bluuebunny/crossref_metadata_2025 huggingface dataset:
Sulfation of endothelial heparan sulfate mediates Staphylococcus aureus induced hepatotoxicity
Authors: Golden Gregory James , Toledo Alejandro Gómez , Cuello Hector , Nizet Victor , Esko Jeffrey
Published: April 2020

Sepsis is defined as life‐threatening organ dysfunction caused by a dysregulated host response to infection and vascular pathology. Understanding the linkage of vascular pathology to organ failure could provide a strategy for treating the disease. Heparan sulfate proteoglycans are critical components of the vascular endothelial glycocalyx and play a key role in sepsis outcome in mice. To study how heparan sulfate modulates organ‐specific vascular pathology during sepsis, we induced Staphylococcus aureus sepsis in Ndst1f/fTie2Cre mice, which express under‐sulfated heparan sulfate in endothelial and myeloid tissues. Hepatic coagulopathy and infarction induced by S. aureus was reduced in Ndst1f/fTie2Cre mice, but not in Ndst1f/fLysMCre or Ndst1f/fPF4Cre mice. To examine how modifying heparan sulfate structure mitigates hepatic damage, pro‐inflammatory neutrophil trafficking and behavior was monitored in the liver during sepsis. Ndst1f/fTie2Cre mice contained fewer extravasated neutrophils in the liver early in sepsis. Intravital microscopy in the liver further verified that Ndst1f/fTie2Cre mice have neutrophil trafficking deficiencies after both sterile and septic injury. Further studies will focus on the role of heparan sulfate in neutrophil trafficking and downstream neutrophil‐driven vascular dysfunction in sepsis.

Support or Funding Information

This work is supported by NIH P01 HL131474 and the UCSD Microbial Sciences Graduate Research Initiative Fellowship.
A Realistic View of Proteins : Solution Scattering at the Life Sciences X‐ray Scattering Beamline (LIX)
Authors: Byrnes James , Chodankar Shirish , Lazo Edwin , Qian Kun , Shea-McCarthy Grace , Antonelli Stephen , Yang Lin
Published: April 2020

Small angle X‐ray scattering is an important tool that reveals the behavior of proteins in their native environment. The scattering pattern produced from a SAXS experiment represents an average of all conformations present in the solution. Since most proteins in our body function in a solution environment, SAXS data can reveal additional biologically relevant states of a molecule or molecular complex. Furthermore, SAXS provides data about the shape and size of the molecule of interest and is a complementary technique to other structural methods. Combining SAXS data with X‐ray crystallography, NMR or Cryo‐EM can lead to a more robust understanding of the species being studied. High quality SAXS data can be used to build ab initio low resolution bead models, which provide a 3D envelope of your molecule. Here at the life sciences X‐ray scattering beamline (LiX), we collect data by flowing sample through the X‐ray path in a 3 channel flow cell. As the sample enters the X‐ray path, the simultaneous collection of SAXS and wide angle X‐ray scattering (WAXS) is obtained via a 3 detector setup. The detector setup has a q‐range of 0.006Å−1 to 3.2 Å−1, primarily to obtain scattering data from the water peak to normalize buffer subtraction. Automated sample handling at LiX allows for collection of up to 360 samples, packaging of data and processing. One challenge in obtaining high quality SAXS data is preparation of pure, monodisperse samples. For these complicated samples that have multiple species, our in‐line HPLC system seamlessly integrates with our beamline for SEC‐SAXS (size‐exclusion chromatography‐SAXS), and increases the chances of obtaining a more pure, monodisperse sample immediately before data collection.

Support or Funding Information

LiX beamline is part of the Life Science Biomedical Technology Research resource, co‐funded by the National Institute of General Medical Sciences (NIGMS) under grant P41 GM111244 and by the DOE Office of Biological and Environmental Research under grant KP1605010, with additional support from NIH under grant S10 OD012331. The operation of NSLS2 is supported by US Department of Energy, Office of Basic Energy Sciences, under contract No. DE‐SC0012704.
New parasite treatment with old alcohol drug targeting COP9 signalosome
Authors: Ghosh Swagata , Farr Laura , Singh Aditya , Leaton Laura Ann , Padalia Jay , Sullivan David J. , Moonah Shannon N.
Published: April 2020

The Ubiquitin‐Proteasomal System (UPS) is essential for cell survival because it is responsible for the large majority of protein degradation within the cell. Thus, targeting the UPS has emerged as an attractive approach for combating protozoan parasites. Yet, the regulation of UPS‐mediated protein degradation remains poorly understood in clinically important protozoans. We identified COP9 signalosome to be conserved in multiple pathogenic parasites including Leishmania, Trypanosoma, Toxoplasma and Entamoeba. Here, we show that COP9 signalosome is an essential and druggable parasite target that regulates protein degradation. Using Entamoeba histolytica as a model parasite, we found that genetic disruption of COP9 subunit 5 (CSN5) by two independent but complementary approaches, inhibited parasite cell proliferation and viability. Both CSN5 knockdown and dominant negative mutation trapped cullin1 in a neddylated state disrupting UPS activity and protein degradation. Using virtual screening by molecular docking, we identified zinc dithiocarbamate (ZnDTC), a metabolite of the FDA approved alcohol drug disulfiram, as a potential COP9 inhibitor. We found that ZnDTC acts in a COP9‐dependent manner, phenocopy CSN5 gene disruption, and is active against E. histolytica parasites at nanomolar concentrations. In addition, in‐vivo studies revealed that ZnDTC had oral efficacy in clearing E. histolytica parasites in a mouse model that mirrors human amebic colitis. Parasite clearance was assessed by live bioluminescent imaging, amebic culture and immunohistochemistry. Also, ZnDTC treatment promoted resolution of inflammation and tissue damage. Our findings provide insights into how COP9 signalosome regulates parasite protein degradation, and supports COP9 inhibition as a potential anti‐parasite therapy.
Adrenergic signaling‐induced ultrastructural strengthening of intercalated discs via PG is crucial for positive adhesiotropy in murine cardiomyocytes.
Authors: Yeruva Sunil , Kempf Ellen , Egu Desalegn Tadesse , Kugelmann Daniela , Waschke Jens
Published: April 2020

