bluuebunny/crossref_metadata_2025 huggingface dataset:
Does the Use of Virtual Microscopy Enhance Student Collaboration and Content Mastery in an Undergraduate Human Anatomy Course?
Authors: O'Loughlin Valerie Dean , Braun Mark W.
Published: June 2021

Virtual microscopy allows students to review microscopic slides via a computer interface that simulates a standard optical microscope. A number of studies have been published examining the use of virtual microscopy in medical and professional schools. However, little is known about the use of the virtual microscopy in undergraduate anatomy courses, an academic setting where learners may be less self‐sufficient than their medical school counterparts.

We implemented the use of virtual microscopy in Indiana University's Basic Human Anatomy (A215) course beginning the summer of 2008. This undergraduate course enrolls over 400 students per semester, has many graduate teaching assistants (TAs), and until the summer session 2008 had used standard optical microscopes for histology. This study reports academic success and student satisfaction following implementation of virtual microscopy. Subject mastery was assessed by comparing aggregate lab exam scores before and after introduction of this technology. Student satisfaction was assessed by means of an online survey. Additionally, TAs were interviewed regarding how their instruction and students' learning may have been affected by the use of virtual microscopy.

Students who utilized virtual microscopy performed much better on lab exams than students from previous semesters who used standard optical microscopes. There was increased student and TA satisfaction with virtual microscopy and more time was spent learning the material (versus learning how to use the microscope). However, student collaboration was not necessarily increased, unless the TAs were proactive and inspired collaboration among students. Our study indicates virtual microscopy is a valuable teaching tool in the undergraduate anatomy setting, but instructors may have to guide their students in utilizing this tool to its fullest potential.
Separating genetic and hemodynamic defects in neuropilin 1
Authors: Jones Elizabeth A.V. , Yuan Li , Breant Christiane , Watts Ryan , Eichmann Anne
Published: June 2021

Formation of the cardiovascular system is crucial for embryo growth and survival and the importance of proper blood flow even during these early stages is becoming increasingly clear. In recent years, there has been a debate regarding which aspects of early vascular formation are genetically predetermined, and which are governed by external stimuli, such as mechanical and biochemical signaling. Though the initial vascular network is patterned without knowledge of the functional requirements, it makes biological sense that a feedback mechanism with respect to physiological function would set in as soon as this information becomes available. We recently showed that physical forces created by blood flow are necessary for proper vascular development and remodeling. In this study, we show how to distinguish between genetic defects and biomechanical defects during vascular development. Using mouse embryo culture, we study hemodynamic defects in Nrp1−/− mice together with an analysis of the emerging vascular defects. The geometry of yolk sac blood vessels is altered and remodeling into yolk sac arteries and veins does not occur. To separate flow‐induced deficiencies from those caused by the nrp‐1 mutation, we arrested blood flow in cultured wild‐type and mutant embryos and followed their vascular development. This method allows genetic defects caused by loss‐of‐function of a gene important for cardiovascular development to be isolated even in the presence of hemodynamic defects.

Grant Funding Source

NSERC
NITRIC OXIDE SIGNALING IN BLOOD‐SUCKING INSECTS, HORNWORMS AND HUMANS
Authors: Montfort William R.
Published: June 2021

We are investigating the molecular mechanisms underlying the wonderful biology of nitric oxide signaling using ultra‐high resolution X‐ray crystallography, rapid transient kinetic measurements, spectroscopy and model cellular systems. Our studies include both heme‐based NO carriers and sensors, cytosolic proteins involved in S‐nitrosation, and transmembrane proteins of importance in transmitting regulatory signals from outside to inside the cell. We have been particularly successful with the nitrophorins, NO transport proteins from the kissing bug and the bed bug, soluble guanylyl cyclase (sGC) from the tobacco hornworm, human thioredoxin and transmembrane protein CD47, which may be a non‐canonical G‐protein coupled receptor. Here, I will describe our efforts to understand both NO‐dependent and independent regulation of sGC, the primary heme‐based NO receptor in animals, and how this relates to the conformational dynamics displayed by the nitrophorins. I will also describe how binding of benzylindazole‐related compounds such as YC‐1 to sGC alters the heme environment, serving to trap NO and CO gas molecules in the pocket through geminate recombination. YC‐1 and related compounds act both independently and synergistically with NO and CO binding and provide a new path for the discovery of drugs targeted to cardiovascular disease.
Growth mechanism of amyloid fibrils
Authors: Thirumalai Dave
Published: June 2021

The process by which a monomer adds onto a fluctuating oligomer or a growing fibril is largely unknown. Using molecular simulations and kinetic models we propose a multi‐stage process by which fibrils grow. The growth mechanism can be roughly described a dock‐lock mechanism. In the initial stage a monomer docks onto the underlying fibril, and much longer time scale structural changes in the monomer occur. After the lock stage the monomer adopts the β‐conformation that is commensurate with the underlying lattice. Surprisingly, growth of oligomers occurs by a similar mechanism except that the preformed oligomer fluctuates greatly to accommodate the growing monomer. The implications of these findings for experiments will be discussed.
Two Crystal Structures of the beta2‐Adrenergic Receptor
Authors: Weis William I.
Published: June 2021

