bluuebunny/crossref_metadata_2025_split huggingface dataset:
Organophosphate Hydrolase (OPH) Designed as a Functional Monomer
Authors: Baker Cetara , Garcia Jaffet Santiago , Sweeney Richard , Kirby Stephen 
Published: June 2021

Organophosphate hydrolase (OPH) mutants haveshown potential use as a medical countermeasure against a range of differentorganophosphorus compounds (OPs). OPH is typicallyexpressed in bacteria as a homodimer. Two separate monomers (~37kDa each) self‐assemble throughnon‐covalent bonding at the enzyme face close to the putative active site, andenzyme activity is dependent upon dimer formation. OPH homodimers do notsecrete expediently from mammalian cells and are often misfolded. This causespotential problems when trying to express the protein from a heterologous plasmidor viral delivery system. To enhance secretion of OPH from mammalian cells, wesought to increase protein solubility without catastrophic detriment toactivity and without addition of fusion proteins. To this end, we designed OPH tobe expressed as a functional monomer by joining two OPH subunits with a poly‐glycinelinker and expressing it as a single polypeptide in bacteria to evaluateactivity. We created the single polypeptide OPH with a tether 10 or 35 aminoacids in length between the two halves, and named them T10 and T35, respectively. Western blot analysis and paraoxon hydrolysis assays revealedthat T10 was being produced and there was a 71% loss in specific activity comparedto an untethered homodimer positive control. This was a surprise as we expectedT10 to have no enzymatic activity since the tether was too short to allowproper dimer formation. The T35 functional monomer (~76 kDa) was produced showing 29% loss of specific activity. Both T10 and T35 OPH showed high molecular weight aggregate formation greater than 250 kDa in dynamic light scattering and native polyacrylamide gels. Construction of a functional OPH monomer with activity against OPs is instrumental in designing secretable OPHs for adeno‐associatedviral expression. It may also prove useful in the development of chimeric OPHs for maintaining control over the composition of each OPH half, allowing blended activities against a combination of organophosphorus substrates. This is the first attempt known for producing OPH as a functional monomer.Support or Funding InformationThe views expressed in this abstract are those of the author(s) and do not reflect the official policy of the Department of Army, Department of Defense, or the U.S. Government. This research was supported by the Defense Threat Reduction Agency – Joint Science and Technology Office, Medical S&T Division. This research was supported in part by an appointment to the Postgraduate Research Participation Program at the U.S. Army Medical Research Institute of Chemical Defense administered by the Oak Ridge Institute for Science and Education through an interagency agreement between the U.S. Department of Energy and USAMRMC.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Roles of Galectins in Infection: First barrier or Trojan Horse?
Authors: Vasta Gerardo R. 
Published: June 2021

Carbohydrate structures on the cell surface encode complex information that through specific recognition by carbohydrate‐binding proteins (lectins), modulates interactions between cells, cells and the extracellular matrix, or mediates recognition of potential microbial pathogens. Galectins are a family of β‐galactoside‐binding lectins characterized by a unique sequence motif in the carbohydrate recognition domain, and evolutionary and structural conservation from fungi to invertebrates and vertebrates, including mammals. Since their discovery in the 1970s, their biological roles, initially understood as limited to recognition of endogenous (“self”) carbohydrate ligands in embryogenesis and early development, dramatically expanded in later years by the discovery of their roles in tissue repair, cancer, adipogenesis, and regulation of immune homeostasis. In recent years, however, evidence has also accumulated to support the notion that galectins can bind (“non‐self”) glycans on the surface of potentially pathogenic microbes, and function as recognition and effector factors in innate immunity. Thus, this evidence has established a new paradigm by which galectins can function not only as pattern recognition receptors but also as effector factors, by binding to the microbial surface and inhibiting adhesion and/or entry into the host cell, directly killing the potential pathogen by disrupting its surface structures, or functioning as opsonins, by promoting phagocytosis and pathogen clearance from circulation. Strikingly, some viruses (HIV, Nipah, IHNV), bacteria (Chlamydia, Streptococcus), and protistan parasites (Trichomonas, Leishmania, Perkinsus) have the capacity to take advantage of the aforementioned recognition roles of the vector/host galectins, for successful attachment or invasion. The recent findings mentioned above have not only revealed an extraordinary functional diversification in this otherwise structurally conserved lectin family, but also suggest unique evolutionary adaptations of the surface glycome of some microorganisms to use host recognition for cell surface adhesion and infection. Thus, it is proposed that galectin‐mediated innate immune recognition and effector mechanisms, which throughout evolution have remained effective for preventing or fighting viral, bacterial and parasitic infection, have been “subverted” by certain pathogens to gain entry into the host.Support or Funding InformationSupported by grant 5R01GM070589 from the National Institutes of Health, and grants IOS‐0822257 and IOS‐1063729 from the National Science FoundationThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Sustained mentorship promotes the development of active learning strategies in undergraduate biology classrooms: Evidence gained from the Promoting Active Learning and Mentoring (PALM) Network
Authors: Wolyniak Michael J , Wick Susan 
Published: June 2021

