bluuebunny/crossref_metadata_2025_split huggingface dataset:
Loss of HAND2 and HOXD13 Binding Sites in the ZPA Regulatory Sequence (ZRS) is not Sufficient to Disrupt Activity in the Limb
Authors: Ball Kathryn F. , Underhill Abbie K. , Pira Charmaine U. , Oberg Kerby C. 
Published: April 2020

The ZPA regulatory sequence (ZRS) is a limb‐specific enhancer that localizes Sonic Hedgehog (SHH) expression to the zone of polarizing activity (ZPA), which consequently regulates anteroposterior patterning in the developing limb. ZRS deletion lacks SHH expression in the limb resulting in loss of posterior limb structures, while ZRS single nucleotide variants (SNVs) can lead to ectopic SHH expression causing preaxial polydactyly (PPD). However, the ZRS mechanism of action is not well characterized. HAND2 and HOXD13 are key transcription factors in SHH regulation that bind to the ZRS, potentially modulating SHH transcription. The present study’s objective was to determine the role of HAND2 and HOXD13 binding sites in ZRS function. To evaluate ZRS enhancer activity, ZRS was cloned into an enhancer‐GFP reporter construct. HAND2 and HOXD13 binding sites were altered using site‐directed mutagenesis, and contructs were electroporated into presumptive limb buds in Hamburger‐Hamilton stage 14 chicken embryos. GFP expression was evaluated 48 hours post‐electroporation. ZRS with mutated HAND2 and HOXD13 binding sites retained activity in the limb bud in a pattern that overlapped ZRS activity. This is unexpected since HAND2 and HOXD13 are known to be critical for SHH expression. Future work is needed to clarify the binding site(s) critical to activity and to characterize the factors involved in ZRS function.Support or Funding InformationSupported in part by a grant from the Department of Pathology, Loma Linda University.
SPARC: Neural elements mediating side effects during cervical vagus nerve stimulation in the pig
Authors: Nicolai Evan Neil , Settell Megan L. , Knudsen Bruce E. , Pelot Nicole A. , Grill Warren M. , Williams Justin C. , Ludwig Kip A. 
Published: April 2020

Vagus nerve stimulation (VNS) is FDA‐approved and CE‐marked for a diverse set of conditions including epilepsy, depression, heart failure, and others. Despite widespread adoption and perceived efficacy, a post‐hoc analysis of patients treated for heart failure using VNS revealed that only 12% of patients showed stimulation evoked heart rate changes one‐year post‐implant. This finding is not unexpected given that A‐type nerve fibers have the lowest activation thresholds, and they innervate neck muscles, causing contractions that patients will not tolerate. Therefore, muscle responses at lower amplitudes may have prevented application of higher amplitudes required to achieve cardiac effects in clinical trials of VNS for heart failure. Understanding therapy‐limiting side effects to VNS and finding ways to mitigate them are critical for optimizing VNS for clinical therapies.We developed a swine model of VNS which closely approximates the size and fascicular organization of the cervical vagus nerve observed in humans. The clinical LivaNova stimulation electrode was used as advised by a practicing neurosurgeon. We hypothesized that neck muscle responses are mediated by somatic branches of the vagus nerve as opposed to direct electrical activation of the muscle fibers. Further, we hypothesized that stimulation evoked responses depend on the location of the stimulation electrode on the vagus nerve. We measured compound nerve action potentials using intrafascicular electrodes placed within the vagus to identify activation of different nerve fiber types by conduction velocity. The downstream effects of nerve fiber activation, i.e. changes in heart rate and neck muscle responses (EMG), were measured using an arterial catheter and intramuscular electrodes, respectively. We then used a neuromuscular junction blocking agent, as well as systematic transection of somatic vagus nerve branches, to specifically identify the pathways causing neck muscle contractions.Two pathways contributed to VNS‐evoked neck muscle responses: activation of nerve fibers in the recurrent laryngeal branch of the cervical vagus nerve at low amplitudes and in the superior laryngeal branch at higher amplitudes. Lesser activation of the superior laryngeal branch could be achieved by moving the stimulation electrode caudally down the length of the vagus. This work represents a step towards mitigating intolerable side effects of VNS, thereby allowing maximization of intended target engagement and therapeutic benefit.Support or Funding InformationThe Defense Advanced Research Projects Agency (DARPA) Biological Technologies Office (BTO) Targeted Neuroplasticity Training Program under the auspices of Doug Weber and Tristan McClure‐Begley through the Space and Naval Warfare Systems Command (SPAWAR) Systems Center with (SSC) Pacific grants no. N66001‐17‐2‐4010, and the NIH SPARC Program Award OT2 OD025340.
Understanding the Conformational Changes of Kinesin‐5
Authors: Robinson Kingston , Griffin Jessica , DeVry Jenelle , Chaney Joseph 
Published: April 2020

