bluuebunny/crossref_metadata_2025 huggingface dataset:
Mass Spectrometry of Bacterial Outer Membrane Enzymes within the Native Lipid Environment
Authors: Esmaili Mansoore , Li Jun , Bishop Russell , Klassen John.S , Overduin Michael
Published: June 2021

Lipid complexity of bacterial membranes and the dynamic interaction of lipophilic substrates with membrane‐embedded enzymes reflect a demand for a robust detection approach that unravels the local lipids and hydrophobic substrates/products either bound or within the milieu of membrane enzymes. In this study, we used styrene maleic acid lipid particle (SMALP) nanodiscs and electrospray ionization‐ion mobility spectrometry–mass spectrometry (ESI‐IMS) mass spectrometry to address the stability of lipid‐bound states of two bacterial β‐barrel enzymes.

We overexpressed and purified different oligomeric states of two model β‐barrel enzymes with their co‐associated lipids (i.e. enzyme substrates/products) from the outer membrane of E.coli using various polymer formulations. We further examined the stability of the intact lipid‐protein nanodiscs in solution as well as in the gas phase. Our results, thus far, has demonstrated the higher efficacy of the native nanodiscs over conventional detergents in isolating lipid populations bound to the enzymes, also an improved thermal stability of the resulting β‐barrel complexes in solution. We have, interestingly, observed that some polymers are more compatible with the required conditions in mass spectrometry hence overcoming limitations (e.g. low pH which is known as destabilizing factor for the original SMALP nanodiscs) in the structural analysis of lipid‐bound states of membrane proteins in the gas phase.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Amyloids and pre‐amyloids from the PDB
Authors: Grolmusz Vince , Takacs Kristof , Varga Balint
Published: June 2021

Using solely geometric conditions for beta‐sheets, we have prepared and maintain an automatically updated list of amyloid structures from the Protein Data Bank (PDB). Our list contains the insoluble amyloid structures, and, additionally, those globular proteins, which contain amyloid‐like sub‐structures. We assume that these globular proteins are transformed more easily to insoluble amyloid fibrils than other proteins. Several observations from the literature show the validity of this assumption.

The list of amyloid‐ and pre‐amyloid structures are publicly available at https://pitgroup.org/amyloid.

Support or Funding Information

Kristóf Takács was partially supported by the European Union, co‐financed by the European Social Fund (EFOP‐3.6.3‐VEKOP‐16‐2017‐00002).

Vince Grolmusz was partially supported by the NKFI‐126472 grant of the National Research, Development and Innovation Office of Hungary.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
An RNA‐Sequencing Approach to Understanding the Genetic Influences on Fetal Alcohol Syndrome
Authors: Bingham Stephanie , Gallagher Alayna , Flores Annalliam , Trinidad Jennyvette , Arboleda Vania , de Almeida Rego Julia , Villada Daniel
Published: June 2021

Ethanol is a known teratogen, though the precise mechanisms by which it exerts this effect are not well understood. In the current study, we aimed to get a handle on the genetic pathways that are disrupted as a result of embryonic ethanol exposure. Using an RNA sequencing approach, we scanned the genome of embryonic zebrafish and performed a comparison of gene expression levels in treated and untreated embryos. The whole‐genome analysis revealed 86 gene sequences that were significantly up‐ or down‐regulated following embryonic exposure. Through prioritization, we narrowed this number down to 40. Using genome search engines, the genes were classified according to spatial expression and function and distinct gene categories were revealed. The most abundant gene category (21%) was that of metal‐binding proteins, followed by actin‐binding (14%) and oxidoreducatase and kinase functions (both 7%). Follow‐up studies are now being performed on the first two of the genes of interest: TyrP1 and CENPF. TyrP1 is an integral membrane protein with diverse roles including melanocyte differentiation. CENPF, on the other hand, is a nuclear matrix protein and regulator of the cell cycle. Further analysis of the latter and its function may reveal an answer to the age‐old question of whether the reduced brain size, a hallmark of fetal alcohol syndrome, is a result of reduced cell division, increased cell death, or a combination of the two.

Support or Funding Information

This research was funded by the Department of Biology and a Faculty Senate research minigrant, Barry University.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
PGF and VEGF‐C secreted by preosteoclasts induce migration and differentiation of Mesenchymal stem cells
Authors: Huh Jeong‐Eun , Yi Sol , Lee Soo Young
Published: June 2021

Osteoblast‐mediated bone formation is coupled to osteoclast‐mediated bone resorption. Several studies suggested that the molecular mechanisms by which osteoblasts control osteoclastogenesis and bone degradation. But, the recruitment of mesenchymal stem cells (MSCs) and their differentiation to osteoblasts is poorly understood. Here we show that preosteoclasts secrete placental growth factor (PGF) and vascular endothelial growth factor C (VEGF‐C) to induce migration and osteogenesis of MSCs. Conditioned media from preosteoclasts increased migration ability of MSCs and alkaline phosphatase activity and mineralization in osteoblast cultures. Osteoclast precursors induced PGF and VEGF‐C expression in response to receptor activator of nuclear factor‐κB ligand (RANKL), an essential cytokine for osteoclast differentiation. In vitro experiments revealed that migration was stimulated by rhPGF and rhVEGF‐C in a dose‐dependent manner. Furthermore, these factors increased osteoblast differentiation. Vascular endothelial growth factor receptor (VEGFR) inhibitor treatment reduced these responses. Taken together, PGF and VEGF‐C secreted by preosteoclasts may enhance the recruitment of MSCs and further induce osteogenesis.