Here we report on the work of the Promoting Active Learning and Mentoring Network (PALM), a National Science Foundation‐supported project started in 2016 and dedicated to leveraging the expertise found in life science professional societies to provide long‐term mentorship in developing active learning techniques in the classroom. PALM mentees work one‐on‐one with mentors that are seasoned in active learning strategies and travel to their mentor's institution to observe classes, plan modifications for their own classes, and strategize the best active learning techniques that are suited to their class' particular needs. A monthly online journal club and regular opportunities to meet in conjunction with the Society for the Advancement of Biology Education Research (SABER) and ASCB meetings provide a dynamic teaching and learning community for mentees and mentors alike. After their mentorship, mentees are encouraged to work with their colleagues on active learning strategies and to become a PALM mentor to further the spread of effective active learning strategies to an ever‐wider audience.We have assessed the efficacy of PALM mentorship through the use of the Classroom Observation Protocol for Undergraduate STEM (COPUS) protocol (Smith et al., 2013) on pre‐mentorship and post‐mentorship videos of mentee teaching. The COPUS results have revealed a consistent shift in mentee teaching practices from a strict focus on lecture to a more diffuse combination of classroom activities in which the student is more directly active and engaged in the class and the instructor is more directly engaging students in a more personalized manner. While the specific changes in classroom practice vary between individual mentees due to the distinct needs and environments of each class, there is a consistent picture of more student‐centered classrooms that are more engaging and dynamic for both student and instructor. Overall, the COPUS data suggests that the long‐term mentoring strategies brought forth by PALM are an effective and sustainable means to promote the development of active learning techniques among undergraduate biology instructors.Support or Funding InformationNational Science Foundation #1624200This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Functional Relevance of Unstructured Regions of AcrA, the Periplasmic Adaptor of the Major Multidrug Efflux System in E. coli
Authors: Ojo Isoiza , Wei Yinan 
Published: June 2021

Efflux pumps, combined with low permeability of the outer membrane of gram negative bacteria play a crucial role in bacteria multi‐drug resistance. There are five classes of efflux pumps but the major and most studied of the efflux system in E. coli is the resistance nodulation cell division (RND) pump. This pump is a complex of three proteins: the inner membrane transporter AcrB, the periplasmic adaptor AcrA, and the outer membrane protein channel TolC. Of the three proteins, the structure and dynamic behavior of AcrA remain to be fully elucidated. In this study the functional roles of the unstructured regions at the N‐ and C‐termini of the protein were examined. These structurally unresolved regions include residues 26–37 at the N‐terminus, and residues 376 to 397 at the C‐terminus. We conducted truncation mutation to systematically remove residues from the C‐terminus of AcrA, and found that residues after Q376 are indeed not functionally important. The truncated AcrA, in which E377 is replaced by a stop codon, is fully active. On the N‐terminus, we created a mutant in which four amino acid residues (Q31QGG34) were deleted. The mutant was nonfunctional. To understand the mechanism of function disruption, we modified the membrane anchoring of AcrA, and determined the role of these first 13 residues at the N‐terminus of the mature AcrA sequence.Support or Funding InformationNSF grant CHE‐1709381This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Automated Evaluation of Microvascular Blood Flow and Oxygenation following Trauma and Hemorrhage in Rats
Authors: Torres Filho Ivo P , Barraza David , Williams Charnae E , Hildreth Kim , Dubick Michael A 
Published: June 2021

