bluuebunny/crossref_metadata_2025 huggingface dataset:
Beta‐Arrestin‐1 Alters Glucocorticoid Signaling and the Glucocorticoid Receptor Transcriptome
Authors: Petrillo Maria G. , Diaz-Jimenez David , Oakley Robert H. , Cidlowski John A.
Published: April 2020

The glucocorticoid receptor (GR) is a transcription factor essential for life which regulates gene expression following glucocorticoid (GC) binding. Ligand‐activated GR regulates transcription of genes involved in development, metabolism, and immune system function. Interestingly, GC‐mediated transcriptional profile is cell‐type specific, but the mechanism regulating selective gene transcription in a cell‐specific manner is still unclear. Selection of target genes, directionality of regulation, and magnitude of the response are largely thought to be dictated by the interplay between the GR and co‐regulatory elements as well as chromatin remodelers. We have discovered that β‐arrestin‐1, a known regulator of G‐protein‐coupled receptor signaling, is a protein binding partner of the GR. Upon glucocorticoid exposure, GR‐β‐arrestin‐1 complexes were detected in the nucleus of cells where GR is transcriptionally active. Moreover, depletion of β‐arrestin‐1 expression altered the GR‐mediated transcription profile by reducing gene responsiveness to glucocorticoids. This protein partnership raised the possibility that β‐arrestin‐1 may be a regulator of the GR transcriptional activity. To investigate if β‐arrestin‐1 contributes to the glucocorticoid‐dependent gene regulation, we performed RNA‐seq and Assay for Transposase‐Accessible Chromatin (ATAC)‐seq from control and β‐arrestin‐1 knockdown A549 cells treated with or without the synthetic glucocorticoid dexamethasone (Dex). RNA‐seq analysis identified three patterns of gene regulation: I) glucocorticoid‐responsive genes whose regulation depends on β‐arrestin‐1 expression (group 1; 720 genes), II) glucocorticoid‐responsive genes that are independent of β‐arrestin‐1 expression (group 2; 1886 genes), and III) genes whose transcription is blocked in control cells but become de‐novo glucocorticoid‐responsive genes upon β‐arrestin‐1 depletion (group 3; 986 genes). For group 1 genes, integration of RNA‐seq with ATAC‐seq analyses revealed that glucocorticoid responsive elements (GREs) near glucocorticoid‐induced genes became inaccessible when β‐arrestin‐1 expression was depleted. In contrast, evaluation of the chromatin state at the levels of the GRE regions of group 3 genes showed that β‐arrestin‐1 maintained the chromatin in a closed state, thus impeding GR from binding. Consistent with this finding, β‐arrestin‐1 depletion facilitated chromatin opening at those regions leading to GR‐dependent transcription. In conclusion, our study demonstrates that β‐arrestin‐1 is a novel coregulator of the GR that contributes to defining the cell‐type specificity of glucocorticoids by modulating the chromatin architecture.

Support or Funding Information

This work was supported by the Intramural Research Program of the NIEHS, National Institutes of Health.
Kv1.3 channel is a potential marker for B acute lymphoblastic leukemia
Authors: Valle Juan Salvador , Castellanos Ramon , Olivas Miguel Ángel , Pottosin Igor , Dobrovinskaya Oxana , Schnoor Michael
Published: April 2020

Acute lymphoblastic leukemia (ALL) is a public health problem worldwide and is the leading cause of death in children under 14 years. Current ALL treatment is efficient in 80% of cases, but 5% of patients may relapse, with a life expectancy less than 5 years. Thus, the search for new therapeutic targets and diagnostic biomarkers needs to be continued. In ALL, different mutations have been identified providing leukemic cells with high metabolic rate, survival due to evasion of apoptosis, proliferation, and drug resistance. Many of these events are directly or indirectly related to Ca2+ signaling that relies on plasma membrane Ca2+ channels. The magnitude and duration of Ca2+ signals are regulated by the function of K+ channels in the same membrane. In healthy lymphocytes, two K+ channels are expressed, the voltage‐sensitive Kv1.3 and the calcium‐activated KCa3.1 ones. Kv1.3 channel in healthy B and T cells regulate their membrane potential, cell volume and entry into the cell cycle, whereas KCa3.1 channel activity is associated with cytokine secretion, differentiation and proliferation. T‐ALL cell lines CCRF‐CEM and MOLT‐3 are reminiscent of activated T‐cells, with a high density of Kv1.3 and KCa3.1 currents. Much less is known about K+ channels expression in B‐ALL. Here, we analyzed the expression of Kv1.3 channel in B‐ALL cells lines, REH and RS4:11, and freshly isolated CD19+ cells from healthy donors, activated with 1 μg/mL of PMA and 500 nM of ionomicin. Kv1.3 mRNA levels were similar in all cell models as revealed by real time RT‐PCR. Western blot assays confirmed the presence of the Kv1.3 protein in all cell models. Immunofluorescence assays localized Kv1.3 channel in the plasma membrane of CD19+ as well as of REH and RS4:11 cells. Patch‐clamp recordings in whole cell configuration revealed typical voltage‐dependent Kv1.3 activity in CD19+ cells, which was totally abolished by 1 nM of margatoxin, a specific blocker of Kv1.3 channel. However, zero Kv current was detected in REH and RS4:11. Thus, albeit being present in the plasma membrane in B‐ALL cells, Kv1.3 channel protein does not mediate K+ fluxes. It may play additional non‐canonical roles, e.g. in signaling. Its loss of ionotropic function may be linked to the leukemic event and could serve as a reliable diagnostic tool, whereas conventional RT‐PCR method will be ineffective.

