bluuebunny/crossref_metadata_2025 huggingface dataset:
Impact of Ovariectomy on Arterial Stiffness
Authors: Wong Tristen J. , Ogola Benard O. , Kilanowski-Doroh Isabella M. , Harris Nicholas R. , Clark Gabrielle L. , Miller Kristin S. , Lindsey Sarah H.
Published: April 2020

Arterial stiffness predicts cardiovascular mortality independent of blood pressure, and women have lower arterial stiffness than men until menopause. Therefore, the lower prevalence of cardiovascular disease in women may be attributed to the impact of estrogen on vascular compliance. The current study evaluated the impact of ovariectomy (OVX) on systolic blood pressure and carotid artery stiffness. We hypothesized that loss of estrogen via OVX would increase blood pressure and induce arterial stiffness. Female mice underwent OVX at 8–9 weeks of age (n=9) or were left ovary‐intact (n=7). Blood pressure was measured via tail cuff plethysmography twice a week, and pulse wave velocity was obtained via high resolution ultrasound. At 15–17 weeks of age, carotids were excised and subjected to biaxial pressure myography under passive conditions to measure large artery distensibility. OVX induced a significant increase in body weight (21.1 ± 0.7 vs. 23.0 ± 0.6 g, unpaired t‐test, p=0.046) and decrease in uterine weight (107 ± 9 vs. 31 ± 13 mg, unpaired t‐test, p=0.0005). Despite similar systolic blood pressures (139 ± 6 vs. 139 ± 4 mmHg; unpaired t‐test, p=0.98), pulse wave velocity was significantly increased by OVX (1.25 ± 0.07 vs. 1.67 ± 0.09 m/s, unpaired t‐test, p=0.02). Pressure myography experiments showed a significant interaction between pressure and distensibility (repeated measures two‐way ANOVA, interaction p=0.0007) with OVX vessels having decreased distensibility at higher pressures. In comparison with carotid cross‐sections from intact mice, OVX vessels had larger external diameters (453 ± 11 vs 495 ± 14 μm, unpaired t‐test, p=0.04) but similar internal diameters (216 ± 17 vs 237 ± 11 μm, unpaired t‐test, p=0.31) . In conclusion, OVX induced an increase in pulse wave velocity without altering blood pressure. The increase in elastic artery external diameter without changes in internal diameter suggests outward vascular remodeling. Additional studies will assess other aspects of vascular geometry as well as changes in components of the extracellular matrix.

Support or Funding Information

NIH HL133619
Anatomy Education Across the Health Professions: A Comparison of Student Perspectives on Body Donation
Authors: Noel Geoffroy P.J.C. , Claveria Alexandra , Bachour Dona , Dube Joseph , Antonacci Rosetta , Ventura Nicole
Published: April 2020

Cadaver‐based learning provides students with the opportunity to not only acquire the visual and tactile experiences necessary to properly apply anatomical knowledge but more importantly, an opportunity for students to reflect upon fundamental values of humanism, such as respect, empathy and compassion. Differences in students' perspectives across the health professions on body donation and the use of body donors as a component of their education has not yet been explored. The goal of the study was to qualitatively analyze the commonalities and differences in student perspectives on body donation across varying health professional programs including nursing, physical and occupational therapy and medicine. One‐page reflections written by nursing (N=37), physical and occupational therapy (N=49) and medical students (N=66) regarding their experiences in the McGill University anatomy laboratory were collected and qualitatively analyzed by deductive coding methods using Atlas.ti software. All of the data were thematically coded using a deductive approach, by 2 independent coders, according to the themes and sub‐themes described by Stephens et al., 2019 (How does Donor Dissection Influence Medical Students’ Perceptions of Ethics? A Cross‐Sectional and Longitudinal Qualitative Study, ASE 2019). IRB# A06‐B42‐17A. Despite the students having different curricula, there were only a few differences in what the nursing, physical and occupational therapy and medical students discussed in their reflections about body donation and cadaveric‐based learning, suggesting that the anatomy laboratory had similar effects on the different users. The nursing students were not reflective about the idea that working with different body donors provided them with unique learning experiences in the anatomy laboratory. In addition, the physical and occupational therapy students did not discuss the idea of maintaining the body donors' privacy by keeping parts of their bodies covered during the anatomy lab. The majority of the students from all three healthcare profession groups have mentioned that, as a result of their positive anatomy lab experience, they would like to give future generations the same privilege they had by donating their bodies to science. However, there were two nursing students that have concluded that they have decided not to donate their bodies to science. This qualitative study is the first of its kind because it directly compares student perspectives on body donation and cadaveric‐based learning, amongst three different health profession groups: nursing, physical and occupational therapy and medicine. This new information can help design interprofessional activities, which require mutual respect of another profession’s perspective, such commemorative services.

Support or Funding Information

This work was generously supported by the McGill University Faculty of Medicine Research Bursary Program.
Very Low‐Density Lipoprotein and a Sphingosine‐1‐Phosphate Receptor Agonist Regulate Aldosterone Production via Lipin‐1 in Human Adrenocortical Cells
Authors: Chowdhury Shinjini , Choudhary Vivek , Choudhary Mrunal , Chen Xunsheng , Bollag Wendy B.
Published: April 2020

