bluuebunny/crossref_metadata_2025 huggingface dataset:
Nondestructive Measurement of Regenerated Esophageal Biaxial Mechanical Properties Utilizing Sonometry For Determination of Directional Esophageal Regeneration
Authors: Aho Johnathon M , Barron Matthew R , Qiang Bo M , Wigle Dennis A , Tschumperlin Daniel M , Urban Matthew W
Published: June 2021

Esophageal diseases often necessitate resection of tissue. Current reconstruction options using autologous conduits such as stomach, colon, and small bowel have significant morbidity. Given these limitations in current therapy, we have developed a large animal model to regenerate short segments of esophageal tissue after circumferential full thickness resection. This is performed by implantation of an electrospun polyurethane tissue engineered graft which results in esophageal regrowth. These implantations have been performed both with cell based seeding the polymer scaffold and without with comparable regenerative results. It is possible that esophageal regeneration in this model has unequal contributions from either the proximal or distal esophagus. To determine preferential directionality of regeneration we aim to utilize biomechanical markers to suggest that there are unequal proximal and distal contributions to regenerated tissue. Given limited availability of regenerated tissue, use of a non‐destructive method to perform this testing would have significant utility. Through using our previously validated non‐destructive sonometric ex vivo method we aim to determine differences in mechanical behavior of the regenerated tissue in comparison to proximal and distal esophageal tissues. Using this method we are able to determine longitudinal and circumferential moduli and derive strain energy functions and parameters. Within this work we demonstrate that there are differences in mechanical behavior and that the regenerated tissue behaves more comparably to proximal esophageal tissue suggesting preferential regrowth and unequal contribution to regenerated tissue.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Mitochondrial Impairment Upregulates MICOS Expression in a Human Microglial Cell Model
Authors: Carot Alejandra Bargues , Malovic Emir , Jin Huajun , Anantharam Vellareddy , Kanthasamy Arthi , Kanthasamy Anumantha
Published: June 2021

Mitochondrial dysfunction and oxidative stress have been implicated as key pathological mechanisms underlying the dopaminergic neurodegenerative process of Parkinson's disease. The mitochondrial contact site and cristae‐organizing system (MICOS) complex, formed by two subcomplexes with a total of 7 subunits, is associated with the formation and maintenance of mitochondrial cristae structure and respiratory complexes as well as in the regulation of mitochondrial import machinery. Alterations of MICOS subunit protein levels, mutations, and protein modifications have been associated with distinct human diseases, including Parkinson's disease, diabetic cardiomyopathy, epilepsy, and cancer. Previous studies using the classic mitochondrial complex‐1 inhibitor rotenone have shown that overexpression of Mic60 attenuates the rotenone‐induced cell death and increased mitochondrial respiration and spare respiratory capacity in PC12 and SH‐SY5Y dopaminergic cell models. We recently reported that mitochondrial impairment in microglia amplifies the neuroinflammatory response in cell and animal models of rotenone‐induced neurotoxicity. To further understand the cellular mechanism underlying mitochondrial impairment and neuroinflammation, we investigated the effect of rotenone on MICOS subunits, specifically Mic27 and Mic60, in the novel C20 human microglia cell model. Our immunocytochemical, qPCR and Western blot analyses revealed that exposing C20 cells to rotenone for 24 hours induced the upregulation of Mic27 and Mic60. Microglia are known to be extremely resilient cells whose metabolic states differ greatly in comparison to neurons. Therefore, our future studies will determine if the upregulation of MICOS subunits in microglia represents a compensatory mechanism to relieve mitochondrial stress.

Support or Funding Information

(NIH grants NS100090, NS088206 and ES027245)

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Investigating the Kinetic Effects of the Allosteric Interaction Between Newly‐Discovered Dynamic Hydrogen‐Bonded Residues and the HINT1 Active Site
Authors: Wang David , Strom Alexander , Wagner Carston R.
Published: June 2021

Histidine Triad Nucleotide binding proteins (HINTs) are an ancient class of enzyme notable for their acyl‐AMP hydrolase and phosphoramidase activity. This class of protein is of research significance due to its involvement in antiviral prodrug activation, as well as its natural role in opioid tolerance. Previously, the Wagner Lab has published the substrate specificity profile, full kinetic mechanism, and kinetics assay for the HINT isozyme HINT1. However, the active site dynamics of this enzyme have yet to be elucidated.

Recent insights into the structural dynamics of HINT enzymes found via CPMG‐NMR and discovered by the Wagner Lab have revealed a potential site of regulation for the kinetic rates of HINT1. We hypothesize that a subtle allosteric interaction exists between the active site of HINT1 and the previously unrecognized distant site through a channel of conserved waters, which plays a key role in the active site dynamics and kinetics.