Support or Funding Information

Supported by CINVESTAV (MS) and CONACyT grants CB‐238689 (OD), CB‐220793 (IP), Fronteras in Ciencia 140 (OD).
Mutants in mitochondrial DNA repair genes in Arabidopsis thaliana generated by using CRISPR‐Cas9
Authors: Campbell David , Wright Janet , Christensen Alan
Published: April 2020

The genomes of plant mitochondria exhibit unique mutation rates and structural variability. Compared to their animal counterparts, plant mitochondria have low mutation rates, high recombination rates, and non‐circular genomes. Recent evidence suggests the prevalence of double‐strand break repair (DSBR) in plant mitochondria as one possible cause of these traits. Interestingly, all of the genes required for plant mitochondrial DSBR are located in the nucleus.

In order to study DSBR, it is necessary to be able to reliably generate single and double‐mutants for DSBR genes. Using traditional methods, such as T‐DNA insertions, double mutant generation has proven cumbersome due to the occasional difficulty in PCR‐based identification of single mutants, as well as the low map distance between several genes of interest. CRISPR‐induced knockouts allow us to overcome some of these difficulties and generate a greater variety of mutants in a shorter period of time. In this study, we selected five proteins related to the processing of double strand breaks (DSBs) in plant mitochondria for mutagenesis: MSH1, RECA3, RNaseH1, SUV3, and OTP87. We prepared CRISPR Cas9 vectors to produce knockouts for each of these genes in Arabidopsis thaliana. The reliable production of mutants for these 5 genes will allow for in depth study of DSBR in the future. CRISPR Cas9 constructs were designed to produce two guide RNAs for each gene, targeting two different exons in the gene of interest, to give a better chance of successful knockouts, or deletions, between the two sites. These constructs were then transformed into Agrobacterium. Successful transformants were selected using a spectinomycin resistance gene in the construct. We then used a standard floral dip protocol to generate mutant Arabidopsis plants. PCR analysis confirmed Arabidopsis knockout mutations in all five lines.

The mutants generated in this study will allow us to investigate several avenues of research on DSBR. We plan to expose plants with knockouts in either one or two DSBR genes to ciprofloxacin, an antibiotic which induces double strand breaks in plant mitochondrial DNA. From there we can observe the mitochondrial genome recombination rates using PCR and Illumina sequencing for each of the mutants.

Support or Funding Information

This work was supported in part by grants from the National Science Foundation to A.C.C. (MCB‐1413152 and MCB‐1933590) and a grant from the UNL UCARE program (26‐0101‐8001‐001)
Homing Endonuclease and Protein Splicing Activity of Inteins from Thermophilic Archaea
Authors: Tarrant Seanan P. , Jankowski Bella J. , Comeau Hannah Y. , Wibben Amanda C. , Mills Kenneth V.
Published: April 2020

Protein splicing is a post translational, self‐catalyzed reaction by which an intein removes itself from flanking polypeptides and ligates those polypeptides together. We are interested in the splicing and homing endonuclease activities of the intein that interrupt the DNA polymerase II in the extreme thermophiles Thermococcus barophilus and Thermococcus kodakarensis. Given that T. kodakarensis is a surface‐dwelling organism whereas the T. barophilus is a piezophile that lives in deep‐sea thermal vents, yet both inteins are highly similar in sequence, we are interested in how temperature and pressure may affect enzyme activity. Additionally, we are interested in the role of the two conserved C‐terminal residues of the intein. We have found that the homing endonuclease domain of the T. kodakarensis intein is more active at a lower concentration and lower temperatures than that of the T. barophilus intein. Protein splicing of both inteins can be induced by in vitro incubation of an isolated, unspliced precursor at elevated temperature, which may also be influenced by hydrostatic pressure.

Support or Funding Information

This work was supported by NSF grant MCB‐1517138, a Henry Dreyfus Teacher‐Scholar Award, and by NIH Grant 1R15GM132817‐0
Exercise Training Restores K+ Channel Contributions to Hydrogen Peroxide‐Mediated Dilation Independent of PKG Dimerization in Collateral‐Dependent Coronary Arterioles
Authors: Johnson Kalen A. , Ramirez Cristina R. , Bray Jeffrey F. , Heaps Cristine L.
Published: April 2020

In coronary artery disease (CAD), the ability of the coronary circulation to deliver blood to meet oxygen demands of the heart is often impaired. Exercise training has been established to mitigate the negative effects of CAD by preserving reactivity of the coronary microcirculation. We have previously reported that exercise increases the contribution of hydrogen peroxide (H2O2) to enhanced endothelium‐dependent vasodilation in collateral‐dependent coronary arterioles. Others have shown that cGMP‐dependent protein kinase (PKG) can be activated via oxidation by H2O2, inducing dimer formation. The PKG dimer has been shown to act on substrates, such as K+ channels, to contribute to vascular smooth muscle relaxation and vasodilation. In the current study, we tested the hypothesis that exercise training‐enhanced H2O2‐induced vasodilation is mediated through enhanced PKG dimerization and subsequent activation of Kv and BKca channels in coronary arterioles isolated from ischemic myocardium. An ameroid occluder was surgically placed on the proximal left circumflex coronary artery of female Yucatan miniature swine to induce gradual occlusion while the left anterior descending artery was left unoccluded to serve as a control. Eight weeks postoperatively, pigs were assigned to sedentary (n=14) or exercise training (n=13; progressive treadmill training for 13 weeks) regimens. Coronary arterioles (75–150 μm) and small arteries (250–400 um) were isolated from myocardium of the left circumflex and left anterior descending regions. Immunoblot analysis indicated that H2O2 induced PKG dimer formation in both porcine coronary arterioles and small arteries. Chronic coronary artery occlusion appeared to diminish H2O2‐mediated PKG dimer formation in collateral‐dependent arterioles and the reduced dimerization persisted with exercise training. H2O2‐mediated vasodilation tended to be impaired in arterioles isolated from the collateral‐dependent regions of sedentary pigs and was partially restored with exercise training. It also appears that both Kv and BKCa channels contributed to H2O2‐mediated dilation after exercise training. Our data demonstrate that H2O2 stimulates PKG dimer formation in both porcine coronary arterioles and small arteries. Contrary to our hypothesis, H2O2‐induced PKG dimerization is not enhanced by exercise training in either nonoccluded or collateral‐dependent arterioles. However, both Kv and BKCa channels appear to contribute to exercise training‐enhanced H2O2‐mediated dilation in collateral‐dependent coronary arterioles.

