bluuebunny/crossref_metadata_2025 huggingface dataset:
Effect of CaS nanostructures in growth and proliferation of melanoma and benign skin cells in vitro
Authors: Pérez‐Caro Cristina Ivelisse , Narváez‐Ramos Michelle Angelie , Rodríguez‐Martínez Olga , Muñoz‐Muñoz Keyshla , Suarez‐Martínez Edu B , Diffoot‐Carlo Nanette , Castro‐Rosario Miguel
Published: June 2021

One person dies of melanoma skin cancer every hour according to the American Cancer Society. The Skin Cancer Foundation highlights that melanoma is the most dangerous form of skin cancer. The standard treatment involves the surgical removal of the tumor with follow up treatments depending on the cancer stage. Once skin cancer melanoma metastasizes, it is very difficult to treat. We have extended our earlier work on the use of calcium sulfide nanostructures to skin cancer melanoma in vitro. We studied the effect of CaS nanostructures in the growth and proliferation of the melanoma ATCC CRL‐7637 skin cancer cells as well as the corresponding pre‐control ATCC CRL‐7636 benign cells and control ATCC CRL‐2106. The results were consistent with our previous studies on mammary adenocarcinoma and lung cancer cells where the malign cell growth and proliferation were limited by the addition of CaS nanostructures with no effect on the growth and proliferation of benign cells over a period of 0, 24, 48 and 72 hours. Work in progress addresses the effect of these nanostructures in the cell life cycle.

Support or Funding Information

Financial support from the NIH RISE‐E‐BASE under grant number 1 R25 GM127191‐01 is gratefully acknowledged.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Comparison of Different Methodologies and Cryostat versus Paraffin Sections for Chromogenic Immunohistochemistry
Authors: Hira Vashendriya , de Jong Annique Loncq , Ferro Klea , Khurshed Mohammed , Molenaar Remco , Van Noorden Cornelis J.F.
Published: June 2021

Immunohistochemistry (IHC) specifically localizes proteins in cells and tissues, but methodologies vary widely. Therefore, we performed a methodological IHC optimization and validation study. First, we compared advantages and disadvantages of cryostat sections versus paraffin sections. Second, we compared and optimized antigen retrieval in paraffin sections using citrate buffer and Tris/EDTA buffer. Third, aminoethyl carbazole (AEC) and 3,3′‐diaminobenzidine (DAB) were tested as horseradish peroxidase (HRP) substrates to obtain a water‐insoluble coloured end product to visualize antigens. Fourth, secondary antibodies conjugated with either mono‐HRP or poly‐HRP were compared. The study was performed using serial sections of human tonsil. IHC was performed with primary antibodies against endothelial cell marker CD31, smooth muscle actin (SMA), chemokine stromal‐derived factor‐1α (SDF‐1α) and its receptor C‐X‐C receptor type 4 (CXCR4), macrophage marker CD68 and proliferation marker Ki67. DAB rather than AEC, and cryostat sections rather than paraffin sections gave optimum staining at highest primary antibody dilutions, whereas tissue morphology in paraffin sections was superior. Loss of antigenicity in paraffin sections by formaldehyde fixation, heat and/or masking of epitopes was counteracted by antigen retrieval but not for all antigens. Two out of six antigens (CD31 and CD68) could not be retrieved irrespective time and type of retrieval. Tris‐EDTA was superior to citrate buffer for antigen retrieval. The use of mono‐HRP or poly‐HRP depended on the affinity of the primary antibody for its antigen. We conclude that IHC methodology optimization and validation are crucial steps for each antibody and each research question.

Support or Funding Information

KWF; UVA 2014‐6839

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Systemic Inflammatory Activation of Astrocytes in Postnatal Development
Authors: Alves Michele Joana , Blackbourn Jessica , Melo e Silva Talita , Alzate Diego , Jones Mikayla , Otero Jose , Czeiler Catherine
Published: June 2021

Systemic inflammation during the postnatal period triggers neuroinflammation which can have a multitude of deleterious effects on the newborn, including increased susceptibility to hypoxia. Astrocytes in the brainstem are capable of sensing oxygen levels and modulating the respiratory response accordingly. Astrocytes are also crucial players in neuroinflammation by regulating immune responses in the central nervous system. Upon activation, astrocytes promote the release of cytokines, which trigger immune cell infiltration and contribute to the exacerbation of existing inflammation. Paradoxically, astrocytes can also exert neuroprotective effects during activation. Therefore, it is important to understand the mechanism whereby astrocytes become activated and the factors affecting astrocyte behavior during inflammation. One of the hallmarks of astrocyte activation is a change in morphology and an increase in migratory behavior. It is unclear how this global change in morphology and migratory behavior affects the astrocyte's ability to sense and react to changing oxygen levels. In this study, we investigate the effects of systemic inflammation on the morphological/migratory pattern and oxygen sensing capacity of astrocyte populations derived from the hindbrain of 5‐day‐old mice. Results of this study increase our current understanding of astrocyte functional and morphological responses to an inflammatory milieu.

