bluuebunny/crossref_metadata_2025 huggingface dataset:
The Effect of Intracellular pH on Myocardial Calcium Sensitivity in the Anoxia‐Tolerant Painted Turtle
Authors: Fanter Cornelia E , Warren Daniel E , Campbell Kenneth S
Published: June 2021

The anoxia‐tolerant western painted turtle, Chrysemys picta belli, has the ability to survive without oxygen for over 170 days at 3°C due to extreme metabolic responses that minimize energy usage. Under such conditions, the heart minimizes rate and contractility, resulting in a decreased cardiac output. The onset of anoxia is accompanied by a substantial decrease in intracellular pH, which suggests that the reduction in contractility results mostly from mechanical changes in the myofilament sensitivity to Ca2+ associated with intracellular H+ accumulation. To test this hypothesis, the cellular mechanical properties of turtle myocardium were studied at different pH's from turtles exposed to either 24 hours of anoxic or normoxic conditions at 20°C. Multicellular preparations of turtle ventricles were chemically permeabilized and subsequently attached to a motor and force transducer. Preparations were exposed to an array of pCa solutions varying in pH from 7.4 to 6.6 at 22 °C. The concentration of calcium required to trigger 50% of maximum tension (pCa50) decreased stepwise: pCa50 was 6.2, 5.9 and 5.6 for pH's 7.4, 7.0 and 6.6, respectively. The shift in pCa50 indicates that as pH decreases, 50% tension production requires higher calcium levels. Additionally, maximum contractile force decreased as pH decreased. These data indicate that an increased concentration of H+ results in reduced myofilament sensitivity to calcium and, therefore, a decreased ability for force development. Comparisons between normoxic and anoxic tissues indicated no mechanical differences, suggesting that the metabolic changes seen at the onset of anoxia are likely to be a response of intracellular compositional changes of the chemical environment, rather than structural changes to the contractile apparatus. Overall, pCa50 the painted turtle myocardium is intrinsically more sensitive to calcium concentrations (5.9 at pH 7.0) when compared to human and rat (5.67 and 5.2 at pH 7.0), but is similar to other ectotherms.

Support or Funding Information

This work was supported by a CAREER award from the National Science Foundation.
Glyceroporin HC‐9 in the Freeze Tolerance Mechanism of Cope's Gray Treefrogs
Authors: Stogsdill Brian , Frisbie James , Tsou Pon Ti , Goldstein David
Published: June 2021

Cope's gray treefrogs Hyla chrysoscelis accumulate glycerol in response to cold as part of their freeze tolerance mechanism. This glycerol may derive from glucose stores in the liver that are broken down and released through glycerol transporters. We therefore hypothesized that these treefrogs would express aquaporin 9 (AQP9), a transmembrane protein from the aquaglyceroporin family that facilitates glycerol transport, predominately in the liver. We also hypothesized that this protein would be up‐regulated during cold acclimation to promote glycerol permeability. To test this hypothesis, we sequenced the AQP9 homolog HC‐9, analyzed RNA and protein expression, and characterized water and glycerol permeability through a Xenopus oocyte expression system.

HC‐9 has 315 amino acids which include 2 conserved NPA motifs characteristic of aquaporins, 5 conserved residues typical of glyceroporins, and high sequence homology to human AQP9 (69%) and to AQP9 from Hyla japonica (97%). The mRNA strand also possesses a long untranslated tail that appears to be unique to amphibians and necessary for translation. Characterization using the Xenopus oocyte expression system indicates that HC‐9 is permeable to glycerol but has a poor water permeability compared with human AQP1.

In warm‐acclimated treefrogs, HC‐9 mRNA (RT‐PCR) and protein (Western blotting) were expressed in many tissues, with highest levels in liver, stomach, ventral skin, and muscle. Across most tissues, realtime mRNA expression (qRT‐PCR) decreased in cold‐acclimated (5°C) and frozen (−2.5°C) treefrogs, with return to levels near those in warm acclimated animals after return to 5°C for 24 hours. In contrast, protein expression increased in the liver and ventral skin of cold and frozen treefrogs, but remained unchanged or decreased in all other tissues.

HC‐9 expression is clearly regulated as a part of the Cope's gray treefrog freeze tolerance mechanism. Contrary to our hypothesis, most tissues have decreased expression of HC‐9 in the cold, which may reduce glycerol permeability to protect cells during the freezing process. Liver is a notable exception by increasing protein expression from warm to cold to frozen conditions. Liver appears to play a critical role in the distribution of the cryoprotectant glycerol during the freeze‐thaw cycle, and this study suggests HC‐9 facilitates in this role.

