bluuebunny/crossref_metadata_2025 huggingface dataset:
Interactions of Pesticides with Drug Efflux Transporter P‐glycoprotein in the European Honeybee
Authors: Pouv Amara , Nicklisch Sascha
Published: April 2020

Modern agriculture depends on pollinators such as the European Honeybee, Apis mellifera, to produce crops. It is estimated that almost $20 Billion a year in US agricultural production can be contributed to honeybees. However, global bee populations have been in recent decline and the high use of agricultural pesticides have been suggested to directly or indirectly affect bee colony health. Yet, little is known about the types and levels of pesticides accumulation in bees and the underlying mechanism of uptake and distribution. Multidrug resistance proteins from the ABC family, such as P‐glycoprotein (P‐gp), are a first line of defense in biological barriers and responsible for the efflux of drugs and toxins in all organisms.

This study aims to determine the molecular interactions of certain prevalent pesticides with the honeybee P‐glycoprotein to predict the bioaccumulation potential of these compounds, including neonicotinoids, glyphosate and fipronil. We are currently cloning honeybee P‐gp from forager bee specimen. Next, we will express and purify honeybee P‐gp and use an optimized ATPase activity assay to define inhibitors, activators and non‐interacting compounds. Compounds identified as P‐gp inhibitors or non‐interacting chemicals will be infused into sugar water and fed to living bees. Total body burden of these compounds will then be determined using LC‐ and GC mass spectrometry. Finally, we will dissect bees and separate the digestive system (foregut, midgut, hindgut) to validate P‐gp expression using Western immunoblotting. Our preliminary data has shown that a commercially available human P‐gp epitope antibody can recognize honeybee P‐gp.

Future experiments will aim at studying behavioral changes upon P‐gp inhibitory pesticide exposure, including changes in flight paths and waggle dance in living colonies. In addition, we will determine the interactions of these pesticides with another key efflux transporter in bees, the multidrug resistance protein 1 or MRP1.

The results of this study will help to identify the bioaccumulation potential of currently used agricultural pesticides and guide the rational design and use of compounds that are better eliminated by bees. Finally, it will inform farmers, stakeholders and governmental agencies about non‐lethal combinations of pesticides often used as mixtures for proper crop protection.

Support or Funding Information

This study is supported by the Principle Investigator Start‐up funds from UC Davis.
Expanding the Use of Tyrosine Phenol Lyase in the Enzymatic Synthesis of Unnatural Amino Acids
Authors: Munozvilla Jubilee H. , Loo Christian E. , Schwans Jason P.
Published: April 2020

The use of enzymes in the synthesis of pharmaceuticals and biochemical tools offers an attractive approach to generating complex structures, as enzymatic reactions often lead to the formation of specific products in high yields. Tyrosine phenol lyase (TPL), an enzyme biologically involved in the degradation of tyrosine, has been used to synthesize a variety of substituted tyrosines including fluorotyrosines for biochemical studies. In addition to fluorotyrosines, expanding the scope of substrates to naphthol‐based compounds offers an attractive target for enzymatic synthesis of complex molecules, as substituted naphthols can be used as biochemical probes and as building blocks in potential pharmaceuticals. Using Citrobacter freundii TPL we enzymatically synthesized the naphthol‐analog of tyrosine, but the unnatural amino acid was generated in low yield. The simplest model for the low conversion is that the larger substrate is poorly accommodated by the enzyme active site and this poor binding leads to the low yield in the enzymatic reaction. To test this hypothesis, we mutated several bulky residues near the substrate‐binding site (M288, M397, F448) to open space for substrate binding and facilitate synthesis of the unnatural amino acid. A series of mutant plasmids were generated using site‐directed mutagenesis and sequenced to confirm the mutation. The wild type and mutant enzymes were recombinantly expressed in E. coli, purified by affinity and ion‐exchange chromatography, and purity was analyzed by gel electrophoresis. Enzyme activity with tyrosine in an NADH‐dependent coupled enzyme was first used to determine that the wild type and mutants are active, and initial results for the first mutants generated (M228S and M379A) indicate that the mutants are active. We are currently surveying reaction conditions using high‐performance liquid chromatography (HPLC) to evaluate the reactions of the TPL mutants with naphthols as substrates. HPLC is being used to determine the generation of product to calculate yields and provides a method to test if additional products are formed. While a goal of this study is aimed at the more efficient production of tyrosine derivatives, a larger goal is to better understand the features important for substrate binding in enzymatic reactions. This understanding may aid in the development of enzymes with a broad range of substrates efficiently converted to products in the biochemical generation of complex molecules.

Support or Funding Information

Funding by NIH/NIGMS T34GM008074
Not all cartilages are the same! Differential effects in the cranial vs caudal skeleton after BMP inhibition in zebrafish
Authors: Franz-Odendaal Tamara Anne , Zinck Nicholas
Published: April 2020

Several signaling factors are known to be involved in cartilage morphogenesis via the regulation of condensation size, shape, and location. Of these signaling factors, the bone morphogenetic proteins (BMPs) are known to be crucial in regulating the formation of condensations, cellular differentiation, and in the expression of the downstream transcription factors. Furthermore, interference with the BMP signaling pathway results in disrupted bone and cartilage development in various vertebrates. This study aims to elucidate the effects of BMP inhibition on cartilage morphology in the zebrafish. We utilise a pharmaceutical BMP pathway inhibitor at specific early time points of development. We focus on two cartilages, one cranial and one caudal, that develop concurrently but which have different developmental origins. Our data shows that the cranial cartilage, namely the scleral cartilage, is highly robust and unaffected by the BMP inhibitor. This is in stark contrast to the caudal cartilages, for example the hypurals, which are dramatically affected. These effects include fusion of elements, missing elements and altered morphology. Understanding these variable effects provides insight into the mechanisms that limit versus enable cartilages from altering their shape and/or size. The robustness of the cranial skeleton is particularly noteworthy given the multitude of disorders that affect this part of the skeleton.

