bluuebunny/crossref_metadata_2025 huggingface dataset:
Pharmacopeia Potency Equated Sulodexide Produces Comparable Anticoagulation as Studied in Whole Blood
Authors: Daravath Bharathi , Bontekoe Emily , Kouta Ahmed , Iqbal Omer , Lattimer Christopher , Fareed Jawed
Published: April 2020

Introduction

Sulodexide is a widely used glycosaminoglycan derived drug for oral (antithrombotic) and parenteral (anticoagulant) indications and is composed of fast moving heparin (80%) and dermatan sulfate (20%). In view of the current shortage of porcine heparin supply sulodexide may be a potential substitute for heparin for surgical and interventional anticoagulation. The purpose of this study is to compare the anticoagulant activities of sulodexide with heparin in simulated studies to mimic surgical and interventional anticoagulation.

Materials and Methods

Powder versions of sulodexide were obtained from Alfasigma (Bologna, Italy). Powder versions of porcine mucosal heparin were obtained from Medefil (Glendale Heights, IL, USA). Stock solution of both heparin and sulodexide were prepared in sterile saline at 10mg/ml. Sterile solutions were made at 1.0 and 0.1mg/ml. USP potency for both drugs were measured, cross referenced against US pharmacopiel standard and expressed as units per milligram. Whole blood activated clotting time was measured in healthy human volunteers at graded concentrations of both drugs. Plasma based anticoagulant assays such as activated partial thromboplastin time (APTT) and thrombin time (TT) were measured. Protamine neutralization studies were also carried out in the different assays used.

Results

The USP potency of sulodexide range from 90 – 105 U/mg whereas the porcine heparin exhibited a potency range of 180 – 200 U/mg as measured by the anti‐Xa activity. In the ACT assays USP potency adjusted sulodexide and heparin produced concentration dependent prolongation of this test which were comparable to heparin. Both the sulodexide and heparin produce concentration dependent anticoagulant effects in the APTT and TT assays. Protamine sulfate produced effective neutralization of the anticoagulant effects of both the sulodexide and heparin.

Conclusions

These results suggest that at USP potency equated levels sulodexide is capable of producing similar anticoagulant effects as unfractionated heparin. The USP potency of sulodexide can be adjusted at a comparable potency to porcine heparin to obtain comparable anticoagulant responses in the in vitro setting. Additional in vivo studies are warranted to compare the potency adjusted sulodexide with heparin.
Functional roles of neuromedin B and gastrin‐releasing peptide in regulating itch and pain in the spinal cord of primates
Authors: Ding Huiping , Kiguchi Norikazu , Park Sun Hee , Kishioka Shiroh , Shiozawa Yusuke , Peters Christopher M , Ko Mei-Chuan
Published: April 2020

Neuromedin B (NMB) has been demonstrated to elicit itch and pain in rodents. However, the functional role of NMB and its cognate receptor (NMBR) in regulating itch and pain in the primate spinal cord is unknown. The aim of this study was to determine the functional profile of spinal NMB and compare its behavioral effects with those elicited by spinal gastrin‐releasing peptide (GRP) in rhesus monkeys (Macaca mulatta). Following intrathecal administration, GRP (3–10 nmol) dose‐dependently elicited robust scratching responses. In contrast, intrathecal NMB (30–200 nmol) only elicited mild scratching responses. Intrathecal GRP‐ and NMB‐elicited scratching activities were attenuated by a GRP receptor (GRPR) antagonist (RC‐3095) and a NMBR antagonist (PD168368), respectively. Unlike intrathecal substance P (100 nmol) producing thermal allodynia manifested as a reduced tail‐withdrawal latency in 46C water, intrathecal administration of GRP or NMB (1–100 nmol) did not change the thermal nociceptive threshold of primates. In addition, intrathecal administration of a mu opioid receptor agonist, DAMGO (3 nmol), inhibited capsaicin‐induced thermal allodynia. Unlike DAMGO, both RC‐3095 and PD16838 (100–300 nmol) intrathecally did not produce antiallodynic effects. Taken together, these pharmacological findings indicate that spinal NMB‐NMBR and GRP‐GRPR systems have a minimum role in the neurotransmission of pain and both spinal GRPR and NMBR function independently to elicit itch/scratching responses in primates.

Support or Funding Information

The US‐PHS grants AR064456 and AR069861
Halotolerance in the Southeastern Michigan Soil Microbiome
Authors: Bowen John , Zwiesler-Vollick Julie , Siddiq Fauzia
Published: April 2020

Many communities in the Midwest and Northeastern United States use salt to treat icy roads in the winter. Studies have shown that the many years of salt application have had harmful effects on freshwater ecosystems, roadside flora and fauna, and overall wetland biomes. We were interested in investigating the impact that road salt use has had on the soil microbiome. Specifically, we sought to determine if microbes that reside near salted roads have become more tolerant of salt, or more halotolerant, than microbes that reside far from roads. We accomplished this by first collecting soil samples from near roads (exposed to road salt) and far from roads (not exposed to road salt) from various parks across Southeastern Michigan. Salinity measurements were taken by mixing 1 g soil per 1 mL distilled water. We then
used these soil samples to compare the number of colony forming units on 5% NaCl nutrient agar plates and standard nutrient agar plates, investigate growth rate differences between these microbes in 5% NaCl nutrient broth, and sequence various salt intolerant, halotolerant, and halophilic microbes. This allowed us to uncover relationships between the soil salt concentrations, the microbiome distances from roads, and the halotolerance of the microbiomes, as well as to gain some insight into the microbiome population makeup. Preliminary data suggests that there may be a correlation between the salt concentration in soil (ppm) and the overall soil microbiome halotolerance. This indicates that microbiomes closer to roads (which we have shown to have greater soil salt concentration) are likely more halotolerant than microbiomes further from roads (which we have shown to have lesser soil salt concentration). We will investigate the growth rate differences between halotolerant and nonhalotolerant strains as well as determine the genus of the microbes to provide further insight into the composition of the microbiome. This study suggests that the application of road salt is not only affecting wetland biomes but may also be having an effect on the soil microbiome as well.