Aldosterone plays an important role in blood pressure homeostasis, and hyperaldosteronism can result in hypertension. Aldosterone is considered to be a link between hypertension and obesity; obese individuals have high serum levels of very low‐density lipoprotein (VLDL). VLDL has been shown to stimulate aldosterone production in multiple zona glomerulosa cell models via phospholipase D (PLD), an enzyme that hydrolyzes phosphatidylcholine to phosphatidic acid (PA), a lipid second messenger. In addition, sphingosine‐1‐phosphate (S1P), a bioactive sphingolipid also elevated in obesity, has been reported to be a novel stimulator of aldosterone secretion and PLD activity. The mechanisms underlying the action of these non‐conventional secretagogues of aldosterone remain to be elucidated. Angiotensin II, a classical aldosterone secretagogue, not only activates PLD but also elevates the expression of lipin‐1, an enzyme that converts PA to another lipid second messenger, diacylglycerol (DAG), in human adrenocortical carcinoma (HAC15) cells. However, it is unclear which of the two lipid signals, PA or DAG, underlies PLD’s role in aldosterone production. Herein, we used HAC15 cells to determine whether VLDL and an S1P1 receptor (S1PR1) agonist (SEW2871) stimulate aldosterone production by increasing steroidogenic gene expression via lipin‐1‐mediated metabolism of PA to DAG. We overexpressed lipin‐1 using an adenovirus or inhibited it using propranolol followed by treatment with or without VLDL or SEW2871 for 24 h. The expression of steroidogenic genes and aldosterone secretion were monitored using qRT‐PCR and radioimmunoassay. We demonstrated that lipin‐1 overexpression enhanced the VLDL‐stimulated 55‐fold increase in CYP11B2 expression by 75% while lipin‐1 inhibition decreased the VLDL‐stimulated 21‐fold increase in CYP11B2 expression by 66%. A parallel trend was observed with aldosterone secretion levels: VLDL‐stimulated increase in aldosterone production was enhanced by lipin‐1 overexpression (182%) and was decreased by propranolol (80%). Similar results were obtained with SEW2871. Our results are, therefore, suggestive of DAG being the key lipid signal since lipin‐1 regulates VLDL‐ and S1PR1 agonist‐stimulated expression of steroidogenic genes and ultimately, aldosterone production. Our study warrants further investigation into these steroidogenic signaling pathways which can lead to the identification of novel therapeutic targets such as lipin‐1, or its downstream pathways, to potentially treat obesity‐associated hypertension.

Support or Funding Information

This work was supported in part by the Augusta University Adrenal Center. WBB was also supported by VA Merit Award #BX001357.
Characterization of Biofilms Produced by Bacillus Wild Isolates
Authors: Wacker Sarah A. , Torres Ryan , Lara-Garcia Juan , Bendesky Andrés
Published: April 2020

Many bacteria form complex multicellular communities known as biofilms. Biofilms are a common source of human infections and are associated with chronic infections and with infections resulting from biomedical implants. Bacillus subtilis has been used as a model organism to study biofilms, leading to important insights into how biofilms are regulated and form. However, most research has examined biofilm formation in a single laboratory strain of B. subtilis. We sought to examine differences in biofilm formation between twenty wild isolates that had been published by others to belong to the species B. subtilis. We characterized the biofilm phenotypes of the strains on several types of media and also sequenced the whole genome of each of the strains. We found a wide array of biofilm phenotypes with most being more intense than biofilms of the laboratory strain. Furthermore, biofilm phenotypes and intensity of the biofilm were media‐dependent. Our genetic results indicated that nine of the twenty strains are not actually Bacillus subtilis, but are likely to be a related species, Bacillus velezencis. Interestingly, biofilm strength did not correlate with species. Among the B. subtilis strains, several coding genetic variations were identified in genes important for biofilm formation. Together our study provides insight into variation in biofilm formation among related bacteria and creates a list of possible variations that can be tested for their impact on biofilm formation.

Support or Funding Information

Research was supported by the National Institute Of General Medical Sciences of the National Institutes of Health under Award Number R15GM135861. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
Differential Augmentation of Thrombin Generation by Andexanet Alfa in Lymphoma Patients
Authors: Siddiqui Fakiha , Antic Darko , Bontekoe Emily , Hoppensteadt Debra , Gerotziafas Grigoris , Tafur Alfonso , Fareed Jawed
Published: April 2020

Introduction

The prevalence of thrombosis in lymphoma patients is reportedly high and ranges from 3–10%. Vascular malfunction and inflammatory processes further contribute to the thrombotic activation processes in these patients. Andexanet alfa is an antidote for factor Xa inhibitors and its usage has been reported with thrombotic complications. This study was designed to compare effect of andexanet alfa (AA) on the thrombin generation potential and its relevance to the generation of thrombin.

Materials and Method

Citrated blood samples from 78 patients with confirmed diagnosis of non‐Hodgkin lymphoma (NHL), Hodgkin lymphoma (HL) and Chronic lymphocytic leukemia/Small lymphocytic lymphoma (CLL/SLL) were collected from the Clinic of Hematology Unit, University of Belgrade, Belgrade, Serbia. 50 samples of normal human plasma (NHP) was obtained from George King Biomedical (Overland park, KS). NHP was prepared for referencing purposes. Individual samples were supplemented with andexanet alfa at 100 ug/ml. Thrombin generation studies were carried out using a commercially available a kinetic fluorogenic substrate method (calibrated automated thrombogram; CAT). Thrombin generation parameters such as peak thrombin (PT), lag time (LT) and area under the curve (AUC) were compiled. Results were compiled in terms of mean ±SD.

Results

On a cumulative basis, lymphoma patients showed an increase in LT (2.9 1.15) in comparison to NHP (2.06 .02) which decreases with AA (1.94 0.1). The PT levels were decreased (120.34 44.3) in comparison to NHP (159.20 4.8) and increases with AA (153.70 37.2). The AUC was also decreased (668.85 284.3) in comparison to NHP (756.46 34.5) which increases with AA (758.32 226.4). When the lymphoma patients were sub‐grouped, peak thrombin levels were similar in HL (128.16), NHL (113.99) and CLL (129.77) were decreased compared to normal human plasma (159.2) which increases with AA, in HL (180.75), NHL (154.16) and CLL (142.46) compared to AA control (153.7). The AUC was increased in the HL (769), decreased in NHL (642.4) and was comparable for CLL (715.4) compared to normal human plasma (756.46). However, with AA the AUC values increases for HL (938.31) and decreases for NHL (744.52) and CLL (725.26) in comparison with AA control (758.32). Variations in lag time were noted in the three groups.

