bluuebunny/crossref_metadata_2025_split huggingface dataset:
Does Clopidogrel Decrease the Efficacy of Carboxylesterase‐1 Prodrugs?
Authors: Laizure Steven C. , He Hui , Parker Roert B. 
Published: April 2020

It has previously been reported that clopidogrel is a potent inhibitor of the carboxylesterase‐1 (CES1) mediated hydrolysis of oseltamivir to its active metabolite based on in vitro inhibition studies. However, clopidogrel undergoes extensive first‐pass hepatic metabolism resulting in 85% of the dose being metabolized to clopidogrel carboxylate prior to reaching the systemic circulation. Thus, despite its potency as an inhibitor of CES1 enzyme activity, it is unlikely to affect the formation of oseltamivir or any other CES1‐substrate prodrug due to the low level of systemic exposure. We studied the inhibition of clopidogrel and its major metabolite, clopidogrel carboxylate, on another CES1‐substrate prodrug used in the treatment of multiple sclerosis, dimethyl fumarate. Our finding corroborates the previous study demonstrating potent inhibition of CES1 enzyme activity by clopidogrel, but also demonstrated a similar potency in the inhibition of enzyme activity by its major metabolite, clopidogrel carboxylate. In human recombinant CES1 enzyme 50 μM of clopidogrel or clopidogrel carboxylate inhibited the CES1‐mediated hydrolysis of dimethyl fumarate to monomethyl fumarate by 90%. Given that the maximum plasma concentration of the clopidogrel carboxylate metabolite is over 1000‐fold greater than clopidogrel after normal therapeutic doses, the primary inhibition of CES1 enzyme activity will be due to clopidogrel carboxylate and not clopidogrel. Furthermore, the maximum concentration of the clopidogrel carboxylate metabolite reported after a 600 mg clopidogrel loading dose was 71.4 μM, which would be in excess of the concentration required to achieve a 90% reduction in CES1 enzyme activity. Therapeutic dosing with clopidogrel is likely to inhibit the formation of the active metabolite of CES1‐substrate prodrugs resulting in therapeutic failure.Support or Funding InformationNone
Impact of freeze‐thaw on isolation of viable CD34+ cells from human cadaveric bone marrow
Authors: Bender Shannon , Gu Dongsheng , LaFontaine Michael , Johnstone Brian , Woods Erik 
Published: April 2020

IntroductionThe CD34 surface protein is a marker used to distinguish hematopoietic stem and progenitor cells (HSPCs). CD34+ cells are currently used in stem cell transplantation to treat hematological malignancies following myeloablation of bone marrow (BM). Ossium Health is creating a large and genetically diverse bank of cryopreserved deceased donor BM for medical purposes such as hematopoietic stem cell transplantation (HSCT) as well as biomedical research. Given the growing diversity of the bank, it would be advantageous to cryogenically store whole BM for future isolation of CD34+ cells possessing desirable genetic and demographic attributes. While current protocols are designed to select cells from fresh BM aspirated from live donors, there are no reports of selecting these cells from either previously frozen or cadaveric BM. Therefore, the purpose of this study was to determine (1) whether cadaveric BM is an acceptable source of CD34+ cells, (2) whether functional CD34 cells could be selected from previously frozen BM, and (3) the integrity of CD34+ cells following multiple freeze‐thaw cycles.MethodsWhole BM was obtained from vertebral bodies recovered from deceased donors consented for research use. CD34+ cells were selected from fresh and thawed BM by Ficoll separation followed by immunomagnetic bead isolation (EasySep, Stem Cell Technologies). Selected cells were subjected to further freeze‐thaw cycles and analyzed by flow cytometry and colony forming unit (CFU) assays to enumerate and evaluate CD34+ cell functionality, viability, purity, and population dynamics.Results and discussionResults thus far indicate no difference in CFU counts between fresh and thawed BM. Furthermore, 7‐AAD staining indicated a CD34+ average cell viability of 99+0% from fresh and 98+0% from thawed BM. Finally, long‐term hematopoietic stem cells (LT‐HSC) as defined by the cell surface protein markers CD34+ CD38‐CD90+CD45RA‐CD49f+, comprised 1.94% and 2.18% of CD34+ cells selected from fresh and previously frozen BM, respectively.Conclusions and future directionsTogether, these results indicate the suitability of deceased donor BM and the comparability of CD34+ cells selected from fresh and previously frozen BM. Ongoing experiments include evaluation of BM from additional donors as well as xenotranplantation studies in an irradiated immunocompromised mouse model. Validation of previously frozen cadaveric BM as an acceptable substrate for viable CD34+ cell isolation will enable a large and genetically diverse source of HSPC for both medical and research uses. Furthermore, due to the large volumes obtainable, BM from a single donor yields >100 million CD34+ cells, which is vastly greater than available from living donors, thus, providing a consistent source for study and to be used in therapeutic development.Support or Funding InformationThank you to the Marian University College of Osteopathic Medicine for their financial support of this project.
Perception of Pharmacology Education by Medical Sophomore students from 2000 to the present: impact of digital technology and educational policy
Authors: Arai Amy 
Published: April 2020