Support or Funding Information

Supported by NSF IOS‐1121457
Effects of Hyperosmotic Stress on Cellular Proteome and Phenotype of Tilapia (Oreochromis mossambicus) OmB Cells
Authors: Wiegers Toni Nicole , Kültz Dietmar
Published: June 2021

Fish that inhabit an environment of fluctuating salinity (e.g. estuaries, intertidal zones) are frequently exposed to osmotic challenges. These euryhaline fish must cope with unpredictable salinity changes, and thus must respond quickly to alterations in environmental osmolality. Some euryhaline fish, e.g. tilapia, also face irregular and longer periods of high salinity, for instance during seasonal droughts in arid areas. Even though teleosts are osmoregulators, hyperosmotic stress leads to a significant increase in plasma osmolality. To prevent the detrimental effects that result from hyperosmotic stress if unaccounted for, cells must compensate for increased plasma osmolality. A previously immortalized Mozambique tilapia (Oreochromis mossambicus) cell line derived from the brain (OmB) was utilized to expose the mechanisms individual cells employ to survive this stress. Cultured cells were exposed to either control isosmotic media (300mOsm/kg), 450mOsm/kg, which is physiologically relevant for fish plasma, or maximum cellular tolerance levels of 700mOsm/kg for 8 days. Hyperosmotic medium was prepared by increasing NaCl concentration. Cell proliferation and time to confluency, as well as passage numbers, were monitored in treatment and control cells. The morphology and general appearance of experimental and control cells were also periodically monitored via phase contrast microscopy. In addition, proteins that are altered in their abundance by hyperosmolality were identified by label‐free quantitative proteomics to explain cellular phenotypes associated with hyperosmotic stress acclimation. Based on our results we conclude that the tilapia proteome is extensively and specifically modulated during hyperosmotic stress to adjust cellular physiology, stress tolerance, and morphology.

Support or Funding Information

This work was supported by National Science Foundation, IOB 1355098.
Acute Hypoxia and Ryanodine Receptor Activity in Pulmonary Arterial Myocytes of High Altitude Acclimatized Fetal and Adult Sheep
Authors: Goyenaga Geraldine , Yoo Timothy , Romero Monica , Puglisi Jose , Longo Lawrence D , Wilson Sean M
Published: June 2021

Exposure to high altitude long term hypoxia (LTH) during the gestational period causes reprogramming of the fetal lung, increasing the likelihood of pulmonary hypertension. There is also regional acute hypoxia in the lung that causes hypoxia‐induced pulmonary vasoconstriction (HPV). This is an important process necessary to sustain arterial oxygenation. Further, ryanodine receptors (RYRs) play an important role in acute hypoxia through HPV and their function is reduced by LTH. In this study, we hypothesized that LTH would alter the activation of RyR by acute hypoxia. This was tested by measuring Ca2+ spark activity in pulmonary arterial (PA) myocytes of fetal and adult sheep, where PAs were isolated from sheep that resided at either low (335m, LA) or high (3801m, HA) altitude for <100 days. Ca2+ imaging was performed by loading cells with Fluo‐4 and imaging en‐face using line‐scan techniques on a Zeiss 710 confocal microscope. Images were analyzed using a customized software that allowed for detailed measurements of the frequency and spatial‐temporal aspects to the Ca2+ sparks. Results showed that acute hypoxia affected Ca2+ signaling in several ways. Specifically, the number of cells firing sparks increased in fetal myocytes at both elevations and in HA but not LA adults. In addition, Ca2+ spark frequency significantly decreased in LA and HA fetuses. Spark frequency decreased in LA adults; which contrasts a frequency increase in HA adults. Aside from these results, distinct differences in the spatial and temporal relationships of Ca2+ sparks were also observed. In conclusion, these findings support the proposition that high altitude fetal reprogramming affects RyR activity and acute hypoxia modifies their activation.

Support or Funding Information

Support from NIH, NSF, and the Walter E. Macpherson Society
Investigating the Half‐site Reactivity of a Member of the PNDOR Family of Proteins
Authors: Li Wuyi , To Daniel , Lee Jerry , Crane EJ , Sazinsky Matthew
Published: June 2021

Half‐site reactivity is an enzyme characteristic in which the active sites of a multimeric enzyme “fire” alternately, which means that one‐half of the identical subunits is active at any one time (1). Many metabolically important enzymes, such as alcohol dehydrogenase (ADH) and glyceraldehyde 3‐phosphate dehydrogenase (GAPDH), are known to exhibit half‐site reactivity (2). Two models, induced‐fit (or alternating sites cooperativity model) and preexistent asymmetry, were proposed in early literature to explain this half‐site reactivity (3). The pyridine nucleotide disulfide oxidoreductases (PNDORs) are members of a flavoprotein family that use NADH/NADPH as their reductive substrate, with most members of the PNDOR family being found in the form of homodimers. It has previously been observed that members of the PNDOR family show half‐site reactivity (via an alternating sites cooperativity model), as evidenced by subunit asymmetry (4,5): when their bound flavin coenzyme is reduced by NAD(P)H, only one subunit reduces, with the other subunit remaining oxidized. Recent data from our laboratory suggests that the CoADR from Pyrococcus horikoshii, a member of the PNDOR family, also shows half‐site reactivity.

Although many enzymes with half‐site reactivity have been characterized kinetically and structurally, the cause of half‐site reactivity and its effect on enzymatic catalytic efficiency and mechanism remains unclear. We have designed asymmetrical dimers of a member of the PNDOR family which disrupts the ability of the subunits to react in an alternating fashion. Preliminary kinetic data suggests that in an asymmetrical dimer, in which one subunit has been inactivated by mutation of its redox‐active cysteine (with the other subunit remaining active), the phCoADR actually exhibits a 3‐fold increase in catalytic efficiency when compared to the wild‐type enzyme for a range of different substrates. In addition, a higher background oxidase activity is observed in the asymmetrical dimer. Our results suggest that subunit communication, in the induced fit model of half‐site reactivity, appears to slow the overall rate of the enzyme, perhaps acting as a control on enzyme activity. The mechanisms of this control will be discussed.