Support or Funding Information

This work was supported by NIH grant R01HL132355.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Measurement of sedation in individual Drosophila
Authors: Clowes Katherine Rose , Petersen E. Nicholas , Hansen Scott
Published: June 2021

Though widely used, the mechanisms of sedation from compounds like ethanol and volatile anesthetics are not well understood. Drosophila melanogasterhas been identified as an ideal model organism for studying phenotypic responses to ethanol and other anesthetics due to its low cost, similar phenotypic response, and many available genetic tools. Previously, measuring the effects of anesthetics on Drosophila has required labor‐intensive methods often using large cohorts of flies, making them cumbersome and inefficient. Here, we describe the setup and experimental protocol for Volatile Anesthetic Position Recording (VAPR), a high‐throughput technique to measure the effect of ethanol vapor and other volatile drugs on Drosophila behavior. VAPR utilizes flow meters to adjust the relative concentration of aerosolized drugs which are then passed through chambers containing individual flies. Automated video tracking records the position and movement of the flies over time, which we analyze to quantify hyperactivity, sedation, and other treatment related behavioral changes. Using VAPR, we found flies lacking the enzyme phospholipase D (PLD) are insensitive to ethanol sedation but appear unaffected in their sensitivity to the anesthetics chloroform and isoflurane.

Support or Funding Information

National Science Foundation (NSF) Summer Undergraduate Research Fellowship (SURF) grant 1359369.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
APP21 transgenic rats develop age‐dependent cognitive impairment and microglia accumulation within white matter tracts
Authors: Liu Qingfan , Weishaupt Nina , Shin Sheojung , Singh Ramandeep , Agca Yuksel , Agca Cansu , Hachinski Vladimir , Whitehead Shawn
Published: June 2021

Background

Most of the animal models commonly used for preclinical research into Alzheimer's disease (AD) largely fail to address the pathophysiology, including the impact of known risk factors, of the widely diagnosed sporadic form of the disease. Here, we use a transgenic rat (APP21) that does not develop AD‐like pathology spontaneously with age, but does develop pathology following vascular stress. To further the potential of this novel rat model as a much‐needed pre‐clinical animal model of sporadic AD, we characterize APP21 transgenic rats behaviorally and histologically up to 19 months of age.

Methods

The open field test was used as a measure of activity; and the Morris water maze was used to assess learning, memory, and strategy shift. Neuronal loss and microglia activation were also assessed throughout the brain.

Results

APP21 transgenic rats showed deficits in working memory from an early age, yet memory recall performance after 24 and 72 h was equal to that of wildtype rats and did not deteriorate with age. A deficit in strategy shift was observed at 19 months of age in APP21 transgenic rats compared to Fischer wildtype rats. Histologically, APP21 transgenic rats demonstrated accelerated white matter inflammation compared to wildtype rats, but interestingly no differences in neuron loss were observed.

Conclusions

The combined presence of white matter pathology and executive function deficits mirrored what is often found in patients with mild cognitive impairment or early dementia, and suggests that this rat model will be useful for translationally meaningful studies into the development and prevention of sporadic AD. The presence of widespread white matter inflammation as the only observed pathological correlate for cognitive deficits raises new questions as to the role of neuroinflammation in cognitive decline.

Support or Funding Information

We appreciate funding support from the Ontario Mental Health Foundation, and funding from the Canadian Institutes for Health Research, Canadian Consortium on Neurodegeneration in Aging and Canadian Foundation of Innovation.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Research on the standardized training mode of clinical medical talents based on clinical pathway
Authors: Duan Hubin , Hao Chunyan , Wang Suping , Zhang Xuefeng
Published: June 2021

Objective

To study the effect of clinical pathway on the ability of standardized treatment of patients for medical students.

Methods

450 undergraduates were divided into group A and group B, Group A was the traditional practice group, group B was the clinical pathway teaching group. 225 students in each group. group A students according to the traditional practice teaching method, finished the ward round and case discussion with their teachers, consulted the attending physician's advice for their patients. On that basis, tried to give the simulation of the doctor's advice after choosing the same disease but different patients, recording their history, according to their own understanding; Group B students studied the clinical pathway in advance, based on the method of Group A, followed the clinical pathway, combined with their own understanding, tried to give the simulation of the doctor's advice. Two high qualificated experts inspected the completeness and correctness of the simulation of the doctor's advice giving by group A and group B students. At the same time , students' confidence in independent treatment of same disease were assessed by themselves.

Results

the integrity of students' medical advice in group A and group B was 85.68±3.10 vs 92.05±2.96‐P<0.01; The correctness was 86.55±3.88 vs 95.59±4.31‐P<0.01; The degree of confidence in the treatment of the doctor's order was 75.19±6.90 vs 90.45±5.65‐P<0.01.

Conclusion

The students in the clinical pathway‐teaching group are better than the traditional teaching group students in the confidence, the integrity and the correctness of the doctor's orders.

