bluuebunny/crossref_metadata_2025_split huggingface dataset:
Aligned Nanofibrillar Scaffolds as a Platform for the Treatment of Volumetric Muscle Loss Injury
Authors: Miles Lindsey M. , Raymond-Pope Christiana J. , Corona Benjamin T. , Paukshto Michael V. , Zaitseva Tatiana S. , Greising Sarah M. 
Published: April 2020

Volumetric muscle loss (VML) injury is the traumatic loss of skeletal muscle fibers, along with associated vascular and neural structures, resulting in long‐term functional disability. To date, several strategies have been developed to target skeletal muscle and vascular regeneration and recovery of muscle function, with only limited improvements reported. Our objective, using a standardized unilateral VML injury to the rat tibialis anterior (TA) muscle, was two‐fold: (1) examine the rate of cell population and distribution of cell phenotypes in a bioengineered aligned collagen scaffold; and (2) evaluate the in vivo transfection efficiency of aligned collagen scaffolds loaded with firefly luciferase (Fluc) using bioluminescence imaging. We hypothesize that a modified mRNA (mmRNA)‐incorporated aligned collagen scaffold could be an ideal platform to stimulate vascular and muscle fiber regeneration following VML injury. In total, 25 adult male Lewis rats were used across studies. In Study 1, following unilateral VML injury to the TA muscle (~82mg removed), rats received implantation of an aligned collagen scaffold (6mm in diameter, 0.5–3.0mm thickness), consisting of two interconnected bundles of BioBridge threads that were cross‐linked, lyophilized, and sterilized. The TA muscle was evaluated histologically at 1, 2, and 4 weeks post‐VML to examine cell population and distribution, fibrotic content, and extent of scaffold degradation within the remaining TA muscle and VML defect area. In Study 2, in vivo bioluminescence imaging quantified localized RNA‐transfected cell expression in rats up to 2 weeks post‐Fluc injection within the TA muscle, with or without VML injury, or implantation of an aligned collagen scaffold loaded with Fluc‐RNA following VML injury to evaluate transfection efficiency for potential mmRNA cargo. Scaffolds exhibited minimal degradation through 4 weeks following implantation. Quantification of cellular distributions within the VML defect area indicated significant macrophage infiltration and inflammation up to 4 weeks post‐VML. Intramuscular Fluc injections indicated peak bioluminescence signal (3.0x105 p/s/cm2/sr) occurs at 7 days post‐injection. These results serve as a reference for our ongoing development of a mmRNA‐loaded scaffold delivery system. Our long‐term goal is to evaluate candidate growth factors to co‐deliver in the mmRNA‐loaded aligned collagen scaffolds to facilitate organized, functional skeletal muscle and vascular regeneration following VML injury.Support or Funding InformationThis work is supported by: US Army Medical Research and Materiel Command under Contract Number W81XWH19C0021; T32AR050938; P217A170051.
Ferric Reductases CG1275 and Nemy in Iron Uptake in Drosophila S2 cells
Authors: Lewis Keyata , Holst Jessica , Kane Gregory , Castaneda Mirella , Gorman Maureen J. , Ragan Emily 
Published: April 2020

Iron plays key roles in humans and insects, including in the electron transport chain and other electron transport processes. There is still uncertainty about how iron enters the cell in insects. Currently our group is investigating two cytochrome b561 family members, CG1275 and nemy, as possible ferric reductase components in a Drosophila S2 cell iron uptake pathway. Nemy is known to have ferric reductase properties in Drosophila cells, but its main function occurs in the insect’s brain where it is expressed in neuroendocrine neurons. Nemy has not yet been directly linked to how flies intake iron, but we do hypothesize that a ferric reductase such as nemy is needed in order for the cell to uptake iron. We hypothesize that Fe3+ is reduced to Fe2+ using a ferric reductase (CG1275 or nemy), which will then pass through the malvolio protein for entry into the cytosol. Our experiment involves RNA interference (RNAi) gene silencing of CG1275 and nemy. By adding dsRNA with a sequence matching CG1275 or nemy mRNA, we cause selective degradation of mRNA. After RNAi we extract total RNA and use quantitative PCR to analyze mRNA levels to see if degradation of targeted mRNA has been achieved. We will also measure cellular iron content using a ferrozine‐based assay. If we detect a decrease in iron content in the cells then we will have support for our hypothesis.Support or Funding InformationNSF IOS‐1656407
Neuraminidase 1 and 2 Active Site Comparison for Viral Drug Resistance Analysis
Authors: Sieradzki Guinevere , Mierswa Sabrina , Sabin Amber , DeBruine Anna , Koprek Jessica , Perez Victoria , Minx Connor , Persaud Krishna , Selvik Josephine , Smith Behrgen , Zieman Joshua , Kanungo Muskan , Berkowicz Alexandra , Cabrera-Brito Bella , Pung Benjamin , Alexander Anne 
Published: April 2020