Support or Funding Information

This project is supported by NSF award MCB 1518306.
Structure‐Function Studies of the Thermotoga maritima ADPGlucose Pyrophosphorylase: Probing the role of the C‐terminus of the GlgD subunit
Authors: Tiet Jan , Kuipers Andrea , Vu Crystal , Orry Andrew , Meyer Christopher R
Published: June 2021

ADP Glucose pyrophosphorylase (ADPG PPase) catalyzes the rate‐limiting step of glycogen and starch biosynthesis in plants and bacteria. Structure‐function studies of the enzyme from different sources will allow the engineering of a more active enzyme to produce more renewable and biodegradable carbon. In Thermotoga maritima (T.ma) there are two genes, glgC and glgD, which code for two subunits of this ADPG PPase. The recombinant His‐tagged subunits were successfully purified using a heat step and nickel chromatography. Previous studies performed at 37ºC (pH 7.5) had shown that the wild type (WT) glgD alone had no activity, while the glgC protein displayed a specific activity of ~ 3 Unit/mg with S0.5 values for ATP and magnesium (Mg2+) of 5.39 mM and 18.66 mM, respectively. However, the WT glgC/D complex at subsaturating substrate conditions was stimulated ~20‐fold compared to glgC alone and was also activated ~2‐fold by fructose‐1,6‐ bisphosphate (FBP). The apparent affinity for ATP and Mg increased ~2 and ~3 fold in the complex while the Vmax increased 4 fold. In the presence of FBP, the apparent affinity for ATP increased ~6‐fold for the complex with a modest ~30% increase in Vmax. Previous alignment studies and molecular modeling principally focused on the functional role of the N‐terminus. Therefore the truncation of 73 amino acids at the C‐terminal end of glgD was generated to test its potential functional role in the complex. In the absence of FBP, the truncated complex exhibited a modest decrease in ATP apparent affinity for ATP and Mg2+ with ~20‐fold and ~2 fold decreased in Vmax, respectively, compared to the WT complex. It was noted that in the presence of the truncated glgD, the Vmax was substantially lower than for glgC alone, indicating an inhibitory role. In the presence of FBP, the truncated complex displayed a ~6‐fold decrease in apparent affinity for ATP, ~3‐fold for Mg and a ~6‐fold lower Vmax compared to the WT complex. However, the Vmax of the truncated complex is close to the Vmax of the glgC alone indicating that FBP diminishes the inhibition by the truncated glgD. The primary effect of the truncation appears to be on the Vmax of the complex as well as a reduction in the K‐type activation by FBP observed with the WT. Complete kinetic and physical characterization of the altered enzyme complexes in the absence and presence of FBP, as well as crystallization of WT GlgC/WT GlgD are still underway.

Support or Funding Information

Supported in part by NSF and NSF BIO MCB grant #0448676.
Getting the Word Out on Effective Ways to Teach: the Promoting Active Learning & Mentoring (PALM) Research Coordination Network
Authors: Wolyniak Michael J , Engen Katie , Peifer Mark , Prunuske Amy J , Ruedi Elizabeth , Wick Susan M
Published: June 2021

The American Society for Cell Biology (ASCB), along with the American Society of Plant Biologists (ASPB) and the Genetics Society of America (GSA), has received NSF funding for an incubator year of a network to promote best practices in active learning. The goal is to achieve sustained biology education reform in diverse institutions through a program that goes beyond short‐term training or individual workshops to develop long‐term teaching mentorships. As we outlined in the proposal overview, “The Promoting Active Learning & Mentoring (PALM) Research Coordination Network program allows faculty and postdoctoral fellows to gain hands‐on experience and long‐term mentorship (at least one semester) in bringing evidence‐based, effective active learning strategies into their own classrooms. PALM Fellows pair with mentors who have already reformed their classrooms, visit their mentors to observe and participate in redesigned classes, and develop an active learning based model for one of their classes with guidance from their mentors.” This initiative builds and expands on the ASCB Education Committee's current MALT (Mentoring in Active Learning and Teaching) program, which likewise pairs mentees with mentors experienced in active learning. Our goal is to use the network to reach instructors in the biological sciences from all types of institutions, particularly instructors of under‐represented students. We encourage EB society members who are interested in being a mentee, as well as members who have experience in active learning and could serve as mentors, to contact us.

Support or Funding Information

National Science Foundation #1539870
Examination of Sociodemographic Correlates of Plate Waste in Early Adolescent Girls
Authors: Panizza Chloe , Boushey Carol , Delp Edward , Lim Eunjung , Banna Jinan C
Published: June 2021

Background

Studies have reported that the proportion of food that is plated, uneaten and thrown away is high among youth in the school environment. To our knowledge, no studies have collected data on food waste over a 24‐hr period in early adolescents in the U.S. Further, few studies have investigated differences in food waste according to demographic characteristics. The mobile food record (mFR) has been found to be a feasible and acceptable method for measuring dietary intake in youth and may be used to collect data on food waste over the course of a day in real time.

Purpose

To examine differences in food waste according to age, race/ethnicity, total household income and mother's education level in early adolescent girls 9 to 13 years in Oahu, Hawaii using the mFR.

Methods

Participants (n=72) used the mFR for 3 days to capture food eaten and any leftovers. A trained analyst met with each girl to review images from the mFR to classify the outcome of food remaining after each eating occasion. Participants’ mothers provided demographic information. The amount of food wasted was estimated. Data were entered into the RapidCalc nutrition database to quantify amount of food waste by food groups. Differences in the proportion of each food group wasted according to demographics were examined using Wilcoxon or Kruskal‐Wallis nonparametric tests.

