bluuebunny/crossref_metadata_2025 huggingface dataset:
Curcumin Improves Autoimmunity in Female Mice with SLE
Authors: Dent Elena L. , Taylor Erin B. , Ryan Michael J.
Published: April 2020

Systemic lupus erythematosus (SLE) is a chronic multi‐system autoimmune disease characterized by the presence of circulating autoantibodies, inflammation, hypertension, renal injury, and cardiovascular disease. Current immunosuppressive therapies often impose unwanted side effects leaving patients searching for alternative therapeutic options. Curcumin has been used in Eastern medicine for its natural anti‐inflammatory and antioxidant properties. We hypothesize that the administration of curcumin will lessen renal inflammation and injury in female mice with SLE.

Female NZBWF1 mice, an established experimental model of SLE, and female NZW (control) mice were administered curcumin (500 mg/kg/day) or corn oil (vehicle) by oral gavage for 14 days. This design is based on previously published experimental data using curcumin in mice. Plasma blood urea nitrogen (BUN), a clinical marker of renal function, was assessed at baseline (26 weeks of age) and again at the end of the study (28 weeks of age). To assess immune activity, we measured circulating anti‐dsDNA IgG autoantibodies, spleen weight, and used flow cytometric analyses of peripheral blood leukocytes to assess circulating B lymphocytes at the same time points.

Compared with vehicle treated SLE mice, curcumin treated mice with SLE have significantly lower BUN at 28 weeks of age (13.7 ± 0.6 mg/dL SLE‐curcumin, n=14 vs. 36.2 ± 11.4 mg/dL SLE‐vehicle, n=5) (P = 0.0030). As expected, spleen weights are increased in female mice with SLE. However, curcumin treated SLE mice at 28 weeks of age have lower spleen weights compared to SLE vehicle treated mice (0.114 ± 0.009 g SLE‐curcumin, n=14 vs. 0.212 ± 0.03 g SLE‐vehicle, n=5) (P = 0.0008). In addition, the percentage of circulating CD45R+ B cells are decreased at 28 weeks of age in SLE curcumin treated mice compared to vehicle treated SLE mice (5.6 ± 1.6% SLE‐curcumin, n=4 vs. 36.4 ± 9.5% SLE‐vehicle, n=10) (P = 0.068). Despite fewer circulating CD45R+ B cells, plasma levels of anti‐dsDNA IgG autoantibodies are unchanged in curcumin treated mice with SLE compared to vehicle treated mice with SLE. These data suggest that curcumin modulates autoimmune activity and may lessen renal injury in female mice with SLE.

Support or Funding Information

Research reported in this publication was supported by the NIGMS of the NIH (5U54GM115428 and P20GM104357), NHLBI of the NIH (P01HL051971), VA Merit (BX002604‐01A2), and AHA pre‐doctoral fellowship (19PRE34380830).
Sex Difference In Kidney Electrolyte Transport III: Impact of Low K intake on Thiazide‐Sensitive Cation Excretion in Male and Female Mice
Authors: Xu Shuhua , Li Jing , Yang Lei , Wang Claire J. , Liu Tommy , Weinstein Alan M. , Palmer Lawrence , Wang Tong
Published: April 2020

The thiazide sensitive Na‐Cl co‐transporter (NCC) in the distal tubule can be regulated by dietary potassium intake and is critical for electrolyte homeostasis. Previously we reported that there was higher NCC activity and expression in female over male mice and high‐K intake preserved the sex‐difference of NCC‐mediated sodium absorption due to comparable reductions of NCC activity and expression in both sexes. However, with high‐K intake hydrochlorothiazide (HCTZ)‐induced kaliuresis was greater in males than in female mice, suggesting a more powerful male adaptation to high‐K intake. In this study, we explored the role of NCC in adaptation to a low‐K (LK) diet. We measured HCTZ‐induced changes in urine volume (UV), glomerular filtration rate (GFR), absolute (ENa, EK) and fractional (FENa, FEK) cation excretion in male and female mice on control‐K (CK, 1% KCl) and LK (0.1%, KCl) diets for 7 days. We also examined LK‐induced changes in NCC, Na/H‐exchanger isoform 3 (NHE3), and ENaC protein expression by Western blotting. With CK, NCC‐dependent ENa and FENa were larger in females than males as we observed previously. However with LK HCTZ‐induced ENa and FENa increased in males but not in females. Thus K restriction abolished the sex differences in NCC function. Changes in total NCC protein followed a similar pattern. Under CK conditions females expressed more NCC protein, as previously reported. LK increased NCC abundance in both males (by 100%) and females (by 40%). The larger effect in males reversed the sex‐dependence of NCC expression, consistent with the measurements of function. LK intake did not change either NHE3 or NHE2 expression, but reduced the amount of cleaved (presumably active) forms of α and γENaC expression in males but not in females. Thus decreased Na reabsorption through ENaC may compensate for increased Na transport through NCC in males. Despite large diuretic and natriuretic responses to HCTZ, EK was only slightly increased in response to the drug when animals were on LK. This suggests that the K‐secretory apparatus in the distal nephron is strongly suppressed under these conditions.