Pharmacology is one of the disciplines that medical students find difficult to study. Reasons are that there are numerous drug names to remember with a variety of side effects; even within a given class, each drug may have unique advantages or disadvantages with regard to drug‐drug interaction, pharmacokinetics, and contraindications. To make things worse, a deep anxiety about the medical licensing step 1 exam induces students to learn pharmacology through simple rote memorization by relying predominantly on pharm cards and online visual‐mnemonics interfaces, and too little time is spent trying to understand drug action in the context of physiology and cellular biochemistry. Thus, the goal of Pharmacology education seems not to be achieved during the basic science years. Our SIU School of Medicine medical curriculum has introduced unique curriculum methods. This includes problem‐based learning modules (PBLM) and group discussions as major curricular activities in Year 1 and 2 basic science teaching, with optional attendance at resource sessions. In this study, AAMC Graduation Questionnaires and students’ comments on Pharmacology curriculum at SIU School of Medicine are analyzed for the academic years 2001 to 2019. One question addressed if the basic science curriculum provides a sufficient foundation for clinical clerkship. The percentage of ‘Good’ and ‘Fair’ remained similar (35–38%) across 18 years. However, ‘Disagree’ was notably reduced from 16% to less than 10% around 2003, whereas the national average remained about the same. The nature of students’ perceptions on Pharmacology Education and their performance will be discussed in relation to the incorporation of digital technologies like online interfaces, cloud and social media into the curriculum, as well as the introduction of innovative teaching methods.Support or Funding InformationNone
Childhood Cancer Drug Development: Why Alternative Designs for Drug Development are Required
Authors: Houghton Peter J. 
Published: April 2020

In the US, the regulatory environment, through the Research to Accelerate Cures and Equity for Children Act (RACE for Children Act) requires FDA to develop a list of molecular targets and molecular targets of new drugs and biologics in development, that are determined to be substantially relevant to the growth and progression of pediatric cancer, and that may trigger the requirement for pediatric investigations. The intent is to engage Pharma in pediatric testing at an early stage in drug development. Under the RACE for Children Act, FDA may now require pediatric assessments when molecular targets under FDA review are substantially relevant to childhood cancer. This may require evaluation of up to 200 entities each year. However, it is clear that because a cancer has a particular ‘molecular driver’ it does not necessarily predict tumor response to a targeted agent. For example, some patients with low‐grade BRAF‐driven glioma have dramatic responses to MEK inhibitors, whereas others have some disease stabilization. Thus, factors other than the driver‐mutation appear important to tumor response. Consequently, to encompass clinical heterogeneity (genetic/epigenetic) that may determine response to treatment, a large number of models having similar molecular characteristics will have to be developed to allow adequate pre‐clinical evaluation of new targeted entities. Previously, the Pediatric Preclinical Testing Program (PPTP) conducted a retrospective analysis of over 2100 studies using 83 PDX and xenograft models and 67 drug entities undertaken using conventional testing (10 mice/group for solid tumor PDX models, 8 mice/group for leukemia PDX models). This analysis showed that use of one mouse per treatment group would accurately predict the group median response in 78% of experiments. Allowing a deviation of + or − one response category, the accuracy was 95% (Murphy B. et al, Cancer Res. 2016). Here we have prospectively tested the ‘single mouse’ experimental design to evaluate both small molecule and antibody‐drug conjugate drugs. We conclude that the ‘single mouse’ approach is highly accurate, and yields more information than traditional experimental approaches, particularly with respect to identifying biomarkers that associate with tumor response. Further, within the current resource constraints, this design allows up to 20 PDX models to be used for every one model used in the conventional testing design. Thus, the single mouse design potentially allows incorporation of disease heterogeneity that may be required for evaluating new agents under the RACE for Children Act.Support or Funding InformationNO1‐CM42216, UO1CA199297, CA165995 from NIH, and CPRIT RP160716
Cervical Vagal Nerve Stimulation Restores Ventilatory Pattern Variability in an E. Coli Sepsis Rodent Model.
Authors: He Zehong , Nethery David , Campanaro Cara , Dick Thomas E. , Jacono Frank J. , Hsieh Yee-Hsee Hsee 
Published: April 2020