Thus, with the goal of investigating the kinetic properties of this potential allosteric interaction, two genetic mutants of the enzyme HINT1 were produced to eliminate or alter the interactions between the highly dynamic residues Q62 and E100. Both steady‐state and stopped flow pre‐steady state kinetic analyses were performed on the mutants to determine any significant changes to their kinetics. Furthermore, circular dichroism analysis of both mutants was performed to ensure that both mutants were structurally sound, with no significant enzyme misfolding.

Through these analyses, it was found that the kcat and Km of the HINT1 mutants were altered significantly and predictably through the pair of successive mutations. Through process of elimination, these findings informed us that these mutations to the dynamic residue Q62 must be altering the rate of intermediate hydrolysis at the HINT1 active site, a site previously thought unlikely to be altered.

Support or Funding Information

Partial funding of this study by the University of Minnesota Office of Undergraduate Research is gratefully acknowledged.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Identifying an Allosteric Switch Region in Human SIRT1
Authors: Huynh Angelina , Amaya Carina Araceli , Montermoso Saira , Lustig Brooke , Wang Ningkun
Published: June 2021

SIRT1 is an NAD+‐dependent lysine deacetylase that is implicated in age‐related diseases and is a potential therapeutic target for drug development. The N‐terminal domain is highly disordered and is usually removed prior to crystallization of the enzyme. However, it has been known to physically interact with the catalytic core to increase enzyme activity. The mechanism of this remains unknown. STAC binding domain (SBD), within the N‐terminal region, is the binding site for sirtuin‐activating compounds (STACs) such as resveratrol, which is known to enhance SIRT1 activity towards peptide substrates including p53W. An allosteric switch is hypothesized as a single or multiple residues responsible for a change in secondary structure essential for allosteric activity and mostly found in disordered regions. Detailed information on switch regions remains unexplored due to the challenges of crystallizing the disordered regions. Our project aims to identify and confirm an allosteric switch region within the N‐terminal domain of SIRT1. The first aim is to computationally identify a switch region in SIRT1 using sequence‐and homology‐based descriptors. A possible switch region is hypothesized as having (1) high secondary structure variability, (2) low sequence entropy, and (3) low sequence disorder propensity, inferred by a combination of predictors. Applying these predictors to the full sequence of SIRT1 showed that such a switch may occur within residues 186–193 (TFVQQHLM) of the SBD. The second aim is to experimentally confirm these predictions. We have mutated the predicted region to serine residues: 186–189S, 190–193S, 188S, and 192S. The enzyme kinetics of the mutants remain relatively similar to the wild‐type SIRT1. We then tested these mutants for susceptibility to allosteric modulations by assaying their enzyme activity towards p53W with and without resveratrol. No increase in activity with the addition of resveratrol would confirm that these residues are involved in an allosteric switch. These results will inform the accuracy of the computational descriptors and provide a better understanding of the regulation of this enzyme in the cellular system.

Support or Funding Information

California State University Program for Education and Research in Biotechnology; Louis Stokes Alliance for Minority Participation

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
A Securinine Derivative as a Novel Treatment for Acute Myeloid Leukemia
Authors: Oldford Natasha R. , Karunanithi Sheela , Liu Ruifu , Hou Youngchun , Stetson Lindsay , Roe Anne , Lee Grace , Goyco Juan Valentin , Moreton Stephen , Wald David
Published: June 2021

Acute Myeloid Leukemia (AML) is a rapidly developing cancer of the bone marrow, with a five‐year survival rate of 27.4%. Current methods of treatment are toxic to older patients, creating a need for new treatment options that are more optimal for patient's health. One potential new treatment is with securinine, a natural compound from the Securinega suffruticosa plant that inhibits AML cell growth. Novel securinine derivatives were tested through cell viability assays, and compound 250 was found to exhibit anti‐cancer properties at a higher potency and lower toxicity than the parent drug. 250 was tested in in vivo AML models that exhibited a decrease in the AML burden on the mouse. Further experiments showed that 250 was able to inhibit thioredoxin reductase, an enzyme which maintains redox homeostasis within the cell. This resulted in negatively affecting AML cell metabolism. Nicotinamide add back experiments with 250 treatment allowed the cells to overcome the cytoxicity and decreased the cell death compared to 250 alone. When analyzing various cancers, it was found that AML cell lines had much lower amounts of Thioredoxin reductase compared to solid cancer lines, leading to 250's potent effect on AML cell lines. Future studies may focus on the effect of 250 on p‐38 activation, and p‐38's relevance in 250 mediated AML cell death.

