bluuebunny/crossref_metadata_2025 huggingface dataset:
PKA Inhibition Mimics Dopamine 2 receptor‐driven Inhibition of VEGF‐induced Proliferation of Human Retinal Microvascular Endothelial Cells
Authors: Fortepiani Lourdes A. , Adams Kaelyn , Varela Crystal , Akimov Nikolay P. , Sohn Jeong-Hyeon , Renteria Rene C.
Published: April 2020

Diabetic retinopathy (DR), a leading cause of blindness, results in part from chronically increased expression of vascular endothelial growth factor (VEGF) in the eye in the diabetic state. VEGF increases proliferation of endothelial cells, a critical step in abnormal angiogenesis that contributes to the pathophysiology of DR. We have previously found that activation of dopamine receptors, specifically using a D2‐type receptor (D2R) agonist, inhibits VEGF signaling on human retinal microvascular endothelial cells (HRMECs) and that HRMECs express the dopamine receptor genes DRD1, 2, 3, and 5, but not DRD4. D2R activation normally inhibits the activity of PKA via activation of Gi‐alpha subunits. Here, we determined whether inhibition of PKA, the canonical downstream target of D2R‐agonist activation, suppresses VEGF‐induced proliferation of HRMECs.

HRMECs, obtained from CSC, Inc. (Kirkland, WA), were plated in CSC 10% serum complete media in 96‐well plates. After overnight starvation in 0.5% serum media, wells were pretreated with either the D2R agonist quinpirole (Q: 50 nM), the PKA inhibitor 14–22 Amide (PKI: 1 μM), or only 0.5% serum media (C). This and all consecutive steps were performed in 0.5% serum media. After 15 min, VEGF (V: 25 ng/mL) was added to some wells pre‐treated with Q or PKI, resulting in the following groups: C, V, V+PKI, V+Q, Q, and PKI. Proliferation as a percent of control was determined using a WST‐1 assay after 48 hours. As anticipated, VEGF induced proliferation of HRMECs compared to control (1.21 ± 0.13 vs 1, normalized values). This proliferation was blocked by either Q or PKI treatment (0.96 ± 0.12 and 0.94 ± 0.12 vs 1.21 ± 0.13 respectively, p<0.05). Treatment with Q or PKI without VEGF had no effect on proliferation compared to control (1.10 ± 0.13 and 0.97 ± 0.18 respectively).

In conclusion, application of the D2R agonist quinpirole or of the PKA inhibitor 14–22 Amide, suppressed VEGF‐induced HRMEC proliferation to control levels. These results suggest that targeting not only specific dopamine receptor activation but also cAMP and PKA signaling systems in HRMECs may be a useful adjunct treatment for blocking VEGF‐induced HRMEC activation in DR.

Support or Funding Information

NIH R01 EY023290 and UIWRSO Faculty Development Grant
Phytoestrogen Coumestrol as an Anticancer Therapy Against Triple‐Negative Inflammatory Breast Cancer
Authors: Rodriguez-Martir Keishla M. , Rivera Colon Jose E. , Colon Ortiz Ayeisha N. , Peterson Esther A.
Published: April 2020