Support or Funding Information

This work was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (NRF‐ 2015R1D1A4A01020104).

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Arl4D modulates microtubule dynamics via its interaction with EB1
Authors: Lee Fang‐Jen Scott , Lin Shin‐Jin , Huang Chun‐fang , Wu Tsung‐Sheng , Li Chun‐Chun
Published: June 2021

ADP‐ribosylation factor (Arf)‐like 4D (Arl4D), one of Arf small GTPases, functions in cell morphology, cell migration, and actin cytoskeleton remodelling. EB1 is a microtubule plus end– tracking proteins that preferentially localized at the tips of growing microtubule plus ends and centrosome. Depletion of EB1 is known to result in many centrosome‐related defect. Here, we report that Arl4D binding to EB1 modulates microtubule plus‐end dynamics at the centrosome. In vivo and in vitro binding assay showed that Arl4D interacts with EB1 in a GTP‐dependent manner and C‐terminal EBH region of EB1 is both required and sufficient for this interaction. We found that Arl4D co‐localizes with γ‐tubulin in centrosomes and depletion of Arl4D resulted in centrosomal microtubule regrowth defect in COS‐7 cell. Arl4D knockout mouse embryonic fibroblasts (MEF) cells were also observed microtubule regrowth defect, which can be rescued by expressing human Arl4D. We also show that Arl4D‐EB1 interaction is essential for microtubule regrowth at the centrosome. In addition, we demonstrate that Arl4D binding to EB1 increases the association of the p150Glued subunit of dynactin to EB1, which is important for microtubule stabilization. Together, our results indicate that Arl4D modulates microtubule dynamics through regulating EB1‐p150 association at the centrosome.

Support or Funding Information

National Health Research Institutes, Taiwan (NHRI‐EX106‐10601B1)

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
NLRP12 binds to Hck potentially regulating the NF‐kB pathway
Authors: Zhang Yue , Okamoto Curtis
Published: June 2021

The nod‐like receptor family pyrin domain containing 12 (Nlrp12) is a cytoplasmic protein that belongs to the Pattern Recognition Receptor family (PRR) of proteins, an important component of the innate immune system for identification of Pattern Associated Molecular Patterns (PAMPs) and Damage‐Associated Molecular Patterns (DAMPs). Nlrp12 is comprised of 1062 amino acids and has three functional domains: an N‐terminal pyrin domain, a Nucleotide‐ Binding Domain (NBD), and a C‐terminal leucine‐rich repeat region. Nlrp12 is mainly expressed in peripheral blood leukocytes, including granulocytes, eosinophils, monocytes, and dendritic cells. Nlrp12 has been shown to function as a negative regulator of inflammation through canonical and non‐canonical NF‐kB pathways. However, the detailed mechanism of how Nlrp12 inhibits NF‐kB is not clear.

The Nlrp12 pyrin and NBD domain was used as bait in a yeast two‐hybrid screen for potential interacting partners in a human leukocyte cDNA library. The top hit was the C‐terminal fragment of Hck; it was shown to interact strongly with Nlrp12. Detailed mapping of the binding between Nlrp12 and Hck was performed. In subsequent yeast two‐hybrid assays, Nlrp12 was shown specifically to bind to Hck. The C‐terminal 30 amino acids of Hck was sufficient to bind with Nlrp12. This fragment carries the negative regulatory phosphotyrosine residue Y521. However, the binding did not require phosphorylation of Y521 residue of Hck. That is, Nlrp12 preferentially bound to unphosphorylated Hck (i.e., active Hck). Functional NF‐KB luciferase reporter assays were performed with 293T cells overexpressing either Nlrp12, or Hck, or Nlrp12 plus Hck. Overexpression of Nlrp12 alone decreased NF‐kB activity, while overexpression of Hck rescued the inhibition of NF‐kB activity by overexpressed Nlrp12. Overexpression of Hck alone had no effect on NF‐kB activity.

We conclude that Hck may be a novel regulator of Nlrp12 activity in its inhibition of NF‐kB function.

Support or Funding Information

University of Southern California School of Pharmacy Intramural Research Funds 2017‐ 2021

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Yeast RNA polymerase I Core Factor binds to the ribosomal DNA promoter through the GC minor groove
Authors: Glover Danea M. , Jackobel Ashleigh J. , Knutson Bruce A.
Published: June 2021

In Saccharomyces cerevisiae, Core Factor (CF) is a key evolutionary conserved transcription initiation factor that helps recruit RNA polymerase I (Pol I) to the ribosomal DNA (rDNA) promoter. Upregulated Pol I transcription has been linked to many cancers, and targeting Pol I is an attractive and emerging anti‐cancer strategy. Using the yeast model system, we characterized how CF binds to the Pol I promoter by electrophoretic mobility shift assays (EMSA). Synthetic DNA competitors along with anti‐tumor drugs and nucleic acid stains that act as DNA groove blockers were used to discover structural characteristics of CF binding. Our results show that CF employs a unique mechanism where it prefers the GC‐rich minor groove of the rDNA promoter, which may offer a new avenue to more specifically target upregulated Pol I activity in cancer.

