bluuebunny/crossref_metadata_2025 huggingface dataset:
Comprehensive proteome profile of the zebrafish (Danio rerio) gill: applications to physiological and biomarker discovery studies
Authors: Goss Greg G , MacCormack Tyson , DeSouza Andrea , Li Liang
Published: June 2021

Zebrafish are an important model in vertebrate genetics, developmental biology, physiology, and toxicology. In this study, we established the first near comprehensive proteome profile of a teleost fish tissue using two‐dimensional liquid chromatography electrospray ionization tandem mass spectrometry. Coverage was significantly improved with the application of novel sequential solubilization and precursor ion exclusion methods. Six thousand one hundred seventy‐one proteins were identified and the proteome exhibited excellent coverage of important biochemical pathways relevant to the function of the gill in respiration, ion and acid‐base homeostasis, and energy metabolism. Numerous established and potential biomarkers of stress, disease, and environmental contamination were also expressed in the gill. Annotation information was completely lacking for greater than 30% of the expressed proteins, highlighting the need for advancements in bioinformatic analysis techniques to complement this research. Nevertheless, the results provide important insights into the physiological function of the gill as well as its role as an environmental interface. We discuss the significance of these findings in the context of exploratory physiological and toxicological studies. This research was supported by the Alberta Ingenuity Centre for Water Research.
A project based introduction to neuroscience laboratory course as a model for experiential learning in science
Authors: Barney Christopher C. , Chase Leah A. , Fraley Gregory S.
Published: June 2021

Important goals of undergraduate science courses are to provide students with experiences in thinking and working like a scientist. We used a project based laboratory in an Introduction to Neuroscience course to help achieve these goals. In this course the students carried out eight independent lab exercises and a seven‐week project aimed at demonstrating the effects of gonadal steroid hormones on the brain and behavior of Long Evans rats. Students performed ovariectomies/hormone implants on the rats, examined sexual behavior, and studied spatial learning using the Morris Water Maze. The students then investigated the effects of the treatments on the expression of LTP in hippocampal slices, the size of the sexually dimorphic nucleus, and the phosphorylation of NMDA receptor in different brain regions of these rats. Students utilized statistical techniques to test their hypotheses and were required to use the scientific literature in this field. Student work was evaluated by lab notebook content, weekly lab reports and a final manuscript‐style report that detailed their findings from this project. Students reported significant gains in posing questions and using evidence from one or more disciplines, in determining what valid scientific evidence is, and in communicating their results. The project also had a strong impact on the ability of the students to utilize the scientific literature. Supported by HHMI.
Teaching Ethical, Experimental, and Quantitative (TEEQ) Biology through Problem‐Based Writing with Peer Review
Authors: Stanley Jennifer Anne , Sinak Kelly Marie , Pelaez Nancy
Published: June 2021

Teaching Ethical, Experimental, and Quantitative (TEEQ) Biology is a project to enhance undergraduate students' understanding of bioscience research and thereby foster development necessary for entrance into dynamic careers. The target for this project is Biology 131, a large‐enrollment lecture course on organismal biology that serves biology majors. TEEQ engages students in critical review of scientific research by using the Calibrated Peer Review (CPR) program to structure writing assignments about experimental methods and quantitative approaches in biology. CPR delivers reading background information and a writing assignment that is followed by anonymous peer‐ and self‐ review of the assignments. Student gains were measured both qualitatively and quantitatively. The students completed a Participant Perception Inventory (PPI) to survey their knowledge, experience, and confidence about ethical, experimental, and quantitative aspects of research. Results pointed the way for improving students' confidence in their abilities to understand a historical continuum of changing theories, views, and methodologies in biology. The project also opened opportunities for faculty to interact with students in a way that should lead to better retention of students in the major and to students who are better prepared to make decisions about bioscience research and careers.
APS Undergraduate Summer Research Fellowship program: exposing undergraduates to physiology research leads to a research‐focused career
Authors: Lowy Melinda E. , McHugh Nansie A. , Galey William R. , Matyas Marsha Lakes
Published: June 2021

The APS Undergraduate Summer Research Fellowship (UGSRF) program seeks to excite and encourage undergraduate (UG) students worldwide to pursue a career as a basic research scientist. The program has 5 objectives: develop a research project, collect/analyze data, write up results; give a presentation on results at a scientific meeting; attend a national meeting; commit to a research career; and enroll in a graduate/professional program in basic biomedical research.