Breast cancer is the second leading cause of death in females in the United States, being Inflammatory Breast Cancer (IBC) the most aggressive subtype. The 5‐year survival rate is 40% for IBC as compared to 85% survival rate in patients with other subtypes. The poor prognosis for patients with IBC emphasizes the need to better understand the molecular signature of this disease with the goal of developing effective targeted therapeutics. To this date, there is no effective targeted therapeutics, especially for those patients that account for approximately 20–40% of IBC. Those cases, triple negative IBC (TN‐IBC), express an alternative isoform of estrogen receptor alpha, estrogen receptor alpha‐36, and GPR30. Importantly, several studies have shown that estrogen can activate non‐genomic signaling pathways involved in the acquisition of oncogenic phenotypes such as increased motility and invasion in triple negative (TNBC) and TN‐IBC. In fact, recent studies have shown that phytoestrogen like Cou, structurally similar to estradiol (E2), induces cell cycle arrest and apoptosis in TNBC. We hypothesize (1) that estrogen non‐genomic signaling has an active role in the aggressive metastatic phenotype of IBC, and (2) that Cou has anticancer activity by inhibiting estrogen non‐genomic signaling in IBC. To test this hypothesis, TN‐IBC cells were treated with E2 or Cou and the effect on phosphorylation of downstream kinases (ERK and AKT) was analyzed by Western blot. Also, functional assays had been performed to determine the effect of treatment with E2 alone, Cou alone, and in combination on migration, proliferation and tumor emboli formation. Immunofluorescence in TN‐IBC and Western Blot of breast cancer cell lines showed expression of estrogen receptors. In addition, preliminary observations demonstrated that upon E2 treatment there was an activation of ERK and AKT kinase in both TN‐IBC and TNBC cell lines. Finally, treatment using previously reported IC50 (50μM) of Cou caused a decrease in cell viability in breast cancer cell lines. Also, in comparison to E2, Cou decrease migration, proliferation, and tumor emboli formation in TN‐IBC. In summary, TN‐IBC cells are responsive to E2 treatment making this signaling pathway attractive for the development of innovative therapeutic strategies. Importantly, these Cou preliminary studies show a potential anticancer activity in TN‐IBC cell lines.

Support or Funding Information

UPR Fondos Institucionales para la Investigación (FIPI) and RISE program (5R25GM061151‐18)
Evidence for vascular microbleeds in brains following repeated mild traumatic brain injury
Authors: Nahirney Patrick Charles , Trivino Juan , Christie Brian R.
Published: April 2020

Mild traumatic brain injury (mTBI) is produced by the rapid movement of the brain within the skull. It is believed that each mTBI leaves the brain in a vulnerable state to subsequent injuries (repeated‐mTBI; r‐mTBI), and can precipitate more serious neurological conditions. However, a mechanism responsible for this vulnerable state is currently lacking, and the mTBI process itself remains poorly understood. The current experiments used an Awake Closed Head Injury (ACHI) model we have recently developed to study r‐mTBI. For each ACHI procedure (n = 8 per animal) a neurological assessment was performed to assess basic levels of consciousness, balance, and sensory perception. Our data demonstrate that administering the ACHI protocol 8 times produces robust, but non‐cumulative neurological deficits. One day following mTBI, rats were perfusion fixed, vibratome sliced (50 μm), and processed for immunohistochemistry. Confocal and light microscopic imaging revealed that r‐mTBI is accompanied by diffuse microbleeds in cortical and subcortical regions that ranged in size from 20 to 100 μm. These microbleeds were characterized by the presence of red blood cells and the blood protein, fibrinogen, in the surrounding perivascular space. The parenchyma around these microbleeds was positive for reactive and phagocytic microglia. In addition, the brains of animals following r‐mTBI were unique in that they also contained ‘rod’ type microglia, similar to those normally observed in Alzeheimer‐like dementias. These observations indicate that repeated concussions produce ruptures in small blood vessels in the brain and these likely contribute to both immediate and long‐term cognitive deficits.

Support or Funding Information

CIHR, NSERC
Habitual Exercise and ETB Receptor Responses in Postmenopausal Women
Authors: Welti Laura M. , McGinty Shane J. , Kuczmarski Andrew V. , Nathaniel Sangeetha , Shoemaker Leena N. , Wenner Megan M.
Published: April 2020

Age‐related declines in endothelial function can be reversed with habitual exercise (HEx) in older men by attenuating the vasoconstricting effects of endothelin‐1 (ET‐1), attributable in part by enhanced ETA receptor activity. Our laboratory has recently demonstrated that ET‐1 contributes to age‐related endothelial dysfunction in postmenopausal women (PMW) via the ETB receptor (ETBR). Although there is evidence for sex‐specific differences in ET‐1 receptor activity with aging, the impact of habitual exercise (HEx) on ET‐1 receptor responses in PMW has yet to be established.