Support or Funding Information

NIH NIDDK RO1 DK099284
Curcuminoid analogs, a novel class of drugs for the treatment of Glioblastoma multiforme
Authors: Shivachar Amruthesh C. , Selvam Chelliah , Bhaskaran Mahendran , Hosseini- Zare Maryam Sadat , Ranganna Kasturi
Published: April 2020

Glioblastoma multiforme is one of the glial‐derived malignant primary brain tumors in adults with a poor prognosis. The average survival period still remains between 14–16 months despite various treatment modalities involving combination of surgery, radiotherapy and chemotherapy with temozolomide, an imidazotetrazine derivative. Curcumin (#1) is a plant derived dietary constituent, and has been shown to play a role in preventing many cancers. We investigated for the first time the potencies and the molecular mechanisms of action of #1 and its three synthetic analogs (#7, #20, and #32) that were prepared through an acid catalyzed condensation with various hydrazine derivatives. Cultures of grade IV glioblastoma multiforme (GBM) MG118 cells (5,000/cm2) were treated with various concentrations (0.01–100μmole) of #1, #7, #20 or #32 for 24 hr. along with temozolomide (0.1–300μmole) as positive control. Cell viability/toxicity was assessed by Cell Titer Glo 2.0 Assay, and by the fluorescent dyes Hoechst 33342/Propidium iodide‐based live‐dead cell assay. Apoptosis was directly assessed by TACS Annexin V‐FITC Apoptosis detection kit. Concurrently, we measured the effects of these analogs on the expression of pro‐angiogenic HIF1α, VEGF, and chemokine receptor 4 (CXCR4) by western blotting analyses and quantitated using GAPDH loading controls. Additionally, we formulated curcuminoid‐loaded nanoparticles & optimized it using Design Expert (DoE) software and then evaluated the prepared formulation for determining the cytotoxic effects in GBM. Our results show that the tested analogs dose‐dependently caused apoptosis, decreased cell viability and inhibited cell proliferation in the order #32> #20> #7> #1>>temozolamide, suggesting that synthetic cucuminoids 32 and 20 are more potent than their parent compound and the current drug temozolomide. Additionally, we noted that these two analogs down‐regulated the expression of VEGF, and anti‐hypoxic HIF1α, and the chemokine receptor CXCR4 a known mediator of cancer cell proliferation and invasion. Thus our preclinical results demonstrate that compounds 32 and 20 may be two novel class of drugs for the treatment of GBM by down‐regulation of key targets that are critical for GBM survival and metastasis.

Support or Funding Information

Partial support from an Institutional grant (ACS), and a grant from NIMHD/NIH #2G12MD007605 (KR).
Exploring the Interaction of Msh2‐Msh6 with the Holliday Junction Through ATPase Activity
Authors: Joshi Meera , Li Yan , Lombardo Zane , Hingorani Manju , Mukerji Ishita
Published: April 2020

DNA mismatch repair systems (MMR) are important in reducing post replicative errors and preventing recombination between homologous sequences. Mismatch repair is highly conserved in most organisms and is initiated by the MutS homodimer in prokaryotes. In eukaryotes, small insertion/deletion loops (IDL), and single base pair mismatches are recognized by the MutS homolog Msh2‐Msh6. Prior research in our lab and by others (Marsischky G. T., Lee S., Griffith J., and Kolodner R. D. (1999) J.Biol.Chem. 274: 7200–7206 doi:10.1074/jbc.274.11.7200) determined the binding affinity of the junction with Msh2‐Msh6 with a Kd = 12.9 ± 1.3 nM, which is similar to the binding affinity for a G:T duplex, suggesting that the Msh2‐Msh6 Holliday Junction interaction is specific. Our investigation aims to understand the Msh2‐Msh6‐junction binding interaction by studying nucleotide utilization upon binding and determining any conformational changes associated with complex formation and ATP hydrolysis. We use the well‐characterized J3 Holliday junction with 17 base pair arms to characterize binding. Malachite green assays measuring inorganic phosphate were performed to obtain the rate of ATP hydrolysis in the presence of Msh2‐Msh6, J3 junction, and other DNA substrates. Msh2‐Msh6 alone has a Kcat = 0.27s−1 in the presence of 100mM NaCl, with the Kcat weakly increasing with a G:T mismatch 34‐mer duplex and the J3 junction to 0.47s−1and 0.6s−1 respectively. We will also report on junction conformational changes associated with protein binding and how protein affinity for the junction is modulated by nucleotide binding.

Support or Funding Information

ASBMB Undergraduate Research Grant.
Link between Nutritional Status and Gene Expression in Regulatory Associated Protein of Mammalian Target or Rapamycin mRNA (RPTOR) and Rapamycin‐insensitive Companion of Mammalian Target of Rapamycin (RICTR) messenger RNA
Authors: Kostner Danica LeAnn , Spainhour Rebekah , Wertz Blaine LeAnn , Schmidtberger Abigail , Dougherty Megan , Abernathy Oaklee LeAnn , Ball Jenna , Kobayashi Yass
Published: April 2020

RPTOR and RICTR form a complex with Mammalian Target of Rapamycin (mTOR) called mTORC1 and 2, respectively and activates the transcription of genes associated with different metabolic pathways, including autophagy. Previous studies showed the expression of mTOR mRNA in muscle was increased in response to reduced food intake in channel catfish. However, whether RPTOR and RICTR mRNA expression is influenced by changes in food intake in catfish is unclear. The objective of the current study was to explore the relationship of RPTOR and RICTR mRNA expression and food intake in the brain, liver, and muscle. For the first study, channel catfish were fed daily (control) or fasted for 28 days (fasted). Fish in the third treatment were fasted for the first 14 days then fed daily for 14 days (refed). For the second study, catfish were fed once every 12 (overfed), 24 (control), or 48 hours (underfed, n=4 tanks per treatment) for 28 days. Brain, liver, and muscle expression of RPTOR and RICTR mRNA was measured from the samples collected on day 28. Muscle expression of RPTOR and RICTR mRNA was unaffected by changes in food intake. In contrast, fasting increased brain expression of RICTR mRNA, and increased feeding frequency decreased RPTOR mRNA in the liver. Our results showed that changes in the expression of RPTOR and RICTR mRNA did not coincide with changes in the expression of mTOR mRNA. The exact relationship between food intake and expression of these mRNA species needs further investigation.