Conclusion

Lymphoma patients represent a heterogenous group of patients in which both the hypercoagulable state and inflammatory responses simultaneously occur. Thrombin is constantly generated in some of these patients leading to the formation of fibrin. The observed decrease in thrombin generation potential in these patients may be due to the consumption of prothrombin and other coagulation factors. Differential increase in thrombin generation in post andexxa supplemented samples suggest that the use of this agent may potentially be associated with thrombotic complications.
Adenosine Protects Zebrafish Photoreceptors from Apoptosis and Damage in a Dose‐Dependent Manner in a Light‐Induced Retinal Degeneration Model of Age‐Related Macular Degeneration
Authors: Khan Amanda S. , Stella Salvatore L.
Published: April 2020

Purpose

Adenosine has been shown to be neuroprotective in the CNS and in retinal ganglion cells in the inner retina. However, the neuroprotective effects of adenosine on outer retinal neurons, in particular photoreceptors, is not known. Age‐related macular degeneration (AMD) is a multifactorial disease characterized by the impairment or loss of central vision due to macular dropout and loss of photoreceptors. Adenosine has been shown to inhibit photoreceptor excitability by suppressing Ca2+ influx through voltage‐gated Ca2+ channels, which could lead to cell death. The purpose of this study was to see if adenosine would reduce photoreceptor damage and death in a light‐induced retinal degeneration (LIRD) model in pigmented zebrafish.

Methods

Adult wild‐type and Tg(nrd:GFP) zebrafish (used to GFP label rod photoreceptors) were dark‐adapted for 24 hours, followed by intravitreal injections of ~ 0.5 ml of 0.1 mM, 1 mM, or 5 mM (final conc: 1.67 to 167 mM) of adenosine in both eyes. Controls were injected with saline in both eyes. All zebrafish were exposed to full spectrum high‐intensity light (≥ 20,000 lux) for four days and collected at 0 and 96 hours post dark‐adaptation. Immunohistochemical analysis was performed on vertical retinal sections in order to visualize cones, rods, and the outer nuclear layer of the retina. Retinal damage was assessed using confocal microscopy with a TUNEL assay and cell specific markers.

Results

Photoreceptors in control retinas collected at 96 hours exhibited severe truncation or loss of the outer segments, and swelling of the cell body. Additionally, there was an increase in TUNEL‐positive cells and a significant decrease in rod and cone photoreceptors in the control retinas. Photoreceptors from retinas treated with higher concentrations of adenosine (1 mM and 5 mM) lacked swelling, exhibited less cell death, reduced truncation of the outer segments, and had cell morphology similar to retinas collected prior to light exposure. Photoreceptors from retinas treated with 0.1 mM adenosine exhibited no significant differences in cell death in comparison to control retinas at 96 hours, and had similar levels of outer segment truncation and cell body morphology. However, there was a significant reduction in the number of cones and rods lost in the 0.1 mM adenosine‐treated retinas at 96 hours in comparison to control retinas at 96 hours.

Conclusion

These findings show that adenosine can prevent light‐induced structural damage to rod and cone photoreceptors, prevent photoreceptor death and loss, and provide evidence of a potential novel therapeutic target for the treatment of the “dry” form of AMD.

Support or Funding Information

HMC CURE Zebrafish Pilot Grant from Penn State University College of Medicine, Pennsylvania Department of Health Tobacco CURE Fund
The FSGS protein Actinin‐4 (ACTN4) is expressed in Thick Ascending Limbs and interacts with NKCC2 and ALMS1 to regulate NKCC2 trafficking.
Authors: Maskey Dipak , Liao Tang-Dong , Ares Gustavo R , Ortiz Pablo A.
Published: April 2020

Thick ascending limb (TAL) of the loop of Henle reabsorbs 30% NaCl via the apical Na/K/2Cl cotransporter (NKCC2). Maintenance of steady‐state surface NKCC2 levels is results from a balance between endocytosis, exocytosis and recycling. These trafficking steps are controlled by signaling and protein‐protein interactions. Very little is known about how NKCC2 endocytosis is controlled and only a handful of proteins that mediate NKCC2 internalization and trafficking are known. To identify new NKCC2 interacting partners we immunoprecipitated NKCC2 from TAL lysates using a C‐terminus antibody and identified interacting proteins by mass spectrometry. We identified Actinin‐4 (ACTN4), a protein studied in podocytes, that when mutated causes Focal segmental glomerulo‐sclerosis (FSGS) in humans. We hypothesized that in addition to podocytes, ACTN4 is expressed along the nephron and is part of protein complex that binds apical NKCC2 and other proteins such as Alstrom Syndrome 1 (ALMS1) and promotes its endocytosis. To begin studying these mechanisms we used a targeted proteomics screen to identify new binding partners for ACTN4 in the TAL. We confirmed that glutathione‐S‐transferase (GST) ACTN4 (full length) pulled down NKCC2 and ALMS1 from TAL lysates identified by mass spectrometry (LC‐MS) or Western blot. GST‐ALMS1 also pulled down ACTN4, identified by LC‐MS. Because there are no studies on the role of ACTN4 in NaCl reabsorption along the nephron, we studied its localization in the kidney. ACTN4 was abundant in glomeruli but also localized in cells along the nephron in a punctate vesicular pattern. ACTN4 was expressed in TALs and partially co‐localized with NKCC2 the subapical space. To study if ACTN4 mediates NKCC2 trafficking, we generated adenoviruses expressing ACTN4 shRNA. In vivo transduction of the rat renal medulla decreased ACTN4 expression in TALs by 66±9% (p<0.04). Silencing ACTN4 in rat TALs increased the surface to total NKCC2 ratio by 49±16% (p<0.01) but did not affect total NKCC2, suggesting that ACTN4 regulates NKCC2 trafficking. To study the relationship of ACTN4 with ALMS1 and NKCC2, we generated nephron‐specific ALMS1 knockout mice and measured surface NKCC2 and ACTN4. Deletion of ALMS1 increased surface to total NKCC2 ratio by 55±19% (p<0.05), but also decreased ACTN4 expression by 45±9% (p<0.05). We conclude that ACTN4 binds NKCC2 and ALMS1 and it is required for proper NKCC2 trafficking. It is likely that a large protein complex containing ACTN4 and ALMS1 mediates NKCC2 endocytosis to control renal salt reabsorption.