Human Kinesin‐5 (Eg5), an anticancer drug target, is key to the assembly of the bipolar spindle during mitosis. Its promise as a clinical target comes from the fact that it is involved only in metaphase and not interphase like some of the leading clinical drugs; however, the overall mechanism of inhibition needs further exploration. Inspection of the crystal structures of Kinesin‐1 dimers reveal the addition of strands to the central β‐sheet is found in one head and absent in the other; this previously unrecognized structural asymmetry between the two heads may suggest a regulatory mechanism involving the N‐terminal Neck‐Linker (NL) and the C‐Terminal Cover‐Neck. These structural changes in the neck‐linker and cover‐neck of Kinesin‐5 have not been reported. Thus, the importance of establishing this conformational switch in Kinesin‐5 requires additional experiments for understanding and molecular validation. But first, we must understand how changes in the neck‐linker modulate kinesin function. We have inserted three residues (+DAL) into the middle of the neck‐linker of dimeric Kinesin‐5 (1‐513) and KHC (1‐560). Our results indicate these neck‐linker mutants can allosterically regulate kinesin ATPase activity. Our future goal is to test the response to inhibition by current anticancer inhibitors such as S‐trityl‐L‐cysteine.Support or Funding InformationBUILD Technicians supported under grant #RL5GM118966 and BUILD undergraduate students under grant #TL4GM118968. An Institutional Development Award (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health under grant #5 P20 GM103424‐15 and 3 P20 GM103424‐15S1. The Louisiana Cancer Research Consortium (LCRC) and National Center for Research Resources RCMI program Grant #8 G12MD007595 and the Louisiana Biomedical Research Network Grant #8P20GM103424. The National Institute of Minority Health and Health Disparities of the National Institutes of Health under award #G12MD007595. Students are supported by GRAD Star Scholar Mini Grants through Center for Undergraduate Research and Graduate Opportunity (CURGO).
Assessing Sialic Acid Uptake in an In Vitro Model of GNE Myopathy
Authors: Goines Briana N. , Sattler Kristina M. , Crowe Kelly E. 
Published: April 2020

Mutations in the GNE gene leads to GNE myopathy (GNEM), a rare autosomal disease that causes skeletal muscle weakness. The GNE gene encodes an enzyme for sialic acid (SA) biosynthesis, causing a reduction of SA when biallelic GNE mutations are present. Currently, gene therapies are in development to replace the mutated GNE gene and restore SA production. Determining that SA can be used as a biomarker would allow researchers to quantify the success of the gene therapy. Our lab is attempting to take a panel of SA‐binding proteins called lectins that are known to show a binding difference in muscle cells from GNEM patients as compared to cells from their unaffected family members and determine which lectin best reflects SA changes in GNEM patients. Preliminary data does not show the expected difference in SA levels between cells derived from GNEM patients and their unaffected family members. As this could be from SA scavenging from serum in the media, we have cultured these GNEM patient‐derived myotubes in serum‐free media prior to fixation to assess SA scavenging from serum in this model. Here, we show the extent to which SA scavenging from serum affects cell surface sialylation in GNEM patient cells in vitro.Support or Funding InformationThis work was supported by funds from the Beta Beta Beta Research Scholarship Foundation Fund.
Ketone Bodies Attenuate Wasting in Diverse Models of Atrophy
Authors: Koutnik Andrew Paul , Poff Angela M. , Ward Nathan P. , Deblasi Janine M. , Soliven Maricel A. , Romero Matthew A. , Roberson Paul A. , Fox Carl , Roberts Michael D. , D’Agostino Dominic P. 
Published: April 2020

