapeutic potential for this growth factor in preeclampsia by interfering in anti‐angiogenic pathways.

Support or Funding Information

SBIR 1R43HD082657, T32HL105324‐01, P01HL051971
Leg Length and the Evolution of Human Stature: Insights from Evolutionary Studies of Tail Length in Snakes
Authors: Lillywhite Harvey B. , Sheehy Coleman M.
Published: June 2021

Gravity imposes potentially significant constraints on blood circulation in tall or elongate animals during upright posture or climbing. Intravascular pressures increase in the lower limbs or body during upright posture in tall and erect animals, which potentially reduces blood flow to the head because of edema and pooling of blood in dependent, distensible vessels. Such gravitational disturbance is related to the absolute height of the blood columns, other factors being equal. Hence, adaptation of physiology, morphology and behavior have evolved to reduce compliance in the lower body. A recent and robust evolutionary analysis of data for 226 species of snakes (representing 15 families having diverse habits) demonstrates that tail length relative to total length has elongated significantly in scansorial species that are adapted to arboreal habitats. The compliance of caudal tissues is relatively low, and is reduced sixfold in arboreal compared with ground‐dwelling species of snakes. Therefore, for any given total length of snake that climbs vertically in trees, a greater length of tail relative to total length will proportionally mitigate the tendency for edema and blood pooling during vertical head‐up posture. We note that in humans the legs have become proportionately longer during evolutionary increases in stature. Venous compliance in the upper body has been shown to exceed that in the lower body by a factor of about 6 in humans. Thus, similarly to upright snakes, humans with relatively longer legs have a greater portion of height that is characterized by low compliance and thereby a lesser tendency to pool blood in the lower limbs. We suggest that enhanced orthostatic tolerance might be one adaptive countermeasure associated with changes of leg length and stature during human evolution.

Support or Funding Information

Supported, in part, by NSF grant IOS‐0926802 to HBL and a Stolarz Postdoctoral Fellowship to CMS.
Chronic Calorie Restriction Downregulates Skeletal Muscle mTORC1 Signaling Independent of Dietary Protein Level and Associated microRNA Expression in Male Rats
Authors: Margolis Lee M , Rivas Donato A , Berrone Maria , Ezzyat Yassine , Young Andrew J , McClung James P , Fielding Roger A , Pasiakos Stefan M
Published: June 2021

Short‐term (5–10 days) calorie restriction downregulates muscle protein synthesis, with consumption of a high protein‐based diet attenuating this decline. Benefit with increased protein intake is likely due to maintenance of amino acid‐mediated anabolic signaling through the mechanistic target of rapamycin complex 1 (mTORC1) pathway. However, the effects of chronic caloric restriction and high protein diets on mTORC1 signaling and the expression of microRNA (miR) that regulates this pathway remain unclear. This study determined skeletal muscle mTORC1 and miR responses to a 16 week ad libitum (AL; ~25 g/d) or calorie restricted (CR; 40%; ~15 g/d) standard (10%; ~96 g/kg) or high (32%; ~327 g/kg) protein milk‐based diet in 12‐wk old male Sprague Dawley rats. Crude and clear mixed gastrocnemius homogenate protein concentrations (BSA Protein Assay) were used to calculate protein content, an index of hypertrophy. Immunoblotting was performed to quantify total protein and phosphorylation status of mTORC1 signaling proteins. Expression of miR‐99a‐5p, miR‐100‐5p, miR‐128a‐3p, miR‐133a‐3p, miR‐199a‐3p, and miR‐221‐3p were determined using RT‐qPCR. Data were expressed as fold change (mean ± SEM), compared to AL 10% as the study control. Protein content was higher (P < 0.05) for AL (21.9 ± 0.9 mg) compared to CR (17.3 ± 0.9 mg) rats, regardless of dietary protein intake. Total AKT, mTOR, rpS6 and p70S6K were 1.85 ± 0.04, 1.71 ± 0.08, 2.57 ± 0.05, and 2.23 ± 0.05 fold lower (P < 0.05) for CR than for AL, with rpS6 positively associated with protein content (r = 0.611, r2 = 0.372, P < 0.05). Phosphorylation status of AKTSer473, mTORSer2448, rpS6Ser235/236 and p70S6KThr389 were 1.72 ± 0.11, 1.41 ± 0.08, 3.47 ± 0.05, and 1.95 ± 0.05 fold lower (P < 0.05) for CR than for AL. No effects were observed for dietary protein level on mTORC1 signaling protein, and neither energy nor protein intake altered skeletal muscle miR expression. These data show that prolonged caloric restriction downregulates mTORC1 intracellular signaling, independent of skeletal muscle miR known to regulate mTORC1 protein expression. Consuming a high protein does not appear to attenuate the negative effects of chronic caloric restriction on the intracellular regulation of skeletal muscle mass.

