bluuebunny/crossref_metadata_2025 huggingface dataset:
miRNA profiling in blood and tissue samples in non‐human primates in response to Yersinia pestis infection using next generation sequencing approach
Authors: Dimitrov George , Kumar Raina , Moyler Candace , Gautam Aarti , Miller Stacy‐Ann , Hammamieh Rasha , Jett Marti
Published: June 2021

MicroRNAs (miRNAs) are a class of small non‐coding RNA molecules (20–30 nucleotides long) that regulate gene expression negatively by transcriptional inhibition or post‐translational degradation. It is increasingly clear that miRNAs are an important part of the host response to microbes. In the current study, African green monkeys (Cercopithecus aethiops) were challenged intranasally with aerosolized Yersinia pestis strain CO92. Blood and tissue samples were collected post‐exposure from 6 hr through 42 hr time points. We used a next generation sequencing approach to profile miRNA expression patterns in different tissues and blood samples. Y. pestis is a gram‐negative facultative anaerobe and the causative agent of plague, which throughout history has had devastating effects on human populations. Our data enabled us to examine miRNA expression profiles during infection and identify tissue‐specific expression patterns during progression of the disease. We observed 180 upregulated miRNAs and 272 downregulated miRNAs across all time points in blood samples. At the 24 hr time point there were 52 differentially expressed (DE) miRNAs in blood samples that targeted 5381 genes. Functional analysis of DE miRNAs and their targets showed activation of TGF‐β and STAT3 pathways. TGF‐ β activated kinase 1 (TAK1, also called MAP3K7) is a known key regulator that inhibits innate immune signaling. In the kidney tissues there were a total of 32 DE miRNAs across all time points. At the 24 hr time point there were 11 unique mature DE miRNAs in exposed animal tissues as compared with non‐exposed animal tissues. In the lung tissues there were 67 DE miRNAs across all time points. At the 24 hr time point there were seven mature DE miRNAs when compared with non‐exposed animal tissues. These seven miRNAs were also DE in the blood samples. In the mediastinal lymph node tissues we had 52 DE miRNAs, eight of which were in common with blood samples. In the submandibular lymph nodes there were 25 DE miRNAs, three of which were in common with blood. We found that blood, lung, submandibular lymph and mediastinal lymph tissues shares two common miRNAs: mml‐miR‐590‐5p, which has 301 gene targets, and mml‐miR‐451, which has 17 gene targets. Both of these miRNAs are known to contribute to cytokine and grown factor activity, and miR‐590‐5p is a known circulating biomarker miRNA in cardiovascular and inflammatory diseases. Follow‐up comprehensive investigations are currently underway to further elucidate disease progression.

Support or Funding Information

This work was supported by the Defense Threat Reduction Agency, contractTMTI0029_09_WR_T
Translocation of Gold Nanoparticles in Model Epithelial Cells (Caco‐2 Monolayers)
Authors: Yao Mingfei , He Lili , McClements David Julian , Xiao Hang
Published: June 2021

At present, there is limited knowledge about the gastrointestinal fate and potential cytotoxicity of ingested inorganic nanoparticles. In this study, we determined the influence of particle size and concentration of gold nanoparticles (AuNPs) on their intestinal absorption, accumulation and cytotoxicity. AuNPs with different sizes (15, 50, and 100 nm) were subjected to an intestinal epithelium monolayer model to establish their patterns of absorption, accumulation and cytotoxicity. Results from inductively coupled plasma mass spectrometry (ICP‐MS) analysis revealed that as the mean particle diameter of the AuNPs decreased, their rate of absorption by the intestinal epithelium cells increased, but their cellular accumulation in the epithelial cells decreased. Moreover, fluoresce microscopy analysis indicated that accumulation of AuNPs caused cytotoxicity in the intestinal epithelial cells, which was evidenced by depolarization of mitochondria membranes. Our results demonstrated that particle size play an important role in dictating intestinal absorption, accumulation and cytotoxicity of AuNPs.

Support or Funding Information

USDA (2011‐67021, 2014‐67021)
Sex‐specific and Obesity‐specific Association of Serum Uric Acid with Cognitive Function in Older Adults
Authors: Cheatham Carol L , Vazquez‐Vidal Itzel , Turski Christa L , Medlin Amanda , Millsap Grace , Voruganti V. Saroja
Published: June 2021