Sepsis is a life‐threatening disease characterized by dysregulated systemic inflammation and organ failure. We have associated an increase in predictability of the ventilatory pattern variability (VPV) with progressive development of systemic inflammation leading to sepsis. Vagal nerve stimulation (VNS) is a proposed electro‐therapeuty that reduces circulating pro‐inflammatory cytokines, like interleukin‐1 beta (IL‐1β), that are associated with systemic inflammation. We hypothesize that the anti‐inflammatory effects of VNS will be reflected in reduced predictability of VPV. Urethane‐anaesthetized adult Sprague Dawley male rats (350–400g n=17) were instrumented for blood pressure (BP), heart rate (HR), and airflow (pneumotach). The left cervical vagus was dissected, isolated and prepared for electrical stimulation via nerve cuff. Each rat was subjected to a staircase protocol of monophasic electrical stimulus pulses (0.6 mA, 0.1 ms pulse width, 10 Hz) to identify the maximal current that was subthreshold for cardiac and respiratory responses. Four experimental cohorts were then established: 1) (+stim/+E. coli) (n=4), 2) (−stim/+E. coli) (n=5), 3) (+stim/−E. coli) (n=2), 4) (−stim/−E. coli) (n=6). In +stim groups, a stimulus train was initiated 10 min before and continued for 20 min after an E. coli solution (60 μg/kg rat; 50,000 cells suspended in normal saline) was injected intravenously (IV). The effects of stimulation on the systemic inflammatory response were determined by comparing serum cytokine levels prior to stimulation to those 90 min after inoculation and the effects on VPV was quantified from non‐linear complexity index (NLCI) prior to the blood draws. Cervical VNS reduced expression of IL‐1β from 102.33 ± 49 pg/ml to 69.50 ± 28 pg/ml and reduced NLCI (0.57 ± 0.04 bits to 0.48 ± 0.03 bits; p < 0.001). VPV became more predictable with the infusion of E. Coli and VNS in the presence of E. coli infusion reduced IL‐1β expression and decreased NLCI. These data suggest that VNS impacts both peripheral and central inflammation during E. Coli infusion.Support or Funding InformationSupported by U01 EB021960, and VA I01BX004197
Uncovering the DNA binding properties of the GATA4 and TBX5 transcription factor complex
Authors: Rodríguez Emili Patricia Rosado , Ríos Jessica Rodríguez , Martínez José Rodríguez 
Published: April 2020

Transcription factors (TFs) are essential master regulators of gene expression. These DNA binding proteins are fundamental for cellular differentiation during development. Eukaryotic TFs are notorious for not working alone and they often bind DNA as multimeric protein complexes to regulate gene expression. GATA4 and TBX5 are transcription factors that are central components of the gene regulatory network of human heart development and function. Recent studies have determined the binding specificity of these transcription factors as monomers. However, the DNA‐binding properties of the cooperative complex between GATA4 and TBX5 remain undetermined. Based on this, we want to know the intrinsic DNA binding preferences and the emergent properties that result from the cooperative complex formed by GATA4 and TBX5. We used a cell‐free protein expression system to produce GATA4 and TBX5, and we validated their monomeric and complex DNA‐binding activity through an Electrophoretic Mobility Shift Assay (EMSA). Subsequently, we successfully determined the in vitro DNA‐binding specificity of the GATA4:TBX5 complex using Systematic Evolution of Ligands by Exponential Enrichment (SELEX‐seq). For further studies, we will use this data to computationally predict genome‐wide recognition sites of this transcription factor complex. The findings of this study will help understand the DNA‐recognition rules of the GATA4:TBX5 complex and its potential roles in normal heart development.Support or Funding InformationThis research was supported by:NIH SC1GM127231NSF PR‐LSAMP #1400868
The Role of AMPK in Neuromuscular Junction Biology
Authors: Ng Sean Y. , Desjardins Eric M. , Steinberg Gregory R. , Ljubicic Vladimir 
Published: April 2020

