bluuebunny/crossref_metadata_2025 huggingface dataset:
The Function of Hepatocellular Uptake Transporters is Affected by Free Cholesterol
Authors: Idowu Jessica Y. , Hagenbuch Bruno
Published: April 2020

Background

We have previously shown that several transporters located in the basolateral membrane of human hepatocytes e.g. NTCP, OCT1, OATP1B1, and OATP1B3 interact with each other. These interactions are due in part to their co‐localization in detergent resistant membrane domains and impact both function and expression. Understanding how transporter interaction and altered membrane cholesterol content impact the function of these drug uptake transporters has implications regarding pharmacological studies, drug development, and drug‐drug interaction predictions.

Aim

We wanted to test how both the addition and removal of free cholesterol to and from the plasma membrane alters the function and localization of drug transporters.

Methods

The function of NTCP or OCT1 was measured using HEK293 Flp‐In cells stably expressing either transporter. We treated the cells with methyl‐b‐cyclodextrin (MCD) to remove cholesterol or with a cholesterol‐MCD complex to add cholesterol. Lipid rafts were isolated from HEK293 Flp‐In cells or frozen human liver samples by homogenization in 1% Triton X‐100 and sucrose gradient centrifugation. Gradient fractions were analyzed by western blotting using positive and negative lipid raft markers as well as transporter specific antibodies.

Results

Treatment with MCD stimulated uptake by NTCP while inhibiting uptake by OCT1. Subsequent treatment of the MCD treated cells with the cholesterol‐MCD complex restored function in part for both NTCP and OCT1. Removal and restoration of membrane free cholesterol also impacted the co‐localization of the transporters within the detergent resistant membrane domains. We confirmed the differential localization of transporters in detergent resistant membranes comparing samples of normal and fatty human livers.

Summary and Conclusion

Both adding or removing cholesterol alters transport function in a transporter‐dependent way. Furthermore, the localization of transporters in lipid rafts is different in normal and in fatty human livers. These results suggest that drug disposition is affected by fatty liver disease and that some drugs may be cleared faster or slower in patients with increased free cholesterol in the liver, due to NAFLD or NASH.

Support or Funding Information

R01GM077336 and P30GM118247
Creation and Curation of an Improved, Custom MALDI‐TOF Edwardsiella Library
Authors: Freese Kaitlin K. , Lawrence Jim , Rogge Matt
Published: April 2020

Edwardsiella is a Gram‐negative rod, mannitol‐negative, hydrogen sulfide producing member of the Enterobacteriaceae family. E. tarda was the initial member of the Edwardsiella genus until E. ictaluri, E. hoshinae, E. piscicida, and E. anguillarum were identified as individual species distinct from E. tarda. The five known species of Edwardsiella are genetically and phenotypically similar to one another. We used Matrix‐Assisted Laser Desorption Ionization‐Time of Flight (MALDI‐TOF) mass spectrometry to create a library of spectral entries containing 5 E. tarda, 5E. ictaluri, 4 E. piscicida, and 5 E. anguillarum isolates. Nineteen Mass Spectral Profiles (MSPs) were assembled from 101 E. tarda, 139 E. ictaluri, 123 E. piscicida, and 112 E. anguillarum MALDI‐TOF spectra. This new library, named the UWSP Edwardsiella library, was challenged with the original 19 Edwardsiella isolates. The UWSP Edwardsiella library correctly identified 19 out of 19 isolates. In contrast, currently available commercial libraries identified the same 19 isolates as E. tarda. Scores generated with the current commercial library averaged just over 2.0. The UWSP Edwardsiella Library scores ranged between 2.50 to 2.75. After initial library creation, additional Edwardsiella isolates were obtained and used to challenge the UWSP Edwardsiella library. All 16 of those isolates were correctly identified with identification scores between 2.51 and 2.81. Here we report the creation of a custom Edwardsiella MALDI‐TOF library with improved ability to accurately differentiate and identify individual Edwardsiella species.

Support or Funding Information

UWSP Office of Student Creative Activity and Research
Age‐Associated Changes in Cerebrovascular Function and BK Channel Function are Sex‐Specific
Authors: Reed Joseph , Romero Damian , Sriramula Srinivas , Pabbidi Mallikarjuna
Published: April 2020

Large conductance potassium (BK) channels are known to regulate the myogenic reactivity, yet, it is not clear if aging alters the myogenic reactivity via the BK channel in males and females. Thus we hypothesize that age‐associated changes in BK channel subunits modulate the myogenic reactivity in a sex‐specific manner. We used vascular reactivity, patch‐clamp, and biochemical methods to measure the myogenic reactivity, BK channel function, and expression, respectively, in cerebral vessels of adult and aged male and female Sprague Dawley rats. The results suggest that aging and ovariectomy (OVX) exaggerated the myogenic reactivity of middle cerebral arteries (MCA) in females but attenuated it in males. Aging induced outward eutrophic remodeling in females but inward hypertrophic remodeling in males. Aging decreased total, Kv, BK channel currents, and spontaneous transient outward currents (STOC) in vascular smooth muscle cells isolated from females, but not in males. Aging increased BKα subunit mRNA and protein content both in males and females. However, aging decreased BKβ1 subunit protein and mRNA levels in females only. In males, BKβ1 mRNA is increased, but protein is decreased. Iberiotoxin‐induced MCA constriction is lower in aged females but higher in aged males. OVX decreased, but chronic supplementation of estradiol (OVX+17βE rats) restored BK channel expression and function. BKβ1 mRNA is decreased in cerebral vessels of OVX rats that is consistent with decreased protein content. However, supplementation with 17βE did not increase mRNA, but protein is not decreased suggesting that post‐translational mechanisms contribute to the preservation of the protein. Overall the results suggest that aging and OVX‐associated downregulation of the BKβ1 expression and function in females exaggerates the myogenic reactivity of MCA. However, age‐associated increase in BK channel function in males attenuates the myogenic reactivity of MCA.