Neuraminidase is an important protein on the surface of influenza and is an antiviral target. There are several types of neuraminidase, but this project focuses on neuraminidase 1 (N1) and 2 (N2) because of their prevalence in human influenza A infections. The amino acid residues in the active sites of N1 and N2 are 94% identical. However, there are several key differences that affect how N1 and N2 interact with the same antiviral drug. The purpose of this project is to gain a deeper understanding of viral drug resistance by modeling the active sites of N1 and N2 and their interactions with oseltamivir (Tamiflu). Two PDB files have been selected for N1 (2HU4) and N2 (4GZP). These PDB files will initially be overlaid in Jmol to identify the level of structural similarity, then active site boxes will be 3D printed for N1 and N2. Active site boxes model only the active site of a molecule, giving a detailed view and understanding of this specific location. Within the models for N1 and N2, Jmol will be used to highlight amino acids E276, I222, and H274Y to investigate their effects on inhibitor interactions. Additionally, a model of the inhibitor oseltamivir (Tamiflu) will be 3D printed to compare its interactions with N1 and N2. These active site boxes will enhance the scientific education and study of these active sites.Support or Funding InformationThis work is part of the CREST program, supported by National Science Foundation Grants I022793, I323414, and 172s940.
Deducing the Origin of Hsp70 Binding Selectivity by Structural Study
Authors: Jansen Rachel M. , Clerico Eugenia M. , Ozden Can , Gierasch Lila M. 
Published: April 2020

Hsp70s are a family of molecular chaperone proteins that aid in the folding of other proteins under physiological and stress conditions. Chaperones prevent protein misfolding and aggregation, characteristic of many neurodegenerative diseases. Hsp70s perform their chaperoning functions through interactions between their substrate binding domain (SBD) and short hydrophobic sequences of their non‐native protein clients that are exposed to the solvent. The details of the interaction between the chaperone and diverse sequences remain poorly understood. Much of the work on the interaction between Hsp70s and client proteins has been done using DnaK, the E.coli Hsp70 and short peptides as surrogates for the client protein. In 1996, the 3D structure of the SBD of DnaK bound to one model peptide was determined using x‐ray crystallography (Zhu et al., 1996), and more recently, the forward and reverse binding between the SBD and a few small peptides has been observed (Zahn el al., 2013). Therefore, it is known that the SBD can accommodate numerous sequences but is nonetheless selective in which sequences it binds. This project aims to understand this “promiscuous selectivity” through analyzing the atomic resolution structures of SBD bound to various peptides. To begin dissecting the selective promiscuity of SBD binding to various sequences we are using proPhoA, an endogenous substrate of DnaK, that has been shown to have five DnaK binding sites. We have determined structures of the SBD of DnaK bound to three of the five binding sites in proPhoA using x‐ray crystallography. These structures reveal a variety of binding modes despite a uniformity of the backbone conformation for the bound peptides. Using this detailed atomic resolution information, we are dissecting how the peptides bind in the SBD and if the SBD remodels to accommodate these unique but specific sequences. This information is shedding light on the basis of Hsp70/substrate recognition.Support or Funding Information1. NIH R35 GM118161 MIRA Grant "Protein folding in the Cell: Challenges and Coping Mechanisms" 6/1/16 – 5/31/20212. University of Massachusetts ‐ Amherst ‐ Biochemistry and Molecular Biology Department
Role for NADH‐sensitive Kv channels in the myocardial‐vascular signaling axis.
Authors: Nystoriak Matthew A. , Ohanyan Vahagn , Dwenger Marc M. , Chilian William M , Bhatnagar Aruni 
Published: April 2020