Support or Funding Information

Special thanks to Lawrence Technological University for laboratory space, equipment, and funding to carry out this study.
Subcellular Localization of Prohibitin 1 is Mediated by Akt Phosphorylation at Thr258
Authors: Thompson Austin , Martin Cole , Dong Christopher , Snyder Zachary , McClung J. Keith
Published: April 2020

The overexpression of intratumoral Prohibitin 1 (PHB1), an evolutionarily conserved gene, has been associated with a 5‐year survival rate of less than 35% in muscle‐invasive bladder cancer patients. When localized to the mitochondria, PHB1 promotes tumor cell proliferation by supporting mitochondrial integrity, whereas nuclear PHB1 assists in inducing cell cycle arrest. As the subcellular localization of PHB1 dictates its function, the mechanisms governing the change in PHB1’s subcellular localization could be exploited to develop novel cancer therapeutics. PHB1 has a mitochondrial signal sequence, but previous work suggested that phosphorylation of PHB1 by Akt at Thr258 is required for mitochondrial localization. Our study confirms and expands this work by using immunofluorescence microscopy to visualize the subcellular location of PHB1 in a muscle‐invasive bladder cancer cell line (5637) after the use of Akt inhibitors. Akt inhibition liberated PHB1 from the mitochondria causing a significant increase in nuclear localization. However, blocking Akt disrupts multiple cellular processes, including those involving the function of PHB1, presenting a potentially confounding explanation for these results. To negate the off‐target effects of Akt inhibition, site‐directed mutagenesis was used to create two mutant PHB1 cDNA plasmids. In the first plasmid, Thr258 was mutated into an aspartate, T258D, which mimics Akt phosphorylation. The second mutation created an alanine, T258A, non‐phosphorylatable. Transfection of T258D showed elevated mitochondrial localization of PHB1, while non‐phosphorylatable T258A pooled in the cytoplasm and failed to enter the mitochondria. Therefore, we concluded that phosphorylation of Thr258 by Akt increases the mitochondrial localization of PHB1.

Support or Funding Information

R.J. McElroy Trust Student/Faculty Research FundWartburg College Undergraduate Research FundWartburg College Biology DepartmentMany generous donors to the Group Alpha Research Fund
Metabolic Regulation of Stem Cell Fate and Function
Authors: Shapira Suzanne N. , Christofk Heather R.
Published: April 2020

The overarching goal of this project is to understand how diet‐derived nutrients regulate stem cell metabolism and downstream fate decisions in the setting of normal development and disease. Quiescent adult stem cells typically favor glycolytic metabolism over the oxidative metabolism utilized by differentiating cells. Importantly, manipulating stem cell metabolism is sufficient to alter cell fate decisions. While the serum concentrations of endogenous metabolites such as glucose and lactate are under homeostatic constraints, diet can cause vast fluctuations in circulating levels of certain metabolites that serve as co‐factors for chromatin and DNA modifying enzymes. As such, exogenous nutrients can control the epigenome and gene expression programs, thus altering differentiation and cell fate. However, little is known about how changing nutrient levels will affect the metabolism and chromatin status in the adult stem cell populations. To address this question, we have adapted a recently described mouse liver organoid system. The liver coordinates whole‐body metabolism and detoxification, and therefore is acutely tuned to respond to dietary changes. Liver organoids generate 3D assemblies that mirror the native physiology of the tissue and self‐renew in vitro due to the activity of Wnt‐responsive stem cells that reside in the hepatic ductal compartment. We have found that treatment with physiologically attainable concentrations of ascorbic acid inhibits hepatic organoid expansion in a dose‐dependent manner. Ascorbic acid, commonly known as Vitamin C, is a water‐soluble antioxidant that is obtained exclusively through the diet in humans. Ascorbic acid is an essential co‐factor for the Fe‐ and α‐ketoglutarate dependent dioxygenase (α‐KGDDs) class of enzymes, which includes collagen hydroxylases, prolyl hydroxylases, and epigenetic erasers such as DNA and histone demethylases. To determine if activation of α‐KGDDs enzymes suppresses organoid expansion, we treated hepatic organoids with a cell permeable form of α‐KG and observed growth inhibition identical to that observed with ascorbic acid treatment. α‐KG/succinate ratios and expression of the ascorbate transporters increases in differentiated hepatic organoids compared to cultures maintained in expansion media, suggesting that ascorbic acid may preferentially accumulate and function during terminal differentiation. Taken together, we propose that ascorbic acid acts as a cofactor for α‐KGDD enzymes in order to restrain stem cell activity and may play a role in promoting differentiation to the hepatic lineage.

Support or Funding Information

S.N.S is supported by a postdoctoral grant from the Eli and Edythe Broad Stem Cell Research Center at UCLA. H.R.C is supported by a CTSI seed grant from the David Geffen School of Medicine at UCLA.
Investigation of Post‐Translational Modification on NHE1 Transport Function
Authors: Hamel Riley P. , Provost Joseph John
Published: April 2020