Support or Funding Information

K‐INBRE P20GM103418
Kinetic control of MERS‐CoV 3CLpro is mediated by non‐conserved residues distal to the active site
Authors: Anson Brandon James , Daczkowski Courtney Marie , Mesecar Andrew David
Published: April 2020

Middle‐East Respiratory Syndrome (MERS) Coronavirus is a re‐emergent zoonotic pathogen with a 30% fatality rate in humans. The MERS positive‐sense single‐stranded RNA genome is translated into two polyproteins that must be processed by two viral cysteine proteases (3CLpro and PLpro) into 16 non‐structural proteins (nsp) before viral replication can occur. Attempts to drug either of these cysteine proteases have proven difficult. MERS 3CLpro functions as dimer and is unique among all of the coronavirus 3CLpro enzymes studied to date because competitive inhibitors activate the wild‐type MERS 3CLpro enzyme at low inhibitor concentrations and inhibit at higher concentrations. This mechanism of substrate/inhibitor‐induced dimerization seems to be mediated by non‐conserved residues in structural regions distal to the catalytic site. We have identified unique residues that are involved in the inhibitor‐induced dimerization mechanism and we show that these ‘competitive’ inhibitors activate and inhibit in a dose‐dependent manner. In an attempt to provide a structural basis for this mechanism, we crystallized the apo and inhibitor‐bound forms of this enzyme and diffracted them to 1.5 – 2.2 Å. We also show that residues in distinct structural regions synergize to modulate the intrinsic kinetic parameters of this family of enzymes. These parameters include increased turnover (kcat) and a lower dimer dissociation constant (KD). Additionally, a rapid‐equilibrium kinetic mechanism was developed to model the kinetic response of MERS 3CLpro to inhibitors that act via substrate/inhibitor induced activation/inhibition mechanisms.

Support or Funding Information

This project has been funded in whole or in part with Federal funds from the National Institute of Allergy and Infectious Diseases,
National Institutes of Health, Department of Health and Human Services, under Contracts No. HHSN272200700058C, HHSN272201200026C, and HHSN272201700060C. This research used resources of the Advanced Photon Source, a U.S. Department of Energy (DOE) Office of Science User Facility operated for the DOE Office of Science by Argonne National Laboratory under Contract No. DE‐AC02‐06CH11357.Use of the Lilly Research Laboratories Collaborative Access Team (LRL‐CAT) beamline at Sector 31 of the Advanced Photon Source was provided by Eli Lilly Company, which operates the facility.
Inhibition of JNK Signaling During Mast Cell Differentiation Promotes Altered Cytokine Secretion and Degranulation Profiles
Authors: Hicks Natalie J. , Crozier Robert W.E. , Den Hartogh Danja J. , MacNeil Lori A. , MacNeil Adam J.
Published: April 2020

Mast cells are large granulated leukocytes active in both innate and acquired immunity. These cells differentiate from hematopoietic stem cells, found in the medullary cavity of long trabecular bones. Increased inflammatory activity and numbers of mast cells contribute to allergic disease, which is of increasing concern worldwide. C‐jun N‐terminal kinase (JNK) is a major signaling node in the map kinase pathway that has been shown to contribute to IL‐3 signaling, known to promote murine mast cell differentiation. The aim of this research is to determine the role of JNK signaling in mast cell differentiation. Primary bone marrow‐derived mast cell (BMMC) cultures were established from C57BL/6 mice and differentiated for 7 weeks in the presence or absence of a JNK inhibitor (JNK‐IN‐8). Following removal of the inhibitor for 5 days, phenotypic and functional characteristics of BMMCs were probed using flow cytometry, ELISA, and β‐hexominidase release assays. Flow cytometric analysis of surface expression of the mast cell marker c‐kit, and levels of IgE bound to FcɛRI, a measure of mast cell sensitization, were unaffected by impaired JNK signaling compared to cells grown in the absence of inhibitor over mast cell differentiation, suggesting a normal mast cell phenotype with the potential to be activated via the allergen‐bound IgE‐dependent pathway. Cells differentiated under JNK inhibition showed a significant impairment in the secretion of IL‐6 (p<0.05) and IL‐13 (p<0.05) and a non‐significant impairment in TNF and CCL1 secretion at 24 hrs post‐activation compared to cells grown in the absence of inhibitor. Degranulation was also significantly decreased (by 25%; p<0.05) in BMMCs grown under JNK inhibition. To probe an epigenetic mechanistic role for histone acetylation changes in JNK‐impaired differentiation, expression of HDAC and HAT enzymes were considered by qPCR and western blotting, but were not modulated by JNK. These results suggest that JNK plays an important role in the development of a fully functional mast cell in the context of IgE‐mediated allergic inflammation and may warrant consideration as a strategy to regulate mast cell development in associated pathologies.

