bluuebunny/crossref_metadata_2025_split huggingface dataset:
Ketone Ester Supplementation Does Not Impair Cognitive or Motor Performance and Delays CNS Oxygen Toxicity in Male Sprague‐Dawley Rats
Authors: Stavtizski Nicole M. , Landon Carol S. , Hinojo Christopher M. , Poff Angela M. , Rogers Christopher Q. , D’Agostino Dominic P. , Dean Jay B. 
Published: April 2020

Oxygen acts as a drug at increased atmospheric pressures, such as occurs during exposure to hyperbaric oxygen (HBO) during hyperbaric and undersea medicine. Use of HBO is limited by the risk of developing CNS oxygen toxicity (CNS‐OT) which manifests as seizures. The ketogenic diet (KD) is used clinically as an anticonvulsant therapy for refractory epilepsy but requires significant dietary restrictions to maintain ketosis. Studies suggest that exogenous ketone ester (KE) induces ketosis and delays the onset of CNS‐OT without the need for dietary restrictions; however, an optimal dose of KE that provides neuroprotection and delays the onset of CNS‐OT without hindering cognitive or motor performance has yet to be elucidated. Sprague Dawley rats received either no gavage, vehicle gavage, or 7.5 g/kg KE gavage and were subjected to a battery of behavioral tests: DigiGait (DG), Light/Dark (LD), open field (OF), and novel object recognition (NOR). No significant differences were observed in motor performance on the DG tests in all treatment groups, suggesting that there is no effect of KE on motor performance. No differences in the time spent in the light or dark zones in the LD test were observed between groups, nor in the latency time to the dark zone. KE‐treated rats exhibited reduced distance traveled in the dark zone, suggesting the KE may be anxiolytic, a property that has been reported previously. No differences in time spent in the perimeter or center zones between the groups in the OF test were noted; however, KE‐treated rats exhibited decreased distance moved and speed, similar to what was seen in the DigiGait test which again may reflect an anxiolytic effect. To further explore this result, we evaluated overall activity level of the rats using the OF test. KE‐treated rats exhibited increased overall activity, indicating that the animals were exploring the areas and were less anxious than vehicle controls. Although there were no significant differences between the groups in the NOR test, KE‐treated rats exhibited a trend in both exploratory preference and discrimination index suggesting that the animals learned and were able to recall that a novel object was present. Additional experiments are currently being conducted in which vehicle‐ or KE‐treated rats are implanted with radio‐telemetry modules, allowing for real‐time monitoring of EEG, ECG, and respiratory EMG during an HBO dive protocol. These experiments are underway but have thus far shown that the KE increases latency time to seizure over vehicle (24.9±6.7; 15.5±6.5, n=8). Together, these findings suggest that a single administration of KE will delay onset of CNS‐OT without hindering performance. This work is funded by the Office of Naval Research.Support or Funding InformationOffice of Naval Research
Reconsidering the Role of the RecF Protein in Repair of Post‐replication Gaps
Authors: Beyer Hope A. , Wood Elizabeth A. , Chitteni-Pattu Sindhu , Henry Camille , Cox Michael M. 
Published: April 2020

In Escherichia coli, the RecA protein is an important factor in homologous recombination and DNA repair. When a post‐replicative gap is formed, RecA is loaded onto the single strand (ss) DNA coated by SSB, a process facilitated by recombination mediator proteins (RMP), RecF, RecO, and RecR. Contrary to current models, RecF, RecO and, RecR do not form a complex. In vitro, RecR can interact with either RecO or RecF but they compete for RecR binding. In vivo, RecF and RecO have distinct locations within the cell. Only RecF colocalizes with the replisome after UV stress, suggesting that RecF and RecO have distinct functions. Moreover, in some conditions only RecF or RecOR appear to be required. The RecOR complex is believed to load RecA onto ssDNA. Nevertheless, the RecF’s role and the function of its ATPase activity remains enigmatic.Building evidence suggests that RecF may have an unanticipated function at the replication fork. Present understanding has been achieved by using a variety of techniques including biochemical assays, phenotypic characterization and single molecule imaging microscopy, all of which confirm and build upon the hypothesis that RecF has a unique and novel role independent of RecOR in gap repair. To further investigate this hypothesis, we use a combination of overexpression and deletion mutant experiments. We hope to further distinguish RecF’s role in gap formation by working with the RecA V79L mutant, which possesses an enhanced ability to bind and remove SSB from the ssDNA.
Molecular Viscometer Identified a Population of Mucin Granules of Higher Viscosity in Cystic Fibrosis Cells
Authors: Ponomarchuk Olga , Boudreault Francis , Brochiero Emmanuelle , Gryczynski Ignacy , Dzyuba Sergei V. , Fudala Rafal , Gryczynski Zygmunt , Grygorczyk Ryszard 
Published: April 2020

