bluuebunny/crossref_metadata_2025 huggingface dataset:
Investigating carbon metabolisms in the marine subsurface environment of the Juan de Fuca Ridge Flank
Authors: Gourdine Tylisha , Freel Kelle , Choi Jessica , Gajigan Andrian , Spotkaeff Cherise , Perez-Rodriguez Ileana , Nigro Olivia , Rappe Michael , Carr Stephanie
Published: April 2020

Microorganisms are the most diverse and abundant life on earth. They are responsible for recycling the elements of our planet and keeping our world habitable. Despite their importance, genome‐based analyses estimate that 90% of the microorganisms on our planet have not been cultured for laboratory investigations. The crustal biosphere of the Juan de Fuca Ridge Flank is a warm, subsurface environment, which has been shown to contain many uncultured organisms. Some of these uncultured groups harbor functional genes of evolutionary and biogeochemical significance, suggesting that further study will increase our knowledge of microbial survival on early Earth, as well as in this hydrothermal, nutrient‐limited crustal environment. In previous work the recovery of mono‐ and bifunctional carbon monoxide dehydrogenase genes suggested that carboxydotrophy may be a viable lifestyle in this environment. In May of 2019, the RV Atlantis traveled to the Juan de Fuca Ridge Flank to test this hypothesis. Equipped with custom fluid samplers the ROV Jason‐II was used to collect pristine crustal fluids from subsurface borehole observatories for chemical analyses and microbial culturing efforts. Our main objective was to measure potential carbon substrates, including carbon monoxide and methane, and to cultivate unique organisms from this environment to better understand carbon‐related metabolisms in this important but understudied aquifer.
Is Integration Within Anatomical Sciences Important? Assessing Medical Student Learning of Histology and Embryology via Interdisciplinary Causal Mechanisms
Authors: Cholack George , Lisk Kristina , Venuti Judith M. , Attardi Stefanie M.
Published: April 2020

Curriculum integration is increasingly recognized as a critical component of undergraduate medical education. One approach to curricular integration is through the use of causal mechanisms, which are links that guide students in making purposeful connections between two distinct disciplines. Woods et al. have shown that integrating basic science with clinical content in various contexts using causal mechanisms results in improved diagnostic performance in learners. It remains unknown whether utilizing a similar integrated approach during instruction of different basic sciences, such as embryology and histology, results in better understanding and application of those disciplines. The aim of this study was to determine the effects of integrating histology and embryology using causal mechanisms on medical students’ immediate and delayed (1‐week) recall and application. Second‐year medical students at Oakland University William Beaumont School of Medicine (n=52) participated in this study and were randomized to either an experimental (n=27) or control group (n=25). The experimental group watched a video about pituitary histology and embryology that explicitly integrated these disciplines through causal mechanisms. The control group watched a video covering the same content, but lacked interdisciplinary causal mechanisms. Counterbalanced immediate and delayed post‐tests assessed recall and application of the content (Blooming Anatomy Tool level 1 and 3 questions, respectively). No significant differences (2‐way ANOVA with Bonferroni correction) were observed between the groups on immediate or delayed tests in terms of overall score (p=0.48), histology subscore (p=0.42), embryology subscore (p=0.78), recall subscore (p=0.64), and application subscore (p=0.61). The lack of significant differences between the two learning groups may be due to the close proximity (temporal integration) of embryology and histology instruction afforded to all participants, despite the control group not receiving instruction with causal mechanisms. It is possible that the explicit integration of causal mechanisms does not provide added value when simultaneously learning embryology and histology. Further research is warranted to identify the effect of temporal integration on medical students’ learning of distinct disciplines within the anatomical sciences.

Support or Funding Information

OUWB Fellowship in Medical Education
Roles of C‐C motif chemokine ligand (CCL) 2 in aggressive phenotypic change of non‐neoplastic breast cells by tumor‐associated macrophages
Authors: Moon Aree , Lee Seungeun , Lee Eunhye , Ko EunYi , Ham Mina , Lee Hye Min , Kim Eun-Sook , Koh Minsoo , Lim Hyun Kyung , Jung Joohee , Park So Yeon
Published: April 2020

Tumor‐associated macrophages (TAMs) are major components of tumor microenvironment that promote invasion and metastasis of cancer cells. In this study, we investigated the effect of TAMs on phenotypic conversion of non‐neoplastic MCF10A human breast epithelial cells using an indirect co‐culture system. Co‐culture with TAMs induced epithelial‐to‐mesenchymal transition, invasive phenotype, and MMP‐9 upregulation in MCF10A cells. Comparative proteomic analysis revealed that endoplasmic reticulum oxidoreductase (ERO)1‐α was increased in MCF10A cells co‐cultured with TAMs compared to that in mono‐cultured cells. ERO1‐α was crucial for TAMs‐induced invasive phenotype and MMP‐9 upregulation involving transcription factors c‐fos and c‐Jun. Cytokine array analysis showed that levels of interleukin (IL)‐6, C‐X‐C motif ligand (CXCL)1, C‐C motif ligand (CCL)2, growth‐regulated oncogene (GRO), IL‐8, and granulocyte‐macrophage colony‐stimulating factor (GM‐CSF) were increased in conditioned media of co‐cultured cells. Among these cytokines increased in conditioned media of co‐cultured cells, CCL2 was secreted from TAMs, leading to induction of ERO1‐α, MMP‐9 upregulation, and invasiveness in MCF10A cells. Our findings elucidated a molecular mechanism underlying the aggressive phenotypic change of non‐neoplastic breast cells by co‐culture with TAMs, providing useful information for prevention or treatment of recurrent breast cancer.