Support or Funding Information

This project is supported by the teaching reform program of shanxi higher education (No. 2013‐ 033), The project of "131" leading talents of shanxi higher education, the education reform project of postgraduate students in Shanxi Province (No. 2017‐027), the education reform project of shanxi medical university, the National Natural Science Foundation Youth Fund (30600637). China Postdoctral Foundation(2014M561207)‐Shanxi Scholarship Council of China

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Ozone‐Induced Systemic Metabolic Alterations are Regulated by Circulating Stress Hormones in Rats
Authors: Snow Samantha J , Henriquez Andres R , House John S , Schladweiler Mette C , Miller Colette N , Kodavanti Urmila P
Published: June 2021

Several recent studies from our lab and others have demonstrated widespread metabolic effects following an acute ozone exposure. Although these effects are similar to those induced by the fight‐or‐flight response involving adrenal‐derived stress hormones, little data are available linking these metabolic changes to activation of neuroendocrine pathways. In this study, we used surgical and pharmacological interventions to understand the influence of adrenal‐derived stress hormones in mediating ozone‐induced metabolic alterations. Male Wistar‐Kyoto (WKY) rats underwent bilateral total adrenalectomy (ADREX) or sham surgery prior to their exposure to filtered air or ozone (0.8 ppm), 4h/day for 1 day. In the sham animals, ozone exposure induced hyperglycemia and glucose intolerance as well as increased levels of circulating total cholesterol, free fatty acids (FFA), and leptin. ADREX and the resultant depletion of adrenal‐derived stress hormones, adrenaline and corticosterone, significantly attenuated these ozone‐induced metabolic effects. Furthermore, ozone‐induced global transcriptional changes in the liver from sham and ADREX rats were assessed by total mRNA sequencing. The upstream regulator assessment within Ingenuity Pathway Analysis revealed that ozone‐induced changes in gene expression were similar to those induced by corticosterone, methylprednisolone, and forskolin, suggesting glucocorticoids and adrenergic mechanisms significantly contributed to the ozone‐induced changes in the liver. These same regulators were not predicted in the ADREX animals exposed to ozone. In a follow up study, we utilized a non‐specific beta‐adrenergic receptor antagonist (propranolol) and/or glucocorticoid receptor antagonist (mifepristone) to understand the specific contribution of these stress hormones in mediating metabolic effects following ozone exposure. Ozone‐induced hyperglycemia, glucose intolerance, and increased FFA levels were significantly attenuated only by the combination of antagonists, suggesting that both adrenergic and glucocorticoid pathways were involved in the regulation of these processes. However, increases in total cholesterol and leptin levels following ozone exposure were blocked by mifepristone alone and in combination with propranolol, suggesting these ozone effects are glucocorticoid‐dependent. Collectively, these data suggest adrenal‐derived stress hormones play a significant role in mediating ozone‐induced systemic metabolic effects. (Does not reflect the US EPA policy)

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Macrophage‐Specific Phenotypes Direct Fibroblast Expression of Matrix Metallopeptidases
Authors: Julian Dana Rose , Johnson Zariel , LoPresti Samuel , Brown Bryan , Yates Cecelia
Published: June 2021

Introduction

During wound repair, fibroblasts secrete abundant extracellular matrix (ECM) components and participate in remodeling of the provisional ECM. Macrophages and other immune cells play a critical role in all stages of wound healing and are primarily responsible for directing ECM‐producing cells into the wound site to replace it with the new scar tissue. Classically activated macrophages, M1M□, contribute to inflammation early on, whereas alternatively activated macrophages, M2M□, secrete ECM‐degrading enzymes, including matrix metallopeptidases (MMPs), during resolution. Macrophages instruct fibroblast activities, largely via secreted factors. However, the differential effects of M1M□vs M2M□ on fibroblasts remains unknown. This study is designed to evaluate the various roles of differentially polarized macrophages through secreted factors on fibroblast activities that relate to wound resolution.

Method

Naïve macrophages(M0) were differentiated into either M1M□ using IFN‐gamma and LPS, or M2M□ using IL‐4. To study the effects of secreted factors and cell‐contact dependent factors, we used indirect (condition media) and direct macrophages‐fibroblast co‐culture systems, respectively with and without TGFβ. Morphologic, phenotypic, and transcriptomic characterization, and cytokine analysis was performed. Cultures were also evaluated for relative gene expression of extracellular matrix genes (MMP8, MMP9, TIMP1, and TIMP2).

Results

We have shown that M1M□ increases gene expression of matrix degrading enzymes, MMP8 and MMP9, while M2M□ enhanced fibroblast expression of matrix signaling proteins ERK, p38 and AKT. We further have shown that M1M□ highly expresses chemokine CXCL10, a known inhibitor of fibroblast matrix production. We use CXCR3−/− fibroblasts, as CXCR3 is the only known receptor of CXCL10, and treated the cells with both M1M□ vs M2M□ condition media. The fibroblasts showed no change in MMP8 gene expression. However, when we knocked out CXCR3 in M1M□ and cultured them with fibroblasts, fibroblast MMP8 and MM9 expression was diminished.

Conclusion

These data suggest that macrophage‐induction of fibroblast expression of MMP8 and MM9 is independent of CXCR3 expression on fibroblasts, but requires the expression of CXCR3 on macrophages. These data points to complex macrophage‐fibroblast crosstalk coordination and the dependency in tissue repair.