The cystic fibrosis (CF) disease is characterized by abnormally thick and sticky mucus. Normal airway mucus has bulk viscosity (macroviscosity) in the range of 12–15×103 cP increasing to 14–110×103 cP for CF patients. At micro‐length scale (≤1 μm), due to the presence of fluid‐filled spaces between mucin fibers, mucus microrheological properties are similar to that of water (~1 cP). However, they are only slightly higher in CF mucus (~3 cP). Our study was designed to verify if similar viscosity differences exist between normal and CF mucin granules and secreted mucus.We used a custom‐designed membrane‐permeant molecular viscometer (phenyl‐BODIPY) and fluorescence lifetime imaging microscopy (FLIM) to study the microviscosity of mucin granules and secreted mucus. FLIM microviscosity measurements were validated in calibration experiments with sucrose (low microviscosity range), glycerol (high microviscosity range), agarose (gel‐like structure) and reconstituted mucus of isolated bovine mucin proteins in solution. Human bronchial epithelial (HBE) cells, isolated from tissues of 4 normal and 5 CF donors, were seeded on filters and cultured at the air‐liquid interface.FLIM measurements revealed that median values of microviscosity for normal granules are 41±7 cP (nfilters=30) and 49±11 cP (nfilters=31) for CF granules (p<0.001, Mann–Whitney U test). Next, we classified mucin granules in three categories based on their microviscosity: low (1–30 cP), medium (30–60 cP) and high (>60 cP). The quantity of granules with medium microviscosity does not differ between normal (74%) and CF (77%) donors. However, normal phenotype was characterized by the presence of a larger fraction of low microviscosity mucin granules (19%) compared to CF (4%), while CF phenotype displayed a larger proportion of high microviscosity granules (19%) compared to normal (7%). Raising the pH inside granules by blocking H+‐ATPase with 10 nM bafilomycin lowered microviscosity values of CF granules towards normal phenotype. Microviscosity of secreted mucus covering HBE cells was measured in situ and reached values of 147±54 cP (nfilters=7) for normal donors and 198±75 cP (nfilters=7) for CF donors.Our study demonstrates the presence of a subpopulation of mucin granules with higher microviscosity in CF that is less pronounced in normal patients. FLIM microviscosity measurements of secreted mucus show much higher values than expected for a water‐filled interfiber spaces in a gel. This may indicate that mucus covering HBE cells resembles an amorphous colloid rather than a well‐structured hydrogel. Relative differences in mucus microviscosity between normal and CF, however, are much smaller than those reported for a bulk viscosity of mucus collected from patients.Support or Funding InformationSupported by the Cystic Fibrosis Canada (R.G.) and CIHR PJT148593 (E.B.).
The Horse & Rider: A Dynamic 3D Model for Studying the Role of Chronic Postural Asymmetries in Degenerative Osteoarthritis
Authors: Lee Alexander K. K. , Uhl Elizabeth W. , Osborn Michelle L. 
Published: April 2020