BackgroundCancer anorexia cachexia syndrome (CACS) is a distinct atrophy disease negatively influencing multiple aspects of clinical care and patient quality of life. Although it directly causes 20% of all cancer‐related deaths, there are currently no model systems that encompass the entire multifaceted syndrome, nor are there any effective therapeutic treatments.MethodsA novel model of systemic metastasis was evaluated for the comprehensive CACS (metastasis, skeletal muscle and adipose tissue wasting, inflammation, anorexia, anemia, elevated protein breakdown, hypoalbuminemia, and metabolic derangement) in both males and females. Ex vivo skeletal muscle analysis was utilized to determine ubiquitin proteasome degradation pathway activation. A novel ketone diester (R/S 1,3‐butanediol acetoacetate diester) was assessed in multifaceted catabolic environments to determine anti‐atrophy efficacy.ResultsHere, we show that the VM‐M3 mouse model of systemic metastasis demonstrates a novel, immunocompetent, logistically feasible, repeatable phenotype with progressive tumor growth, spontaneous metastatic spread, and the full multifaceted CACS with sex dimorphisms across tissue wasting. We also demonstrate that the ubiquitin proteasome degradation pathway was significantly upregulated in association with reduced IGF‐1/insulin and increased FOXO3a activation, but not TNF‐α‐induced NF‐κB activation, driving skeletal muscle atrophy. Additionally, we show that R/S 1,3‐butanediol acetoacetate diester administration shifted systemic metabolism, attenuated tumor burden indices, reduced tissue catabolism, and mitigated comorbid symptoms in both CACS and cancer‐independent atrophy environments.ConclusionsOur findings suggest the ketone diester attenuates multifactorial CACS skeletal muscle atrophy and inflammation‐induced tissue catabolism, demonstrating anti‐catabolic effects of ketones bodies in multifactorial atrophy.Support or Funding InformationThis work was supported by Disruptive Nutrition (Grant #: 61431150), Florida High Tech Corridor (Grant #: FHT 18‐15), Donner Foundation (Grant #: 61431151), and Office of Naval Research (Grant #: N00014‐18‐1‐2701). Additional funding for muscle molecular analysis were provided by discretionary laboratory funds of MDR.
Using Edpuzzle as a Method to Improve Student Engagement and Performance in the Introductory Biochemistry Laboratory
Authors: Shelby Shameka J. , Fralish Zachary 
Published: April 2020

Various engagement tools have been commonly used within biochemistry courses to increase student engagement and maximize student exposure to material within the time constraints of a class session. These time constraints also extend to the laboratory component of these courses which often leads to increased levels of anxiety for students. One method that has been used successfully in the past to increase student understanding and performance during laboratory experiments is the utilization of pre‐laboratory video lectures. While the use of pre‐laboratory videos has been shown to be beneficial for students, instructors often have no efficient way of assessing student understanding of the video content prior to the laboratory session. Edpuzzle is a free online service that allows for instructors to upload videos and incorporate questions into videos at various points within the video to assess student understanding. In an effort to increase student performance in an introductory biochemistry laboratory, edpuzzle was adapted to engage students with the material in preparation for the laboratory session. Prior to each laboratory session, students viewed an edpuzzle video and answered questions to assess their understanding of the experimental details and concepts that would be covered in the lab. Surveys were conducted to determine student satisfaction, learning outcomes, and level of anxiety associated with the laboratory as a result of using edpuzzle as a laboratory preparation tool. Students reported high satisfaction rates, increased confidence during laboratory experiments, and decreased anxiety about the laboratory overall. The results suggest that students viewed the edpuzzle application as a valuable component of their laboratory preparation routine. Due to the flexibility of edpuzzle, it can be adapted for use in other chemistry or biology laboratories at various levels. In addition, edpuzzle can also be used as a supplementary tool for expanding lecture content.Support or Funding InformationFlorida Southern College Department of Chemistry, Biochemistry, & Physics
Compensatory Enzyme Expression In Response To Chronic Ethanol Intake And Partial Hepatectomy Disrupts Zone‐Specific Organization Of Lobular Metabolism In The Rat Liver
Authors: Barnhart Benjamin , Chopra Chetna , Achanta Sirisha , Hoek Jan B. , Vadigepalli Rajanikanth 
Published: April 2020