Support or Funding Information

The work was supported by B.A.K. grants from the US National Institutes of Health (NIH) NCI (5K22CA184235), a Sinsheimer Scholar award from the Alexandrine and Alexander L. Sinsheimer Fund, Central New York Community Foundation, Joseph C. George Fund, and Virginia Simons & Dr. C. Adele Brown Fund.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Pasteurella Multocida Toxin Mediated ECM Expression Is Independent of mTOR signaling pathway
Authors: Oubrahim Hammou , Leong Megan , Chock Boon P
Published: June 2021

Bacterial virulence factors that interfere with cell signaling and result in disruption of normal cell division and apoptosis could facilitate cancer metastasis via unknown mechanisms. Pasteurella multocida toxin (PMT) is an intracellular acting bacterial protein known for its potent mitogenic properties in vitro and in vivo and its ability to induce strong anchorage‐independent growth for certain type of cells. These properties suggest that PMT might have the potential to act as a tumor promoter especially in the case of chronic infections. We have shown that rPMT induces cell proliferation in serum‐starved Swiss 3T3 cells by hijacking cellular signal transduction pathways via deamidation of heterotrimeric G‐proteins and leads to a sustained activation of mTOR, MAPK, and membrane associated tyrosine kinase signaling. In addition, further experiments revealed that PMT induced a prolonged upregulation of chromosomal passenger complex (CPC) proteins including survivin and aurora kinase B both at mRNA and protein levels in serum starved Swiss 3T3 cells treated with PMT in a manner dependent on both mTOR and MAPK signaling pathways.

An increasing body of evidence supports the idea that extracellular matrix (ECM) proteins are major players in the global control of intercellular communication and integration of environmental signals. It was not known whether rPMT‐treated cells are able to express and secrete into the medium a substrate(s) capable of activating autocrine and/or paracrine signaling. Microarray analysis identified connective tissue growth factor (CTGF) mRNA as the most upregulated gene in 3T3 cells, along with other genes involved in cell proliferation and cancer biology. The elevation of CTGF, along with other extracellular matrix proteins, were confirmed by RT‐PCR and Western blot analysis. In accord with rPMT‐induced mTOR activation, upregulation of CTGF was mediated by deamidation of Gαq/11, and was independent of TGFβ, a well‐known inducer of CTGF. However, PMT‐induced ECM expression is independent of both mTOR, MAPK and membrane associated tyrosine kinase activation. Taken together, our results show that PMT activates mTOR and membrane associated tyrosine kinase, and MAPK leading to the upregulation of proteins involved in cell proliferation but do not have any effect on the expression of ECM proteins.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Liraglutide improves insulin sensitivity in diabetic mice through reduction of inflammation and induction of thermogenesis.
Authors: Zhou Joseph , Poudel Anil , Li Lixin
Published: June 2021

Glucagon like peptide ‐1 (GLP‐1) is released from the ileal mucosa L cells after food digestion to promote glucose stimulated insulin secretion. Liraglutide, a full agonist of the GLP‐1 receptor, has been known to reduce body weight in obese subjects and patients with type 2 diabetes. However, the underlying mechanisms of this effect remains unclear. Mice were fed with high fat high sucrose diet for 3 months to induce diabetes. Two groups of diabetic mice were injected with liraglutide or vehicle daily for 14 days. Improved glycemic control and intra‐peritoneal glucose tolerance test were observed in liraglutide treated group after 14 days. In addition, liraglutide treatment group showed significant reduction of liver weight in association with reduced expression of the phosphorylated Acetyl‐CoA carboxylase‐2 (ACC2) and upregulation of LCAD, cAMP‐activated protein kinase (AMPK)‐α as well as Sirt‐1 in liver and perigonadal fat. The elevation of fatty acid oxidation induced by liraglutide might be mediated through AMPKα‐SIRT‐1 cell signaling pathway.

Brown adipose tissue (BAT) plays a key role in regulating energy balance via uncoupling of mitochondria ATP synthesis in rodents. Liraglutide induced BAT differentiation in skeletal muscle, including induction of the uncoupling protein‐1 (UCP‐1), a brown fat specific marker, PR domain‐containing 16 (PRDM16) protein expression, a transcription factor that regulates the thermogenic gene program in brown and beige adipocytes. Mitochondrial biogenesis and functional gene such as PPAR‐γ, PGC‐1αand SIRT‐1 were also upregulated by liraglutide in insulin sensitive tissues.

Lastly, liraglutide displayed anti‐inflammatory effect. Liraglutide treatment leads to a significantly reduction of circulating IL‐6 as well as IL‐6 content in liver. Expression of iNOS‐1 and COX2 in insulin sensitive tissues were also reduced followed liraglutide treatment.

Taken together, our study indicates liraglutide improves insulin sensitivity through multiple pathways which includes suppression of food intake, reduction of inflammation, elevation of fatty acid oxidation and induction of thermogenesis.