Lower serum uric acid concentrations have been linked to cognitive dysfunction. Uric acid is an antioxidant that accounts for over half of the free radical scavenging activity in humans. Higher serum concentrations of uric acid have been associated with slower progression of several neurodegenerative diseases. On the other hand, elevated serum uric acid levels have been associated with poorer processing speed and executive functioning and greater white matter atrophy. Our aim in this study was to ascertain the relation between uric acid and cognitive function in older adults with mild cognitive decline (defined at 1–1.5 SD below the mean on the Montreal Cognitive Assessment, MoCA). Cognitive function was assessed using the MoCA and Cambridge Neuropsychological Test Automated Battery (CANTAB). Mean concentrations of serum uric acid in 107 individuals (mean age = 72.7 ± 4.3 years) were 5.9 ± 2.3 mg/dl; males had significantly higher uric acid concentrations (6.3 ± 1.1 mg/dl) compared to females (5.4 ± 1.1 mg/dl). Obese individuals’ concentrations were higher (6.2 ± 1.1 mg/dl) as compared to overweight (6.0 ± 1.1 mg/dl) and normal weight individuals (5.1 ± 1.2 mg/dl). Hyperuricemia (in males, serum uric acid > 7mg/dl and in females, serum uric acid > 6mg/dl) was present in 24% of the individuals. Compared with normouricemic individuals, hyperuricemic had significantly (p < 0.05) elevated serum triglyceride levels (122.1 ± 67 vs. 184.5 ± 152 mg/dl) and higher waist circumference (39.3 ±4 vs. 41.1 ± 3 in). Serum uric acid was positively correlated with BMI, waist circumference, serum creatinine, serum glucose, serum cholesterol ( p < 0.00001). Serum uric acid was negatively associated with MoCA scores (r2 = −0.23, p = 0.02). When stratified by sex, serum uric acid was positively associated with errors made on the CANTAB spatial working memory task (t = 2.6, p = 0.014) in men, but not in women. Similarly, when stratified by BMI, serum uric acid was positively associated with spatial working memory errors (t = 2.9, p = 0.013) and rapid visual processing errors (t = 3.0, p = 0.01) from the CANTAB in obese individuals but not in normal or overweight individuals. In summary, among older adults, higher serum uric acid concentrations are linked to poorer cognitive function in all individuals, poorer spatial working memory in men and obese individuals, and poorer processing in obese individuals compared to other groups. These findings indicate a potential role for serum uric acid concentrations in cognitive function in older adults.

Support or Funding Information

This research was funded by grants from the United States Department of Agriculture (2010‐2342‐02 and 2015‐2809‐01) to the first author and a small grant from the Nutrition Research Institute to the first and last authors.
Susceptibility to sickle cell co‐morbidities is regulated by single nucleotide polymorphisms of the TOLLIP gene
Authors: Agyingi Smith , Donkor Akua , Thomas Bolaji
Published: June 2021

Infectious diseases, despite the availability of vaccines, remain a challenge for sickle cell disease (SCD) patients. This is exacerbated in developing countries with high disease rates, poor hospital infrastructures and preponderance of co‐morbidities, complicating SCD case management. Therefore, elucidating the role of pathogen recognition factors or signaling pathways in immune response is critical in clarifying genes regulating infectious co‐morbidities. We have shown that the dysregulated immune response observed in SCD is not attributable to dendritic cells (CD209 gene promoter polymorphism) or Toll‐like receptor (TLR‐4 polymorphisms). Though there is a significant difference in CD209 gene promoter polymorphism between groups, it does not explain infection overload in African but not African American SCD patients. Toll‐interacting protein (TOLLIP) regulates human TLR signaling pathways, and its polymorphisms have been associated with infectious diseases. To decipher whether TOLLIP polymorphisms affect immune response upstream of TLR‐4 signaling, we examined the genetic diversity of TOLLIP polymorphisms (rs5743899 and rs3750920) within and between control (230, 379 and 159 Africans, African Americans and Caucasians respectively) and disease groups (145 and 331 African and African Americans respectively), utilizing a polymerase chain reaction and restriction fragment length polymorphism assay. We found significant interethnic disparity in the genotypic frequency (15.2%, 21.6% and 2.5%) of rs5743899 mutant variant between Africans, African Americans and Caucasians respectively. In addition, we also found a significant difference for rs5743899 mutant variant between disease and control groups in Africa (22.7% versus 15.2%) but not in the United States (23.3% versus 21.6%), with no difference between sickle cell disease groups. This observation shows that Tollip gene rs5743899 mutant variant is a potential contributor to predisposing sickle cell patients but not control groups in Africa to infectious co‐morbidities, and may explain the high levels of circulating immune complexes in disease groups. This report reveals interethnic susceptibility to infectious co‐morbidities and how this contributes to immune response in sickle cell disease.

Support or Funding Information

RIT Louis Stokes Alliance for Minority Participation Fellowship (NSF Award #25901‐03200‐S01) (SEA)Laboratory Support Fund, College of Health Sciences and Technology (BNT)
After Oxidation Zinc Nanoparticles Lose the Ability to Enhance Responses to Odorant
Authors: Singletary Melissa , Daniels Yasmine , Hagerty Samantha , Pustovyy Oleg , Globa Ludmila , MacCrehan William A , Morrison Edward E , Sorokulova Iryna , Vodyanoy Vitaly J
Published: June 2021

Previous studies have demonstrated that 1–2 nm zinc metal nanoparticles at small concentrations can enhance electroolfactogram (EOG) responses to the odorant. Introducing these particles to an odorant can increase responses up to 3‐fold1. Small zinc nanoparticles are comprised of only ≈60 metal atoms and, therefore, their stability in water is under consideration. The possibility that zinc nanoparticles enhance olfactory responses after oxidation is unknown. Zinc metal nanoparticles were produced from bulk metal rods by an underwater high‐voltage discharge method. Part of the small zinc nanoparticles were oxidized by percolating of air through the water suspension of nanoparticles. NIST zinc oxide nanoparticles of 15 and 70 nm 2 were also used in olfactory experiments. Odorant responses were evoked by the odorant mixture of ethyl butyrate, eugenol, and (+) and (−) carvone combined with metal or metal oxide nanoparticles. The EOG was recorded with isolated rat olfactory epithelium. The metal nanoparticles were characterized by atomic force microscope (AFM) and X‐ray photoelectron spectroscopy (XPS). AFM revealed size distributions of small Zn metal nanoparticles, with average diameters of 1.2 ± 0.3 nm. The XPS high‐resolution 2p3/2 spectra for zinc and zinc oxide revealed 93.9 ± 3.4 % and 5.9 ± 3.7 % of Zn and ZnO, respectively. This data indicated that more than 93 % of Zn atoms were not oxidized. EOG evoked by odorant mixed with small Zn nanoparticles, small and large ZnO nanoparticles showed that only small non‐oxidized Zn nanoparticles enhanced odorant responses. None of the particles delivered without odorant produced olfactory responses. This experimental evidence strongly supports our observations of the influence of Zn metal nanoparticle size on the enhancement of olfactory odorant responses. It has been suggested that the most critical property depending on metal particle size is its ability to donate and accept electrons3. A large number of small stable metal Zn nanoparticles were found in human and animal blood 4. We speculate that Zn nanoparticles function as electron donors in the Turin's model of olfaction5.