Support or Funding Information

NIH
Biased agonists of the chemokine receptor CXCR3 differentially drive formation of Gαi and β‐arrestin complexes
Authors: Zheng Kevin , Smith Jeffrey S. , Choi Issac , Warman Anmol , Gundry Jaimee N. , Pack Thomas F. , Inoue Asuka , Caron Marc , Rajagopal Sudarshan
Published: April 2020

G‐protein‐coupled receptors (GPCRs) are the most common cell surface receptor class and influence nearly every biological process within a cell. Approximately 30% of all FDA‐approved medications target GPCRs. Following receptor activation, G proteins and β‐arrestins are currently appreciated to be the two primary proximal intracellular signaling pathways that regulate GPCR signaling. It has been thought that G protein and β‐arrestin signaling pathways are largely discernable, evidenced in part by ‘biased agonists’ that can preferentially activate one of these pathways relative to the other. However, increased crosstalk between G protein and β‐arrestin pathways is beginning to be appreciated. We recently described the ability of Gα proteins to directly interact with β‐arrestins to form signaling scaffolds, with Gαi/o found to directly interact with β‐arrestin following GPCR activation regardless of canonical GPCR G protein coupling. We previously demonstrated in other work that all GPCRs tested, including the vasopressin type 2 receptor, β2‐adrenergic receptor, neurotensin receptor type 1, and the dopamine D1 and D2 receptors, formed a Gαi:β‐arrestin complex. Now that we appreciate this novel GPCR signaling paradigm, it is unclear if biased agonists that preferentially stimulate either G protein‐ or β‐arrestin‐dependent signaling differentially regulate Gαi:β‐arrestin interactions, if at all. To address this question, we utilized the chemokine receptor CXCR3 that binds three endogenous chemokines with high affinity. We demonstrate that these endogenous biased CXCR3 chemokines, CXCL9, CXCL10, and CXCL11, along with its synthetic biased agonists, VUF10661 and VUF11418, differentially form Gαi:β‐arrestin complexes. The ability of CXCR3 agonists to form Gαi:β‐arrestin complexes does not correlate highly with more well‐established GPCR signaling pathways including G protein recruitment, G protein signaling (e.g., regulation of cAMP), or β‐arrestin recruitment, suggesting that Gαi:β‐arrestin complex formation is distinct from conventional G protein or β‐arrestin signaling events. Additionally, differential Gαi:β‐arrestin complex formation was found to correlate with similar degrees of Gαi:β‐arrestin:CXCR3 “megaplex” formation, suggesting that formation of a long‐lived complex may depend upon close coupling of the two effectors at the receptor initially. This work provides further support for a separable Gαi:β‐arrestin signaling pathway and enhances our understanding of GPCR signaling and biased agonism.

Support or Funding Information

NIH
The role of PHLPP in regulating mitochondrial dynamics in colorectal cancer
Authors: Hasani Sumati R. , Xiong Xiaopeng , Gao Tianyan
Published: April 2020

Colorectal cancer (CRC) is the second leading cause of cancer deaths in men and women combined in the United States. According to the American Cancer Society, approximately 100,000 new CRC cases and 50,000 CRC‐related deaths are estimated in 2019. A better understanding of molecular mechanisms underlying tumor initiation and progression in CRC is urgently needed. We have shown previously that the PH domain leucine‐rich‐repeat protein phosphatase (PHLPP) family of phosphatases function as tumor suppressors by inhibiting several oncogenic signaling pathways, including the PI3K/Akt and RAS/RAF signaling. In our studies to determine the role of PHLPP in regulating cellular metabolism, we uncovered a novel link between PHLPP and Drp1, a member of the dynamin family of GTPases that plays a key role in regulating mitochondrial dynamics. Phosphorylation of Drp1 at S616 has been shown to increase its activity and induce mitochondrial fission. A number of protein kinases have been implicated in phosphorylating S616 site, however, how phosphorylation of Drp1 is regulated by phosphatases remains largely unknown. We found that knockdown of PHLPP increased, whereas overexpression of PHLPP decreased, the phosphorylation of Drp1 at S616, suggesting that PHLPP may negatively regulate Drp1 activity. As a consequence, increased levels of mitochondrial fragmentation were observed in PHLPP knockdown cells. Moreover, bioinformatics analysis of normal and tumor samples in TCGA dataset showed that the expression of Drp1 (DNM1L) gene is significantly increased in colon adenocarcinoma supporting a role of Drp1 in promoting tumorigenesis in CRC. Taken together, we identified PHLPP as a regulator of Drp1‐dependent mitochondrial fission. Results from our studies will provide novel insights into the functional significance of PHLPP‐mediated regulation of mitochondrial dynamics in CRC.