Support or Funding Information

Funding was provided by the Natural Science and Engineering Research Council of Canada
Etiology of disrupted cholesterol metabolism in the liver of young rats submitted to obesogenic diets
Authors: St-Pierre David H. , Saint-Amand Roxane , Ngo Sock Emilienne T. , Lavoie Jean-Marc
Published: April 2020

The purpose of the study was to test the hypothesis that a short period (2 weeks) of a high fat (HFD) and a western (WD) diet leading to an increase in liver fat accumulation will disrupt key markers of liver cholesterol metabolism mainly targeting the LDL‐receptor (LDL‐R) pathway. Rats were submitted to either a standard (SD), HFD (60%) or a WD (40% fat + 35% sucrose) diet for 2 and 6 weeks. Body weight gain and body fat values were higher (p < 0.01) in rats fed the HFD and WD for 6 weeks although food intakes in kcal/d were not different for all groups throughout the experimental period. Liver fat accumulation was significantly (P < 0.01) higher in HFD and WD rats as compared to the SD diet for both feeding durations. On the other hand, liver fat accumulation was higher in WD rats (P < 0.05) than HFD animals after two weeks of treatment. In addition, WD animals displayed the highest plasma cholesterol levels. After 2 and 6 weeks of treatment, hepatic gene expression of LDL‐R, Pcsk9 and SREBP‐2 was lower (p < 0.01) in WD animals than in HFD and SD rats. In addition, mRNA levels of Lrp1 and Acat2 were lower (p < 0.01) in WD rats as compared to those fed with SD. Gene expression of HMGCoA‐r, the main enzyme involved in the regulation of cholesterol synthesis, was lower in HFD (2 weeks) and even more so in rats fed the WD diet (2 and 6 weeks). The WD was associated with a higher (p < 0.01) gene expression of markers involved in the mediation of de novo lipogenesis (ACC and ChREBP). It is concluded that a short‐term WD promptly increased fat accumulation while disrupting cholesterol metabolism in the liver. The present results provide new insights on the mechanisms through which westernized diets rapidly promote increases in plasma cholesterol levels.

Support or Funding Information

NSERC, FRQS, CFI
Review of Riboflavin Transporter Deficiency Syndrome: Sameness in Genotype occurs with Variations in Phenotype.
Authors: Ukpong Christopher , Einstein George P , Tulp Orien Lee
Published: April 2020

Riboflavin Transporter Deficiency Syndrome (RTD) is a rare childhood neurodegenerative disorder. RTD is acquired as an autosomal recessive pattern, which indicates that a child whose parents are carriers of RTD has a 25% chance of having the disease [homozygous for the RTD trait], a 50% chance of being a carrier [a heterozygous for the RTD trait], and a 25% chance of not having the disease [homozygous negative for the RTD trait]. RTD is a sensory motor neuron disease with symptoms such as auditory neuropathy, sensory ataxia, sensorineural deafness, and muscle weakness. These symptoms were first described in 1892 but diagnosis only became established in 2009 following the application of molecular biology investigation to discern the cause. Through exome sequencing and genetic testing, a mutation of the Solute Carrier 52A gene (SLC52A) was found to be implicated in RTD. There is no cure for RTD currently, but dietary Riboflavin supplementation has been shown to be a very effective treatment for most RTD patients. The current treatment is based on mg Riboflavin/kg of Body Weight/day. This approach fails to consider the role of other potential factors in the efficacy of the therapy, as well as in the variations and severity of the symptoms. This research uses case reports and meta‐analysis of available work reports to highlight the potential pathophysiological factors that may contribute to the variations, severity of signs and symptoms reported, and treatment responses in RTD affected patients. This report includes the molecular, cellular, and the phenotypic dynamics of RTD, and ends with a review of reported treatment responses. It is believed that such a hierarchical assessment will better equip the patients, their families, and the attending clinicians with the knowledge to assess and treat each RTD patient individually and with greater accuracy, success and outcomes than in the past.

Support or Funding Information

This research supported by Institutional Resources of the University of Science Arts and Technology, Montserrat, British West Indies and the Einstein Institute, USA.
Ring‐14 Chromosomal Syndrome: A Statistical Analysis and Comparison of Signs and Symptoms
Authors: Ivanoff Athena E. , Tulp Orien Lee , Einstein George P.
Published: April 2020

The parent(s) and/or guardian(s) of Ring‐14 patients all signed appropriate consent form(s) prior to the study.

OBJECTIVE

The purpose of this extensive study was to investigate the manifestations and challenges faced by subjects suffering from the rare, genetic condition known as Ring‐14 Chromosomal Syndrome, caused by an abnormal ring formation of chromosome fourteen (14), by comparing signs and symptoms exhibited, including, but not limited to the following: phenotypic similarities; physical appearances and properties; abnormal oral manifestations (such as delayed development and eruption of teeth, excessive swelling of the oral mucosa (periodontitis and gingivitis), and presence of any atypical oral lesions and /or sores), maxillofacial characteristics; specific types of seizures most commonly experienced; onset, duration and frequency of seizures; most common race(s) affected; comprehension capabilities (such as the ability to speak, read, or write, and IQ/intelligence quotient), gender; age, presence of lymphedema; mobility characteristics; and existence and range of decreased muscle tone. The parent(s) and/or guardian(s) of Ring‐14 patients all signed appropriate consent form(s) prior to the study, which was also approved by the Human Subjects Committee of the University. The information gathered and analyzed from this study is intended to raise an awareness of this extremely rare, yet highly complex genetic condition and allow health professionals to gain familiarity with the signs and symptoms exhibited by these patients to encourage genetic testing early on, upon suspicion of it's potential presence; thus, limiting the occurrence of a late, or misdiagnosis. In addition to educating health professionals and those in training to appropriately examine these patients, as many of them are prone to seizures, behavioral outbursts, and are unable to speak; and finally, to challenge the current belief that this syndrome is limited to pediatric patients, by documenting and introducing current statistics of those affected by Ring‐14 Syndrome, as several cases remain diagnosed, yet undocumented in the current medical literature.