Support or Funding Information

Supported by the Natural Sciences and Engineering Research Council of Canada (NSERC); Canada Foundation for Innovation (CFI); Government of Ontario and Brock University.
Effect of 7‐Day Whey Protein Supplementation on Steroid Hormones and SHBG in Women with Polycystic Ovary Syndrome (PCOS)
Authors: Zumbro Emily L. , Rao Manisha , Balcom-Luker Shenavia , Broughton K. Shane , LeMieux Monique J.
Published: April 2020

Polycystic ovary syndrome (PCOS) is the most prevalent endocrinopathy in women of child‐bearing age and is associated with obesity, infertility, insulin resistance, and hyperandrogenemia. Sex‐hormone binding globulin (SHBG) is suppressed as a result of hyperinsulinemia leading to an increase in free testosterone levels commonly observed in PCOS women. High levels of free testosterone contributes to hirsutism, acne, and irregular menstrual cycles in this population resulting in the need for interventions aimed at increasing SHBG concentration in PCOS women. Subsequently, estradiol and progesterone levels suffer in this population resulting in irregular menstrual cycles and infertility. Current treatments for PCOS target symptoms of the disease but not the underlying mechanisms, and typically are associated with adverse side effects. Nutritional interventions, therefore, that can ameliorate PCOS‐related issues are warranted. Whey protein has recently been shown to have improved glycemic homeostasis, reduced chronic inflammation and enhanced protein anabolism effects in people with or without insulin resistance. The purpose of this study was to examine changes in SHBG and free testosterone concentrations following 7 days of whey protein supplementation and to examine differences in baseline measurements of progesterone and estradiol between PCOS women and healthy controls (CON). Women with PCOS and CON consumed 35g of whey protein for 7 days. Baseline and Day 7 concentrations were measured via plasma and serum collection. Progesterone and estradiol concentrations were measured at baseline only whereas free testosterone and SHBG concentrations were measured at baseline and Day 7. Protocols used in this study were approved by the TWU Institutional Review Broad (IRB). Initial analyses indicate progesterone levels were lower in the PCOS group compared to CON. Currently, no other statistical differences are reported at baseline nor over time in any other variable. Additional analyses are currently being conducted. Seven days whey protein supplementation is not a viable intervention to increase fertility in PCOS women by enabling changes in steroid hormone and globulin binding concentrations. Future studies will include a prolonged intervention period to establish potential changes in the aforementioned hormones following chronic whey protein supplementation.

Support or Funding Information

Glanbia Nutritionals, Inc., TWU Research Enhancement Program, TWU Human Nutrition Research funds
Genetic Influences on Fall Risk in Patients with Type 2 Diabetes Mellitus
Authors: Quinlan Cody Mcquaid , Haji Barrawan Awni , Singleton John David , Raynes Edilberto Alcantara
Published: April 2020

Globally, the prevalence rate of obesity is continuously increasing giving rise to a corresponding increasing prevalence of type 2 diabetes mellitus (T2DM). Reflective of this global increase in the field of physical therapy is increased treatment of patients with T2DM who are at increased risk for falls. The purpose of this study was to explore whether physical therapists should implement fall risk prevention interventions based on genetic variants among patients with T2DM. Utilizing PubMed, Google Scholar, and Research Gate studies were identified then ranked using the Oxford Levels of Evidence. Results identified fall risk factors within the scope of a physical therapist and associated with T2DM include diabetic peripheral neuropathy, osteoarthritis and sarcopenia. Genetic variants in NOS1AP, IL‐1 gene cluster and MSTN were identified to be associated with increased development and progression of the above‐mentioned fall risk factors. It can be argued patients with T2DM who present with variants in these genes are more likely to develop increased fall risk. Therefore, these patients should be treated preemptively by the physical therapist. These physical therapy treatments should include interdisciplinary and preventative care for diabetic peripheral neuropathy, osteoarthritis and sarcopenia leading to a decreased development of fall risk. Future studies should focus on preventative physical therapy treatment of genetically identified fall risk factors in patients with comorbidities associated with fall risk.
Cryo‐EM Structure of CtBP2 Confirms Tetrameric Architecture
Authors: Jecrois Anne M. , Bellesis Andrew , Schiffer Celia , Royer William
Published: April 2020

C‐terminal binding proteins 1 and 2 (CtBP1 and CtBP2) are transcriptional regulators that regulate gene expression. CtBP can act as both an activator and repressor of transcription by interacting with different transcription factors and chromatin modifying enzymes. CtBP is activated under conditions of hypoxia when the NADH level is elevated in the cell, which has direct implication in various forms of cancer. The transcriptional activity of CtBP can confer resistance to apoptosis and/or promotes oncogenesis and metastasis depending on its interacting partners. Due to its role in tumorigenesis and correlation with poor patient prognosis, CtBP is a potential drug target.

NADH exerts its transcriptional effect through promoting CtBP oligomerization. The activated oligomer can then associate with other co‐factors to form the CtBP‐mediated repression complex. Although previous studies have proposed dimeric CtBP as the relevant oligomeric state, our studies with multi‐angle light scattering have shown that the primary effect of NADH binding is to promote the assembly of two CtBP dimers into tetramers. Here, we present the 4.5Å CryoEM structure of CtBP2 corroborating that the native state of NADH bound CtBP2 is indeed tetrameric. Together, these results highlight the tetramer as the functional oligomeric form of CtBP proteins.