Support or Funding Information

National Institutes of Health R01‐HL139903
Anti‐myeloma Effect of Xanthohumol on Multiple Myeloma Cells
Authors: Tucker Michael , Rayalam Srujana , Wang Xinyu
Published: April 2020

Multiple myeloma (MM) is characterized as a type of blood cell cancer, specifically affecting plasma cells within bone marrow. This disease results in approximately 13,000 deaths each year in the United States. The over‐accumulation of malignant plasma cells crowds out normal, healthy blood and immune cells, leaving the host potentially immunocompromised and prone to weakness, fatigue, weight loss and broken bones. Xanthohumol (XN), a prenylated flavonoid derived from the common hop plant Humulus lupulus, has been shown to have profound anti‐obesity, anti‐inflammatory and anti‐cancer effects. However, its effect on MM was rarely evaluated. The purpose of our study was to examine cytotoxic and apoptogenic effects of XN on human MM cells. Our results show that XN decreased MM cell viability in a time (24, 48, 72 hours) and dose (1, 5, 10, 20 and 30 μM) ‐dependent manner. Apoptogenic effect of XN was confirmed by flow cytometry analysis and western blotting. Our data demonstrated that, as the concentration of XN administered increased, the number of apoptotic MM cells increased. Protein expression of apoptosis‐related factors including caspase 3, caspase 8, caspase 9 and poly‐(ADP‐ribose) polymerase (PARP) was examined and showed that the amounts of cleaved caspase 3 and PARP1 were increased corresponding to an increase of XN. In order to determine whether the AMPK pathway is involved in XN‐induced apoptosis of MM cells, we will pre‐treat MM cells with either an activator (AICAR) or an inhibitor (Compound C) of AMPK followed by XN treatment in MM cells to elucidate the role of AMPK on anti‐myeloma effect of XN.
The ASBMB Accreditation Program as a Grass‐roots Educational Effort
Authors: Wolfson Adele J. , Bailey Cheryl , Carastro L. Michael , Dean Diane M. , Del Gaizo Moore Victoria , Dries Daniel R. , Kennelly Peter J. , Loertscher Jennifer , Martin Debra , Offerdahl Erika G. , Tansey John T. , Tyler Ludmilla , Zeidan Quira
Published: April 2020

ASBMB’s accreditation program was launched in 2013 in order to provide a structure for promoting instructional excellence and disseminate best practices. As of fall 2019, the number of accredited programs is 85 and includes those at all types of institutions in 37 states across the US. Accreditation allows institutions to be evaluated by peers, and the criteria include those aspects of programs that members judge to be crucial: inclusion of core concepts and sufficient infrastructure, but also professional development opportunities for all faculty, clear commitment to diversity and inclusion, active learning, and instruction in professional ethics. Criteria for curricula avoid a prescriptive topic‐ or course‐specific set of courses, in favor of foundational concepts in the discipline that also leave room for the continuing evolution of instructional practices. The required curriculum also includes experiential learning, communication skills, and the role of research, cooperative experiences, and internships for student development. We have evaluated the impact of accreditation on a variety of stakeholders – programs/departments (both accredited and non), students, graduate schools, and employers – with a recent survey. Programs reported that the process was worthwhile, and that it led to collection of data that would not otherwise be examined.
Drawing Our Feelings: An Approach to the First Day of Cadaver Dissection
Authors: Zumwalt Ann
Published: April 2020

RATIONALE

The cadaver dissection experience can elicit intense emotions in students who are taking anatomy for the first time. Students are often disinclined to share these feelings with others because they believe they are the only ones experiencing such feelings and/or do not want to appear less able than their student colleagues to cope. Discussing anticipatory feelings about the dissection process can reduce students’ feelings of isolation, imposter syndrome, and simple nervousness by revealing that most students share similar experiences and that there is a range of normal anticipatory feelings.

METHODS

For the last 10 years at Boston University School of Medicine we have employed the following exercise on the first day of anatomy: Students are asked to draw their answers to the question, “How do you feel about the experience of dissecting a human body?” After the drawing exercise the course director presents a slideshow of drawings from the same exercise by previous classes. The slideshow demonstrates that there are common themes exhibited by students in every class: mixed feelings, all alone/nervous, physiologic reactions, journey, knowledge, enthusiasm, and the humanity of their donor. The students are then invited to share comments or ask questions in a large group discussion format. Participation in this session is typically lively, likely because the drawing exercise and slideshow demonstrate to individual students that their responses are normal and expected.