The sodium hydrogen exchanger isoform 1 (NHE‐1) is a sodium hydrogen antiporter that regulates intracellular pH (pHi), cell volume, dynamic actin remodeling processes, and coordination of cell migration in mammalian tissues. NHE‐1 is post‐translationally modified by phosphorylation and lipid modification (palmitoylation). The intracellular C‐terminal domain of NHE‐1 is reversibly phosphorylated at multiple sites byseveral kinases including: extracellular signal‐regulated kinase (ERK), protein kinase B (AKT), B‐Raf, p90 Ribosomal S6 Kinase (p90rsk), RhoA Kinase (ROCK), and other protein kinases. We recently identified that NHE‐1 is reversibly palmitoylated (S‐acetylation), in which a 16‐carbon palmitic acid is covalently attached to a cysteine residue on the C‐terminal domain via a thioester linkage. Such modifications are reported to regulate protein trafficking, membrane micro localization, and protein‐protein interactions. Steady‐state pHi assays incubated with pH‐sensitive dye were conducted to measure the change in pHi of Chinese Hamster Lung Fibroblast Cells due to NHE‐1. Investigating agonists and stimulating factors that increase NHE‐1 palmitoylation allows us to determine the impact of palmitoylation on NHE‐1 transport. Palmitoyl‐transferases (PATs) catalyze the lipidation of proteins and are inhibited by 2‐bromo‐palmitate (2BP). The presence of 2BP causes a loss of palmitoylation to occur, effectively inhibiting LPA‐induced NHE‐1 activity by 0.1 pH units. Fetal Bovine Serum (FBS), Lysophosphatidic acid (LPA), Phorbol 12‐myristate 13‐acetate (PMA), and Insulin effectively stimulate NHE‐1 by increasing pHi by 0.1 pH units. In the presence of kinase inhibitors, NHE‐1 activity significantly decreases compared to agonist or serum treatment alone. The impact of the combination of kinase inhibitors and palmitoylation inhibitors on NHE‐1 mediated pHi change was determined. The data supports the hypothesis that palmitoylation and phosphorylation coordinate to subtly modify NHE1 activity in a novel rheostat of the cell.
Embryonic versus adult cardiomyocyte loss of ULK1 and ULK2 uncover temporally distinct effects on autophagy and cardiac function
Authors: Harris Matthew , Zhang Quan J. , Cochran Cole T. , Ponce Jessica , Fuqua Jordan D. , Alexander Sean , Kronemberger Ana , Harper Tyler , Murry Matthew Larson , Fattal Ranan , Grueter Chad E. , Abel Dale , Lira Vitor A.
Published: April 2020

Autophagy is a conserved cellular process that degrades dysfunctional organelles and long‐lived proteins. Insufficient autophagy contributes to several cardiomyopathies; however, the regulation of cardiac autophagy is still evolving. To this matter, Unc‐51 like autophagy activating kinases 1 and 2 (ULK1 and ULK2) are thought to play a primarily redundant role in autophagy initiation but their function in the heart remains insufficiently understood. Here, we first studied mice with embryonic cardiac‐specific deletion of Ulk1 or Ulk2 (cU1‐KO and cU2‐KO). Unexpectedly, adult cU1‐KO and cU2‐KO mice presented enhanced basal cardiac autophagy despite unchanged expression of essential autophagy machinery proteins. Cardiac function was unaltered in these mice. We then tested if this autophagy overcompensation relied on the remaining ULK protein by studying mice with embryonic cardiac‐specific deletion of both Ulk1 and Ulk2 (cU1/2‐DKO). Indeed, autophagy was impaired in cU1/2‐DKO leading to an aging‐associated cardiomyopathy and reduced survival. Next, we tested if the overall autophagy overcompensation resulting from the loss of a single cardiac Ulk was only feasible at early stages of development by studying mice with adult deletion of either Ulk1 or Ulk2 (i.e., icU1‐KO and icU2‐KO). Interestingly, while adult loss of ULK2 had no discernable effect on cardiac autophagy or function, loss of ULK1 caused a rapidly developing cardiomyopathy, heart failure and early death. Mechanistically, adult loss of cardiac ULK1 impaired autophagy and led to an overall fissed mitochondrial reticulum with deficiencies in respiration and ATP re‐synthesis. Altogether our results demonstrate that the impact of ULK1 and ULK2 loss on cardiac autophagy and function is developmentally determined. Further, although ULK1 and ULK2 appear to have redundant roles in modulating cardiac autophagy during development, only ULK1 is essential for maintenance of basal cardiac homeostasis in adulthood.
Peripheral Blood Mononuclear Cell‐Derived Superoxide Production, Spontaneous Baroreflex Sensitivity, and Vascular Function in Chronic Kidney Disease Patients
Authors: Kaur Jasdeep , Sabnani Manoj K. , Stephens Brandi Y. , Nandadeva Damsara , Weidanz Jon A. , Fadel Paul J.
Published: April 2020

Patients with chronic kidney disease (CKD) have an increased risk of cardiovascular morbidity and mortality. Previous studies have shown that CKD patients have high oxidative stress, which may be a potential underlying mechanism contributing to vascular dysfunction and impaired baroreflex sensitivity in this patient group. However, the exact mechanism(s) leading to elevated oxidative stress in CKD remains unclear. Peripheral blood mononuclear cells (PBMCs) have been identified as a primary source of NADPH oxidase‐derived superoxide production, leading to elevated systemic oxidative stress. PBMCs can cross the blood brain barrier and increase oxidative stress centrally, which could potentially reduce arterial baroreflex sensitivity (BRS) in these patients. Indeed, there is an emerging body of evidence indicating that elevated oxidative stress impairs BRS. Herein, we tested the hypothesis that CKD patients have greater superoxide production in PBMCs that is associated with impaired vascular function and attenuated BRS. To test this hypothesis, we studied 7 CKD patients stage 3 and 4 (age = 62 ± 3 years, BMI = 35 ± 2 kg/m2, estimated glomerular filtration rate (eGFR) = 43 ± 7 ml/min/1.73 m2) and 5 age‐matched controls (age = 57 ± 3 years, BMI = 25 ± 1 kg/m2, eGFR = 80 ± 5 ml/min/1.73 m2). PBMCs were isolated from whole blood and intracellular superoxide production in PBMCs was measured using dihydroethidium fluorescence. Resting heart rate (ECG) and arterial blood pressure (finger photoplethysmography) were measured for 10 minutes and spontaneous cardiac BRS was determined using the sequence technique. In addition, central artery stiffness (pulse wave velocity, SphygmoCor) and brachial artery flow‐mediated dilation (duplex Doppler ultrasound) were measured to assess vascular function. CKD patients had significantly elevated PBMC‐derived superoxide production compared to controls (CKD = 32769 ± 4067 vs. controls = 18421 ± 3515 relative fluorescence units; p = 0.03). However, cardiac BRS was not different between the groups (18.2 ± 3.5 in CKD vs 15.4 ± 1.5 ms/mmHg in controls; p = 0.35). Central artery stiffness appeared greater (CKD = 7.8 ± 0.6 vs. controls = 6.3 ± 0.2 m/s; p = 0.07), and endothelial function lower in CKD patients (CKD = 3.5 ± 0.6% vs. controls = 5.8 ± 1.4%; p = 0.19) but neither reached statistical significance in the cohort studied thus far. These preliminary findings suggest that CKD patients exhibit elevated PBMC‐derived superoxide production, which may be one of the mechanisms contributing to impaired vascular function in this population. However, this elevated oxidative stress was not associated with impaired spontaneous cardiac BRS in CKD patients.