Regulation of liver metabolism is heterogeneous across lobular zones. In response to the acute metabolic challenge of partial hepatectomy (PHx), hepatocytes enact a global transcriptional program both to compensate for and replace lost tissue mass. Chronic ethanol intake has also been shown to rewire transcriptional programs in hepatocytes, with suppressive effects on hepatocyte proliferation and blunted regeneration post PHx. However, it is not clear how zone‐specific metabolism transiently adjusts to ensure PHx‐induced regeneration, or how zone‐specific metabolism is adapted due to chronic insult by ethanol intake.Male Sprague‐Dawley rats were fed a Lieber‐DeCarli liquid diet containing ethanol or carbohydrate for 5–6 weeks prior to 70% PHx in which left lateral and medial lobes were surgically removed. Liver tissue samples were collected at the time of surgery and after 24 hours of recovery. Laser‐capture microdissection was used to collect regions of tissue from eight distinct lobular zones between portal triads and central veins, and high throughput qPCR was used for transcriptional analysis.We show here that regrowth after PHx involves multiple patterns of compensatory gene expression of enzymes from most major metabolic pathways. Genes whose expression is normally restricted to a particular zone responded to PHx in a wide range of patterns, e.g., by increasing expression with no change of zone (e.g., Arg1), expanding expression to nearby zones (e.g., Glul), or transient uniform expression across the whole lobule (e.g., Ldha). We tested whether chronic ethanol intake dysregulates zonation patterns of gene expression by exaggerating, dampening, redistributing, or eliminating preexisting lobular heterogeneity. After PHx, chronic ethanol intake dysregulated zonation of expression patterns in a variety of ways, including a reversal of healthy adaptations (e.g., Pck1). Other ethanol‐induced patterns of expression seen were suppression of PHx‐induced expression (e.g., Ldha) and redistribution of expression (e.g., Arg1).The dysregulation of zonated metabolic gene expression patterns in chronic‐ethanol‐fed rats suggests that zone‐specific compensatory mechanisms may contribute to the ethanol‐impaired liver regeneration post‐PHx. Overall, this work shows the transient changes in zone‐specific transcriptional regulation in response to PHx, and identifies genes whose zone‐specific expression is affected early in ethanol‐induced disease progression. These heterogeneous changes within the hepatic lobule could be a marker of ethanol‐induced adaptation preceding more severe impairment.Support or Funding InformationSupported by NIH NIAAA R01 AA018873, T32 AA007463, and NIBIB U01 EB023224.
Skeletal muscle‐specific PHD2 deficiency improved running endurance in mice
Authors: Kubota Takahiro , Nunomiya Aki , Osana Shion , Takada Hiroaki , Nagatomi Ryoichi 
Published: April 2020

IntroductionHypoxic training is one of the methods supposed to improve the performance of endurance athletes. Under a normoxic condition, prolyl hydroxylase domain containing protein 2 (PHD2) leads hypoxia inducible factor (HIF) to proteasomal degradation. Under hypoxia, PHD2 becomes inactive and let HIF be stabilized. HIF, then, promotes the expression of downstream genes, resulting in enhanced glycolysis, suppressed electron transport system, promotion of red blood cell production and angiogenesis. In PHD2 systemic knock‐out mice, hypoxic responses are elicited even under normoxia due to stabilized HIF. Interestingly, an enhanced endurance training effect was confirmed in PHD2 systemic knock‐out mice (Nunomiya et al. 2017). In PHD2 systemic knock‐out mice, calcineurin dependent increase in the proportion of type I fibers in skeletal muscles has been confirmed. (Shin et al. 2016). Whether the shift in fiber type, and more importantly the training effect is dependent on the hypoxic response within the muscle fibers or on the systemic hypoxic response has not been elucidated. The purpose of this study was to examine the effect of hypoxic response in skeletal muscle on the endurance training effect.MethodIn order to delete PHD2 specifically in skeletal muscle, doxycycline was administered for 18 days to PHD2flox/flox/Acta1rtTA/tetO‐Cre mice. The running time on a rodent treadmill until exhaustion was measured 8–10 days after the final administration of doxycycline before training and after 4 weeks of treadmill training, Endurance testing protocol: treadmill speed was set at 10 m/min for the initial 30 min, then after the speed was increased by 2 m/min every 15 min with no inclination. Training protocol: training was performed 5days per week for 4 weeks. Mice ran for 30 min per day. The running speed was 18m/min with 5 degrees uphill inclination.ResultIn PHD2flox/flox/Acta1rtTA/tetO‐Cre+/− mice, a significant reduction in PHD2 expression in gastrocnemius, tibialis anterior muscle, and soleus was confirmed. Skeletal muscle specific PHD2 knocked out mice had significantly increased endurance time compared to control mice before training. But there was no significant difference in the training effect between skeletal muscle‐specific PHD2‐deficient mice and control mice. There was no significant difference in training effect between skeletal muscle‐specific PHD2‐deficient mice and control mice.ConclusionHypoxic response in the skeletal muscle resulted in a higher endurance capacity without training. There was no additive training effect in the mice the mice with hypoxic response. It was suggested that systemic hypoxic response may be responsible for the additive endurance training effect confirmed in the previous study.
Understanding Sarcospan Protein Function by Exposure of Mice to Beta‐Adrenergic Stress Conditions
Authors: Wacker Amanda L. , Elsheikh Salma , Valera Isela C. , Hwang Hyun Seok , Parvatiyar Michelle S. 
Published: April 2020

