bluuebunny/crossref_metadata_2025_split huggingface dataset:
Teleost eyes: The role of the developing lens in skeletal development
Authors: Dufton Megan M , Hall Brian K , Franz‐Odendaal Tamara 
Published: June 2021

The development of the eye is a complex process involving many signaling events. Previous research has suggested that the developing lens may influence the ocular skeleton in the Mexican tetra, Astyanax mexicanus, which exists as two morphs; one sighted and one blind. In sighted tetras, the sclera ossicles develop from a scleral cartilage template whereas in blind tetras only scleral cartilage is present. We ablated the lens in sighted tetras in order to understand the relationship between the developing lens and ocular skeleton. Two methods of surgical lens removal were used; manual removal with tungsten needles and laser removal. This resulted in one eyed fish. Histological and whole‐mount analyses of these one‐eyed fish suggests that the lens may have the capacity to regenerate, yet, surprisingly the ocular skeleton is still affected. In situ hybridizations with key genes involved in skeletal development within condensations and comparative analyses in zebrafish, Danio rerio, will also be presented. The capacity of a soft tissue to influence the skeleton in this way is intriguing. Funding provided by NSERC, Canada.Grant Funding SourceNSERC
Virtual Osteology: Developing the biological profile of the 3D Visible Human Male Skeleton
Authors: Ford Jonathan M , Decker Summer J , Hilbelink Don R 
Published: June 2021

Three‐dimensional (3D) imaging technology can be used to create accurate virtual computed models of anatomical structures for a wide range of educational and research activities. The purpose of this study was to produce a biological profile based on anthropological assessment of a 3D computed model of the skeleton of the Visible Human Male (VHM). Using previously validated methodologies, a virtual model of the skeleton was computed, measured and a biological profile developed and compared to that of the known information of the VHM.Computed 3D skeletal models were produced from the image data available from the Visible Human Project. Seventy eight anthropometric measurements were selected from an index of standard landmarks used in establishing a profile in skeletal analysis. Non‐metric traits used in the assessment of age, sex and ancestry were also examined. Once the anthropological analysis was complete, the profile (Caucasian male between 35‐45 years old, approximately 70 inches tall) was found to be consistent with the known information from the VHM. Using the virtual model, a life size replica of the skeleton was 3D printed, measured and its accuracy validated using previous results.As a result of the study, both a virtual and an accurate life size model of the VHM skeleton were produced. This resource can be utilized for teaching osteology to a wide variety of biological sciences such as anatomy and anthropology.Grant Funding SourceN/A
Temperature effects on solute transport: In vivo imaging of the growth plate
Authors: Serrat Maria A. , Williams Rebecca M. , Farnum Cornelia E. 
Published: June 2021

Determining how the environment modulates bone elongation is central to understanding skeletal variation. Ambient temperature and physical activity can alter limb length, but the mechanisms by which they do so remain elusive. Solute delivery to cartilaginous growth plates could play a key role since nutrient supply is critical to bone elongation. Advances in imaging technology have revolutionized our ability to visualize growth plates in vivo, but quantitative methods are still lacking. We developed standards for measuring solute delivery ‐‐defined by amount and entry rate of intravenous tracers‐‐ in tibial growth plates of 5 week old mice using multiphoton microscopy. Following IACUC protocols, we employed an acute temperature model (N=6/group) to test the hypothesis that solute delivery is increased or decreased by warm (36C) or cool (23C) limb temperature, holding other physiological parameters constant. Solute delivery differed as predicted with significantly more tracer in growth plates at 36C. These data validate the sensitivity of the method, which we are now using to assess chronic temperature and exercise effects on limb length and nutrient supply to the growth plate. These results are relevant to strategies for delivering therapeutic agents to growth plates of children and have the potential to greatly advance our understanding of skeletal variability in mammals. Supported by NIH‐RO1AR052003‐04.Grant Funding SourceNIH‐RO1AR052003‐04
Crystal structure of HsEg5 in complex with S‐trityl‐L‐cysteine
Authors: Parke Courtney Lane , Buckley Rebecca , Kim Sunyoung , Worthylake David 
Published: June 2021