Support or Funding Information

Supported by NIH R01 HL127071
Effects of post‐exercise lactate administration on glycogen recovery in mouse skeletal muscle
Authors: Takahashi Kenya , Kitaoka Yu , Matsunaga Yutaka , Hatta Hideo
Published: April 2020

During high‐intensity exercise, glycogen content in skeletal muscle and liver decreases, while lactate accumulates in the circulation. Although sports nutrition guidelines recommend carbohydrate intake for glycogen recovery, the metabolic interaction between ingested carbohydrate and circulating lactate after exercise is unclear. In this study, we examined whether elevated circulating lactate additively enhances post‐exercise glycogen restoration. Eight‐week‐old male ICR mice were allocated to one of four groups as follows: PBS + water (PW) group, lactate + water (LW) group, PBS + glucose (PG) group, and lactate + glucose (LG) group. Animals performed treadmill running (20 m/min, 60 min) following overnight fasting (16 hours). Immediately after the exercise, animals received intraperitoneal injection of PBS or lactate (1 g/kg BW), followed by oral ingestion of water or glucose (2 g/kg BW). At 30, 45, 60 minutes of recovery, blood glucose concentration was higher in the LW group than in the PW group (p<0.05). In contrast, blood glucose level at 15 and 30 minutes of recovery and its iAUC were lower in the LG group than in the PG group (p<0.05). At 60 minutes of recovery, glucose ingestion resulted in higher glycogen content in the soleus, plantaris, and gastrocnemius muscles and the liver of the PG and LG groups compared with the PW group, although plasma insulin level at 15 minutes recovery (p<0.01) and its iAUC (p<0.05) were lower in the LG group than in the PG group. Moreover, lactate injection enhanced glycogen content in the plantaris and gastrocnemius muscles of the LW and LG groups (p<0.05), but not in the soleus muscle and the liver. Our current findings suggest that elevated circulating lactate additively enhances post‐exercise glycogen recovery in glycolytic phenotype muscle.
Nuclear Pregnane X Receptor Transduces Glucose Signal to Suppress Estrogen Sulfotransferase Gene in Human Liver Cells
Authors: Hu Hao , Negishi Masahiko
Published: April 2020

Estrogen sulfotransferase (SULT1E1) metabolically inactivates estrogen and SULT1E1 expression is tightly regulated by multiple nuclear receptors. Previously, it was found that nuclear receptors HNF4a and RORa form as complexes binding to the SULT1E1 enhancer (−943/−922 bp) to activate gene expression in high glucose (450 mg/dL) treated human liver cells. However, the mechanism by which low glucose signal (40 mg/dL) mediated SULT1E1 suppression in human liver cells remain mysterious. Here, we have demonstrated that low glucose signal stimulates PXR phosphorylation at Ser350 on its ligand binding domain, preventing its heterodimerization with RXR and enabling it to interact with the HNF4a/RORa binding complexes on the enhancer, thereby suppressing SULT1E1 gene transcription. SULT1E1 was significantly repressed at both mRNA and protein levels in low glucose cultured human liver cells and this suppression was found to be PXR dependent. Vaccinia‐related kinase 1 (VRK1) was found to phosphorylate PXR at Ser350 in low glucose treated cells. Transient transfection analysis of SULT1E1 promoter‐luciferase reporter genes revealed that phosphorylated PXR significantly suppressed SULT1E1 promoter activity via the enhancer. Chromatin immunoprecipitation and gel shift assays elucidated that phosphorylated PXR bound to the SULT1E1 enhancer and disrupted the formation of the HNF4a/RORa binding complexes on it at low glucose condition. The physical interactions among these nuclear receptors were subsequently demonstrated by a series of co‐immunoprecipitation assays. Our study has shown that xenobiotic sensing nuclear receptor PXR transduces low glucose signal by its conserved phosphorylation to suppress SULT1E1 expression in human liver cells.