Support or Funding Information

The present study was supported by the National Research Foundation of Korea (No. 2013R1A2A2A04013379, No.2015M3A9B6074045, and No. 2016R1A6A1A03007648).
An Organoid Approach to Target Her2/PD‐L1 Positive Gastric Cancers
Authors: Chakrabarti Jayati , Koh Vivien , Chan Shing Leng , Wang Jiang , Ahmed Syed , Ito Yoshiaki , So Jimmy Bok Yan , Yong Wei Peng , Zavros Yana
Published: April 2020

Background

Tumors can evade immune surveillance by expressing ligands such as programmed cell death 1 (PDL‐1) which interacts with PD‐1 or CTLA‐4, respectively, on CD8+ cytotoxic T lymphocytes (CTLs). These interactions inhibit CTL proliferation, survival, and effector function subsequently leading to immune evasion and cancer persistence. Despite that 40% of gastric cancers express PD‐L1, the response rate to immunotherapy is only 30%. Human epidermal growth factor receptor2 (HER2) is one of the critical regulators of several metastatic cancers, including gastric. Half of PD‐L1‐positive gastric cancers co‐express HER2, but whether HER2‐induced PD‐L1 sustains tumor cell immune evasion has not been determined.

Hypothesis

The current research tests the hypothesis that targeted anti‐HER2 and immune checkpoint inhibition may provide synergistic effects in gastric cancer treatment.

Methods

Patient‐derived gastric cancer organoids, generated from resected tumor tissues or biopsies were co‐cultured with cytotoxic T lymphocytes (CTLs) and tumor antigenloaded dendritic cells and myeloid‐derived suppressor cells (MDSCs).

Results

While PD‐L1‐positive tumor organoids co‐expressed HER2, pharmacological inhibition and gene knockdown of HER2 resulted in decreased PD‐L1 expression in tumor organoids. Within the organoid/immune cell co‐culture, decreased PD‐L1 expression in patient‐derived gastric cancer organoids correlated with a significant increase in CTL proliferation and survival, leading to cancer cell death. Similar results were within the co‐culture were observed in response to Nivolumab treatment. MDSCs within the co‐culture counteracted the inhibitory effects of Her2 inhibition by Mubritinib and Nivolumab.

Conclusion

Thus, HER2‐induced PD‐L1 may contribute to tumor immune evasion. Organoid/immune cell co‐cultures may be used to effectively screen for targeted therapy in individual gastric cancer patients. Therapeutic anti‐HER2 antibody and, immune checkpoint inhibition, as well as depletion of MDSCs, may provide synergistic effects in gastric cancer treatment.

Support or Funding Information

• NIH NIDDK R01DK083402‐08 (Zavros)

NIH 1U19AI116491‐01, (PIs: Weiss and Wells, Zavros Project Leader 1
Dynamic Phosphorylation and Dephosphorylation of CAP1 (Cyclase‐Associated Protein 1) by Antagonistic Signaling through CDK5 and cAMP are Critical for the Protein Functions in Actin Filament Disassembly and Cell Adhesion
Authors: Xiao Yitong , Zhang Haitao , Ramsey Auburn , Karki Uddhab , Akana Lokesh , Xie Jennifer Y. , Xu Jianfeng , Kelly Thomas , Ono Shoichiro , Zhou Guolei
Published: April 2020

CAP1 (
Cyclase
Associated
Protein 1) is a conserved actin‐regulating protein that enhances actin filament dynamics and also regulates adhesion in mammalian cells. We previously found that phosphorylation at the Ser307/Ser309 tandem site controls its association with cofilin and actin, and is important for CAP1 to regulate the actin cytoskeleton. Here, we report that transient Ser307/Ser309 phosphorylation is required for both CAP1 functions in actin filament disassembly and cell adhesions. Both the phospho‐mimetic and non‐phosphorylatable CAP1 mutants, which resist transition between phosphorylated and dephosphorylated forms, had defects in rescuing the reduced rate of actin filament disassemblyin the CAP1‐knockdown HeLa cells. The phosphor mutants also had defects in alleviating the elevated FAK (
Focal
Adhesion
Kinase) activity and the enhanced focal adhesions in the knockdown cells. In dissecting further phosphor‐regulatory cell signals for CAP1, we found that CDK5 (
Cyclin‐
Dependent
Kinase 5) phosphorylates both Ser307 and Ser309 residues, whereas cAMP signaling induces dephosphorylation at the tandem site, through its effectors PKA (
Protein
Kinase
A) and Epac (
Exchange
proteins directly
activated by
cAMP). No evidence supports involvement of activated protein phosphatase in executing the dephosphorylation downstream of cAMP, whereas preventing CAP1 access to its kinase CDK5 appears to underlie CAP1 dephosphorylation induced by cAMP. Therefore, this study provides direct cellular evidence that transient phosphorylation is required for both CAP1 functions in actin filament turnover and adhesion, and the novel mechanistic insights substantially extend our knowledge on the cell signals that function in concert to regulate CAP1 by facilitating its transient phosphorylation.

Support or Funding Information

This project was supported by an Institutional Development Award(IDeA) from the National Institute of General Medical Sciencesat the National Institutes of Health [Grant number: P20GM103429].
Evaluation of Osteoblastic Gene Expression in Human Mesenchymal Stem Cells
Authors: MacDonald Amber F. , Bow Austin , Masi Thomas , Dhar Madhu
Published: April 2020

Background

Osteoblastic differentiation is a primary pathway to stimulate bone remodeling within regenerative medicine. Human mesenchymal stem cells (hMSCs) are an attractive model for bone regeneration due to their ability to differentiate into osteoblasts, thus promoting an osteogenic environment. With the development of bone biomaterials, it is important to establish a solid baseline of the genes involved in osteoblast differentiation. Once established, the genes regulating osteoblastic differentiation can be compared to any biomaterial product that may potentially speed up the process of bone regeneration. Therefore, the purpose of this study is to examine gene expression as hMSCs undergo osteoblastic differentiation.