Support or Funding Information

Grant from the National Institute of Standards and Technology: 70NANB14H324
Intermittent Hypoxia Enhances Connectivity between Cervical Spinal Interneurons
Authors: Streeter Kristi , Sunshine Michael , Patel Shreya , Reier Paul , Baekey David , Fuller David
Published: June 2021

Intermittent hypoxia is a potent initiator of spinal plasticity and a promising rehabilitative modality to enhance respiratory and non‐respiratory motor function in certain cases of neurologic dysfunction. Current models of intermittent hypoxia‐induced spinal plasticity have focused exclusively on motoneurons, and nothing is known about how cervical spinal interneurons are impacted by intermittent hypoxia. Prior work has established that most cervical spinal interneurons alter discharge rates during acute hypoxia, and that some spinal interneurons are synaptically coupled with phrenic motoneurons. We hypothesize that acute intermittent hypoxia triggers “network level” spinal plasticity which manifests as an increase in the prevalence of temporally related discharge of spinal interneurons. To test this hypothesis, bilateral phrenic motor output was recorded in N=12 anesthetized and ventilated adult rats and a 16‐channel multi‐electrode array was used to record mid‐cervical neuron activity during and following three, 5‐min episodes of hypoxia (11% O2). To evaluate “functional connectivity” among spinal interneurons, cross‐correlation histograms were constructed for all possible pairs of simultaneously recorded neurons. A positive correlogram “peak” > 3 stdev above the mean was taken to indicate an excitatory connection. The current analysis focused on post‐hypoxia discharge, and data were included for 319 neuronal pairs in which a correlogram peak was detected during any post‐hypoxia period. A correlogram peak was present in 24% of neuronal pairs during the pre‐hypoxia baseline. This value tended to increase during hypoxia (Hypoxia‐1: 34%; Hypoxia‐2: 30%; Hypoxia‐3: 34%), but this did not reach statistical significance (all P>0.05 vs. baseline). In contrast, the number of excitatory connections, as reflected by positive correlogram peaks, were significantly increased following each bout of hypoxia (Hypoxia‐1: 60%; Hypoxia‐2: 53%; Hypoxia‐3: 50%) (all P<0.05 vs. baseline). This is the first demonstration that intermittent hypoxia alters the connectivity of spinal networks and we therefore conclude that the spinal impact of intermittent hypoxia extends beyond phrenic motoneurons. These data support the hypothesis that neuroplasticity in spinal networks contributes to sustained changes in respiratory and/or autonomic output following intermittent hypoxia.

Support or Funding Information

NIH: 1F32NS095620‐01 (KS); 1R01NS080180‐01A1 (DF); and T32‐ND043730 (MS). Department of Defense W81XWH‐14‐1‐0625 (PR).
Sugar‐Sweetened Beverage Consumption in Young Adults: Understanding Eating Occasion Contextual Factors
Authors: McNaughton Sarah A , Leech Rebecca M , Pendergast Felicity J , Worsley Anthony
Published: June 2021

Background/Objectives

Young adulthood represents an influential transitional period, marked by poor dietary habits and excess weight gain. Sugar‐sweetened beverages (SSBs) are a major source of excess caloric intake among young adults, yet little is known about the correlates of SSB consumption. This study examines the correlates of SSB consumption including eating occasion contextual factors, using real‐time assessment of young adults' eating occasions (EOs).

Methods

Data were collected during the Measuring Meals in Everyday Life (MEALS) study (n=557, adults 18–30 years). Participants reported all foods and beverages consumed over 3–4 non‐consecutive days using a food diary recorded via a Smartphone app. Contextual characteristics such as eating location, purchase location, presence of others and activities while eating were documented in the app at each EO. Information on sociodemographic factors and health behaviours were collected via an online questionnaire. An “eating occasion” was defined as an occasion when any food or beverage was ingested, while a “sugar‐sweetened beverage occasion” was defined as any EO where a SSB was consumed (with or without other items). SSB consumers were categorised as low or high SSB consumers based on the median frequency of consumption. Associations for individual level and EO level characteristics with level of SSB consumption were analysed using multivariate logistic regression, accounting for the clustered nature of the data.

Results

Overall, 30% of participants consumed SSBs. Most SSBs were consumed: either alone (43%) or with family/partner (42%); at home (50%), and while undertaking screen‐based activities (46%). Most SSBs consumed were purchased from the supermarket/grocery store (58%). Compared to non‐consumers, SSB consumers had a significantly higher BMI (mean [SD]: 23.1 [5.7] v 21.8 [3.9], P<0.001). Being a current/past smoker, being overweight/obese and participating in screen‐based activities were associated with being a high SSB consumer (P<0.01). After adjustment for covariates, smoking status (OR [95% CI]=5.69 [1.98,16.31]; P<0.01) and weight status (2.31 [1.06,5.01]; P<0.05) remained significantly associated with high SSB consumption.