Support or Funding Information

This work was supported by National Institutes of Health intramural research program Z01ES1005‐01.
High Altitude Hypoxia Induces Cellular Immaturity of Pulmonary Arteries in the Fetal Lamb: Assessment of Protein Biomarkers
Authors: Jones Breanna , Leslie Eric , Alvarez Rafael , Dasgupta Chiranjib , Zhang Lubo , Wilson Sean M.
Published: April 2020

Pulmonary Arterial Hypertension (PAH) in newborns can be mediated by intrauterine hypoxia and which can cause significant aberrations on vascular structure and function. Previous evidence suggests that long term hypoxic stress can induce phenotypic modifications to various vascular beds, including those of the lung. We are now seeking to interrogate the influence of intrauterine hypoxia on pulmonary vascular phenotypes and to test the hypothesis that intrauterine hypoxia causes the vasculature to shift from a mature to an immature phenotype. This was evaluated by examining protein expression using proteomic analyses on pulmonary arteries isolated from near term fetal lambs that were exposed to intrauterine high altitude hypoxia (3801 m) for the latter 110+ days of pregnancy or those gestated near sea level. Protein networks were created via Ingenuity Pathway Analysis (IPA) and Search Tool for the Retrieval of Interacting Genes/Proteins (STRING). Intrauterine hypoxia caused a number of significant changes including reducing the expression of smooth muscle myosin, fibronectin, and collagen along with upregulation of proteins involved in nucleotide synthesis, mitosis/cell proliferation, as well as cell adhesion and migration. The observed changes are consistent with a hypoxia‐induced phenotypic shift towards cells being more synthetic, proliferative, and migratory. Such findings provide insight into the mechanistic underpinnings related to the development of pulmonary hypertension due to gestational hypoxia. This work opens new avenues of investigation to determine common signaling pathways affected by intrauterine hypoxia with the aim of discovering the fundamental mechanisms driving hypoxia‐induced remodeling and senescence.

Support or Funding Information

Supported in part by NIH grants HD083132 (LZ), HD098477 (SMW), RA was a Apprenticeship Bridge to College summer research fellow through the LLU Center for Health Disparities Research with funding from P20MD006988 (M. DeLeon) from the National Institute of Health Disparities and Minority Health of the National Institutes of Health.
Control of Initiation and Aggressiveness of DMBA‐Induced Breast Cancer in Rats by Dietary Consumption of Hinute‐AM.
Authors: Das Salil K. , Sharan Chakradhari , Maebuchi Motohiro , Mukherjee Shyamali K.
Published: April 2020

We reported earlier that the consumption of soy protein controlled not only the proliferation of dimethylbenz [a] anthracene (DMBA)‐induced breast cancer in rats but also its aggressiveness. Soy protein is hydrolyzed in the gut to small peptides which are easily absorbed and are involved in various cellular functions. Fuji Oil Company, Osaka, Japan, has commercially marketed a soy protein hydrolysate, termed Hinute‐AM, composed of primarily di‐ and tripeptides, to be used as a health food, and it has anti‐inflammatory property. The purpose of the present study was to investigate whether dietary intake of Hinute‐AM can delay the development of DMBA‐induced breast cancer and its aggressiveness in rats. For this study, we obtained 22‐days old female Sprague Dawley rats from Charles River (Wilmington, MA). Animals were divided into 5 groups, each containing 5 animals. Group 1 received standard AIN‐76A diet (Envigo – Teklad Diets, Madison, WI) containing 20% casein and 0% Hinute‐AM; Group 2 received the same diet containing 15% casein + 5% Hinute‐AM; Group 3 received the same diet, but containing 10% casein + 10% Hinute‐AM; Group 4 received the same diet but containing 5% casein + 15% Hinute‐AM; and Group 5 received the same diet but containing 0% casein + 20% Hinute‐AM. For induction of mammary tumors, all groups received a single intragastric administration of DMBA (Sigma Chemical Co., St. Louis) dissolved in sesame oil (80 mg/kg b.wt.) at 50 days of age. At 120 days post DMBA injection, the animals were euthanized by CO2 asphyxiation. All tumors were weighed and measured for volume, and a section of tumor was subjected to histological grading by a certified pathologist. Our preliminary data revealed that replacement of casein by Hinute‐AM as a source of dietary protein caused a dose‐dependent delay in the development of breast tumor, decrease in breast cancer incidence, a decrease in tumor volume, and tumor grade.

Support or Funding Information

This work was supported by grants from Fuji Oil Company, Osaka, Japan and by the Meharry Translational Research Center grant NIH 5U54MD007593
Involvement of PDZ‐SAP97 Interactions in Regulating AQP2 Translocation in Response to Vasopressin in LLC‐PK1 cells
Authors: Bahouth Suleiman W. , Nooh Mohammed M.
Published: April 2020

Arginine‐vasopressin‐ (AVP) mediated translocation of aquaporin‐2 (AQP2) protein forming water channels from storage vesicles to the membrane of renal collecting ducts is critical for renal conservation of water. The type‐1 PDZ‐binding motif (PBM) in AQP2 “GTKA” is a critical barcode for its translocation, but its precise role and that of its interacting protein partners in this process remains obscure. We determined that SAP97, a MAGUK protein involved in establishing epithelial cell polarity was an avid binding partner to the PBM of AQP2. The role of the PBM and SAP97 on AQP2 redistribution in response to AVP was assessed in LLC‐PK1 renal collecting cells by confocal microscopy and by cell surface biotinylation techniques. These studies indicated that the distribution of AQP2 and SAP97 overlapped in the kidneys and in LLC‐PK1 and that knockdown of SAP97 inhibited the translocation of AQP2 in response to AVP. The binding between AQP2 and SAP97 was mediated by specific interactions between the second PDZ of SAP97 and the PBM of AQP2. Mechanistically, inactivation of the PBM of AQP2, global delocalization of protein kinase A (PKA) or knockdown of SAP97, inhibited AQP2 translocation as well as AVP‐ and forskolin‐mediated phosphorylation of serine256 in AQP2, which serves as the major translocation barcode of AQP2. These results suggest that targeting of PKA to the AQP2 microdomain via SAP97‐AQP2 interactions in association with cross‐talk between two barcodes in AQP2, namely the PBM and phospho‐Ser256, play an important role in the translocation of AQP2 forming channels in the kidney.