G protein coupled receptors (GPCRs) constitute the largest family of membrane proteins in the human genome, and are responsible for the majority of signal transduction events involving hormones and neurotransmitters across the cell membrane. GPCRs that bind to diffusible ligands have low natural abundance, are relatively unstable in detergents, and display basal G protein activation even in the absence of ligands. To overcome these problems two approaches were taken to obtain crystal structures of the β2‐adrenergic receptor (β2AR), a well‐characterized GPCR that binds catecholamine hormones. The receptor was bound to the partial inverse agonist carazolol and co‐crystallized with a Fab made to a three‐dimensional epitope formed by the third intracellular loop (ICL3), or by replacement of ICL3 with T4 lysozyme. Small crystals were obtained in lipid bicelles (β2AR‐Fab) or lipidic cubic phase (β2AR‐T4 lysozyme), and diffraction data were obtained using microfocus technology. The structures provide insights into the basal activity of the receptor, the structural features that enable binding of diffusible ligands, and the coupling between ligand binding and G‐protein activation.
Effect on blood pressure of diets enriched with different sources of n‐3 fatty acids in healthy adults
Authors: Teo Choon Chew , Grakov Stoyan , Chiu Hsueh‐Ting , Oda Keiji , Sabaté Joan
Published: June 2021

Background

Consumption of n‐3 fatty acids from algal‐oil supplements and plant sources produce health benefits, which may be dependent on the reduction in blood pressure.

Aim and Methods

To determine if plant sources (flaxseed oil and walnuts) of á‐linolenic acid (ALA) produce similar change in blood pressure (BP) as algal‐oil (EPA/DHA) supplements, we performed a randomized 4x3 cross‐over study (4 diets and 3 8‐week study periods) in 24 healthy adults (15 women, 9 men; mean±SE age 42±2.7 years; BMI 25.4±0.9 kg/m2). Under controlled feeding conditions, subjects consumed eucaloric lacto‐ovo vegetarian diets: Control, ALA (6‐7g/2400 kcal/d), EPA/DHA (0.20/0.72g EPA/DHA per 2400 kcal/d), and, Combination (ALA + EPA/DHA). Under controlled conditions, 6 BP readings, (3/day on 2 days) were taken at the beginning and end of each diet period.

Results

Significant (p < 0.001) reduction in systolic BP were observed in all four diets: Control (‐2.64%), EPA/DHA (‐2.42%), ALA (‐1.64%) and Combination (‐1.85%) while non‐significant (p > 0.05) reduction were observed in the diastolic BP: Control (‐2.03%), EPA/DHA (‐2.51%), ALA (‐0.05%) and no change for the Combination diets (0.0%). No significant differences in BP between diets were observed.

Conclusion

Following diets enriched with ALA, EPA/DHA or a combination of these fatty acids for 8 weeks do not seem to affect BP when compared with the control diet.
Remodeling of data in the 2004 Canadian Community Health Survey 2.2 (CCHS) using actual rather than mandated levels of folic acid fortification suggests a lower prevalence of inadequacy of folate in women of reproductive age
Authors: Shakur Yaseer A , O'Connor Deborah L
Published: June 2021

Folic acid (FA) fortification of grain products has been mandatory in Canada for 10 years. Previously we reported that foods are over‐fortified by approximately 50% of the values in Canada's main food composition database, the Canadian Nutrient File. We used SIDE to calculate usual intakes of folate based on mandated and actual levels (based on 50% overage) of FA fortification from 24‐hour dietary recall data of approximately 35,000 respondents in the CCHS. In women of reproductive age, the prevalence of inadequacy (intakes below the Estimated Average Requirement) based on actual levels decreased by more than half when compared to that based on mandated levels (27.3% to 12.4% in 14‐18y; 24.5% to 10.2% in 19‐30y; 28.1% to 14.2% in 31‐50y). A similar decline was observed in lactating females (27.7% to 12%), while in pregnant females, the decline was more pronounced (50.2% to 18.4%). In sum, given its protective role in the prevention of neural tube defects, women capable of becoming pregnant should consume a FA supplement; however, at actual levels of FA fortification in the Canadian food supply, the prevalence of inadequacy in women of reproductive age is lower than estimated using mandated levels.

Grant Funding Source

Research Training Award
Upregulation of CMO1 as possible link between β‐carotene supplementation and increased risk of lung cancer
Authors: Gong Xiaoming , Marisiddaiah Raju , Afeld Jamiee L , Wiener Doris , Rubin Lewis P
Published: June 2021

In several large clinical trials, administration of high doses of β‐carotene has been associated with increased lung cancer risk in chronic smokers. The mechanism for this association remains unknown. We hypothesized that regulation of β‐carotene 15,15′‐monooxygenase (CMO1) expression may be a potential mechanism for supra‐physiologic β‐carotene dosing and increased lung cancer risk. We demonstrate that human alveolar epithelial (A549) cells robustly express CMO1 and metabolize β‐carotene to several biologically active retinoic acid isomers. Exposure of A549 cells to β‐carotene initially (<6 h) induces, and then suppresses cellular CMO1 mRNA and protein levels. β‐carotene suppresses CMO1 promoter activity in a dosage‐dependent manner. Treatment with supra‐physiologic levels of b‐carotene decreases cellular levels of PPARγ and reduces formation of functional PPARγ/RXRa heterodimers, resulting in inhibition of CMO1 promoter activity. We also show that β‐carotene treatment antagonizes PPARγ activity in HEK293 cells that stably express CMO1, but not in cells that express the mutant (non‐enzymatic) CMO1. Immunohistochemical analysis of non‐small cell lung carcinomas shows greater CMO1 staining than in adjacent normal lung, which is consistent with an increased expression of PPARγ in lung carcinogenesis. These findings suggest that β‐carotene suppresses CMO1 gene expression through downregulation of PPARγ in alveolar epithelial cells. Increased CMO1 expression in lung cancer may lead to accumulation of retinaldehyde from cleavage of β‐carotene and inhibition of PPARγ which, in turn, promotes carcinogenesis. This pathway may explain adverse effects of supra‐physiologic b‐carotene supplementation in lung cancer trials. (NIH HD42174, Muma Family Endowment)
Comparison of nutrient intakes from three self‐reported measures of dietary supplement use: results from the SURE Study
Authors: Murphy Suzanne , Wilkens Lynne , Monroe Kristine , Steffen Alana , Albright Cheryl , Yonemori Kim , Morimoto Yukiko
Published: June 2021