Support or Funding Information

• 1 F31 GM129988‐01

• R01 GM119014
5‐Aminoethylbenzimdazole Suppresses Lipopolysaccharide (LPS)/Interferon Gamma (IFNγ)‐Induced Inflammatory Responses in Macrophages
Authors: Chen Xin , Smith Amanda , Plummer Christopher , Lei Wei
Published: April 2020

Tec family non‐receptor tyrosine kinases, including TEC, BTK, ITK/EMT/TSK, BMX and TXK/RLK, are expressed in a variety of immune cells. Previous studies have demonstrated that Tec kinases play pivotal roles in inflammatory disorders such as allergic asthma and atopic dermatitis. Several 5‐aminomethylbenzimdazoles (5‐AMBD) have been reported as highly potent inhibitors of ITK as well as other TEC kinases and showed strong activities on reducing IL‐2 and IL‐4 production. However, the impact of 5‐AMBD on macrophage‐mediated inflammatory responses remains unclear. The goal of this study was to determine the effect of 5‐AMBD on the inflammatory responses in macrophages stimulated with lipopolysaccharide (LPS) and interferon‐gamma (IFNγ). RAW 264.7 cells (a murine macrophage cell line) were stimulated with 10 ng/mL LPS and 1 ng/mL IFNγ for 10 min, then treated with various concentrations of 5‐AMBD. The cell viability and production of nitric oxide (NO) were measured 20 hr after treatment. The total RNA was extracted from cells treated for 4 hr, and then the expression of inducible nitric oxide synthase (iNOS), tumor necrosis factor α (TNFα), and interleukin 1 beta (IL‐1β) were evaluated using quantitative real‐time PCR. The impact of 5‐AMBD on the activation of nuclear factor kappa B (NF‐κB) was investigated using the RAW 264.7 cells stably expressing the NF‐kB luciferase reporter. 5‐AMBD dose‐dependently reduced the NO production with no significant toxicity to the macrophages. Treatment of 100 nM and 1 μM 5‐AMBD significantly inhibited the expression of iNOS by 50% and 95%, respectively. Meanwhile, 5‐AMBD (1 μM) also reduced the expression of IL‐1β and TNFα. Furthermore, we found that 5‐AMBD dose‐dependently diminished the activation of NF‐κB at 4 hr and 20 hr after treatment. These findings demonstrate that 5‐AMBD has a strong anti‐inflammatory activity in macrophages, and suggest that TEC kinases could be potential drug targets for inflammatory diseases.

Support or Funding Information

Work supported by Small Pharmacy Awards for Research and Collaboration from the Presbyterian College School of Pharmacy.
Assembling Three‐Dimensional Models of the Human Brain to Study Development and Disease
Authors: Pasca Sergiu P.
Published: April 2020

A critical challenge in understanding the programs underlying the development, assembly and dysfunction of the human brain is the lack of direct access to intact, functioning human brain tissue for direct investigation and manipulation. In my talk, I will describe efforts in my laboratory to build functional cellular models and capture previously inaccessible aspects of human brain function and dysfunction. To achieve this, we have been developing methods for deriving in vitro, from pluripotent stem cells, self‐organizing 3D tissue structures called brain region‐specific spheroids or organoids that resembles specific regions of the human brain. We have shown that these brain‐region specific organoids, such as the ones resembling the cerebral cortex, recapitulate many features of in utero neural development, can be derived with high reliability across dozens of cell lines and can be maintained for years to capture advanced stages of neural and glial maturation and function. Moreover, we demonstrated that regionalized brain organoids can be put together to form fused structures called brain assembloids, which can be subsequently applied to investigate cell migration, long‐distance connections and the formation of neural circuits. Lastly, I will illustrate how our modular, stem‐cell derived 3D platform can be used to model disease and to study the cellular and molecular consequences of mutations associated with neuropsychiatric disorders.
Zebrafish‐inspired strategies for spinal cord repair.
Authors: Mokalled Mayssa H. , Zhou Lili , Mcadow Ryan , Burris Brooke
Published: April 2020

Spinal cord injury (SCI) causes irreversible loss of sensory and motor functions in mammals. In contrast to mammals, adult zebrafish are capable of efficient, spontaneous recovery after SCI. Whereas glial scarring and neuron loss accentuate spinal cord (SC) damage and inhibit regeneration in mammals, zebrafish are capable of pro‐regenerative glial bridging and effective adult neurogenesis post‐injury. Emerging evidence suggests that in mammals these pro‐regenerative processes are either inefficient or overshadowed by anti‐regenerative effects. We propose that developing efficient SCI treatments will require a deeper understanding of endogenous SC regenerative capacity, and that adult zebrafish present a valuable model to uncover fundamental, evolutionarily concealed mechanisms of mammalian SC regeneration.

Although glial bridging and neurogenesis are hallmarks of the robust SC repair observed in zebrafish, the mechanisms that direct these processes are underexplored. Using expression profiling and genetic loss‐of‐function, we screened for and identified a suite of molecular factors and cell populations that are activated after injury and required for SC regeneration. These studies revealed that discrete lineage‐restricted niches of progenitor cells differentially contribute to glial bridging and neurogenesis after SCI in zebrafish. Using a battery of genetic and molecular tools, we identified yap‐ctgfa‐twist1a as a central glial bridging signaling axis, generated the first transcriptional profiles of zebrafish bridging glia, and elucidated glial cell injury responses at the single cell level in zebrafish. We are currently using our understanding of the bridging glial cell fate in zebrafish to perform molecular comparisons with mammalian glial cells, and to reprogram mammalian glia into scarless, pro‐regenerative scaffolds that promote SC regrowth. Taken together, our studies elucidate fundamental mechanisms that direct innate SC regeneration in zebrafish, and use natural SC regeneration as a platform to enhance mammalian SC repair at the molecular and cellular levels.