Support or Funding Information

This work was supported by, in part, by a grant from the Intramural Research Support Program from UMMC and AHA Scientist Development Grant 13SDG14000006 (M. R. Pabbidi).
‘The Good Place’, Body Donation, and Frida Kahlo: A Perfect Match? A qualitative evaluation of a novel workshop series
Authors: Dueñas Angelique N. , Whiting Demian , Finn Gabrielle M.
Published: April 2020

There has been a recent shift in focus on emphasizing the overlap of professionalism and ethics (P&E) with anatomy education. There is the sense that topics related to anatomy, such as the relationship between the dead and living, lend well to discussions of philosophy, ethics, professional development, and humanities‐related focus. However, with a general decrease in contact hours for anatomy‐related subjects, a renewed focus on P&E requires innovative teaching methods and interdisciplinary collaboration.

With this in mind, a 9‐week workshop series entitled ‘Body and Self’ was designed for first year, undergraduate medical students in a P&E module (course). Designed by an interdisciplinary team, and facilitated by an anatomist, in these workshops, students participated in activities related to topics such as: philosophy, culture, body donation, organ donation, art‐based approaches in medicine, presentation of medicine in lay culture, and medical ethics. The present work evaluates initial student reactions to this novel workshop series, via weekly qualitative survey evaluation. Students also completed an end‐of‐term evaluation of the workshops.

Content analysis was completed to code responses and analyze overall student views of these workshops, and how the Body and Self stream was constructively aligned.

Preliminary analysis of evaluations indicate that students received most sessions positively, with greater appreciation for topics that students viewed as directly anatomy‐ and medical‐related. The workshops encouraged reflection and growth of professional skills, such as interpersonal skills, personal reflective practice, patient and peer communication. Many students reflected on the “deep thinking” workshop content encouraged, and many viewed sessions as “insightful.” They also responded positively to hearing varied peer perspectives, and considering cultural views on topics such as death, dying, and body donation, as well as the medical ethics surrounding such topics. The majority of negative comments related to the actual structure of the workshop, such as the request for more time. Several students also frequently questioned the link between the workshop content and clinical experiences, and many negative comments correlated with the concept of ‘fixed mindsets.’

Initial evaluation of the Body and Self workshops suggest that students react quite positively to ethical discussions surrounding the nature of the body, particularly when these are linked to practical anatomical and clinical presentation. Follow up analysis are planned with consideration of the Kirkpatrick Model of evaluating learning experiences, in order to gauge student learning and possible changes in behavior. However, such a model of workshop, supported by evaluation and feedback, has early indications of being a powerful tool for further embedding anatomical sciences into P&E practice.
A Microglial Subset at the Tumor‐Stroma Border in High Grade Glioma
Authors: Caponegro Michael D. , Oh Ki , Sterge Nicholas , Moffitt Richard A. , Tsirka Stella E.
Published: April 2020

Standard of care for Grade IV Glioblastoma (GB) currently results in a very short 14–16 month survival window for newly diagnosed patients. Even with maximal surgical resection, rates of recurrence, progression, and mortality are virtually absolute. The invasive nature of GB into the surrounding tissue is thought to be a major factor of disease progression/recurrence. Thus, there is great interest to characterize the GB tumor microenvironment (TME) to target non‐cancerous, stromal cells via immunotherapeutic avenues. The TME in CNS tumors such as GB is unique in that it contains an abundance of neural tissue and specialized glial cells, including the resident innate immune cells, microglia. In GB, glioma‐associated microglia and peripherally infiltrating monocytes/macrophages (GAM) accumulate within the neoplastic lesion where they facilitate tumor growth and drive immunosuppression. Previously, it has been difficult to accurately differentiate microglia versus macrophage roles in GB progression/recurrence. Additionally, the consequences of spatial organization of these cells have not been studied. Here we characterize the tumor‐stroma border and identify peripheral glioma associated microglia (PGAM) at the leading edge as a unique cellular subpopulation of bona fide GAM. Using data mining and analysis of several scRNAseq datasets derived from patients with varying glioma diagnoses, we show that PGAM exhibit a pro‐inflammatory and chemotactic phenotype. We cross‐validate these findings in a murine model of GB, and propose that PGAM may constitute a novel cellular target in the TME.
Testosterone induces higher carotid body response to hypoxia in older adult male rats
Authors: Kinkead Richard , Janes Tara Adele , Soliz Jorge
Published: April 2020