Support or Funding Information

Support provided by the University of Science Arts and Technology, Montserrat, British West Indies.
ECM1: A CTGF Binding Protein Inhibiting Ductular Reaction and Liver Fibrosis
Authors: Pi Liya , Ran Gai
Published: April 2020

Introduction

Extracellular matrix protein 1 (ECM1) has inhibitory roles against transforming growth factor (TGF)β activation and liver fibrosis. We have identified ECM1 as binding protein for connective tissue growth factor (CTGF) in yeast two‐hybrid analysis. This study aims to determine whether ECM1 binding inhibits CTGF‐mediated ductular reaction and liver fibrosis.

Methods

Mice were fed with 3,5‐diethoxycarbonyl‐1,4‐dihydrocollidine (DDC) to induce ductular reaction and liver fibrosis. ECM1 was overexpressed in adenovirus and intravenously injected into animals before DDC feeding. ECM1 interaction with CTGF, and their effects on liver injury after DDC feeding were analyzed.

Results

ECM1 downregulation was correlated with CTGF upregulation during DDC‐induced liver injury. ECM1 overexpression after DDC feeding caused low levels of EpCAM and Sox9 mRNAs, small areas of EpCAM+ cells, and few proliferating ductular epithelial cells in comparison to controls that received GFP adenovirus. Weak fibrogenesis was also observed in the Ecm1 overexpressing livers, as evidenced by low levels of fibrosis‐related genes, small fibrotic areas in staining with α smooth muscle actin antibody and Sirius red as well as low collagen production determined by hepatic hydroxyproline content. Finally, we found that ECM1 could interact with CTGF and integrin αvβ6 leading to low TGFβ activation in vitro and in vivo.

Conclusion

These observations indicate that ECM1 is a novel binding protein for CTGF. Its overexpression significantly reduces DDC‐induced ductular reaction and fibrogenic reaction, supporting anti‐fibrotic potentials of this molecule for the treatment of chronic liver disease.

Support or Funding Information

This study is supported by National Institutes of Health NIAAA KO1AA024174 grant and Children Miracle Research Foundation grant awarded to L Pi.
Antihyperglycemic Potential of Butterfly Pea Leaf (Clitoria ternatea) Extract in Alloxan‐Induced ICR Mice
Authors: Malbas Fedelino Fernandez , Mondoy Melissa Undan , Trigo Blyth Barroga , Geronimo Josefina Garcia
Published: April 2020

The present study was conducted to evaluate the antihyperglycemic potential of the hydromethanolic extract of Butterfly pea leaf in alloxan‐induced ICR mice.

Thirty female ICR mice were grouped into six: normal, diabetic, positive control groups and three experimental groups treated with varying dosages of Butterfly pea leaf extract (100 mg/kg. b.w., 250 mg./kg b.w., and 500 mg/lg b.w.). All groups, except the normal control received alloxan monohydrate (200mg/kg b.w.) to induce diabetes. ICR mice were gavaged with their respective treatment’s once daily for 14 days and blood samples were collected weekly for the determination of blood glucose levels. On the 14th day, ICR mice were sacrificed and pancreases were extracted for histopathological examination.

The administration of Butterfly pea leaf extract on alloxan‐induced ICR mice elicited a blood glucose lowering activity throughout the course of the experiment. The 500 mg/kg dose of the extract exhibited the highest percent blood glucose reduction at the two time intervals (69.79% and 81.92%). The three varying dosages of the extract showed statistically no difference (P>0.05) when compared with the positive control group/ Histopathological examination of pancreas reported no significant difference (P>0.05) in the pancreatic damage between the Butterfly pea leaves treated groups and the diabetic control group, thus may suggest a different mechanism of action responsible for the antihyperglycemic activity of Butterfly pea leaves . The above results may propose that leaf extract is a potential candidate as an antihyperglycemic agent.

Support or Funding Information

Research Institute for Tropical Medicine Veterinary Research Department, Alabang Muntinlupa City, Philippines; De La Salle University Laboratory Animal Research Center, Dasmarinas Campus, Philippines and Department of Education, Quezon City, Philippines
High Throughput siRNA Screening for Sulfur Mustard‐Induced Corneal Epithelial Cell Injury
Authors: Gomez Amber , Koenig Jeffrey , Ruff Albert
Published: April 2020