Results

All participants’ mothers completed at least 4 years of college. Participants whose mothers only completed college had at least twice the amount of waste with regards to total energy (p=0.008), dairy (p=0.033) and grains (p=0.029) than those whose mothers had a higher level of education. Compared to girls who identified as white or Asian, girls in the other race category (Native Hawaiian or Other Pacific Islander, American Indian or Alaska Native, Black or African American, Some Other Race) had at least twice the amount of waste with regards to total energy (p=0.018), grains (p=0.012) and dairy (p=0.006). No differences in waste according to age and total household income were found.

Conclusion

Differences were observed in food waste over 3 days among the girls according to mother's education level and race/ethnicity. No differences were found with regards to age and total household income. Findings suggest that educational campaigns to reduce food waste may benefit from tailoring to specific groups.

Support or Funding Information

Supported by the USDA National Institute of Food and Agriculture, Hatch project, HAW02026‐H, Accession no. 1000090. Also supported by NIH grant U54MD007584.
Participants in the Shaping Healthy Choices Program Increased the Intensity of Physical Activity
Authors: Fetter Deborah S , Scherr Rachel E , Linnell Jessica D , Dharmar Madan , Schaefer Sara E , Brian Kelley M , Zidenberg‐Cherr Sheri
Published: June 2021

Regular physical activity (PA) has been shown to aid with weight control and improve physiologic, metabolic, and immunologic processes, along with quality of life. The objective of this study was to determine whether intensity of PA improved in 4th‐graders participating in the multi component intervention Shaping Healthy Choices Program (SHCP). At pre‐ and post‐intervention assessments, students at the control and intervention schools were asked to wear a PolarActive monitor on their non‐dominant wrist 24 h/d for 5 consecutive days. The PolarActive used accelerometer technology to measure 3 dimensions of motion capturing time (minutes) spent in different activity intensity levels (very vigorous, vigorous, moderate, easy, and very easy). A total of 54 students at the intervention school and 38 students at the control school used the accelerometers for at least 2 days during both the pre‐ and post‐assessments. Analyses were conducted using STATA 14.0. Tests performed were Student's t‐test and paired t‐test, as appropriate. Mean moderate to vigorous physical activity (MVPA) significantly increased at the intervention school from 81.43 ± 29.36 min/d at pre to 103.70 ± 40.76 min/d at post (P=0.01) and at the control school from 70.10 ± 29.42 min/d at pre to 100.03 ± 63.15 min/d at post (P=0.01). There were no significant differences for the change in MVPA between the intervention and control schools. Students at the intervention school significantly decreased mean time performing very easy activity from pre to post by 78.53 ± 119.12 minutes compared to the controls (16.55 ± 138.42 minutes; P=0.02). While there was no significant difference in the change of very vigorous PA between the control and intervention schools, mean very vigorous PA significantly increased in students at the intervention school from 5.57 ± 4.37 min/d at pre to 7.80 ± 9.23 min/d at post (P=0.04). This significant increase in very vigorous PA was not observed at the control school (P=0.44). Taken together, these results suggest students at the intervention school decreased very easy PA and increased very vigorous PA from pre to post; while these changes were not observed at the control school. These improvements in PA, concomitant with other positive behavioral outcomes, may have contributed to the significant decrease in BMI percentile observed in students participating in the SHCP. Due to the small sample size, significant changes in intensity of PA for those students that improved their BMI percentile were not evaluated. These data support that PA is an important area to target within a multi‐component nutrition intervention aimed at preventing childhood obesity.

Support or Funding Information

Funding was provided by UCANR competitive grant #11‐1018, USDA 2011‐38420‐20082, and USDA NIFA Hatch project 221082.
Sensation Seeking Behaviors and Dietary Supplement Usage in Warfighters
Authors: Hatch Adrienne M , DiChiara Adam J , Sepowitz John J , Cole Renee E , Merrill Ellen P , Lieberman Harris R , Bukhari Asma S
Published: June 2021

Military personnel use dietary supplements (DSs) for various reasons. Despite lack of evidence of effectiveness, safety concerns and increasing cost, Soldiers continue using DSs.

PURPOSE

To characterize how sensation seeking behaviors, which have been repeatedly associated with illicit drug abuse, smoking, and alcohol use, are related to DS use and other demographics factors in military populations.

METHODS

Background and behavioral questionnaires were administered to 260 Army Soldiers (mean ± SD, 28 ± 6 y, 26± 3 kg/m2 BMI, 16% female) at three installations. The Brief Sensation Seeking Scale (40 total points (pts) with four 10‐point subscales: experience seeking, boredom susceptibility, thrill seeking, and disinhibition) and the Arnett Inventory of Sensation Seeking (80 total points with two 40‐point subscales: novelty and intensity) were used to assess Soldiers’ sensation seeking behaviors. One‐way ANOVA's were conducted to compare sensation seeking subscale scores of DS user and non‐users as well as tobacco products users, Army Physical Fitness Test score, and assignment type.