The Supplement Reporting (SURE) Study measured dietary supplement use using a unique inventory method: interviewers visited the participants¡‐ homes five times across one year to count the pills in all supplement containers. Participants also completed three self‐reported measures of dietary supplement use: a one‐month diary, a recall of the past month, and a short frequency questionnaire covering the past year (SFQ). For 402 participants who completed the study, we calculated nutrient intakes from all supplements and compared the diary and recall intakes to those from the corresponding inventory. We present here the results for five common components of supplements: vitamins A, C, E, folic acid, and calcium. Both the recall and the diary methods over‐estimated intakes of all five nutrients, with the diary estimates even higher than those for the recall (on average, intakes were over‐estimated by 40% for the diaries vs. 20% for the recalls). The SFQ under‐estimated intakes of vitamin C, folic acid, and calcium, and over‐estimated vitamins A and E, but the average absolute difference was similar to the recalls (19%). However, weighted kappa statistics were much higher for the recall and diary methods (0.69 to 0.83) than for the SFQ (0.44 to 0.59). We conclude that a four‐week recall of supplement use is the preferred method, both for its increased accuracy and decreased subject burden. Supported by NCI grant R01 CA106744.
Breast milk vitamin B12 concentrations in Guatemala: relationship to maternal and infant intake and status
Authors: Allen Lindsay Helen , Deegan Kathleen L , Jones Katherine M , Zea Manuel Ramirez , Zuleta Clara , Lildballe Dorte L , Nexo Ebba
Published: June 2021

There are no studies of breast milk vitamin B12 in groups with a range of B12 status, and previous methods are invalid due to failure to remove apohaptocorrin (apoHc) prior to analysis. B12 was measured in breast milk collected at 12 ± 0.5 mo postpartum from 129 women in a low income Guatemalan community. Complete data on maternal and infant plasma B12 and intakes were available for 83 mother‐infant pairs. Plasma B12 was measured (competitive protein‐binding radioassay) and cut‐offs designated Deficient (<148 pmol/L), Marginal (148‐220 pmol/L) and Adequate (>220 pmol/L) maternal and infant status. Milk B12 was analyzed by chemiluminescence after removing apoHc by cobinamide‐coated EAH sepharose gel. One third of women were B12 Deficient, one third had Marginal status, and 88% of infants were Deficient or Marginal. Both infant plasma and breast milk B12 were sig. higher in Adequate vs Deficient or Marginal status women. Breast milk B12 was below the detectable limit of 50 pmol/L in 47% of samples, none from Adequate mothers. In the Adequate group milk B12 averaged 128 pmol/L, vs. 870 pmol/L in 21 healthy Californian women, providing only ≈0.1 ug/d for exclusively breastfed infants. Maternal plasma B12 correlated with milk and infant plasma B12 (both r=0.36, P<0.001), and milk correlated with maternal intake. Since high B12 doses to mothers did not improve infant status, interventions are needed before/during pregnancy.
Compound A from Plantago asiatica essential oils downregulates the expression of SREBP‐2
Authors: Cho Sungyun , Hong Sung‐Jin , Kim Jin‐Young , Lee Sung‐Joon
Published: June 2021

We previously reported the hypocholesterolemic effects of Plantago asitica essntial oils in vivo by suppression of HMG‐CoA reductase. Based on the GC‐MS analysis and in vitro tests, we identified an active hypocholesterolemic compound (compound A) from the essential oils and investigated its cellular mechanism further. The HepG2 cells were treated with the compound A at various concentrations (0, 0.05, 0.1, 0.2, 0.5, 1mM) for 24 hours, then total RNA and proteins were assayed for RT‐PCR and immunoblotting. The expressions of HMG‐CoA reductase and SREBP‐2 were significantly reduced by the treatments, however, the expression of genes in the posttranslational activation of SREBP‐2, including Insig‐1 Insig‐2, SCAP, S1P, and S2P, were marginally altered. Both membrane‐bound and soluble forms of SREBP‐2 were reduced in immunohistochemistry and immunoblotting. In chromatin immunoprecipitation, the compound A decreased the binding of SREBP‐2 on the promoter of HMG‐CoA reductase. These suggest that the compound A suppressed the HMG‐CoA reductase expression by direct downregulation of SREBP‐2.
Lymphomonocyte subpopulations, activation and cytokine production in Alzheimer's Disease (AD)
Authors: Pellicanò Mariavaleria , Colonna‐Romano Giuseppina , Bulati Matteo , Barbagallo Mario , Di Prima Anna , Misiano Gabriella , Di Carlo Marta , Vasto Sonya , Candore Giuseppina , Caruso Calogero
Published: June 2021

AD is a neurodegenerative illness characterized by accumulation of β‐amyloid (A β‐42) and inflammation. To investigate the systemic signs of immune processes in AD we examined lymphomonocyte subpopulations (PBMCs) of 17 patients and 7 age‐matched healthy controls (HC). No differences were observed in T, B and NK subsets between the two groups, whereas an increase of both CD4+ and CD8+ naïve T cells was observed in AD patients. We analyzed AD and HC PBMCs after "in vitro" activation with Aβ‐42 and no differences were observed in the expression of early activation markers CD25 and CD69 on T and B cells, although AD cells show a basic activation. We also evaluated, on monocytes, the expression of the activation markers CD80 and HLA‐DR and of the receptor for fibrillar Aβ‐42, CD36. Here again no differences were observed between the two groups, even if there is a basic high expression of CD36 on AD cells. The evaluation of cytokines production after "in vitro" culture with Aβ‐42 shows an increased production of pro‐ and anti‐inflammatory cytokines and chemokines, in AD when compared to HC. These results, although puzzling, demonstrate an involvement of systemic immunity in AD patients.