Methods

hMSCs were extracted from human adipose tissue and cultured in DMEM‐F12 medium, 10% FBS. For experimental conditions, cells were cultured in either a growth medium (GM) or an osteogenic induced medium (OIM), supplemented with β‐glycerophosphate, ascorbic‐2‐phosphate, and dexamethasone. Cells were stained with Alizarin Red S staining to confirm calcium deposits of cells cultured in GM or OIM. hMSCs cultured in OIM were extracted for total RNA at days 3, 7, and 21, followed by cDNA synthesis for RT2 Profiler PCR Human Osteogenesis Array (Qiagen, Hilden, Germany). Gene expressions were analyzed using Qiagen Gene Globe software to determine the relative fold change.

Results

hMSCs appear to express alkaline phosphatase (ALPL) and bone gammacarboxyglutamate protein (BGLAP) early throughout OIM culture. In contrast, Sp7 expression increases over time, while Sox9 peaks at Day 7 in comparison to the control at Day 3.

Conclusion

Our data collectively show a change in gene expressions associated with osteoblastic differentiation such as ALPL, BGLAP, Sp7, and SOX9. Sp7, a master osteoblastic transcription factor, increases over time suggesting hMSCs favoring into osteoblastic transformation. Interestingly, Sox9 is another transcription regulator known to commit hMSCs into osteo‐chondrogenic progenitors, and our data show its expression to increase before diminishing near control levels. However, further studies are needed to confirm the genetic pattern of hMSCS undergoing osteoblastic differentiation as well as the upstream mechanisms controlling their activation. This information will be useful in the application of bone biomaterials as a therapeutic treatment for bone regeneration.
Reimagining dissection lab preparation ‐ the role of digital media in anatomy education
Authors: Ng Nicole , Javadian Farnaz , Tse Shirley , Jiang Andy , Doroudi Majid
Published: April 2020

Introduction

Learning materials provided to students prior to anatomy dissection labs have classically been in the form of written instructions and images from prosections. However, these materials can be difficult to interpret, especially for beginner students. With the advancement of technology, educational curricula are exploring the integration of digital platforms to supplement more traditional teaching methods.

Goal

To investigate the utility of a video‐based guide for approaching dissection.

Methods

A video dissection guide was created demonstrating the dissection of the superficial back. The video outlines the anatomy, technique, procedure, and includes review questions. The video was made available online through YouTube, along with a feedback survey. A secondary video dissection guide highlighting the dissection of the deep back was subsequently produced and made available in the same avenue.

Results

After two months, the superficial back dissection video has received more than 1,400 views. Feedback was received from 68 respondents; a majority of whom were female (63.2%), aged 20–24 (60.3%), and had a current education level of a postgraduate degree or professional degree (54.4%). The majority of respondents agree or strongly agree that the video presented the anatomy in a clear and organized fashion (95.6%), enhanced their learning of the anatomy (100%), familiarized them with dissection tools and how to use them (89.7%), familiarized them with methods and techniques for dissection (92.6%), was more effective than a dissection guide (97.1%), and was more effective for learning anatomy than a textbook (88.2%). A poll of 54 respondents found that most agree or strongly agree that the video is a valuable resource for review/test preparation (88.9%).

Preliminary survey results for the deep back dissection video indicates that, of 15 respondents, the majority agree or strongly agree that the video presented the anatomy in a clear and organized fashion (100%), enhanced their learning of the anatomy (93.3%), was more effective than a dissection guide (93.3%), and is a valuable resource for initial learning of dissection/anatomy (100%) and review/test preparation (86.7%).

Conclusion

In summary, digital media in the form of video‐based dissection guides may be a useful tool to incorporate into educational curricula for teaching gross anatomy of both superficial and deep structures. Future goals include the incorporation of clinically relevant information to dissected anatomical structures.
Leucine‐Rich Repeat Kinase 2 Promotes β‐Oxidation via the Regulation of CPT1a in Nonalcoholic Fatty Liver Disease
Authors: Lin Chiao-Wei , Peng Yu-Ju , Ding Shih-Torng
Published: April 2020

Nonalcoholic steatohepatitis (NASH) occurs in about 30% patients of the nonalcoholic fatty liver disease (NAFLD), and these patients have high risk to liver cirrhosis and liver cancer. However, the etiology of the NASH development in NAFLD patients has not been clearly elucidated. In the NAFLD mice induced by high‐fat diet, we identified that leucine‐rich repeat kinase 2 (LRRK2) was down‐regulated in the livers accompanied with inflammation. Moreover, the treatment of palmitic acid (PA), a fatty acid that induces inflammation, led to down‐regulation of LRRK2 in HepG2 cells. The in vivo and in vitro data suggested the down‐regulation of LRRK2 may be correlated to liver inflammation. In addition, in PA‐treated HepG2 cells, the overexpression of LRRK2 significantly decreased the intracellular level of non‐esterified fatty acids as a result of the up‐regulation of CPT1a, which acts as the rate limiting step of beta‐oxidation. Accumulated studies have demonstrated the increase of CPT1a can enhance the beta‐oxidation to inhibit the PA‐induced inflammation. Interestingly, the overexpression of LRRK2 suppressed the NF‐kB‐mediated pro‐inflammatory pathway and inhibited the secretion of the pro‐inflammatory cytokine TNFa induced by PA treatments. Therefore, our results suggested the anti‐inflammatory effect of LRRK2 may also work through the promotion of beta‐oxidation in the liver. In this study, we firstly observed that LRRK2 was down‐regulated in the livers of NAFLD mice. Furthermore, the overexpression of LRRK2 enhanced the beta‐oxidation and inhibited the inflammation in HepG2 cells. Conversely, the decrease of LRRK2 resulted in the inflammation by suppressing beta‐oxidation in the livers. Therefore, LRRK2 may be a therapy target to improve NAFLD in human.
Increased activation of HDAC1, 2, and 6, and Sp1 underlies therapeutic resistance and tumor growth in glioblastoma
Authors: Chuang Jian-Ying , Yang Wen-Bin , Hsu Che-Chia
Published: April 2020