RESULTS

Overall, 79% of soldiers reported the use of DSs ≥1 time/week; 34% used sports beverages, 8% used sports bars or gels, and 6% used meal‐replacement beverages. The most commonly used DSs were protein powders (55%), multivitamin/mineral supplements (44%), amino acid mixtures (22%), and herbal supplements (10%). Vitamin/mineral users had higher experience seeking (8.1 ± 1.6 vs. 7.6 ± 2.0, P < 0.05) and novelty (13.8 ± 3.5 vs. 12.4 ± 4.3, P< 0.001) scores compared to non‐users. Protein/sport users had higher boredom susceptibility (7.2 ± 1.6 vs. 6.7± 1.7 P< 0.05), thrill seeking (7.4 ± 2.2 vs. 6.4 ± 2.1, P < 0.001), novelty (13.6 ± 3.8 vs. 12.3 ± 4.1, P< 0.05), and intensity (14.2 ± 4.3 vs. 12.6 ± 3.8, P < 0.05) scores than non‐users. Tobacco users reported greater susceptibility to boredom (7.5 ± 1.4 vs. 6.7 ± 1.7, P < 0.001), disinhibition (5.9 ± 2.3 vs. 5.1 ± 2.1, P < 0.001), novelty (14.0 ± 3.5 vs. 12.7 ±4.1, P < 0.05), and intensity (15.0 ± 3.7 vs. 13.0 ± 4.5, P < 0.001) scores compared to non‐users. Soldiers who scored higher on their (APFT) had greater thrill seeking scores. Soldiers assigned to combat arms units had greater disinhibition and intensity scores.

CONCLUSION

These data support previous studies, showing high supplement use among soldiers that may reflect the unique demands and stressors of military service. Soldiers with higher sensation seeking behavior scores are more likely to be users of DSs. Unit leaders and healthcare professionals can assess soldiers’ lifestyle factors to identify potential use of DSs and safety education.

Support or Funding Information

Supported by USAMRMC.

The opinions or assertions contained herein are the private views of the author(s)and are not to be construed as official or reflecting the views of the Army or the Department of Defense.
Promotora‐academic partnership to reduce nutritional health disparities among Mexican‐heritage families in Texas border colonias
Authors: Sharkey Joseph , Meyer Renee Umstattd , St. John Julie
Published: June 2021

The burden of obesity disproportionately affects marginalized populations, such as Mexican‐heritage (MH) children who reside in impoverished communities along the U.S.‐Mexico border. These areas are occupied by a growing population of people who share a similar Mexican heritage, language, and socioeconomic standing; have unacceptably high rates of poverty and financial stress, adult and childhood obesity, food insecurity, and physical inactivity; and limited access to affordable, healthy foods and physical activity opportunities. There has been success with traditional approaches: 1) top‐down, researcher‐driven approaches, 2) disregard for contextual dimensions (culture, sociocultural, network, and personal), 3) not reaching most in need, 4) language and literacy, and 5) lack of trust by the priority population. Promotoras de Salud (community health workers) are trusted members of underserved, at‐risk Hispanic communities experiencing social and health inequities. Promotoras have the unique ability and opportunity not only to provide outreach and education but also to be actively engaged in conducting research in their communities and serve as a cultural bridge between the community and researchers. The purpose of this study was develop an understanding of needs and assets of priority communities regarding opportunities for affordable healthy foods and physical activity. Data collected included neighborhood/community assessment (ground‐truthed enumeration of all food venues and physical activity opportunities, environmental infrastructure, neighborhood characteristics, assessment of physical activity places, and informal and formal policies). This was followed by separate panel series of focus groups with MH children (age 7–11 y) and mothers. Data documented colonia and community needs and assets and the voices of children and mothers. The constellation of health behaviors and settings that place children at increased risk for poor nutrition and obesity must be simultaneously addressed.

Support or Funding Information

USDA‐NIFA (#2015‐68001‐23234)
Nutrition Education for Low‐Income Population through Social Marketing: Insight from SNAP‐Ed Eligible Participants
Authors: Hagues Rachel , Childers Austin , Lee Jung Sun , Phua Joe
Published: June 2021

Social Marketing (SM) has been used to deliver public health interventions, and has the potential to deliver nutrition education interventions targeted to large, low‐income population quickly at low‐costs. The purpose of this study was to conduct focus group interviews with those that qualify for Supplemental Nutrition Assistance Program Education (SNAP‐Ed) (185% of poverty) to learn their perspectives on benefits, barriers to accessing messages and food, suggested content, and media channels most useable for them to be able to best segment nutrition social marketing messages. Two focus groups in each quadrant of Georgia were conducted for a total of 8 focus group interviews. Groups were held in both rural and urban areas; settings ranged from community centers, to housing authorities, to schools. Sixty‐nine individuals participated, representing a range of rural – urban counties. The research team developed a semi‐structured interview guide. Focus groups were tape‐recorded and transcribed; a thematic analysis was conducted using Atlas.ti. To ensure findings were accurate and credible, a summary was distributed to each group to be given to all participants for member checking. Data from each group were compared across urban/rural and north/south/east/west. Participants understood some of the benefits of eating healthy foods and wanted to know more, such as how to cook for someone with chronic conditions (e.g., diabetes). Participants identified many barriers to healthy eating including culture, high costs and lack of availability of healthy food. Participants in the Atlanta area talked about having a lack of time due to long commutes. Many participants wanted to learn ways to reduce sodium in food, as well as ideas for cooking southern traditional foods they have typically fried through healthier means. Several African American participants emphasized the need to make messages culturally relevant. Participants expressed their interest and need for more education on serving sizes, measurement conversions, and healthy substitutions The majority of participants reported they can get online and access nutrition information via mobile devices, especially smart phones, unless they are out of data. While participants reported having Internet access, it is not always as accessible from home in rural areas as urban. Sometimes rural participants reported needing to go to public places to access it. Participants reported they search and obtain nutrition and health information often through Google and Facebook. In conclusion, a segmented marketing approach may help reach Georgians with low‐income, particularly if segmented by urban/rural and across the various cultures that exist within the State. Because participants have more access to the Internet than anticipated, social media marketing could be an effective intervention.