This work was supported by grants from the Italian Ministry of Education, University and Research (MIUR, ex 60%)
Inducible nitric oxide synthase contributes to MAPK‐phosphatase induction and abundance of cyclooxygenase‐2 expression in an inflammatory setting
Authors: Lamon Brian David , Upmacis Rita K. , Deeb Ruba S. , Hajjar David P.
Published: June 2021

Cyclooxygenase (COX)‐2 and inducible nitric oxide synthase (iNOS) are responsive to inflammatory stimuli and have been localized to vascular smooth muscle cells (SMCs) in pathophysiological settings, e.g. atherosclerosis. Furthermore, the production of, and subsequent impact of COX products appear to be correlative with the status of NO synthesis and bioavailability. This study examined the role of inflammation‐driven NO synthesis on COX‐2 expression in rat aortic SMCs. Concurrent stimulation of quiescent SMCs with lipopolysaccharide (LPS) and interferon (IFN)‐γ induced iNOS and COX‐2 expression maximally at 2‐4hr. Inhibition of NO synthesis (LNMMA) significantly reduced nitrite (140±8 vs. 19±7 nmol/mg protein) and enhanced LPS+IFN‐γ‐mediated COX‐2 mRNA (156‐ vs. 43‐fold increase) and protein expression (5‐fold) at 30 and 60min. COX‐2 induction was associated with activation of the ERK, p38 and JNK pathways. Importantly, LNMMA enhanced ERK and p38 phosphorylation, and suppressed MAPK‐phosphatase (MKP)‐1 induction. Consistent with these data, exposure of SMCs from iNOS‐/‐ mice to LPS+IFN‐γ produced a more robust induction (p<0.05) of COX‐2 and a dramatically attenuated upregulation of MKP‐1 vs. WT. These findings provide pharmacological and genetic evidence implicating an inhibitory influence of iNOS‐derived species on phosphorylation cascades associated with COX‐2‐induction in SMCs.
IGF‐I and EGF promote synergistic increases in proliferation, enhances IGF‐IR activation and increases nuclear β‐catenin in intestinal epithelial cells
Authors: Newton Victoria A , Simmons James G. , Lund P. Kay
Published: June 2021

Insulin‐like growth factor I (IGF‐I) and epidermal growth factor (EGF) increase risk and promote pathogenesis of colon cancer in humans. Studies suggest that these pathways may interact to promote synergistic effects on colon cancer cells. We tested the hypothesis that combined IGF‐I and EGF treatment will additively or synergistically increase proliferation of intestinal epithelial cells and activate tumor‐promoting pathways, such as the β‐catenin pathway. IEC‐6 cells, a non‐transformed intestinal epithelial cell line, were treated with IGF‐I and EGF, alone or in combination. DNA synthesis was assayed using H3‐thymidine. EGF stimulated DNA synthesis more potently than IGF‐I, but combined IGF and EGF treatment synergistically increased DNA synthesis. EGF pretreatment, followed by IGF‐I treatment increased IGF‐IR tyrosine phosphorylation. EGF alone increased total IGF‐IR and this was enhanced by EGF pretreatment and subsequent IGF‐I treatment. EGF increased nuclear β‐catenin and synergistically enhanced IGF‐I‐induced nuclear β‐catenin. Ongoing studies will determine whether inhibition of both IGF‐IR and EGFR signaling decreases tumorigenesis, in vivo. These studies suggest that interactions between IGF‐I and EGF may reduce the effectiveness of anti‐cancer therapies that specifically target these pathways in colon cancer. [NIH‐DK40274‐16 RO1 and NIH‐DK44769‐10 CCFA].
Delta opioids attenuate pro‐inflammatory responses in human vascular cells
Authors: Stoll Lynn , Denning Gerene M , Romig‐Martin Sara A , Ackermann Laynez W , Weintraub Neal L , Dickson Eric W
Published: June 2021

Cells of the vascular wall, including perivascular adipocytes and smooth muscle cells (SMC), are poorly characterized sources of proinflammatory mediators that contribute to metabolic syndrome, diabetes, and atherosclerosis. Because of their unique properties and location, we hypothesize that perivascular adipocytes overlying the coronary arteries, along with coronary artery SMC, play an important role in transducing inflammatory responses in the coronary arteries. Enkephalins are endogenous delta opioids that improve myocardial ischemic tolerance. Both Met5‐enkephalin‐Arg6‐Phe7 (MEAP) and PCP‐enkephalin inhibited LPS‐ and TNF‐induced IL‐8 release by ¡Ý 50% in perivascular adipocytes. Real‐time PCR microarray analysis revealed that MEAP blocked LPS‐induced up‐regulation of a number of immunomodulatory adipocyte genes. Parallel studies with human coronary artery SMC showed that the delta opioid receptor agonist p‐chloro‐DPDPE caused attenuation of LPS‐induced up‐regulation of many of the same genes, including MCP‐1, RANTES, IL‐1alpha, CSF2, ICAM‐1, MMP‐1, MMP‐3, and CFLAR. Together, these data suggest that activation of the delta opioid receptor may blunt proinflammatory responses in cells of the human coronary artery wall, thus playing a protective role in coronary artery disease. Funding was provided by the Department of Emergency Medicine, The University of Iowa.
Stem Cell Transplantation for Cardiac Repair is Improved by Mechanical Preconditioning
Authors: Drowley Lauren , Cassino Theresa , Okada Masaho , Keller Bradley , Tobita Kimimasa , LeDuc Philip , Huard Johnny
Published: June 2021