Support or Funding Information

We acknowledge support from NIH (R01‐NS113915), the Missouri Spinal Cord Injury/Disease Research Program, the McDonnell Center for Cellular and Molecular Neuroscience, the Institute of Clinical and Translational Sciences, and from Washington University School of Medicine to M.H.M.
Intracellular Localization Patterns of Cancer‐associated Thyroid Hormone Receptor Mutants
Authors: Silva Carolina Lopez , Allison Lizabeth A.
Published: April 2020

The thyroid hormone receptor (TR) is a nuclear receptor that plays a critical role in human development and metabolism, regulating thyroid hormone interactions. TR rapidly shuttles between the nucleus and cytoplasm but is primarily localized to the nucleus at steady state, where it acts as a hormone‐dependent transcription factor. This process of nuclear import and export is mediated by two nuclear localization signals (NLSs) and multiple nuclear export signals (NESs). Previous studies have shown that TR may act as a tumor suppressor by regulating key genes in cell growth and proliferation in response to thyroid hormone. However, if TR shuttling is misregulated, it will not be able to perform its transcriptional regulatory functions. In this project, we analyzed the impact of NLS and NES amino acid substitutions on the intracellular localization patterns of TRα1 mutants previously associated with hepatocellular carcinoma (G24E, M256V, E343A, P269L) and thyroid cancer (K29T, C97A). We transfected mammalian cells with expression plasmids for green fluorescent protein (GFP)‐tagged and mCherry‐tagged TRα1 mutants and examined localization patterns through quantitative fluorescence microscopy. Here, we show that both TRα1 mutations exhibit increased cytosolic distributions. Further, mutant TRα1 (K29T, C97A) had an increased tendency to form nuclear and cytosolic protein aggregates. In co‐transfection experiments, TRα1 (K29T, C97A) failed to co‐localize with LC3, a well‐characterized autophagy marker, but showed significant co‐localization with GFP‐170, a marker for aggresome formation. These data suggest that aggregates formed by TRα1 (K29T, C97A) are actively recruited to cellular aggresomes as a cellular stress response. Overall, our study provides evidence that even minor changes in TR’s primary structure can have a significant impact on its intracellular localization and folding patterns.

Support or Funding Information

This project was funded by the National Institutes of Health. Grant information: 2018–2021, P.I., “Thyroid Hormone Receptor Mislocalization and Pathogenesis,” National Institutes of Health (NIH‐NIDDK) Research Grant # 2 R15 DK058028‐05: $405,273
Blunted Autophagy and Heat Shock Responses in Peripheral Blood Mononuclear Cells of Elderly Adults During Prolonged, Extreme‐Heat Exposure
Authors: McCormick James J. , Meade Robert D. , McGarr Gregory W. , Notley Sean R. , Fujii Naoto , Kenny Glen P.
Published: April 2020

With rising global temperatures and accompanying increases in the incidence and severity of extreme heat events, there are growing concerns about the impact of heat stress on human health. Increases in the activation of autophagy and the heat shock response are essential to cellular survival under stressful conditions, such as extreme heat. Accumulating evidence from various animal models suggests an age‐related decline in the autophagy and heat shock response pathways during heat stress; however, this has not yet been assessed in humans. We therefore sought to evaluate the hypothesis that the autophagy and heat shock response would be blunted in elderly compared to young adults following a prolonged heat exposure. To assess this hypothesis, peripheral blood mononuclear cells (PBMCs) were harvested from 10 young (23 [SD 3] years; 3 women) and 9 elderly adults (71 [SD 3] years; 2 women) prior to and following 9‐hours of resting exposure to extreme heat (40°C, ~15% relative humidity). Changes in microtubule associated protein 1 light chain 3 beta (LC3)‐II (indicative of autophagy) and heat shock protein 70 were analysed via Western blot, while their associated genes (LC3B and HSPA1A, respectively) were measured via quantitative reverse transcription polymerase chain reaction. All values were normalized to β‐actin and reported as fold changes relative to baseline. Core temperature (indexed by rectal temperature) was measured throughout the exposure. Data are presented as means and standard deviations (SD) and were compared between‐groups using unpaired (2 tailed) t‐tests (α=0.05). After 9‐h of heat exposure, the elevation in core temperature from baseline values were greater in the older (1.0°C [SD 0.3]) vs. young adults (0.6°C [SD 0.2]; p<0.01). Despite this, LC3‐II protein was lower in elderly (2.02 [SD 2.00]) when compared to young adults (4.45 [SD 5.93]; p=0.04). This was further revealed by a reduction in LC3B gene expression in elderly (1.39 [SD 0.39]) compared to young (2.57 [SD 3.78]; p=0.04) adults suggesting a blunting in the autophagic response during heat stress. In elderly, HSP70 protein and HSPA1A gene expression were both lower when compared to young (1.21 [SD 0.43] vs. 1.80 [SD 0.65]; p=0.02 and 1.58 [SD 1.64] vs. 2.88 [SD 4.29]; p=0.04, respectively) suggesting an age‐associated decline in the heat shock response. These preliminary findings demonstrate that despite greater elevations in core temperature, elderly adults experience blunting in both the autophagy and heat shock response during a prolonged extreme‐heat exposure.