Since the UGRSF program began in 2000, 109 undergraduates have participated. Most students (92%) attended EB meeting and made 99 presentations, with some students having more than one abstract or presentation. Over 35% of the students published a paper on their APS‐funded research.

From surveys 2 years post‐Fellowship, 85% of the UGSRFs had graduated. Of these, 46% applied to graduate school (PhD or Master's programs) and 74% were accepted. As well, 58% applied to a science‐related professional school (MD, etc.) and 75% were accepted. At the time of the survey, most (78%) were already enrolled in a graduate or professional degree program.

In 2007 and 2008, the program added an online interactive component where UGSRFs discussed professional networking, structure of research studies, and career options in biomedicine. Results indicated the online interaction provided a powerful tool for UG students to explore critical professional development topics.
Defect in Vascular Endothelial Growth Factor Expression Leads to Vessel Loss in the Lungs of Mitogen‐Activated Protein Kinase Phosphatase‐1 Deficient Mice
Authors: DAS MITA , Shields Kristin M , Burns Nana , Zawada Wojciech M
Published: June 2021

Role of MAP kinase phosphatase‐1 (MKP‐1), a critical regulator of neovascularization and angiogenesis, in the regulation of vascular endothelial growth factor (VEGF) expression is unknown. We tested the hypothesis that VEGF expression and subsequent vessel development in the lung are regulated by MKP‐1 using MKP‐1 deficient (MKP‐1−/−) and wilt‐type (MKP‐1+/+) mice. Animals (4‐5 weeks old) were exposed to sea level (SL), Denver altitude (DA; 5280 feet) and severe altitude (HYP; 17,000 feet) for 6 weeks. Substrate of MKP‐1, p38 MAP kinase levels were greatest in vessels, respiratory epithelium, and interstitial cells of the lungs of MKP‐1−/− mice. Smooth muscle actin expression was increased in the lungs of MKP‐1 null mice. Vessel (<50µm in diameter) numbers as measured by vessel to alveoli ratio was markedly decreased whereas wall thickness of the vessel was augmented in hypoxia‐exposed lungs of MKP‐1 deficient mice. Immunoreactivities against anti‐VEGF as well as anti‐Ki67 antibodies were greatest in the lungs of MKP‐1+/+ mice. To evaluate the mechanisms of hypoxia‐induced loss of vessel in the lungs of MKP‐1 deficient mice, embryonic fibroblasts (MEFs) were isolated and VEGF level was evaluated by ELISA. Hypoxia‐stimulated increase in VEGF expression (8‐fold) was maximally inhibited by the JNK1/2 antagonist (SP600125) in wild‐type MEFs. However, VEGF expression was upregulated by < 1‐fold only upon exposure to hypoxia in MKP‐1 deficient MEFs. Our data strongly suggest that MKP‐1 plays a critical role in lung vessel development through the regulation of hypoxia‐stimulated VEGF expression.
Anti‐proliferative Properties of novel D‐Peptide‐VEGF‐Antagonists: Plausible Role in Anti‐angiogenic and Anti‐tumor Formation
Authors: Rajashekhar Gangaraju , Renwick Abby , Cook Todd , Vemula Sasidhar , Kapur Rueben , Schürmann Gregor , Wagner Peter , Clauss Matthias
Published: June 2021