Support or Funding Information

Funding Source: American Society of Investigative Pathology‐ Summer Research Opportunity in Pathology Program (SROPP), and University of Pittsburgh School of Nursing supported by grants from NIAMS and NIGMS (AR68317 CCY), and support in kind from and the School of Nursing.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Sex Differences in Local Nucleus Accumbens Circuitry and Motivated Behavior
Authors: Brady Lillian J , Johnson Amy R , Tat Jennifer , Calipari Erin S
Published: June 2021

Drug addiction is a chronic, relapsing brain disease characterized by compulsive drug seeking, high levels of drug intake, and repeated cycles of abstinence and relapse. Historically, preclinical addiction research has focused almost entirely on male subjects; however, in humans, women take and use drugs in larger quantities, transition to addiction faster, and are more likely to relapse, thus representing a particularly vulnerable population. There are a variety of factors that could contribute to these sex differences – including estrous cycle dependent ovarian hormone fluctuations ‐ however the precise neural circuits and neurobiological mechanisms that underlie these differences in reward, motivation, and addiction vulnerability is largely unknown. The mesolimbic dopamine pathway, which connects the ventral tegmental area (VTA) to the nucleus accumbens (NAc), is an essential component of this process as it controls motivation and reward‐seeking behavior. To understand how these phenotypic differences in addiction vulnerability could be driven by estrous cycle‐dependent neurochemical mechanisms we took a multipronged approach to outlining sex‐differences in local circuit regulation of VTA dopamine terminals in the NAc. Using fast‐scan cyclic voltammetry (FSCV) paired with site‐specific pharmacology we measured subsecond dopamine kinetics in male and female mice in either diestrus (low circulating hormones) or estrus (high circulating hormones) and defined sex‐differences in local circuit regulation of dopamine release in the NAc. We found an enhanced dynamic range of calcium and presynaptic D2 autoreceptor control of dopamine release in females compared to males. During estrus, D2 autoreceptors were less effective at inhibiting dopamine release. Further, the relationship between calcium and dopamine release was enhanced in estrus. Finally, cholinergic regulation of dopamine signaling in the NAc was altered in a way that promotes and enhances dopamine release in estrus females. Together, these factors converge to increase the responsivity of dopamine terminals in the NAc to incoming stimuli and likely act to drive enhanced motivation and drug effects on this system. Moving forward it will be critical to directly link these sex‐differences to addiction vulnerability.

Support or Funding Information

Funding was provided by startup funds from Vanderbilt University School of Medicine Department of Pharmacology (E.S.C, L.J.B.), as well as funding from the National Institutes of Health (NIH). Funds from the National Institute of Drug Abuse (NIDA) DA042111(E.S.C), National Institute of Mental Health (NIMH) MH065215 (A.R.J), the Brain and Behavior Research Foundation (to E.S.C), Whitehall Foundation (to E.S.C), Edward J Mallinckrodt Jr. Foundation (to E.S.C) also supported this work.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Effects of CF102, an Adenosine A3 Receptors Agonist, on Memory Deficits and Oxidative Stress in a Mouse Model of Streptozotocin‐Induced Dementia of Alzheimer's Type
Authors: Andrade Geanne Matos , Bezerra Jessica Rabelo , de Souza Nascimento Tyciane , Tavares Juliete , Alves Amanda Aragão , Carmo Marta Regina Santos do
Published: June 2021

Alzheimer's disease (AD) is an age‐related neurodegenerative disease characterized by a progressive decline in cognitive functions. Adenosine receptors play a pivotal role in disease progression, however, adenosine A3 receptors are understudied and their effects vastly unknown. We now investigated the effects of the activation of adenosine A3 receptors on memory deficits and oxidative damage in a mouse model of streptozotocin‐induced experimental dementia of Alzheimer's type. Male Swiss mice received bilateral intracerebroventricular injections of streptozotocin (STZ, 3 mg/kg) dissolved in artificial cerebrospinal fluid. Two days after the first STZ administration, injections were repeated, and the animals were treated with the A3 receptor agonist, CF102 (200 μg/kg intraperitoneally), or vehicle (2% DMSO in saline), 1 h after surgery and once a day until the last day of behavioral evaluation (18th after the first STZ injection). The animals subjected to STZ administration showed significant recognition memory deficits evaluated by the object recognition task, spatial memory deficits evaluated by water maze test, and early and late memory deficits in the passive avoidance test. No differences of locomotor activity in the open field test were observed between groups. STZ also increased malonaldehyde and nitrite contents in the hippocampus. The treatment with CF102 significantly prevented the early and late memory deficits and object recognition memory deficits. CF102 treatment also reduced the increase of nitrite production in 37% in the hippocampus when compared to STZ group. These data highlight the therapeutic potential of A3 agonists in AD, however the role of these receptors on STZ‐induced AD model requires more investigation.

Support or Funding Information

Supported by Brazilian National Research and Development Council (CNPq), Coordination for the Improvement of Higher Education Personnel (CAPES) ‐ Brazil, and the Research Support Foundation of Ceará (FUNCAP) – Brazil.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Cognition Assessment in APPPS1 Transgenic Mice
Authors: Theobald Robert James , Han Byung Henry
Published: June 2021