RESULTS & CONCLUSIONS

The combination of exercises illustrates for students that there is a variety of normal reactions to the dissection experience. The exercise opens opportunities for dialogue that are otherwise less likely to occur. The overall result is a culture within the class that is open to acknowledging and discussing the complex feelings elicited by dissection.
DMSO inhibits human cancer cells and downregulates the expression of cdk2 and cyclin A
Authors: Villarroel Ariana , Duff Angela , Hu Tang
Published: April 2020

Dimethyl sulfoxide (DMSO) has many applications in basic research. For example, it has been broadly used as an efficient solvent for many chemicals with poor solubility in water and as a common cryoprotectant (5–10%) that prevents cellular damage during freeze‐thaw process. DMSO is also a prescription medicine that provides rapidly, temporary relief of pain in patients with headache, arthritis, and skeletal tissue injuries. However, little known is its effect on cell viability and proliferation. In this study, we found that DMSO significantly inhibited proliferation of human myeloid leukemia (TF‐1a and MV4‐11) and epithelia cancer (Hep‐G2) cells in culture, measured by XTT assays and microscope examinations. The initial inhibition was observed when DMSO was added to the cell culture at a concentration of 2%. The inhibition was substantial and appeared dose‐dependent, with a maximal inhibition of the cell growth being observed in the cells treated with 5–6% of DMSO. It is a little surprising that more than 6% DMSO (up to 12%) did not further inhibit the cell proliferation and cell viability, generating a hyperbolic dose‐response curve. DMSO at 1% or less had no significant inhibitory effect on the cells. Cytology examination showed that both MV4‐11 and TF‐1a cells have a relatively homogeneous population with smooth cytoplasmic border. These cells have a large nucleus to cytoplasm ratio. Addition of DMSO induced clear cellular damages: cytoplasm shrank or broken; the nuclei appeared as fragments or dots; and the cell body decreased in size. Western blot analysis showed that DMSO downregulated the expression of CDK2 and cyclin A in TF‐1a and MV4‐11 cells after 24 h incubation. The inhibition of CDK2 and cyclin A is also dosedependent. Thus, our data demonstrate that DMSO has a significant inhibitory effect on the growth and viability of the cancer cells tested. This inhibition is linked to cell cycle control and apoptotic molecules. The outcome from this study also suggests that as a solvent the final concentration of DMSO in cell culture should be no more than 1%. In summary, our study provides a supplementary information on both function and mechanism by which DMSO affects human cancer cells.

Support or Funding Information

Barry University
FoxO1 inhibition attenuate diabetes‐related vascular remodeling by reducing chronic inflammatory states
Authors: Xie Xiang , Xia Zhengyuan , Irwin Michael. G
Published: April 2020

Diabetic vascular complications result in poor outcomes, even after optimized interventions. Forkhead transcriptional factor(FoxO1) contributes to diabetic cardiomyopathy, yet the role of FoxO1 in diabetic vascular complications is poorly understood. We found that arterial wall hypertrophy occurred in the carotid artery of diabetic rats, which was associated with significant increases in wall thickness and cross‐sectional area as compared to non‐diabetic control group. The immunofluorescence results showed that α‐smooth muscle specific protein expression was downregulated in diabetic carotid media which was restored by treatment with the selective FoxO1 inhibitor AS1842856. The mRNA expressions of pro‐inflammatory biomarkers IL‐1 and TNF‐α in diabetic carotid arteries were increased as compared with control and were decreased following AS1842856 treatment. Also, the NLRP3 protein expression in diabetic carotid arteries was increased as compared with control group and reverted by AS1842856 treatment. It is concluded that our findings identify FoxO1 as a critical trigger for diabetes‐related adverse vascular remodeling and define the FoxO1 as a novel checkpoint for NLRP3 inflammasome‐dependent low‐grade chronic inflammatory states during diabetic adverse vascular remodeling progression, which may provide a promising therapeutic target to treat the diabetic cardiovascular disease.

Support or Funding Information

The work was supported by the NSFC grant (81770831) and in part by the General Research Fund (17118619M, 17123718M, Research Grants Council of Hong Kong).
Application of Kahoot! as a Teaching and Learning Tool in PharmD Curriculum
Authors: Ofori Edward , Abulaila Yazen , Al-Kurdi Diane , Jacob Sherin , Miserendino Monica , Faridi Hafeez
Published: April 2020

Millennials approach things differently compared to earlier generations, and this includes the manner in which they learn. The absence of approaches best suited for teaching this generation can hinder their learning abilities. This can be particularly challenging in professional programs such as doctor of pharmacy where students are not only expected to have mastery of content but also demonstrate high level of knowledge retention. In recent years, there have been significant technological advances and an educational approach that takes advantage of these will be well received by this young generation of students. In a bid to capitalize on such teaching and learning approaches to better connect with the students of today, there has been a proliferation in gamification tools for teaching. In this study we employed a game‐based student response system known as Kahoot! to assess PharmD students’ perception on gamification and its influence on learning outcomes. We introduced Kahoot! as a formative assessment tool in the classroom for pharmacy students in professional years one to three (P1 – P3) at Chicago State University. We administered a questionnaire to the students and their responses were collated and analyzed using Qualtrics. Results of the study show that over 84 % of the sampled PharmD students indicated that Kahoot! was fun and easy to use. Overall, the findings reveal that using Kahoot! enriches the quality of student learning in the classroom, with 73 % of respondents reporting that gamification gives them a better understanding of topics. The same percentage of students responded positively about employing the gamification tool to assess students’ understanding of topics. About 71 % of participants reported that the tool was able to highlight areas that required more attention. The study also revealed that 73% of respondents indicated that the use of games in the classroom kept them engaged and helped them score well on tests given at the end of the lecture. Of note, about 80% of students stated that the tool has given their confidence a boost. Taken together, gamification has improved the quality of teaching and learning beyond what is provided in conventional classrooms where normal PowerPoint slides and “chalk and talk” are used.
The Effects of Quercetin on Apoptosis and Cell Proliferation in A375 Melanoma Cells
Authors: Martinez P.J. , Byrd Shere Kuss
Published: April 2020