Conclusions

Almost a third of young adults in this study consumed SSBs and most SSBs were consumed alone or with family/partner, at home and while undertaking screen‐based activities. However, individual factors, rather than EO contextual factors, were more strongly associated with being a high SSB consumer. Future research is needed to explore how SSB occasions differ from other beverage consumption occasions in relation to their accompanying foods and contextual characteristics.

Support or Funding Information

Australian Research Council, Victorian Health Promotion Foundation, National Health and Medical Research Council
Depletion of Dietary Bovine Milk Exosomes Impairs Sensorimotor Gating and Spatial Learning in C57BL/6 Mice
Authors: Mutai Ezra , Zhou Fang , Zempleni Janos
Published: June 2021

Background

Bovine milk exosomes contain cargos such as miRNAs, mRNAs, DNA and proteins. We have demonstrated that dietary bovine exosomes deliver functional RNA species and regulate genes and metabolism in humans and other non‐bovine species. Preliminary studies suggest that RNAs obtained through milk exosomes accumulate in the brain of mice.

Hypothesis

Depletion of dietary bovine milk exosomes affects cognitive performance in C57BL/6 mice.

Methods

C57BL/6 mice were fed AIN‐93G based, bovine milk exosome‐defined diets for up to 20 weeks. Exosome‐depleted diets are denoted E−; exosome‐sufficient controls are denoted E+. Spatial learning and memory were assessed using the Barnes maze and Morris water maze. Sensorimotor gating was assessed using an acoustic startle response (ASR) system. The statistical significance of differences was assessed by unpaired, two‐tailed t‐test.

Results

The time needed to locate the escape hole in the Barnes maze increased by up to 130% in mice fed the E− diet compared to E+ controls (Fig. 1). Likewise, the time needed to locate and reach the submerged escape platform in the Morris water maze was significantly greater in female mice fed E− diet than female controls (Fig. 2). Prepulse inhibition (PPI) of the ASR is a measure of sensorimotor gating, and was significantly lower in female mice fed the E− diet than in female controls (Fig. 3). Diet did not affect PPI in male mice (p>0.05).

Conclusion & future plans

Diets defined by their content of bovine milk exosomes affect sensorimotor gating and cognitive performance in mice. Future studies will investigate which regions and cells in the brain accumulate dietary milk exosome RNA cargos, and whether a distinct species of RNA is responsible for the phenotypes reported here.

Support or Funding Information

Support: NIFA 2015‐67017‐23181, NIFA 2016‐67001‐25301/NIH DK107264, NIH 1P20GM104320, the Gerber Foundation, and USDA Hatch Act and W3002.
Functional characterization of three GH10 xylanases
Authors: Park Jesslyn , Glasgow Evan , Fox Brian G
Published: June 2021

Decreased availability of fossil fuels has driven an increased demand for sustainable energy sources such as biofuels. One area of bioenergy research involves development of biocatalysts able to efficiently degrade biomass “waste”‐products, such as substrates derived from corn stover, rather than easily‐fermentable biomass used for food, such as starch from corn kernels. Xylanases are of interest for their ability to hydrolyze hemicellulosic sugars found in plant cell walls, allowing utilization of waste‐derived biomass. In this study, three xylanases of glycoside hydrolase family 10 (GH10) are functionally characterized and optimized for temperature and pH, as well as kinetically analyzed for xylose monomer and oligomer production. These enzymes, GH10‐4, GH10‐9, and GH10‐15, demonstrate activity upon substrates xylan and arabinoxylan. For GH10‐4, optima are 50°C at pH 8 on xylan and 45°C at pH 8 on arabinoxylan. For GH10‐9, optima are 70°C at pH 5 on xylan and 70°C at pH 5 on arabinoxylan. For GH10‐15, optima are 50°C at pH 8 on xylan and 45°C at pH 5 on arabinoxylan. Kinetic analysis reveals GH10‐4 produces xylose (X1), xylobiose (X2), xylotriose (X3), xylotetraose (X4), and xylopentaose (X5) from xylan, while GH10‐9 and GH10‐15 produce X1, X2, and X3. From arabinoxylan, GH10‐4 produces X1, X2, X3, X4, while GH10‐9 and GH10‐15 produce X1, X2, X3. Conclusive kinetic data may suggest applicability of these enzymes for industrial use within biocatalysts and contribute further insight to the functionality of enzymes within GH10.

Support or Funding Information

The authors are grateful for financial support by the Integrated Biological Sciences Summer Research Program (IBS‐SRP, NSF) at the University of Wisconsin‐Madison and the Great Lakes Bioenergy Research Center (GLBRC) of the U.S. Department of Energy. Presentation travel support is provided by the Ronald E. McNair Scholars Program of Winthrop University. The authors also thank Lai Bergeman, Craig Bingman, Bob Smith, Emily Beebe, Kirk Vander Meulen, Amber Smith, David Wassarman, and John Greenler for professional support.
Temperature‐Induced Changes to Aquaporin Localization in the Freeze‐Tolerant Cope's Gray Treefrog
Authors: Stogsdill Brian , Frisbie James , Krane Carissa , Goldstein David
Published: June 2021

Cope's gray treefrogs alter their physiology in response to cold as part of their freeze tolerance mechanism. One such change involves the accumulation, both extracellularly and intracellularly, of cryoprotective glycerol, which likely crosses cellular membranes using glycerol‐permeable channels called aquaglyceroporins (GLPs). Three GLPs (HC‐3, HC‐7, and HC‐9) have been identified in the Cope's gray treefrog, representing homologs to the mammalian AQP3, AQP7, and AQP9, respectively.