Background

Glioblastoma is associated with poor prognosis and high mortality. Although the use of first‐line temozolomide can reduce tumor growth, therapy‐induced stress drives stem cells out of quiescence, leading to chemo‐resistance and glioblastoma recurrence. The Sp1 transcription factor is known to protect glioblastoma cells against temozolomide; however, how tumor cells hijack this factor to gain resistance to therapy is not known.

Methods

Sp1 acetylation in temozolomide‐resistant cells and stem‐like tumorspheres was analyzed by immunoprecipitation and immunoblotting experiments. Effects of the HDAC/Sp1 axis on malignant growth were examined using cell proliferation–related assays and in vivo experiments. Furthermore, integrative analysis of gene expression with ChIP‐seq as well as the recurrent glioblastoma omics data were also used to further determine the target genes of the HDAC/Sp1 axis.

Results

We identified Sp1 as a novel substrate of HDAC6, and observed that the HDAC1, 2, 6/Sp1 pathway promotes self‐renewal of malignancy by upregulating BMI1 and hTERT, as well as by regulating G2/M progression and DNA repair via alteration of the transcription of various genes. Importantly, HDAC1, 2, 6/Sp1 activation is associated with poor clinical outcome in both glioblastoma and low‐grade gliomas. However, treatment with azaindolylsulfonamide, a potent HDAC6 inhibitor with partial efficacy against HDAC1 and HDAC2, induced G2/M arrest and senescence in both temozolomide‐resistant cells and stem‐like tumorspheres.

Conclusions

Our study uncovers a previously unknown regulatory mechanism in which the HDAC6‐Sp1 axis induces cell division and maintains the stem cell population to fuel tumor growth and therapeutic resistance.

Support or Funding Information

This work was financially supported by research grants from the Ministry of Science and Technology in Taiwan
Pilates Exercise in Hypoxia Increases Vascular Endothelial Function in Professional Pilates Players
Authors: Jung Kyounghwa , Seo Jongbeom , Jung Won-Sang , Park Hun-Young , Lim Kiwon
Published: April 2020

Purpose

The purpose of this study was to investigate the pilates exercise in hypoxia increases vascular endothelial function in professional Pilates players.

Methods

Ten healthy Pilates expert female (age: 26.9 ± 2.7, weight: 50.8 ± 5.2, BMI: 19.3 ± 1.4) completed two different trials on different days, consisting of exercise in normoxia (N trial) and exercise in 3,000 m simulated altitude (526 mmHg) hypobaric hypoxia (H trial). After 30 minutes of exposure in each environment, they performed tubing Pilates exercise for 50 minutes under normoxia and hypoxia. Pilates exercise using tubing were composed of 25 types, each performed for 2 minutes. Pilates motion is configured as follows : roll up & down, biceps, arm circles, teaser, rolling, spine twist, mermaid, cobra, swimming, double kicks, swan, cat, thigh stretching, hug a tree, squat, row, saw, hip pull, hundred, lats pull, leg arc, scissor, helicopter, right side leg pull, left side leg pull. Metabolic parameters (minute ventilation; VE, oxygen uptake; VO2, carbon dioxide excretion; VCO2, respiratory exchange ratio; RER, carbohydrate oxidation; CO, fatty acid oxidation; FAO, energy expenditure; EE) and cardiac function (heart rate; HR, stroke volume; SV, cardiac output; CO, end‐diastolic volume; EDV, end‐systolic volume; ESV, ejection fraction; EF) were measured every minutes during Pilates exercise. The sum of the values measured for 50 minutes was used as the result (exceptionally, RER uses average values). Blood pressure (BP), branchial ankle pulse wave velocity (baPWV) and flow mediated dilation (FMD) in branchial artery were measured as vascular function before and after exercise.

Results

In metabolic parameters, H trial showed significantly higher VE, VCO2, RER and CO than N trial. However, FAO was lower in H trial than in N trial. VO2 and EE. VO2 and EE presented no significant difference between the two trials. In cardiac function, HR showed a significant increase in H trial compared with N trial, however, the remaining cardiac function parameters did not show significant difference. In vascular function, BP (systolic blood pressure, diastolic blood pressure, pulse pressure and mean arterial pressure) and baPWV did not show significant interaction effects between exercise and environment. However, FMD presented a significant interaction effects between exercise and environment. As a result of the post‐hoc, both trial showed a significant increase in FMD via exercise and the increase rate in FMD was significantly higher in H trial (48.5 ± 22.9) than in N trial (34.9 ± 18.3). Also, H trial showed a significantly higher FMD before exercise than N trial.