Studies evaluating blood flow and oxygen partial pressure (PO2) may not directly measure both parameters and may depend on investigator's selection of measuring sites. In addition, relevant systemic parameters may not be simultaneously recorded. Therefore, we implemented a new automated system for blood flow and PO2 acquisition in large tissue areas while collecting systemic information. In 10 animals anesthetized with isoflurane, cardio‐respiratory parameters were continuously recorded, in experiments lasting up to 4 h. Other data were collected at baseline, and after laparotomy and 30 min hemorrhage (40% of total blood volume). A cremaster muscle was spread over a thermostatically‐controlled pedestal fixed to a motorized stage. Sixteen noninvasive PO2 measurements using oxygen‐dependent phosphorescence quenching and fiber‐optics were performed during a computer‐controlled tissue scan. In the same areas used for PO2 measurements, microvascular blood flow was estimated employing laser speckle contrast imaging. Blood was sampled for extensive biochemistry and coagulation profiles. The system was used successfully by different operators. One set of flow and PO2 measurements was completed in less than 90 s. Changes in muscle flow correlated with local PO2 but several systemic parameters did not correlate with blood flow and PO2, emphasizing the importance of performing both local and systemic evaluations. System advantages include integration between local and multiple systemic parameters, unbiased data collection/analysis, easy implementation, improved performance, no need for customized programming, and simplified training compared to intravital microscopy.Support or Funding InformationSupported by US Army Medical Research and Materiel Command.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Acute Spinal Cord Injury Causes Cardiac Contractile Dysfunction that when Treated Aids Spinal Cord Oxygenation
Authors: West Christopher , Williams Alex , Manouchehri Neda , Tauh Keerit , Poormasjedi‐Meibod Malihe , Boushel Robert , So Kitty , Shortt Katelyn , Streijger Femke , Kwon Brian 
Published: June 2021

ObjectiveIn chronically injured humans and rodents with spinal cord injury (SCI) cardiac function is impaired. It is unknown whether such dysfunction is present acutely post‐SCI and if so whether it contributes to reduced spinal cord oxygenation. The aims of the present study, therefore, were to 1) define the acute cardiac and hemodynamic response to SCI, and 2) test the efficacy of a cardiac‐centered hemodynamic management strategy (β1‐receptor agonist dobutamine, DOB) vs. a traditional vasopressor‐based strategy (norepinephrine, NE) on intraparenchymal spinal cord oxygenation.DesignTo address aim 1, 7 Yucatan mini‐pigs received a T2 contusion SCI with 2 hours of compression and underwent measures of left‐ventricular (LV) function and spinal cord oxygenation pre‐ and up to 4 hrs post‐SCI. Pigs were instrumented with 1) a left‐ventricular (LV) pressure‐volume catheter for assessment of contractility (i.e., end‐systolic elastance, Ees) and LV hemodynamics, 2) a Swan‐Ganz pulmonary artery catheter for assessment of cardiac output (Q), 3) an inferior vena cava occlusion catheter, 4) a femoral arterial catheter to monitor mean arterial pressure (MAP), and 5) an intraparenchymal spinal cord oxygen sensor. To address aim 2, an additional 14 pigs were evenly assigned to either dobutamine (DOB, cardiac‐centered therapy), or norepinephrine (NE, vasopressor therapy) and were instrumented in the same way as aim 1. Drug infusions began 30 mins post‐SCI, and were continuously titrated to attain a targeted “normal” pre‐SCI Ees of ~2.5–2.9 mmHg/ml (DOB) or pre‐SCI MAP of ~85–90 mmHg (NE).ResultsEes and MAP were significantly reduced 4 hrs post‐SCI vs. pre‐ SCI (all p<0.048), while Q was unchanged. When titrating DOB infusions to a pre‐established range of “normal” LV contractility, 3 animals received higher doses (≥3 mcg/kg/min) and 4 received lower doses (≤1 mcg/kg/min). Relative to their own pre‐drug baseline, animals receiving high‐dose DOB had significant increases in intraparenchymal spinal cord oxygenation at 4hr post‐SCI (p=0.014), whereas those treated with low‐dose DOB, NE or CON did not.ConclusionsSCI is associated with reductions to cardiac contractility in the acute setting. Normalizing reduced contractility with high‐dose DOB increased spinal cord oxygenation, whereas low‐dose DOB, NE or CON did not alter cord oxygenation. These findings have important clinical implications as a reduction in spinal cord oxygenation post‐SCI is known to be a major contributing factor the development of the secondary injury and poor neurological recovery.Support or Funding InformationUS Department of Defense, Craig Nielsen Foundation, Rick Hansen Institute, Michael Smith Foundation for Health ResearchThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The ERG1a K+ Channel Increases Intracellular Calcium and Calpain Activity in C2C12 Myotubes
Authors: Pond Amber Lynn , Whitmore Clayton L. , Pratt Evan E. , Anderson Luke Brian , Rantz Emily , Salyer Amy , Carraro Ugo , Zampieri Sandra , Weilbaecher Rod , Davie Judy K. , Hockerman Gregory H 
Published: June 2021