Support or Funding Information

Canadian Institutes of Health Research
Microglial Targeting as a Therapeutic Strategy for Social Stress Susceptibility
Authors: Pope Brittany S. , Harrington Evelynn N. , Bouknight Samantha J. , Martinez- Muniz Gustavo A. , Augenblick Lee M. , Morgan C. Grant , Wood Susan K.
Published: April 2020

Social stress is a common risk factor for anxiety and cardiovascular disease (CVD). Women are more likely than men to suffer from comorbid anxiety and CVD. While the underlying neural mechanisms linking these pathological conditions remain unclear, the locus coeruleus (LC) is a stress‐sensitive region known to facilitate both behavioral and cardiovascular responses to social stress. Importantly, neuroinflammation has been shown to play a causative role in generating anxiety‐like behavior, yet whether pharmacological manipulation of microglia represent a strategy to regulate stress susceptibility in a female population remains to be determined. Here, we utilize mannosylated liposomal clodronate, a small hydrophilic molecule toxic to microglia. Both the liposomes and the drug clodronate (CLD) are selective for macrophages/microglia, making this intra‐LC administration a targeted depletion of microglial cells. Two separate studies were conducted to test the hypothesis that microglial targeting is effective in regulating susceptibility to social stress. In study #1, following recovery from surgical implantation of cardiovascular transmitters (HD‐S11, DSI) and bilateral LC cannulae, rats were treated with liposomal CLD injections (5 or 25 μg/side) or empty‐liposomal vehicle (0 μg/side). Female rats were then subjected to witnessing an aggressive social defeat encounter between a male intruder and a novel male resident for 15 min on 5 consecutive days. This study identified that witness stress exposure increased anxiety‐like burying compared with controls and that partial microglial depletion in the LC attenuated this burying response. Interestingly, intra‐LC CLD had no cardiovascular effect upon the first exposure to stress, yet promoted the pressor response following repeated stress exposure (day 5). To further examine whether microglia are controlling the behavioral and cardiovascular responses to stress, female rats were treated with intra‐LC LPS (0, 1, 3 ug/rat) with or without a TLR‐4 selective microglial inhibitor (LPS‐RS and (+)‐ naloxone) prior to witness stress exposure. While these treatments had no effect under non‐stress conditions, witnesses treated with LPS alone exhibited greater burying duration compared with those treated with vehicle, and this was blocked by the microglial inhibitor. Taken together, these studies reveal a novel role for microglia in regulating behavioral and cardiovascular responses to social stress and demonstrate that selective targeting of microglia may be a therapeutic strategy to alter stress susceptibility.

Support or Funding Information

Support provided by 1R01MH113892‐01A1, NARSAD Young Investigator Grant 26809, I21 BX002085.
Regulation of Neutrophil Function by Selective Targeting of Glycan Epitopes Expressed on the Integrin CD11b/CD18
Authors: Brazil Jennifer Catherine , Kelm Matthias , Lehoux Sylvain , Azcutia Veronica , Cummings Richard D. , Nusrat Asma , Parkos Charles A.
Published: April 2020

Polymorphonuclear neutrophils (PMN) play a critical role in the innate immune response to invading pathogens. However, dysregulated mucosal trafficking of PMN and associated epithelial tissue damage is a pathological hallmark of numerous inflammatory conditions including inflammatory bowel disease and chronic obstructive pulmonary disease. The glycoprotein CD11b/CD18 plays a well described role in regulating PMN transepithelial migration and PMN inflammatory functions. Previous studies have demonstrated that targeting of the N‐linked glycan Lewis X on CD11b blocks PMN transepithelial migration (TEpM). Given evidence of glycosylation dependent regulation of CD11b/CD18 function, we performed MALDI TOF Mass Spectrometry (MS) analyses on CD11b/CD18 purified from human PMN. MS analyses revealed a surprising lack of sialylation on CD11b glycans. Unusual glycan epitopes identified on CD11b/CD18 included high Mannose oligosaccharides recognized by the Galanthus Nivalis (GNA) lectin and biantennary galactosylated N‐glycans recognized by the Phaseolus Vulgaris erythroagglutinin (PHA‐E) lectin. Importantly, we show that selective targeting of glycans on CD11b with such lectins results in altered intracellular signaling events that inhibit TEpM and differentially affect key PMN inflammatory functions including phagocytosis, superoxide release and apoptosis. Taken together, these data demonstrate that discrete glycan motifs expressed on CD11b/CD18 such as biantennary galactose could represent novel targets for selective manipulation of CD11b function and reduction of PMN associated tissue damage in chronic inflammatory diseases.

Support or Funding Information

This work was supported by the Crohn’s and colitis foundation (SRA # 544596)
The balance of life and death may hinge on the intracellular localization of the small GTPase, Rap1b in the megakaryocyte cell line (DAMI)
Authors: Wisinski Jaclyn , McElfresh Regan , Muntifering Grace , Nguyen Tam , Veleke Carter
Published: April 2020

Platelets are specialized cell fragments that circulate in the bloodstream and clot to prevent blood from leaving damaged vessels. The number of circulating platelets is governed by the balance of platelet production by mature megakaryocytes and clearance of platelets from the bloodstream. Normal platelet function requires the signaling protein, Rap1b, whose abundance also increases as megakaryocytes mature. The intracellular location of Rap1b is determine by its phosphorylation state. When Rap1b is not phosphorylated, it associates with membranes; conversely when Rap1b is phosphorylated, it detaches from membranes and reside in the cytosol. We hypothesize that Rap1b, specifically in its phosphorylated, cytosolic state, promotes megakaryocytes survival and proliferation. To address this hypothesis, the human megakaryocyte cell line (DAMI cells) have been modified 1) by disrupt the rap1b gene creating a Rap1b‐knockout cell line, 2) stable overexpression of a Rap1b phospho‐mimetic mutant, 3) stable overexpression of a Rap1b phospho‐deficient mutant or 4) stable overexpression of wild‐type Rap1b (to be used as a control). Using the modified DAMI cell lines we are beginning to understand the impact of Rap1b phosphorylation on proliferation on in MTS survival assays. Changes in cell survival can be attributed to changes in cell cycle progression and/or susceptibility to apoptosis. To address possible changes in cell cycle progression, we have evaluated each cell line based on the percentage of cell undergoing DNA synthesis at a given time by BrdU incorporation assays. To evaluate apoptosis susceptibility, mitochondrial membrane integrity and activation/cleavage of Caspase 9 was determined in the presence or absence of the apoptotic inducer ABT‐737. The analysis of the role of Rap1b in megakaryocytes has the potential to increase our collective understanding of regulation of platelet count and could be a novel target to combat thrombocytopenia (low platelet count) or thrombocythemia (high platelet count).