We have previously shown that muscle‐derived stem cells (MDSCs) are significantly better than myoblasts at maintaining cardiac function after a myocardial infarction, which is likely due to higher survival and vascular endothelial growth factor secretion (VEGF) after transplantation. VEGF has been shown to be critical for cardiac repair, and mechanically stimulated MDSCs have increased VEGF secretion. We hypothesized that transplanting mechanically preconditioned MDSCs would result in increased repair of a myocardial infarction. After injection with mechanically stretched MDSCs, cardiac function at all time points is improved above unstimulated cells and saline. The improvement is likely due to an increase in angiogenesis and a small decrease in scar tissue formation in the mechanically preconditioned group. To clarify the mechanisms we explored cell behavior in vitro, where we saw increased VEGF secretion. Results from our VEGF secretion study also demonstrate that the effect of mechanical stimulation is time‐dependent, indicating that loading history control may be important to achieve ideal regeneration. The tight regulation and timing demonstrated by these results suggest that the mechanical loading history can predispose the MSDCs toward specific responses that are advantageous for cell transplantation.
Contribution of the N‐glucuronidation pathway to the overall in vitro metabolic clearance of midazolam in humans
Authors: Klieber Sylvie , Hugla Sebastien , Ngo Robert , Arabeyre‐Fabre Catherine , Meunier Viviane , Sadoun Freddy , Fedeli Olivier , Rival Martine , Bourrie Martine , Guillou Francois , Maurel Patrick , Fabre Gerard
Published: June 2021

Midazolam is one of the most commonly used in vivo and in vitro CYP3A4 probe substrate for drug‐drug interactions studies in humans based on the CYP3A4‐mediation of its 1′‐hydroxylation, a major metabolic pathway. In the present work, the in vitro metabolic pathways of midazolam in humans were studied in details in order to elucidate whether or not an alternative metabolic route such as direct N‐glucuronidation could be responsible for a metabolic shift between CYP3A4/5 and UGT. Indeed, following incubation of midazolam with human liver microsomes supplemented with UDPGA, two metabolites were identified as quaternary direct β‐N‐glucuronides of midazolam. Molecular modelling experiments showed that both glucuronide derivatives were in an atropoisomerism equilibrium. Only isoform UGT1A4 can catalyze this direct N‐glucuronidation which follows Michaelis‐Menten kinetics. Midazolam‐N‐glucuronide was formed in human hepatocytes incubated with and without ketoconazole. Direct midazolam‐N‐glucuronidation may partly compensate for the decrease in midazolam metabolic clearance caused by ketoconazole thus potentially leading to a certain extent of misinterpretation of CYP3A4‐mediated DDI study results both in vitro and in vivo.
The +ACA BRCA1 Promoter is Functionally Deficient and Subject to FAC1 Repression
Authors: White Kristen K. , Rivenbark Ashley G. , Bowser Robert , Coleman William B.
Published: June 2021

We previously identified a +ACA insertional polymorphism at ‐600 bp in the BRCA1 promoter that creates a FAC1 transcriptional repressor binding site (AACAACAC). FAC1‐negative NIH‐3T3 cells were transfected with luciferase vectors containing the 847 bp wild‐type (WT) or 850 bp +ACA minimal BRCA1 promoter sequence. The WT BRCA1 promoter‐driven luciferase construct produced 64,448 ± 9125 units of luciferase activity (relative light units) and the +ACA BRCA1 promoter‐driven luciferase construct produced 30,567 ± 2568 units. This result indicates that the +ACA BRCA1 promoter is functionally deficient compared to the WT BRCA1 promoter (53% reduction, p=0.004). Exogenous FAC1 protein did not significantly alter the luciferase activity from the WT BRCA1 promoter. In contrast, we observed a 79% reduction in luciferase activity from the +ACA BRCA1 promoter in the presence of FAC1 (p<0.0001). The FAC1‐mediated repression of the +ACA BRCA1 promoter reflects a 90% decrease in activity compared to the WT BRCA1 promoter (p<0.0001). Thus, the +ACA BRCA1 promoter is functionally deficient and subject to repression (silencing) by FAC1. Individuals that are homozygous for the +ACA polymorphism will exhibit compromised BRCA1 function and may become BRCA1‐null in response to FAC1 activation. These studies identify an alternative mechanism for loss of BRCA1 leading to breast cancer susceptibility. Support: NIH CA78343.
Energy metabolism during chronic REM sleep deprivation: the start of something big
Authors: Koban Michael
Published: June 2021