Support or Funding Information

The research was supported by the NIH grant R43CA22870.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Mice lacking membrane‐localized estrogen receptor 1 (mESR1) are partially protected from reproductive pathologies resulting from developmental diethylstilbestrol (DES) exposure
Authors: Ortega Madison Taylor , Cooke Paul S. , Medrano Theresa I. , Mesa Ana M. , Nanjappa Manjunatha K. , Caldo Paul D. , Mao Jiude , Kinkade Jessica A. , Levin Ellis R. , Rosenfeld Cheryl S.
Published: June 2021

Both membrane and nuclear fractions of estrogen receptor 1 (ESR1) mediate 17β‐estradiol (E2) actions. Mice expressing nuclear (n)ESR1 but lacking membrane (m)ESR1 (nuclear‐only estrogen receptor 1 [NOER] mice) show reduced E2 responsivity and reproductive abnormalities culminating in adult male and female infertility. Using this model, we investigated whether reproductive abnormalities caused by the synthetic estrogen diethylstilbestrol (DES) are mitigated by mESR1 ablation. To test this, homozygous and heterozygous wild‐type (WT and HET, respectively) male and female mice and NOER mice were subcutaneously injected with DES (1 mg/g BW) or vehicle daily from postnatal day (PND) 1–5. Uterine histology was assessed in select DES‐treated femalesa at PND 5, whereas others were ovariectomized at PND 60 and treated with E2 (10 ng/g BW) or vehicle 2 weeks later. In WT females, DES resulted in ovary‐independentepithelial proliferation in the vagina and uterus. These changes were absent in DES‐treated NOER females. Uterine gene expression paralleled cell proliferation differences with neonatal DES inducing ovary‐independent adult expression of classical E2‐induced transcripts (e.g., Lf, Ezh2) in WT but not NOER mice. Males were examined around PND 90. DES‐treated WT and heterozygous males showed smaller testes and increased incidence of bacterial pyogranulomatous inflammation encompassing the testes, epididymis and in some cases the ductus deferens with spread to lumbar lymph nodes; such changes were largely absent in NOER males. Results indicate male and female NOER mice are protected from deleterious effects of neonatal DES and thus, mESR1 signaling is required for adult manifestation of DES‐induced reproductive pathologies.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Multiple Persistent Environmental Chemicals at Low Concentrations Differentially Regulates Fatty Acids/Lipids in Wild Type and PPARAa−/− Mice
Authors: Gonnabathula Pavani K , Yakubu Momoh A
Published: June 2021

Many chemicals are detected in the environment at very low levels, they were viewed as being incapable of causing harm. Their risks to human health and associated environment are now emerging. But effects of their multiple interactions at cellular and molecular levels is unknown. We have investigated the effects of treatment of wild type and PPARα knockout mice on fatty acid contents in different organs. Mice were treated with low concentrations of multiple persistent environmental chemicals: atrazine, dieldrin, endrin, endosulfan and anthracene (1–100ng/L) in drinking water for six weeks and brain, liver, kidney, heart and spleen were collected, processed for fatty acid/lipid contents determination by GC/MS. The percentage changes in the levels of the fatty acid following treatment were calculated for wild type (WT) and PPARα −/− (KO). The changes (WT and KO) were compared to find the effects of treatment and the genetic (PPARα) influence on the regulation of fatty acid levels. Our results show differential presence of fatty acids: hexadecanoic acid, hexadecanoic acid methyl ester, hexadecanamide, 9‐octadecanemaide, pentadecanoic acid, octadecanoid acid, arachidonic acid, and cholesterol in the different organs as well as their differential regulation in the WT and KO. Results are summarized in Tables 1 and 2. Briefly, treatment increased 9‐octadecanemaide by 145% (WT) and 38% (KO) in the heart, reduced in the kidney 17% (WT), 29% (KO); hexadecanoic acid, octadecanoic acid was increased 0% (WT) but by 178% and 110% respectively (KO) (kidney), octadecanoic acid went up by 334% (KO) with not change in WT, hexadecanamide increased by 92% in KO Liver with 5% in WT. Generally, more perturbations in the regulation of the levels of fatty acid species in the KO than in the WT. These results indicate that multiple persistent environmental chemicals at low concentrations have limited effects on fatty acid dynamics in the WT but enhanced possible dysregulations in the PPARAα−/− mice.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
PKCα Mediates mTORC1 Activation in Non‐Small Cell Lung Carcinoma Cells with EGFR Deletion Mutation
Authors: Salama Mohamed Fouda , Liu Mengling , Clarke Christopher , Espaillat Mel Pilar , Haley John , Obeid Lina , Hannun Yusuf
Published: June 2021

Constitutively active epidermal growth factor receptor (EGFR) mutations play critical roles in the development and progression of non‐small cell lung cancer (NSCLC). Patients with EGFR mutations are initially sensitive to EGFR‐tyrosine kinase inhibitors; however, they eventually develop drug resistance. Therefore, thorough understanding of mutant EGFR signaling is crucial in order to avoid such resistance and develop novel control therapeutic strategies. In the present study, we observed high PKCα expression in lung adenocarcinomas and in NSCLC cells with EGFR exon 19 deletion mutation. Next, we examined the role of the highly expressed PKCα in the signaling pathways downstream of mutant EGFR. Our results demonstrated that targeting PKCα either by pharmacological inhibition or downregulation impaired mTORC1 signaling, decreased proliferation, and induced caspase‐3 dependent apoptosis in cells with EGFR mutation. Moreover, PKCα gain of function was sufficient to induce proliferation of cells with wild‐type EGFR when grown under low serum conditions that was further increased when co‐expressed with mutant EGFR. Our results clearly demonstrate important roles of PKCα in regulating mTORC1 activity in NSCLC cells with mutant EGFR, whereby a switch occurs from a PKCα‐independent to a PKCα‐dependent signaling. The results suggest that PKCα could be synergistically targeted for the treatment of NSCLC with constitutively active mutant EGFR.