Quercetin, a bioflavonoid, is known to have various health benefits and has been used as a treatment for several forms of cancer. The aim of our research was to understand quercetin effects on cell growth and apoptosis pathways in a melanoma cell line. A dose/toxicity curve was done to determine cell viability in relation to a 24 hr treatment with quercetin concentrations ranging from 25–150μM. To test the effects of quercetin on cell proliferation, melanoma cells were grown to confluence then wounded while being treated for 24 hr with 50μM quercetin. DMSO was used as a vehicle control. Average initial wound distance was measured at three sites along the wound. Wound closure (cell proliferation) was re‐measured after 24 hr and the rate of closure was used as an index of cell proliferative ability. Results of the wound assay showed 40% less regrowth for the cells treated with quercetin, indicating inhibition of proliferative ability. To determine whether the inhibition of proliferation was due to an increase in quercetin‐induced apoptosis, we used Annexin V/PI and flow cytometry to quantify early and late apoptosis following treatment and found that the increase in apoptotic cells mirrored the loss in proliferative ability. These results show that quercetin should be considered as an adjunct treatment to prevent proliferation of melanoma.

Support or Funding Information

Colorado Alliance for Minority Participation
Terbutaline, Forskolin and cAMP Reduce Secretion of Aqueous Humor and Inhibit Intracellular Calcium Release in Bovine Cultured Ciliary Epithelium
Authors: Shahidullah Mohammad , Rafiq Kazi , Sikder Mahmudul Hasan , Ferdous Jannatul , Wilson William Stuart , Delamere Nicholas A.
Published: April 2020

In order to elucidate the involvement of cyclic AMP and intracellular Ca2+ concentration [Ca2+]i, in the modulation of aqueous humor (AH) secretion, we studied the effects of terbutaline, forskolin and 8‐Br‐cAMP on AH secretion in the isolated intact bovine eye and on ATP‐induced increase in [Ca2+]i in cultured ciliary epithelial (CE) cells. Drug effects on AH secretion were measured by fluorescein dilution. Drug effects on [Ca2+]i were studied by the Fura‐2 fluorescence ratiometric measurement. Terbutaline (100 nmol–100 μM), forskolin (30 nM–100 μM) or 8‐Br‐cAMP (100 nM – 10 μM), administered at fixed concentrations in the arterial perfusate of the intact eye, produced significant reductions in AH secretion. The AH reducing effect of terbutaline was blocked by a selective inhibitor of protein kinase A (KT‐5720). ATP (100 μM) caused a rapid, transient (peak) increase in [Ca2+]i followed by a small but sustained plateau phase lasting more than 5 minutes. Preincubation of the cells (6 min) with terbutaline, forskolin or 8‐Br‐cAMP significantly reduced the peak calcium response to ATP. Each of the drugs, on the other hand, augmented the sustained plateau phase of the calcium response. KT‐5720 partially reversed the inhibitory effect of terbutaline on the peak and totally inhibited its effect on the plateau phase. Taken together, our data support the notion that AH secretion in the bovine eye can be manipulated through cyclic AMP, operating through protein kinase A and that interaction of [Ca2+]i and cAMP may play a role in modulating AH secretion.

Support or Funding Information

Fight for Sight, London
Radiation‐induced Changes in Stimulant Pharmacokinetics Relevant to Long‐distance Space Travel
Authors: Hambuchen Michael D. , Mazur Anna , Findley David L. , McGill Mitchell R.
Published: April 2020

A round trip to Mars or a 10 year stay on the planet’s surface is estimated to be associated with 1 or 3 Gray (Gy) whole body radiation exposure, respectively. Radiation exposure is known to alter the pharmacokinetics of some medications. The objective of these experiments was to determine how radiation exposure may alter drug pharmacokinetics in male Wistar rats using methamphetamine (METH) as a probe. METH was chosen due to its well established pharmacokinetics in rats and the relevance of stimulant administration for alertness in space travel. Rats were exposed to 0, 1, or 3 Gy X‐ray radiation followed by measurement of clearance‐related serum biomarkers (e.g., alanine aminotransferase, serum creatinine, etc.), 1 mg/kg subcutaneous (SC) METH pharmacokinetics, and 1 mg/kg SC METH‐induced locomotor activity (which is correlated with brain METH concentrations). While radiation dose‐dependently reduced rat total body weight and spleen weight, there were no significant changes in serum biomarkers, METH pharmacokinetic parameters, or METH‐induced locomotor activity. In conclusion, acute Mars travel relevant radiation exposures did not alter METH disposition, but longer‐term studies of gradual radiation exposure on stimulant pharmacokinetics are needed.

Support or Funding Information

NASA West Virginia EPSCoR Research Seed Grant
Astrocytes play an active role in persistence of respiratory augmentation in the recovery phase after hypoxic exposure
Authors: Fukushi Isato , Kono Yosuke , Takeda Kotaro , Yokota Shigefumi , Onimaru Hiroshi , Pokorski Mieczyslaw , Okada Yasumasa
Published: April 2020