Support or Funding Information

This project was funded by USDA SNAP‐Ed
CpG Methylation Differences Between Neurons and Glia are Highly Conserved from Mouse to Human
Authors: Kessler Noah J. , Van Baak Timothy E. , Baker Maria S. , Laritsky Eleonora , Coarfa Cristian , Waterland Robert A.
Published: June 2021

Understanding epigenetic differences that distinguish neurons and glia is of fundamental importance to the nascent field of neuroepigenetics. A recent study (Lister & Mukamel, et al., Science 2013) used genome‐wide bisulfite sequencing to survey differences in DNA methylation between these two cell types, in both humans and mice. That study minimized the importance of cell type‐specific differences in CpG methylation, claiming these are restricted to localized genomic regions, and instead emphasized that widespread and highly conserved differences in non‐CpG methylation distinguish neurons and glia. We reanalyzed the data from that study and came to markedly different conclusions. In particular, we found widespread cell type‐specific differences in CpG methylation, with a genome‐wide tendency for neuronal CpG‐hypermethylation punctuated by regions of glia‐specific hypermethylation. Alarmingly, our analysis indicated that the majority of genes identified by the primary study as exhibiting cell type‐specific CpG methylation differences were misclassified. To verify the accuracy of our analysis, we isolated neuronal and glial DNA from mouse cortex and performed quantitative bisulfite pyrosequencing at nine loci. The pyrosequencing results corroborated our analysis, without exception. Most interestingly, we found that gene‐associated neuron vs. glia CpG methylation differences are highly conserved across human and mouse, and are very likely to be functional. In addition to underscoring the importance of independent verification to confirm the conclusions of genome‐wide epigenetic analyses, our data indicate that CpG methylation plays a major role in neuroepigenetics, and that the mouse is likely an excellent model in which to study the role of DNA methylation in human neurodevelopment and disease, and effects of nutrition on these processes.

Support or Funding Information

This work was supported by grants from the National Institutes of Health Roadmap Epigenomics Program [grant number U01DA025956] and the U.S. Department of Agriculture (USDA) [grant numbers CRIS 6250‐51000‐055 and CRIS 3092‐5‐001‐059].
Aquaporin 9 in the Freeze Tolerance Mechanism of Cope's Gray Treefrogs
Authors: Stogsdill Brian , Frisbie James , Goldstein David
Published: June 2021

Cope's Gray Treefrogs Hyla chrysoscelis accumulate glycerol in response to cold as part of their freeze tolerance mechanism. This glycerol may derive from glucose stores in the liver that are broken down and released through glycerol transporters. We therefore hypothesized that these treefrogs would express aquaporin 9 (AQP9), a transmembrane protein from the aquaglyceroporin family that facilitates rapid glycerol transport, predominately in the liver. We also hypothesized that this protein would be up‐regulated during cold acclimation to promote glycerol permeability. To test this hypothesis, we sequenced the AQP9 homologue HC‐9, analyzed RNA and protein expression, and characterized water and glycerol permeability through a Xenopus oocyte expression system.

HC‐9 has 315 amino acids which include 2 conserved NPA motifs unique to aquaporins, 5 conserved residues typical of glyceroporins, a high homology (69%) to human AQP9, and a nearly identical homology (97%) to the hyla japonica. The mRNA strand also possesses a long untranslated tail unique to amphibians that appears to be necessary for translation. Furthermore, characterization using the Xenopus expression system has shown that HC‐9 is permeable to glycerol but has a poor water permeability compared to human AQP1.

In warm‐acclimated animals, HC‐9 mRNA (RT‐PCR) and protein (western blotting) were expressed in many tissues, with some of the highest levels in liver, stomach, ventral skin, and muscle. Across most tissues, realtime mRNA expression (qRT‐PCR) decreased in cold‐acclimated (5ºC) and frozen (−2.5 ºC) treefrogs, with recovery in treefrogs returned to 5ºC for 24 hours. Meanwhile, protein expression increased in the liver of cold and frozen treefrogs, but remained unchanged or decreased in all other tissues.

HC‐9 expression is clearly regulated as a part of the Cope's Gray Treefrogs freeze tolerance mechanism. Contrary to our hypothesis, most tissues have decreased expression, which may reduce glycerol permeability to protect cells during the freeze process. Liver is a notable exception by increasing protein expression, which may facilitate the re‐uptake of glycerol during ice formation. Liver appears to play a critical role in the distribution of the cryoprotectant glycerol, and this study suggests HC‐9 facilitates in this role.