Support or Funding Information

1. This work was supported by NCI grant CA97132

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Annexin V and gaussia luciferase fusion protein studies for bioluminescence apoptosis detection
Authors: Perez Andres
Published: June 2021

Programmed cellular death, otherwise known as apoptosis, is a hallmark of many prominent diseases ranging from cancerous to neurodegenerative. Due to this common cellular fate found in a variety of diseases, it has become a goal to develop and construct a mechanism through which apoptotic cells can be detected with a high selectivity. Herein, we describe the development of a class of biosensors are capable of identifying these cells destined for apoptosis through the use of Annexin V alongside a bioluminescent reporter expressed as a single fusion protein. In this work, the bioluminescent protein used is Gaussia luciferase (GLuc), natively expressed by the copepod Gaussia princeps. Annexin V is a protein innately found in the human body that will bind to phosphatidylserine (PS), a membrane phospholipid that shifts towards the outer membrane when the cell has become apoptotic, a key marker of regulated cell death. By combining Annexin V with Gaussia luciferase, we developed a a fusion protein (AgFP), and demonstrated it to be stable, show correlating results when testing for bioluminescence. Analyses through in vitro studies show the potency that the fusion protein, AgFP, does not inhibit either component of the construct from either binding PS or generating bioluminescent signals. Future prognosis of this Annexin‐Gaussia fusion protein includes testing using in vivo models with certain pathologies and determining whether bioluminescence is seen in the target area at a level of affinity and selectivity that can be statistically significant in detecting apoptosis. End stage applications of the fusion protein construct would be an effective and reliable way to screen various areas of the human body for abnormal rates of apoptosis and possibly intervene before some of these lethal diseases become too advanced.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Effects of Transplanting an Exercised or Sedentary Microbiota into Gnotobiotic Mice on Global Gene Expression in Gut, Muscle, and Brain Tissue
Authors: Mailing Lucy J. , Allen Jacob M. , Wang Selena S. , Kashyap Purna , White Bryan A. , Woods Jeffrey A.
Published: June 2021

Introduction

The gut microbiota is intricately connected with host health and is known to influence gene expression across multiple organ systems. Both animal and human longitudinal studies have demonstrated that exercise has independent effects on the composition and functionality of the gut microbiota. Previous studies performed by our group have also shown that transplant of an “exercised” microbiota into germ‐free mice confers enhanced protection against colitis compared to transplant of a “sedentary” microbiota. In this study, we assessed how an “exercised” microbiota transplant impacts global gene expression in gut, brain, and muscle tissue compared to a “sedentary” microbiota transplant.

Methods

Donor mice received access to a running wheel (n=10) or remained without access (n=9) for six weeks prior to euthanasia. Donor cecal contents were pooled by activity group and transplanted into two separate groups of recipient germ‐free mice (n=16 for each group). After five weeks of gut microbial colonization, recipient mice were sacrificed, and tissues were collected. Total RNA was isolated from the distal colon, soleus muscle, and hippocampus. Four representative mice were chosen from each group for whole transcriptome analysis (RNAseq) of these tissues.

Anticipated Results

We hypothesize that mice that received an “exercised” microbiome will have different global gene expression profiles than those that received a “sedentary” microbiome. We predict that genes related to immune function, cellular metabolism, and gut barrier function may be particularly affected by transplant of the “exercised” microbiome.

Support or Funding Information

This work supported by a grant from the Mayo Clinic‐University of Illinois Alliance for Technology‐based Healthcare.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Shocking! The Effect of TSST‐1 in Toxic Shock Syndrome
Authors: Arnholt Mark , Munzenmaier Diane H , Anderson Evelyn , Benson Cassidy , Brown Julia , Colwell Margaret , Colwell Theodore , Flanagan Michael , Hautala Lauren , Hennes Delaney , Hoffmann Gabriel , Kennedy Cara , Lisowski Leah , Stoeckmann Tate , Witt Emma , Zuern Rachel
Published: June 2021