Conclusion

Our results suggest that Pilates exercise under moderate hypoxia shows greater metabolic and cardiac response by relatively higher exercise intensity, and it also elicit synergy effect on vascular endothelial function than under normoxia in professional Pilates players.
Identification of early candidate urine biomarkers for measure Escitalopram treatment response from major depressive disorder
Authors: Huan Yuhang , Wei Jing , Zhou Jingjing , Liu Min , Yang Jian , Gao Youhe
Published: April 2020

Major depressive disorder (MDD) is a common mental disorder that can cause substantial impairments in quality of life. Clinical treatment is usually built on trial and error method, which lasts about 12 weeks to evaluate whether the treatment is efficient or not, leading some inefficient treatment measures. We therefore identify early candidate urine biomarkers to predict efficient treatment response in MDD patients. In this study, urine samples were collected from 10 respondent and 10 non‐respondent to escitalopram MDD patients with 0‐, 2‐, and 12‐week treatment. Differential urinary proteins were then analyzed by liquid chromatography coupled with tandem mass spectrometry (LC‐MS/MS). On week 2, there were 14 differential proteins identified in response group when compared with week 0 and these proteins can’t be enriched by random allocation analysis, 10 among which have been associated with the mechanisms of MDD. While in non‐response group, there were 35 differential proteins identified on week 2, and 18 among which have been associated with the mechanisms of MDD. In addition, differential urinary proteins on week 2 between respondent and non‐response group can be distinguished clearly by using Orthogonal Partial Least Squares Discrimination Analysis (OPLS‐DA). Our small pilot results indicated that the urine proteome can reflect early changes between respondent and non‐respondent group to escitalopram therapy since on week 2, which will provide potential early candidate urine biomarkers to predict efficient treatment measures in MDD patients.

Support or Funding Information

This research was supported by the National Key Research and Development Program of China (2018YFC0910202, 2016 YFC 1306300), the Key Basic Research Program of the Ministry of Science and Technology of China (2013FY114100), the Beijing Natural Science Foundation (7173264, 7172076), the Beijing cooperative construction project (110651103), the Beijing Normal University (11100704), the Peking Union Medical College Hospital (2016–2.27), and Beijing Biobank of Clinical Resources‐‐Mental Disorders, BBCR‐MD.
Deletion of Elavl1 in renal collecting duct cells is associated with a specific gene expression pattern resulting in major functional changes
Authors: Bornkessel Nicola Marcia , Chernyakov Dmitry , Groß Alexander , Gallwitz Lisa , Becker Franziska , Fischer Annika , Edemir Bayram
Published: April 2020

Introduction

The action of vasopressin and aldosterone on water and sodium reabsorption in the renal collecting duct includes the action of the RNA binding protein human antigen R also known as Embryonic Lethal Abnormal Vision Like 1 (Elavl1). Elavl1 modifies the protein expression of the epithelial sodium channel EnaC by stabilizing the mRNA. Under hypertonic conditions it down regulates the expression of the mineralocorticoid receptor (MR), the endogenous target of aldosterone. So far, the role of Elavl1 on the expression of other factors has not been studied, nor whether hypertonicity has an effect on Elavl1 mediated gene expression. Within this study we identified hundreds of genes that show differences in expression upon Elavl1 knock out.

Methods

We used the CRISPR‐Cas9 technology to delete Elavl1. First we cloned specific guideRNAs targeting Elavl1 and scrambled gRNA into the lentiV2‐vector and used them to produce virus particles in HEK293T‐cells. With those particles we transduced a mouse cortical collecting duct cell line (mpkccd). The successful induction of mutation in the Elavl1 locus was verified by Sanger‐Sequencing. Single cell clones were selected and the deletion on protein level was confirmed by immunofluorescence analysis. The Elavl1‐deficient cells were used for functional analysis like cell proliferation, migration and colony forming capacity. Using next generation sequencing we analyzed the gene expression profile comparing Elavl1 deficient to control cells and identified signaling pathways, which were deregulated by deletion of Elavl1.

Results

The successful deletion of Elavl1 was functionally associated with significantly slower proliferation, migration and lower colony forming capacity. As expected, Elavl1 deficiencies lead to down regulation of the MR gene. Beside MR the expression of more than 440 genes is significantly (p<0,001) up and more than 2700 genes are down regulated. Classification by gene ontology terms showed that gene members of the ontology categories like “enzyme binding”, „rna binding”,„cell stress” and „cell death” are significantly enriched. KEGG pathway analysis showed that the differentially expressed genes are involved in metabolic and cancer associated pathways, such as „PI3K‐AKT‐Signaling pathway” or „HIF1A Signaling pathway”.

Discussion

Within this study we showed a comprehensive analysis of Elval1 function in renal collecting duct cells. Beside mayor physiological differences, Elavl1 deficiency is associated with massive changes in gene expression. Further analyses are needed to investigate its role for in vivo renal function.
Role of Microglia in Ventilatory and Blood Pressure Responses to Acute Hypoxia
Authors: Okada Yasumasa , Yoshizawa Masashi , Fukushi Isato , Takeda Kotaro , Kono Yosuke , Hasebe Yohei , Koizumi Keiichi , Toda Takako
Published: April 2020

Microglia induces neuroinflammation in response to intermittent and chronic hypoxia, and enhances ventilatory augmentation and blood pressure elevation during hypoxic exposure. However, the role of microglia in ventilatory and blood pressure responses to acute hypoxia, especially to brief period of hypoxic exposure, has not been well investigated. In the present study we aimed to clarify the role of microglia in ventilatory and blood pressure responses to short duration acute hypoxia. To investigate this issue we conducted experiments using adult rats. A blood pressure telemeter was implanted intraperitoneally with a pressure transducer tip in the abdominal aorta. After at least one week recovery period, ventilatory and blood pressure responses to hypoxia were measured in an unanesthetized freely moving condition by whole body plethysmography and blood pressure telemetry, respectively. Measurement was first conducted without pretreatment with minocycline which is an inhibitor of microglial activation. Then, in more than one week after this control measurement, the animal was pretreated with minocycline for three consecutive days, and ventilatory and blood pressure responses to hypoxia were measured under the suppression of microglial activation. To test hypoxic responses, mild (13% O2, 4 min) and severe (7% O2, 5 min) hypoxia was loaded with a sufficient interval. In results we did not find differences ether in ventilatory or blood pressure response to either level of hypoxia between the conditions before and after pretreatment with minocycline. These results indicate that microglial activation does not affect ventilatory or blood pressure response to short duration acute hypoxia, which is not in agreement with previous reports, and this disagreement may be attributed to the different period of hypoxic loading between the previous reports and our present study, which could you too short to induce neuroinflammatory action by activated microglia.