Eg5, a motor kinesin involved in the formation of the bipolar mitotic spindle, is essential for the successful completion of mitosis. Monastrol and S‐trityl‐L‐cysteine (STC) are two well‐characterized inhibitors of Eg5, with STC being the more potent inhibitor. We have determined the 2.5 Åresolution crystal structure of the Eg5 motor domain in complex with STC and Mg ?ADP. STC interacts with Eg5 via a pocket formed by helices a2, a3 and loop L5, and induces conformational changes within the Eg5 necklinker and switch I and II regions similar to those seen with bound monastrol. However, there are two distinctions between the STC‐Eg5 and monastrol‐Eg5 structures: the nature of the interactions between the allosteric ligand and Eg5 and solvent accessibility of residues in the allosteric binding site. Monastrol contacts with Eg5 are largely mediated by nonpolar drug surfaces. During STC binding, contact between the motor domain and polar surfaces of the drug is increased in relation to that of monastrol by over 20 Å2. STC binding occludes ~80 Å2 more of the Eg5 surface from solvent access than does monastrol. Site‐directed mutagenesis and coupled biochemical assays monitoring ATP hydrolysis were used to test the hypothesis that several residues altered in solvent accessibility upon STC binding to Eg5 were critical to the increased efficacy of inhibition by this small molecule.Supported by Louisiana Board of Regents Grant
Alternative splicing of scleroderma‐associated lysyl hydroxylase 2 mRNA is regulated by Fox proteins
Authors: Seth Puneet , Walker Linda Clippard , Yeowell Heather Nina 
Published: June 2021

Scleroderma (SSc) is a complex skin disease with heterogeneous components. No treatment against scleroderma is available and many questions about its biology are unanswered. We focus on extracellular component of SSc and towards understanding the regulation of lysyl hydroxylase 2 (LH2) alternative splicing that plays a significant role in SSc biology.We report that Fox2 RNA binding protein regulates the splicing of SSc‐associated LH2(long) mRNA that includes an extra 63bp exon 13A (E13A). Using an LH2 minigene, we show that overexpression of Fox2 in skin A431 cells increased E13A inclusion in LH2(long) mRNA. As control, overexpression of the Fox2 RNA binding motif deletion mutant did not increase E13A inclusion. Knockdown of Fox2 with siRNAs in Murine Embryonic Fibroblast (MEFs) decreased E13A inclusion. Our data suggests that the Fox2 role in regulating LH2 alternative splicing is neither cell nor species specific. We also knocked down Fox2 in primary cells derived from SSc patients. This decreased the LH2(long) mRNA that is increased in SSc. There are 2 Fox2 motifs in each of the introns flanking E13A. We mutated the Fox2 motifs individually and in combination to determine the relative effect on LH2 splicing. Identification of regulatory factors that play roles in inclusion of E13A in SSc‐associated LH2(long) mRNA offers important insights to therapeutic approaches against SSc.
Decreased expression of microRNA miR‐133 but not miR‐1 is associated with impaired cardiac performance
Authors: Danowski Nina , Frey Ulrich , Kottenberg‐Assenmacher Eva , Jakob Heinz Günther , Manthey Iris , Siffert Winfried , Peters Jürgen 
Published: June 2021

MicroRNAs (miRs) post‐transcriptionally regulate gene expression. miR‐1 and miR‐133 can evoke opposing effects on apoptosis in cardiomyocytes with miR‐1 acting pro‐apoptotically and miR‐133 anti‐apoptotically. In 86 patients undergoing coronary artery bypass grafting (CABG), we tested the hypothesis that cardiac miR‐1 and miR‐133 expression are associated with altered cardiac performance. Cardiac index and vascular pressures were determined during general anesthesia and right atrial appendages were obtained during cannulation for extracorporal circulation. miR expression was quantified by RNase protection assay and by Real‐Time PCR.Decreased miR‐133 expression was significantly associated with diminished exercise performance as indicated by worsened NYHA functional class (p=0.0045), and higher pulmonary artery occlusion pressures (p=0.0042). Furthermore, patients with NT‐proBNP concentrations >1800 pg/ml showed a 60% decrease in miR‐133 expression compared to patients with concentrations <300 pg/ml (p=0.036). In contrast, no association could be detected between miR‐1 expression and variables of cardiac performance.This study shows for the first time a link between decreased miR‐133 expression and development of cardiac failure in CABG patients, possibly indicating a role of miR‐133 in regulating cell fate by inhibiting apoptosis.
Three‐dimensional Structure of a Prion‐remodeling Machine
Authors: Tsai Francis T. , Sielaff Bernhard , Lee Sukyeong 
Published: June 2021