Support or Funding Information

T32 NIDDK training grant # 5T32DK062032‐23, USDA, USAMRMC, and the Dairy Research Institute
Evaluation of Resuscitation Fluids for Maintaining Human Endothelial Cell Antioxidant Status In Vitro
Authors: Dubick Michael A , Gibson Ruby S , Barr Johnny L , Bowman Phillip D
Published: June 2021

With hemorrhage a major cause of death in trauma patients, infusing resuscitation fluids is still considered an important treatment strategy. The endothelial cell lining all blood vessels is the first cell type exposed to these intravenous solutions and at the highest concentrations. Crystalloid resuscitation fluids (RF) such as Ringer's solution, normal saline (NS), lactated Ringer's (LR) and colloids such as 5% albumin (Buminate 5%, Baxter Healthcare, Westlake Village, CA) and Hextend (BioTime, Berkeley, CA) have shown similar hemodynamic responses in animal hemorrhage models. However, choosing the most efficacious resuscitation fluid might be more rationally based on maintenance of endothelial antioxidant status, which may be adversely affected by restrictions of cellular metabolites that occur with fluids intended mainly to maintain intravascular volume. In this study, human umbilical vein endothelial cells (HUVEC) were exposed to various resuscitation fluids, and total antioxidant status was assessed. Pooled gender‐mixed (HUVEC) were cultivated on 75‐cm2 culture flasks in Medium 131, and endothelial supplements. Stock cultures were cultivated at 37°C in a humidified atmosphere of 3% oxygen and 5% CO2 with medium changes every 2 days until confluent. Prior to an experiment, cells were subcultivated with Trypsin/EDTA onto Costar® 6‐well or 96‐well multiplates at 5000 cells/cm2 and used when confluent. Ringer's, LR, NS, 5% Alb, Hextend, and also 10% human plasma (George King Bio‐Medical, Overland, KA), were buffered with 10 mM Hepes (pH 7.3) and made 0.025% in phenol red to monitor pH. Replicate cultures in multiplates were exposed to each fluid for 3 hrs sealed in a humidified box at 37°C. ATP levels in the cells were measured at the end of exposure utilizing the luciferase reaction from the CellTiter‐Glo Luminescent Cell Viability Assay. The number of cells per well was monitored with the PicoGreen DNA assay. Reactive Oxygen Species (ROS) Generation was estimated with CellROX® Deep Red Reagent (Life Technologies, Grand Island, NY). Incubation with NS resulted in about a 20% reduction in cellular DNA compared with control cells, however ATP levels were generally maintained but reduced 40% in albumin treated cells compared with controls grown in complete medium. Morphologically it appeared that apoptosis occurred in cells treated with Hextend. A marked elevation in ROS was observed in albumin treated cells compared to the other groups, whereas ROS production in other cells was about 50% of controls. Taken together these data suggest that RF appear to affect mitochondria in endothelial cells as they regulate apoptosis and ATP production and they are the principal site of ROS production.