Hypoxia induces shortness of breath, and augments respiration, and the respiratory augmentation persists for a while even in the post‐hypoxic recovery phase. However, the mechanism of posthypoxic persistent respiratory augmentation has not been fully elucidated. We propose a hypothesis that astrocytes are involved in the central mechanism of post‐hypoxic persistent respiratory augmentation. To investigate this hypothesis, we conducted two kinds of experimental analyses. First, we examined astrocytic involvement in hypoxic ventilatory responses in the in vitro experiments. In this experiment, we used the medulla‐spinal cord preparation isolated from newborn rats. Briefly, the medulla and spinal cord were together isolated from neonatal rats under anesthesia. The preparations were fixed in a recording chamber and superfused firstly with oxygenated (95% O2, 5% CO2) artificial cerebrospinal fluid (aCSF), secondly with hypoxic (95% N2, 5% CO2) aCSF for 5 min, and thirdly again with oxygenated aCSF for 20 min. We compared respiratory frequencies in the post‐hypoxic recovery phase in preparations without and with administration of arundic acid (500 μM), a blocker of astrocytic activation. Second, we examined astrocytic involvement in hypoxic ventilatory responses in the in vivo experiments. In these experiments, unanesthetized adult mice were exposed to various levels of hypoxia, and ventilatory responses were analyzed by whole body plethysmography. Effects of arundic acid on ventilation were investigated. In results, in in vitro experiments, hypoxia increased respiratory frequency in both groups. During superfusion with oxygenated aCSF after hypoxic exposure, respiratory frequency in the group without arundic acid was still persistently increased. On the other hand, respiratory frequency in the group with arundic acid was gradually decreased in the post‐hypoxic phase, and it was eventually depressed and less than that of the pre‐hypoxic phase in the later post‐hypoxic phase. In in vivo experiments, moderately strong hypoxia increased ventilation, and ventilatory augmentation persisted even after cessation of hypoxia loading. Arundic acid blocked persistence of post‐hypoxic ventilatory augmentation. Stronger hypoxia induced biphasic responses, i.e., initial ventilatory augmentation followed by ventilatory depression. Arundic acid strengthened hypoxic ventilatory depression. We conclude that astrocytes play an important role in persistence of post‐hypoxic respiratory augmentation.

Support or Funding Information

JSPS KAKENHI numbers 17H05540, 17K08559 and 18K17783
Montelukast Potentiates the Antiinflammatory Effect of NSAIDs in the Rat Paw Formalin Model and Simultaneously Minimizes the Risk of Gastric Damage
Authors: Abdelhady Sherien A. , Ali Mennatallah A. , Al-Shafie Tamer A. , Abdelmawgoud Ebtsam M. , Yacout Dalia M. , El-Mas Mahmoud M.
Published: April 2020

Although the cysteinyl leukotriene receptor antagonist montelukast has been recognized for its antiinflammatory action, little information is available on whether this effect would be intensified if montelukast is co‐administered with nonsteroidal antiinflammatory drugs (NSAIDs). Here, we tested the hypothesis that simultaneous exposure to montelukast and NSAIDs elicits more efficacious control of histopathological and inflammatory signals in the rat paw formalin model. We also determined whether montelukast could improve gastric damage caused by NSAIDs and associated inflammatory insults. The individual treatment of rats with montelukast (20 mg/kg), diclofenac (nonselective COX1/COX2 inhibitor, 10 mg/kg), or celecoxib (selective COX2 inhibitor, 10 mg/kg) significantly reduced the formalin‐induced increases in (i) paw edema, (ii) necrotic debris and keratinized stratified squamous epithelium, (iii) paw protein expressions of iNOS and COX2, and (iv) serum IL‐6 and LTB4. These favorable histopathological and molecular effects were accentuated in rats treated with montelukast plus diclofenac or celecoxib. Alternatively, montelukast reduced the diclofenac‐evoked gastric damage (lesions and hyperemia) and rises in gastric TNFα levels, but it failed to alter the concomitant reductions in gastric COX1 expression and PGE2 content. Unlike diclofenac, normal gastric mucosal and COX1/PGE2 patterns were observed in celecoxib‐treated animals. Overall, the current data provide evidence that montelukast boosts the antiinflammatory action of NSAIDs while diminishing the potential of gastric damage and inflammation.
Valeric Acid, a Gut Microbiota Product, Lowers Arterial Blood Pressure in Rats.
Authors: Ufnal Marcin , Onyszkiewicz Maksymilian , Gawrys-Kopczynska Marta , Aleksandrowicz Marta , Sawicka Aneta , Kozniewska Ewa , Samborowska Emilia
Published: April 2020

Valeric acid (VA), a short‐chain fatty acid (SCFA), is produced by gut bacteria. The aim of the study was to evaluate if VA affect arterial blood pressure (BP) in rats. Furthermore, we investigated physiological concentrations of VA, and distribution of VA‐D9 to organs involved in the control of the circulatory system after the administration of VA‐D9 into the colon, a major site of microbiota metabolism. Hemodynamics were recorded in anesthetized, male, 14‐week‐old Wistar rats. A vehicle (0.9% NaCl), VA, or 3‐hydroxybutyrate, an antagonist of SCFA receptors GPR41/43 (ANT) were administered into the colon (IC) or intravenously (IV). Reactivity of mesenteric (MA) and gracilis muscle (GMA) arteries was tested in ex vivo conditions. SCFAs concentration was measured using ultra performance liquid chromatograph with mass spectrometer.

Physiological concentration of VA in the colon content was ≈ 650μM and ≈ 0.4μM in systemic blood. VA‐D9 was detected in the liver, the heart, the brain and kidneys 5 min after the administration into the colon. The vehicle did not affect BP and heart rate (HR). VA acid produced a dose‐dependent decrease in BP, and at higher doses decrease in HR. The hypotensive effect of VA was inhibited by the ANT. In vitro, VA dilated GMA and MA. In MA the dilatory effect was reduced by the ANT.

In conclusion, VA rapidly penetrates from the colon to systemic tissues exerting a hypotensive effect. The latter is mediated by vasodilation. The hypotensive effect of colon‐derived VA may have a therapeutic potential.