Almost 20% of children who die of Sudden Infant Death Syndrome have a protein called Toxic Shock Syndrome Toxin‐1 (TSST‐1), produced by Staphylococcus aureus. This protein is also responsible for toxic shock and food poisoning. S. aureus produces this superantigen TSST‐1 to divert the immune system and multiply rapidly inside the body. TSST‐1 contains 194 amino acids and three domains. Identical domains A, B, and C consist of a larger alpha‐helix, residues 152‐168, along with two beta‐sheets, and three shorter alpha helices. The long alpha helix defines the protein as a superantigen. Antigen‐presenting cells, such as macrophages, contain Major Histocompatibility Complex class II. These domains are bound to T‐cell receptor beta sheets by TSST‐1, bypassing the normal interface, and activating up to 24% more than the average immune response. Despite TSST‐1 having no disulfide bridges, therefore lack of thermal denaturation resistance, TSST‐1 thrives in the human body due to an immune system distraction. TSST‐1 can move through mucous membranes, allowing S. aureus to disrupt the immune response without being inside the body. In the wild form of TSST‐1, His‐135 located adjacent to the long alpha‐helix is mutated to Alanine, creating mutant TSST‐1, making the superantigen inactive for reasons unknown. The HUHS SMART Team (Students Modeling A Research Topic) Team has designed a model of the wild form of TSST‐1 with 3D printing technology to investigate structure_function relationships. By studying how TSST‐1 interacts with the immune system, advances can be made to control the spread of these pyrogenic toxin superantigens.

Support or Funding Information

The MSOE Center for BioMolecular Modeling would like to acknowledge and thank the National Institutes of Health Clinical and Translational Science Award (NIH_CTSA UL1RR031973) and the Milwaukee School of Engineering for their support in funding the 2018_2019 SMART Team program.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Murine Mouse Models of Metastatic Colorectal Cancer in Lungs: Foci Counts in Three Dimensions
Authors: Monteith Corey , Jackson Hunter , Mechanic Jordan , Vemuri Venkata , Daniel Cody , Parker Kristy , Martinez Morgan , Scott Katherine , Farahani Navid , England Steve
Published: June 2021

In standard histological slide preparations, only a few representative samples are taken, which introduces sampling bias and necessarily eschews information about the full tissue sample. However, it is possible to obtain a full 3D data set of an entire tissue sample using the Knife Edge Scanning Microscope (KESM), an automated simultaneous sectioning and imaging microscope. In this study, we performed manual counts of metastases of colorectal cancer in mouse model lungs stained with India ink and hematoxylin and eosin, which were then sectioned and imaged in their entirety. We were able to get more accurate count of tumor foci than the current standard of surface nodule count can allow; multiple tumor foci were buried hundreds of microns deep into the parenchyma and hence not grossly visible from the surface. Volumetric analysis of whole samples also revealed unusual tumor morphology and permitted the visualization of unique architectural features where multiple tumor foci had grown together. Following the manual count of tumor foci, we developed a semi‐automated algorithm that could increase the efficiency of screening for foci, and create the opportunity for digital 3D reconstruction and morphology interrogation. Without access to a full dataset of the lungs, it is impossible to determine true foci number or morphology.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Bile Acid Excess Impairs Thermogenic Function in Brown Adipose Tissue
Authors: Zhou Weinan , VanDuyne Philip , Riessen Ryan , Akinrotimi Oludemilade , D'Souza Shawn , Anakk Sayeepriyadarshini
Published: June 2021

Obesity is a major epidemic and is associated with metabolic dysfunction leading to excess accumulation of fat. Bile acids (BAs) are body's natural detergents that facilitate fat digestion. Additionally, BAs act as signaling molecules regulating metabolism and can protect against obesity. Despite, several studies have demonstrated that elevated BAs promote energy expenditure, it remains unknown if BAs are present in brown adipose tissue (BAT) and can they directly affect BAT function. Here, we show that BAs, BA transporters and de novo synthetic enzymes are detected in BAT. Notably, the global farnesoid X receptor; small heterodimer partner double knockout (DKO) mice, a model for BA overload, exhibit higher BAs levels in BAT than the control mice. DKO mice exhibit lower body temperature, decreased mitochondrial DNA content, and altered activities of oxidative phosphorylation enzyme complexes. Consistent with this phenotype, we find that excess BAs impair BAT mitochondrial function and can cause thermogenic dysfunction. Importantly, when low physiological BA levels are used to treat brown adipocytes, we do not find mitochondrial defects. However, upon treatment with pathological BA concentrations, differentiated primary brown adipocytes in vitro show reduced mitochondrial membrane potential and concomitant reduction in thermogenic gene expression profile. We have shown that DKO mice are resistant to fat accumulation both on normal chow and when challenged with a high‐fat diet. This protection is significantly lost when the mice are housed at thermoneutral conditions indicating that by having leaky mitochondria and poor thermoregulation, DKO mice are protected against accumulating fat. Overall, we show for the first time the presence of BA metabolic machinery in the BAT and identify that elevated levels of BAs can cause thermogenic dysfunctions. Weinan Zhou and Philip VanDuyne contributed equally to this work.