Interfering with VEGF binding to its endothelial receptor, VEGFR‐2 inhibits angiogenesis in tumors. Existing anti‐angiogenic peptide candidates, which efficiently compete with VEGF in binding of the VEGFR‐2 have been shown to demonstrate immunogenic and thromboembolic complications in clinical trials. D‐peptides against VEGF rather than the natural L‐form have the potential to overcome these problems faced by existing antibody‐based therapeutics. In this study, we attempted to screen for such a candidate peptide which has both antiangiogenic and anti‐tumor properties. A number of peptides were initially screened for anti‐proliferative capacity, a key step in angiogenesis using HUVEC cells both by coulter counter measurements and further confirmed with thymidine incorporation assays. Two candidate peptides were found to be dose dependently decreased the HUVEC proliferation in the presence of 1nM VEGF. When compared to Bevacizumab at 166nM, 07‐81‐1 and 07‐82‐1 at 30nM demonstrated a significant 35% and 27% reduction in proliferation respectively. Furthermore, these two peptides demonstrated a significant (p<0.05) inhibition at 300nM in an in vitro capillary sprout angiogenesis assay. Pending the analysis of these two candidate peptides in vivo for anti‐tumor properties, we will discuss the outcome in light of the fact that these peptides could be used to improve antiangiogenic tumor therapy.
Lymphangiogenesis in the heart
Authors: Watanabe Michiko , Karunamuni Ganga , Yang Ke , Doughman Yong Qiu , Wikenheiser Jamie , Bader David , Barnett Joey , Austin Anita , Parsons‐Wingerter Patricia
Published: June 2021

Our goal was to follow the development of the lymphatic system in the developing heart using markers that identify the lymphatics in adult tissues. The transcription factor Prox‐1 identified a branching network of vessels in the avian embryonic heart that first appeared on the outflow tract at Embryonic Day 5 (Hamburger and Hamilton Stage 25‐26). Prox‐1 was also expressed in cardiac myocytes and cardiac valves throughout development. In the mouse embryo heart, the lymphatic marker LYVE‐1 was expressed in epicardial cells and PECAM‐positive/VEGFR‐3‐positive myocardial vessels that were Prox‐1‐negative. LYVE‐1 co‐localization with Prox‐1 was not observed in the epicardial lymphatics until after ED 13.5. Lymphatic markers were also present in adult rat, embryonic mouse, and embryonic avian epicardial cell lines. According to Western Blot analysis, Prox‐1 has two isoforms in the embryonic heart, including one that is sensitive to phosphatases. Our data are consistent with the hypothesis that the cardiac lymphatics arise from at least two sources: they migrate into the embryonic heart and may also originate from epicardial precursors. Source of Research Support: AHA 0715153B and grants from the National Institutes of Health R01 ES013507, HL65314, HL0775436, HL083048, and NASA GRC IR&D 04‐54.
Lipid raft‐associated signaling pathways are involved in polychlorinated biphenyls‐induced disruption of tight junction in human brain endothelium
Authors: Eum Sung Yong , Choi Yean Jung , Radakovic Masa , Andras Ibolya E , Hennig Bernhard , Toborek Michal
Published: June 2021

Polychlorinated biphenyl (PCB) congeners exhibit a broad range of adverse biological effects including neurotoxicity. The blood‐brain barrier (BBB) is a physical and metabolic barrier separating brain microenvironment from the peripheral circulation and is mainly composed of endothelial cells connected by tight junctions (TJs). In the present study, we have explored whether lipid rafts‐associated signaling can contribute to 2,2',4,4',5,5′‐hexachloro‐biphenyl (PCB153)‐induced alternations of TJ proteins, such as zonula occludens‐1 (ZO‐1) and occludin, in human brain endothelial cells. Lipid rafts‐disrupting agent, methyl‐beta‐cyclodextrin (MβCD), efficiently blocked PCB153‐induced disruption of TJ proteins. Similar effects were mediated by inhibition of lipid raft‐related signaling molecules, such as matrix metalloprotease‐2 (MMP‐2), the Src family kinase, or EGFR. In addition, pretreatment of HBMEC with MβCD attenuated PCB153‐induced activation of Src kinase. These data suggest that lipid rafts‐mediated activation of EGFR, Src kinases and MMP‐2 may be involved in PCB‐induced disruption of the BBB integrity and thus contribute to the development of neurotoxic effects of these compounds. Supported in part by P42 ES 07380, MH63022, MH072567, and NS39254.
Component analysis of change in fluid shear stress (Δτ) stimulus and basal capillary hydraulic conductivity (Lp)
Authors: Williams Donna A. , Flood Mary H.
Published: June 2021