Support or Funding Information

This work was supported by the National Science Centre, Poland grant no. UMO‐2016/21/B/NZ5/02544.
Mechanisms of Phosphorylation‐Mediated Regulation of the RNAi Effector Protein Argonaute 2
Authors: Bibel Brianna , Elkayam Elad , Joshua-Tor Leemor
Published: April 2020

Argonaute (Ago) proteins play a central role in gene regulation through a process called RNA interference (RNAi). Ago binds to small RNAs (sRNAs) such as small interfering RNA (siRNA) and microRNA (miRNA) to form the functional core of the RNA Induced Silencing Complex (RISC), which targets mRNAs with complementary sequences, ultimately leading to down‐regulation of the corresponding genes. It was previously shown that phosphorylation of a cluster of residues (824–834) in the “eukaryotic insertion” (EI) of Ago by CK1α can alleviate this repression in a variety of cell types. However, the mechanisms of such regulation were unknown. Using in‐vitro phosphorylation assays, we show that binding of miRNA‐loaded Ago2 to target RNA primes the EI for phosphorylation. Furthermore, we show that this phosphorylation reduces the affinity of the Ago‐miRNA complex to its target mRNA. While the EI of Ago2 contains 5 potential phosphorylation sites, we propose that specific residues in the EI are more susceptible to phosphorylation and dictate the pattern and sequence of the phosphorylation events. We also examined the structural requirements for the target to induce phosphorylation. These findings shed light on the manner in which phosphorylation of the EI acts to regulate the RNAi pathway central to gene regulation. The high conservation of potential phosphosites in the EI (both among Ago homologs and throughout eukaryotes) suggests that such a regulatory strategy may be a shared mechanism with broader impact.

Support or Funding Information

This work was supported by the NSF Graduate Research Fellowship Program and Cold Spring Harbor Laboratory’s Watson School of Biological Sciences. L.J. is an Investigator of the Howard Hughes Medical Institute.
Antihypertensive effect of Launeae taraxacifolia on L‐Nitro Arginine Methyl Ester (L‐NAME) Induced Hypertension
Authors: Hassan Fasilat Oluwakemi , Oyagbemi Ademola Adetokunbo , Ogunpolu Blessing Seun , Falayi Olufunke Olubunmi , Omobowale Temidayo Olutayo , Ola-Davies Olufunke Eunice , Saba Adebowale Benard , Adedapo Adeolu Alex , Yakubu Momoh Audu
Published: April 2020

Launeae taraxacifolia popularly known as African Wild lettuce is rich in flavonoids, cardiac glycosides, saponins, β carotenes and also some valuable minerals. It has been reported as a local remedy for tooth pains, vomiting and diabetes. Hypertension was induced by non‐selective endothelial nitric oxide synthase (eNOS) inhibitor L‐Nitro Arginine Methyl Ester (L‐NAME). The antihypertensive effect of methanol leaf extract of LT was examined. Fifty (50) male Wistar rats were divided into 5 groups of 10 animals per group, Group A (Distilled water), Group B (L‐NAME 40 mg/kg), Group C (100 mg/kg extract, 40 mg/kg L‐NAME), Group D (200 mg/kg extract, 40 mg/kg L‐NAME), Group E (10 mg/kg of Lisinopril, 40 mg/kg L‐NAME). They were orally administered for five weeks. Blood pressure parameters, urinalysis and markers of oxidative stress were determined. Histology of the kidney was also carried. Immunohistochemistry of mineralocorticoid receptor (MCR), angiotensin converting enzyme (ACE) activity and kidney injury molecule 1 (Kim‐1) expressions were determined in the kidney. Findings from this study showed that blood pressure parameters, urinary sodium and markers of oxidative stress increased significantly while the in vivo antioxidant defence system decreased significantly in hypertensive rats. Histopathology revealed the presence of lesions in the kidney and the testes of hypertensive rats. Further, immunohistochemistry revealed significant increases in MCR, ACE, and Kim‐1 expressions in kidney of hypertensive rats, respectively. However, treatment with Launeae taraxacifolia normalized blood pressure parameters, urinary sodium, reduced oxidative stress, improved antioxidant defence system, serum nitric oxide bioavailability levels, and ultrastructure of the kidney. In conclusion, Launeae taraxacifolia as a vegetable could be used for the prevention of hypertension. This plant also holds promise in the nearest as novel drug candidate for treatment of hypertension in poor‐resource setting like Africa
Mechanisms of Myosteatosis in Obesity and the Effects of Muscle Hypertrophy
Authors: Corley Zachary L. , Padgett Caleb A. , Mintz James D. , Fulton David J. , Stepp David W.
Published: April 2020

Objective

To determine the mechanism of ectopic lipid deposition in skeletal muscle in obesity and if any impact is conferred by increasing muscle mass.

Methods

Skeletal muscles from db/db mice, a well characterized model of human obesity, were isolated and myosteatosis assessed for lipid content via NMR, chemical assay and transmission electron microscopy. Genes involved in lipogenesis, lipid storage and oxidation were assessed by qPCR. To determine the effects of hypermuscularity, db/db mice were crossed with mice lacking myostatin, a myokine that inhibits muscle growth, to generate lean, obese, and hypermuscular obese mice.

Results

Obese mice exhibited a 3‐fold increase in muscle triglyceride content, which appeared on electron microscopy as diffuse and heterogeneous lipid droplet accumulation in individual muscle fibers. Lipogenic pathways, assessed by expression of sterol regulatory element binding protein (SREBP) and its downstream targets stearyl CoA desaturase‐1 (SCD1), acetyl‐CoA carboxylase (ACC) and fatty acid synthase (FAS), were increased in obesity as was expression of perilipins. Muscle from obese mice was characterized by accumulation of mitochondria and increased expression of carnitine palmitoyltransferase I (CPT1). Deletion of myostatin produced a 40% increase in skeletal muscle mass and reversed the observed change in lipid content and gene expression.

Conclusion

Obesity increases both lipogenesis and lipid oxidation in the skeletal musculature of obese mice. The balance of these processes favors lipid accumulation as evidenced by droplet formation and enhanced expression of droplet‐related proteins. Deletion of myostatin produced muscle hypertrophy and clearance of ectopic lipids. Specific links between muscle hypertrophy and excess muscle lipids remain to be defined.