Support or Funding Information

Startup funds from the University of Illinois at Urbana‐Champaign, and R01 DK113080 from NIDDK.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Analysis of an Omega 6:3 Balanced Diet Impact on Hibernation Behavior and Cortisol of Captive Arctic Ground Squirrels
Authors: Mikes Monica L , Rice Sarah A , Bibus Doug , Drew Kelly L
Published: June 2021

Arctic Ground Squirrels (AGS) are widely studied in laboratories for their capability to sustain low metabolic rates and temperatures without enduring debilitating bodily harm. A specific range of omega 6 polyunsaturated fatty acids (PUFAs) has been shown to improve captive hibernation by increasing torpor bout length and depth of hibernation across multiple species (Geiser 1993, Frank 2008). To our knowledge, only three studies have investigated the impact of omega 3 fatty acids on hibernation behavior with varying results (Giroud 2018, Frank 2004 and Hill and Florant 2000). Our prior findings show wild AGS plasma have a balanced omega 6 to 3 fatty acid profile (1.3:1), but no one, to our knowledge, has tested the impact of this specific PUFA ratio on captive AGS. We hypothesize that substituting a high omega 6:3 (8:1) diet for a balanced diet (1.3:1) would increase the length of torpor bouts, entrance timing into hibernation and decrease cortisol.

Wild juvenile AGS (12 f, 12 m) were evenly distributed to two groups: a balanced omega 6:3 ratio diet (1.3:1) and a control diet of Mazuri rodent chow with an omega 6:3 ratio of (8:1). Animals were fed 47g daily of their diets until first torpor bout and food was withdrawn. Animals were instrumented with abdominal temperature I‐buttons in early August. White adipose tissue (WAT) and plasma were sampled longitudinally at fall (early August at the time of surgery) and winter (mid‐December at time of euthanasia). Animals were euthanized in three stages of the torpor bout cycle: early torpor, late torpor, and full arousal.

WAT was analyzed a modified lipid dense tissue protocol using solid phase extraction. Cortisol was measured using RIA. Early August levels of cortisol were 1.4× higher in the animals supplemented with the balanced omega 6:3 diet (p<0.0001). Preliminary temperature data indicates that the balanced diet and control diet AGS do not significantly differ in the number of hibernation days, entrance timing into hibernation or torpor bout length. Additionally, preliminary data indicates a significant increase in body weight of females on a balanced omega 6:3 diet during fall (p<0.013) and winter (p<0.049).

Preliminary data indicates the balanced diet does not negatively impact hibernation behavior. The unexpected increase in cortisol in the balanced omega 6:3 ratio fed diet may reflect normal physiological levels rather than stress. Cortisol functions as a stress indicator, but also enhances fat deposition, which is an essential task prior to hibernation.

Support or Funding Information

This work was supported by NIH 20GM103395, UL1GM118991, TL4GM118992, or RL5GM118990. This work was performed in conformance with the FASEB Statement of Principles for animals used in research.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
LPS‐induced mTORC1 Signaling Activates Lipid Raft‐Actin Cytoskeletal Interactions for Phagocytosis through SREBP‐1a
Authors: Lee Jae‐Ho , Kim Do Young , Lee Sun Hee , Song Dae‐Kyu , Bae Jae‐Hoon , Im Seung‐Soon
Published: June 2021

There is a growing appreciation for a fundamental connection between lipid metabolism and the immune response. Macrophage phagocytosis is a signature innate immune response to pathogen exposure and cytoplasmic membrane expansion is required to engulf the phagocytic target. The sterol regulatory element binding proteins (SREBPs) are key transcriptional regulatory proteins that sense the intracellular lipid environment and modulate expression of key genes of fatty acid and cholesterol metabolism to maintain lipid homeostasis. In this study, we show that TLR4 dependent stimulation of macrophage phagocytosis requires mTORC1‐directed SREBP‐1a dependent lipid synthesis. We also show that the phagocytic defect in macrophages from SREBP‐1a deficient mice results from decreased interaction between membrane lipid rafts and the actin cytoskeleton presumably due to reduced accumulation of newly synthesized fatty acyl chains within major membrane phospholipids. We show that mTORC1 deficient macrophages also have a phagocytosis block downstream from TLR4 signaling, and interestingly, the phagocytosis deficiency in both SREBP‐1a and mTORC1 mutant macrophages can be restored by ectopic SREBP‐1a expression. Taken together, these observations indicate SREBP‐1a is a major downstream effector of TLR4‐mTORC1 directed interactions between membrane lipid rafts and the actin cytoskeleton that are required for pathogen‐stimulated phagocytosis in macrophages.

Support or Funding Information

The National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIP)(2016R1A2B4008516). The National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIP) (2014R1A5A2010008).