One δτ stimulus consists of two components: flow increase and flow plateau. Previously, we reported lower Lp following acute, ramp versus square wave δτ, consistent with rate sensing capability (Microvasc Res 66:2003). Here, basal capillary Lp following δτ was evaluated in terms of rate and magnitude of change. We hypothesized that rate and magnitude of δτ would correlate with Lp. Frogs (Rana pipiens, N=26) were pithed and the mesentery was exteriorized and superfused with frog Ringer's (14‐16°C). Either one or two true capillaries/frog were cannulated at 10 cm H2O and perfused with 10 mg.ml−1 BSA dissolved in Ringer's. After 2 min of low flow each capillary was stimulated with ramp δτ (steady infusion pump) and a plateau was established (2 min). Lp was assessed at 30 cm H2O using the modified Landis technique. Range for acceleration (a) was 27.1 to 202.6 μm▸s−2 and 9.5 to 82.6 dynes▸cm−2 for δτ. Mean±SE Lp was 3.1±0.2 x 10−7 cm.s−1.cm H2O−1 (n=31). Lp correlated negatively with a (slope=‐0.020; R2=0.45; P=0.0002) and positively with magnitude of δτ (slope=0.048, R2=0.37, P=0.0003). δτ /a strengthened the relationship (slope=2.95, R2=0.63, and P<0.0001). These data are consistent with a transducer/signaling mechanism in the capillary wall that senses both components of δτ, integrates them, and adjusts Lp accordingly. Both rate and magnitude of change in blood flow likely impact tissue hydration. Supported by RO1 HL63125
Cardiac Birth Defects Caused by Lifestyle and Their Potential Prevention by Nutritional Molecules
Authors: Memon Samreen , Pratten Margaret Kathryn
Published: June 2021

Heart defects are responsible for many neonatal deaths. Changing life styles, (exposure to retinoic acid, excessive alcohol or nicotine) could be a cause of congenital malformations. Supplementation with vitamins during the periconceptional period has been shown to prevent neural tube defects. In this study embryonic hearts were dissected from 5 day old White Leghorn chick embryos and the isolated cells cultured in DMEM medium. The cells were exposed to 100µl/ml ethanol or 10µM nicotine only, or with the addition of 100µM vitamin C. Similarly, cells were exposed to 10µM retinoic acid, or retinoic acid plus 1mM folic acid. Cultures were incubated at 37oC in 5% CO2 in air, and observations made for cardiomyocyte differentiation at 24, 48, and 144 hours, cell viability assessed using the resazurin reduction assay and cell number by the kenacid blue assay. Statistical analyses were via ANOVA and Kruskal Wallis tests. Ethanol and retinoic acid dramatically reduced differentiation, viability and protein content (p <0.01). Vitamin C administered concurrently with ethanol, or folic acid with retinoic acid, could significantly improve all parameters to those comparable with the control. Nicotine only had an effect on the rate of beating, this being reversed by the addition of Vitamin C. It may be that environmentally induced teratogenic effects could be prevented by vitamin supplementation during pregnancy.