Chronic lamenesses in horses are often managed but not effectively treated. We hypothesize that (1) a causal relationship exists between the forces created by aberrant biomechanical positions and movements, including those caused by the rider, and degenerative joint diseases; and (2) adjusting the aberrant biomechanics will be therapeutic. To test our hypotheses, we constructed a dynamic 3D computer model using equine CT data and human CT data to model full body positions of horses with riders that can then be related to sites of tissue damage in equine lamenesses. The bones of the horse and human were reconstructed from CT data using the 3D visualization and analysis software, Avizo®. The individual bones were then imported into Autodesk® Maya® for re‐positioning into their respective organism, creating a 3D human model and a 3D equine model. A 3D rig was developed for each model separately to allow for accurate positioning and animation for each. The ribbon spine technique was used for the neck and spine on both models for increased accuracy in positioning and movement with the ability to rotate and bend the spines and necks into natural positions. The rigging used for both the horse and human were adopted to be manipulated using controls for quicker and easier positioning. Once the two rigs were completed, they were imported into a single file. The human rig and its matching skeleton were scaled in reference to the equine rig and its matching skeleton; and the human rigged model was positioned atop the rigged equine model to simulate a rider on its horse. The combined model was used to analyze the chronic postural abnormalities of a horse with cervical osteoarthritis using information from photographs of the horse being ridden at the trot. Findings included a markedly asymmetric stride, a dorsiflexed lower limb that indicates overloading of the front legs, and over‐flexion of the neck that correlated with sites of compressive degeneration in the cervical spine. Our long term goal is to expand the models for biomechanical analysis and to further develop the rigging protocol in order to expand its usefulness for both animal and human studies.Support or Funding InformationLouisiana State University School of Veterinary Medicine Charles V. Cusimano Equine Health Studies Program
Bioprospecting for Novel Natural Products in Ancient Non‐Actinobacteria
Authors: Hamrick Grayson S. , Hoang June , Kokona Bashkim , Charkoudian Louise K. 
Published: April 2020

Polyketides are a particularly important category of secondary metabolite, and alone comprise 20% of the top‐selling drugs around the world.
1 Polyaromatic (type II) polyketides incorporate aromatic rings into their structures and are synthesized in vivo by a cohort of enzymes called a synthase. Three enzymes remain constant in all type II polyketide synthases: ketosynthase (KS), chain length factor (CLF), and acyl carrier protein (ACP). These three enzymes are known as the minimal polyketide synthase (PKS) and form the basis for the production of all type II polyketides. The most well‐studied of these polyketides are produced by a phylum known as Actinobacteria. Some of the polyketides that these bacteria produce include tetracycline, erythromycin, and lovastatin. Unfortunately, Actinobacteria are difficult to work with in the lab and researchers have historically been unsuccessful in expressing their KS‐CLF complexes in robust heterologous hosts such as E. coli. In 2015, a study by the Charkoudian and Hillenmeyer labs showed that the gene clusters responsible for the transcription and translation of the minimal PKS are present in some anciently diverged strains of non‐Actinobacteria.
2 These strains are known as orphan strains; the sequences of the genes are known but the enzymes and the product they manufacture remain uncharacterized. Many of the uncharacterized products include those made by non‐Actinobacterial species. Additionally, many of the identified non‐Actinobacterial species originate in phyla such as the Firmicutes and Proteobacteria, which means that their gene products could be expressed in tractable, non‐Actinobacterial heterologous hosts, such as S. pyogenes (a Firmicute) or E. coli (a Proteobacteria). Thus, non‐Actinobacterial ancient strains seemed ripe for bioprospecting.One of the first non‐Actinobacterial species that this research considered was the strain Gloeocapsa sp. PCC 7428, a cyanobacteria whose genome was found to contain a putative PKS gene cluster in the original 2015 study. Herein we show that the Gloeocapsa KS‐CLF can be expressed and purified in E. coli in high titers. Further characterization has shown this KS‐CLF to have several valuable qualities. The successful expression of such a protein may open doors to solutions to the antibiotic resistance problem that were previously closed.Support or Funding Information1. This work was supported by the Arnold & Mabel Beckman Foundation Scholar Award to G.S.H. and NSF CAREER grant number CHE‐1652424 to L.K.C.
Characterization of an Acyl‐CoA:retinol Acyltransferase (ARAT) Activity in the Hepatopancreas of the American Lobster (Homarus americanus)
Authors: Dew Stephanie E. , Wells Collin K. , Honaker Olivia M. , Li Yuzhang 
Published: April 2020