Chemical injuries to the eyes are true ocular emergencies due to the potential to inflict significant tissue damage that can lead to vision impairment or eventual loss of vision. Sulfur mustard (HD) is a chemical warfare agent that was originally used in World War I and has been used as recently as 2016 in the Syrian conflict. As with many potential chemical threats, no specific treatment for HD ocular exposure is currently available. We previously utilized a high throughput small inhibitory RNA (siRNA) screening model to identify potential therapeutic targets for two different toxicant‐induced ocular injuries: chloropicrin and hydrogen fluoride. Several targets for these injuries are now being evaluated in mouse models of injury. We proposed utilizing the same high throughput screening (HTS) method to identify potential therapeutic targets involved in the response to HD‐induced ocular injury. The model utilized SV40 large T antigen immortalized human corneal epithelial cells (SV40‐HCEC). Selected endpoints were cell viability and IL‐8 expression, as targets of interest should mediate cell health and/or the inflammatory response. Primary screen results were used to down‐select 300 targets from the original 3,027. Deconvolution of these 300 targets narrowed the target list down to 25, which are currently undergoing in vitro validation. Various pathways are implicated by the current target list, including ubiquitination, transcription regulation, signal transduction, and membrane transport. We anticipate that approximately ten validated targets will be transitioned to study in a mouse model to further determine potential therapeutic efficacy for HD‐induced ocular injury. Although work for HD is still in progress, we have compared the target lists that entered in vitro validation studies for all three toxicants evaluated thus far. Only one target was found in common: NR3C1, a glucocorticoid receptor. Additionally, there were two targets in common between chloropicrin and hydrogen fluoride: KMT5A and RELA. These findings show little similarity in the early molecular responses to these toxicants and support the hypothesis that specific therapeutics will be needed to treat different chemical‐related injuries.

Support or Funding Information

Disclaimer: The views expressed in this abstract are those of the authors and do not reflect the official policy of the Department of Army, Department of Defense, or the U.S. Government.

Funding Sources: This research was supported in part by an appointment to the Postgraduate Research Participation Program at the U.S. Army Medical Research Institute of Chemical Defense administered by the Oak Ridge Institute for Science and Education through an interagency agreement between the U.S. Department of Energy and U.S. Army Medical Research and Materiel Command. Support is also provided by a CounterACT inter‐agency agreement between the NIH/NINDS (Y1‐O6‐9613‐01) and USAMRICD (A120‐B.P2009‐2).
Effects of Personalized Precise Exercise Training with Holistic Integrative Protocol (PPETHIP) on Patients with Chronic Heart Failure: Summary Report of Controlled Clinical Trial
Authors: Sun Xingguo
Published: April 2020

BACKGROUND

The effects of Personalized Precise Exercise Training with Holistic Integrative Protocol (PPET‐HIP) in patients with chronic heart failure (CHF) were less reported previously, we hypothesized that PPET‐HIP would improve exercise capacity, facilitate to manage co‐morbidities and decrease the average re‐hospitalization rate(ARR) among CHF patients.

METHODS

30 CHF patients were recruited and randomly divided into Control group (n=15) and PPET‐HIP group (n=15).Before and after 12‐week intervention, general evaluations (blood pressure, glucose, and lipid profiles), cardiopulmonary exercise testing (CPET), echocardiogram, 6‐min walking distance (MWD), quality of life (QoL) score, and the adjustment of drugs treated co‐morbidities of CHF patients in 2 groups were recorded respectively. The ARR of CHF patients in 3 years before and after intervention was analyzed respectively.

RESULTS

After 12‐week PPET‐HIP, the parameters including CPET parameters, left ventricular ejection fraction, 6MWD and QoL in PPET‐HIP group were improved significantly than those in control group or those before PPET‐HIP (P<0.05). The levels of blood pressure, glucose and lipid in PPET‐HIP group maintained stable after the dosages of drugs treated co‐morbidities were adjusted. Surprisingly, a few patients stopped taking their some drugs treated co‐morbidities. The ARR of cardiovascular‐related events in PPET‐HIP group was decreased obviously than that in control group (P<0.05).

CONCLUSIONS

Based on Holistic Integrative Physiology and Medicine, PPET‐HIP can improve exercise capacity and QoL, and can facilitate to manage the co‐morbidities of CHF, and to reduce ARR.

Support or Funding Information

National Natural Science Foundation of China (81470204); Fuwai Hospital, National Cardiovascular Institute of Chinese Academy of Medical Sciences (2012‐YJR02); Research on Clinical Characteristics of the Capital (Z141107002514084); Research and Outcome Promotion of Clinical Characteristics in the Capital (Project No. Z161100000516127)
PECAM‐1 and VEGF‐Dependent Permeability
Authors: Abraham Valsamma , Zoga Kristi , Desai Mitan , Massa Alyssa , DeLisser Horace
Published: April 2020

VEGF has long been recognized as a potent permeability factor and PECAM‐1 has been implicated in endothelial barrier function. However, the role of PECAM‐1 in VEGF‐mediated permeability is unclear. We observed in studies of wild type and PECAM‐1‐null mice that the loss of PECAM‐1 was not associated with an increase in the baseline permeability of non‐stimulated vessels. Consistent with this was the finding that the junctional architecture of quiescent vessels was preserved in the PECAM‐1‐deficient mice. In contrast, VEGF‐dependent permeability was significantly impaired in the skin of PECAM‐1‐null mice compared to wild type animals, while the dermal responses to two other factors (PAF and histamine) were preserved in the mutant mice. In line with these in vivo findings, in vitro permeability induced by VEGF (but not that of PAF) was reduced in PECAM‐1‐null endothelial cells (ECs). Further, VEGF receptor phosphorylation and downstream activation of PI3 kinase, Akt and eNOS following VEGF stimulation were blunted in PECAM‐1‐null ECs. Together these data implicate the involvement of PECAM‐1 in VEGF‐mediated permeability.