Support or Funding Information

University of Wisconsin ‐LaCrosse Faculty Research Grant
The Early Progression of Renal Injury in Obese Dahl Salt‐Sensitive Rats is Associated with Increased M2 Macrophage Infiltration
Authors: Poudel Bibek , Shields Corbin A. , Cornelius Denise C. , Williams Jan M.
Published: April 2020

Previously, we reported that, prior to puberty, the obese Dahl salt‐sensitive leptin receptor mutant (SSLepRmutant) strain displays proteinuria and renal injury independent of hyperglycemia and elevations in arterial pressure. Recently, we observed that the early development of renal injury in the obese SSLepRmutant strain was associated with a 5‐fold increase in renal infiltration of macrophages compared to SSWT rats. In this study, we examined whether depletion of macrophages with clodronate would reduce renal injury in prepubescent obese SSLepRmutant rats. Four week‐old SSWT and SSLepRmutant rats were separated into four groups (n=8/group): (1) SSWT and (2) SSLepRmutant rats treated with vehicle and (3) SSWT and (4) SSLepRmutant rats treated with clodronate (50 mg/kg, i.p., twice/week) for 4 weeks. While administration of clodronate did not alter the macrophage population in SSWT rats, clodronate reduced macrophages in the kidneys from SSLepRmutant rats compared to vehicle‐treated rats (8±4 vs. 33±10 % gated, respectively; p<0.05 vs. vehicle) without altering the monocyte population. Interestingly, we did not observe any effects on MAP in animals chronically treated with clodronate. At baseline, protein excretion was significantly higher in SSLepRmutant rats compared to SSWT rats (70±14 vs. 10±3 mg/day, respectively; p<0.05 vs. SSWT) that further increased over the course of the study (335±82 vs. 45±14 mg/day, respectively; p<0.05 vs. SSWT). After 2 weeks of treatment with clodronate, protein excretion was markedly reduced in the SSLepRmutant strain (159±32 vs. 303±52 mg/day; p<0.05 vs. vehicle). However, after 4 weeks of treatment, the effect of clodronate was no longer observed in the SSLepRmutant strain (346±195 vs. 399±50 mg/day). Chronic treatment with clodronate significantly decreased glomerular injury and renal fibrosis in the obese SSLepRmutant strain. Since clodronate depletes all macrophages: M1 (pro‐inflammatory) and M2 (anti‐inflammatory or pro‐inflammatory), we also examined macrophage polarization in the vehicle treated lean SSWT and obese SSLepRmutant strain prior to puberty at 8 weeks of age. We found that the M1/M2 ratio was significantly higher in the lean SSWT versus obese SSLepRmutant rats (1.60±0.05 vs. 0.54±10, respectively; p<0.05 vs. SSWT), demonstrating more M2 macrophages in the kidneys of the obese SSLepRmutant strain. Overall, these data show that the early development of renal injury in the obese SSLepRmutant strain is associated with increased renal M2 macrophages and suggests that M2 macrophages may be a therapeutic target in obesity‐induced renal injury.

Support or Funding Information

This study was supported by DK109133.
The Effects of Flipped Teaching and Retrieval Practice in a Graduate Level Physiology Course
Authors: Rever Anna Lee , Gopalan Chaya
Published: April 2020

Retrieval practice is a deliberate recalling exercise that has been shown to enhance student learning and engagement. Yet another teaching method, the flipped classroom (FC), is gaining interest as it allows active learning instead of traditional lecture‐based teaching (TT). These three teaching methods were compared in a graduate‐level physiology course for Certified Registered Nurse Anesthetist (CRNA) students over four semesters using exam grades. The TT method involved classroom lectures during the entire class period with periodic quizzes and group activities over the lecture content. In the FC method, the lecture content was shifted outside of the classroom for students to review on their own prior to be engaged in active learning activities during class time. An online individual assessment was assigned to ensure student participation in completing the review of lecture content. The in‐class time was used for mini‐lectures for the clarification of content and team‐based learning activities (TBL), where the students could use resources to solve problems. Subsequently, FC was combined with retrieval practice (FCR). In the FCR teaching method, students were still given study materials to review prior to scheduled class meeting. However, the individual assessment was shifted to be completed during class where students were not allowed to use any of the resources, in order to enhance retrieval exercise. Similarly, the TBL assessment was completed in class without the use of any study materials. The FCR teaching method resulted in higher exam scores (M = 91.13, SD = 0.089) than the TT method (M = 89.57, SD = 0.549). Thus, students learning seems to be more enhanced with the use of the FCR teaching method than the TT or the FC methods. Also, when the exam scores were divided into the upper and lower half of the class, the upper half benefited more than the lower half in both the FC and FCR methods. On the other hand, when male and female students were compared between the three teaching methods, there was no statistical difference. Typically, CRNA students are highly motivated and maintain a very high class average (89.57% in the TT method). Thus, any further increment will require a major shift, such as the FCR teaching approach. The upper half of the class demonstrating significantly higher scores in both the FC and FCR teaching methods suggests that they utilize pre‐class content more efficiently in their preparation for their in‐class activities compared to the lower half of the class. Overall, in conclusion, FTR appears to be the best among the three teaching methods, TT, FC, and FCR studied.