In rodent models of chronic and unremitting sleep deprivation, hyperphagia develops after 5‐6 days. If the regimen is taken to 10‐20 days, food intake increases substantially irrespective of whether rats are fed chow or a palatable calorie‐dense liquid diet (Ensure); body weight does not increase. Moreover, in a new model of chronic sleep restriction, sleep deprivation is enforced for 20 hours followed by 4 hours of recovery in home cages, and this also leads to hyperphagia. Of importance is that despite greatly increased food intake in all paradigms, rats consistently fail to gain body weight. This can only happen if there is an increase in energy metabolism. Indeed, the resting metabolic rate of sleep‐deprived rats becomes elevated, mediated by robust up‐regulation of uncoupling protein 1 in brown adipose tissue. Additionally, gene expression of uncoupling protein 3 in skeletal muscle is strongly stimulated, although the role of this protein in thermogenesis remains controversial. Chronically sleep‐deprived rats show rapid mobilization of hepatic and muscle glycogen, followed by reduction in abdominal white adipose tissue. Despite a lack of weight gain, there is evidence to suggest that sleep deprivation/restriction leads to glucose dysregulation. For example, sleep‐deprived rats have a higher glucose:insulin ratio, and when challenged with an intraperitoneal glucose tolerance test, blood glucose of sleep‐restricted rats is higher than in controls. In summary, rat models of sleep deprivation/restriction may be useful tools in understanding the etiology of hyperphagia and suggest a dissociation between weight gain and glucose dysregulation and insulin resistance.

Supported by NCRR RCMI and a Pilot and Feasibility grant from the Clinical Nutrition Research Unit of Maryland, P30DK072488.
Conserved Links Between Nutrient Signaling, Translation and Aging
Authors: Kennedy Brian , Steffen Kristan , Stanfel Monique , Burtner Chris , MacKay Vivian , Kaeberlein Matt
Published: June 2021

Invertebrate model organisms have proven invaluable for aging research, leading to the identification of hundreds of aging genes. But why should we expect orthologs of worm or yeast aging genes to have similar effects in mammals? We have addressed this question, taking a quantitative approach to identify the extent of conservation between C. elegans and S. cerevisiae, which diverged over a billion years ago. Yeast orthologs of reported worm aging genes were identified and tested for their effects on replicative aging. We find that these orthologs are statistically more likely to modulate yeast aging than randomly chosen yeast genes. Ortholog pairs that modulate aging map to known pathways involved in nutrient responses, and mammalian counterparts to these genes are prime candidates for mammalian aging genes. We propose that this conservation exists not because genes have evolved to regulate aging but rather the organismal response to nutrient restriction, and longevity is tightly linked to this response.

Dietary restriction extends lifespan in nearly all model organisms for aging research and delays the onset of a spectrum of age‐related diseases. We have attempted to define the pathways modulating the longevity‐specific effects of dietary restriction again using a multi‐organism approach. Studies in yeast and worms are consistent with reduced TOR signaling and altered translational control being a primary consequence of dietary restriction. Current studies are focused on achieving a mechanistic understanding of the links between altered translational control and aging, as well as testing these models in mice. Since these longevity pathways appear to be conserved, a better mechanistic understanding will likely define molecular targets in efforts increase human healthspan.
Autophagy and aging: connecting nutritional‐regulated catabolism and cellular quality control
Authors: Cuervo Ana Maria
Published: June 2021

Autophagy is a basic catabolic cellular process by which intracellular components are delivered to lysosomes for degradation. Activation of autophagy obeys to two different cellular needs, as an internal source of energy and as a mechanism for removal of damage or no longer functional structures. Malfunctioning of autophagy has been described with age. Our studies have focused primarily in one type of autophagy known as chaperone‐mediated autophagy (CMA), which mediates selective degradation of cytosolic proteins. Maximal activation of CMA is attained during prolonged nutritional stress, mild‐oxidative stress or toxin exposure. CMA activity declines with age due to a decrease in the levels of the lysosomal receptor that mediates substrate translocation. Interestingly, we have found that caloric restriction results in constitutive activation of this form of autophagy. As part of our ongoing studies, we have developed a transgenic mouse model in which we can preserve normal CMA activity until advanced ages in liver by correcting the age‐related defect in this pathway in this organ. Old mice with preserved CMA activity display improved cellular homeostasis, better resistance to stress and a general improvement in basic liver functions. We will use similar approaches to evaluate the importance of maintaining proper energetic balance and adequate protein removal until advanced ages in different organs.
Hypoxia and acidosis independently regulate cell viability and cell death of immortal neuroblastoma (Neuro 2‐A) cells
Authors: Mazzio Elizabeth A , Kolta Malak G , Soliman Karam F
Published: June 2021

Hypoxia and acidosis are hallmarks of tumors as well as critical determinants of response to treatments. In present study, we investigate the extent to which changes in O2/CO2 gas ratio may affect the pH and subsequently initiate cell death in malignant neuroblastoma. The data indicate that cell death occurred in either a closed chamber containing 100% oxygen or atmosphere (.05%CO2) after 24hrs of exposure at 37°C. These effects were also accompanied by a rise in pH within cell culture medium. Under both conditions, toxicity was potentiated by the addition of sodium bicarbonate but prevented by neutralization with acetic acid. In contrast, cells grown in a closed chamber containing 100% pure CO2 underwent cell death tantamount to a drop in pH resulted from the build up of carbonic acid. Under these conditions, toxicity was exacerbated by acetic acid and reversed by neutralization with sodium bicarbonate. These data clearly indicate that while O2 appears to be toxic to cancer cells, the rise in alkalinity is the determining factor in cell death. Moreover, the pH range to which cells thrived was 5.6 to 7.4, where a slight rise in basic pH from 7.4 to 8.0, corresponded to dose dependent cell death mediated by higher levels of O2. These findings suggest a paramount role for alkalinity rather than O2 itself in mediating adverse effects on tumor cells. (Supported by NIH grant NCRR: RR 03020)
NERO: A Nanoscience Research and Education Outreach Program
Authors: Mills David , Ramsey Linda , Decoster Mark , Mainardi Daniela
Published: June 2021