Voltage‐gated potassium (Kv) channels expressed in the coronary vasculature play a pivotal role in coupling oxygen delivery with myocardial metabolic demand. Despite their importance in maintaining adequate perfusion to support proper cardiac function, the processes by which these channels respond to metabolic cues from the myocardium to elicit smooth muscle hyperpolarization and rapid relaxation are unclear. We tested the hypothesis that the heteromeric auxiliary Kvβ subunit (Kvβ1:Kvβ2) complex facilitates increases in Kv1 channel activity and coronary vasodilation during periods of elevated myocardial oxygen consumption. The Kvβ proteins are functional aldo‐keto reductases, and thus catalyze nicotinamide cofactor‐ (i.e., NAD(P)H) dependent reduction of a range of aldehyde substrates. Intriguingly, these subunits also differentially modify the voltage‐sensitivity of Kv1 channels in a manner that depends upon the redox state of bound cofactor. Imaging mass spectrometry revealed that lactate:pyruvate (used as a surrogate for NADH:NAD+) was significantly increased in intramyocardial arteries in hearts from mice acutely treated with dobutamine to drive elevated cardiac workload versus those from vehicle‐treated mice. Elevation of NADH:NAD+ ratio in coronary arterial myocytes following perfusion of sodium lactate reversibly increased whole cell Kv currents and shifted the voltage‐dependence of activation by ~−15 mV. Moreover, application of 1 mM NADH, but not NAD+, increased single Kv1 channel currents in excised membrane patches from coronary arterial myocytes of wild type mice. In contrast, ablation of Kvβ2 abolished the NADH sensitivity of Kv1 activity, whereas ablation of Kvβ1 had little effect. Consistent with this, we found that vasodilation of coronary arteries to reduced bath oxygen (to ~3%) and the relationship between cardiac work and myocardial blood flow was significantly blunted in Kvβ2−/− arteries/mice when compared with wild type arteries/mice. Together, our results suggest that the Kvβ complex is a functional sensing unit that modulates native coronary Kv1 channel activity upon changes in pyridine nucleotide redox ratio, which may impart a profound influence on the control of myocardial blood flow.Support or Funding InformationThis work is supported by grants from the National Institutes of Health (HL142710, GM103492).
TEV‐FRET: High‐Throughput Assay for Determining Protease Activity
Authors: Johnson Maddi , Page Richard C. 
Published: April 2020

The production of proteins for structural studies has been greatly aided by the use of fusion tags to increase expression yields or facilitate preparative‐scale purification with high yields and high purity. Isolating the desired protein construct from the fusion protein is commonly performed using tobacco etch virus (TEV) protease. Here we develop a high‐throughput assay for testing fusion protein cleavage by TEV protease. Initial results with a TEV‐targeted peptide containing a FRET pair allow for rapid assessment of cleavage efficiency and activity using FRET. Future versions of the assay will capitalize on ELISA with specific N‐ and C‐terminal targets for identifying cleavage efficiency and activity. These results are outcomes of a project executed by members of the Miami University ASBMB Undergraduate Student Chapter.Support or Funding InformationNIGMS R35 GM128595
Mode of Protein Targeting to the Proteasome Determines Substrate Fate
Authors: Braganca Christopher Eric , Kraut Daniel Adam 
Published: April 2020