IntroductionSarcospan (SSPN) is a core component of the dystrophin‐glycoprotein complex (DCG) located at the sarcolemma of muscle fibers. SSPN has been shown to play a major role in membrane stabilization of the heart and other striated muscles, however many of its additional functions remain unknown. Previous studies have shown that SSPN deletion diminishes cardiac β‐adrenergic responsiveness, however it elicits increases in cardiac mass (JAHA, 2015 Dec 23;4(12). Blunted β‐adrenergic responsiveness in SSPN‐null hearts appears to be part of a complex phenotypic presentation that occurs downstream of alterations in sympathetic drive, and a propensity toward metabolic syndrome and insulin resistance.MethodsHere, we document the whole‐body effects of ß‐adrenergic stimulation in three‐month‐old WT and SSPN‐null male and female mice after treatment with an isoproterenol (ISO) dosing regimen (8 mg/kg of body weight/day for 5 days) with tissues collected 2 weeks after treatment and examined for alterations in histology, gene and protein expression. After the treatment regimen, morphometric measurements were recorded and cardiac function assessed by echocardiography.ResultsAfter ISO treatment significant differences were found in sympathetic and cholinergic receptor expression in SSPN‐null cardiac and white adipose tissue (WAT) that were distinct from WT. The response of SSPN‐null mice to ISO treatment displayed distinct sex‐differences. After ISO treatment, WT and SSPN‐null male mice exhibited similar levels of WAT lipolysis, evidenced by loss of body and reduced size of epidydymal fat pads. However, female SSPN‐null mice were protected from ISO treatment induced loss in body weight. Furthermore, WT females had reduced spleen weights after ISO treatment, indicative of normal immune cell migration to other tissues, whereas spleen weights were unchanged in SSPN‐null females after ISO treatment suggesting suppressed immune responsiveness.DiscussionThe goal of this study is to better understand how the cardiac function of SSPN‐null mice appears largely unaltered by β‐adrenergic stimulation, whereas other tissues display overall normal responsiveness to ISO treatment. Since lipolysis is induced through activation of β‐adrenergic receptors, we will examine associated markers in untreated and β‐adrenergic‐stimulated WT and SSPN‐null mice of both sexes.Support or Funding InformationResearch Support: Florida State University, FSU CRC FYAP Award, and AHA 16SDG29120002.
Durable Changes to Airway Mucus Secretion Following a Single Cholinergic Challenge
Authors: Collins Emily , Liao Yan-Shin , Guevara Maria Valentina , Schurmann Veronica , Atanasova Kalina , Reznikov Leah R. 
Published: April 2020

Cholinergic signaling in the airway elicits robust airway smooth muscle contraction and mucus secretion. In our previous work, we showed that re‐exposure of the airways to a cholinergic agonist 48 hours post initial challenge exaggerated airway narrowing. Here we test the hypothesis that re‐exposure to a cholinergic agonist exaggerates mucus secretion. To test this hypothesis, we challenged male and female pig airways with the synthetic cholinergic agonist bethanechol. Forty‐eight hours later, we re‐exposed the airways and assessed mucus secretion ex vivo using jacalin lectins. We found that re‐exposure mildly increased mucus secretion. The increase in mucus secretion was not associated with an impairment or alteration in mucociliary transport. We also stained tracheal cross sections with antibodies directed against mucin5B and mucin5AC, the major secreted glycoproteins that comprise mucus. Our ongoing studies suggest that cholinergic challenge had no major effect on the amounts of mucin5B or mucin5AC. Critical next steps include measuring the amount of mucins in the lung lavage fluid, as well as examining key components of the cholinergic signaling cascade.Support or Funding InformationHL119560, OD023854