Intercalated discs (ICD), which connect adjacent cardiomyocytes, are composed of desmosomes, adherens junctions (AJ) and gap junctions (GJ). Previous data demonstrated that adrenergic signaling enhances cardiac myocyte cohesion, referred to as positive adhesiotropy, via PKA‐mediated phosphorylation of plakoglobin (PG). Here, we further investigated the role of PG in adrenergic signaling‐mediated ultrastructural changes in ICD of cardiomyocytes. Quantitative transmission electron microscopy analysis of ICD demonstrated that cAMP elevation caused significant elongation of area composita and thickening of the ICD plaque, paralleled by enhanced cardiomyocyte cohesion, in WT but not PG‐deficient cardiomyocytes. STED microscopy analysis supported that cAMP elevation ex vivo enhanced Dsg2 and N‐cadherin (N‐cad) staining in ICDs of WT but not PG‐deficient cardiomyocytes. For dynamic analyses we utilized HL‐1 cardiomyocytes, in which cAMP elevation induced translocation of desmoglein‐2 (Dsg2) and PG but not of N‐cad to cell junctions. Nevertheless, depletion of N‐cad but not of Dsg2 resulted in a decrease in basal cell cohesion whereas positive adhesiotropy was abrogated in monolayers depleted for either Dsg2 or N‐cad. Moreover, AFM experiments showed that the cAMP elevation‐induced increase in Dsg2 binding events close to cell junctions was abolished by depletion of either Dsg2 or N‐cad. Our data demonstrate that in murine cardiomyocytes, adrenergic signaling via PG enhances N‐cad and Dsg2 in the ICD leading to strengthening of ICDs and thereby positive adhesiotropy.

Support or Funding Information

This work was supported by the Ludwig‐Maximilians‐Universität Munich with the Föfole program; and the Deutsche Forschungsgemeinschaft DFG grant number WA2474/11‐1 to Jens Waschke
Inflammation and Immune Markers in Psychological Health
Authors: Scholl Jamie L. , King Zach , Potter Kari , DeCino Daniel A. , Hertel Danielle , Pearson Kami , Graack Eric , Zeng Erliang , Brown-Rice Kathleen , Forster Gina L.
Published: April 2020

There has been a growing emphasis on the relationship between inflammatory responses, gut microbiota and the immune system, and their influence on mental health outcomes. Stress can influence the composition of gut microbiota which, in turn, influences the central nervous systems response to future stressors. C‐Reactive Protein (CRP) is a biomarker for inflammation that has been linked to depression, anxiety disorders and PTSD that is released in response to pro‐inflammatory cytokines. The goal of this study was to test a direct association between behavior profiles such as anxiety and depression, the composition of the gut microbiota, basal levels of CRP and cytokines, cortisol and genetic predisposition. Measures of anxiety, depression, PTSD, substance use and overall health were obtained from adults with no recent illness or surgery, or exposure to antibiotics. Biological measures focused on pro‐ and anti‐inflammatory cytokines from plasma, cortisol levels from hair, and gut microbiome; as well as single nucleotide polymorphisms (SNPs) were assayed. Using factor analysis we determined a shared mental health panel including depression, anxiety and PTSD measures that was negatively correlated to related cytokines. Regression modeling was used to related quantitative traits to demographic and genetic factors, revealing significant relationships between inflammatory markers and mental health. Understanding these relationships will help to determine direct future research in assessing causal direction between all of these factors that can promote mental well‐being, and could in the future be used as biomarkers for vulnerability to mental illness, and even help re‐think how we treat mental health disorders.

Support or Funding Information

The project described was supported by the National Institute Of General Medical Sciences, 1U54GM115458‐01; The Center for Brain and Behavior Research (CBBRe), USD; Basic Biomedical Sciences, USD; and the South Dakota Governor’s Center for Genetics and Behavioral Health.
Tracing the Cellular “Life Cycle” of Medical Education: Histology as a Theme in Medical Education
Authors: Macnow Alexander Stone Renner
Published: April 2020

As students pass through the various phases of their “life cycle” in medical education—from undergraduate or post‐baccalaureate student to medical student to residency training and beyond as a healthcare practitioner—cellular structure and function underlie a thorough understanding of mechanisms of disease and are recognized as appropriate launching‐off points to explore the subjects of pathology and therapeutic interventions.

Starting in undergraduate and post‐baccalaureate education, students are exposed to cellular structure and function as a corollary to physiology. Research conducted by the Association of American Medical Colleges (AAMC) in anticipation of the Medical Colleges Admissions Test (MCAT®) redesign in 2015 highlighted the importance of histology, cellular physiology, and molecular biology—and the connections between these disciplines—as necessary for success in medical curricula of the twenty‐first century (AAMC MR5 Committee, November 2011).