In skeletal muscle increased intracellular calcium concentration ([Ca2+]i; uM range) is necessary for excitation‐contraction coupling; however, smaller increases in [Ca2+]i (nM range) can modulate other physiological processes in non‐contracting muscle. Indeed, fluctuations in localized calcium concentration can serve as a second messenger. In contrast, inappropriate changes in [Ca2+]i can have detrimental effects on muscle tissue and are associated with numerous skeletal muscle pathologies, for example muscular dystrophy, amyotrophic lateral sclerosis, malignant hyperthermia, cancer cachexia, and atrophy. Therefore, [Ca2+]i must be tightly regulated in terms of time, space and amplitude for cellular processes to occur in a properly coordinated fashion. In earlier reports, we showed that the ERG1a K+ channel is upregulated in atrophying skeletal muscle and contributes to increased ubiquitin proteasome proteolysis. Here, using the ratiometric Ca2+ indicator fura‐2AM, we show that ERG1a expression in C2C12 myotubes produces an increase in basal [Ca2+]i as well as a transient increase in [Ca2+]i as a consequence of depolarization. We explored this transient increase in [Ca2+]i using pharmacological agents. The data demonstrate that the ERG1a‐induced increase is not sensitive to the L‐type calcium channel blocker nifedipine, suggesting that it does not result from modulation of Cav1.1 channels. To further support this data, immunoblots reveal that there is no change in Cav1.1 channel abundance. However, the data do demonstrate that the increase in [Ca2+]i is sensitive to the SERCA blocking agent thapsigargin, suggesting that the source of the calcium is the sarcoplasmic reticulum stores. Additionally, the data reveal that ERG1a expression also increases basal calpain activity. In summary, to date the data show that ERG1a increases [Ca2+]i levels and suggest that this increase could occur, at least in part, as a result of release of calcium from sarcoplasmic reticulum stores. Additionally, the data reveal that ERG1a expression also increases calpain activity, suggesting that the increase in intracellular calcium results in increased calpain activity and possibly contributes to the increased proteolysis that occurs in atrophic skeletal muscle.Support or Funding InformationU.S. Department of Defense, PRMRP Grant# PR170326This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Quadrupole ICPMS Determination of Iron in Drosophila melanogaster S2 Cell Cultures using Collision Cell and Desolvation Strategies
Authors: AL‐Ogaidi Hussein A , Ketterer Michael E , Ragan Emily J 
Published: June 2021

Inductively coupled plasma mass spectrometry (ICPMS) has become the preferred technique for trace and ultra‐trace determination of elemental concentrations in a wide variety of sample matrices. In our studies of iron uptake and metabolism in insect cells, we evaluated different ICPMS procedures to measure picogram to microgram quantities of iron in whole insects and Drosophila melanogaster S2 cells. While a well‐known spectrophotometric iron assay with ferrozine suffices for microgram amounts of the elements, ICPMS is imperative for lower level work.The determination of iron by ICPMS faces several significant challenges: samples must be decomposed to mineralize organic matter; the separation of iron from the matrix may be required in some circumstances; all sample preparation and analytical processes must be conducted under sufficiently “clean”, low‐blank conditions; and the interference of polyatomic ions (ArN+, ArO+, ArOH+) upon iron ions must be addressed. To address these challenges, samples were digested and mineralized by heating with nitric acid at 90°C in fluoropolymer containers. “Trace metal” grades of HNO3 were found to be satisfactory for minimizing reagent contributions to the overall blank. As quadrupole mass analyzers cannot distinguish between ions of the same nominal mass (e.g., 40Ar16O+ vs. 56Fe+), alternative strategies must be used to suppress the formation of polyatomic ions so that Fe+ ions can be observed above background signal. We used two different approaches. The first is de‐solvation of the sample aerosol, where water vapor is removed by a membrane dryer with countercurrent Ar gas flow. The second is passage of the extracted ions through a “collision cell” prior to mass analysis, which selectively attenuates the polyatomic ions via ion kinetic energy discrimination. A combination of both approaches lowered solution detection limits for Fe from ~ 50 – 100 micrograms/liter to << 1 microgram/liter. We present optimized procedures for the routine determination of Fe in digested cell and insect tissue samples.Support or Funding InformationNSF IOS‐1656407This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Ferrozine‐Based Assay for Determining Iron Content in Insect Cells
Authors: Castaneda Mirella , AL‐Ogaidi Hussein , Gorman Maureen J. , Ragan Emily J. 
Published: June 2021

We are optimizing a ferrozine‐based assay to analyze the iron content in Drosophila S2 cells. We hypothesize that putative ferric reductases CG1275 or CG8399 may be involved in reducing iron in the hemolymph and allowing uptake into cells. We have modified a ferrozine‐based assay, in which ferrozine complexes with ferrous ions in solution and forms a stable magenta complex species that can be detected at 562 nm. Using this method with an Ultraspec II spectrophotometer we obtained a limit of detection of 0.30 μg/mL. This method provides a reliable, inexpensive and simple way of analyzing and quantifying the iron content within the S2 cells, allowing us to analyze iron content after RNA interference experiments to knock down CG1275 and CG8399.Support or Funding InformationNSF IOS‐1656407This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.