Regulation of transmembrane glycerol diffusion depends on GLP expression in the plasma membrane resulting from changes to transcriptional and translational output of the GLPs and/or by changes in protein trafficking to the plasma membrane. We have previously reported that GLP mRNA and protein expression are modulated during cold acclimation and freezing; however, localization of GLP proteins in the plasma membrane has not been studied thoroughly. We hypothesized that changes in acclimation temperature would alter the localization of the GLPs HC‐3 and HC‐9 in the plasma membrane and confirmed this with immunofluorescence, plasma membrane fractionation, and biotinylation.

For HC‐9 in liver, quantitation of protein expression (by immunoblot) showed a 4‐ to 6‐fold increase in the higher molecular weight band of frozen (−2.5°C for 24 hours) and thawed (5°C for 24 hours after freezing) over warm (20°C) treefrogs. In immunohistochemistry images, HC‐9 signal appeared to be localized to the plasma membrane of hepatocytes of warm‐ and cold‐acclimated animals but dispersed intracellularly in frozen treefrog hepatocytes. In preliminary studies, fractionation of cellular protein (Biovision, Milpitas, CA) indicated that between freezing and thawing, the nominal (~34 kDa) molecular weight band of HC‐9 diminishes in the plasma membrane fraction while the (putatively) glycosylated high molecular weight band of HC‐9 increases.

For HC‐3 in erythrocytes, protein expression is enhanced by cold acclimation and by incubation in medium containing glycerol. We incubated erythrocytes taken from warm‐acclimated animals at 4°C for 48 hours and biotinylated the surface proteins. In immunoblots of the biotinylated fraction probed with anti‐HC‐3 primary antibody, the high molecular weight (glycosylated) band was enriched and the nominal (~32 kDa) molecular weight band was diminished. These results suggest that water and glycerol permeability during cold acclimation and freezing can be modulated by protein trafficking, which may be influenced by the post‐translational modification of glycosylation.

Support or Funding Information

Supported by NSF IOS‐1121457.
Undergraduate Student Research in Quantitative Analysis of Transcription Elongation Perturbation Networks Using Mass Spectrometry
Authors: Bedard Lynn , Boyd Asha , Dyer Nichole , Golay Zachary , Smith‐Kinnaman Whitney , Alakhras Nada , Mosley Amber L
Published: June 2021

The main goal of this project is to identify how cells respond and adapt to genetic changes that alter the process of RNA synthesis. Using baker's yeast as the study system, experimental approaches provide undergraduate students with early exposure to cutting‐edge research technologies that are broadly applicable in both academia and industry. The project introduces students at all levels of training to the importance of quantitative methods to interrogate biology. The specific focus of the research is to understand the consequences of genetic perturbations that knock out or reduce expression of non‐essential elongation factors on RNA polymerase II‐catalyzed RNA synthesis (transcription). Preliminary data suggest that such genetic perturbations lead to significant adaptations within the RNA polymerase II protein‐protein interaction networks. These adaptations may include (but are not limited to) disruption of interactions with RNA polymerase II, compensation leading to novel protein recruitment, and activation of rescue pathways to dispose of arrested RNA polymerase II. The laboratory portion of an undergraduate course in molecular biology was redesigned to generate elongation factor knock‐out yeast strains with a focus on key molecular biology concepts and techniques including: genomic DNA isolation, oligonucleotide design, PCR, genotyping, protein isolation, and western blotting. In addition to these key concepts, we introduced students to the theory and practice of proteomics analysis through mass spectrometry including discussions of bioinformatics analysis of proteomics data, calculation of false discovery rates, and affinity purification mass spectrometry approaches. Thus far in this study, 9 elongation factor knock out strains have been made and verified by DNA sequencing and are in the process of being analyzed by quantitative mass spectrometry. Data from pre‐ and post‐instructional tests will be presented which demonstrate increased student understanding of mass spectrometry. Additionally, students participated in an 8 week summer internship held at the IU School of Medicine where they extended the project from the classroom lab to performing quantitative mass spectrometry analysis of their elongation factor knock‐outs.

Support or Funding Information

DePauw University Student Faculty Research Fund, Showalter Fund (IU), NSF Award Number:1515748 PI Amber Mosley, PhD
Effects of Isotopic Substitution in Enzyme and Co‐factor on Enzyme Catalyzed Hydride Transfer
Authors: Ranasinghe Chethya , Pagano Philip , Guo Qi , Cheatum Christopher , Kohen Amnon
Published: June 2021

Enzyme dynamics range from seconds to femtoseconds. Previous computational, theoretical, and experimental studies have shown an imperative role of slow enzyme dynamics in its function. It is equally important to unravel the origin of fast enzyme dynamics, which are more likely to play a role in catalyzing chemical bond cleavage. Isotopically labeled (heavy) enzymes and subsequently heavy cofactors were employed to experimentally study the role of fast dynamics. Here, we used nicotinamide adenine dinucleotide (NAD+) dependent formate dehydrogenase (FDH, EC 1.2.1.2) as the model system. Heavy enzyme studies were carried out with 13C; 15N and 13C; 15N; 2H labeled FDHs. Temperature dependence of kinetic isotope effects (KIE) were compared between light and heavy FDHs. The results showed that perturbation of fast enzyme dynamics by mass modulation, alters the chemical step at the tunneling ready state (TRS, a diffused transition state) of the catalyzed hydride transfer. Studies are underway to examine similar trend using heavy cofactor (NAD+). Temperature dependence of KIE would give insight to the extent of contribution of the ring motions in sampling the donor‐acceptor distances at the TRS.