Funded by Government of Pakistan
Mesenchymal regulation of vascular invasion and matrix remodeling during intramembranous ossification
Authors: Butcher Kristin D. , Jheon Andrew H. , Smith Francis J. , Schneider Richard A.
Published: June 2021

With the goal of devising new treatments for skeletal disease and injury, there is much need to discover mechanisms through which mesenchyme makes bone. Many studies have pinpointed molecular factors that induce the osteogenic differentiation of mesenchyme in vitro, but given that a clinical objective is to engineer mesenchyme for applications in vivo, more work is required to identify critical interactions between osteogenic mesenchyme and surrounding tissues. We investigate the extent to which mesenchyme regulates vascular invasion and matrix remodeling. In the developing jaws and face, all bone comes from neural crest mesenchyme (NCM) whereas angioblasts and osteoclasts arise from mesoderm. We take advantage of these distinct embryonic origins and transplant faster developing quail NCM into slower developing duck embryos. Previously, using this quail‐duck chimeric system, we have shown that NCM makes bone by executing autonomous molecular and histogenic programs. Here, we assess the effects of NCM on host‐derived blood vessels and osteoclasts using gene expression, histology, and whole‐mount staining analyses. We find that quail donor‐NCM induces premature vascular invasion by the duck host during ossification but not at earlier stages. NCM also influences osteoclast activity. We conclude that NCM plays an important role in synchronizing the timing of vascular invasion and matrix remodeling.
Analyses of the craniofacial skeleton of a zebrafish lens degeneration mutant
Authors: Franz‐Odendaal Tamara Anne
Published: June 2021

The zebrafish mutant, bumper, is characterized as having a lens defect, which begins at 4dpf. Previous work has shown that in the blind morph of Astyanax mexicanus, which also has a lens defect, the ocular skeleton is affected. The ocular skeleton of sighted Astyanax mexicanus and wildtype Danio rerio consists of two scleral ossicles joined by scleral cartilage. In the blind tetra morph, however, the scleral cartilage is persistent and is not replaced by bone. In this study, homozygous and heterozygous bumper mutants of Danio rerio were analyzed. Whole‐mount analyses show that the ocular skeleton is unaffected in this mutant, unlike in blind tetras. Surprisingly, however, several other regions of the craniofacial skeleton are affected; these include the jaws and skull vault. The significance of these results is discussed with respect to the nature and timing of eye degeneration in the two species analyzed. Funding: NSERC, Canada

Grant Funding Source

NSERC, Canada
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 Source

NSERC
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 Source

N/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 Source

NIH‐RO1AR052003‐04
Yeast High Mobility Group Protein HMO2, a subunit of the chromatin remodeling complex INO80, binds preferentially to DNA ends
Authors: Ray Sreerupa , Grove Anne
Published: June 2021

High Mobility Group Proteins (HMG) are non‐histone chromosomal proteins which are common in eukaryotes. They bind to DNA in a non sequence specific manner. HMO2 from Saccharomyces cerevisiae is an HMGB protein that is a component of the DNA‐damage‐dependent chromatin remodeling complex INO80, which is involved in double strand break repair. The gene encoding HMO2 was amplified from yeast genomic DNA. It was expressed in E. coli Rosetta Blue cells and purified using nickel affinity chromatography. DNA binding assays indicate that HMO2 binds to supercoiled DNA with higher affinity than linear DNA and that it can restrain DNA supercoils. Like other HMGB proteins it has a preference for distorted DNA such as four‐way junctions (4WJs) and DNA with loops and abasic sites. Analysis of DNA constructs of increasing length suggests that HMO2 may bind distorted DNA as a dimer. DNA end‐joining and exonuclease protection assays show that it binds preferentially to the DNA ends, both blunt and cohesive ends, and that the sequence of the single‐stranded overhangs plays in DNA end‐binding. Since HMO2 has been reported to recruit the INO80 complex to the region of a double strand break, and since it effectively protects DNA ends against exonucleolytic cleavage, it is possible that it contributes to DNA repair beyond its role in INO80 recruitment.
Regulation of intrachromosomal homologous recombination in human cells by BLM helicase
Authors: Waldman Alan S. , Waldman Barbara Criscuolo , Wang Yibin
Published: June 2021