Support or Funding Information

This work was supported by JSPS KAKENHI Grant Numbers 17K08559, 17H05540, 18K17783, 19K17386 and 19K17620 and a Grant‐in‐aid of the Cardiovascular Research Fund, Tokyo, Japan.
Arterial Baroreflex Function in Patients with Parkinson’s Disease: a Pharmacological Approach
Authors: Sabino-Carvalho Jeann L. , Cartafina Roberta A. , Guimarães Gabriel M. N. , Brandão Pedro R. P. , Vianna Lauro C.
Published: April 2020

Parkinson’s disease (PD) is a neurodegenerative disorder that is known for its motor dysfunction. Moreover, PD also causes non‐motor impairments, and approximately 40% of the patients experience coexistent abnormalities, like orthostatic hypotension (OH). The OH induces incapacitating symptoms that can contribute to an increased risk of falls, which have a great impact on patients’ quality of life. The arterial baroreflex system plays a pivotal role in counteracting transient falls in blood pressure during the execution of activities of daily living. Although a plethora of studies have indicated that an impaired baroreflex function is a clinical feature in patients with PD, none of the human studies have used the modified oxford method, which is considered the gold standard approach for assessing arterial baroreflex sensitivity (BRS). Additionally, since the OH and arterial BRS have an age‐dependent effect, studies with younger patients with PD is lacking. In this sense, the aim of the present study was to test the arterial baroreflex function in patients with PD with and without OH and aged‐matched control during rapid changes in blood pressure induced by sequential boluses of vasoactive drugs (Modified‐Oxford Technique). Beat‐to‐beat HR (electrocardiography) and BP (finger photoplethysmography) were obtained during 10‐min of supine rest preceding a modified‐Oxford [bolus injection of 65–100μg sodium nitroprusside (NTP) followed by 65–100μg phenylephrine hydrochloride (PHE) 1 min afterwards] in five patients with PD (50±6yr; Hohen and Yhar: <3a.u.; diagnose time: 8.8±3.7yr) and seven age‐matched controls (48±7yr). Arterial BRS was analyzed using the slopes of the linear portions of the R‐R interval‐systolic BP relationship. Patients with PD had lower BP at rest compared to controls (82±7 vs. 94±5mmHg, P=0.01) and a trend for higher HR (72±13 vs. 59±8bpm, P=0.06). The average of falling (ΔSBP: −37±0.65 vs. −31±0.43, P=0.26) and rising systolic BP (ΔSBP: −27±0.76 vs. −20±0.58, P=0.38) was similar between patients with PD and controls. Our preliminary results showed that arterial BRS was lower in patients with PD compared to controls for falling pressure (NTP trial: 4.52±0.34 vs. 9.81±0.23ms/mmHg, P=0.01) and for rising pressure (PHE trial: 5.08±0.27 vs. 13.60±0.55ms/mmHg, P=0.03). Of note, only one of the patients presented OH, and the baroreflex function was characterized by a marked blunted arterial BRS for failing pressure (NTP trial: 1.83ms/mmHg) and an averaged BRS for rising in pressure. Our preliminary results indicated that the ups and downs of baroreflex function are impaired in patients with PD, and the downs appear to be further impaired in patients with OH. These findings provide a better understanding of the neural control of blood pressure in patients with PD, and future studies should focus on OH.

Support or Funding Information

CNPq, FAPDF and CAPES.
Early Life Stress Induces Anxiety‐like Hyperactivity but Not Altered Ethanol Drinking in Outbred Heterogeneous Stock Rats
Authors: Deal Aaron , Cooper Nicholas , Weiner Jeffrey , Woods Leah Solberg
Published: April 2020

Early life stress (ELS) during adolescence is known to have lasting effects impacting vulnerability to psychopathological disorders in adulthood, including post‐traumatic stress disorder (PTSD) and alcohol use disorder (AUD). The mechanisms by which ELS affects an individual’s susceptibility to these psychopathological outcomes are not fully understood. There is evidence suggesting that genetics may play a role in susceptibility and resistance to ELS‐induced disorders, though few specific genes have been identified. Therefore, we investigated the outbred Heterogeneous Stock (HS) rat as a model to study the genetics contributing to ELS‐induced PTSD and AUD‐like behavior. The HS population has high genetic diversity and more closely resembles the range of phenotypes expected within a human population than inbred rodent strains. We hypothesized that adolescent social isolation (ASI) of HS rats would increase anxiety‐like behavior, induce hyperactivity in response to a novel environment, and increase ethanol self‐administration.

In this study, we isolated (n=32) or group‐housed (n=32) male HS rats at 4 weeks of age. After 5 weeks of isolation (ISO) or group‐housing (GH), behavioral outcomes were measured via elevated plus maze (EPM), open field test (OFT), and forced swim test (FST). Following behavioral testing, all rats were individually housed and assessed for voluntary ethanol self‐administration.

We found no differences between ISO and GH rats in EPM anxiety‐like behavior as measured via open arm time or closed arm time. However, we did find ISO increased hyperactivity in response to a novel environment compared to GH in the OFT, including increased movement time and distance, increased beam breaks, and increased rearing. Interestingly, we found that ISO rats demonstrated decreased despair‐like behavior in the FST, including decreased immobility and increased wall climbing. When presented with 20% ethanol, we found no difference in self‐administration between ISO and GH animals. At increasing concentrations of ethanol, GH rats appeared to consume more ethanol than ISO, though results did not reach significance. We observed high variability in individual propensity to self‐administer ethanol in both ISO and GH groups which may be playing a larger role than ELS in this population.