Support or Funding Information

Start Up Fund ( Central Michigan University)

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Subnormothermic Regulated Hepatic Reperfusion Preserves Mitochondrial Function in Swine Liver Procured after Cardiac Death
Authors: Stowe David F , Yang Meiying , Mishra Jyotsna , Kim Joohyun , Heisner James S , Zimmerman Michael , Camara Amadou KS , Hong Jonny
Published: June 2021

Livers procured after cardiac death can have an extreme degree of ischemia‐reperfusion injury (IRI) and are at increased risk of graft failure after liver transplantation. Reductions in respiratory control index (RCI) and Ca2+ retention capacity (CRC) are markers of mitochondrial dysfunction and cell apoptosis. Regulated hepatic reperfusion (RHR) is a novel organ resuscitation therapy that may facilitate hepatic mitochondrial recovery from IRI using an energy substrate‐enriched, leukocyte‐depleted, oxygen‐saturated perfusate delivered in a pressure, and temperature‐controlled milieu. We used a swine donation after cardiac death (DCD) model, in which all livers were subjected to 60 min of in situ warm ischemia and 120 min of cold static (CS) preservation. All livers were subsequently reperfused and monitored for 2 h in the ex vivo perfusion machine. Four methods of reperfusion were compared with 5 animals in each treatment group. Group I: Sanguineous‐Subnormothermic (SS); Group II: RHR‐Subnormothermic (s‐RHR); Group III: Sanguineous‐Normothermic (NS); Group IV: RHR‐Normothermic (n‐RHR). Liver biopsies were collected at baseline (pre‐ischemia), at 2 h of CS following 1 h of in situ warm ischemia, and after 30 min of ex vivo reperfusion. We isolated mitochondria via differential centrifugation and measured mitochondrial bioenergetics, i.e. the ability to utilize nutrients and O2 (respiration) to generate energy (state 3 (added ADP)/state 4 (depleted ADP) respiration = RCI) and CRC, i.e. the capability of mitochondria to tolerate a challenge of 20 μM CaCl2 boluses every 90 s until mitochondrial bioenergetic failure. We found that with substrate glutamate/malate (GM), RCI with s‐RHR was 60% higher than its baseline and 2.3‐fold higher than after NS reperfusion. With substrate succinate (SC), RCI with s‐RHR reperfusion was 99% of its baseline and 2‐fold higher than NS. These changes were due mostly to the lower state 4 respiration rate in the s‐RHR group, indicating less proton leaking compared to other groups. Reperfusion lowered CRC by about 33% across all groups; however, with substrate GM, CRC after s‐RHR was 2‐fold larger than in the SS group; with substrate SC, CRC after s‐RHR was 3‐fold larger compared to other groups. Mitochondria likely play a critical role in control of hepatocyte function and viability during liver transplantation; thus preservation of mitochondrial function is critically important. Ex vivo subnormothermic‐regulated hepatic reperfusion best preserved mitochondrial function and viability. This novel strategy has potential applicability to human liver transplantation.

Support or Funding Information

MCW Institutional Funding

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Differential Expression of PPARγ and CHOP‐10 During Adipogenic Differentiation of Human Bone Marrow Derived Mesenchymal Stem Cells
Authors: Rony R M Imtiaz Karim , Hull Barbara E. , Excoffon Katherine , Ramirez‐Vick Jaime E
Published: June 2021

Mesenchymal stem cells (MSCs) hold great promise for cell‐based therapy and regenerative medicine owing to their multilineage differentiation potential that ranges from osteogenic to cardiac lineages. Osteogenic and adipogenic differentiation of MSCs in particular is of great interest since any imbalance that perturbs the regulation of osteogenesis and adipogenesis is typically associated with diseases such as obesity, osteoporosis, and other age‐related diseases. In our project, we sought to profile the gene expression pattern of key osteogenic and adipogenic transcription factors during differentiation using time course experiments. Adipogenesis was induced in passage 4 human bone marrow derived mesenchymal stem cells (BM‐MSCs) seeded with a density of 6579 cells/cm2 and RNA was collected at Day 0 (control), Day 3, Day 7, and Day 14 after differentiation to analyze the expression level of master adipogenic transcription factor peroxisome proliferator activated receptor‐γ (PPARγ) and negative adipogenic factor CHOP‐10. Our preliminary data suggests that compared to control (Day 0, undifferentiated) samples, PPARγ expression increases about 20‐fold, 22‐fold, and 168‐fold at Day 3, 7, 14 respectively. In contrast, expression of CHOP‐10, which is a negative regulator of adipogenesis, is upregulated approximately 2.4‐fold at Day 3, then downregulated 1.13‐fold at Day 7, and again increased 1.1‐fold at Day 14 respectively. Future work will compare these results and expression pattern of other adipogenic transcription factors to MSC differentiation in a 3D culture system.