Mutation of BLM helicase causes Blooms syndrome, an autosomal recessive disorder associated with genome instability and predisposition to cancer. In vitro studies of BLM have suggested that BLM may modulate homologous recombination (HR). We studied the influence of BLM on intrachromosomal HR events by stably transfecting normal human fibroblast cell line GM637 with pLB4 which contains a thymidine kinase (tk)‐neo fusion gene disrupted by insertion of the 18 bp recognition site for endonuclease I‐SceI. Also contained on pLB4 is a functional tk gene. A double‐strand break (DSB) was introduced within the tk‐neo gene by expression of I‐SceI endonuclease. DSB repair events were recovered by selection for cells that gained resistance to G418 either via HR or by nonhomologous end‐joining (NHEJ). When DSB repair was examined following transfection of siRNA directed against BLM expression, 89 out of 123 HR events recovered had resolved as crossovers. In controls, 43 out of 123 HR events were crossovers. This difference was highly statistically significant (p=5.8 X 10−9 by a Fisher exact test). Our data also suggested that a greater portion of DSB repair occurred by HR (versus NHEJ) when BLM expression was knocked down. Our work provides the first direct evidence that BLM deficiency may contribute to genome instability by promoting the resolution of bona fide HR events as crossovers. Supported by NIH grant R01GM081472 to ASW.
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
Regulation of Cpt1b Gene Transcription in liver and muscle cell lines
Authors: Liu Li , Park Edwards A. , Cook George A.
Published: June 2021

Regulation of mitochondrial fatty acid oxidation resides at the outer membrane enzyme carnitine palmitoyltransferase‐1 (Cpt1). The Cpt1b isoform is expressed in skeletal muscle cells while liver cells express Cpt1a. Fatty acids stimulate expression of Cpt1 genes through PPARa response elements (PPRE). We found a similar expression spectrum of Cpt1b‐luciferase activities in C2C12, L6 and H4IIE cell lines. WY‐14,643 (WY) stimulated expression in H4IIE cells, but not in C2C12 or L6 cells. Measurement of PPARa mRNA by real‐time PCR indicated the muscle cell lines had low levels of PPARa as well as PGC1a. After cotransfection of L6 cells with PPARa and PGC1a expression vectors, luciferase activity was increased by PPARa and additionally enhanced by PGC1a. WY further stimulated expression in cells with PPARa overexpression. Mutation of the PPRE in the ‐391/+80 Cpt1b construct eliminated WY stimulation of transcription, but co‐transfection with the PGC1a still increased luciferase activity in the mutant construct. Results show that activation through PPRE is highly dependent on PPARa binding, but PGC1a can stimulate through PPARa and independently of PPARa.
Nucleoside modifications modulate activation of the protein kinase PKR in an RNA structure‐specific manner
Authors: Nallagatla SubbaRao , Bevilacqua Philip C
Published: June 2021

Protein kinase PKR is a key component of innate immunity. It is activated by long stretches of dsRNA and functions to inhibit translation initiation. Many cellular and viral transcripts contain nucleoside modifications which could affect PKR activation. For example, a 5′‐triphosphate confers the ability of relatively unstructured transcripts to activate PKR. Herein, PKR activation by ssRNA and dsRNA containing internal nucleobase, sugar, and phosphodiester modifications is analyzed. We find that for 5′‐triphosphate‐containing ssRNA, most base and sugar modifications abrogate activation, although 2′‐fluoro‐modified ssRNA does not, indicative of a critical role for hydrogen bonding at the ribose sugar. In the case of dsRNA, a more limited set of nucleoside modifications affect PKR activation. Surprisingly, GU wobble pairs also largely abrogate dsRNA‐mediated activation when present at modest levels. Overall, the findings indicate that nucleoside modifications and wobble pairing may serve to discriminate self‐RNA and pathogenic RNA in innate immunity.
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.
Decapping of mRNA containing the histone 3′‐stem loop requires recruitment of stem loop binding protein (SLBP)
Authors: Su Wei , Grudzien‐Nogalska Ewa , Lerner Rachel S. , Kowalska Joanna , Jemielity Jacek , Darzynkiewicz Edward , Marzluff William F. , Rhoads Robert E.
Published: June 2021