Bacterial ClpB and its yeast ortholog Hsp104 are ATP‐dependent molecular machines, which have the remarkable ability to rescue proteins from a previously aggregated state. In addition to its role in thermotolerance, Hsp104 is also essential for the inheritance, maintenance and elimination of [PSI+], a yeast prion that increases translational read‐through of nonsense codons. The prion‐remodeling activity is unique to Hsp104 and, unlike its protein disaggregating function, is not shared with ClpB.The cryo‐EM structure of Hsp104 was determined recently and was used to generate a 3D model of the Hsp104 hexamer (Wendler et al., CELL 2007). Surprisingly, the proposed Hsp104 model differed dramatically from the 3D structure of ClpB (Lee et al., CELL 2003; MOL. CELL 2007). This of course has significant mechanistic implications, including substrate interaction and the disaggregation of previously aggregated proteins.To address this question we set‐out to determine the 3D structure of Hsp104 using innovative, multi‐pronged structural and biochemical approaches. Our new results suggest that ClpB and Hsp104 are more similar in structure than previously proposed. The mechanistic implication of our new structural insight will be discussed at this meeting.
New calpain inhibitor preserves brain architecture in 3‐nitropropionic acid (3‐NP) model of Huntington disease
Authors: Kesner Leo , Yang Lichuan , Beal M. Flint , Stracher Alfred , Hassen Getaw Worku , Shulman Abraham 
Published: June 2021

The 3‐nitropropionic acid model Of Huntington's disease produces a variety of biochemical anomalies. One of these, is the activation of the protease calpain. Excess calpain activity is probably responsible for major cell death expressed as striatial degeneration. Protease inhibitors designed to reduce damage from calpain should have qualities such as the ability to cross the blood‐brain barrier, have some selective targeting ability to nervous tissue and be non‐toxic in the concentrations used. We have designed a new inhibitor (Gabadur) with many of these qualities. The targeting end is an analog of the anti‐epileptic compound "pregabalin" and the calpain inhibitor end is argininal, similar to leupeptin.Twelve week old Lewis rats were treated with 3‐nitropropionic acid (3NP) 50mg/kg/day for 5 days. In addition some of these rats were treated with either saline or Gabadur (10mg/rat/day IP). Brain slices taken from these rats, and treated only with saline, showed focal degeneration and necrosis with complete loss of architecture. Brains from Gabadur treated rats showed about 50% preservation of the architecture. Calpain activity was similarly depressed, as measured by Western blot techniques. Alpha‐2 Spectrin degradation products were almost normal in Gabadur treated rats, but increased three fold in the saline treated rats.
Hepatic gene expression and proteomic profile of mice with nonalcoholic fatty liver disease due to high fat diet
Authors: Kirpich Irina , Cave Matt , Deaciuc Ion , McClain Craig 
Published: June 2021

IntroductionAlthough nonalcoholic fatty liver disease (NAFLD) is the most common chronic liver disease, the responsible molecular mechanisms are incompletely understood. Here, we performed hepatic gene expression and proteomic analysis in mice with NAFLD due to a high saturated fat diet. Materials and Methods: Male C57/BL6 mice were fed 60% lard or control diet for 8 weeks. Those on the high fat diet developed hepatic steatosis. Hepatic gene expression was analyzed by microarray technology, and the proteome was analyzed by two‐dimensional difference gel electrophoresis with mass spectrometry.ResultsCompared to control diet, high fat diet changed the expression of 473 genes (256 up‐ and 217 down‐regulated) by 2‐fold or greater. Multiple genes encoding proteins involved in fatty acid oxidation were up‐regulated. Several genes encoding extracellular matrix proteins, adhesion molecules, and xenobiotic metabolizing enzymes were down‐regulated. Proteomic analysis revealed eleven proteins which were differentially expressed. Of these, glutathione S‐transferase mu1 and pi, and selenium binding protein 2 were decreased on both gene chip and proteomic analysis.ConclusionNAFLD due to a high saturated fat diet in mice is associated with specific gene expression and proteomic changes. This work was supported by the NIH (IVD, CJM), the AASLD (MC) and the VA (CJM).
Science in the Cinema Summer Film Festival
Authors: Fuchs Bruce A , Knorr Debra 
Published: June 2021