We have shown that HS rats acquire hyperactivity in response to a novel environment following ELS but are resistant to ELS‐induced despair‐like behavior and alcohol consumption. There is literature to support ELS as a protective event in certain environmental situations. These results support the use of the HS rat as a model to investigate the genetics involved in resistance to ELS‐induced AUD.

Support or Funding Information

Funding for this study was provided by the Wake Forest Center for Research on Substance Use and Addiction Pilot Grant with support from the Wake Forest Alcohol Center NIH/NIAAA award P50 AA26117.
A Novel Hybrid Silk Fibroin/Polyurethane Arteriovenous Graft for Hemodialysis: Proof‐of‐principle Animal Study in an Ovine Model
Authors: Acocella Fabio , Tozzi Matteo , Bagardi Mara , Ravasio Giuliano , Cigalino Giorgio , Crippa Luca , Piccolo Solange , Nahal Emma Amal , Spandri Mattia , Catto Valentina Ilaria Maria , Tironi Matteo , Remuzzi Andrea , Greco Francesco Giovanni , Riboldi Stefania Adele
Published: April 2020

BACKGROUND

In an attempt at answering the need for a valuable and durable hemodialysis vascular access, a novel semi‐degradable hybrid vascular graft, manufactured by electrospinning using silk fibroin and polyurethane (Silkothane®), has been developed and characterized in vitro according to standards.

OBJECTIVES

In this proof‐of‐principle animal study, we aimed at evaluating the performances of Silkothane® grafts in a sheep model of arteriovenous shunt, with respect to patency and short‐term graft remodeling.

MATERIALS AND METHODS

Nine Silkothane® grafts (6mm inner diameter, ≈7cm long, ≈400μm thick) were surgically placed into the left cervical fossa of nine female sheep, between the common carotid artery and the external jugular vein. During the housing period the animals were treated with an inhibitor of platelets aggregation. Grafts were examined by palpation three times per week, and by duplex ultrasound every two weeks to exclude significant stenosis, dilation, presence of thromboses, neointimal proliferation or other complicating features. The sheep were euthanized at 30, 60 and 90 days (N=3 for each group). At sacrifice, grafts were harvested, fixed, and submitted for histologic, immunohistochemical and Scanning Electron Microscopy examination.

RESULTS

No cases of graft‐related complications (e.g. seroma formation, transgraft bleeding, aneurismal dilation, calcification, structural yielding) were recorded in this study. One graft failed due to a thrombotic event likely related to a technical error (11%). One sheep had post‐operative wound infection, surgically resolved (11%). Four out of nine sheep (44%) showed post‐operative edema, usually disappearing in 7 days. Eight of nine sheep (89%) showed 100% primary unassisted patency at the respective time of sacrifice, as confirmed by palpation and ultrasound evaluation (average flow rate 1.76 ± 0.61 l/min). Histological, immunoistochemical and SEM analysis evidenced signs of inflammation and formation of neointimal hyperplasia at the venous anastomosis, as it typically happens with arteriovenous fistulae and synthetic grafts.

CONCLUSIONS

In our ovine model of arteriovenous shunt, Silkothane® grafts provided evidences of safety and efficacy in granting 100% patency up to 90 days. Further studied need to be conducted in order to directly compare the novel grafts with commercially available synthetic grafts.
Sex differences in cholinergic regulation of behavioral responses to rewarding and aversive stimuli
Authors: Tat Jennifer , Brady Lillian J. , Zachry Jennifer E. , Kutlu Munir Gunes , Calipari Erin S.
Published: April 2020

Substance use disorder (SUD) is a major public health concern, characterized by dysregulation in drug taking, drug seeking, and compulsive behavior, as well as deficits in the valuation of drugs and alternative reinforcers. Determining neurobiological factors that contribute to individual variability of SUD is critical for identification of at‐risk populations as well as the use of preventative measures and personalized medicine in treatment. Women represent a particularly vulnerable population when it comes to the development and trajectory of SUD, therefore understanding the sex‐specific mechanisms underlying the differential control of reward and motivation will be critical for improving treatment outcomes in women. At the core of this SUD vulnerability is an enhancement in reward processing and motivation for both natural and drug rewards. Nicotinic acetylcholine receptors (nAChR) have been directly linked to the control of reward‐related behavior for both natural and drug reinforcers, thus, here we aimed to understand how these receptor populations differentially control motivated behavior for positive and negative stimuli in a sex‐specific fashion. Mice were trained to acquire positive (response = reward delivery) and negative (response = shock removal) reinforcement in the presence of discrete antecedent auditory cues. We found sex differences in performance at baseline, with females showing reduced rates of acquisition for negative reinforcement and increase motivation for positive reinforcers. Next, the non‐selective nAChR antagonist, mecamylamine, was administered systemically to assess the effect of nAChRs on task performance. Blocking nAChRs reduced discrimination for auditory cues in both sexes regardless of what task type they were predicting, demonstrating that its effects on reinforcement are not stimulus specific. Further, while we saw sex‐differences in reinforcement at baseline, we observed no sex differences following mecamylamine treatment. Together this data shows that nAChR modulation of reward‐related behavior eliminates basal sex differences in reinforcement behavior and suggests that the cholinergic system may be a crucial factor in sex differences in motivation.