Excessive carotid body responsiveness to chemical stimuli contributes to respiratory instability and apneas during sleep. In hypogonadal men, testosterone supplementation increases the risk of sleep‐disordered breathing; however, the site of action is unknown. The present study tested the hypothesis that exposure to elevated levels of testosterone potentiates the carotid body’s response to hypoxia. Because testosterone levels decline with age, we also determined whether these effects are age‐dependent. To do so, carotid bodies were isolated ex vivo from young (70 days old) and aging (185 days old) male Sprague‐Dawley rats. Isolated organs were maintained in oxygenated Tyrode solution (95% O2; 5% CO2) in the presence of testosterone (5 nM) or vehicle (<0.001% DMSO) for 90–120 min. Preparations were then mounted in the recording chamber and a suction electrode was placed on the carotid sinus nerve to record activity (# of impulses/sec). Activity was recorded at rest and during exposure to hypoxia (0% O2, 5% CO2, 95% N2; 15 min). Basal carotid body activity did not differ between age groups; however, the responses to both stimuli were greater in young rats. Testosterone exposure augmented the responses to hypoxia in aging rats only. In vivo experiments performed on anesthetised rats show that testosterone injection (2 mg/kg) 30 min prior hypoxic exposure (12%O2; 20 min) potentiates the hypoxic ventilatory response in an age‐dependent fashion. We conclude that age‐dependent potentiation of the carotid body’s O2 chemosensitivity by testosterone may lead to ventilatory adjustments that exceed the organism’s need. In aging animals, this may predispose to respiratory instability and apnea during sleep.

Support or Funding Information

Supported by a discovery grant from the Natural Sciences and Engineering Research Council of Canada (RK). Dr Janes was supported by a scholarship from the Réseau en Recherche Respiratoire du Québec.
The Multi‐Organ Activity of an Lmx1b‐Associated cis‐Regulatory Module Suggests a Global Role in Regulating Lmx1b‐Mediated Development
Authors: Wysong Kenrick E. , Clark Tatianna C. , Pira Charmaine U. , Oberg Kerby C.
Published: April 2020

Lmx1b is a LIM homeodomain transcription factor important for normal development of many organ systems including the cerebellum, eyes, limbs, and kidneys. In humans, haploinsufficiency of Lmx1b results in a Nail‐Patella syndrome, a disorder characterized by hypoplastic nails, bone deformities, glaucoma, and even kidney failure in severe cases. Despite its importance in developing these organ systems, the induction and regulation of Lmx1b is poorly understood. Previously using ChIP‐seq analysis, we identified two upstream, highly conserved Lmx1b‐associated regulatory modules (LARM1/2) bound by Lmx1b that were found to be associated with limb‐specific auto‐amplification of Lmx1b expression vital for limb dorsalization. We have subsequently identified another highly conserved region 10 kb upstream to these limb‐specific cis‐regulatory modules (CRMs), although it is not bound by Lmx1b. Yet, due to its active chromatin marks and close proximity to the Lmx1b locus, we hypothesized that this third regulatory module (LARM3) may also play a part in modulating Lmx1b expression. We generated a GFP‐reporter construct containing the isolated LARM3 sequence from mouse genomic DNA to test this hypothesis. To determine limb activity, this LARM3 construct was electoporated into the presumptive limb of Hamilton‐Hamburger stage 14 (HH14) chicken embryos. To determine activity in other organ systems such as the kidney and eye, the LARM3 construct was electroporated into a whole embryo (HH4) and in the presumptive lens placode (HH10), respectively. These embryos were viewed by fluorescent microscopy a few days after transfection to determine enhancer activity. LARM3 was found to be active in the limb mesoderm without a dorsoventral bias, showed punctate activity in the intermediate mesoderm that was consistent with mesonephric development (kidney), and was also active in the presumptive lens placode. The multi‐organ enhancer activity of LARM3 suggests a global role in regulating Lmx1b‐mediated development.

Support or Funding Information

Funded in part by the National Organization for Rare Diseases
Crystal structure of the wheat dwarf virus Rep domain
Authors: Litzau Lauren Anne
Published: April 2020

The wheat dwarf virus Rep domain is an HUH‐endonuclease and is involved in rolling‐circle replication. HUH‐endonucleases, or HUH‐tags, form covalent protein‐ssDNA adducts by coordinating a divalent metal ion to cleave a specific ssDNA sequence and form a phosphotyrosine linkage. This protein‐ssDNA fusion is useful for various biotechnology applications such as cellular imaging, cellular barcoding, DNA‐guided protein localization, and single molecule manipulation of DNA‐tethered proteins. Solving the structure of the Rep domain in complex with DNA could present necessary information regarding HUH‐tag sequence specificity and allow for rational engineering of protein‐DNA interactions. Here, the structure of WDV Rep domain in the apo state was solved with a crystal diffracting to 1.24 Å. While ssDNA soaks were attempted, they proved ineffective. However, the solved structure represents a step towards solving protein‐ssDNA complex.