In addition to the kinetic measurements, vibrational spectroscopy will be used to test the effect of heavy protein and cofactor on dynamics at the enzyme's transition state. Azide ion, which is a transition state analog of the FDH catalyzed reaction, is a good IR chromophore. This enables the use of vibrational spectroscopic methods such as two dimensional IR (2D‐IR), to directly probe the femtosecond‐picosecond timescale motions in the enzyme active site. Together, these studies should shed light on the molecular origin of fast motions that enhance catalytic efficiency of enzymes.

Support or Funding Information

NIH GM065368 and GM79368, NSF CHE‐1149023, BSF 2012340
Young adults physical activity behavior and chronic disease prevention knowledge across Appalachia
Authors: Barr Makenzie L , Beller Erika L , Colby Sarah E , Morrell Jesse , Kidd Tandalayo , Riggsbee Kristin , Olfert Melissa Diann
Published: June 2021

Chronic disease refers to a host of diseases ranging from pulmonary diseases to cardiovascular diseases, obesity, diabetes, metabolic disorders, and cancer. Within the United States population, the Appalachian region (New York, Ohio, Pennsylvania, West Virginia, Kentucky, South Carolina, North Carolina, Georgia, Mississippi, and Alabama) has some of the highest incidence rates within the country. Conditions such as these are on the rise, and intervening is critical. Specifically, the knowledge of chronic disease prevention is important to understand early in the life span to decrease incidence in older adulthood. Additionally, previous research has shown that physical activity is a primary preventative action for chronic disease. In a sample of 18–24 year olds from eight different universities, which are predominately Caucasian (55%), non‐Appalachian (83.7%, Appalachian N=949, non‐Appalachian N=132), and female (66%), chronic disease prevention knowledge (CDK) and physical activity (PA) were summarized to identify the differences in scores across Appalachian status. CDK was broken into two categories: knowledge of fat and fiber in foods as it related to prevention and knowledge and beliefs of diet and cancer risk. These categories were scored on a scale of 1=low knowledge, 2=moderate knowledge, and 3=high knowledge. PA was scored from the International Physical Activity Questionnaire (IPAQ) of 1=not active, 2=moderately active, and 3=very active. Participants who reported as identifying as Appalachian status had their highest proportion of individuals in the low knowledge level for fat and fiber knowledge (52.5%) and moderate level for diet and cancer knowledge (54.1%). For PA, Appalachian status individuals had their highest proportion in the ‘very active’ category (52.8%). Likewise, in the non‐Appalachian group, individuals were also highest proportion in the low knowledge level for fat and fiber knowledge (54.5%), and moderate level for diet and cancer knowledge (57.9%). Further, in IPAQ category, they were also in the ‘very active’ category (50.7%). For the current results, across both Appalachian and non‐Appalachian status, young adults from the eight universities are reported as being very active but have low knowledge of fat and fiber in foods for a prevention measure and moderate knowledge and beliefs of how diet impacts cancer risk. Further exploration is needed in the education of young adults on prevention of diet and chronic diseases.

Support or Funding Information

Approval to use the data set was granted by the University of Tennessee Institutional Review Board prior to study implementation. This material is based upon work that is supported by the National Institute of Food and Agriculture, U.S. Department of Agriculture, under award number 2014‐67001‐21851.
INDIVIDUAL VARIABILITY IN SYMPATHETIC NEURAL RESPONSES TO ALTITUDE EXPOSURE: RELATIONSHIP TO SEA LEVEL SYMPATHETIC NERVE ACTIVITY
Authors: Mitchell Katherine , Bradbury Karleigh , Beidleman Beth A. , Fulco Charles S. , Muza Stephen , Posch Alexander , Charkoudian Nisha
Published: June 2021

Acute or chronic altitudeexposure causes increased sympathetic nerve activity (SNA), which may persist after the exposure ends. However, it is unknown if an individual's level oftonic SNA at sea level (SL, 50 m) is associated with the change in SNA after chronic altitude exposure.

PURPOSE

To evaluate muscle SNA (MSNA) at SL before and after 12 days of altitude residence at the summit of Pikes Peak, CO (PP, 4300 m). We hypothesized that: (1) the altitude exposure would result in increased MSNA which would persist into the first 24 hours after return to SL; and (2) the magnitude of individual increases in MSNA after exposure would be inversely related to their SL baseline levels of MSNA.

METHODS

Supine MSNA (via peroneal microneurography), heart rate (HR), and blood pressure (BP) were measured pre‐ and post‐residing on the summit of PP for 12 days in 9healthy humans (7 M, 2 F; 24 ± 8 years). Paired t‐tests were used to compare pre‐ and post‐altitude responses.

RESULTS

MSNA was higher within 24 hours of return from altitude (27 ± 5 bursts/min; 51± 10 bursts/100 heart beats [hb]) vs. SL (17 ± 6 bursts/min; 33 ± 14 bursts/100hb)(p < 0.01 for both). Individuals with higher SL baseline MSNA had smaller increases in MSNA after altitude exposure than those with lower SL MSNA (r = − 0.69,p < 0.05). Altitude exposure resulted in no significant differences in HR or BP (p > 0.05 for both).

CONCLUSION

After 12 days of altitude residence, (1) MSNA levels remain elevated during the first 24 hrs after return to SL, and (2) there is individual variability in the magnitude of the MSNA increase that is inversely related to SL baseline MSNA. Funded by USAMRMC; author views not official US Army or DOD policy.