Support or Funding Information

Supported by NSF IOS‐1121457.
Effects of High‐Fat Diets on Mortality and Feeding in the Tobacco Hornworm Caterpillar, Manduca sexta
Authors: Greenlee Kendra J , Cambron Lizzette D , Thapa Gita , Baldwin Maxwell
Published: June 2021

Nutritionally‐balanced diets are important for fitness, but for insects the high energy value of fat storage is crucial for reproduction and development. Although studies have shown that insects fed high carbohydrate diets have increased fat storage, and excess fat storage in insects may have a fitness cost, little is known about how insects regulate fat consumption when presented with diets containing varying lipid content. To determine how insects respond to high fat diets, we fed caterpillars of different ages high (5.6%), medium (3.4%), or low (0.4%) fat diets. Young Manduca sexta larvae reared on high fat diet had 80% mortality and 43% lower body mass compared to those reared on medium or low fat diets. Older larvae showed no difference in mortality, but those fed a high fat diet had a lower average body mass by the third day of the fifth instar. Growth rates and development time also differed. High fat‐fed caterpillars wandered later than those fed low and medium fat diets. To test the hypothesis that the increased survival of older larvae was due to increased lipid transport, we measured mRNA expression of Apolipoprotein I and II (APO1 and 2), proteins responsible for transporting lipids to the fat body and to various tissues. APO1 and 2 expression did not differ with dietary fat content. In addition, we tested the hypothesis that caterpillars preferentially metabolize fat during the fifth instar. We measured oxygen consumption and carbon dioxide emission of caterpillars fed a high or low fat diet and calculated the respiratory quotient. To determine the mechanism underlying the decrease in body size, we tested the hypothesis that the high fat diet altered feeding. Caterpillars fed a high fat diet indeed ate less, as indicated by a decrease in food consumption and the number and mass of fecal pellets produced. These results suggest that increased dietary fat results in cessation of feeding in these insects. Due to conserved fat metabolism pathways in vertebrates and invertebrates, better understanding of the role of lipids in insects could contribute to knowledge of human metabolic diseases and provide support for using insect models for these studies.

Support or Funding Information

This research was funded in part by NSF EPS‐0447679 and IOS‐0953297 to K.J.G. and NIH National Center for Research Resources 2P20RR0l5566. The contents of this study are solely the responsibility of the authors and do not necessarily reflect the views of the NIH.
Endothelial Cell DNA Integrity following Anoxia/Reoxygenation Injury
Authors: Barr Johnny L , Gibson Ruby S , Dubick Michael A , Bowman Phillip D
Published: June 2021

Ischemia/reperfusion injury (I/RI) is associated with trauma and various diseases including stroke, heart attack and hemorrhagic shock. This study investigated the extent of DNA damage in an in vitro system of hypoxia/reoxygenation that may be useful for screening for drugs to reduce this form of injury. The usual method of subjecting cells to reduced oxygen (O2) by purging with nitrogen is a slow process and does not reproduce the rapid local reduction in O2 that occurs in vivo during ischemia. Glucose oxidase (GOX), however, rapidly oxidizes extracellular glucose producing a very low O2 level (< 0.4 μM) within 5 min with only minor reductions in glucose levels. We previously showed 10 min of anoxia followed by reoxygenation of human umbilical vein endothelial cells (HUVEC) damaged various biomolecules, such as lipids, proteins, and DNA. In the present study, human umbilical vein endothelial cells (HUVEC) were subjected to GOX‐induced hypoxia for 10 min and returned to medium and normoxia for up to 6 hrs. The most commonly used biomarker of DNA oxidation, in epidemiological as well as experimental studies, is 8‐oxo‐7,8‐dihydro‐2′‐deoxyguanosine (8‐oxodGuo), however this methods requires large amounts of DNA. Here we used a high‐throughput assessment of DNA damage utilizing a high‐sensitivity long‐run real‐time PCR technique for DNA‐damage quantification in both the mitochondrial and nuclear genome. Following hypoxia cells were provided normoxia (3% oxygen) for up to 6 hours, fixed in 50% isopropanol and DNA extracted with DNAzol (MRC, Cincinnati, OH). The isolated DNA was subjected to real‐time polymerase chain reaction (RT‐PCR) with primers designed to amplify large and small regions of mitochondrial and nuclear DNA where the extent of template amplification is inversely proportional to the lesion frequency within a given DNA sequence. Detected lesions per 10kb DNA were determined. Compared to hydrogen peroxide induced DNA damage, the test conditions produced extensive damage to both mitochondrial and nuclear DNA that could readily be detected by 4 and 6 hours. These results were also correlated with HPLC analysis of 8‐oxoguanine that indicated that this biomarker to oxidative stress was enhanced about 50%. This in vitro system of hypoxia/reoxygenation can be used to further understand responses of cells to I/RI and identify drugs and potential therapeutic windows in vitro that may be translated to ameliorate ischemia/reperfusion injury in vivo.

Support or Funding Information

Funded by US Army Medical Research Materiel Command
Influence of social isolation on serotonergic fiber density in the auditory midbrain
Authors: Sandlain Rebecca L , Keesom Sarah M , Wise Bradley M , Hurley Laura M
Published: June 2021