Support or Funding Information

1. National Institutes of Health/National Institute of General Medical Sciences (grant No. GM119483); NIH (grant No.242 NIGMS R35‐GM118047)
Collaborative group testing increases intrinsic motivation but does not impact competition in medical students
Authors: Augustyniak Robert A. , Lujan Heidi , DiCarlo Stephen E.
Published: April 2020

It is increasingly recognized that the practice of medicine is a “team sport” with different medical professionals working together toward the common goal of providing health care. Collaborative group testing, where students work together to answer test questions models this team environment. The act of collaborating to achieve a challenging task may be intrinsically rewarding because one feels like they contributed to the “greater good”, which for the same reason may reduce competition. Therefore, we hypothesized that prolonged participation in collaborative group testing in first and second year medical students would increase intrinsic motivation and decrease competition. We assessed intrinsic motivation and competition via administration of the Intrinsic Motivation Inventory and the Competitive Attitude Scale, respectively. Students completed the surveys at baseline and after participating in thirteen collaborative group tests in a medical physiology course over a time‐period of 10 months. We found that all measures of intrinsic motivation were significantly increased after participation in collaborative group testing, while levels of competition were not changed. We conclude that medical student participation in collaborative group testing provides the opportunity to address the core psychological needs required to increase intrinsic motivation. Further studies are needed to understand why there was no effect on competition.
Formative Assessments Predict Performance and Promote a Positive Learning Environment
Authors: Schneider Brandt , Kaur Gurvinder , Bishop Keith , Gore Megh , Higgins Katie , Vories Bridget , Williams Simon , Lee Vaughan
Published: April 2020

At Texas Tech University Health Sciences Center School of Medicine, our curriculum begins with a rigorous and fast‐paced Clinically Oriented Anatomy course. Over the past four years, we have increased and improved the use of formative quizzes and exams to promote higher learning and performance outcomes, clarify examination expectations, and reduce student stress. Prior to starting our curriculum students are offered the opportunity to take a 25 question comprehensive formative pre‐test that previews examination content and USMLE vignette style questions. In the past year, we found that 93% of the incoming class utilized this voluntary resource. An average score of 23% on this pre‐test is consistent with a lack of familiarity of incoming students with human anatomy. To evaluate the effectiveness of our curriculum to bridge this informational gap, we provide students with two formative assessments (practice practical and practice written exam) prior to each summative exam. Computer based laboratory practice exams use digital images of prosected cadavers while didactic exams encompass a range of first, second and third order questions. Post‐assessment question/answer rationales provide students with immediate feedback to help identify areas of strength and weakness. Using this formative assessment approach, we have investigated the ability of formative assessments to predict exam performance and identify “at risk” students early in the course (i.e. after taking the first formative practice exam). Analysis of formative assessment data revealed a strong correlation with graded exam performance (p values ranging from 0.01–0.001 and r values from 0.44–0.70). Furthermore, overall graded summative exams correlate strongly with NBME performance (p = 0.001 and r = 0.92). Indicative of a successful educational experience, students scored an average of 233% higher on the repeat post‐course formative exam. Furthermore, the student’s perception of these formative assessments is very favorable and increases each year. In the past year, we had significantly fewer “at risk” students, and NBME performance was a record high. Thus, we find that formative assessments improve the educational experience, boost exam performance, and reduce student stress.

Support or Funding Information

(Sponsored by the Department of Medical Education at the TTUHSC SOM.)This study is approved by TTUHSC‐QIRB (approval#17008)
Human Intestinal AIEC Strains Alter the Mucosal Microbiome and Establish a Niche for AIEC and Non‐AIEC Strains in Il10−/− Mice
Authors: Bleich Rachel , Barlogio Cassandra , Franks Adrienne , Zarmer Sandra , Broberg Christopher , Bulik-Sullivan Emily , Dogan Belgin , Simpson Kenneth , Carroll Ian , Gharaibeh Raad , Arthur Janelle
Published: April 2020

Inflammatory bowel disease (IBD) affects over one million people in the United States and costs billions of dollars in direct medical expenses annually. This chronic inflammatory condition, which includes Crohn’s disease, currently has no medical cure. Additionally, patients with IBD are at a greater risk of developing inflammation‐associated colorectal cancer (I‐CRC). IBD and I‐CRC are associated with an increase of mucosally‐adhered bacteria, particularly Adherent‐Invasive Escherichia coli (AIEC). This proximity to the host can promote host‐directed pro‐inflammatory and pro‐carcinogenic activities by AIEC and co‐colonizing microbes. The overall aim of the project is to assess how colonization with Crohn’s‐associated clinicalE. coli strains alters the mucosal microbial community in the inflamed and non‐inflamed intestine.

To this end, we have developed a polymicrobial colonization strategy that uses a novel barcoding technology to easily distinguish genetically similar, but functionally distinct sub‐strains of E. coli in a complex community. We have developed this strategy using a collection of well‐characterized clinical AIEC and non‐AIEC strains isolated from the intestinal mucosa of IBD and healthy patients. These strains are administered to formerly germ‐free mice (either inflammation susceptible Il10−/− or resistant WT) also with a murine fecal microbial transplant (FMT) to simulate physiologically‐relevant conditions. Our in vivo modeling has revealed that colonization with a pool of clinical isolates in the presence of the FMT enhances inflammation and promotes higher levels of E. coli colonizing the mucosa in inflammation‐susceptible mice. Importantly, colonization with these strains very significantly alters the composition of the mucosal microbiome, assessed by 16S rRNA sequencing, in inflamed but not uninflamed mice, including expansion of a co‐occuring pro‐inflammatory taxon and contraction of multiple protective species. These data suggest that colonization of the chronically inflamed with high levels of E. coli influence host health through both host‐microbe and microbe‐microbe interactions.