Support or Funding Information

Funding for these projects are supported by Wright State University.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Analysis of experimental feed impacts on fatty acid profiles of captive and wild Arctic Ground Squirrels
Authors: Mikes Monica L , Rice Sarah A , Bibus Doug , Drew Kelly
Published: June 2021

Arctic Ground Squirrels (AGS) are widely studied in laboratories for their capability to hibernate for months without any bodily repercussions while dormant. Hibernation is the ability to decrease metabolism, core body temperature and activity to conserve energy. Despite being heavily studied, information on the impact of the laboratory diet, a basic component of their husbandry protocol, lacks in scientific research. Polyunsaturated fatty acids are renowned for their effect on hibernation. The increase of omega 6 PUFAs has been shown to improve captive hibernation (Geiser 1993, Frank 2008). In contrast, when omega 3s were given in abundance compared to omega 6s hibernation was inhibited (Florant 1999). No literature, to our knowledge, has tested feeding natural omega 3:6 ratios to captive AGS. Our preliminary data spotlights a significant difference in the ratio of omega 3 and omega 6 PUFA levels between wild and captive AGS. The captive diet does not represent the natural ratio of omega 3 to omega 6. We hypothesize that substituting captive AGS with a Test Diet comparable to the natural diet will mirror wild AGS fatty acid profiles. To test this hypothesis we fed a pilot study Test Diet comprised of a 1:1 ratio of omega 3 to omega 6s. The Test Diet significantly reduced some divergent fatty acid profiles between wild and captive AGS. Further work, however, needs to continue to adjust specific fatty acids in the Test Diet and measure the impact of the diet on hibernation.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
High Throughput Validation of Orphan Riboswitch Candidates
Authors: Arachchilage Gayan Mirihana , Sherlock Madeline E , White Neil , Panchapakesan Shanker SS , Breaker Ronald R
Published: June 2021

Riboswitches are cis‐acting gene controlling RNA elements often found in 5′ UTRs of bacterial mRNAs. The discovery of riboswitches and identification of their cognate ligands have led to the revelation of several important biological pathways and processes. At least 40 different riboswitch classes have been experimentally validated, but many more are waiting to be validated (orphan riboswitches). Computational approaches including comparative genomics have proven to be very successful in identifying riboswitch candidates with a high level of confidence. However, these riboswitch candidates need to be experimentally validated by identifying their cognate ligands which can be challenging and may take much more time. Our recent bioinformatics approach to discover rare riboswitch classes by focusing on GC‐rich, long intergenic regions (IGRs) in bacteria continues to add new riboswitch candidates to the growing collection of orphan riboswitches. Therefore, a high throughput and systematic method for the validation of orphan riboswitches is crucial to keep up with the pace of new riboswitch discovery by computational approaches. We are developing a higher throughput strategy to experimentally validate orphan riboswitches. This systematic process of validation includes using bioinformatics to update consensus structure models, biochemical investigation by in‐line probing, and genetic validation using reporter assays. For the initial investigation, we subjected five different orphan riboswitch candidates, SLH, chrB‐a, algC, aceE, and Bifido‐metK to this systemic validation process. We identified Bifido‐metK as the sixth class of riboswitches (SAM‐VI) that sense either S‐adenosylmethionine (SAM) or S‐adenosylhomocysteine (SAH), and the results from the other riboswitch candidates have provided valuable information about their structures and functions. Thus, our improved validation methods should help to expedite the experimental validation of riboswitch candidates and make the riboswitch discovery more productive.

Support or Funding Information

M.E.S. was supported by an NIH Cellular and Molecular Biology Training 13 Grant (T32GM007223). This work was also supported by NIH grants to R.B. (GM022778 and DE022340). R.R.B. is also supported by the Howard Hughes Medical Institute.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Reproducibility of Phosphoinositide Lipid Extraction & Presentation in Bioanalytical Assays
Authors: Branch Angie M. , Neilsen Paul O.
Published: June 2021

Phosphatidylinositol 4‐phosphate (PI4P) produced by Phosphatidylinositol 4‐Phosphate kinases is an abundant Golgi phosphoinositide functioning in lipid exchange and vesicle transport within mammalian cells. Researchers wanting to quantify PI4P use the Bligh and Dyer method for extracting and separating phospholipids in cells and biological tissues. This common lipid extraction procedure is known to have inherent variation. Using a human promyelocytic leukemia cell line and an ELISA that is sensitive and specific for PI4P, we examine sources of variation in bioassays including sample (HL‐60 cells), the extraction procedure, and lipid detection. We determined a PI4P Mass ELISA has low coefficient of variation (<10%) if the extracted sample volume is held constant and samples are prepped and lipids presented with standardized procedures. Thus, the organic solvent extraction process is the major source of variation in typical Bligh‐Dyer preparations.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The Use of Pharmacogenetics in Targeting SGLT2 and its Variants
Authors: Fassler Amy
Published: June 2021

When the contents of the bloodstream are filtered by the kidney, glucose is reabsorbed. Familial glycosuria, a benign disorder often attributed to genetic variants in the gene SLC5A2 which encodes the protein Sodium Glucose Transporter 2 (SGLT2), prevents reabsorption of glucose leading to an excess amount in urine. SGLT2 is used for glucose reabsorption, and when functioning properly, about 90% of glucose from the urine is reabsorbed into the bloodstream. Upon glucose and sodium binding, SGLT2 undergoes a conformational change, where the structure shifts from an outward to an inward facing orientation, allowing glucose to be released. Recently, two novel variants in SLC5A2 have been identified in the Marshfield Clinic patient population; further research is being conducted to determine whether these variants impact glucose transportation and how they affect physiological output. To research this, homology models of WT SGLT2 were constructed and analyzed based on a previously solved structure of a bacterial SGLT protein (vSGLT). It can be concluded that SGLT2's structure is composed of 14 transmembrane helices which surround both galactose/glucose and sodium binding sites. Recently, SGLT2 has become a popular target for Type II diabetes therapeutics. These drugs inhibit SGLT2, as they prevent glucose to be reabsorbed; thus, glucose is excreted out of the body. Further understanding how genetic variants effect SGLT2 structure and function may allow practitioners to properly prescribe medications and prevent adverse drug reactions when treating patients with Type II diabetes.