Support or Funding Information

Title: Circadian Origins of Vascular Disease in Obesity | Investigator(s): Stepp, David W. and Fulton, David J., Augusta University, Augusta, GA | Agency: National Institute of Health (NIH) | Institute: National Heart, Lung and Blood Institute (NHLBI) | Type: Research Project (RO1) | Project #: 1RO1HL147159‐01
The Effects of TNF‐α on TTX Resistant NaV Current in Muscle Dorsal Root Ganglion Neurons after Femoral Artery Occlusion
Authors: Li Qin , Qin Lu , Li Jianhua
Published: April 2020

Background/Aim

Femoral artery occlusion in rats has been used to study human peripheral artery disease (PAD). Using this animal model, a recent study suggests that increases of TNF‐α and its receptor lead to the exaggerated responses of sympathetic nervous activity and arterial blood pressure as metabolically sensitive muscle afferents are activated. Note that NaV1.8 channels are predominately presented in chemically sensitive thin fiber sensory nerves. The purpose of this study was to examine the role played by TNF‐α in regulating activity of NaV1.8 currents in muscle dorsal root ganglion (DRG) neurons of rats with PAD induced by femoral artery occlusion.

Methods

DRG neurons from control and occluded limbs of rats were labelled by injecting the fluorescence tracer DiI into the hindlimb muscles five days prior to the experiments. A voltage patch clamp mode was used to examine TTX‐resistance (TTX‐R) NaV currents.

Results

Seventy‐two hours of femoral artery occlusion increased peak amplitude of TTX‐R and NaV1.8 currents in muscle DRG neurons. Likewise, after application of A803467 blocking NaV1.8 component of TTX‐R, TTX‐R currents were attenuated to a greater degree in DRG neurons of occluded limbs than in DRG neurons of control limbs. The inhibitory effects of A803467 on TTX‐R currents in muscle DRG neurons were 59 ± 2% in control and 75 ± 5% after occlusion (P<0.05 between two groups; n=6 in each group). TNF‐α exposure amplified TTX‐R and NaV1.8 currents in DRG neurons of control limbs and occluded limbs in a dose‐dependent manner. Notably, amplification of TTX‐R and NaV1.8 currents induced by TNF‐α was attenuated in DRG neurons with pre‐incubation of respective inhibitors of intracellular signal pathways p38‐MAPK, JNK and ERK.

Conclusion

Our data suggest that NaV1.8 is engaged in the role of TNF‐α in amplifying muscle afferent inputs as the hindlimb muscles are ischemic; and p38‐MAPK, JNK and ERK pathways are likely necessary to mediate the effects of TNF‐α.

Support or Funding Information

This study was supported by NIH P01 HL134609 & R01 HL141198
One‐Time Acute Elevation of Muscle Temperature Is Effective for Attenuation of Exaggerated Exercise Pressor Reflex in Rats with Peripheral Artery Disease
Authors: Qin Lu , Li Qin , Li Jianhua
Published: April 2020

Background

The blood pressure (BP) response is augmented during static exercise in peripheral artery disease (PAD). Heating treatment was found to be effective for the attenuation of the BP response in PAD patients. However the underlying mechanism for the muscle temperature (Tm) on BP responses in PAD is still unclear.

Aims

We first determined the effect of one‐time acute Tm elevation on the exaggerated exercise pressor reflex in PAD rats with 72 hours of femoral artery occlusion. We further determined if P2X receptor was engaged during this process because a lower Tm in occluded muscle can alter expression and function of P2X.

Methods

The BP response to static muscle contraction was recorded to examine the exercise pressor reflex. Also, αβ‐methylene ATP (αβ‐me ATP) was injected into the arterial blood supply of the hindlimb muscles to stimulate P2X receptors in the muscle afferent nerves. To elevate the Tm, a heating pad was placed locally on the hindlimb and the Tm was increased by ~1.5 °C. Then the Tm was stabilized and maintained for 5 min.

Results

The BP response to static muscle contraction was examined 20 minutes before (pre‐heat), immediately following (acute heat) and 20 minutes after the end of elevation of Tm (recovery). Compared with control rats, a greater mean arterial pressure (MAP) response was observed in PAD rats in the pre‐heat session (27.50±2.07 mmHg in PAD rats/n=12 vs. 18.00±1.67 mmHg in control rats/n=9; P<0.05). However, following elevation of the Tm, the amplification of MAP response was similar in PAD rats and in control rats (21.25±3.00 mmHg in PAD rats/n=12 vs. 18.00±2.56 mmHg in control rats/n=8; P>0.05). During the recovery, a greater MAP response remained in PAD rats (27.54±0.90 mmHg in PAD rats/n=11 vs. 20.14±1.43 mmHg in control rats/n=7; P<0.05). Note that no significant difference in developed muscle tension during contraction was seen in control rats and PAD rats (P>0.05). When αβ‐me ATP (0.125mM) was given, the MAP responses were higher in PAD rats (n=7) than in control rats (n=7) (56.03±4.70 mmHg in PAD vs. 39.12±4.59 mmHg in control for pre‐heat, 59.63±3.55 mmHg in PAD vs. 39.92±5.20 mmHg in control after heating, and 63.33±2.51 mmHg in PAD vs. 35.57±4.38 mmHg in control for recovery; P<0.05 for all sessions between two groups).

Conclusion

One‐time acute elevation of the Tm inhibits the exaggeration of the exercise pressor reflex in PAD rats. The effects of one‐time heat may not be through alternating muscle afferent nerves’ P2X receptors.

Support or Funding Information

This study was supported by NIH P01 HL134609 & R01 HL141198