Support or Funding Information

Supported by K01 award to JCA and the SPIRE NIH IRACDA program.
Using Near Infrared Spectroscopy, Diffuse Correlation Spectroscopy, and Intravital Video Microscopy to Monitor the Skeletomuscular and Cerebral Microcirculation
Authors: Mawdsley Laura , Rajaram Ajay , Yip Lawrence , Ellis Christopher G. , Diop Mamadou
Published: April 2020

Introduction

The microcirculation is the primary facilitator of oxygen delivery in tissue, and dysfunction in the microvasculature is often the first indication of a disease state. The microcirculation can be studied using a variety of methods, both invasive and non‐invasive. Two non‐invasive optical methods are near infrared spectroscopy (NIRS) and diffuse correlation spectroscopy (DCS). NIRS is sensitive to changes in tissue hemoglobin concentration and oxygen, while DCS is sensitive to changes in blood flow. However, neither method can directly visualize changes on a capillary by capillary basis. Intravital Video Microscopy (IVVM) allows us to do so, but is an invasive technique that requires surgery to expose the microcirculation. By using IVVM we can compare a direct visualization of the microvasculature to the signal received by NIRS/DCS in an equivalent microvascular bed. In addition, we can compare the NIRS/DCS signal from the skeletal muscle to the signal from the brain, allowing us to simultaneously monitor change in these two organs in response to disease progression or in response to stimulus.

Methods

Data is collected from Sprague Dawley rats (n=4) using both a dual wavelength Olympus inverted microscope (with 2 Rolera XR cameras and a beam splitter) and an in‐house NIRS/DCS device. Rats are anaesthetized and have their right extensor digitorum longus (EDL) muscle, found in the hind limb, exposed and reflected over the objective. Probes for the NIRS/DCS device are located on the left hind limb and the top of the skull. IVVM is used to collect microvascular velocity, hematocrit, and oxygen saturation from the right EDL, and NIRS/DCS will be used to collect microvascular hematocrit, oxygen saturation, and blood flow data from the left hind limb and the brain. Microvascular blood flow and oxygen saturation data from the left and right hind limbs will be compared, then the NIRS/DCS‐gathered data from the left hind limb will be compared to NIRS/DCS‐gathered data from the brain.

Discussion

This study will be the first to make a direct comparison between NIRS/DCS and IVVM measurements of the microcirculation, and will provide insight into the sensitivity of NIRS/DCS and its ability to accurately monitor the microvasculature. In addition, this study will be the first to make a direct comparison between skeletomuscular and cerebral NIRS/DCS measurements. Future work includes using this approach to determine the differences in effect of a phenylephrine bolus or an inspired oxygen challenge on these two microvascular beds. Further studies will determine whether disease progression occurs on the same timeline in the brain as it does in skeletal muscle.

Support or Funding Information

NSERC Discovery Grant to C.G. Ellis.
Mitochondria‐targeted scavenger of reactive isolevuglandins, mito2HOBA, protects from mitochondrial dysfunction and inflammatory injury
Authors: Mayorov Vladimir , Uchakin Peter , Amarnath Venkataraman , Panov Alexander , Bridges Christy , Uzhachenko Roman , Zackert Bill , Dikalova Anna , Moore Christy , Davies Sean , Dikalov Sergey
Published: April 2020

Hypertension is linked to oxidative stress and inflammation producing highly reactive dicarbonyl lipid peroxidation products, isolevuglandins (IsoLG) which covalently modify and cross‐link proteins via lysine residues and promote end‐organ‐damage. Mitochondrial dysfunction has been associated with inflammation; however, its molecular mechanisms and pathophysiological role are still obscure. We hypothesized that inflammation‐induced isolevuglandins contribute to mitochondrial dysfunction and mortality. To test this hypothesis, we have (a) investigated the mitochondrial dysfunction in response to synthetic 15‐E2‐isolevuglandin (IsoLG) and its adducts; (b) developed a new mitochondria‐targeted scavenger of isolevuglandins by conjugating 2‐hydroxybenzylamine to the lipophilic cation triphenylphosphonium, (4‐(4‐aminomethyl)‐3‐hydroxyphenoxy)butyl)triphenylphosphonium (mito2HOBA); (c) tested if mito2HOBA protects from mitochondrial dysfunction and mortality using a lipopolysaccharide model of inflammation. Acute exposure to either IsoLG or IsoLG adducts with lysine, ethanolamine or phosphatidylethanolamine inhibits mitochondrial respiration and attenuates Complex I activity. Complex II function was much more resistant to IsoLG. We confirmed that mito2HOBA markedly accumulates in isolated mitochondria and it is highly reactive with IsoLGs. To test the role of mitochondrial IsoLGs, we studied the therapeutic potential of mito2HOBA in lipopolysaccharide mouse model of sepsis. Mito2HOBA supplementation in drinking water (0.1g/L) to lipopolysaccharide treated mice increased survival by 3‐fold, improved complex I‐mediated respiration, and histopathological analyses supported mito2HOBA‐mediated protection of renal cortex from cell injury. Depletion of Cyclophilin D partially attenuated IsoLG‐induced mitochondrial dysfunction suggesting that mitochondrial IsoLG can affect both Cyclophilin D and Complex I functions. These data support the role of mitochondrial IsoLG in mitochondrial dysfunction and inflammation. We conclude that reducing mitochondrial IsoLGs may be a promising therapeutic target in inflammation and conditions associated with mitochondrial oxidative stress and dysfunction.