Support or Funding Information

This work was supported by the National Institutes of Health grant: HL‐085848 and by Bridge Funding support by the University of Tennessee‐HSC.
Fragment‐Based Screening to Identify Thioesterase Superfamily Member 2 (Them2) Inhibitors for the Management of Obesity‐Related Disorders
Authors: Krumm Christopher S. , Adura Carolina , Liu Xu , Li Yingxia , Bracken W. Clay , Ginn John D. , Glickman J. Fraser , Ortlund Eric A. , Cohen David E.
Published: April 2020

Background

The prevalence of obesity‐related disorders continues to increase, but current management options remained limited. In the setting of chronic overnutrition, excessive tissue concentrations of free fatty acid (FFA) contribute to the pathogenesis of obesity‐related disorders. Thioesterase superfamily member 2 (Them2) is an acyl‐CoA thioesterase (Acot) that hydrolyzes long‐chain fatty acyl‐CoAs into free fatty acids and CoASH. Them2 is enriched in oxidative tissues, and Them2−/− mice are protected from diet‐induced obesity, diabetes, and non‐alcoholic fatty liver disease. These findings suggest that chemical inhibition of Them2 could be leveraged in the management of obesity‐related disorders.

Aim

This study was designed to develop a small molecule inhibitor that targets the enzymatic activity of Them2.

Methods

Recombinant His‐tagged human Them2 was expressed in E. coli, and purified by affinity chromatography and size exclusion chromatography. Two fragment‐based screens (FBS) were performed: 1) A 1056 fragment library was screened by differential scanning fluorimetry (DSF), and 2) a 500 19F‐labeled fragment library was screened by 19F‐nuclear magnetic resonance (19F‐NMR). Secondary orthogonal biochemical (microscale thermophoresis; MST) assays confirmed binding interactions between fragment compound hits and Them2. A microplate‐based fluorometric Acot activity assay was used to measure the Acot activity of Them2.

Results

30 DSF‐derived fragment compounds (2.8% hit rate) that decreased the melting temperature of Them2 by greater than 4°C (i.e. Them2 de‐stabilizers) were identified. Deconvoluted NMR chemical shifts revealed binding of 15 19F‐labeled fragment compounds to Them2 (3% hit rate). The most promising fragment compound hits exhibited Kd values in the μM range by MST. The most potent fragment compound hits were those that suppressed Acot activity.

Conclusions

Complementary biochemical approaches identify fragment compounds capable of both binding and inhibiting Them2. Following structural characterization of ligand binding by x‐ray crystallography, medicinal chemistry strategies can be leveraged to improve potency. Lead compounds that inhibit Them2 should prove attractive in the management of obesity‐related disorders.

Support or Funding Information

This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants R01 DK056626, R37 DK048873, and T32 DK116970 (to D.E.C).
The Impacts of Sublethal Nitrite Exposure on Goldfish (Carassius auratus) Microbiomes
Authors: Ortiz Whitney , Carlos-Shanley Camila , Huertas Mar
Published: April 2020

Around 50% of fish consumption comes from aquaculture. An increase in world population and food demands requires fish production to dramatically increase over the next decade. One of the major obstacles of increasing aquaculture production is disease. Thus, a key aspect of aquaculture research is understanding immune health and the spread of disease within farmed populations and into wild stocks. A major portal for pathogen entry in fish is the nose; nasal infections tend to be severe and result in damage to the olfactory and nervous systems. Immune health is impacted by numerous systems including the microbiome. The microbiome trains the immune system, provides a barrier for pathogens, and helps maintain homeostasis. Therefore, dysbiosis, a disruption of the microbiome due to changes in diet, exposure to pollutants, antibiotic consumption, and physiological stress, can contribute to disease. One common pollutant in water is agricultural runoff containing nitrates and nitrites, which are known to cause stress in fish and can be deadly in some concentrations. However, it is unknown how nitrites impact the microbiomes of sensitive tissues and how these changes impact immune health. I hypothesize that increased nitrite concentrations will cause dysbiosis of the microbiome resulting in changing abundance and diversity of the microbiome. This work aims to determine the impacts of sublethal levels of nitrite on the nasal microbiome and compare it to the microbiomes of the gills and gut. For 2 months goldfish (10 fish/30L tank) were held under various nitrite concentrations, 0.0mM (control), 0.01mM, 0.1mM, and 1.0mM. The system utilized continuous aerated water flow at 24°C to maintain the desired concentrations of nitrite. Tissue samples were collected from each fish with water collected to serve as a control to determine the host‐specific microbiome. The DNA from the tissues were extracted, amplified, and prepared as a library for sequencing using 16S rRNA gene primers for Illumina MiSeq. The sequences were analyzed using the DADA2 pipeline in R and MaAsLin from Galaxy. The gill microbiome showed no significant changes in abundance compared to the control treatment. The nose microbiome appears to show an increase in diversity compared to the control microbiome; however, these changes were not determined to be significant. Both the gut and water microbiomes showed significant changes in abundance compared to their control levels, with the gut microbiome changing the most. Sublethal levels of nitrite exposure significantly increase the diversity of the microbial communities of the water and the gut microbiomes. There are also some indications that the nose and gill microbiomes are impacted, however, small sample sizes could be obscuring the changes. These impacts imply that sublethal nitrite concentrations can cause dysbiosis of the fish microbiomes and could have negative impacts of fish health.
Sphingosine‐1‐Phosphate Receptor 1 Mediated Vasodilation in Human Arterioles
Authors: Katunaric Boran , Schulz Mary E. , Freed Julie K.
Published: April 2020