Support or Funding Information

Competitive Pilot Project Fund, VA Health Network VISN4
Pansteatitis in the Nile Crocodile: A Glimpse into the Pathophysiology using RNA‐Seq and bioinformatics
Authors: Azeez Odunayo Ibraheem , Myburgh Jan , Sibeko-Matjila Kgomostso , Bosman Ana-Mari , Featherstone Jonathan , Oosthuizen Marinda , Chamunorwa Joseph
Published: April 2020

Concerted efforts to identify the pathogenesis and mechanism(s) involved in pansteatitis, that was attributed to the recent crocodile die off in the Kruger National Park, South Africa have been in the forefront of investigation in recent time. As part of the efforts, molecular characterization of healthy and pansteatitis adipose tissue was carried out by RNA sequencing (RNA‐Seq) using Next Generation Sequencing (NGS) and de novo assembly of the adipose transcriptome, followed by differential gene expression analysis.

Healthy adipose tissue consisting of fifty samples was collected from the subcutaneous, visceral, intermuscular adipose tissues and the abdominal fat body of ten 4 years old juvenile crocodiles from a local crocodile farm in Pretoria, South Africa. Ten pansteatitis samples were collected from visceral and intermuscular adipose tissues of five crocodiles that were dying of pansteatitis.

Seventy genes were differentially expressed in pansteatitis. They include genes coding for extracellular matrix (ECM) signaling ligands, inflammatory cytokines and tumour necrosis factor alpha (TNFα) receptors, fatty acid synthase and fatty acid binding proteins, peroxisome proliferator‐activated receptor gamma (PPARγ), nuclear factor and apoptosis signaling ligands, and mitogen activated protein kinase enzymes among others. Majority (88.6%) of the upregulated genes were found to be involved in hypoxia inducible pathways for activation of NFkβ and inflammation, apoptosis, Toll‐like receptor pathway and PPARγ. Bicaudal homologous 2 Drosophila gene (BICD2) associated with spinal and lower extremity muscle atrophy was also upregulated in pansteatitis while Sphingosine‐1‐phosphate phosphatase 2 (SGPP2) involved in Sphingosine‐1‐phosphate metabolism was downregulated. Futhermore, Doublesex –mab‐related transcription factor 1 (DMRT1) responsible for sex gonad development and germ cell differentiation was also downregulated.

Thus, from the present study, based on differentially expressed genes in pansteatitis, affected Nile crocodiles might have died partly due to their inability to utilize stored triglycerides as a result of inflammation induced insulin resistance, leading to starvation in the midst of plenty. Affected animals may have also suffered muscular atrophy of the lower extremities and poor fertility.
Comparable Blood Velocity Changes in Middle and Posterior Cerebral Arteries During and Following Acute High‐Intensity Exercise in Young Fit Women
Authors: Labrecque Lawrence , Drapeau Audrey , Rahimaly Kevan , Imhoff Sarah , Billaut François , Brassard Patrice
Published: April 2020

The influence of high‐intensity exercise training (HIIT) on cerebral blood flow (CBF) regulation remains unclear. HIIT induces surges in mean arterial pressure (MAP), which could be transmitted to the brain, especially early after exercise onset. The aims of this study were to 1) describe regional CBF changes during and following 30 s of high‐intensity exercise and; 2) examine whether dynamic cerebral autoregulation (dCA) is associated with CBF changes. Ten women (age: 26 ± 6 yrs; VO2max: 48.6 ± 3.8 ml×kg×min−1) cycled for 30 s at the workload reached at VO2max followed by 3 min of passive recovery. dCA was characterized using transfer function analysis of forced oscillations induced by repeated squat‐stands (0.05 and 0.10 Hz). Middle (MCAvmean) and posterior cerebral artery mean blood velocities (PCAvmean; transcranial Doppler), MAP (finger photoplethysmography) and end‐tidal carbon dioxide partial pressure (PETCO2; gaz analyzer) were measured. MCAvmean (+19 ± 10%) and PCAvmean (+21 ± 14%) increased early after exercise onset, returning toward baseline values afterwards. MAP increased throughout exercise (p<0.0001). PETCO2 initially decreased by 3 ±2 mmHg (p<0.0001) before returning to baseline values at end‐exercise. During recovery, MCAvmean (+43 ±15%), PCAvmean (+42 ± 15%) and PETCO2 (+11 ± 3 mmHg; p<0.0001) increased. TFA gain was higher in the MCA (p < 0.0001). Other dCA metrics were comparable between arteries and unrelated to exercise‐induced cerebral blood velocity changes. In young fit women, blood velocity changes during and following a 30‐s high‐intensity exercise are comparable between MCA and PCA and unrelated to dCA.

Support or Funding Information

L.L. and S.I. are supported by a doctoral training scholarship from the Fonds de recherche du Québec – Santé (FRQS).
Nicotine‐Induced Glomerular Injury is Ameliorated in NLRP3 Gene Knockout Mice
Authors: Puchchakayala Goverdhan , Li Xiang , Koka Saisudha , Boini Krishna M.
Published: April 2020