Support or Funding Information

This work was supported by the National Institutes of Health grants (R01HL124116 and R01HL144943) and Navicent Health Foundation Research & Education Grant.
Genetic Inhibition of Nfatc2 Attenuates Asparaginase Hypersensitivity in Mice
Authors: Rathod Sanjay , Ramsey Manda , Finkelman Fred D. , Fernandez Christian A.
Published: April 2020

Introduction

The development of a T cell‐dependent antibody response to asparaginase (ASNase) is the most common adverse effect of treatment with this chemotherapeutic agent for pediatric acute lymphoblastic leukemia (ALL). Anti‐ASNase antibodies decreases ASNase levels and drug efficacy and can mediate anaphylaxis. Transcription factors of the nuclear factor of activated T cells (NFAT) family play a critical role in Th cell activation, differentiation, and effector function. Here we show that a member of this family, Nfatc2 (NFAT1), upregulates Th2 responses, with Nfatc2‐deficient ASNase‐immunized mice being completely protected from anaphylaxis. Consistent with this, our clinical data demonstrate increased risk of ASNase hypersensitivity in pediatric ALL patients who express the NFATC2 rs6021191 intronic variant, which increases Nfatc2 expression, whereas the risk of ASNase hypersensitivity is decreased in ALL Down syndrome patients, who overexpress NFAT pathway inhibitors.

Hypothesis

Nfatc2 deficiency protects mice against ASNase‐induced hypersensitivity by suppressing type 2 immunity.

Methods

Nfatc2‐deficient mice and WT littermates were immunized with native E. coli ASNase using aluminum hydroxide adjuvant, after which immune cell phenotype and levels of basophil activation, cytokines, and ASNase‐specific antibody were determined. Hypersensitivity reactions to ASNase were assessed by detecting hypothermia with rectal thermometry. β‐hexosaminidase release was used for in vitro analysis of the degranulation of peritoneal mast cells (PMCs) obtained from naïve and ASNase‐immunized mice, while passive cutaneous anaphylaxis, passive systemic anaphylaxis, and active systemic anaphylaxis models were used for in vivo determinations.

Results

Nfatc2‐deficient mice were protected against ASNase hypersensitivity reactions. Naïve Nfatc2‐deficient mice had fewer CD4+IL‐4+ cells and lower IL‐4 and IL‐13 levels than wild‐type mice. ASNase‐immunized Nfatc2‐deficient mice had increased levels of Treg cells and the regulatory cytokines, IL‐10 and TGF‐β1, along with decreased plasma IgG and IgE anti‐ASNase and total IgE levels. Consistent with this, basophils/peritoneal mast cells from Nfatc2‐deficient mice had decreased FcɛRI expression and IgE‐mediated activation.

Conclusion

Nfatc2 deficiency decreases ASNase hypersensitivity by increasing regulatory responses and attenuating type 2 immune responses to ASNase immunization. Nfatc2 may be a promising target for the prevention of ASNase‐induced hypersensitivity.

Support or Funding Information

University of Pittsburgh School of Pharmacy & NIH Grants RO1 CA216815
Regulators Of G‐Protein Signaling 2 And 4 Differentially Regulate Nicotine‐Induced Affective Behavioral Responses
Authors: DSouza Manoranjan S. , Snyder Madison , Ho Sheng-Ping , Yi Tsai , Seeley Sarah , Rorabaugh Boyd R.
Published: April 2020

High rates of tobacco smoking are observed in individuals suffering from anxiety or depression. Nicotine is a major psychoactive component of tobacco smoke and previous work has shown that nicotine can produce both anxiolytic‐ and antidepressant‐like effects. Regulators of G protein (RGS) signalling proteins negatively regulate intracellular signaling pathways of neurotransmitters (e.g. adrenaline, dopamine and serotonin), which mediate the antidepressant‐ and anxiolytic‐like effects of nicotine. The objective of the study was to determine the role of the RGS2 and RGS4 in the anxiolytic‐ and antidepressant‐like effects of nicotine. Antidepressant‐ and anxiolytic‐like effects were assessed using the tail suspension model and elevated plus maze, respectively. We used both male and female mice lacking either RGS2 (i.e. RGS2 KO) or RGS4 9i.e. RGS4 KO) and their wildtype counterparts. Both male and female RGS2 KO mice showed anxiolytic effects to nicotine (0.1 mg/kg; i.p., base) compared to respective saline controls. In contrast, no nicotine‐induced (1 mg/kg; i.p., base) antidepressant‐like effects were observed in male and female RGS2 KO mice compared to respective saline controls. Together, these data suggest that RGS2 plays a role in nicotine‐induced anxiolytic effects but not nicotine‐induced antidepressant‐like effects. In contrast, nicotine‐induced antidepressant‐like effects were observed in male RGS4 KO mice, but not female RGS4 KO mice compared to respective saline controls. Also, nicotine‐induced anxiolytic effects were not observed in either male or female RGS4 KO mice. Together, these data suggest that RGS2 and RGS4 differentially mediate nicotine‐induced affective responses. Clinically, these data support a role for targeting these proteins in treating nicotine addiction in patients suffering from comorbid anxiety and/or depression.