Science in the Cinema is a free summer film festival for the public sponsored by the National Institutes of Health (NIH) Office of Science Education (OSE). It is intended to promote public understanding of science and medicine and increase visibility of the 27 institutes and centers that comprise the NIH.The program was launched on the NIH campus in 1994, and was later relocated to the American Film Institute Silver Theatre in Silver Spring, Maryland. Once a week for six weeks, a film with a medical science theme is screened. The evening begins with brief remarks from the series host. Following the screening, a guest speaker provides commentary on the topic, identifies factual and fictionalized elements of the film, and answers questions from the audience. More than 35,000 Washington, DC area residents have attended Science in the Cinema since its inception.At the end of each program, attendees complete a questionnaire to capture their thoughts about how the film impacted their knowledge, addressed misconceptions, increased knowledge of the NIH, and demographics (optional).The festival includes feature‐length films that include recent releases such as The Diving Bell and the Butterfly (2007) and classics such as The Quiet Duel (1949). Films are shown so that they are accessible to persons with disabilities. A detailed list of films shown in the series and speakers can be found at the OSE Web site: http://science.education.nih.gov/cinema.
A novel approach to bioremediation of chromium using bacterial strains resistant to oxidative stress
Authors: Frederick Tamlyn , Woodruff Peter 
Published: June 2021

Bioremediation is a process by which microorganisms reduce or prevent the negative effects of pollutants and contaminants in the environment. It has emerged as an effective and relatively inexpensive method utilized to clean up toxic waste sites and polluted environments. Given its powerful applications, an important focus in this field is the genetic engineering of microorganisms to increase the rate of bioremediation. Many naturally occurring bacteria have been found to detoxify the environment by reducing Chromium (VI) to Chromium (III). Chromium (VI) is a carcinogen that enters the environment as a byproduct from factories and industrial waste sites. Reducing this contaminant from the hexavalent form to its trivalent form makes its less soluble in water and less likely to contaminate water supplies. A problematic byproduct of the reduction process is the generation of reactive oxygen species. Therefore, we hypothesize that protecting bacteria involved in bioremediation from this oxidative stress will improve their capacity for bioremediation. The aim of this research is to genetically engineer bacteria such as E. coli that have improved resistance to the oxidative stress associated with chromium (VI) reduction. These heightened defenses against oxidative stress should increase viability of bacteria and improve bioremediation of Chromium (VI).This research was supported by a Faculty Research Award from Bowdoin College (PW) and a Howard Hughes Medical Institute fellowship (TF)
Notch/RBP‐J signaling is necessary for astrogliogenesis and the expression of astrogliogenic genes NFIA and Sox9
Authors: Janssens Derek , Johnson William , Sarah Sarala , Van derKlay Tiffany , Taylor Merritt 
Published: June 2021

As neural stem cells (NSC) divide they can give rise to three classes of cells: neurons, glia and more stem cells (a process called self renewal). During development NSCs first primarily give rise to neurons (neurogenesis) and later give rise to glia (gliogenesis). In early development the Notch/RBP‐J signaling pathway allows NSCs to self renew by suppressing neuronal differentiation. As development progresses, Notch/RBP‐J signaling regulates the differentiation of NSCs into glia. Although it is known that Notch regulates self renewal through the expression of the Hes genes, the mechanism for Notch‐dependent gliogenesis has not been characterized.We are determining if Notch signaling controls the expression of the astrogliogenic genes NFIA and Sox9. We have identified putative binding sites for RBP‐J, a transcriptional co‐regulator of canonical Notch signaling, in the promoter regions of both genes. Using quantitative PCR and immunofluorescence techniques we found that disrupting Notch signaling in neural progenitors of the developing CNS by deletion of the gene for RBP‐J (called Nestin Cre Rbpsuh fl/fl mice) causes a significant decrease in both NFIA and Sox9 expression. This demonstrates that Notch/RBP‐J signaling is necessary for the expression of these astrogliogenic genes, and suggests that Notch employs different signaling pathways for NSC self‐renewal and for gliogenesis.
Biochemical and structural characterization of innate immunity kinase complex proteins
Authors: Call R. Jason , Ghirlando Rodolfo , Bell Jessica K. 
Published: June 2021