Nicotine, a major compound of tobacco and the key component of the vapor inhaled via electronic cigarettes, has been shown to worsen kidney injury in animal models of acute kidney injury, diabetes, subtotal nephrectomy. However, it remains unknown how nicotine‐induced glomerular injury. Hence, the present study tested whether NLRP3 inflammasome mediates the nicotine‐induced glomerular injury. Uninephrectomized Nlrp3 knockout (Nlrp3−/−) and wild type (Nlrp3+/+) mice were fed a normal chow for 5 weeks with or without nicotine treatment to produce glomerular injury. Nicotine treatment significantly increased the colocalization of Nlrp3 (Nod‐like receptor protein 3) with ASC (apoptosis‐associated speck‐like protein) or Caspase‐1 in glomeruli of Nlrp3+/+ mice than in vehicle treated mice. However, such nicotine‐induced increases in colocalization of Nlrp3 with ASC or Caspase‐1 was attenuated in Nlrp3−/− mice. In consistency with decreased inflammasome formation, the caspase‐1 activity and IL‐1β production was significantly attenuated in glomeruli of Nlrp3−/− mice. Morphological examinations showed that nicotine‐induced profound injury in glomeruli of Nlrp3+/+ mice which was markedly attenuated in Nlrp3−/− mice. The decreased glomerular damage index in Nlrp3−/− mice was accompanied by attenuated proteinuria. Immunofluorescence analysis showed that the nicotine treatment significantly decreased the expression of podocin but increased desmin levels in glomeruli from Nlrp3+/+ mice but not in those from Nlrp3−/− mice. Furthermore the nicotine‐induced hypertension was significantly attenuated in Nlrp3−/− mice. In conclusion, these observations disclose a pivotal role of Nlrp3 inflammasomes in mediating the glomerular sclerosis associated with smoking.

Support or Funding Information

R01DK104031 and R56HL143809
Chemotherapy Stress Induces Translocation of Mitochondrial Enzymes to the Nucleus and a Nuclear Metabolomic Shift in Cardiomyocytes Derived from Human Induced Pluripotent Stem Cells
Authors: Srivastava Shubhi , Miyamoto Hiroe , Ong SangGing , Rehman Jalees
Published: April 2020

Background

Doxorubicin is a highly effective chemotherapeutic agent which can induce severe cardiomyopathy and heart failure in more than 30% of the patients receiving this treatment. Human iPSC‐derived cardiomyocytes (iPSC‐CMs) can be used to model chemotherapy‐induced cardiotoxicity as well as identify adaptive stress mechanisms. We studied the metabolic responses of iPSC‐CMs to doxorubicin in order to identify mechanisms of metabolic injury as well as metabolic adaptation to stress.

Methods and Results

We studied iPSC‐CMs following short (6 h) and prolonged (24 h) doxorubicin treatment. We observed that doxorubicin stress induces the translocation of mitochondrial TCA cycle enzymes to the nucleus during the course of treatment with ~15 % of the TCA cycle enzymes translocate to the nucleus in 6 h and 30 % in 24 h (p value <0.0001). The compartment specific metabolomic analysis of the nuclear fraction showed that the enzyme translocation induced a metabolic shift in the nuclear compartment, indicating that the translocated enzymes were metabolically active in the nucleus. Super‐resolution microscopy demonstrated that the translocation of TCA cycle enzymes was mediated by carrier vesicles originating from mitochondria. We observed that the metabolic rewiring in the nucleus alters the nuclear NAD+ levels and therefore fuels the activity of cardioprotective nuclear sirtuins by ~2 fold in 6 h of doxorubicin treatment which further increases to ~2.7 fold in 24 h (p value <0.0001).

Conclusion

The recent discovery that mitochondrial TCA cycle enzymes can be found in the nuclei of cells where they generate locally acting nuclear metabolites and modulate the epigenome has raised intriguing possibilities regarding mitochondrial‐nuclear crosstalk during stress. Our findings suggest that mitochondrial enzyme translocation to the nucleus may initiate metabolic adaptation which increases the activity of sirtuin histone deacetylases. In light of the known cardioprotective function of sirtuin activation, stress‐induced mitochondrial enzyme translocation may serve as a potential novel therapeutic target to reduce chemotherapeutic cardiotoxicity.

Support or Funding Information

NIH HL 126516
Electronic‐cigarette Vapor Exposure Causes Impaired Left‐Ventricular Relaxation and an Inflammatory Response in Male Adolescent Mice
Authors: Neczypor Evan William , Gorr Matthew W. , Mears Matthew J. , Schweiterman Neill A. , Anaruma Chadi Pellegrini , Grimmer Jacob A. , Muffler Michael D. , Wold Loren E.
Published: April 2020

Objective

Electronic cigarettes (e‐cigs) are the most commonly used tobacco product by teens and young adults. The aim of the present study was to determine the potential effects of e‐cig vapor exposure on cardiac function in adolescent mice.

Materials and Methods

Four‐week‐old FVB/NJ male mice were separated into three exposure groups: 1) e‐cig exposure with nicotine (EC(+)) (20.2 mg nicotine/1 mL 50:50 propylene glycol:vegetable glycerin), 2) e‐cig exposure without nicotine (EC(‐)), or 3) filtered air exposure (FA) (HEPA filter), and exposed for three months. A puff profile consisting of one 70‐mL e‐cig vapor puff per minute of exposure was administered for 4 h/day, 5 days/week over the course of three months, and filtered air was administered continuously throughout the 4‐hour periods. Cotinine concentration in the serum of mice from either group was determined using a cotinine ELISA. Echocardiography was used to examine global cardiac function after 1, 2, and 3 months of exposure. Following the exposure period, mice were sacrificed within 24 hours of exposure, and heart tissue and serum was collected. Serum was analyzed for 42 cytokines by Ampersand Bioscience’s Rodent MAP 4.0 service. Left ventricular (LV) cardiac lysates were examined for protein expression via western blot.

Results

The average cotinine concentration in the serum of the EC(+) mice after three months of exposure was 189 ng/mL, while there was not a detectable concentration of cotinine in the serum of EC(‐) or FA mice. Echocardiography illustrated significant reduction in the E/A ratio for mitral flow in mice exposed to EC(+) as early as two months into exposure. There were no significant alterations in ejection fraction (%EF), fractional shortening (%FS), or posterior wall thickness (PWT). The cytokine panel showed significant increases in the concentrations of MCP‐1, IL‐18, and MIP‐1β in EC(+) compared to FA, and increases in SCF and VEGF for both EC(‐) and EC(+) compared to FA. Cardiac fibrosis was observed via an increase in collagen I levels in LV tissue of EC(+) compared to EC(‐) and FA. Additionally, a decrease in phosphophospholamban and phospho‐troponin I was observed in EC(+) LV tissue, suggesting alterations in calcium cycling and myofilament regulation.