Modifications at the 5' and 3' ends of eukaryotic mRNA determine both mRNA stability and translational efficiency. One pathway for mRNA degradation involves decapping by Dcp2. We previously developed a phosphorothioate cap analog, m27.2'−OGppspG (S‐ARCA), that is resistant to Dcp2 and confers a longer in vivo half‐life when incorporated into mRNA, indicating that decapping contributes to degradation of polyadenylated mRNAs (Grudzien et al., RNA 13, 1745, 2007). We have now extended this to the 3′‐terminal stem loop (SL) of histone mRNA. The SL is recognized by the stem loop binding protein (SLBP), which is required for the regulation of mRNA processing, turnover, and translation. Luciferase mRNA containing the 5′‐S‐ARCA and 3′‐SL (S‐ARCA‐Luc‐SL) had a 1.8‐fold longer half‐life when introduced into HeLa cells than ARCA‐Luc‐SL, indicating that degradation of SL‐containing mRNAs also involves decapping. mRNA containing a mutated SL that cannot bind SLBP could not be translated yet had a half‐life that was ~3 fold longer than that of S‐ARCA‐Luc‐SL. Interestingly, the increase in stability was independent of the cap, since both mRNAs had similar half‐lives. This result is consistent with translation being required for rapid degradation of histone mRNA, and with SLBP playing a role in the recruitment of the decapping machinery. (Supported by grants No. GM20818 and GM29832 from the NIGMS and No. 55005604 from the HHMI)
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.
Polyunsaturated Fatty Acids (PUFAs) Inhibit Polyisoprenylated Methylated Protein Methyl Esterase (PMPMEase)
Authors: Duverna Randolph , Taylor Shalina , Lamango Nazarius S
Published: June 2021

Polyisoprenylation is a protein modification with 15 or 20 carbon isoprenes. Polyisoprenylated proteins (PP) undergo S‐adenosyl‐L‐methionine‐dependent methylation of the terminal ‐COOH. PP mediate various intracellular processes such as signaling, cell proliferation, differentiation and apoptosis and mutations of PP are encountered in about 30% of cancers. The methylation by PP methyl transferase (PPMTase) is counteracted by PMPMEase and constitute the only reversible step of the pathway. The relative amounts of the acid and ester forms of PP are determined by the two enzymes. Since PMPMEase and PPMTase may influence functional conformations of PP, a thorough knowledge of these enzymes is essential to the understanding of the significance of PP. Unlike the well studied PPMTase, PMPMEase is less understood. PMPMEase's ability to hydrolyze food‐derived substances has led us to hypothesize a regulatory role of PUFAs on the enzyme activity. A series of saturated and PUFAs were tested for PMPMEase inhibition using an HPLC‐based assay. Shorter chain, more unsaturated, cis fatty acids were the most inhibitory, with an IC50 of 11.6 μM for eicosapentaenoic acid. The anticancer and other benefits of short chain fatty acids and PUFAs may be related to PMPMEase inhibition given the many putative PP substrates that impact various biochemical events. Supported by NIH/NIGMS/SCORE (GM 08111‐35) & NIH/NCRR (G12 RR0 3020).
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.
Identification of non‐substrate amino acid analogs that alter activity and substrate binding in gamma‐glutamylcysteine
Authors: Boettcher Colin , Rozenboom Kelly , Kelly Brenda S.
Published: June 2021