Vitamin A (retinol) is a necessary nutrient in higher animals. In mammals, the pathways by which retinol is transported and metabolized are well described. One important reaction in these pathways is the conversion of retinol into retinyl esters, which occurs in several tissues, including the small intestine, liver, and eye. Little is known about the conversion of retinol into retinyl esters in invertebrates. We have identified an acyl‐CoA:retinol acyltransferase (ARAT) activity in the hepatopancreas of the American lobster (Homarus americanus). This activity shows a linear dependence on time and microsome concentration. It shows temperature dependence and an optimal pH of 6.5. We have characterized its acyl‐CoA preference and determined Km and Vmax values for both retinol and acyl‐CoA.Support or Funding InformationCentre College
Defining the molecular determinants required for RNA binding functions of the Bicaudal‐C (Bicc1) translational repressor protein
Authors: Dolma Sonam , Tonelli Tomasso , Dowdle Megan , Sheets Michael 
Published: April 2020

Bicaudal‐C (Bicc1) is an RNA binding protein that functions as translational repressor in all animal species. Its repression activities are required for a multitude of biological processes such as guiding the earliest steps of embryonic development and organ homeostasis. For example, defects in Bicc1 expression or its activities can give rise to polycystic kidney disease in humans, a disorder that is characterized by an abnormal collection of cysts in the kidney. In addition, Bicc1 functions in vertebrate embryos to control the translation of specific maternal mRNAs that encode key developmental regulatory proteins. During the early stages of vertebrate development defects in Bicc1 trigger cell fate changes that lead to embryonic malformations similar to some human birth defects. A general feature is that in all of its biological contexts Bicc1 binds to specific mRNAs and it is through this binding that it selects specific mRNAs for translational repression. My project is focused on defining the molecular determinants of the Bicc1 protein required for that specific mRNA binding. Recent results from the Sheets lab identified a 70 amino acid region of the Bicc1 protein, the KH2 domain that is required for mRNA binding. However, the particular molecular features within the KH2 domain important for RNA binding activity are unknown. To address this issue I am generating mutations of the KH2 domain that change evolutionarily conserved amino acid residues in all Bicc1 proteins. These mutant proteins will be analyzed with gel shift RNA binding assays in vitro as well as in vivo RNA binding experiments performed in Xenopus embryos. My goal is to define the amino acid residues in the KH2 domain that are responsible for providing specificity for Bicc1 binding to specific mRNAs, guide its repression activities and mediate its biological functions.Support or Funding InformationThis work is supported by a Hilldale Undergraduate Research Fellowship to SD and funding from NICHD (R01HD091921) to MDS. Megan Dowdle is supported by a SciMed GRS Advanced Opportunity Fellowship and Biotechnology Training Program of the National Institutes of Health (NIGMS T32GM008349).
The Impact of Aging on Bone Regeneration in Mice
Authors: Stewart Kennon , Watt James , Busse Emily , Simkin Jennifer , Hoffseth Kevin , Sammarco Mimi 
Published: April 2020

Older people are projected to outnumber children for the first time in U.S. history by 2030 (U.S. Census Bureau, 2017), creating a tsunami of people with reduced bone strength who are more susceptible to bone injury and fracture. Decreased bone quality has a multivariate causal path including loss of progenitor cells, reduced anabolic and catabolic capabilities of cells, and increased senescence. The age‐related cellular decline of bone begins prior to the onset of bone diseases such as osteoporosis. Thus, aging models exclusive of disease can be immensely beneficial in finding novel angles of approach for better bone healing during aging. With this in mind, our study uses the well‐characterized mouse digit amputation model. After amputation of the digit tip young mice are able to regrow patterned bone via direct ossification and soft tissue. However, regeneration in aged mice is delayed, and the regenerated bone is architecturally inferior showing decreased trabecular thickness, increased trabecular spacing, and an attenuated distal growth of the bone. Here we explore the impact of aging on regeneration and bone architecture, introduce a new method of analysis for bone architecture and discuss the advantages to alternative analysis for bone regeneration in the digit model.Support or Funding InformationCOBRE Grant ‐ 2P20GM103629 in addition to CELT LS‐LAMP Grant
Beta‐lactam Antibiotic Potentiation in MRSA Using Small Molecule Adjuvants
Authors: Miller Heather , Blackledge Meghan 
Published: April 2020