The Ubiquitin Proteasome System (UPS) is the canonical pathway for protein degradation in eukaryotic cells. Proteins targeted for degradation are first tagged with a polyubiquitin chain through a series of enzyme‐catalyzed reactions before degradation. After polyubiquitination, the 26S proteasome hydrolyzes ATP to processively unfold substrates before they are degraded in the 20S core particle. Not all substrates are degraded well by the proteasome, and many factors underlying the proteasome’s unfolding ability remain unknown. We set out to investigate the role of targeting by comparing a linear Ub4 proteasome‐binding tag with the UBL domain from yeast Rad23, which is also commonly used in degradation experiments. We found that the UBL domain allows for degradation of stable sGFP‐containing substrates, while the Ub4 tag does not. Replacing sGFP with a less stable variant allows degradation in either case. Our results suggest that different proteasome‐binding tags, which are presumably recognized by different ubiquitin receptors, can lead to different proteasome unfolding abilities.Support or Funding InformationCEB is a Beckman Scholar. This material is based upon work supported by the National Science Foundation under Grant No. 1515229 to DAK.
Improving Protein Translation in an Attenuated Vaccinia Virus System using Translation Enhancing Elements
Authors: Juba Amber N. , Wellensiek Brian P. 
Published: April 2020

Developing effective and safe vaccines without the necessity for booster vaccinations is essential to providing lasting immunity for global populations. The vaccinia virus, which was successfully used as a vaccine in the elimination of small pox, is an attractive agent for vaccine development given the ease with which it can be modified. One major drawback to vaccinia virus use however, is the potential for harmful systemic vaccinia reactions. To improve safety, the virus used as an immunogen can be attenuated. Yet, while attenuation can improve safety concerns, it also reduces the efficacy of the vaccine. This reduction is largely attributed to a decreased amount of protein delivered to the immune system. In an effort to improve vaccines exhibiting less immunogenicity, we aim to increase protein translation in an attenuated virus system by using translation enhancing elements (TEEs) previously discovered from the human genome. One in particular, termed 5d5, acts as a vaccinia virus promoter and a TEE. Here we explore combining other TEEs demonstrating high levels of activity with 5d5 to determine whether we can increase protein expression in an attenuated virus system. The effect of each combination on protein expression is measured by transfection/infection assays using luciferase based plasmids. We use wild‐type and attenuated vaccinia virus strains (Copenhagen and NYVAC, respectively) in our model system to compare luciferase expression levels between the two for each of the constructs. Although we did not find any significant increases in translation when combining TEEs with 5d5, we did show for the first time at least a 2‐fold increase in translation when using 5d5 in the NYVAC system when compared to expression when unmodified virus is used.Support or Funding InformationMidwestern University intramural funding
Foliar Fungicides Reduce Short Term Non‐Target Soil Microbial Activity and Community Structure
Authors: Kosel Kelby , Collins Harold 
Published: April 2020

Pesticides are essential in modern agricultural practices and are used frequently for the protection of crops. Fungicides are a group of pesticides that inhibit the establishment of fungal pathogens. Our systematic knowledge of the different affects fungicides have on non‐target organisms, unfortunately, is inadequate. It is difficult to determine the range of organisms affected by fungicides because of the variability and sensitivity of both eukaryotes and prokaryotes. A laboratory study was initiated to assess non‐target effects of fungicides on soil microbes. This study examined the effect of the common fungicides Tilt and Trivapro at different concentrations and incubation periods for their non‐target effects on soil microbial communities and selected activities. Three rates, 0X, 1X (0.0058 μL/cm2) and 2X (0.0116 μL/cm2) of each fungicide were applied to 100 g of Bastrop soil, common to the Blackland Prairie of Central Texas. Significantly lower CO2 was respired from amended soils compared to the control. Additionally, nitrate mineralization concentration decreased over the 30‐day trial period compared to the control. Low carbon mineralization and nitrogen mineralization rates are both attributed to a decrease in soil microbial activity. Plate counts indicate fungi (PDA) and bacteria (TSA) showed variability at two sampling dates indicating that treatment effects could not be determined solely on population counts. Our results suggest these fungicides at 1X and 2X rates have significant effects on non‐target organismal activity and community structure. Future studies will examine field applications and reassess results.Support or Funding InformationUSDA Agricultural Research Service CRIS #3098‐13610‐008‐00D
ROLE OF SPINAL AT1, IONOTROPIC GLUTAMATERGIC AND GABAERGIC RECEPTORS ON CONTROL OF RENAL SYMPATHETIC NERVE ACTIVITY IN 2K‐1C RATS
Authors: de Oliveira Milanez Maycon Igor , Nishi Erika Emy , Rocha Antônio Augusto , Bergamaschi Cássia Toledo , Campos Ruy Ribeiro 
Published: April 2020