So what role does histology play, then, in medical curricula themselves? As also discussed by the other members of this panel, recognition of histology as a physical correlate of proper cellular function permits the identification of morphologic changes as correlates of cellular dysfunction. Indeed, it is only through the assimilation of a paradigm of “normal” that the student can distinguish “abnormal” and apply or infer the effects of disease states in tissues.

Finally, as only a small minority of graduating medical students will select the fields of Anatomic or Clinical Pathology as their career paths, how does histology remain relevant for practitioners during and after graduate medical training? Exploration of analytical and post‐analytical communication between diagnostician and clinician highlights several of these key roles of histology (and pathology) in medical communication and in the discourse of disease. These include improving classification and nomenclature of neoplasms; providing rapid on‐site cytologic diagnoses; and carefully choosing language to trigger or eschew specific interventions, improving patient safety. In these myriad ways, cellular histology (though perhaps often “hidden” behind the microscope) remains relevant throughout the life cycle from student to practitioner.
A new approach for preparing SC‐514 loaded PLGA particles by single emulsion method
Authors: Famuyiwa Toluleke Oloruntobi , Diaka James Kumi , Asghar Waseem , Bowers Zoey , Jebelli Joubin , Bentley Austin , Subtil Paulynice
Published: April 2020

Poly (lactic‐co‐glycolic acid) (PLGA) is a non‐toxic or non‐injurious subdivision of biodegradable polymer that is not capable of causing immunological rejection. PLGA has been successfully used to encapsulate drugs and deliver drugs in various applications. This high success rate can be linked to the fact that PLGA has been approved by FDA (Food and Drug Administration). SC‐514 is a relatively new hydrophobic dug, which has been shown to have anti‐cancer effects via inhibition of NF‐KB dependent gene expression in cancer cells. SC‐514 was encapsulated in PLGA nanoparticles via single‐emulsion method. The SC‐514 loaded PLGA nanoparticles (diameter=49.4nm) synthesized has the potential to increase the bioavailability of SC‐514 drug in prostate cancer treatment. Hence, increasing the therapeutic effect of SC‐514 in prostate cancer treatment.

Support or Funding Information

Fellowships and Scholarships from FAU
Endothelial Dysfunction is Transferable from Humans to Germ‐Free Mice via Fecal Microbiota Transplantation
Authors: Trikha Raj , Lee Dustin M. , Ecton Kayl E. , Wrigley Scott D. , Vazquez Allegra R. , Litwin Nicole S. , Thomas Keely N. , Battson Micah L. , Johnson Sarah A. , Weir Tiffany L. , Gentile Christopher L.
Published: April 2020

Introduction

Endothelial dysfunction and arterial stiffness are commonly observed in obese individuals and are both independent risk factors for cardiovascular disease. Recent preclinical data from our laboratory suggest that alterations in the gut microbiota may be an important factor linking obesity to endothelial dysfunction and arterial stiffness. The purpose of this study was to begin to translate these preclinical data to a human population by examining whether vascular phenotypes in humans are transmissible by fecal microbiota transplantation to germ‐free mice. We hypothesized that germ‐free mice receiving transplantation of an obesity‐associated microbiota would display impaired vascular function compared to germ free mice receiving transplantation of a lean microbiota.

Methods

Twenty lean and obese participants were screened for vascular dysfunction and stool samples were collected. One representative obese and one lean age‐ and sex‐matched participant with disparate vascular function were selected as donors for fecal microbiota transplantation. Following transplantation, mice consumed a maintenance diet for 12 weeks. During weeks 11 and 12, mice underwent a glucose tolerance test and a FITC‐dextran test to assess intestinal permeability. After 12 weeks, vascular phenotypes were determined via in vivo aortic pulse wave velocity and ex vivo endothelium‐dependent and ‐independent dilation. 16S rRNA sequencing was used to analyze the microbiota in all samples and sequence data was analyzed using QIIME2 and myPhyloDB.

Results

Principal Component Analysis of mouse and donor samples revealed separation of global microbiota profiles with species (human vs mouse) being the factor accounting for the greatest variability (Axis 1; ~24%) in the microbiota, followed by vascular phenotype of the donor (Axis 2; ~19%).

Endothelium‐dependent dilation was significantly impaired (p<0.05) in mice receiving microbiota from the obese donor relative to those with lean donor microbiota (maximal dilation to ACH: 50.1% vs 76.9%, p=0.013). Glucose tolerance was also impaired in mice receiving the obese microbiota (AUC: 1577.3 vs 1168.13 mg/dL, p=0.064; minute 90: 372.0 vs 219.2 mg/dL, p=0.043; minute 120: 371.7 vs 211.5 mg/dL, p=0.032). No significant differences were seen with endothelium‐independent dilation, aortic pulse wave velocity, or body weight.

Conclusions

These data provide further evidence that the gut microbiota is an important regulator of vascular function, and suggest that directly targeting the microbiota may be a novel therapeutic strategy for altering vascular phenotypes.