In a previous study, the effects of various anti‐muscarinic drugs were tested on wild type mice using a Barnes Maze to assess cognition. With the availability of the APPPS1 transgenic mouse model of Alzheimer's disease, this study plans to use the Barnes Maze protocol to assess these drug's effects on wild type and transgenic mice in this strain. However, there are no baseline data using the Barnes Maze with this strain, either wild type or transgenic, and the age of onset of cognition deficits in the transgenic mice has not been fully characterized. Therefore, this study was performed to determine if the Barnes Maze was an effective assessment tool in this strain, and if it could be used to determine the age of onset of cognition problems in these mice. Newly born mice were genotyped and both APPPS1 and littermate wild type mice were used in this study. Once the mice reached 3 months of age, they were trained and tested using the Barnes Maze. There were 10 mice tested, 7 wild type (WT) and 3 transgenic mice (APP/PS1 Tg). The mice were tested 3 times per week, Monday, Wednesday, and Friday for approximately 6 months, to establish a pattern of learning the location of an escape box (EsB), and if or when they manifested signs of cognitive failure. Parameters measured included, time to locate the EsB, considered latency, distance traveled to the EsB, and time to enter the EsB. After completion of the behavioral tests, amyloid plaque load in the brain was assessed with methoxy‐ X04 staining. Results indicate that the APPPS1 Tg mice experienced some cognitive failure during the testing period, about 50 days into the testing period. This is manifested as an increase in all three parameters. The WT data shows some variability in performance, but their performance was reasonably consistent after an initial ‘learning’ phase. The data suggests that the Barnes Maze protocol can be used to study the development of cognition failure in the APPPS1 Tg mice, and may be useful in assessing effects of various drugs in these mice.

Support or Funding Information

No extramural funding

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The Role of Glutamatergic Antagonism in the Antidepressant‐like Activity of the Rapid‐Acting Antidepressant RO‐25‐6981 and Its Analogs
Authors: Laymon Kristen , Suriyachottakul Jiratchaya , Ham Ahrom , Yu BaoLing , Shim Bora , Valls Danielle C , Kirsh Robert D , So Christopher H , Rawlins David B , Talbot Jeffery N
Published: June 2021

The glutamatergic antagonist, RO‐25‐6981, has previously been shown to exert both rapid and sustained antidepressant‐like activity. The purpose of the current study is to delineate its putative antidepressant mechanisms of NR2B‐selective NMDA receptor antagonism and monoamine reuptake transporter inhibition. RO‐25‐6981 and structural analogs with varying degrees of NR2B binding affinity were tested for antidepressant‐like behavior in wild‐type mice and animals deficient in NR2B subunit expression. In the tail suspension test, four RO‐25‐6981 analogs (TR‐2, TR‐4, TR‐5, and TR‐6) were found to exhibit antidepressant‐like activity in wild‐type mice following acute administration (30 mg/kg, i.p., 30 min) with maximal reductions in immobility by approximately half compared to vehicle‐treated controls. By contrast, RO‐25‐6981 (10 mg/kg, i.p., 30 min) reduced immobility by approximately 90%, an effect comparable to that exhibited by the traditional monoaminergic antidepressants fluoxetine and desipramine. However, unlike RO‐25‐6981, TR‐2, TR‐4, TR‐5 and TR‐6 profoundly limited generalized locomotor activity suggesting increased activity in the tail suspension test was related to psychotropic vs. generalized drug effects. In contrast, other TR analogs tested showed no antidepressant‐like activity in the tail suspension test, despite possessing robust NMDA receptor antagonist activity via mid‐ to low‐nanomolar binding affinity at the NR2B subunit. Interestingly, RO‐25‐6981 and TR‐5 exhibited similar antidepressant‐like activity in wild‐type and NR2B‐deficient mice, despite possessing low‐nanomolar NR2B binding. In addition, cellular serotonin transport assays showed functional inhibitory activity of both agents. Taken together, these data suggest that the antidepressant‐like activity of RO‐25‐6981 and its analogs does not correlate with the degree of NMDA receptor antagonism. Furthermore, these data point to monoamine reuptake inhibition contributing to the overall antidepressant‐like activity of RO‐25‐6981 in animal models of mood.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Protein kinase C‐beta isoform as a mechanism promoting chronic pain in sickle cell disease
Authors: Wang Zaijie Jim , He Ying
Published: June 2021

Pain is a hallmark symptom in sickle cell disease (SCD). Besides acute painful vaso‐occlusive crises, SCD is also accompanied by intractable chronic pain. This persistent, and often unrelieved, pain starts early in childhood and continues throughout life. The neurobiological mechanisms of chronic pain in SCD remain unclear, which limits effective pain management and the quality of life in patients with SCD. Taking advantage of a humanized mouse model of SCD, this study aimed to investigate protein kinase C (PKC)‐beta isoform mechanism for chronic pain in SCD. We characterized pain phenotypes in the Townes' mouse model (TOW) of SCD that exclusively expresses human alleles encoding normal α‐ and sickle β‐globin. TOW mice exhibited ongoing spontaneous pain as well as hypersensitivity to evoked pain stimuli compared with littermate control mice. We then examined spinal PKCbeta‐mediated signaling and behavioral consequences. Prominent activation of PKCbeta was identified in the superficial laminae of the spinal cord in TOW sickle mice. Functional inhibition and genetic silencing of the PKCbeta attenuated sickle cell pain phenotypes, abolishing PKCbeta activation, spontaneous pain, mechanical allodynia, and heat hyperalgesia in the sickle cell mice. Taken together, these data suggest PKCbeta may be one of the mechanisms for the generation and maintenance of ongoing and evoked pain in SCD. These findings offer new insights into sickle cell pain mechanisms, which may offer new pharmacological interventions.