Support or Funding Information

Marshfield Clinic Research Foundation

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Constructing a 3‐D Molecular Model to Highlight the Conversion of the Normal Protein PrPC into the Mutated PrPSC in a Prion Disease
Authors: Schmitt Lavin Emily F. , Barraza Allan , Bui Hoang (Gwen) , Speth Robert
Published: June 2021

Gerstmann‐Straussler‐Scheinker (GSS) is a rare, genetically inherited prion disease caused by a mutation in the prion protein gene (PRNP). Patients who are carriers begin to experience GSS symptoms (similar to Parkinson's and Alzheimer's) during their 30s and 40s. This prion protein was modeled to reflect the changes in protein structure that leads to this disorder. While molecular details concerning the progression of this condition remain largely unknown, the alteration in shape of the prion protein could be directly involved in the aggregation of plaques contributing to the progression of GSS. The Milwaukee School of Engineering (MSOE) Center for Biomolecular Modeling ‐ Students Modeling A Research Topic (CBM‐SMART) team at Nova Southeastern University used 3‐D modeling and printing technology to examine structure and function relationships between this normal and mutated prion protein. Details of the normal GSS prion structure (PrPC) from the Protein Data Bank (PDB) File, 3HAK, were imported into Jmol, a protein visualization software. Next, the mutated form of the GSS prion protein, PrPSC (PDB file 3HER) was imported into Jmol. The differences between the two proteins were studied. Jmol commands were used to highlight these differences and a 3‐D molecular model was printed to explain the transition from normal to mutated protein. The transitions highlighted included a mutation in which phenylalanine becomes serine at positon 198, a loop from residues 193–197 which becomes deleted, and the alpha helix structure at residues 164 – 170 which becomes malformed due to the formation of a salt bridge between arginine 164 and aspartic acid 167. The N‐ and C‐ termini of the prion protein were also highlighted. This model can be used by genetic counselors, physicians, and researchers to educate the public on the molecular changes associated with prion diseases like GSS.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Hemolymph Glucose Levels as a Measure of Crayfish Stress: A Methodology Using a Human Glucometer
Authors: Caldari‐Torres Cristina , Banta‐Long Wendy , Bruss Andrew , Choi Edric , Fiegel Haley , Jollis Margot Sherman , Ly Stephanie , Viswanathan Sruthi
Published: June 2021

The objective of this study was to develop a methodology for measuring hemolymph glucose levels in crayfish of the genus Orconectes using a human glucometer. A secondary objective was to confirm through the use of this methodology that subjecting crayfish to acute stress in the form of a short‐term exposure to a basic solution or to chronic stress in the form of prolonged exposure to a nitrate‐containing solution, would increase hemolymph glucose levels. To measure the effects of acute exposure to a basic solution on crayfish hemolymph glucose levels, crayfish were placed in a beaker containing a basic solution (Ringer's buffer, pH=8) or a control solution (Ringer's buffer, pH=7) for a period of 10 seconds, after which time they were removed from the beakers and hemolymph was collected with a syringe. A ReliOn Prime glucometer (Wal‐Mart Stores, Inc.) was used to measured glucose levels (mg/dl) directly from the hemolymph. In order for the glucose levels to be within the sensitivity of the glucometer, hemolymph was mixed with high glucose Dulbecco's Modified Eagle Medium (DMEM; Gibco) in a 3:1 ratio. Glucose levels were determined by subtracting the levels of glucose measured in the DMEM solution from the glucose levels measured in the DMEM+hemolymph solution and correcting for the dilution factor. Crayfish acutely stressed through immersion in a basic solution had significantly higher hemolymph glucose levels compared to control crayfish (45.8 ± 5.9 vs 12.4 ± 3.8 mg/dl, two‐tailed t‐test; p<0.0001). To measure the effects of chronic exposure to a solution containing nitrate on crayfish hemolymph glucose levels, crayfish were housed in control tanks (no nitrate) or tanks containing 14 mg/L nitrate for one week, after which time hemolymph was drawn and glucose levels measured using the methodology described above. Crayfish chronically stressed through exposure to a nitrate‐containing solution had significantly higher hemolymph glucose levels compared to control crayfish (45 ± 14.3 vs 28.4 ± 8.8 mg/dl, two‐tailed t‐test; p=0.045). These results show that implementing this methodology human glucometers can be used to determine crayfish hemolymph glucose levels, making them an easy, inexpensive, and reliable alternative for this type of measurement. We were also able to show that hemolymph glucose levels are increased in both chronically and acutely stressed crayfish compared to control crayfish. These experiments can be used in physiology classrooms to demonstrate and discuss the role of the autonomic nervous system in an animal's response to stressful environmental conditions.