Support or Funding Information

This work was supported by a Bower, Bennett and Bennett grant awarded to Dr. Manoranjan S. D’Souza by the Raabe College of Pharmacy, Ohio Northern University (ONU), Ada, Ohio.
Dietary Effects on Fiber Type Plasticity in the Masseter Muscle
Authors: LaRocque Holly , Rossiter Jeffrey A. , Menegaz Rachel A.
Published: April 2020

Clinical and experimental studies have shown the masticatory complex (the skeletal and soft tissues associated with feeding) to be phenotypically plastic with regard to dietary material properties. Harder, tougher diets generally produce larger skeletal elements with greater bone density. However, less is known about the plasticity of the chewing musculature. Muscles are composed of two primary fiber types: slow‐twitch (type I) and fast‐twitch (type II). Fast‐twitch fibers produce rapid, powerful contractions but fatigue more quickly than slow‐twitch fibers. Here we use a rodent model to examine the plasticity of the masseter muscle fibers in response to inter‐individual variation in dietary material properties. We hypothesize that rats raised on hard, tough diets will have more fast‐twitch muscle fibers with greater cross‐sectional area, representing an increase in force production by the masseter in response to the dietary material properties.

Male Sprague‐Dawley rats were raised from weaning (3 weeks) to adulthood (16 weeks) in two dietary treatment groups: a “hard/tough” pellet diet and a “soft” meal diet. Post‐sacrifice, fixed muscles were embedded in paraffin and divided into anterior, middle, and posterior regions. To visualize fast‐twitch fibers, 6 sections per region per individual were stained with a primary antibody for the fast isoforms of myosin heavy chain (MHC) and a fluorescent secondary antibody. Images were collected at 4× using a fluorescent confocal microscope. ImageJ was used to collect total muscle cell area, fast‐twitch cell area, and slow‐twitch and fast‐twitch cell counts. Mann‐Whitney U tests were used to compare cell count and area between the treatment groups (n=5/group, α = 0.05).

Preliminary results indicate that hard/tough diets tend to be associated with a decrease in slow‐twitch fiber count and an increase in fast twitch fiber cross‐sectional area in the rat masseter. This allows for a greater proportion of fast‐twitch muscle fibers in a given muscle volume. We propose that this change reflects an increase in muscle force production during feeding on mechanically challenging food items. The results of this study allow for greater understanding of skeletal muscle growth postnatally through adulthood, and provides insight into the consequences of material properties of diet for the function and dysfunction of the human masticatory complex. Future studies are needed to address how muscle fiber plasticity changes with age and with intra‐individual variation in the material properties of diet.

Support or Funding Information

Funding was provided by the NSF (BCS‐1061368), the Wenner‐Gren Foundation, the American Society of Mammalogists, and the American Association for Anatomy Innovations Program.
The Regulatory Role of Phosphorylation of Mitochondrial Transcription Factor B2 (TFB2M)
Authors: Moya Gonzalo , Bostwick Alicia M. , Senti Mackenna L. , Dittenhafer-Reed Kristin E.
Published: April 2020

Mammalian cells contain genetic information in two compartments, the nucleus and the mitochondria. Mitochondrial DNA (mtDNA) encodes thirteen protein subunits required for oxidative phosphorylation. The remaining mitochondrial proteome is encoded by the nuclear genome; which includes additional oxidative phosphorylation subunits, as well as the proteins necessary for mtDNA replication, expression and stability. Therefore, to respond to metabolic changes, mitochondrial gene expression must be coordinated with nuclear gene expression. While the control of nuclear gene expression is widely studied, there is a need to understand the regulation of mtDNA transcription. Our central hypothesis is that reversible protein post‐translational modifications of the mtDNA transcriptional machinery is a means to tune mtDNA transcription in response to changes in cellular metabolism. To address this gap in knowledge, this research focuses on a member of the mtDNA transcription initiation complex, mitochondrial transcription factor B2 (TFB2M). TFB2M melts mtDNA at the promoter to allow for the RNA polymerase (POLRMT) to access the DNA template and initiate transcription. Three phosphorylation sites have been previously identified on TFB2M by mass spectrometry: threonine 184, threonine 313 and serine 197. To mimic the behavior of phosphorylation on these three sites, individual amino acids were mutated to glutamate, an amino acid that mimics the size and charge of the phosphoryl group. WT and phosphomimetic TFB2M were expressed in E. coli and purified. The purified proteins were analyzed for their ability to bind mtDNA through a pull‐down assay and fluorescence polarization was used to determine the binding dissociation constants for the light strand promoter. Interactions between TFB2M phosphomimics and POLRMT were also assessed. The data support the hypothesis that the phosphorylation at these sites may decrease the ability of TFB2M to bind to mtDNA. In vitro transcription assays were performed to determine the impact of these modifications on TFB2M’s role in transcription initiation. It is expected that these findings will lead to a more complete understanding of the dynamics and coordination of mitochondrial and nuclear gene transcription, as well as adding to the understanding of the importance of protein post‐translational modifications in regulating aspects of mitochondrial function.