Support or Funding Information

Funded by USAMRMC
Expression, purification, and characterization of an intrinsically disordered Late Embryogenesis Abundant (LEA) protein from Artemia franciscana utilizing Escherichia coli and Nicotiana tabacum
Authors: Karim Md Fazlul , Yordanov Yordan S. , Menze Michael A.
Published: June 2021

Anhydrobiosis is an astounding strategy that allows certain species (both in animals and plants) to survive severe environmental conditions such as desiccation, extreme cold, or heat in the habitat. Among different molecular strategies, expression of highly hydrophilic polypeptides termed LEA proteins has been linked to the survival of plants and animals during periods of water stress such as freezing and drying. Several classification schemes for LEA proteins have been proposed and the brine shrimp, Artemia franciscana, is the only known animal that naturally expresses LEA proteins from three different classification groups (groups 1, 3, and 6). LEA proteins occur in several subcellular compartments including the cytosol and mitochondria. To investigate the biochemical properties of LEA proteins, it is important to characterize their structure. LEA proteins are intrinsically disordered in aqueous solution and the exact structure and function of these proteins is still poorly defined and understood. We hypothesized that LEA proteins will show different protective properties depending on the target enzyme (e.g. lactate dehydrogenase vs. succinate dehydrogenase). We found, that a purified group 1 LEA protein from A. franciscana (AfrLEA 1.1) helped to retain enzyme activity after desiccation of lactate dehydrogenase (LDH) and dry storage of the enzyme for 1, 3 and 7 days in the presence or absence of bovine serum albumin or trehalose. Increased concentration of purified AfrLEA 1.1, increased the percentage of LDH activity retained after desiccation. To further characterize AfrLEA 1.1, we cloned, expressed, and purified the protein in E. coli. We purified untagged AfrLEA 1.1 protein by affinity chromatography via intein mediated purification with an affinity based chitin‐binding system; a novel protein purification system which utilizes the inducible self‐cleavage activity of protein splicing elements to separate the target protein from the affinity tag. Furthermore, AfrLEA1.1 was expressed in Nicotiana tabacum to investigate if the protein increases drought tolerance of this model plant.

Support or Funding Information

Supported by NSF IOS‐1456809
Shifting Sands: Modulating Interactions and Outputs of the CHIP‐Hsp70/HOP‐Hsp70 Protein Quality Control Complexes
Authors: Page Richard C , Zhang Huaqun , Cottingim Kelsey , VanPelt Jamie , Nix Jay C , Schisler Jonathan
Published: June 2021

The 70‐kilodalton heat shock protein (Hsp70) interacts with tetratricopeptide repeat (TPR) domains of CHIP (C‐terminus of Hsp70 Interacting Protein) and HOP (Hsp Organizing Protein) using a two‐carboxylate clamp binding mechanism. Differential affinity for the TPR domains of CHIP and HOP are achieved through interactions with the six residues preceding the carboxy‐terminal Asp641 of Hsp70. In particular, phosphorylation of Hsp70 Thr636 produces a dramatic swing in binding affinities that shift preferential formation of CHIP/Hsp70 complexes to preferential formation of HOP/Hsp70 complexes. Given that CHIP‐Hsp70 complexes promote ubiquitin‐mediated degradation of Hsp70‐chaperoned clients while HOP‐Hsp70 complexes promote refolding of Hsp70‐chaperoned clients, phosphorylation of Hsp70 Thr636 acts as a switch that directs the output and function of cellular protein quality control. Herein, through biophysical assays and new structures of CHIP‐Hsp70 and Hop‐Hsp70 we identify the molecular mechanism that enables phosphorylation‐driven switching that directs interactions within the CHIP‐Hsp70/HOP‐Hsp70 protein quality control complexes. We also present a structure‐guided mutation of Hsp70 that bypasses Thr636 phosphorylation‐driven switching by recovering affinity of CHIP for phosphorylated Hsp70, thereby redirecting Hsp70 to preferentially bind to CHIP. This structure‐guided mutation corrects a binding defect present in wild type CHIP and provides a system for studying the effect of shifting output of the CHIP‐Hsp70/HOP‐Hsp70 protein quality control complexes toward chaperoned ubiquitination via the CHIP‐Hsp70 complex.

Support or Funding Information

The authors acknowledge financial support from the US National Science Foundation (Award No. MCB 1552113 to RCP), the American Heart Association (Award No. 16SDG26960000 to RCP), the Burroughs Wellcome Fund (Award No. 1014031 to RCP), and institutional support from Miami University through the Robert H. and Nancy J. Blayney Professorship (to RCP). The Advanced Light Source is supported by the US Department of Energy under contract number DE‐AC03‐76SF00098 at Lawrence Berkeley National Laboratory.
Interactions of Rat Embryonic Hippocampal Neuronal Stem Cell on Au‐nanowire Arrays and Gold Nano‐Film Surface
Authors: Islam Asiful
Published: June 2021