Support or Funding Information

This research was funded by the Denison University Department of Biology.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Engaging Students in Authentic Research in Lab‐based Courses Increases Student Competency in Applying the Scientific Method and Increases Collaboration Between Teaching and Research Faculty
Authors: Parks Lisa , Meitzen John
Published: June 2021

In response to our TH!NK program, designed to engage students in critical and creative thinking across the campus, and the need to provide more students with authentic research experiences, we have designed and integrated several course‐based research labs into our curriculum. These courses have allowed undergraduates to engage in meaningful research beyond the classroom without taxing the space, time, and resources of the current research faculty. Our newest cell biology lab, using cell culture, Western blots, and immunofluorescent chemistry allows students to learn advanced lab techniques and data analysis typically reserved for faculty research labs. Course‐based research allows student to receive credit toward their degree and allows them the opportunity to design and implement original experiments within a framework for potential publication. Since the development of these courses, students who have taken one or more of these labs have scored higher on post assessments related to applying scientific methods and scientific communication – a key departmental learning outcome ‐ than students who did not take these labs. (92% vs 74% competency on post‐assessments).

An added, and somewhat unforeseen benefit, has been the strengthening of the faculty learning community, particularly between teaching‐focused and research‐focused faculty. At many R1 institutions, the teaching mission has taken a backseat to the research‐dominated culture. As these labs have been developed, teaching and research faculty have engaged with each other ‐ becoming a more involved community that has led to increased team teaching, research projects, and publications. Teaching faculty has had another mechanism for staying current and engaged in research and literature, making them better instructors and giving them another outlet for potential scholarly work. Research faculty has had another mechanism for exploring side projects that they may not have had the time or funds to pursue in their labs. In some cases, research faculty have provided a one page proposal for a research project to pursue in class along with necessary protocols. This is modified for a student lab of up to 24 students working in groups of 4. We are hopeful that these labs will eventually increase in number to accommodate all students who wish to enroll in a research‐based lab course and that numbers of publications from undergraduates and collaborations between teaching and research faculty will increase.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Instruction Across an Educational Spectrum as a Tool for Building Teaching Skills in the Biomedical Sciences
Authors: Gwathmey TanYa M. , Howlett Allyn C.
Published: June 2021

The ability to convey knowledge to individuals at diverse levels of education is an important skill that is requisite for effective teaching, but often lacking in science educators. Post‐doctoral scholars supported by an NIGMS‐funded Institutional Research and Academic Career Development Award were first trained in current pedagogical techniques and educational philosophy through a semester‐long course, coupled with workshops on innovative and transformative instruction. These activities were essential in helping the scholars to develop skills necessary to teach in undergraduate, graduate, medical and allied health professional schools. However, in these settings, the audiences often possess a basic understanding of the foundational principles being presented, and may be capable of filling in “gaps” of information not presented by the instructor, in order to complete the learning process. In an effort to develop well‐trained teachers who are highly effective communicators of biomedical and health sciences, we developed an arrangement with several K‐12 institutions within the local area, where post‐doctoral teaching scholars provided instruction to students through the development of teaching modules that were modified for age‐appropriate instruction at high‐school, middle‐school and elementary‐school levels. These activities required scholars to communicate complex technological concepts to individuals who lacked the basic understanding of the relevant foundational principles being presented, and challenged the teaching scholars in their communication of scientific principles to lay‐person populations. Improvements in communication skills, augmentations in lecture style and testing strategies, as well as the ability to create an active learning environment were reassessed by the scholars' teaching mentors at the completion of these activities.

Support or Funding Information

K12‐GM102773

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Skeletal muscle non‐shivering thermogenesis in hibernating Arctic Ground Squirrels
Authors: Oliver Scott Ryan , Krishnan Jishnu , Rogers Jace , Hunstiger Moriah
Published: June 2021

All mammals depend on heat producing mechanisms to maintain homeostasis. Skeletal muscle shivering and brown adipose tissue (BAT) uncoupling through the expression of uncoupling protein 1 (UCP‐1) are the identified major sources of heat production, though recently, skeletal muscle has also been highlighted as a thermogenic source. Skeletal muscle nonshivering heat production occurs via sarcolipin (SLN) uncoupling of sarcoendoplasmic reticulum (SR) calcium ATPase (SERCA)‐mediated ATP hydrolysis. SLN may play a significant role in hibernating animals due to the wide temperature range that requires regulation. A thermoregulatory mechanism needs to be in place to suppress heat production during hibernation and also initiate rapid thermogenesis during arousal. We hypothesized that skeletal muscle uncoupling driven by SLN activity is a significant source of thermogenesis and inhibiting this pathway can alter the metabolic rate during hibernation in Arctic ground squirrels (AGS). Specifically, this work aimed to elucidate the role of skeletal muscle SLN uncoupling and its connection with BAT heat production in thermoregulation and metabolic control during temperature fluctuations. AGS were treated with inhibitors of BAT‐uncoupling or skeletal muscle‐uncoupling during hibernation to assess skeletal muscle and BAT ability to aid in heat production. Rate of temperature increase and changes in VO2 were compared to internal controls during inter‐bout arousals to quantify the effect of suppressing BAT or skeletal muscle uncoupling throughout hibernation. Results show that suppressing skeletal muscle uncoupling by SLN causes a significant reduction in metabolic rate during AGS rewarming. Interestingly, inhibition of BAT thermogenesis did not reduce metabolic rate. Inhibition of thermogenic sources highlighted the importance of SLN uncoupling and metabolism in thermogenesis and systemic thermoregulation. Thermogenesis control is directly linked to metabolic rate and energy loss regulation, and thus influencing thermoregulation processes has the potential for translation to innovative treatments for obesity and other diseases and conditions relevant to metabolism.