Support or Funding Information

5R01CA133429‐12 THE ROLE OF PHLPP IN COLON CANCER
IPA Software Analysis Reveals Inflammation and Apoptosis as Major Enrichment Functional Process in Normal Lung During Aging
Authors: Calyeca Jazmin , Balderas-Martinez Yalbi , Mora Ana , Selman Moises , Pardo Annie
Published: April 2020

Aging is an irreversible biological process characterized by progressive functional decline and represent an independent risk factor for many chronic‐degenerative diseases. Several hallmarks have been associated to aging including genome instability, telomere attrition, epigenetic alterations, loss of proteostasis, deregulated nutrient‐sensing, mitochondrial dysfunction, cellular senescence, stem cell exhaustion, and altered cellular communication. In lungs, accelerated aging has been associated to some diseases such as idiopathic pulmonary fibrosis and chronic obstructive lung disease. However, the consequences of physiological aging on the lungs remain uncertain.

In this study, we aimed to identify the dysregulated genes and pathways occurring in mice lung transcriptome during aging. For this purpose, lungs from 3 young (1‐month‐old) and 3 old (18‐month‐old) C57BL/6 mice were examined. We used Ingenuity Pathway Software analysis on a previous reported transcriptome evaluation performed in our Lab by Microarray technology (GeneChip™ Mouse Gene 2.0 ST Array of Affymetrix).

In old lungs, 576 genes were differentially expressed compared with their young counterparts. In the top 10 upregulated pathways revealed by IPA analysis, the highest over‐expressed pathway in old WT lungs was the Triggering receptor expressed on myeloid cells‐1 (TREM‐1) followed by Toll‐like receptor (TLR) signaling and p38 MAPK. According to these findings, the expression of the upstream regulators INFγ, TNF, TLR2, and TLR4 were increased in the lungs of old mice. In addition, among the top five functions from the “diseases and disorders category” we found Respiratory Disease and Inflammatory Response as one of the higher enriched pathways. Finally, extended analysis of the top five functions from the “molecular and cellular functions category” reveals Cell Death and Survival, including Apoptosis of several lung cell populations, as one of the most enriched pathways.

Support or Funding Information

Supported by CONACYT 281074
Behavioral Effects of d‐Methamphetamine and sin vivo microdialysis in rats
Authors: Desai Rajeev I. , Honrao Chandrashekhar , Dawes Monica , Ma Xiaoyu , Solakidou Eleana , Makriyannis Alexandros , Bergman Jack
Published: April 2020

The present studies were conducted to examine the relationship between locomotor stimulant effects of d‐methamphetamine (d‐MA) and its ability to increase extracellular concentrations of d‐MA, its active metabolite d‐amphetamine (d‐AMP), dopamine (DA), serotonin (5‐HT), norepinephrine (NE), γ‐aminobutyric acid (GABA), and glutamate (GLU) in dialysate samples obtained from the rat nucleus accumbens (nAcc) shell. First, the effects of cumulatively administered d‐MA (0.3–5.6 mg/kg) on locomotor activity were determined in rats. In other studies, in vivo microdialysis and LC/MS analysis of dialysate samples were used to determine the effects of cumulatively administered d‐MA (0.3–5.6 mg/kg) on extracellular levels of d‐MA, d‐AMP, DA, 5‐HT, NE, GABA, and GLU in the nAcc shell. d‐MA produced dose‐related effects on locomotor activity, characterized by an inverted‐U dose‐response curve; 0.3–3 mg/kg increased activity, whereas 5.6 mg/kg suppressed activity (induced stereotypy). Results from microdialysis studies show that levels of d‐MA (0.3–5.6 mg/kg i.p.) in the nAcc shell increased in a dose‐ and time‐dependent manner with concentrations ranging from 12–319 nM, respectively; peak levels (334 nM) detected 20 min after 5.6 mg/kg slowly declined over the next hour. An increase in d‐AMP was also observed with concentrations ranging from 4.5–130 nM after 0.3–5.6 mg/kg d‐MA; peak levels of d‐AMP were detected 20 min (164 nM) after 5.6 mg/kg d‐MA and remained at this level for more than the next hour 100 min. d‐MA produced dose‐ and time‐dependent increases in DA, 5‐HT, and NE efflux in the nAcc shell to maxima of approximately 642% (17 nM), 582% (1.2 nM), and 422% (0.7 nM) of control values, respectively. In contrast, d‐MA did not produce significant changes in GABA or GLU levels. Time course and dose‐response analysis show that increases in d‐MA levels after 0.3–3.0 mg/kg are concordant with increases in DA, 5‐HT, and NE efflux. However, levels of d‐MA after 5.6 mg/kg appear to reflect increases in DA and 5‐HT but not NE. Correlation analysis revealed an inverse relationship between d‐MA‐induced increases in locomotor activity and in vivo extracellular levels of 5‐HT and d‐AMP. Although not statistically significant, a trend towards an inverse relationship was also observed between d‐MA‐induced increases in locomotor activity and extracellular levels of d‐MA and other neurotransmitters in the nAcc shell. Taken together, the ability to detect and correlate the presence and clearance of in vivo brain levels of d‐MA, and its active metabolite d‐AMP in the nAcc shell of rats to neurochemical and behavioral effects can provide insight into the neuropharmacological effects of d‐MA.
A human‐origin probiotic cocktail therapy for aging‐related leaky gut and inflammation by modulating microbiota‐taruine‐tight junction axis
Authors: Yadav Hariom , Ahmadi Shokouh , Wang Shaohua , Nagpal Ravinder , Wang Bo , Jain Shalini , Razazan Atefeh P. , Mishra Sidharth P.
Published: April 2020