Support or Funding Information

NIH R01 HL14411; American Heart Association #18TPA34170585; NIFA AES COL00766
A Randomized, Placebo‐Controlled, Double‐Blind Clinical Investigation on a Patent‐Pending KD120 Metabolically Activated Enzyme Formulation, N‐SORB, in Healthy Young Volunteers
Authors: Nair Sreejayan , Smith Derek , Nair Rama , Downs Berrnard W. , Kushner Steve W. , Bagchi Debasis , Bagchi Manashi
Published: April 2020

Impaired digestive competence and consequential sluggish immune and metabolic sequela are epidemic‐sized health problems in modern cultures. Since digestion is the first step of metabolism, implementing novel strategies to restore digestive and metabolic competence are important health priorities. N‐SORB is a patent‐pending KD120 multienzyme complex composed of metabolically activated natural digestive‐type enzymes encapsulated in a high‐potency phosphatidylcholine non‐GMO soy lecithin SK713 SLP absorption technology (Prodosome®). A randomized, double‐blind placebo‐controlled study was conducted in 46 male and female healthy volunteers (age: 25.8 ± 12.1 years) to assess the safety and efficacy of N‐SORB. Subjects received either N‐SORB (1 mL, twice daily) or the same amount of placebo for 90 consecutive days. Body weight, blood pressure, physical health, and complete blood chemistries including blood glucose, liver enzymes, and lipid profiles; and immune health, physical health, sleep quality and quality of life parameters were assessed pre‐ and post‐intervention. Serum cytokine levels were determined by using a Bio‐Plex Pro Human Cytokine 8‐plex assay and ELISA. Whole body composition analysis was performed by dual‐energy X‐Ray absorptiometry (DEXA) to determine body fat mass, lean mass and android and gynoid fat. Quality of life (QOL) and sleep quality were assessed using WHOQOL‐BREF and Pittsburgh Sleep Quality Index (PSQI) questionnaire. Adverse events were strictly monitored. 40 subjects completed the study. Hematocrit, hemoglobin, mean corpuscular volume, platelets and lymphocytes provide evidence indicating consistent trends of improvement in the N‐SORB group. QOL parameters showed a small but significant improvement in the N‐SORB group. A significant increase was observed in AST levels in the placebo group at the end of 90 days of treatment, indicating increased liver distress. However, no increase was observed in the N‐SORB group. No significant changes in BUN, serum creatinine, ALP, ALT, and lipid profile were observed between the placebo and treatment groups before and after the 90‐day intervention. No adverse effects were reported. This 90‐day intervention study with N‐SORB clearly exhibited trends of improvement in RBC and immune parameters and found improvements in QOL and sleep quality scivnin already healthy volunteers. The N‐SORB treatment group experienced significant improvement in gastrointestinal functions, immune and metabolic parameters and other systemic improvements. No adverse events were reported.

Support or Funding Information

The Project was partially sponsored by VNI Inc, Lederach, PA
Vacuole Membrane Protein 1 Deficiency Promotes the Development of Pancreatitis Through Autophagy Impairment and Endoplasmic Reticulum Stress
Authors: Wang Shaogui , Chao Xiaojuan , Ma Xiaowen , Ni Hong-Min , Ding Wen-Xing
Published: April 2020

Pancreatitis is a potentially fatal inflammatory disease with significant morbidity and mortality but the exact molecular mechanisms remain largely obscure. We investigated the role of vacuole membrane protein 1 (VMP1), an endoplasmic reticulum (ER) resident protein, in the pathogenesis of pancreatitis. We examined VMP1 expression and ER stress in human pancreatitis samples. We also determined VMP1 level and ER stress in experimental pancreatitis induced by cerulein as well as in pancreatic acinar cell‐specific VMP1 knockout (VMP1Δacinar) mice. We found that VMP1 expression was significantly lower in human pancreatitis samples associated with extensive ER stress. In a mouse experimental pancreatitis model, treatment with ceruleint markedly decreased VMP1 expression but increased levels of several ER stress markers such as p‐elf2α, XBP1s and CHOP. Western blot and immune histochemical analysis of human and experimental mouse pancreatitis samples showed impaired autophagy with accumulation of p62, an autophagy substrate protein that generally is degraded by autophagy. Intriguingly, VMP1Δacinar mice developed spontaneous pancreatitis with pathological features of chronic pancreatitis. In addition, VMP1Δacinar caused accumulation of p62 as well as selective ER‐phagy receptor FAM134B. Moreover, VMP1Δacinar mice had increased ER tress accompanied with massive cell death, suggesting VMP1 may serve as a key mediator of pancreatic ER homeostasis. Collectively, these data indicate a critical role of VMP1 in maintaining ER homeostasis in acinar cells and decreased VMP1 expression may contribute to the development of pancreatitis.

Support or Funding Information

R01 AA020518, R01 DK102142, U01 AA024733 and P30GM118247 (W.X.D).
Hemodynamic Effects of Acute Over‐Ventilation in a Swine Hemorrhage Model
Authors: Blackburn Megan B. , Hudson Ian L. , Rodriguez Cassandra M. , Ryan Kathy L.
Published: April 2020

Introduction

Airway compromise is the second leading cause of potentially survivable death on the battlefield, indicating a need for improved airway management approaches. Over‐ventilation of the lungs is a complication associated with prolonged use of mechanical ventilation, but few studies have examined the consequence of acute, pre‐hospital over‐ventilation. Therefore, the goal of this study was to determine the effect of acute hyperventilation on the morbidity of hemorrhaged swine.