Support or Funding Information

Research reported in this publication was supported by an Institutional Development Award (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health under grant number P20GM103395. The content is solely the responsibility of the authors and does not necessarily reflect the official views of the NIH.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
A Research about Single‐Point Ligated Ischemic Heart Failure Rabbits in Physiology and Biomechanics
Authors: Zhao Dongliang , Huo Yunlong , Tan Wenchang
Published: June 2021

Objective

Animal models are important to study basic mechanism and biomechanics of ischemic heart failure (HF). Among a four heart chambers, the left ventricular (LV) plays the most significant mechanical part of pumping blood to the entire body. Biomechanical and morphological analyses can reflect the function of LV. The single‐point ligated HF model is a typical rabbit heart failure model, which can reveal the mechanism of heart failure. Left ventricular myofiber stress and strain analyzing is an effective biomechanical method in HF study. We aim at studying the physiological and biomechanical behave of the ischemic heart failure model.

Method

By single‐point ligation method, we establish the ischemic heart failure model. We ligate the left circumflex coronary arteries of 6 New Zealand white rabbits. The rabbits are scanned by Computed Tomography (CT), with short‐axis slice separated by 1.25 mm, after 2‐weeks and 4‐weeks. To determine regional stress and strain distributions in rabbit ventricular myocardium, an anatomically detailed nonlinear finite element model was used, which can solve the equations of stress equilibrium during passive filling of the left ventricle. At same time, the paraffin sections have be surveyed in pathology.

Result

The volume can be calculated by 3‐D reconstructed model, which range from 1528.33 mm3 (SD 286.23) at 2‐weeks to 1990.72 mm3 (SD 178.71) at 4‐weeks. The strain and stress get bigger from 2‐weeks to 4‐weeks. The huge change can be found at the pathology sections.

Conclusions

This research find the changing regulation of the strain and stress. Based on biomechanics and physiology, a new method for evaluating the function of the left ventricular is presented.

Support or Funding Information

This research is supported in part by the National Natural Science Foundation of China Grant 11372010(Y Huo); Shenzhen Science and Technology Innovation Institution (China) Grant JCYJ20160427170536358 (Y Huo).

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Exercise in a warm condition enhances plasma volume expansion compared to a cool condition with an increased skin but not core temperature
Authors: Okazaki Kazunobu , Takeda Ryosuke , Imai Daiki , Kawai Eriko , Ota Akemi , Hanno Genta , Morita Emiko , Yokoyama Hisayo
Published: June 2021

Plasma volume (PV) expansion is a fundamental physiological adaptation to exercise‐heat acclimation. We investigated the effects of an increased body core temperature and skin temperatures on PV expansion after intense exercise.

METHODS

Eight healthy young men underwent two identical trials which differs only ambient temperature (Ta) during exercise. On day 1, baseline blood sample was taken in a thermoneutral condition, then they performed a 72‐min intense‐intermittent exercise (8 sets of 4 min at 80% VO2max – 5 min at 20% VO2max) under cool (Ta of 20°C) and warm (Ta of 30°C) conditions. On day 2, after 23 hours after exercise, blood sample was taken again in the thermoneutral condition. We measured hematocrit, hemoglobin concentration, and plasma albumin and protein concentrations, and from these data, percent change in PV (Δ%PV) and in plasma albumin and total protein content (Δ%Albcont and Δ%TPcont, respectively) from baseline were determined. Esophageal temperature (Tes) and mean skin temperature (Tsk) were measured during exercise.

RESULTS

During exercise, Tsk were higher (p < 0.05) in warm than cool condition while Tes was comparable between trials. Δ%PV was higher in warm condition than cool condition (6.8 ± 1.3 % and 3.3 ± 1.3 %, means ± SE, p < 0.05), Δ%Albcont and Δ%TPcont were also higher in warm condition than cool condition (both, p < 0.05).

CONCLUSIONS

Exercise in a warm condition enhances plasma volume expansion with plasma protein content after intense exercise compared with a cool condition, which is associated with an increased skin temperatures but not core temperature. Thus, increase in skin temperatures would be a possible target to enhance physiological adaptation to exercise‐heat acclimation.

Support or Funding Information

This work was supported by a Grant‐in‐Aid for Scientific Research (B) and a Grant‐in‐Aid for Challenging Exploratory Research (K. Okazaki) from the Ministry of Education, Culture, Sports, Science and Technology of Japan.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.