Support or Funding Information

This material is based upon work supported by the National Science Foundation Division of Molecular and Cellular Biosciences under Grant No. 1814845.
Kv1.3 Constitutes a Valid Target for Reducing Neuroinflammation in the Wake of Ischemic Stroke in Male, Female and Aged Mice
Authors: Chen Yi-Je , Nguyen Hai M. , Cui Yanjun , Wulff Heike
Published: April 2020

The voltage‐gated Kv1.3 channel was first discovered in human T cells in 1984 and has since then been pursued as a target for autoimmune diseases. Kv1.3 is further expressed on pro‐inflammatory microglia and our laboratory therefore evaluated the small molecule Kv1.3 blocker PAP‐1 in models of ischemic stroke. In both male C57BL/6J mice and male Wistar rats, treatment with PAP‐1 started 12 hours after reperfusion reduced infarction and inflammatory brain cytokine levels and improved neurological deficit on day‐8 after reperfusion MCAO (Y.‐J. Chen et al. 2018). In order to test the therapeutic hypothesis that Kv1.3 inhibition is equally effective in both sexes, we established 60‐min MCAO with reperfusion in female C57BL/6J mice. Females have smaller infarcts, but 4‐months old animals of both sexes showed qualitatively similar pathologies with similar IBA+‐microglia/macrophage and T cell densities and a GFAP+ glial scar. Based on patch‐clamp recordings performed on acutely isolated CD11+ cells Cx3cr1GFP/+ male and female mice exhibit similar Kv1.3 current densities on microglia/macrophages from the infarcted area. Immunofluorescence revealed that in both male and female Cx3cr1GFP/+ mice Kv1.3 expression increases with time after MCAO, peaks around day‐7 and is localized to GFP+ microglia/macrophages and CD3+ T cells. Since these findings suggest that both sexes would equally benefit from Kv1.3 inhibition, we subjected 4‐month and 20‐month old male and female WT and Kv1.3−/− mice to 60 min‐MCAO with reperfusion. T2‐weigthed MRI revealed that Kv1.3‐deletion produces a significant reduction in infarct volume on day‐8 in both sexes. The reduction in infarction was most effective in aged Kv1.3−/− females. Based on these observations and the finding that treatment with PAP‐1 significantly reduces infarction in 4‐months old WT female mice (n=14), we conclude that Kv1.3 inhibition effectively reduces stroke associated inflammation in both sexes independent of age.

Support or Funding Information

Supported by NIH (NS100294).
Reframing Lower‐Level Bloom’s Taxonomy Exam Questions in Applied Anatomy Alters Statistical Outcomes for Item Performance: A Pilot Study
Authors: Stevens Kelsey , Immonen Jessica , Dykstra Emily
Published: April 2020

Background

Bloom’s Taxonomy is a hierarchical model designed to categorize course learning objectives based on the complexity of learning associated with the task. Little is known about performance and abilities of entry‐level allied health students related to Bloom’s Taxonomy within anatomy education.

Objective

The objective of this assessment was to use exam reviews from 2018’s PAS 501 Applied Anatomy course to re‐create exam items at higher levels of Bloom’s learning. Cohort performance at the “Remembering” level of Bloom’s was compared to 2019 reconstructed questions to determine if graduate‐level PA students are most capable of memorizing anatomy or interpreting more detailed applications.

Methods

Four “Remembering” questions were selected from the 2018 PAS 501 Abdomen written exam. The regional concepts in these questions were reframed in clinical scenarios and questions where regional anatomy needed to be applied at higher levels of thinking. Statistics of cohort performance in the form of the DI, PBC and the DisI were used to compare the lower‐ and higher‐level Bloom’s questions

Results

Two questions reframed as clinical scenarios and demonstrated decreases in the DI and an increase in the PBC. Two questions were initially written as basic definition questions and reconstructed to represent a discussion of regional anatomy that required the student to understand and reinterpret the information. One of these questions displayed an increase in the DI and the PBC.

Conclusions

Higher level Bloom’s questions framed as clinical scenarios (N=2) displayed noted increases in the PBC implying these questions are quality discriminators for students who do well overall and those who do not. Reframing “Remembering,” definition questions to “Understanding” level questions may increase item success for clinically‐minded graduate‐level students. Additional work is required to draw meaningful conclusions from this pilot data.

Support or Funding Information

N/A
Inositol polyphosphate multi‐kinase is a novel regulator of reverse‐transsulfuration pathway
Authors: Tyagi Richa , Snyder Solomon H. , Paul Bindu
Published: April 2020

Inositol polyphosphate multikinase (IPMK) is one of the members of inositol phosphate kinase family that generates inositol polyphosphates. IPMK possesses inositol phosphate kinase (IP3‐kinase) as well as phosphatidylinositol kinase (PI3‐kinase) activities. IPMK is a pleiotropic protein and non‐catalytically regulates mammalian target of rapamycin complex 1 (mTORC1), serum response factor, p300, and tumor suppressor protein p53. We report that IPMK regulates expression of cystathionine γ‐lyase (CSE) and 3‐mercaptopyruvate sulfurtransferase (3‐MST), enzymes involved in Hydrogen sulfide production. Protein levels of CSE and MST are increased in IPMK null fibroblasts as compared to wild type fibroblast cells. Regulation of CSE and MST is independent of catalytic activity of IPMK. IPMK regulates CSE expression at the transcriptional level. Since cystathionine β‐synthase (CBS) is expressed at relatively lower levels as compare to CSE in fibroblasts, CSE is the major source of cysteine generation from cystathionine in mouse fibroblasts. Lysates prepared from IPMK null fibroblasts produced more cysteine as compared to wild type cells. Hydrogen sulfide levels are also increased in IPMK null fibroblasts. Expression of CSE is inducible upon condition such as lipopolysaccharides (LPS), and inflammation mediated by tumor necrosis factor α (TNF‐α). We propose that IPMK acts as a repressor of CSE expression.

Support or Funding Information

This work was supported by NIH grants MH18501 to S.H.S.