Materials and Methods

Female Yorkshire pigs (40–50 kg) were anesthetized with isoflurane, instrumented for hemodynamic measurements and blood draws, and switched to IV anesthesia with midazolam and ketamine. Following baseline recording, 25% blood volume by weight was removed, followed by 1 hour of: spontaneous breathing (n=8), “normal” bag ventilation (6 L minute volume, n=9), bag hyperventilation (20 L minute volume, n=8), or mechanical ventilation (n=8). The pigs were then allowed to wake up from anesthesia, recovered for 24 hours, and euthanized for blood and tissue collections.

Results

Hemorrhage significantly decreased mean arterial pressure (P<0.001), cardiac output (P<0.001), and stroke volume (P=0.004), but increased heart rate (P<0.001). Ventilation following hemorrhage did not have any further impact on hemodynamic variables, except a significant increase in heart rate in those that underwent bag hyperventilation as compared to spontaneous breathing, normal bag ventilation, and mechanical ventilation groups (24±5 bpm, vs −7±7, 9±11, and 2±2 bpm), respectively. Following ventilation, blood lactate levels were significantly elevated compared to baseline in all groups. The change was significantly greater in those that underwent hyperventilation than in spontaneous breathing, normal bag ventilation, and mechanical ventilation groups (7.9±0.7 mmol/L, vs 4.1±0.6, 5.9±0.4, and 5.4±1 mmol/L), respectively. Blood lactate levels returned to baseline levels by 24 h in all groups. Wet to dry lung weight ratios were also not significantly different between groups, an indication that any edema caused by over‐inflation dissipates by 24 hours.

Discussion

This study was undertaken on the premise that over‐inflation of the lungs, even acutely, would result in significant barotrauma and negative downstream physiological effects. Preliminary results, however, indicate that hemorrhage followed by acute hyperventilation does not affect hemodynamic variables or increase morbidity in mildly hemorrhaged swine. Ongoing studies include histological evaluation of tissue samples, assessment of blood markers for tissue damage, and measurement of plasma cytokine levels to more fully discern the presence of any lasting detrimental physiological effects to the lung or to systemic physiology.

Support or Funding Information

USAMRDC Tactical Combat Casualty Care Research Program
Recombinase‐mediated Method for Multi‐scale Genomic Rearrangements
Authors: Ahmed Haidar S , O’Connell Ronan , Bashor Caleb
Published: April 2020

With the widespread use of genomic editing tools like CRISPR/Cas9, scientists have gained the ability to modify a host genome with high precision. Although these technological advancements have improved throughout the years, there is still a need for better methods to build more sophisticated multi‐part biological circuits. For this reason, we have employed the use of a genomic integration technique, landing pads, which utilized site‐specific recombinases to insert relatively large DNA assemblies into a well‐defined location the host genome. Using the CRISPR/Cas9 system, we knocked‐in an 8kb landing pad repair template into the AAVSl safe harbor locus of HEK293T cells. After single‐cell sorting and verifying the genotype of our clonal cell line, we used the BxB1 recombinase to integrate a destination plasmid into the landing pad site. Using microscopy, flow cytometry, and PCR genotyping, we demonstrate almost complete replacement of the original landing pad template with our destination vector species within 12 days. Here we present an approach that enables the rapid creation of mammalian cell lines harboring complex, multi‐part regulatory circuits at single copy. This platform allows for precise and tunable control of gene expression profiles, providing an excellent opportunity to engineer safer and more sophisticated cell‐‐based therapies.

Support or Funding Information

National Institute of General Medical Sciences of the National Institutes of Health under linked Award Numbers SC1GM111172, RL5GM118969, TL4GM118971, UL1GM118970, and 5G12MD007592.
SPOP as an Emerging Key Player in Breast Cancer Oncogenesis
Authors: Deml Natalie , Galindo Esther , Burleson Marieke Oldenbroek
Published: April 2020

Breast cancer is currently the second leading cause of cancer deaths among American women thereby highlighting the compelling need to uncover novel and superior treatment regimens for women suffering from this disease. Recent advances in genome‐wide studies have allowed for the identification of cancer‐related mutational landscapes, which allows for the identification of oncogenic alterations that are unique to particular cancer patients. Through this technology it has been uncovered that up to 37% of breast cancer patients have downregulated expression of the E3 ubiquitin ligase substrate binding subunit Speckle‐type POZ Protein (SPOP) thereby prompting our lab to study the mechanistic basis behind breast oncogenesis when SPOP expression is low. Previous findings from our lab and others have shown that SPOP targets GLI3 for ubiquitination and subsequent degradation which subsequently promotes SHH signaling in prostate cancer patients. Since SHH hedgehog signaling is often found to be hyper‐activated in advanced breast cancer, we hypothesize that downregulated SPOP promotes GLI3‐dependent Sonic Hedgehog (SHH) signaling in breast cancer patients to promote tumorigenesis and that this signaling pathway could prove to be an attractive therapeutic target for SPOP downregulated breast cancer. To study the effect of downregulated SPOP, a lentivirus carrying an shRNA against SPOP was generated and infected into MCF‐7 cells. Proliferation assays were utilized to confirm that downregulated SPOP plays a role in the increased proliferation of breast cancer cells. Next, quantitative PCR was performed to determine the effect of downregulated SPOP on the expression of GLI3‐dependent SHH target genes. Finally, a natural compound library was utilized to find a novel therapeutic strategy for SPOP downregulated breast cancer. Our findings confirmed that downregulation of SPOP increases proliferation of MCF‐7 cells in vitro. Furthermore, quantitative PCR confirmed that GLI3 target genes are upregulated in MCF‐7 cells when SPOP expression is low. Finally, our natural compound library screen identified novel therapeutic compounds that specifically target SPOP downregulated MCF‐7 cells in a manner that involves disruption of GLI3‐dependent SHH signaling. These findings therefore provide critical insight into novel treatment strategies for patients suffering from SPOP downregulated breast cancer.
Elevated Phosphate Levels Induce Markers of Systemic Inflammation and Anemia in Murine Hepatocytes
Authors: Cunningham Stephen Eric , Czaya Brian Eric , Faul Christian
Published: April 2020