Inflammation is a major risk factor of morbidity and mortality in older adults. Although its precise etiology is unknown, low grade inflammation in older adults is commonly associated with increased intestinal epithelial permeability (leaky gut) and abnormal (dysbiotic) gut microbiota. The lack of treatments to reduce aging‐related microbiota dysbiosis, intestinal permeability and inflammation, and the increasing older population culminates on a rise in aging‐related comorbidities, constituting a significant public health concern. Here we demonstrated that a human‐origin probiotics cocktail containing 5‐lactobacilli and 5 enterococci isolated from infant gut, prevented high‐fat diet (HFD)‐induced microbiota dysbiosis, intestinal permeability, inflammation, metabolic dysfunctions and physical function decline in older mice. Probiotic‐modulated gut microbiota primarily reduced intestinal permeability by increasing tight junctions, which in turn reduced inflammation. Mechanistically, probiotic modulated microbiota in a way to increased bile salt hydrolase activity, which in turn increased taurine abundance in the gut that stimulated tight junctions and suppressed gut leakiness. Further, in Caenorhabditis elegans, taurine increased life span, reduced adiposity and intestinal permeability, and enhanced physical function. The results suggest that such probiotic therapy could prevent or treat aging‐related intestinal permeability and inflammation in elderly.

Support or Funding Information

This work was supported by funding from Department of Defense, National Institutes of Health and Wake Forest School of Medicine.
A Characterization of Misfolded Alpha‐Synuclein in Parkinson’s Disease
Authors: Shannon Daniel , Bugada John-Paul , Collins Ethan , Fischer Ellie , Hellard Delaney , Hoffman Trevor , Kellogg Nick , Peters Kyle , Poetker Garret , Young Logan , Diao JiaJie
Published: April 2020

The Moeller SMART Team in conjunction with Dr. JiaJie Diao at the University of Cincinnati Medical College and MSOE Center for Biomolecular Modeling used 3‐D modeling and printing technology to study the role of misfolded alpha‐synuclein proteins in Parkinson’s Disease (PD). Neurodegenerative diseases are all characterized by the aggregation of misfolded proteins that form an insoluble substrate. Misfolded alpha‐synuclein is thought to be the protein involved in PD. This protein is natively found as an unfolded monomer that can take on many different conformations. It consists of 140 amino acids and is divided into three regions: the N‐terminal region (residues 1–60), non‐amyloid beta component (NAC) (residues 61–95), and the C‐terminus (residues 96–140). Alpha‐synuclein shows the ability to form amyloid fibrils which are associated with toxicity. Subunits within the fibrils adopt a beta‐strand conformation with hydrogen bonding between adjacent beta strands to form long linear polymers. The N‐terminal and C‐terminal residues display a random coil arrangement that are not involved in hydrogen bonding. The central beta sheet core is comprised of an inner hydrophobic region that interlocks into compact right‐angled spirals. Each fibril can form a dimer with rotational symmetry about the bonding interface. Within the beta sheet region, the NAC and pre‐NAC regions are responsible for fibril formation. The NAC region is the central hydrophobic region that when removed from alpha‐synuclein leads to the inability of aggregation.
Elucidating the Role of Purinergic and Calcium Signaling During Rotavirus Infection
Authors: Engevik Kristen , Chang-Graham Alexandra , Engevik Melinda , Kellen Joseph , Perry Jacob L. , Hyser Joseph
Published: April 2020

Calcium (Ca2+) is a ubiquitous messenger that influences numerous cellular processes, and therefore Ca2+ signaling is tightly regulated by cells. Ca2+ signaling dysregulation results in severe and potentially life‐threatening diseases, which is exemplified by rotavirus (RV) infection. RV is an enteric virus that causes life‐threatening diarrhea in children, resulting in ~198,000 deaths each year. While pathophysiological consequences of RV infection are widely studied, host Ca2+ signaling pathways and the mechanisms by which RV exploits them to cause diarrhea remain incompletely characterized. We have previously found that RV infection increases Ca2+ signaling both within infected enterocytes and in surrounding uninfected cells through paracrine signaling. This manifests as intercellular Ca2+ waves that originate from the infected cell and propagates to surrounding uninfected cells mainly through ADP activation of the P2Y1 receptor, which is expressed in many cell types in the intestine. We hypothesize that RV‐induced paracrine purinergic signaling functionally dysregulates neighboring uninfected enterocytes and modulates innate immune responses from both epithelial and intestinal immune cells. We generated cell lines and human intestinal enteroids (HIEs) stably expressing cytosolic genetically‐encoded Ca2+ indicators to characterize Ca2+ signaling throughout RV infection by time‐lapse imaging. We found that P2Y1‐mediated signaling was critical for activation of secretory epithelial cells, including induction of serotonin secretion of enterochromaffin cells and mucus secretion from goblet cells in human intestinal enteroids (HIEs) and mucin‐producing intestinal cell lines. Further, signaling from RV‐infected MA104 monkey kidney cells chemoattracted mouse bone‐marrow derived macrophages, which was blocked by pharmacological inhibitors of the P2Y1 receptor. Consistent with our in vitro findings, we observed that murine RV infection promoted secretion of serotonin, mucin and accumulation of macrophages. These effects of minimized in the presence of P2Y1 inhibitors and in P2Y1 knock out mice. Furthermore, we found that the RV‐induced increase in NFKb and IL‐1α are significantly attenuated by P2Y1 receptor blockers. Collectively these findings indicate that intercellular Ca2+ waves caused by paracrine purinergic signaling have a broad influence on intestinal pathophysiology during RV infection. Understanding the cellular consequences of P2Y1 activation during RV will generate new mechanistic insights into the homeostatic function of purinergic signaling in the GI tract.