Previous studies performed in our laboratory showed a higher glutamate and angiotensin receptors activation, as well as an alteration in GABAergic pathways in central nuclei involved in the cardiovascular control that may lead to sympathoexcitation in renovascular hypertensive rats (2K‐1C). However, the role of the spinal cord neurons on the renal sympathoexcitation remains unclear in this model. Thus, we aimed to assess the influence of spinal glutamatergic and GABAergic (ionotropic receptors), as well as angiotensin II type 1 receptors (AT1) receptors on the renal sympathetic nerve activity (rSNA) in the 2K‐1C rats. Male Wistar rats (250–300 gr) were used to induce renovascular hypertension by clipping the renal artery with a silver clip (0.2 mm). After six weeks, a polyethylene catheter (PE‐10) was inserted into the subarachnoid space and advanced to the T10–11 vertebrae level in urethane‐anaesthetized rats (1.2 g/Kg, iv). All experiments were approved by the Institutional Ethical Committee of UNIFESP. The effects of intrathecal (i.t.) injection of kynurenic acid (KYN) (160 nmol in 2 μl), losartan (Los) (2nmol in 2 μl) or bicuculline (Bic) (8 mM in 2 μl) on blood pressure (BP) and rSNA were investigated. KYN i.t. injection induced a larger and significant fall on rSNA in the 2K‐1C compared to control rats. Intrathecally Los evoked a larger and significant fall in rSNA in the 2K‐1C compared to control group. KYN decreased BP similarly in both, control and 2K‐1C groups, however, Los significantly decreased BP in the 2K‐1C group only. Intrathecally Bic triggered a more intense BP and rSNA reponses in 2K‐1C compared to control rats. Gene expression analysis by qRT‐PCR showed a spinal (T11–12) AT1 mRNA upregulation in the 2K‐1C rats (1.71‐fold change), while no changes were observed in AT2, ionotropic glutamate and GABA receptors subunits. Therefore, our data show that ionotropic spinal cord excitatory amino acid, GABAergic and AT1 receptors play a significant role in the control of rSNA in the 2K‐1C model that may contribute to the maintenance of hypertension. The participation of spinal AT1 receptors seems to be more important in the establishment of sympathoexcitation in this model. The origin of those projections remains unclear.Support or Funding InformationCNPq, FAPESP (2018/01898‐4) and CAPES
Streamlining FEF25‐75 and Peak Flow Calculations for Use with Physiological Data Acquisition Software
Authors: Tomasson Erik P. , Welper Hannah M.E. , Bates Melissa L. 
Published: April 2020

Our lab has developed novel techniques to quantify the lung‐structure function relationship, but is also interested in how lung structure impacts ventilation during exercise. But, a limitation to evaluating pulmonary function is that the most commonly used data analysis packages in exercise physiology do not include functions to calculate mid‐expiratory flow (FEF25‐75). To date, we have been calculating FEF25‐75 values manually or switching to equipment that includes a spirometry analysis program. We set out to write a program to automate these calculations so that they could be made in the context of exercise physiology studies. The data is first downsampled to yield a flow value every hundred milliseconds. Then, the program makes a running sum of all the flow values in each breath to determine the expiratory volumes of every data point. Flow values within 25% and 75% of the total volume are then averaged to calculate the breath’s FEF25‐75. Every flow value is then passed through python’s built‐in maximum function to determine peak flow. The FEF25‐75 and peak flow values for each of the breaths are then averaged to return a single FEF25‐75 and peak flow for each breath set. We found that nine breaths are necessary to ensure representative accuracy >95%. Upon further testing, with sets of nine breaths, the program calculated FEF25‐75 values and peak flow values consistently to within 3.13% and 3.64% of the manually calculated values respectively. The average deviation from the manually calculated values is 1.23% for FEF25‐75 and 0.92% for peak flow. The increased efficiency that this program brings to small airway analysis can streamline many projects and serve as a platform for more useful research into lung function.Support or Funding InformationThis work was supported by the University of Iowa Old Gold Fellowship Program (Bates)