In chronic kidney disease (CKD), elevated serum levels of fibroblast growth factor 23 (FGF23) and phosphate are associated with various pathologies, including systemic inflammation and anemia. Experimental studies have shown that high phosphate can accelerate CKD‐associated pathologies, but direct effects of circulating phosphate on tissues are not well described. Our objective was to compare the effects of phosphate versus FGF23 on primary murine hepatocytes and to determine their respective contributions to systemic inflammation and anemia in the context of CKD. We postulate that in CKD, elevations in serum phosphate levels contribute to systemic inflammation and anemia by directly targeting the liver and inducing gene programs in hepatocytes, which systemically regulate the inflammatory response and iron metabolism. Our study does not only provide novel insights into the basic pathophysiology of CKD, but might also suggest novel therapeutic strategies to block hepatic effects of phosphate in order to tackle CKD‐associated injuries.

Support or Funding Information

PROmoTE R25 DK 112731
Cardiomyocyte‐Specific Deletion of GCN5L1 Limits Cardiac Contractile Decline in High Fat Diet Fed Mice
Authors: Scott Iain , Thapa Dharendra , Manning Janet R. , Stoner Michael W. , Zhang Manling , Xie Bingxian
Published: April 2020

Healthy hearts use several fuel substrates for energy metabolism, with fatty acid oxidation (FAO) accounting for ~70% of total ATP production. Reduced substrate flexibility, and a shift towards increased fatty acid utilization, is a key feature of obesity and diabetes. We previously reported that increased acetylation of several mitochondrial FAO enzymes, regulated in part by increased abundance of the mitochondrial acetyltransferase GCN5L1, correlated with increased FAO enzyme activity in the heart. The focus of the current study was to investigate whether decreased acetylation, via cardiomyocyte‐specific deletion of GCN5L1 (GCN5L1 cKO), regulates cardiac energy metabolism following exposure to a high fat diet (HFD). Excess dietary fat led to similar cardiac hypertrophy in wildtype and GCN5L1 cKO mice, however animals lacking cardiac GCN5L1 expression were protected from a reduction in cardiac workload. We found that GCN5L1 deletion led to decreased acetylation and FAO activity following a HFD. Furthermore, we found that acetylation of pyruvate dehydrogenase (PDH) was significantly reduced in HFD GCN5L1 cKO animals, which resulted in its increased enzymatic activity relative to HFD wildtype controls. Finally, the acetylation of both electron transport chain Complex I protein NDUFB8 and manganese superoxide dismutase 2 (SOD2) was significantly reduced by cardiac GCN5L1 deletion in HFD animals, resulting in decreased lipid peroxidation. In summary, we demonstrate that GCN5L1 deletion in the heart limits cardiac contractile decline observed in HFD mice, through increased glucose oxidation potential and decreased reactive oxygen species damage.

Support or Funding Information

This work was supported by NIH T32HL110849 to D.T. and J.R.M., and by NIH K22HL116728, R56HL132917, R01HL132917 to I.S.
Cognitive Integration: The Value of Explicitly Communicating the Connections Between Anatomy and Clinical Science
Authors: Lisk Kristina
Published: April 2020

Background

Integration of basic and clinical sciences is a central component of ongoing curricular reform discussions within health professions education. Despite focused efforts, successfully supporting the development of learners’ integrated knowledge remains an educational challenge. A growing number of experimental studies suggest that learners can benefit when instruction is designed to support cognitive integration; this type of integration is supported through micro‐level teaching activities that present the basic and clinical sciences in a causal network. In this research, I examine the effect of different instructional and learning strategies on supporting cognitive integration of anatomy and clinical science in novice learners using diagnostic performance measures.

Methods

In each study, allied health students were taught four musculoskeletal hand pathologies using different instructional approaches (integrated, segregated, or clinical science only) and learning strategies (self‐explanation or feature counting). Integrated instruction involved explicitly linking the clinical features of each disease with its underlying anatomical pathology, whereas in segregated instruction the anatomy and clinical features were separated. In clinical science only instruction, the general anatomy and anatomical pathology was excluded. Diagnostic performance was measured immediately after instruction, and one‐week later.

Results

The findings of these studies show that students who were taught the musculoskeletal pathologies with integrated causal mechanisms had superior diagnostic accuracy and a better understanding of the relative importance of key clinical features of disease categories. The application of additional learning strategies, including self‐explanation or feature counting, combined with integrated or segregated instruction did not result in superior diagnostic performance.

Conclusions

This research highlights the value of designing instruction that supports cognitive integration and emphasizes the importance of explicitly communicating the link between anatomy and clinical science during individual teaching sessions. These findings suggest that educators need to carefully consider not only the structure of a learning strategy, but also the extent to which any given learning tool fosters a holistic understanding of clinical signs and symptoms with causal mechanisms.