Support or Funding Information

R35 HL140031 and K99 HL133590

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The Role of Vesicular Glutamate Transporter 2 in Neuropathic Pain and Its Regulation Mechanism
Authors: Su Rui‐Bin , Yu Gang
Published: June 2021

BACKGROUND

Up‐regulation of glutamate neurotransmission has been proposed as a crucial mechanism of neuropathic pain. Vesicular glutamate transporters (VGLUTs), which transport glutamate into presynaptic vesicles, are fundamental in glutamate storage and release. Several lines of evidence from genetically modified mice suggest a possible association of VGLUT2 with neuropathic pain.

OBJECTIVE

The purpose of this study is to investigate the changes of VGLUT2 expression and function in neuropathic pain and to elucidate its regulatory mechanism, in a mouse spared nerve injury (SNI) model.

METHODS

The changes in VGLUT2 expression were determined by western blot and immunohistochemistry in the spinal cord and pain‐related brain regions at different time points after SNI surgery. The cell distribution characteristics of VGLUT2 in the pathological condition were observed by immunofluorescent staining. RNA interference and VGLUT inhibitors were used to regulate the expression and function of VGLUT2 respectively, and the mechanical pain behavior was observed. The effects of VGLUT2 down‐regulation and inhibition on glutamate release were also investigated. The effects of Wnt/β‐catenin signaling on VGLUT2 expression and pain behavior were investigated by using the Wnt agonist, Wnt1, and Wnt/β‐catenin pathway inhibitor XAV939 in SNI mice.

RESULTS

VGLUT2 protein levels were significantly up‐regulated in the spinal cord, thalamus, periaqueductal gray and amygdala after SNI surgery. In SNI mice, VGLUT2 was mainly expressed in neurons (co‐locolized with NeuN and MAP2), but not glia cells (Iba‐1 and GFAP). Intrathecal or intracerebral administration of VGLUT2 shRNAs or the VGLUT inhibitor CSB6B significantly attenuated mechanical allodynia and decreased glutamate release. Meanwhile, Wnt1/β‐catenin signaling pathway was activated during the development of neuropathic pain. The Wnt agonist or Wnt1 significantly increased VGLUT2 expression, while Wnt/β‐catenin pathway inhibitor XAV939 decreased VGLUT2 expression. Moreover, administration of XAV939 7 days after SNI surgery significantly attenuated mechanical allodynia with a long duration of action, and this effect was in coincidence with VGLUT2 down‐regulation. In naïve mice, intrathecal administration of the Wnt agonist or Wnt1 produced mechanical allodynia, which was abolished by preventive VGLUT2 down‐regulation.

CONCLUSION

Neuropathic pain are associated with the changes in VGLUT2 expression and function, and Wnt1/β‐catenin signaling up‐regulates VGLUT2 in the pathological condition.

Support or Funding Information

This work was supported by National Natural Science Foundation of China (81200850) and Beijing Natural Science Foundation (7123224).

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Calmodulin interaction with GRK5 N‐terminus regulates kinase activation
Authors: Sulon Sarah Marie , Komolov Konstantin , Bhardwaj Anshul , van Keulen Siri Camee , Dror Ron , Benovic Jeffrey
Published: June 2021

GRK5 belongs to a family of G protein‐coupled receptor (GPCR) kinases that mediate phosphorylation and desensitization of GPCRs. In addition, GRK5 has been implicated in a number of diseases through its ability to phosphorylate non‐receptor substrates such as α‐synuclein, HDAC5 and moesin. GRK5 is directly regulated by the calcium‐sensing protein calmodulin (CaM). Ca2+/CaM binds GRK5 and inhibits GPCR phosphorylation, though the precise mechanism of regulation is unclear. Interestingly, GRK5 retains kinase activity when in complex with CaM. To better understand the structural basis of GRK5 regulation by CaM, we utilized crystallography, molecular dynamics simulations, isothermal titration calorimetry, mutagenesis and in vitro kinase assays. The 2.0 Å crystal structure of the GRK5/CaM complex reveals that the far N‐terminal α‐helical region (αN‐helix) of GRK5 interacts with the CaM N‐lobe via a hydrophobic interface. The CaM‐bound structure of GRK5 exhibits key conformational changes that are characteristic of kinase activation. These include disruption of the ionic lock, ordering of the αN‐helix and closure of the catalytic domain. In addition, CaM stabilizes GRK5 in an open, active conformation and stimulates non‐receptor substrate phosphorylation. The stabilization of the αN‐helix by CaM likely serves as the mechanism of activation. Thus, CaM binding to GRK5 via a novel N‐terminal binding site stabilizes an active conformation of the kinase, enhancing phosphorylation of non‐receptor substrates.

Support or Funding Information

ROI HL142310

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Role of Piezo1 in cAMP‐Dependent Calcium Release From ER Stores in Endothelial Cells
Authors: Santana Dianicha , Malik Asrar B. , Mehta Dolly , Komarova Yulia A.
Published: June 2021