Protein‐protein interactions form the communication link from membrane bound receptors to their downstream cellular targets.ObjectiveOur long‐term goal is to examine the protein‐protein interactions responsible for the activation and regulation of the Toll‐like receptor 3 (TLR3) signaling cascade that leads to type I interferon production. In the current study we have targeted the kinase scaffold proteins, TRAF family member‐associated NF‐κB activator (TANK) and NAK‐associated protein 1 (NAP1).MethodsBacterial expression systems have been used to produce recombinant target protein and refolding methods developed for insoluble targets. Initial biochemical characterization of recombinant targets was completed using circular dichroism (CD), gel filtration (GF) and analytical ultracentrifugation (AUC).ResultsThe protein domain comprising the N‐terminal region of NAP1 can be successfully refolded as assessed by CD, while TANK is soluble. Both constructs form homo‐oligomers as determined by GF. AUC experiments show that the NAP1 N‐term behaves as an elongated dimer in vitro. Gel filtration studies of TANK's kinase interaction site show that it elutes as a dimer species.ConclusionsThe scaffold proteins, TANK and NAP1, form stable homo‐oligomers via their N‐terminal regions. Funding from the Am. Cancer Society (RJC) and supported in part by the NIH intramural research program, NIDDK (RG).
Effects of Ziram on Human Natural Killer Cells MAP kinases p38 and p44/42
Authors: Taylor Thyneice Rochelle , Whalen Margaret M 
Published: June 2021

Human natural killer cells (NK) are lymphocytes that can recognize and kill virally infected cells, antibody coated cells, and tumor cells without any prior sensitization. Ziram is used as a fungicide in agriculture and as an accelerating agent in the production of latex. Previous studies have shown that ziram inhibits the ability human NK cells to lyse tumor target cells. Mitogen activated proteins kinases (MAPK) are important signaling enzymes in NK cells. Exposure of NK cells to another environmental contaminant, which also blocks NK lytic function, tributyltin (TBT), induced phosphorylation (activation) of MAPK. Thus, disturbances in MAPK activation state could contribute to TBT (and possibly ziram) ‐induced loss of lytic function. The current study examines whether ziram causes MAPK activation. The MAPKs, p44/42, p38 and JNK were examined. A 10 minute exposure to 10 µM or 5 µM ziram caused a significant increases in phosphorylation of p44/42 (3 fold and 2 fold, respectively). A 10 minute exposure to 10 µM, 5 µM, or 2.5 µM ziram caused a significant increase in phosphorylation of p38 (4 fold, 3 fold, and 2 fold, respectively. These results suggest that MAPK activation may be part of the mechanism by which ziram inhibits NK lytic function, and that MAPK activation may be an effect that is shared by several environmental contaminants that have been shown to decrease NK lytic function.Supported by NIH grant S06 GM008092‐33.
The branched chain fatty acid iso‐14:0 is metabolized by Caco‐2 cells
Authors: Ran‐Ressler Rinat Rivka , Glahn Raymond P , Brenna James Thomas 
Published: June 2021

Background & ObjectiveWe recently showed differences in the distribution of branched‐chain fatty acids (BCFA) in the vernix and meconium of healthy newborns: BCFA with < 16 carbons were detected in vernix but not in meconium. The aim of this study is to test the hypothesis that iso14:0 is taken up and metabolized by the enterocyte, using the colon carcinoma (Caco‐2) cell culture model.MethodCaco‐2 cells were grown in 6 trans‐membrane well plates with 0.4µ inserts in appropriate media. Graded levels of iso14:0 (0.0‐0.5mM) were delivered to the cells as micelles in serum free media. After 4 hours incubation, apical and basolateral media were collected and cells were harvested to analyze the lipid classes and fatty acids.ResultsMicelle iso14:0 taken up by Caco‐2 cells and incorporated into phospholipids and monoglyceride lipid fractions. The BCFA iso12:0 and iso16:0 were detected in treated cells but not in controls. In the 0.5mM treatment, iso14:0 was found in the basolateral media.ConclusionCaco‐2 cells take up iso14:0 and incorporate it into to phospholipids. iso‐14:0 is also oxidized and elongated by the cells.SupportNIH grant GM71534.