The enzyme, gamma‐glutamylcysteine ligase (gamma‐GCL) catalyzes the first and rate‐limiting step in the synthesis of glutathione, an important detoxicant in nearly all eukaryotes that has been implicated in chemotherapeutic resistance. In the present study, non‐substrate amino acid analogs that activate or inhibit activity of E. coli gamma‐GCL in a concentration‐dependent manner were identified. Fluorescence titration studies confirmed that the analogs specifically bind to the enzyme and alter the binding affinity of the biological substrate cysteine or a cysteine‐mimic. Moreover, sigmoidal binding curves suggest that the enzyme exhibits at least two distinct, cooperative binding sites that may be filled with a cysteine substrate or non‐substrate analog. We hypothesize that E. coli gamma‐GCL is monomeric and has an active site that, when bound with a non‐substrate analog, significantly impacts whether cysteine can bind in a productive (activating) or non‐productive (inhibiting) manner. Current and future studies include confirmation of the oligomeric state of the enzyme, identification of structural characteristics that result in high affinity binding, and site‐directed mutagenesis of proposed active site residues in order to gain further insight into the structure of E. coli gamma‐GCL. This study is supported by Sigma Xi and the Langsjoen fund at Gustavus Adolphus College.
Regulation of Manduca sexta hemolymph proteinase 8 by serpin‐1 isoforms
Authors: An Chunju , Ragan Emily J. , Kanost Michael R.
Published: June 2021

Some insect immune responses, including prophenoloxidase activation and Toll pathway initiation, are mediated by serine proteinase cascades and regulated by serpins. Hemolymph proteinase 8 (HP8), a trypsin‐like serine proteinase, is involved in a Toll‐like pathway by cleaving pro‐sp ?tzle, the putative ligand of M. sexta Toll. The M. sexta serpin‐1 gene is expressed as 12 isoforms, which vary in their reactive center loop due to alternative splicing of exon 9. Serpin‐1 isoforms A, E, and J can all inhibit a fungal trypsin‐like protease, PR2, in vitro. To identify the roles of serpins in the Toll pathway in M. sexta, we purified recombinant serpin‐1A, ‐1E, ‐1J and a mutant HP8 (proHP8Xa) in which the activation site was changed from NNDH to IEGR, allowing activation by bovine Factor Xa. Recombinant serpin‐1A, ‐1E, and ‐1J formed SDS‐stable complexes with HP8Xa. Serpin‐1J was the most effective inhibitor of HP8 among the tested serpin‐1 isoforms. The association rate constant of serpin‐1J and HP8 was 1.3×104 M‐1s‐1, indicating that serpin‐1J may contribute to the inhibitory regulation of HP8 in the hemolymph. Injection of serpin‐1J into M. sexta larvae resulted in a significant decrease in bacteria‐induced mRNA levels for the antibacterial peptides attacin and cecropin. These results suggest that serpin‐1J regulates the activity of HP8 to modulate the Toll pathway response in M. sexta. Supported by NIH grant GM41247.
A high throughput colorimetric assay for angiotensin I‐converting enzyme and its inhibitors
Authors: Karakkat Jimsheena V , Gowda Lalitha R
Published: June 2021

Angiotensin I converting enzyme (ACE) converts angiotensin I to the potent vasoconstrictor angiotensin II which regulates blood pressure. ACE is conventionally assayed by measuring the release of hippuric acid (HA) from hippuryl‐histidyl‐leucine (HHL), either spectrophotometrically or by HPLC. These methods are time intensive and not ideally suited for high throughput screening of natural ACE inhibitors. A diagnostic colorimetric assay to measure HA in urine was transformed into an ACE assay which was sensitive and rapid. The released HA is mixed with pyridine, layered with benzene sulfonyl chloride and measured at 420 nm. The yellow color formed is directly proportional to HA released. The limits of detection and quantitation were 1.46 ?10‐10 and 4.43 ?10‐10 moles of HA respectively. The measured ACE activities of different tissues were comparable to the standard HPLC method. The Km and Vmax values of porcine ACE were 30.8±0.1 ?10‐9 M and 1.3±0.01 ?10‐9 moles/min respectively. The IC50 values for captopril and lisinopril were 1.1±0.05 ?10‐9 M and 2.5±0.03×10‐9 M respectively. A 96 well micro‐plate assay was used to screen the ACE inhibitor activity of peptides generated by the enzymatic hydrolyses of arachin, the major storage protein of peanut (Arachis hypogaea). Our results demonstrate that the application of this colorimetric method not only improves the sensitivity but also increases the sample throughput.
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

Introduction

Although 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.

Results

Compared 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.

Conclusion

NAFLD 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.