The NERO program builds on partnerships established though Louisiana Tech's CATALyST Center and a NSF GK‐12 Teaching Fellows program. Targeted schools display similar patterns: poor performance in STEM fields, students from low socioeconomic backgrounds, and high percentages of minority students. NERO program goals are to engage middle and high school teachers in conducting research through a summer research program and follow‐up academic year workshops, provide support for teachers as they translate their research experiences into classroom learning experiences, improve communication of science and the scientific research process to K12 schools and the community, and increase K‐12 students interest in pursuing STEM careers. Since 2007 science teachers, Teaching Fellows, and research faculty have participated in a collaborative learning environment. We have seen growth in teacher confidence and competence in content knowledge areas in molecular biology, material science and nanotechnology. Teaching Fellows have increased their confidence in their ability to work with teachers and students, developed a toolkit of strategies for use in working with K12 schools, and obtained insights into the K‐12 classrooms. Research faculty gained an understanding of appropriate and effective outreach methods and received training in communicating their research. The NERO program "broadens its impact" through linkages with its new NSF GK‐12 'Creating Connections' program designed to provide solid educational content, a strong technical foundation in the molecular sciences and bionanotechnology, and significantly enhance mathematics and science teaching in targeted 6‐12 classrooms through Teaching‐Learning labs, a University‐based K12 Teaching Lab, academic and summer science enrichment activities.
A 5‐year B.S./M.A. program in Pharmacology and Biotechnology at UC Santa Barbara
Authors: Stoyan Tanja , Thrower Doug , Clegg Dennis
Published: June 2021

We have developed a new B.S./M.A.[s1] program that will allow qualified students to complete a bachelor's and a master's degree in Pharmacology and Biotechnology within five years. This program is built on the solid foundation of our undergraduate Pharmacology Program which was established in 1974. Students who complete the pharmacology major receive instruction in general pharmacology that cover physiology, drug‐receptor theory, and a detailed examination of drug mechanisms, as well a laboratory training in tissue physiology, the role of animal model systems in pharmaceutical research, and a wide variety of pharmaceutical assays. The combined degree plan allows undergraduate students to begin taking graduate courses during their 4th year. According to their primary area of interest, students will choose one of three tracks: Molecular and Cellular Biology, Chemistry/Biochemistry, or Psychology. To gain business leadership skills, students can additionally take courses in Technology Management through the engineering department. Finally, the students complete an internship in a pharmaceutical or biotechnology company, thus gaining practical experience. We believe that the multidisciplinary nature of the five year program in pharmacology and biotechnology will enable our students to meet the challenges of drug discovery and development in the twenty first century.
Putting the fire in science education: The muse of fire project focuses student interest in science using region‐specific biology
Authors: Rockhold Robin William , Bordenstein Seth , McKeown James , McGuire Sarah Lea , Reznikoff William , Sullivan Donna , Yates Anne , Team The Muse of Fire
Published: June 2021

The Muse of Fire project hypothesizes student interest in science education will improve by highlighting topics with which students have personal interactions. The fiery sting of the red imported fire ant, Solenopsis invicta, is such a topic for many students in the United States and internationally. Five teams of faculty, high school teachers and undergraduate students developed high school lesson plans investigating aspects of S. invicta biology and venom alkaloids. Ecological relationships; symbiosis with the insect bacterium, Wolbachia; human health impact; toxicological; and microarray analysis of genetic responses to, S. invicta venom alkaloids were targeted. Field activities and advanced science and biomedical topics (eg., PCR, gel electrophoresis, microarray analysis, statistical treatment of data) are introduced in the high schools and supported by university faculty. Each lesson is specifically linked to published curriculum standards. Lessons are currently being piloted in five public school sites in both urban and rural school districts. Evaluation of student outcomes will continue through 2010. The Muse of Fire models inter‐institutional, multi‐disciplinary collaboration in which a topic of regional fascination is utilized to enhancement of inquiry‐based high school science education. (Supported by Howard Hughes Medical Institute)
Murine epidermal progenitor cells have a low redox state
Authors: Carr Wanakee , Zhang Yuping , Oberley Christopher , Oberley Larry W , Dunnwald Martine
Published: June 2021

The epidermis is a stratified cornified epithelium that protects the organism against physical and microbial insults. These environmental exposures can result in oxidative stress and affect cellular proliferation and differentiation. However, very few studies have evaluated the base line levels of reactive oxygen species (ROS) and antioxidant proteins in the epidermis or in the context of stem/progenitor cells, cellular types critical to the homeostasis of the epidermis. Here, we show that ROS are mainly produced by epidermal cells and that antioxidant proteins (MnSOD, GPx1, catalase) are differentially expressed in the epidermis. Using our previously sorted epidermal progenitor cells (side population, EpSPs) and more differentiated cells (non‐SPs), we show that catalase expression and activity are significantly decreased in EpSPs compared to non‐SPs. Furthermore, we found fewer peroxisomes per cell and decreased mitochondrial surface area in the EpSPs compared to non‐SPs. Using dihydroethidium (DHE), we show that EpSPs have less DHE signal compared to more differentiated cells. In summary, our data suggest that EpSPs have decreased levels of antioxidant proteins and ROS. Together, these studies indicate that the intracellular redox environment of EpSPs is reduced relative to their more differentiated progeny.