Support or Funding Information

Funding was provided by startup funds from Vanderbilt University School of Medicine Department of Pharmacology and the National Institutes of Health (NIH) (to E.S.C.) and the Vanderbilt University Academic Pathways Postdoctoral Research Fellowship (to L.J.B.). Funds from the National Institute of Drug Abuse (NIDA) DA042111 and DA048931(E.S.C), the Brain and Behavior Research Foundation (to E.S.C), Whitehall Foundation (to E.S.C), and the Edward J Mallinckrodt Jr. Foundation (to E.S.C) also supported this work.
Reactivation of eNOS in the endothelium rescues eNOS KO mice from endothelial dysfunction and hypertension
Authors: Leo Francesca , Piragine Eugenia , Vornholz Lukas , Suvorava Tatsiana , Isakson Brant , Cortese-Krott Miriam Margherita
Published: April 2020

Production of nitric oxide (NO) by endothelial NO synthase (eNOS; NOS3) plays a key role in regulation of vascular tone and blood pressure. The aim of this study was to establish “loss” and “gain” of function models for eNOS by using tissue specific Cre‐induced gene inactivation or reactivation. To accomplish this, two independent genotypes were created: 1) a conventional floxed eNOS (eNOSflox/flox) against exon 2 or 2) a Cre‐inducible eNOS knock‐in (KI) gene construct (eNOSinv/inv) allowing targeted removal or reactivation of eNOS expression. Both the eNOSflox/flox and eNOSinv/inv were crossed with endothelial cell (EC)‐specific tamoxifen (TAM)‐inducible Cre mice to create EC‐specific knock‐out (EC eNOS KO) and EC eNOS KI mice. We found that Cre‐specific DNA recombination occurred specifically in endothelium of both EC eNOS KO and EC eNOS KI, as assessed by quantitative polymerase chain reaction (qPCR). TAM treatment fully abolished mRNA and protein expression of eNOS in the aorta of EC eNOS KO (eNOSflox/flox Crepos) mice, but not in Cre negative or untreated mice, as determined by retro transcription qPCR and western blot analysis. Functionally, the EC eNOS KO demonstrated inhibition of Ach‐induced vasodilation of aortic rings ex vivo, as well as flow‐mediated dilation (FMD) of the arteria iliaca in vivo (assessed by ultrasound). Moreover, absence of eNOS in the endothelium of EC eNOS KO was correlated with spontaneous hypertension, assessed by Millar catheter (MAP was 80.37 ± 2.135 mmHg in EC eNOS KO against 67.88 ± 2.723 mmHg in control mice). Next, we tested blood pressure of eNOSinv/inv which fully lacked eNOS expression in all tissues and was hypertensive. In EC eNOS KI (eNOSinv/inv Crepos treated with TAM) the expression of eNOS was completely restored in endothelium, leading to a significant decrease of the blood pressure as compared to eNOSinv/inv Creneg mice. In conclusion, we report the first inducible “loss” and “gain” of function models of eNOS in the vascular endothelium demonstrating that reactivation of eNOS rescues from endothelial dysfunction and hypertension. These mice models can further be applied to non endothelial tissues to further investigate the role of eNOS in different tissues in modulating blood pressure and systemic hemodynamics.

Support or Funding Information

This work was supported by the German Research Council (DFG CO 1305/2‐1 to M.M.C.‐K.; F.L. is a scholar of the IRTG1902 to M.M.C.‐K. and B.E.I. and by a research grant of the Forschungskommission to M.M.C.‐K., Medical Faculty, Heinrich Heine University of Düsseldorf.
Endothelial prolyl hydroxylase 2 is necessary for Angiotensin II‐mediated renal fibrosis and reactive oxygen species formation
Authors: Zeng Heng , Zhao Yongzhen , Chen Jian-Xiong
Published: April 2020

Angiotensin II (Ang‐II) is one of the major contributors to the progression of chronic kidney disease and renal fibrosis. The aim of present study is to investigate the role of endothelial oxygen sensor prolyl hydroxylase‐2 (PHD2) in the Ang‐II‐mediated renal fibrosis and reactive oxygen species (ROS) formation. Wild type (WT) mice and PHD2 endothelial cell‐specific knockout (PHD2ECKO) mice were infused with Ang‐II (1000ng/kg/min) for 28 days. Renal fibrosis, ROS formation, and iron contents were measured by Masson’s trichrome, dihydroethidium, and Prussian blue staining, respectively. In the WT mice, infusion with Ang‐II caused a significant increase in the fibrosis area of kidney. Ang‐II infusion also significantly increased the ROS formation and iron contents. Furthermore, the levels of hypoxia‐inducible factor (HIF)‐1α, HIF‐2α, transforming growth factor‐β1 (TGF‐β1), p47phox and gp91phox, heme oxygenase‐1 (HO‐1), ferroportin, caspase‐3, p53 and the acetylation of p53 (Acetyl‐p53) in both renal cortex and medulla were significantly upregulated after infusion with Ang‐II in WT mice. Interestingly, knockout of PHD2 in endothelium led to a significant suppression of Ang‐II‐induced renal fibrosis, ROS formation and iron accumulation. The expression of HIF‐1α, HIF‐2α, TGF‐β1, p47phox, gp91phox, HO‐1, ferroportin, caspase‐3, p53 and Acetyl‐p53 was also significantly reduced in the PHD2ECKO mice infused with Ang‐II compared with the WT mice infused with Ang‐II. Our study for the first time demonstrates that the endothelial PHD2 is necessary for Ang‐II‐mediated renal fibrosis and injury. Specific knockout of endothelial PHD2 protects the kidney against Ang‐II‐induced fibrosis and ROS formation.

Support or Funding Information

This work was supported by the National Institutes of Health grant 2R01HL102042‐7 and University of Mississippi Medical Center Intramural Research Support Program to J.X. Chen.