Hippocampal neuronal stem cell culture on the nano‐substrates has introduced new era in nano‐neuroscience research. Neurons were cultured on the gold nano‐wire and nano‐film surfaces to see the viability and interactions between neurons and nanoscale surfaces. Anodization of aluminum templates created nanoporous alumina tubes arranged parallel to each other and electrodeposition using gold solution (AuHCl4) resulted in arrays of gold nanowires (GNWs) inside the pores. For investigation of Au‐nano‐neuronal interactions we have cultured embryonic day 18 rat hippocampal neurons on the surface of gold nanowires and surfaces. For preliminary understanding of the neuronal growth and connectivity we studied the images of nano‐neuronal interactions by using optical, fluorescence and scanning electron microscopy (SEM). For adhesion of neurons to nano‐arrays and nano‐surfaces we used bioconjugating protein coating Poly‐D‐Lysine (PDL). The rat hippocampus tissues were mechanically dissolved into neuronal cells and cultured on top of the gold nano‐wire (GNW) and flim (GNF) surfaces and preserved at 37° C constant temperatures in a 5% CO2 incubator. These neurons on the GNW and GNF surfaces started to grow from day 2 and survived up to 25 days or more. The extracellular protein PDL acted as an intermediate substance for neurites growth on the nano films slips. Without a PDL coating layer showed no or very little growth of neurons on the GNF substrates. We demonstrated well‐defined neuronal networks on Au‐nanowire arrays and GNF. The neurons attached with the nanowires surfaces by PDL, which is a positively charged poly‐peptide and plays a vital role in neurites growth on the nanowire arrays. The pre‐and post‐synaptic effects of neuronal interactions on the surfaces of nanowires were measured by Ca+ imaging in keck microscope. This study is the initial result for implementing the gold nano‐substrates to record the electrical signals from the neurons through gold nano‐films or nano‐wires.

Support or Funding Information

NSF
Facial Imagery BMI Algorithm correlates with Normal and Overweight Measured BMI
Authors: Barr Makenzie L , Famodu Oluremi A , Guo Guodong , Colby Sarah E , Olfert Melissa Diann
Published: June 2021

Efficient procedures to determine health status are in high demand in today's generation. Body Mass Index (BMI) is a global method for determining an individual's health status. This research exploration is to identify if a facial image (photograph) can identify participant's BMI correctly. Sample of 1,210 young adults with a facial image and objective height and weight were used for analysis. Facial BMI (fBMI) was measured through the Local Binary Pattern (LBP)/Active Shape Model (ASM) was used to detect 76 points on each face and measured BMI (mBMI) was calculated by weight in kilograms divided by height in meters squared. Correlation analysis of fBMI to mBMI showed significant correlation between BMIs in the normal and overweight categories (p<.0001). Further analysis indicated the measure to be less efficacious in underweight and obese participants. Matched pairs data for each individual, there was a range of 14.73–49.74 for mBMI and a narrowed spread for fBMI (range = 16.29–28.85) indicating that fBMI detected participant BMI 0.4212 less than mBMI (p<.0004). BMI categories used included: less than 18.5 BMI (0), 18.5–24.9 (1), 25.0–29.9 (2), and equal to or above 30 (3). mBMI had representation of categories across 0–3 BMI, while fBMI representation included just 0–2. This shows less sensitivity of the algorithm to obese individuals. Contingency table analysis indicted 109 participants in the 3rd category of mBMI were placed into a lower category for fBMI. Agreement test for symmetry shows significant disagreement 95% CI [0.0618,0.1446] (p<.0001). Facial imagery identification of health status is a useful measure in human research however; more sensitive measures to identify underweight and obese individuals are warranted.

Support or Funding Information

Approval to use the data set was granted by the University of Tennessee Institutional Review Board prior to study implementation. This material is based upon work that is supported by the National Institute of Food and Agriculture, U.S. Department of Agriculture, under award number 2014‐67001‐21851.
Do Cord Serum Ferritin Concentrations Serve as an Index of Neonatal Iron Status?
Authors: Delaney Katherine M. , Fleming Robert K. , Guillet Ronnie , Queenan Ruth Anne , Pressman Eva K. , O'Brien Kimberly O.
Published: June 2021

Low iron stores at birth have been associated with adverse health outcomes. Although the importance of this issue has been highlighted, few studies have examined the relationship between indicators of iron status and hemoglobin concentrations at birth. To address this issue, we measured a broad panel of iron status indicators and associated regulatory hormones in 471 infants born from two obstetric populations known to be at increased risk of maternal iron deficiency and anemia: pregnant adolescents (n=280) and women with a multiple pregnancy (n=191). Neonatal cord blood and maternal blood samples were obtained at delivery, and used to evaluate concentrations of hemoglobin (Hb), serum iron (Fe), ferritin (SF), soluble transferrin receptor (sTfR), hepcidin, interleukin‐6 (IL‐6), erythropoietin (EPO), folate and vitamin B12. Delivery occurred at average 39 ± 3 weeks of gestation in adolescents, and at 34 ± 3 weeks in women carrying multiples. When analyzed as a whole, cord hepcidin was found to be inversely correlated with hemoglobin (Hb) (p = 0.0134) and with serum iron (p < 0.001), while positively correlated with IL‐6 (p < 0.0001). No significant associations were found between maternal hepcidin concentrations and neonatal markers of Fe status, including cord hepcidin. Despite serving as a marker of iron status in other settings, ferritin concentrations in cord blood were not significantly associated with any of the Fe status indicators evaluated, and were instead inversely associated with Hb (p = 0.0032). Excluding values from neonates with elevated cord IL6 concentrations did not alter these findings. Additionally, no correlation was observed between cord IL‐6 concentrations and Hb. These results challenge the current understanding that serum ferritin can serve as an independent marker of Fe status at birth. Our observations underscore the need for further identification and characterization of biomarkers of fetal and neonatal Fe status, and their relationships with outcomes historically attributed to poor iron status during this developmental time frame.

Support or Funding Information

This work was supported by US Department of Agriculture (USDA) and the Gerber Foundation