Support or Funding Information

Funding for this research was provided by Humber College Institute of Technology & Advanced Learning.
Metabolism as a Target for Age‐Related Muscle Mass and Functional Loss
Authors: Schaar Anne , Balasubramanian Priya , Diffee Gary , Anderson Rozalyn
Published: April 2020

The progressive age‐related loss of skeletal muscle mass and function, known as sarcopenia, increases the risk for impaired mobility, falls, fractures, and mortality. Currently, there are no pharmacological interventions to prevent or treat sarcopenia. Here we show that stimulation of adiponectin signaling in skeletal muscle by AdipoRon, a pan‐adiponectin receptor agonist, stimulates pathways linked to exercise and improves muscle function in old mice.

Intravenous delivery of AdipoRon (1.2mg/kg bw; 3 times per week, 6 weeks) activated the AMPK‐PGC‐1a axis in skeletal muscle and induced changes in fiber type distribution in young (5 months) and old (24 months) male C57Bl6/J mice. In old mice, AdipoRon treatment improved running endurance and muscle contractile force. To explore mechanisms and enhance translational potential, the response to oral delivery through diet of AdipoRon (10, 25, or 50 mg/kg; daily feeding, 16 weeks) was investigated in young male and female C3B6F1 mice. AdipoRon enhanced insulin sensitivity and decreased body mass in females, however, these changes were not displayed in males. Increased expression of Ppargc1a and ACADM were observed in EDL of male mice in response to AdipoRon treatment and a decrease in expression of PDK4 was observed in both gastrocnemius and soleus indicating a shift toward oxidative metabolism in all muscle types. Conversely, these changes at the gene expression level were not observed in the female mice. Overall, AdipoRon treatment induced changes in the relative proportions of oxidative and glycolytic muscle fibers in EDL, gastrocnemius, and soleus; however, the skeletal muscle remodeling response to AdipoRon was muscle type and sex specific. In both sexes, the greatest response to AdipoRon was detected in EDL, which is the more glycolytic of the three muscle groups investigated. These data uncover differences in the underlying biology of skeletal muscle between males and females that may have implications for sarcopenia treatment. Taken together, our studies show that AdipoRon has potential as a therapeutic for sarcopenia in males and although sex dimorphic in its mechanism of action, is likely to also be effective as a treatment for sarcopenia in females.

Support or Funding Information

1I01BX003846‐01A2
NFkB Mediates Renal Sodium Retention via the Sodium Chloride Cotransporter in Zinc Deficiency‐induced Hypertension
Authors: Murta Cindellynn , Schindele Dylan , Wenegieme Tara-Yesomi , Waite Aston , Naraine Meagan , Sonner Martha , Williams Clintoria
Published: April 2020

Background

Zinc deficiency (ZnD) is comorbid with diseases such as kidney disease and diabetes. Individuals in these populations have a higher prevalence of hypertension. Recently, we reported that ZnD promotes hypertension in mice. The blood pressure elevations were accompanied with increased Na+ retention via the renal Sodium Chloride Cotransporter (NCC). Although our published results indicate that zinc plays a critical role in blood pressure and NCC regulation, the mechanisms involved were not investigated.

Hypothesis

Since nuclear factor‐kB (NFκB) mediates ZnD‐induced detrimental effects, we tested the hypothesis that NFκB drives ZnD‐induced NCC upregulation and subsequently renal Na+ retention and hypertension.

Experimental Design

To determine the role of NFκB in ZnD‐induced renal Na+ retention and hypertension, adult male C57Bl/6 mice were fed a ZnD diet (5 weeks) followed by administration of the NFκB inhibitor ‐ caffeic acid phenethyl ester (CAPE; 1 week). Systolic blood pressure and urinary Na+ concentrations were examined. To examine if NFκB mediates ZnD‐induced NCC upregulation, mouse distal convoluted tubule (mDCT) cells were treated with the zinc chelator N, N, N′, N′‐tetrakis(2‐pyridinylmethyl)‐1,2‐ethanediamine (TPEN) ± CAPE, a NFκB inhibitor. After 24 hours, NCC mRNA and protein expressions, as well as activation, were assessed by qRT‐PCR, Western blot and immunofluorescence, respectively.

Results

In mice, ZnD promoted hypertension and renal Na+ retention. Further, increased NCC expression was accompanied with enhanced NFκB expression in ZnD mice. However, CAPE administration lowered blood pressure and elevated urinary Na+ concentrations. In mDCT cells, TPEN‐induced NCC upregulation was prevented by CAPE treatment.

Conclusions

Together, these results demonstrate that 1) NFκB is a novel NCC regulator and 2) NFκB mediates ZnD‐induced renal Na+ retention and hypertension. These novel findings indicate that ZnD drives renal NFκB activation thereby stimulating NCC‐mediated Na+ retention and promoting hypertension.

Significance

This study identifies NFκB as a possible pharmacological target to mitigate hypertension.

Support or Funding Information

American Heart Association ‐ Scientist Development Grant #16SDG27080009 National Institutes of Health ‐ R21 Exploratory/Development Grant #R21DK119879
Investigation of Factors that Trigger Essential Conformational Changes in FTT258
Authors: Hossain Asif Ali , Johnson R. Jeremy
Published: April 2020

FTT258 is a close bacterial relative of human acyl protein thioesterase (hAPT), a protein linked to cancer. hAPT removes long chain lipids from key cancer signaling proteins. Removal of these lipids requires their insertion into a pocket within hAPT, which is only exposed after the movement of a flexible loop, a common feature between hAPT and FTT258. However, the causes for the movement of this loop are unknown. I investigated factors that may trigger loop movement in APTs like hAPT and FTT258. In particular, I exposed FTT258 to inhibitors structurally similar to long chain lipids and to membrane‐mimicking surfactants in order to determine if these factors induce loop movement. Loop movement was detected by measuring the change in intrinsic tryptophan fluorescence before and after exposure to these conditions. Ultimately, understanding the factors controlling FTT258 and hAPT function can provide novel mechanisms for inhibiting cancer progression.