Precise regulation of vascular resistance is critical to maintain proper tissue perfusion. Recent data in animal models have shown that sphingolipids, a ubiquitous class of bioactive lipid messengers, can influence vasomotor tone via dilation or constriction. It has been suggested that sphingosine‐1‐phosphate (S1P) elicits vasodilation through generation of endothelial cell nitric oxide (NO) via activation of sphingosine‐1‐phosphate receptor 1 (S1PR1). We therefore examined the hypothesis that S1P is a regulator of human microvascular tone and elicits dilation via S1PR1‐induced increase in NO. Arterioles from healthy human adipose tissue were dissected and prepared for videomicroscopy, equilibrated and pre‐constricted with endothelin 1. Changes in arteriolar luminal diameter were recorded in response to increasing concentrations of S1P (10−12 to 10−6M) in the presence or absence of the S1PR1 receptor antagonist W146 (10−5M), nitric oxide synthase (NOS) inhibitor Nω‐nitro‐
l‐arginine (L‐NAME, 10−4M) or hydrogen peroxide (H2O2) scavenger polyethylene glycol‐catalase (peg‐Cat, 500U/ml). Administration of exogenous S1P induced vasodilation in healthy vessels to a maximal dilation of 63.7%±5.0, n=8 (mean±SEM). As with previous animal studies, vascular constriction was observed with higher, but still physiological levels of S1P (10−6 M). Vasodilation due to S1P was reduced following inhibition of S1PR1 compared to S1P alone (1.8%±4, n=5). In the presence of L‐NAME, S1P‐induced dilation decreased (9%±7.9, n=6). Interestingly, addition of peg‐Cat, an H2O2 scavenger, also decreased S1P‐induced dilation (11.1%±6.6, n=3). Together these data suggest that S1PR1 is a critical mechanistic component of S1P‐induced vasodilation in the human vasculature. Further, the S1P vasodilatory pathway may require formation of H2O2 in addition to NO.

Support or Funding Information

This research was supported by National Institute of Health (NHLBI) K08 HL141562‐02 (JKF)
Bioengineered microRNAs suppress lung cancer cell metabolism and proliferation through targeting of folate cycle
Authors: Chen Yixin , Liu Zhenzhen , Tu Meijuan , Batra Neelu , Yu Ai-ming
Published: April 2020

Lung cancer remains as the leading cause of cancer deaths in the world. Ver recently, we have established a novel technology for the production of bioengineered miRNA agents for the study of cancer biology and new therapies. Using recombinant miRNA molecules produced and folded in living cells, we identified a number of the most effective miRNAs for the inhibition of non‐small cell lung cancer (NSCLC) cell proliferation, among which miR‐22‐3p, miR‐9‐5p and miR‐218‐5p are all predicted to interfere with folate cycle and one‐carbon metabolism. The serine hydroxymethyltransferase‐1 (SHMT‐1), a tumor biomarker and important enzyme in folate biotransformation, was verified as a new target for both miR‐9‐5p and miR‐218‐5p. The methylenetetrahydrofolate dehydrogenase 1 like (MTHFD1L) was proven to be regulated by miR‐9‐5p directly. Furthermore, both methylenetetrahydrofolate reductase (MTHFR) and methylenetetrahydrofolate dehydrogenase‐2 (MTHFD2) were significantly suppressed by miR‐22‐3p in NSCLC cells. As determined by accurate LC‐MS/MS methods, folate metabolites and amino acid metabolome were altered remarkably by individual miRNAs in human NSCLC cells. Consequently, glycolysis rate and mitochondrial function were significantly inhibited in NSCLC cells treated with these miRNAs, accompanied by cell cycle arrest, ROS accumulation, NADP+/NADPH imbalance and other metabolic dysfunctions. In conclusion, our results demonstrated that bioengineered miR‐22‐3p, miR‐9‐5p, and miR‐218‐5p act on folate cycle to suppress NSCLC cell metabolic capacity and exhibit strong antiproliferation activities. These findings provide new insights into mechanistic actions of tumor suppressive miRNAs and development of novel therapeutic strategies.

Support or Funding Information

This study was supported by National Cancer Institute (grant No. R01CA225958) and National Institute of General Medical Sciences (R01GM113888), National Institutes of Health.
Highly effective methods for expression/purification of recombinant human HSP90 and its four distinct (N‐LR‐M‐C) domains
Authors: Thongboonkerd Visith , Aluksanasuwan Siripat , Plumworasawat Sirikanya , Manissorn Juthatip , Peerapen Paleerath
Published: April 2020

Heat shock protein 90 (HSP90) plays essential roles in normal physiology to maintain homeostasis and in many human diseases. It comprises four distinct domains, including NH2‐terminal (N), charged linker region (LR), middle (M), and COOH‐terminal (C) domains, all of which are important for regulating HSP90 biological functions. We reported herein detailed protocols to produce recombinant full‐length (FL) and all these four domains of human HSP90 from Escherichia coli. cDNAs encoding FL, N, LR, M and C domains of human HSP90α were amplified and cloned into pET‐32b(+) expression vector. All HSP90 constructs were expressed as soluble Trx‐His‐S tagged proteins after induction with 0.25 mM isopropyl‐β‐D‐thiogalactopyranoside (IPTG) at 18°C overnight and further purified by affinity chromatography using nickel‐nitrilotriacetic acid (Ni‐NTA) resin. The enterokinase (EK) digestion was optimized for efficient cleavage of the Trx‐His‐S tag from each HSP90 construct by varying concentrations of EK (0.5–1 U) and urea (0–3 M). After rebinding to Ni‐NTA resin, each HSP90 construct was highly purified and approximately 0.1–1 mg proteins were obtained from 100 ml of bacterial culture. All the purified HSP90 constructs were successfully confirmed by tandem mass spectrometry (nanoLC‐ESI‐ETD MS/MS). Our expression and purification protocols would facilitate further structural and functional studies of human HSP90.