Endothelial cells (ECs) lining the vessel wall express various mechanosensing and mechanotransducing molecules that are responsible for converting mechanical cues into chemical signals to elicit specific cellular responses. The recently discovered mechanosensitive Ca2+ channel Piezo1 plays an important role in sensing hydrostatic pressure and shear stress by ECs. However, specific mechanisms involved in Piezo1‐mediated mechanotransduction remains elusive. Using intracellular and endoplasmic reticulum (ER) calcium sensors to respectively monitor the changes in Ca2+I and Ca2+ ER concentrations, we have shown that pharmacological activation of Piezo1 induces cAMP‐evoked Ca2+ release from ER stores. Ca2+influx through Piezo1 leads to a transient activation of soluble adenylyl cyclase (sAC) which, in turn, activates cAMP‐dependent Ca2+ release via Inositol TrisphosphateReceptor 2 (IP3R2), a calcium channel located in ER. Depletion of either sAC or IP3R2 markedly reduces the Piezo1‐mediated increase in cytosolic Ca2+ I indicating that Ca2+ ER release represents a positive feedback loop amplifying Ca2+ signals in ECs. Our data, for the first time, demonstrate that mechanosensitive channels induces Ca2+ release from the ER. Further work will establish the role of sAC and IP3R2 in regulating the adaptive cellular responses of ECs to mechanical cues.

Support or Funding Information

This work is supported by the National Institutes of Health T32HL007829 to DS and 2R01HL045638‐30 to ABM and YK.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Caffeic Acid Phenethyl Ester Improves Diabetic Mice Cutaneous Wound Healing
Authors: Salles Jeanine , de Souza Bruna Romana , Costa Andréa Monte Alto
Published: June 2021

Diabetes mellitus is a chronic condition that causes several complications. Studies indicate that around 1/3 of the diabetic patients in USA suffer cutaneous complications, resulting in a higher morbidity and mortality. In the last years, scientific studies have used the active principle of the natural products to treat acute and chronic wounds. As an example we have the caffeic acid phenethyl ester (CAPE), one of the propolis active principles, that presents potent antioxidant and anti‐inflammatory action. Due the great performance of CAPE to improve the burn healing in rats, as observed in our laboratory, we seek to investigate if this agent can also be beneficial in wound healing of chronic lesions. Diabetes was induced in Swiss mice (males) by multiple streptozotocin injections. After one week, hyperglycemia was confirmed and two excisional wounds (8 mm2 each) were made on the dorsum of these animals (day 0). Mice were divided in two groups, the diabetic mice group (DB group), that received only the vehicle and the diabetic mice CAPE treated‐ group (DB+CAPE group), that received 10μM of CAPE dissolved in the vehicle. The treatment started on the day of the wounds creation and extended until the day of euthanasia, 12 days after wounding, when the wounds were collected. CAPE treatment improved wound inflammatory and oxidative profile; in this group the tumor necrosis factor α (TNF‐α) and phosphorylated nuclear factor kappa B (NFkBp65) protein, nitric oxide synthase 2 (NOS2) and cyclooxygenase‐2 (COX‐2) expression was reduced. These results reflected in the inflammatory cell infiltrate, which was reduced. The reduction in inflammatory cell amount in DC+CAPE group influenced the reduction of the protein carbonylation and lipid peroxidation. In DB+CAPE group, the granulation tissue presented a greater collagen fiber density, with more collagen type I. The improvement in inflammatory parameters influenced the macroscopic parameters. The DB+CAPE group presented a strong tendency towards greater contraction and reepithelization when compared to DB group. These results lead us to believe that the treatment of diabetic lesions with CAPE may be a good option for a therapeutic treatment, with the benefit of being a product of natural origin, which could reduce side effects.

Support or Funding Information

Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro ‐ FAPERJ

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Oral Administration of a Vermiculite‐Nanocellulose Hybrid Boosts Sperm Parameters and Gonadotropin Levels in Rats
Authors: Otuechere Chiagoziem A. , Adewuyi Adewale , Oluwabayo Tanitoluwa , Afolayan Folashade , Avwioroko Oghenetega , Abazuh Uche
Published: June 2021

The safety of vermiculite‐nanocellulose (VEN) hybrid on the male reproductive system is not known. Hence we investigated the effects of VEN on the biochemical and hormonal indices of rats. Animals were orally exposed to VEN at 0, 5, 10 and 20 mg/kg body weight for 14 consecutive days. Administration of VEN had no effect on organo‐somatic index of the testes. Although Gamma –glutamyl transferase, catalase and superoxide dismutase activities and hydrogen peroxide level remained unaffected, exposure of rats to VEN significantly depleted myeloperoxidase, glutathione peroxidase and malondialdehyde levels in the testes of treated rats, when compared to control. The spermiogram of VEN‐exposed rats showed a significantly higher sperm count and lower sperm morphology rates than control group. Treatment with VEN also significantly increased circulatory concentrations of follicle‐stimulating hormone and luteinizing hormone, and decreased testosterone levels at the 20 mg/kg dose. Histoarchitecture of the testes, in the treatment groups, except at the 20 mg/kg dose, revealed normal looking Leydig and Sertoli cells. The epididymis presented with tubules completely filled with spermatids across all treatment groups. Taken together, our study show that VEN could play an important role in male reproduction in rats, especially in redox regulation and stimulation of the gonadotropins.