Grant Funding Source

National Institute of Health
Decellularized native matrices + primitive adult stem cells + donor islets form islet organoids for the treatment of type‐I diabetes
Authors: Fenley Caroline L. , Jones M , Long G F , Fenley J L , Collins J A , Black A C , Young H E
Published: June 2021

This study was designed to test the ability of decellularized native matrices and primitive pluripotential stem cells to provide optimal conditions for native islets to secrete insulin in response to a glucose challenge. Adult rats were euthanized following the guidelines of MU‐IACUC. Adult rat pancreases were decellularized using detergents. Control cultures consisted of native matrices, naïve stem cells, and native islets, all cultured individually. Experimental groups consisted of native islets co‐cultured with naïve stem cells seeded on top of decellularized native matrices. Control cultures and experimental cultures were challenged with glucose. The culture media were assayed using a RIA specific for rat insulin. No detectable insulin was secreted by naïve stem cells or decellularized pancreatic matrices. The native pancreatic islets secreted ng insulin per ng DNA. Pancreatic islets co‐cultured with naïve stem cells and native matrices demonstrated increased insulin secretion in the range of mg insulin per ng DNA. The current study shows that co‐cultures of decellularized native matrices, naïve adult stem cells, and native islets exhibited greater than a 250‐fold increase in insulin secretion in response to a glucose challenge compared to native pancreatic islets alone. Funding was provided by LM & HO Young Estate Trust and Dragonfly Foundation.
Pax3 expression by satellite cells of avian skeletal muscle spindles
Authors: Kirkpatrick Lisa J , Rosser Benjamin WC
Published: June 2021

Pax3 protein is usually co‐expressed with Pax7 by embryonic dermomyotome cells which give rise to skeletal muscle fibers. Following myogenesis, it is held that Pax3 expression becomes restricted to a few satellite cells (SCs) of select mature muscles. Intrafusal fibers of muscle spindles are thought to persist in an immature state as, unlike extrafusal fibers, they maintain small diameters, developmental myosins, Myf5 expression and high concentrations of SCs. Thus, we hypothesize that Pax3 expression is maintained in intrafusal fibers. Immunohistochemical methods were used to quantify nuclear numbers in chicken anterior latissimus dorsi muscles excised at 9, 30, 62, and 138 days post‐hatch. SCs were identified by their Pax7 expression. In intrafusal fibers, the percentage of SCs expressing Pax3 decreased from 76% at 9 days to 16% at 138 days post‐hatch and, at each age studied, was higher in intrafusal than extrafusal fibers. In extrafusal fibers, after 14% at 9 days, only 0‐2% of SCs expressed Pax3. Muscle spindles offer a novel model for the study of Pax3 expression in SCs. Funded by the Natural Sciences and Engineering Research Council of Canada.

Grant Funding Source

NSERC
MMP‐1 Reduced in Organ Cultured Human Skin and Dermal Fibroblasts by Ginger and Curcumin
Authors: DASILVA MARISSA , WARNER ROSCOE L , BHAGAVATHULA NARASIMHARAO , DEMB JOSHUA B , WEBER STEPHANIE L , JOHNSON KENT J , VARANI JAMES
Published: June 2021

Recent studies have shown that ginger extract and curcumin work synergistically to promote wound healing in Temovate‐treated rats. With this in mind, the current study focused on the effects of combined ginger and curcumin on organ cultured human skin and primary dermal fibroblasts. Dermal fibroblasts were treated for a 72‐hour period with a range of ginger and curcumin concentrations, both individually and combined, in low calcium and high calcium media. Biopsy punches from the hip of adult human subjects were organ cultured for 6 days in supportive media and treated with a similar dose range of ginger and curcumin as the fibroblasts. Findings were parallel in fibroblast monolayer and organ cultured skin. The combination of ginger and curcumin protected fibroblasts from cytotoxicity under conditions in which survival did not occur in their absence. Taken together with the previous wound‐healing study, these data support the synergistic action of ginger and curcumin and indicate their potential benefit in "at‐risk" human skin. Work supported by NIH HL07097
Biodegradable polymers useful in wound repair requiring negative pressure wound therapy
Authors: Wagner William D. , Liu Jie , Morykwas Michael , Argenta Louis
Published: June 2021

Treatment of wounds using negative pressure wound therapy (NPWT) currently utilizes non‐degradable sponges of polyurethane or polyvinyl alcohol (PVA), which require removal. The purpose of this study was to construct and test biodegradable combinations of chitosan, Type I collagen, poly (lactic‐co‐glycolic acid) (PLGA) 50:50, and poly (ε‐caprolactone) (PCL) as substitute sponges. Polymer sponges were porogen leached, cross‐linked, and hydrated in phosphate buffered saline prior to use in NPWT. Sponges were tested on the uncut surface of pig liver maintained in a hydration chamber continuously replenished with saline. PLGA and PCL sponges were used under conditions of negative 25mm Hg to 75mm Hg for 72 hours and removed fluid from the hydrated liver. Other sponges were mechanically inferior. Mechanical properties of PLGA: PCL: PVA sponges were as follows; maximum stress (0.51, 0.52, 0.43, MPa); maximum strain (0.94, 0.59, 2.95 mm/mm); Young's modulus (2.74, 2.42, 0.24 MPa). The results demonstrate PLGA and PCL sponges are appropriate substitutes for currently used nondegradable sponges.