Conclusions

E‐cig exposure causes early signs of impaired cardiac relaxation with no observed systolic dysfunction in adolescent male mice. Fibrosis, alterations in calcium cycling, and inflammation may contribute to the observed dysfunction.

Support or Funding Information

Project was supported by a grant from the National Institutes of Health (1R01HL139348‐01) to LEW.
Loss of Evolutionarily Acquired Functions of Atp1b4 leads to Increase Energy Expenditure and Protection Against Diet‐Induced Obesity of Adult Mice
Authors: Modyanov Nikolai
Published: April 2020

Co‐option of vertebrate orthologous ATP1B4 genes in placental mammals radically changed properties of the encoded BetaM‐proteins. These proteins in lower vertebrates are authentic Na, K‐ATPase β‐subunits which in complex with a‐subunits function as ion pumps in the plasma membrane.

In placental mammals BetaM proteins gained entirely different properties through structural changes of N‐terminal domains. As a result of these evolutionary alterations, eutherian BetaM completely lost its ancestral role and became the only currently known skeletal and atrial cardiac muscle‐specific protein of the inner nuclear membrane, which is expressed at highest level during perinatal development and is implicated in gene expression regulation.

Loss of BetaM the Atp1b4 knockout mouse model results in significantly lower body size and weight, growth retardation and high mortality of neonates. Transcriptome analysis by mRNA sequencing of skeletal muscle from neonatal wild type and knockout littermates revealed broad changes in expression of genes regulating lipid metabolism. These results imply that BetaM plays an essential role during a critical period of perinatal and neonatal development of placental mammals and its damage or malfunction can cause birth and growth defects in humans.

Most importantly, Atp1b4 disruption exhibits a somewhat unexpected and profound beneficial effect of on metabolic parameters in adult male mice. BetaM deficient (Atp1b4‐/Y) mice have significantly lower body weight and remarkably low adiposity. They exhibit lower fasting blood glucose, enhanced insulin sensitivity and improved glucose tolerance and are resistant to high‐fat diet‐induced obesity as compared to their wild type littermates. Knockout mice display higher heat production associated with increased food intake, elevated oxygen consumption especially in darkness and higher locomotor activity. Lower respiratory exchange ratio of knockout mice indicates that fat from high‐fat diet is metabolized rather than deposited as storage. These robust changes in mouse metabolic parameters induced by Atp1b4 disruption clearly demonstrate that eutherian BetaM plays the important role in development of pathways regulating metabolism of adult mice.

Finally, ablation of the evolutionarily acquired functions of Atp1b4 gene in mice is in fact a simulation of the alternative pathway of mammalian evolution.

Support or Funding Information

This work was supported by College of Medicine and Life Sciences, Center for Diabetes and Endocrine Research and Department of Physiology & Pharmacology, University of Toledo
Designing Cell Biosensors to Detect Incremental Glucagon Expression
Authors: Ennis Teresa Annmarie , Kolar Grant R. , Samson Willis K. , Yosten Gina L. C.
Published: April 2020

Hypoglycemia is a highly prevalent complication for patients with Type 1 Diabetes. Research into alpha cell function and glucagon release has been hampered because current methods used to measure glucagon, such as ELISA, are limited in sensitivity and specificity. In order to uncover the targets and pathways involved in alpha cell dysfunction in type 1 diabetes, we have designed multipurpose glucagon biosensors to detect changes in glucagon release on a cellular level and in real time. To generate the glucagon biosensors, HEK‐293 cells were transfected with a GCGR (RFP‐Tagged) Human Glucagon Receptor vector, along with GCamP6s calcium reporter (GFP). Once transfected, they were maintained and selected for using media consisting of puromycin and G418. This selection allows the cells with the incorporated vectors to be isolated. We previously found that treatment of these cells with recombinant glucagon led to clearly visible increases in GCamP6s (GFP) fluorescence. To better simulate hypoglycemic conditions, immortalized alpha cells were co‐cultured with the glucagon biosensors, and the glucagon response was measured. This system could provide a novel model for evaluating the regulation of glucagon release from alpha cells as well as the dynamics of glucagon release following exposure to hypoglycemic conditions. In future experiments, we will expose the cells to environmental stressors, such as a pro‐inflammatory cytokines, to determine their effects on glucagon release. In summary, the glucagon biosensors can offer enlightening information about sensitivity along with specificity of cellular interactions in real‐time.