Support or Funding Information

This research was done without specific grant from any funding agency in the public, commercial, or not‐for‐profit sectors.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Regulation of Cataractogenesis in Cultured Bovine Lenses by ATB 343
Authors: Maffofou Leonce N , SegewkalHeruye Segewkal H , Njie‐Mbye Ya Fatou , Ohia Sunny E , Opere Catherine A.
Published: June 2021

Purpose

There is evidence that substrates for the endogenous production of hydrogen sulfide (H2S) such as L‐cysteine and N‐acetylcysteine (Zhang et al., Mol Vis, 14:862–870, 2008) and NSAIDs such as ibuprofen (Robert and Harding, Exp Eye Res, 54:509–518) can mitigate cataractogenesis in vitro and in vivo. However, the role of the hybrid compound consisting of the NSAID, indomethacin and a H2S‐donating moiety, ATB 343 on cataractogenesis has not been elucidated. In this study, we investigated the effect of the ATB 343 on cataractogenesis in cultured bovine lenses.

Methods

Freshly isolated bovine lenses were cultured in a DMEM buffer solution as follows: Group I: Control (DMEM); Group II–III: H2O2 (10 mM or 50 mM); Group IV–VI: ATB 343 (10−8M to 10−6M; Group VII: ascorbic acid (AA; 10 mM) in presence and absence of H2O2 (10 mM or 50 mM). Lenses were incubated in a CO2 chamber and assessed at 3, 6, 24, 48 and 72 hour‐time points. Qualitative assessments were conducted by visual inspection of lenses and photographic images captured against a black grid, while quantitative assessment was conducted by measurement of transmittance using a plate reader (Synergy H1 hybrid reader; Bio Tek Instruments, Inc) at every time point.

Results

DMEM‐cultured lenses exhibited a time‐dependent decrease in transmittance (420nm) and a corresponding loss of lens optical clarity up to 72 hours. Similar to the endogenous antioxidant, AA (10 mM), ATB 343 (10−7M) attenuated time‐dependent lens degradation up to 24 hours. H2O2 (10 mM and 50 mM) decreased light transmittance in a time‐dependent manner, achieving an inhibition of 41.98% (n=12; p<0.0001) and 46.37% (n=12; p<0.0001), respectively after 72 hours. Interestingly, ATB 343 (10−8 M to 10−6 M) partially reversed H2O2 (10 mM)‐induced degradation up to 72 hours. After 48 hours, the rank order of protection was: ATB (10−7M)>ATB (10−6M)>AA (10 mM)>ATB (10−8M). Similarly, ATB 343 (10−7 M and 10−6M) partially reversed H2O2 (50 mM)‐induced lens degradation up to 48 hours. After 24 hours, the rank order of protection was: ATB (10−7M)>ATB (10−8 M)>ATB (10−6M >AA (10 mM).

Conclusion

ATB 343 protected cultured lenses from time‐dependent degradation up to 24 hours and partially protected cultured bovine lenses from H2O2‐induced cataractogenesis.

Support or Funding Information

Creighton University‐SPAHP, Pharmaceutical Sciences Graduate Program

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Expression of Nox4 NADPH Oxidase Splice Variants Generate Hydrogen Peroxide and Modify the Cell Cycle
Authors: Scott Danielle A , Schickling Brandon M , Miller Francis J
Published: June 2021

The NADPH oxidase (Nox) family of proteins generate reactive oxygen species (ROS), which regulate redox‐mediated cell signaling and may lead to oxidative stress. Unlike the other catalytic subunits, Nox4 is mostly constitutively active and regulated by its expression levels. It is reported that Nox4 has splice isoforms, including variant D which retains all FADH and NADPH binding sites of the full length Nox4A, but lacks transmembrane domains. The goal of this study was to characterize expression and function of splice variants Nox4A and Nox4D. We first validated the specificity of qPCR primers used for detection of the splice variants by overexpression of Nox4A and Nox4D in Cos22 cells. Next, we examined expression of these isoforms in cancer cell lines. The relative expression of Nox4A and Nox4D was not significantly different in human epithelial colorectal adenocarcinoma cells (CaCo), human Asian endometrial adenocarcinoma (Ishikawa) glioblastoma (LN229), adenocarcinomic human alveolar basal epithelial (A549) and human umbilical vein endothelial (EA.hy926) cells. We next examined the function of Nox4 splice variants and found that expression of Nox4A and Nox4D both increased ROS levels as compared to control transfected cells. It has been suggested that the subcellular targeting of Nox4A and Nox4D may be different. To test this, we measured hydrogen peroxide levels in the cytosol, mitochondria, and nucleus using the genetically targeted HyPer probes. Whereas Nox4A expression increased ROS in each subcellular compartment of Cos22 cells, Nox4D primarily increased the HyPer signal in the nucleus. We next examined the effect of Nox4 on protein thiol oxidation and found that expression of Nox4A caused oxidation of several protein thiols. Finally, Cos22 cells were transfected with Nox4A, Nox4D, or an empty vector and treated with cytokines. The expression of Nox4 isoforms had a differential effect on the percentage of cells in the G0/G1, S, and G2/M cell cycle phase. In summary, Nox4A and Nox4D splice isoforms are detectable in different cell lines, overexpression of Nox4A and Nox4D increase ROS levels in subcellular organelles and the oxidation of multiple protein thiols, and expression of Nox4A and Nox4D alters the cell cycle. The expression of Nox4 splice isoforms may provide specificity for regulation of subcellular redox signaling.

Support or Funding Information

DAS was supported by the American Society for Pharmacology and Experimental Therapeutics (ASPET) sponsored Duke SURPH program. FJM is supported by the Office of Research and Development, Department of Veterans Affairs [2I01BX001729] and the National Institutes of Health [HL130039].

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.