Support or Funding Information

This study was supported by Mahidol University research grant and the Thailand Research Fund (IRN60W0004).
Anti‐obesity and Anti‐diabetes Effects of Silk Peptide in 3T3‐L1 Adipocytes.
Authors: Lee Kippeum , Jin Heegu , Oh Jae-Hoon , Chei Sungwoo , Oh Hyun-Ji , Lee Boo-Yong
Published: April 2020

Obesity is caused by imbalanced food intake and physical activity. Obesity is associated with severe metabolic diseases such as type 2 diabetes. Silk peptide, derived from Bombyx mori cocoons, is a natural biomaterial that has various applications in biotechnology and bio‐pharmacology in Asian countries. However molecular mechanism of the anti‐obesity and anti‐diabetic effect has not been elucidated. In this study, we investigated the molecular mechanism of adipogenesis inhibition induced by silk peptide on 3T3‐L1 adipocytes. Our data show that silk peptide decreased lipid accumulation and early adipogenic factors in a dose‐dependent manner. Silk peptide inhibited the protein expression of early adipogenic transcription factors, such as CCAAT/enhancer‐binding protein beta(C/EBP α) and peroxisome proliferator‐activated receptor gamma (PPARγ) compared with control. In addition, the anti‐diabetes effect of silk peptide was evidenced by the up‐regulation of GLUT4 expression. Our findings suggest that silk peptide suppresses adipocyte differentiation and promotes glucose uptake ability. Silk peptide may be a safe, effective and economical food grade ingredient for obese patients.
Use of Ion Exchange and Lectin Chromatograpy To Fractionate Proteins from Carcinus maenus
Authors: Johnson Heidi , Myracle Angela D. , Grant Jennifer E.
Published: April 2020

Across many different species of crabs, it is notable that their shells have a high abundance of protein. For instance, the snow crab is 34.2% w/w of protein within its shell (Asunchion, et al., 2011.) And while the blue crab contains about 33.4% w/w protein within its shell (Lee et al 2017), the red crab contains about 16% w/w protein (Aklog 2016). Less is known about the European green crab, Carcinus maenus, which is a species found in Europe and North Africa. This organism has also invaded the coast of Maine.

We have developed an acid extraction and acetone precipitation strategy for isolating proteins from the shells of the European green crab, Carcinus maenus. Typically, roughly 5 to 10 proteins are visible when this extract is analyzed by 12% SDS‐PAGE gel. In this study, cation and anion‐exchange resins, and lectin resins, were utilized to fractionate protein species based on their charge and glycoprotein content, respectively. This work reports on the progress of these studies.

This work is of value to the study of bioactive materials present in shelled marine organisms.
Membraneless Organelles and Viscosity Changes during Desiccation: A New Phase in Anhydrobiosis
Authors: Belott Clinton J. , Janis Brett R. , Sithu Tryphena S. , Menze Michael A.
Published: April 2020

AfrLEA6 is an intrinsically disordered protein found in the anhydrobiotic cysts of Artemia franciscana. AfrLEA6 shares homology with plant seed maturation proteins (SMPs), which have been linked to the longevity of orthodox seeds in the desiccated state. Therefore, it was hypothesized that ectopically expressing AfrLEA6 will increase the desiccation tolerance of a desiccation‐sensitive Drosophila melanogaster (Kc167) cell line. Surprisingly, ectopically expressed AfrLEA6 was found to undergo a liquid‐liquid phase separation (LLPS) in the cytoplasm of Kc167 cells. The overall architecture of the protein is composed of two SMP domains connected with a glycine‐ and proline‐rich spacer to a potential protein‐binding domain. It was determined that AfrLEA6 LLPS is dependent on the SMP domains, regulated by polysome assembly and disassembly, and may best be described as a specialized biphasic stress granule. It is speculated that AfrLEA6 stress granules protect desiccation‐sensitive proteins prior to glassy‐state formation during water loss. This hypothesis was strengthened by the observation that expression of AfrLEA6 in Kc167 cells increases their desiccation tolerance. Unexpectedly, AfrLEA6 also increases intracellular viscosity during desiccation compared to control cells, which reduced plasma membrane fusion between adjacent cells and may protect against internal sheer stresses. These data agree with recently reported cellular dielectrophoretic data, that AfrLEA6 expressed in Kc167 cells underwent a shift to become more entangled when cells were exposed to osmotic stress, thereby suggesting an increase intracellular viscosity. In A. franciscana, a rapid increase in cytoplasmic viscosity at relatively high‐water contents may be key to successfully entering the anhydrobiotic state, while trehalose glass formation at relatively low‐water contents may create a more stable intracellular environment for long‐term dormancy.

Support or Funding Information

Supported by NSF IOS‐1659970.
‘Slepen al the nyght with open ye’: Personal reflections after 35 years of being on-call
Authors: Fitzpatrick Chris
Published: April 2020

The European Working Time Directive (2003) has had a significant impact in reducing the total number of hours worked per week, as well as shift-lengths. There is, however, no agreement on optimal shift-lengths and day/night work balance in different medical specialities. Given the time it takes for clinical events to unfold, particularly in relation to labour, there may be advantages in retaining the 24 hours on-call shift for obstetricians––in the interests of patient care and training. Increasingly seen as a relic of the past, this shift-length merits further research, which should include the impact on clinician well-being.
Ivermectin as a potential COVID-19 treatment from the pharmacokinetic point of view: antiviral levels are not likely attainable with known dosing regimens
Authors: Momekov Georgi , Momekova Denitsa
Published: April 2020

The broad-spectrum antiparasitic agent ivermectin has been very recently found to inhibit SARS-CoV-2 in vitro and proposed as a candidate for drug repurposing in COVID-19. In the present report the in vitro antiviral activity end-points are analyzed from the pharmacokinetic perspective. The available pharmacokinetic data from clinically relevant and excessive dosing studies indicate that the SARS-CoV-2 inhibitory concentrations are not likely to be attainable in humans.