Support or Funding Information

Funding is supported by the Helmsley Charitable Trust
Chronic hypoxia in utero increases PAF receptor expression in lung vessels of fetal lambs
Authors: Ibe Basil Obijiaku , Placenscia Joanna , Foryoung Stacy , Zheng Ruoying , Soremekun Racquel , Raj J. Usha
Published: April 2020

The fetus is chronically in low oxygen environment that maintains a desirable high pulmonary vascular resistance (PVR) such that only 8–10% of total cardiac output passes through fetal lungs. With oxygenation, PVR falls dramatically, lung blood flow increases to accommodate the total cardiac output. Chronic hypoxia in utero leads to increase in vessel wall thickness and media wall area in pulmonary arteries of fetal lambs. Platelet activating factor (PAF) is an endogenous lipid molecule with different physiological and pathological properties. In ovine fetal pulmonary VSMC, hypoxia increases PAF receptor (PAFR) density and PAFR binding, indicating that high PAF level and its activities in fetal lungs are, in part, responsible for maintaining the desirable high fetal PVR in utero, but which postnatally manifest as persistent pulmonary hypertension of the newborn (PPHN). In spite of the tremendous advances in therapy of newborn respiratory diseases, PPHN still accounts for about 10–20% of neonatal mortalities. The magnitude and tissue distribution of PAFR expression in pulmonary vasculature of fetal lambs, in vivo, has not been examined. We hypothesized that chronic hypoxia in utero leads to over‐expression of PAFR and PAFR binding in fetal pulmonary vasculature that maintain high fetal PVR in utero. We obtained two groups of term pregnant ewes. One group was raised under low oxygen saturation (high altitude hypoxia, HAH). The other group was raised at sea level (normoxia). Fetuses were exteriorized from anesthetized ewes. Fetal lungs were harvested and sectioned for immunohistochemical staining and analytical microscopy of PAFR protein expression. Lung tissues and pulmonary vessel smooth muscle cells (SMC) were prepared and assayed for PAFR binding. We found that PAFR was expressed in the pulmonary vascular tissues of both groups of fetal lambs. Comparatively, PAFR protein fluorescence was greater in the HAH lung tissues in the internal elastin, endothelium, the smooth muscle layer and the adventitia. Baseline PAFR binding to vessel SMC of the HAH lambs was also greater. These data show that the greater PAFR expression in fetal hypoxic lung environment is in part, responsible for the high PVR of the fetal pulmonary circulation.
Cocaine receptor identified as BASP1
Authors: Harraz Maged M. , Malla Adarsha P. , Semenza Evan R. , Shishikura Maria , Kang In Guk , Snowman Adele M. , Cortés Pedro , Song Young Jun , Hwang Yun , Snyder Solomon H.
Published: April 2020

Cocaine is a behavioral stimulant with substantial abuse potential related to its positively rewarding actions. Definitive molecular mechanisms of action have not been established, though cocaine is known to inhibit the reuptake inactivation of monoamine neurotransmission at high nanomolar to low micromolar concentrations. There is evidence for significantly more potent neurochemical and behavioral actions of cocaine that could not be attributed to the inhibition of monoamine transporters. Thus, evidence for a specific, high affinity cocaine receptor that mediates its behavioral actions has been lacking. In the present study we report high affinity binding of cocaine to the membrane‐associated protein BASP1, which appears to be a pharmacologically relevant cocaine receptor. Utilizing affinity pull‐down, mass spectrometry and ligand binding assays, we establish that BASP1 is a high‐affinity cocaine binding protein (Kd 7 nM). Our data show that knocking down BASP1 in the nucleus accumbens or striatum (pre‐ and post‐synaptic) inhibits behavioral actions of cocaine and reduces [3H]cocaine binding to striatal synaptosomes respectively. Taken together, our data suggest that BASP1 is a high‐affinity receptor for cocaine.

Support or Funding Information Funding

this work was supported by U.S. Public Health Service Grants DA00266 and DA044123 to SHS and a NARSAD Young Investigator Grant (grant # 25360) from the Brain & Behavior Foundation to MMH.
Gap Junction Intercellular Communication and Coronary Microvascular Disease in Type 2 Diabetes
Authors: Cabrera Jody Tori O. , Si Rui , Makino Ayako
Published: April 2020

Recent reports demonstrate that diabetic patients with coronary microvascular disease (CMD, also known as non‐obstructive coronary artery disease) exhibit higher cardiac mortality than diabetic patients without CMD. However, the molecular mechanisms in which diabetes leads to CMD are not well understood. In this study, we examine the role of gap junction intercellular communication in the development of CMD in diabetes. We used type 2 diabetic (T2D) mice induced by a single injection of low‐dose streptozotocin (75 mg/kg) with a high‐fat diet (60 % kcal). Coronary flow velocity reserve (CFVR) was measured in mice to assess coronary microvascular function and T2D mice showed significant decrease in CFVR compared to control mice, suggesting that T2D mice are suffering from CMD. Mouse coronary endothelial cells (MCECs) isolated from T2D mice and human coronary endothelial cells (HCECs) treated with high‐glucose exhibited the reduction of gap junction activity compared to their controls. Gap junction is composed of connexins (Cxs) and coronary endothelial cells express Cx37, Cx40, Cx43, and Cx45. Among those Cxs, Cx40 mRNA level was significantly decreased in MCECs isolated from T2D mice. Furthermore, Cx40 protein levels were significantly decreased in MCECs from inducible T2D mice, MCECs from spontaneous T2D mice (Tallyho mice), and HCECs from T2D patients compared to those controls. Cx40 knockout mice exhibited lower CFVR which was accompanied by decreased capillary density in the left ventricle (LV), when compared to the wild‐type mice. The potential causes of decreased capillary density include attenuated endothelial migration/proliferation and increased endothelial apoptosis. In ex vivo, high‐glucose treatment impaired capillary network formation in HCECs and Cx40 overexpression restored the level of capillary network in high‐glucose treated HCECs. We also found that Cx40 overexpression in T2D mice increased capillary density in the LV and CFVR. These data suggest that the downregulation of Cx40 decreases capillary density in the heart and leads to CMD in diabetes. Therefore, Cx40 overexpression in ECs could be the potential therapy for CMD in diabetic patients.

Support or Funding Information

This work was supported by grants from the National Heart, Lung, and Blood Institute of the National Institutes of Health (HL142214 and HL146764 to A. Makino).