Animal communication involves both a “sender”, which emits a signal, and a “receiver”, which processes the signal. Signal processing can be shaped by past social experience. In the auditory midbrain, serotonin is a potential mechanism for linking social experience with auditory processing for three reasons: 1) the serotonergic system is sensitive to social experience in other brain regions; 2) changes in serotonin concentration reflect contextual features of social encounters; and 3) serotonin influences single auditory neurons’ response to sounds, such as conspecific vocalizations. Laboratory mice (Mus musculus) provide an excellent model for addressing this potential mechanism, because mice demonstrate sensitivity to social experience via a simple manipulation of social housing conditions, and because laboratory mice are being increasingly studied as a model of auditory communication. In this study, we investigated how social isolation affects serotonergic fiber density in the inferior colliculus (IC), an auditory midbrain region that demonstrates selectivity for species‐specific vocalizations. Male CBA/J laboratory mice were given different social experiences by housing mice for four weeks either individually or in a social group of three mice. Subsequently, immunohistochemistry with florescence was used to label the serotonin transporter (SERT) as a marker for serotonergic fibers. Coronal sections of brain tissue were imaged, and ImageJ was used to quantify fiber density as the percent area of the IC covered by fibers. Mice from both social treatments demonstrated dense coverage (around 30%) of the IC by serotonergic fibers, corroborating with previous studies that demonstrate innervation of the IC by the serotonergic system. Individually housed mice had qualitatively lower percent coverage of the IC by serotonergic fibers than socially housed mice, particularly in sections of the IC that were more caudal; however, there were no statistically significant differences between social versus isolated treatment groups. Whether the smaller qualitative differences over the entire IC could be driven by disparities in the effects of social isolation among subregions of the IC has not yet been explored. These preliminary results suggest that social experience does not affect serotonergic fiber density in the IC. However, this does not rule out influences of social experience on other aspects of serotonergic signaling in the auditory midbrain, including socially induced serotonin release and profiles of serotonin receptors.

Support or Funding Information

NSF grants: IOS 1456298 and DBI 1460949
Inhibition of Endocannabinoid Degradation Improves Recovery of Neurobehavioral Function and Resolution of Neuroinflammation and Synaptic Hyperexcitability in a Rodent Model of Traumatic Brain Injury
Authors: Mayeux Jacques P , Katz Paige , Edwards Scott , Middleton Jason , Molina Patricia
Published: June 2021

Traumatic brain injury (TBI) is an increasingly prevalent condition affecting soldiers, athletes, and motor vehicle accident victims. It is the most common cause of death for people under 30 and currently lacks effective therapeutic interventions. TBI is defined as a primary mechanical insult followed by a secondary cascade involving inflammation, apoptosis, release of reactive oxygen species, and excitotoxicity, all of which can cause neuronal death and poor recovery. Previously we showed that inhibition of monoacylglycerol lipase (MAGL), the enzyme involved in 2‐acylglycerol (2‐AG) degradation, with JZL184 following mild TBI attenuated neuroinflammation and improved recovery of neurobehavioral function during the early 72 h post‐TBI period. The aim of this study was to examine if common pathologies resulting from the injury, including inflammation, neurobehavioral disruption, and synaptic excitability, could be reversed during the acute recovery period up to 10 days post‐injury. Adult male Wistar rats underwent a 5‐mm left lateral craniotomy, and TBI was induced three days later by lateral fluid percussion. Thirty minutes post‐TBI, rats received intraperitoneal injections of vehicle (alcohol, emulphor, and saline; 1:1:18) or JZL184 (16 mg/kg). Neurobehavioral function, neuroinflammation (immunohistochemistry) and synaptic excitability (brain slice electrophysiological recordings) were assessed up to 10 days post‐TBI. JZL184 administered‐TBI animals had improved neurobehavioral recovery compared to vehicle‐injected TBI animals beginning 24 hours post‐injury and persisting for two weeks. JZL184 administration significantly improved both ipsilateral cortex (gray matter) and corpus callosum (white matter) neuroinflammation seven days post‐TBI. JZL184 administration significantly attenuated TBI‐induced increases in mEPSC frequency and amplitude at 10 days post‐TBI. Taken together these results show that EC degradation inhibition 30 minutes post‐TBI has potential therapeutic benefits that persist throughout the acute recovery period up to 10 days post‐injury. Current studies are investigating the mechanisms of EC degradation inhibition‐mediated improvements from TBI.

Support or Funding Information

Support T‐32 AA007577, DOD W81XWH‐11‐2‐0011
Identification of inhibitors of Cytochrome P450 (CYP) 1B1
Authors: Roach Tyler , Kennedy Larry
Published: June 2021

Cytochrome P450(CYP)1B1 is known to be overexpressed in several types of human malignancies. The role of CYP1B1 in estradiol metabolism to catalyze the hydroxylation at the C‐4 position is of specific importance in estrogen‐related tumorigenesis. Finding a safe, potent inhibitor to this critical step could lead to chemoprevention of several forms of human cancer. We initially used the Chembridge online database (www.hit2lead.com) to perform similarity searches, using 2,3′,4,5′‐tetramethoxystilbene (TMS), a compound known to inhibit CYP1B1 activity, as the search target. Using A 50% similarity threshold, 64 compounds were ultimately tested at a single concentration for inhibition of CYP1B1 catalyzed ethoxyresorufin‐o‐deethylase (EROD) activity. Five compounds inhibited EROD activity to an extent equal to or greater than TMS, and were considered positive hits. CYP1‐selectivity studies were performed on these five compounds, establishing selectivity of CYP1B1 over both CYP1A1 and CYP1A2. Kinetic studies were used to determine the Ki and the mechanism of enzyme inhibition. Cell viability studies were performed using the MTT assay on breast cancer cell lines. Additionally, follow on studies have been performed using analogs of the original five compounds to continue the search for safe, potent inhibitors of CYP1B1.

Support or Funding Information

ONR