Support or Funding Information

NSF MCB‐1812971
Genetic ablation of caspase‐2 decreases muscle function in female mice
Authors: Bhattacharya Arunabh , Gonzalez Peter , Valentine Joseph , Hussong Stacey , Martinez Vanessa , Sharma Ramaswamy
Published: April 2020

Caspase‐2, a cysteine aspartate protease, is an important regulator of apoptosis. We have previously shown that caspase‐2 modulates bone homeostasis. Specifically, we found that 2‐month old female and 5‐month old male Casp2−/− mice exhibited significantly reduced bone mass when compared to wild‐type (WT) mice that continued with aging. Bone is intricately linked to muscle mechanics and function; however, the role of caspase‐2 in skeletal muscle is not clear. This study examined the effect of caspase‐2 on muscle mass and function in 6‐mo‐old WT and Casp2−/− C57BL/6 mice of both sexes. Quantitative nuclear magnetic resonance was used to measure whole‐body lean mass and fat mass. Muscle mass was determined by measuring the mass of quadriceps, gastrocnemius and soleus muscles as well as by measuring cross‐sectional area (CSA) of gastrocnemius muscle. Peak isometric torque of ankle plantarflexors (gastrocnemius, soleus, plantaris) was measured in vivo to determine muscle function. We found decreased body weight and lean mass in both male and female Casp2−/− mice. Muscle mass, CSA and function were not significantly different between male WT and Casp2−/− mice when the data was adjusted for total body lean mass. Similarly, muscle mass and CSA were not different between female Casp2−/− and WT mice. However, significant decrement in muscle function (p < 0.0001) was observed in female Casp2−/− mice compared to female WT mice. We confirmed that our findings in female mice were independent of caspase‐2’s effects on sensorimotor function as there was no difference in sciatic or sural nerve conduction velocity between WT and Casp2−/− mice. Together, these findings suggest a gender‐specific role for caspase‐2 in modulating skeletal muscle function.

Support or Funding Information

Owens Foundation
Enzymatic Oxidation of Biological Thiols by MitoNEET
Authors: Skolik Robert A. , Geldenhuys Werner J. , Konkle Mary E. , Menze Michael A.
Published: April 2020

MitoNEET is a mitochondrial [2Fe‐2S] protein known for its involvement in mitochondrial bioenergetics, iron metabolism, and oxidative stress. The protein has been extensively characterized at the biochemical level since its discovery in 2004, but the mechanisms of physiological function(s) remain unresolved. Two observations seem especially relevant for understanding the physiological function of mitoNEET: (1) under anaerobic conditions in vitro thiol compounds such as glutathione (GSH) and cysteine (Cys) reduce the 2Fe‐2S cluster from the Fe+3Fe+3 to the Fe3+Fe2+ state, and (2) mitoNEET expression levels increase in cells when the glutathione redox balance is shifted towards the oxidized state. We hypothesize that mitoNEET’s in vivo function can be elucidated by bridging these two observations and postulate that mitoNEET is an enzyme responsible for the oxidation of thiol groups on either low molecular‐weight compounds or cysteine residues on target proteins involved in redox signaling. To test this hypothesis, we monitored thiol oxidation by following oxygen consumption rates and demonstrate that mitoNEET accelerates thiol oxidation of cysteine and glutathione (GSH). Four distinct experiments comparing the reactivity of Fe3+ in the form of FeCl3 to mitoNEET demonstrate that the observed thiol oxidation by mitoNEET was not due to free Fe3+contamination. These observations were: (1) rates of cysteine oxidation (8 mM) catalyzed by mitoNEET (1.5 mM) or Fe3+ (1.5 mM) show different optima in the pH range 6.4 to 9.4 and the mitoNEET‐catalyzed reaction is 137% faster at pH 8.4 than the rate observed for Fe3+ alone. (2) Oxidation rates of the nonphysiological compound, n‐acetylcysteine (NAC) by mitoNEET are 181% higher compared to Fe3+ at a pH of 9.0. (3) MitoNEET‐mediated glutathione oxidation remained unaffected by addition of cysteine while oxidation catalyzed by free Fe3+ decreased after addition of cysteine. (4) Dimedone, a probe used to inhibit thiol oxidation from sulfenic acid to sulfinic and sulfonic acid, was 25% more effective at inhibiting cysteine oxidation when free Fe3+ was the catalyst compared to mitoNEET. These results support our hypothesis that mitoNEET possesses an active site which functions to oxidize biological thiols. While the in vivo substrate is currently unknown, we have narrowed down potential candidates to small thiol containing compounds and/or cysteine residues on specific target proteins. We are currently investigating how the enzymatic activity may be regulated by endogenous compounds and through pharmacological compounds targeting mitoNEET.

Support or Funding Information

This work was supported by NSF CHE‐1806266 to M.A.M and M.E.K and UofL Mentored Undergraduate Research and Creative Activities Grant to R.A.S.