Support or Funding Information

R01 DK115507 (Hyser)

T32 DK007664 (Engevik)
Examining the Structure of Activation‐Induced Cytidine Deaminase (AID)
Authors: Feng Angela , Alex Katherine
Published: April 2020

In order to respond to any foreign antigen that might present itself, B cells must produce antibodies of great variety. Activation‐Induced Cytidine Deaminase (AID) is a protein that plays a critical role in the diversification of antibodies.1 Discovered by T. Honjo et al. in 2000, AID is a protein consisting of 198 amino acids that creates a U:G mismatch by deaminating cytosine to uracil in transcribed Ig DNA2. AID helps in the process of binding site differentiation, known as somatic hypermutation (SHM), and class‐switching, known as CSR (class switch DNA recombination).3 The Governor’s Academy MSOE Center for BioMolecular Modeling SMART team used 3‐D modeling and printing technology to better understand how several aspects of the structure of AID play a critical role in accomplishing the functions of SHM and CSR. Our model highlights Histidine 56, Cysteine 87 and Cysteine 90, which bind zinc ions forming a zinc finger, and the hotspot recognition loop, all of which are involved in DNA binding.4 The catalytic domain, which is similar to the other members of the apolipoprotein B RNA‐editing cytidine deaminase enzymes (APOBEC) is also shown.5 Unfortunately, two of the residues that are most critical for AID functioning could not be shown on our model. Serine 38, which is an important phosphorylation site, was cut out during the isolation and crystallization process and thus is not included in the protein data bank files. Glutamic Acid 58 is the residue most critical for the deamination reaction and was mutated to an Alanine in order to prevent the reaction from occurring so that AID could be isolated with DNA bound.6 Lastly, we highlighted residues R112 and R24. Mutation of either of these Arginines disrupts the configuration of AID, which undermines the ability of AID to undergo SHM and CSR, causing an immunodeficiency disorder called Hyper‐IgM syndrome type 2 (HIGM‐2)
Elucidating the signaling pathways of CXCR4‐dependent chemotaxis.
Authors: Robleto Valeria Lucia , Zhuo Ya , Marchese Adriano
Published: April 2020

Signaling by the G protein‐coupled receptor CXCR4 is essential to a number of developmental and physiological processes, including stem cell mobilization, organ development, and immune responses. In addition, CXCR4 signaling has been linked to disease, including cancer and immunodeficiency diseases. Despite its importance in health and disease, the mechanisms regulating CXCR4 signaling remain poorly understood. Typically, upon binding to its cognate ligand CXCL12, CXCR4 signaling occurs via G protein‐ and β‐arrestin‐dependent signaling pathways. Recently, our lab showed that β‐arrestin1‐dependent signaling is necessary for CXCR4‐mediated chemotaxis. However, this signaling pathway remains poorly understood. With this in mind, we performed proximity labeling by ascorbic acid peroxidase (APEX2) fused to β‐arrestin1 in order to identify proteins that are part of the β‐arrestin1 interaction network downstream of CXCR4. Labeled proteins from cells treated with vehicle or CXCL12 for 5 or 60 minutes were selectively purified and identified by mass spectrometry. Several proteins of biological relevance were identified and predicted to be part of the β‐arrestin1 interaction network downstream of CXCR4. One of these proteins was selected for further analysis and shown to interact with β‐arrestin1 by glutathione S‐transferase pull down experiments. The goal of this project is to provide further evidence that this protein is indeed part of the β‐arrestin1 interaction network and to determine its role in CXCR4 signaling. This will be accomplished by using cell culture model systems coupled with several approaches, including biochemical, molecular, RNA interference, fixed and live cell confocal microscopy techniques and chemotaxis studies.

Support or Funding Information

Gran Number 10672706A1
Cognitive function during passive heat exposure is not affected by short‐term heat acclimation
Authors: Barry Hadiatou , Gendron Philippe , Behzadi Parya , Gravel Hugo , Gagnon Daniel
Published: April 2020

INTRODUCTION

Hyperthermia affects cognitive performance, particularly decision‐making, the speed of performing a task and the ability to perform complex tasks. Cognitive performance is generally thought to be impaired by hyperthermia, although a u‐shaped relationship may exist between the level of hyperthermia and cognitive performance. Few studies have examined potential strategies to alter the relationship between hyperthermia and cognitive performance. Heat acclimation improves the physiological responses to heat exposure, but its effects of cognitive function remain understudied. The purpose of this study was to evaluate how cognitive performance during acute heat stress is modulated by short‐term heat acclimation.

METHODS

Eight healthy adults (25 ± 4 years, 4 women/4 men) participated in the study. Participants first underwent either (n=6) a 7‐day controlled hyperthermia protocol (internal body temperature ≥38.6°C for 60 minutes) or (n=2) a 7‐day time‐control period. After a minimum 1‐week washout, participants crossed‐over to the other condition. On days 1 and 7, participants performed a digital Stroop test to evaluate their response time to reading, counting, inhibition and overswitch tasks at baseline and after 60 minutes at an internal body temperature ≥38.6°C or after a 60‐minute time‐control period.

RESULTS

On day 1 of the control condition, reaction time during reading (−6 ms [−31, 19], p=0.79), counting (11 ms [−21, 42], p=0.66), inhibition (−31 ms [−95, 34], p=0.44) and overswitch (−38 ms [−82, 6], p=0.09) tasks was not acutely affected by a 60‐minute time‐control period. On day 1 of the heat acclimation protocol, internal body temperature (39.00 ± 0.17°C, p<0.01), mean skin temperature (37.66 ± 0.55°C, p<0.01) and heart rate (110 ± 11 bpm, p<0.01) were greater during the cognitive tasks performed under heat stress. Heat exposure acutely improved response time during reading (−56 ms [−81, −31], p<0.01), counting (−57 ms [−89, −26], p<0.01), inhibition (−84 ms [−149, −20], p=0.01) and overswitch (−68 ms [−113, −24], p<0.01). However, heat acclimation did not affect cognitive performance during each of the cognitive tasks as determined from 3‐way interactions (time × condition × day) for reading (p=1.00), counting (p=0.73), inhibition (p=0.09) and overswitch (p=0.70).