Support or Funding Information

This study was funded by the US Army Medical Research and Materiel Command
Inactivity‐Induced Phrenic Motor Facilitation underlies Spontaneous Recovery of Ventilation and Diaphragm EMG activity Following Cervical Spinal Injury
Authors: Weltman Joel Green , Baker Tracy
Published: June 2021

Cervical spinal injury disrupts neural pathways to phrenic motor neurons, thereby causing diaphragm paresis and paralysis and diminished breathing capacity. Some spontaneous functional recovery in the capacity to generate phrenic nerve or diaphragm activity is observed days‐to‐months post‐injury via mechanisms that not understood. We have previously found that prolonged reductions in spinal synaptic inputs to phrenic motor neurons in uninjured rats results in a PKCz‐dependent, rebound enhancement of phrenic burst amplitude, a form of spinal plasticity known as inactivity‐induced phrenic motor facilitation (iPMF). Little is known regarding the role for iPMF in the control of breathing. Here, we tested the hypothesis that mechanisms giving rise to iPMF underlie spontaneous recovery of ventilation and diaphragm EMG activity following a high cervical spinal injury. Sprague Dawley rats (Harlan colony 217, 12–16 weeks old) chronically instrumented with radiotelemetry for bilateral diaphragm electromyogram (EMG) recordings received small interfering RNAs directed against PKCz or non‐targeting siRNAs in the interpleural space (100pg/side) three days prior to a C3 midline contusion. Three days following contusion, EMG and plethysmography were recorded in unanesthetized rats breathing room air (FIO2/FICO2=0.21/0.0, NX) or with maximal chemoreceptor stimulation (FIO2/FICO2= 0.105/0.07, MCS). In injured rats receiving control injections, tidal volume under both inspired gas concentrations and diaphragm EMG during MCS showed minimal change from baseline conditions (TV NX: 99%, TV MCS: 90%, diaphragm EMG right: 95%, left: 100%). By contrast, in injured rats receiving siPKCz, tidal volume during normoxia and MCS (63% NX, 90% MCS) and electrical activity of the diaphragm during MCS (right: 85%, left: 80%) were reduced from pre‐injury levels, suggesting that impairment of iPMF prevented spontaneous recovery of phrenic motor output (and hence, diaphragm EMG activity) following spinal injury. Thus, we speculate that iPMF is a form of compensatory plasticity that senses and responds to reductions in spinal synaptic inputs to phrenic motor neurons to maintain stable phrenic motor output following spinal injury.

Support or Funding Information

Supported by NIH R01‐HL105511, DoD SC120226 and NIH T32‐OD010423‐09
Gastrointestinal biotransformation of resveratrol in mice
Authors: Li Fang , Sun Yue , Song Mingyue , Wu Xian , Xiao Hang
Published: June 2021

Resveratrol, a stilbenoid compound present in various berries, has been associated with reduced risk of chronic diseases such as cancer, obesity and diabetes. Accumulated evidence indicates that biotransformation plays important roles in the biological activities of dietary compounds. We investigated the gastrointestinal biotransformation of resveratrol in mice fed with resveratrol for 5 weeks. LC‐MS analysis revealed that the major site of biotransformation for resveratrol was the small intestine, although there was a low degree of biotransformation occurring in the stomach. In the small intestine, resveratrol underwent phase I metabolism to produce dihydroresveratrol and piceatannol. Subsequently, resveratrol, dihydroresveratrol and piceatannol all underwent phase II metabolism to yield their corresponding sulfate and glucuronide conjugates. However, in the colon, resveratrol, dihydroresveratrol and piceatannol mainly existed in the non‐conjugated forms, presumably due to the deconjugation by colonic microbiota. Moreover, the level of dihydroresveratrol in the colon was much higher that of resveratrol, suggesting that dihydroresveratrol may be essential for potential beneficial biological effects in the colon, such as anti‐inflammation and anti‐carcinogenesis. In summary, our results provided detailed information on the metabolic fate of resveratrol in the gastrointestinal tract, which is important for better understanding of the health benefits of resveratrol.

Support or Funding Information

This study was supported by funding from USDA.