Methicillin‐resistant Staphylococcus aureus (MRSA) is a major public health concern, as it has developed resistance to our existing arsenal of antibiotics. These bacteria also form robust biofilms that are evident in chronic infections. MRSA expresses a master regulator of both antibiotic resistance and biofilm formation. This protein, Stk1, is a eukaryotic‐like serine/threonine kinase. We have identified novel small molecule inhibitors of Stk1 which combat both antibiotic resistance and biofilm formation. We hypothesized that if cultures were treated with an inhibitor of Stk1 as an adjuvant, then changes in resistance genes would be observed. In order to determine if the adjuvant, the antibiotic oxacillin, and/or cotreatment changed mRNA levels in MRSA, total RNA was purified and RT‐qPCR was used to measure changes in gene expression in key genes such as blaI, blaZ, mecI, mecA and pbp2. We observed differential gene expression that helps explain at the transcript level why treatment of MRSA with an adjuvant is effective at potentiating beta‐lactam antibiotics.Support or Funding InformationResearch reported in this presentation was supported by the National Institute Of General Medical Sciences of the National Institutes of Health under Award Number R15GM134503. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
Assessing Vascularization of the Heart of Young Pigs After Cardiac Injury by Ischemia/ Reperfusion
Authors: Matos-Ortiz Gabriela Nicole , Agnew Emma , Velayutham Nivedhitha , Moore Victoria , Riggs Kyle W. , Baker Richard Scott , Alsaied Tarek , Zafar Farhan , Yutzey Katherine E. 
Published: April 2020

Existing studies in axolotls, river fish, zebrafish and newts demonstrate that the heart has the capacity to self‐repair following injury, with an increase in existing cardiomyocyte proliferation (CM). Studies on neonatal mice show that the heart can repair during the first week of life following injury, suggesting mammals have a transient window of cardiac regenerative capacity. However, how these findings extend to large mammals remains poorly understood. Recent findings from our laboratory show CM cell‐cycling activity occurring up to 2‐months of age in pigs. This suggests large mammals may have an elongated capacity of heart regeneration compared with previously studied organisms. To assess regeneration after injury, we generated a large mammalian heart injury model. Eight postnatal 30‐day (P30) old pigs underwent ischemia (1hr)/ reperfusion (IR) injury via occlusion of the second diagonal branch of the left anterior descending artery (LAD) and controls underwent sham surgery. We hypothesized that blood vessel density would increase as an effort to normalize blood perfusion to the heart after injury. Tissue harvest occurred 4 weeks after injury and subsequent histochemical analyses were performed within 3 specific zones (scar, border, remote) to assess cell cycling, scar formation, and vessel density. Similar patterns of cell‐cycling activity, measured via pHH3 immunohistochemistry, were found between sham and IR models. Vessel density, as measured via lectin‐DAB staining, was also similar between sham and IR pigs; suggesting no evidence of new blood vessel formation after injury. Collagen deposition, suggestive of scar formation, was evident following injury in IR pigs. This novel approach in assessing cardiac repair following injury in young large mammals highlights that scar formation, rather than an alteration in cardiomyocyte proliferation, might be hindering cardiac regeneration in P30 pigs. Hence, targeting scar formation to understand heart repair mechanisms in young pigs might provide a better understanding of the self‐repair capability of the heart, with potential translation to human systems.Support or Funding InformationSURF‐American Heart Association Research Funding, Cincinnati Children’s Hospital Medical Center Research Foundation, Schmidlapp Young Women Scholarship