The neuromuscular junction (NMJ) is the signaling apparatus between a motoneuron and the corresponding muscle fibers that it innervates. While its stability is vital for the neuromuscular system, the NMJ is susceptible to remodeling across the lifespan. Adenosine monophosphate‐activated protein kinase (AMPK) is a primary modulator of skeletal muscle phenotype, however the precise role of AMPK at the NMJ has yet to be investigated. Thus, the purpose of this study was to examine the role of skeletal muscle AMPK on NMJ biology across the lifespan. We employed 3‐ and 12‐month‐old muscle specific AMPKβ1β2 knock out mice (mKO, n = 3), age‐matched wild‐type (WT, n = 3) mice, as well as 22‐month‐old WT animals. Extensor digitorum longus (EDL) and soleus (SOL) muscles were dissected to represent fast glycolytic and slow oxidative tissues, respectively. These muscles were then whole‐mounted and immunolabelled for presynaptic neurofilament and synaptic vesicle 2A proteins, as well as postsynaptic acetylcholine receptors (AChR) revealed by □‐bungarotoxin. Approximately 25 NMJ images per muscle were acquired via confocal microscopy. In total, we conducted 3D analysis on over 400 NMJs to determine metrics of NMJ morphology. Postsynaptic analyses revealed an effect of age (p < 0.05) on the total amount of fragmented and ectopic NMJs in the SOL and EDL muscles of WT animals. Interestingly, mKO animals exhibited a significantly greater count of fragmented and ectopic NMJs in the SOL and EDL muscles, compared to their WT littermates. In fact, NMJs positive for postsynaptic abnormalities in EDL muscles of 12‐month‐old mKO animals were significantly greater than 22‐month‐old WT animals (p < 0.05), while SOL muscles were comparable. An effect of age (p < 0.05) on axonal blebbing and sprouting was observed in the EDL and SOL muscles, respectively. Additionally, NMJs with axonal blebbing were observed more frequently (p < 0.05) in the mKO animals compared to their age‐matched WT littermates. Quantitative real‐time PCR was also utilized to examine genes important for NMJ development and maintenance in the tibialis anterior muscles of 3‐month‐old WT and mKO animals (n = 10). Specifically, AChR□/ɣ, low‐density lipoprotein receptor‐related protein 4, rapsyn, and muscle‐specific kinase mRNA expression was significantly higher in the mKO animals relative to their WT counterparts. Our results suggest that AMPK is critical to the determination and maintenance of a healthy pre‐ and post‐synaptic apparatus in motoneurons and muscles of disparate phenotypes. Additionally, the loss of muscle‐specific AMPK exacerbates age‐induced postsynaptic alterations in faster, and more glycolytic muscle fibers.Support or Funding InformationCanadian Institutes of Health Research, Canada Research Chairs, and Ontario Graduate Scholarship.
Sfrp4 Expression in Thyroxine Treated Calvarial Cells: A Novel Target
Authors: Cray James , Durham Emily , Grey Zachary , Black Laurel , Howie R. Nicole , Barth Jeremy L. , Lee Beth S. 
Published: April 2020

Evidence suggests alterations of thyroid hormone levels can disrupt normal craniofacial development. Most data suggest the major targets of thyroid hormones to be the Htra1/Igf1 pathway. Recent discovery by our group suggests involvement of targets of the WNT pathway, specifically overexpression of antagonist Sfrp4 in the presence of excess thyroid hormone. Here we aimed to model these interactions in vitro to determine if thyroid hormone directly drives Sfrp4 expression in cells relevant to craniofacial development. Affymetrix transcriptional profiling found Sfrp4 to be overexpressed in cranial suture derived cells stimulated with thyroxine in vitro. Bioinformatics interrogation of the SFRP4 promoter identified multiple putative binding sites for thyroid hormone receptors. Experimentation with several cell lines demonstrated that thyroxine treatment induced Sfrp4 expression. However, protein levels did not correlate with induction of gene expression, demonstrating that Sfrp4 mRNA and protein levels are not tightly coupled. Transcriptional and protein analyses demonstrate thyroid hormone receptor binding to the proximal promoter of the target gene Sfrp4. Interestingly functional analysis of stem cells, fibroblasts, or primary culture cells after thyroxine stimulation for alkaline phosphatase activity shows only pre‐osteoblasts are pushed to become positive after thyroid hormone stimulation, suggesting cell type susceptibility. Finally, we added recombinant SFRP4 to pre‐osteoblasts in combination with thyroxine treatment and observed a significant decrease in alkaline phosphatase positivity, suggesting SFRP4 may be a key regulatory molecule that prevents thyroxine driven osteogenesis. The implications for this relationship indicate SFRP4 as a potential diagnostic or therapeutic target for hyperostotic craniofacial disorders.Support or Funding InformationThe authors would like to thank Xing Ming Shi for isolation of the bone marrow mesenchymal cells. The following funding sources were used for this study a grant from the Cleft Palate Foundation, NIH/NIDCR R03DE023350A, 5T32DE017551, and F31DE026684, NIH/NCATS UL1 TR000062, NIH/NIGM P30GM103331, GM103342 and GM103499, and MUSC’s Office of the Vice President for Research.
Cancer mutations inhibit the coactivator activity of p300 core
Authors: Hall Meghan , Shanle Erin K. 
Published: April 2020