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Avoiding a Swelled Head: Aquaporin‐4 and Brain Edema
Authors: Shelton Katelyn , Gross Gabriela , Baca Mackenzie , Kaka Nornubari
Published: June 2021

Aquaporins are integral membrane proteins that serve as water channels. Aquaporin 4 (AQP4) is an aquaporin specific to the brain. Besides working as the “plumbing system” to allow water to diffuse across the membrane, it also prevents the passage of ions and other solutes. Aquaporin‐4 works in a bundle of four aquaporin monomers to form a tetramer and is highly expressed within astrocytes (cells in the CNS that help maintain homeostasis) and within the blood‐brain barrier. The blood‐brain barrier is a lipophilic membrane which permits lipid penetration freely while preventing water diffusion. Aquaporin‐4 regulates water circulation of the brain and does not require any energy to function, allowing for the simple diffusion of water. AQP4 has been shown to facilitate astrocytic edema and is responsible for the reabsorption of extracellular edema fluid. Symptoms of cerebral edema include nausea, vomiting, Aquaporin‐4 is supposed to maintain the fluid levels through the spinal cord but the real question is how does it do that in the structure it appears to be. Instead of explaining the function of the Aquaporin‐4, we are going to show it through a model we have created. Cerebral edema can occur when a clot is preventing blood access to the brain; the immune system begins to attack and exert fluids via AQP4, resulting in excess fluid. That edema therefore increases pressure within the skull preventing blood from flowing to the brain and occasionally leads to a stroke. If AQP4 can be targeted, then edema can significantly be reduced and become less of a risk to injured brain tissue.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The Snow Flea Antifreeze Protein and Cryopreservation
Authors: MacGillivray Brendan , Greig James , Cobo Nicolás Alonso , Chow Alyssa , Han Brian , He Lucy , Mark‐George Matthias , Niu Zimu , Wai Ewan , Wang Yier , Xiao Mason
Published: June 2021

Hypogastrura harveyi (snow fleas) produce a glycine‐rich antifreeze protein which enables it to survive in environments that would otherwise freeze its body fluid. Snow flea antifreeze protein (sfAFP) is a 6.5 kDa monomeric protein made of six alpha helices linked by hydrogen bonds and disulfide bridges. The protein's amino acid sequence has a frequent glycine‐x‐y amino acid pattern, where the x amino acid is also glycine 50% of the time and the y amino acid is either hydrophobic or hydrophilic. The hydrophobic side of sfAFP binds to the surface of seed ice crystals, breaking up the ice surface into smaller areas and preventing the further binding of water molecules due to the Kelvin effect. Other AFP's have been shown to be efficacious in improving the preservation of rat hearts, leading to the yet‐untested hypothesis that sfAFP's are also able to prolong the cryopreservation of organs. The Ashbury MSOE Center for BioMolecular Modeling SMART Team used 3‐D modeling and printing technology to examine structure‐function relationships of sfAFP when bonding to an ice lattice.

Support or Funding Information

MSOE ‐ Center for Biomolecular Modelling ‐ co‐sponsor Diane Munzenmaier

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Qualitative and Comparative Analysis of Antemortem vs. Postmortem Root Hair Banding Use in Forensic Science
Authors: Wiggins Abigail Grace
Published: June 2021

Hair is one of the most important types of evidence in Forensic Science. Hair can be used to place a victim, and an assailant, in the same location. One method utilizes root banding of a hair sample, recovered from a victim or crime scene, that are analyzed using microscopy. Postmortem root banding is observed in samples collected from deceased humans. This methodology is used to determine if the evidence came from a living, or deceased victim. However, postmortem root banding patterns have recently been observed in antemortem anagen hair samples, now referred to as antemortem root banding. Further characterization and comparison between the two types of root banding is needed to assist in determining the type of investigation required in forensic cases; missing persons or body recovery missions. The objective of this research is to better differentiate postmortem and antemortem root banding. Laboratory created antemortem root banding data will be characterized by the banding location on the hair follicle, coloration by stage, and width compared to corresponding postmortem root banding. This work is expected to contribute to advancements in the field of Forensic Science and strengthen the ability to differentiate postmortem and antemortem root banding. The legal argument of antemortem root banding, and postmortem root banding evidence is easily dismissible in a court of law. The ability to differentiate between the two types of root banding patterns will afford more reliable evidence in the judicial system.

Support or Funding Information

Department of Mathematics and Science, Saint Leo University, Saint Leo, FL 33574

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Sox100B is required in the ovary peritoneal muscle sheath development in Drosophila melanogaster
Authors: Melendez Ashley , Biteau Benoit
Published: June 2021

The transcription factor Sox100B is known to have a function in embryonic gonads differentiation and testis development. Drosophila as a model organism enabled the identification of expression patterns of Sox100B in ovaries to establish its role in proper ovarian development and function. Previous results from our lab showed that a decrease in the transcription of the Sox100B gene is associated with reduced fertility and mild defect in ovarian muscle sheath morphology. Additionally, Sox100B is expressed in the apical cells, which are the muscle cell progenitors. These results suggest that Sox100B plays an active role in the ovary muscle by regulating ovarian muscle sheath development during morphogenesis. Based on these experiments we hypothesize that Sox100B is required for the development of the ovarian muscle sheath. We approached this question by knocking down and overexpressing Sox100B using Heartless‐Gal4 (htlGal4), a driver used for studying targeted gene expression in the Drosophila wing heart and the ovary muscle. To acquire a complete understanding of the Sox100B function throughout morphogenesis, we observed ovaries of late larvae (LL3), young adults and mature adults. Experiments with Sox100B overexpression constructs proved to be fatal at embryonic stage, thus preventing the study of the ovarian phenotype in these flies. However, when we crossed the htlGal4 driver with RNAi lines for Sox100B, a significant decrease was observed in egg laying and pupariation assays. Eggs that were successfully fertilized survived to adulthood, suggesting oogenesis is not affected. We utilized immunohistochemistry (IHC) and confocal microscopy to examine adult ovaries from htlGal4>Sox100BRNAi and we observed an intact epithelial sheath but tearing of the peritoneal sheath. Based on these results we concluded that Sox100B is required for the peritoneal muscle sheath development and ovary function.