CONCLUSIONS

These results suggest that heat exposure acutely improves reaction time during reading, counting, inhibition and overswitch cognitive tasks, but that this improvement is not affected by 7 days of heat acclimation.

Support or Funding Information

Natural Sciences and Engineering Research Council (RGPIN‐2017‐04243)
LEW.1WR1 Rats have increased insulin levels and liver lipid lipolytic gene expression during their type 1 diabetes susceptibility window.
Authors: Wimalarathne Madushika M. , Wade Amber M. , Love-Rutledge Sharifa
Published: April 2020

LEW.1WR1(1WR1) rats have increased type 1 diabetes (T1D) onset with the administration of polyinosinic‐polycytidylic acid (PIC). The susceptibility window of inducible T1D in the 1WR1 rat occurs between 21–35 days of life. The susceptibility of this model is related to FAT10 overexpression, a ubiquitin‐like protein that plays an unclear role in disease induction and the regulation of lipid metabolism. We hypothesized that these rodents have altered liver lipid metabolism due to increased FAT 10 gene expression in their livers and increased circulating insulin levels which can lead to decreased liver insulin sensitivity and increased beta‐cell sensitivity to inflammation. The objective of this study was to compare the insulitis status and liver gene expression in both control and treated 1WR1 rats during disease induction with a T1D‐resistant strain of rat, the LEW/SSNHsd.

Using immunohistochemistry we observed higher T cell infiltration in the 1WR1 treated rats compared to the control strain, the LEW/SsNHsd(SsNHsd) PIC‐treated rat. By correlating plasma glucose levels measured by glucometer with this finding we confirmed that the 1WR1 rats were not undergoing significant insulitis at the time point we selected. We also observed that the rats have significantly fewer total islets than the SsNHsd rats but remained normoglycemic. Interestingly the 1WR1 treated rat had significantly increased insulin levels compared to the SsNHsd rats, as previous studies showed a 3 fold increase in the 1WR1 rat compared to Wistar Furth rats in a similar study(unpublished data). Both groups of the 1WR1 rats also had increased liver FAT10 gene expression compared to the SsNHsd. We measured liver lipid lipolytic gene expression to assess the metabolic status of the livers at this time point. We observed 1WR1 rats have increased expression of genes related to lipid catabolism like SREBP‐1 (Sterol Regulatory Element‐binding Protein‐1), ATGL (Adipose triglyceride lipase), DGAT1(diacylglycerol acyltransferase‐1), DGAT2(diacylglycerol acyltransferase‐2) and lower expression of glucose metabolism‐related genes like GLUT2(Glucose transporter 2), suggesting that the 1WR1 rats may have had a liver sensitivity response to the hyperinsulinemia. In conclusion, 1WR1 rats have reduced insulin sensitivity in the context of increased inflammation due to PIC injection which led to beta‐cell sensitivity and eventual T‐cell mediated destruction.

Support or Funding Information

Department of Chemistry, UAH

Department of Biology, UAH

College of Science, UAH

Office of Provost, UAH
The Activation of Macrophage Scavenger Receptors through the Phosphorylation Cascade Generated by Nanomolar Cadmium Doses
Authors: Kumar Wasan Mani , Ribeiro Marta , Balla Andre Kajdascy
Published: April 2020

Nanomolar cadmium concentrations have been shown to increase the risk for atherosclerosis and promote endothelial damage. Atherosclerotic plaque formation is regulated by scavenger receptors, which are cell surface receptors crucial to macrophage apoptosis and foam cell formation. LOX‐1, CD‐36, and SR‐A I/II are the main macrophage and endothelial receptors responsible for internalization of oxidized LDL.

Incubation with CdCl2 caused a four‐fold increase in LDL absorption by RAW 264.7 (murine) macrophages. To study mechanisms of this increase, macrophages were treated with 2 mg/ml of LDL only, 5nM to 200 nM CdCl2 and then both treatments combined, all for a 48 hour period. Cell lysates were then subjected to a phospho‐kinase array, which simultaneously analyzes the rate of phosphorylation for 43 kinases. When exposed to 10 nM CdCl2, Signal Transducer and Activator of Transcription 5 (STAT5) had a 5 fold increase in phosphorylation compared to control (macrophage cells only), which was the highest in the array. 30 kinases had a significant increase in phosphorylation, while 13 kinases showed a significant decrease in phosphorylation, the greatest decrease being p53 which was knocked down to half. Interestingly, the cadmium induced changes in phosphorylation were attenuated by LDL co‐incubation.

In vitro studies were performed to assess levels of LOX‐1, CD‐36 and SR‐A I/II. Macrophage cells were exposed to 200 nM CdCl2, STAT5 inhibitor AC‐4‐130, and 2 mg/ml of LDL for 48 hrs. Lysates were assessed for scavenger receptor expression by ELISA assay. Both the 200 nM CdCl2 and STAT5 inhibitor groups showed significantly increased expression of all 3 scavenger receptors, and the 200 nM CdCl2 STAT5 inhibitor combination resulted in an even higher fold expression.

Overall, cadmium plays a role in the progression of atherosclerosis which could in part be via the phosphorylation cascade. STAT5 inhibits the transcription of CD36, hence the increased expression of CD36 and other scavenger receptors noted in the presence of a STAT5 inhibitor. However, cadmium increases the phosphorylation and subsequent function of STAT5 while also increasing the expression of CD36, LOX‐1 and SR‐A I/II. This implies a non‐STAT5 mediated mechanism. It would be interesting to examine the function of the other cadmium‐modulated highly phosphorylated or dephosphorylated kinases in scavenger receptor expression.