p300 is an important transcription coactivator that enhances gene expression by acetylating histones through its catalytic core domain. p300 is mutated in some cancers, and cancer mutations in the histone acetyltransferase (HAT) domain can inactivate the coactivator function of the protein. However, many mutations in the p300 catalytic core that have been observed in human tumors have not been characterized for functional effects on p300 coactivator activity. The goal of this study was to screen cancer mutations using a previously published system in which the p300 catalytic core is fused to deactivated Cas9 (p300core‐dCas9). Cancer mutations in the bromodomain, PHD/RING domain, and histone acetyltransferase (HAT) domain were created in the p300core‐dCas9 protein. After the addition of guide RNA targeting p300core‐dCas9 to the promoter of a given target gene, quantitative PCR was performed to measure the coactivator function of the wild‐type and mutant proteins. Mutations in the bromodomain showed variable effects on gene expression activation, while mutations in the PHD/RING domain and HAT domain were inhibitory. These results demonstrate that cancer mutations in the p300 core can alter p300 function and provide insight into the effects of cancer mutations that occur outside of the HAT domain.Support or Funding InformationLongwood University Faculty Research and Development Grant
DUAL ACTING COX‐2 AND SOLUBLE EPOXIDE HYDROLASE INHIBITOR ATTENUATES GLOMERULAR INJURY IN FOCAL SEGMENTAL GLOMERULAR SCLEROSIS
Authors: Stavniichuk Anna , Khan Md Abdul H. , Jankiewicz Wojciech K. , El-Meanawy Ashraf , Yeboah Michael M. , Savchuk Olexiy , Hwang Sung H. , Hammock Bruce D. , Imig John D. 
Published: April 2020

Proteinuric glomerular diseases like focal and segmental glomerulosclerosis (FSGS) are the leading cause of chronic and end‐stage kidney diseases. FSGS is characterized by glomerular injury, and, at the clinical level, a classic hallmark of FSGS is proteinuria. Although FSGS is associated with high morbidity and mortality, treatments are limited that emphasize the need for novel therapies. We developed a novel dual modulator, PTUPB that concurrently acts as a soluble epoxide hydrolase (sEH) inhibitor as well as a cyclooxygenase‐2 inhibitor (sEHi/COXi). PTUPB has previously demonstrated robust kidney actions including reduction of glomerular injury in diabetic nephropathy. In the current study, we investigated renal actions of PTUPB in a genetic FSGS model, ROP Os/+ mice. ROP Os/+ mice developed proteinuria and glomerular injury between 16–20 weeks of age (8‐fold higher albuminuria vs. WT). We treated 20 weeks old male wild type (WT) and 20 weeks old ROP Os/+ mice with vehicle or PTUPB (10 mg/kg/d, i.p.; n=4–5/group)for 4 weeks. At the end of the 4 week treatment period urine, blood, and kidney tissue were collected for biochemical and histological analysis. The glomerular injury was assessed from albuminuria, histopathology, mRNA expression of proteins critical for glomerular structural integrity and function. Vehicle treated ROP Os/+ had 10‐fold higher albuminuria compared to WT and PTUPB treatment markedly attenuated albuminuria in ROP Os/+ mice by 70%. The ROP Os/+ mice developed glomerular injury with a 3‐fold higher glomerular injury score compared to WT and PTUPB treatment decreased glomerular injury by 28%. We further demonstrated that there was 20–30% lower renal mRNA expression of nephrin, podocin, and synaptopodin in vehicle‐treated ROP Os/+ mice, and PTUPB treatment improved the increased expression for these glomerular proteins in ROP Os/+ mice. These findings demonstrate that the dual modulator PTUPB that concurrently inhibits sEH and COX‐2 has exciting potential to treat FSGS and related glomerular diseases.Support or Funding InformationDK103616 and Dr. Ralph and Marian Falk Medical Research Trust Bank of America, N.A. to John D. Imig