Support or Funding Information

Supported by the National Institutes of Health under Award Number T34GM008419.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The Effects of CLCN2 Knockout on Epileptic Absence Seizures
Authors: Naik Navin , Beenhakker Mark
Published: June 2021

Epilepsy is a neurological disorder affecting about 1 percent of Americans. Epileptic seizures are caused by nerve cell activity in the brain and can vary from being nearly undetectable to long periods of shaking. Idiopathic generalized epilepsy includes a group of epileptic disorders that are believed to have a strong genetic basis. The seizures of interest here are petit mal seizures, or absence seizures. Absence epilepsy is one of the most common forms of childhood epilepsy. It is characterized by brief, frequent seizures and affects children from the ages of 4 to early adolescence. Absence seizures are also characterized by generalized spike‐and‐slow wave electrical discharges (SWD), which can be detected using electroencephalography (EEG). Due to the prevalence and comorbidities associated with absence epilepsy, it is important to study it further. Based on research published in a 2003 paper by Haug et al, we decided to examine the effects of a chloride channel gene CLCN2 in a mouse model. Data was collected via EEG recordings of mice over a 5‐day period. Two mouse models were used: wild‐type mice and CLCN2 knockout mice. EEG recordings were scored for spike‐and‐wave discharges to determine seizure occurrence. Due to time constraints, only limited data was obtained. This data confirmed the hypothesis that CLCN2 KO mice would have increased seizures. However, the insufficient data prevented us from creating a significant conclusion. Nevertheless, important procedural information was found to aid in future experiments.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Urethral Smooth Muscle Dysfunction in Middle‐aged Male Rats May Affect Micturition
Authors: Alexandre Eduardo Costa , de Oliveira Mariana G , Silva Fábio H , Calmasini Fabiano B , de Carvalho Hernandes F , Yoshimura Naoki , Antunes Edson
Published: June 2021

INTRODUCTION AND OBJECTIVES

Epidemiological studies have shown that age is the most important unmodifiable risk factor of lower urinary tract symptoms (LUTS) in men. Much attention has been given to the involvement of the bladder and prostate in men's lower urinary system, however much less to the urethra. We raised the hypothesis that middle‐aged urethra smooth muscle (USM) impairment may exert a negative influence on lower urinary tract functioning. Here we aimed to evaluate the urethral main contractile and relaxant pathways through functional, biochemical, and molecular studies and their consequences to micturition.

METHODS

Young (3.5‐month) and middle‐aged (10‐month) male Wistar rats were used (CEUA 4404‐1). USM was collected for functional, biochemical, and molecular assays. Urethral contractions were evaluated by electrical field stimulation (EFS) and phenylephrine (PE) and relaxations by sodium nitroprusside (SNP) and glyceryl trinitrate (GTN). cGMP was measured and qRT‐PCR was conducted for different gene targets like NOS enzymes, sGCβ1, PDE5, MRP5, gp91phox, HIF1α, SOD1, and catalase. Anion superoxide, NO, collagen, and smooth‐muscle area were also measured and a cystometric analysis was performed.

RESULTS

Contractile responses elicited by EFS (1–16 Hz) and PE were greater in USM for the middle‐aged group compared with the young group, an alteration that was accompanied by a decrease in NO‐induced USM relaxation by SNP and GTN. cGMP concentration was lower in the middle‐aged USM compared with the young group. In qRT‐PCR, up‐regulation of PDE5, MRP5, gp91phox, and HIF1α, associated with down‐regulation of sGCβ1, SOD1, and catalase were observed in middle‐aged USM. No differences of NOS enzymes expression were found. The anion superoxide and NO levels in the USM of middle‐aged rats were increased and decreased, respectively. This was accompanied by higher USM collagen and smooth muscle area. Middle‐aged rats showed an irregular micturition pattern characterized by significant increases of basal pressure, capacity, and compliance.

CONCLUSIONS

USM dysfunction in middle‐aged rats due to the association with NO‐cGMP pathway impairment and oxidative stress may represent a detrimental factor to micturition and the start of bladder alterations observed in the elderly.

Support or Funding Information

Source of funding: FAPESP (2017/04556‐4 and 2018/03625‐5)

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.