PURPOSE

To test the hypothesis that ETBR responses would be attenuated in HEx PMW.

METHODS

In a retrospective analysis, 30 PMW were grouped by self‐reported weekly exercise. We measured vasodilatory responses to local heating of the skin (laser Doppler flowmetry) during microdialysis perfusions of lactated Ringer’s (Control) and ETBR blockade (BQ‐788, 300nM) in 15 low (LEx: 2±2 d/wk, 57±4 yrs) and 15 high (HEx: 6±1 d/wk, 60±5 yrs) frequency exercisers. Cutaneous vascular conductance (CVC) was calculated during the plateau phase of local heating (42°C), and normalized to maximal vasodilation achieved by perfusion of sodium nitroprusside (28mM) and heating to 43°C. We analyzed the impact of weekly exercise frequency on vasodilatory responses to ETBR blockade using a two‐way ANOVA. Data presented are mean ± standard deviation.

RESULTS

Groups were well‐matched for body mass index (BMI) (LEx: 25±4 vs HEx: 24±2 kg/m2, P=0.31), resting heart rate (LEx: 60±8 vs HEx: 54±9 bpm, P=0.14), and resting mean arterial pressure (LEx: 90±10 vs HEx: 90±8 mmHg, P=0.96). Microvascular endothelial function was similar between LEx (control: 87±9 %CVCmax) and HEx (control: 88±6 %CVCmax, P=0.86). ETBR blockade improved vasodilation in LEx (BQ‐788: 93±7 %CVCmax, P=0.01 vs. control) but not in HEx (BQ‐788: 91±7 %CVCmax, P=0.11). To account for differences in age and time since menopause, we performed a sub‐analysis of age and BMI‐matched early PMW (<5 years since menopause) PMW (LEx, n=4: 53±2 yrs, 24±4 kg/m2; HEx, n=5: 53±3 yrs, 23±2 kg/m2). This sub‐analysis revealed a similar trend of enhanced vasodilation during ETBR blockade in LEx (control: 88±3 vs BQ‐788: 96±4 %CVCmax) but not HEx PMW (control: 87±6 vs BQ‐788: 85±13 %CVCmax; drug*exercise, P=0.07).

CONCLUSION

Blocking ETBR improved microvascular endothelial function in LEx, indicating a removal of the vasoconstricting effects of ETBR in LEx but not HEx PMW. Therefore, these data suggest that exercise may attenuate ETBR‐mediated vasoconstriction in PMW. Additional data are needed to determine the impact of exercise on ETA receptor responses in PMW.

Support or Funding Information

Supported by NIH R01 HL 146558, P20 GM 113125, AHA 16SDG30700015, and University of Delaware Research Foundation
Changes in Splanchnic Hemodynamics Contribute to the Cardiovascular Responses Following Activation of Bradykinin‐Sensitive Pericardial Afferents
Authors: Martin Doug , Roessler Kyle
Published: April 2020

Sympathetic drive is a major controller of cardiovascular function. The heart performs an important sensory function via a variety of cardiac receptors that modulate cardiovascular responses by both neural and humoral reflexes. Bradykinin‐sensitive sympathetic afferent nerves from the heart can trigger marked increases in efferent sympathetic nerve activity to some cardiovascular target organs such as the kidney, arteries and veins. We reported previously that pericardial injection of bradykinin increased venous tone and cardiac output. The splanchnic vascular compartment plays an important role in control of venous return and cardiac output. This work tested the hypothesis that activation of bradykinin‐sensitive pericardial afferents modulates splanchnic hemodynamics.

Sprague Dawley rats were anesthetized with a cocktail of alpha chloralose (80 mg/kg) and urethane (800 mg/kg). Catheters were placed in the femoral artery and vein to record arterial pressure (MAP) and heart rate (HR). A PE10 catheter was placed in the pericardial space for the injection of bradykinin (BK), an agent known to stimulate cardiac sympathetic afferents. The chest was closed and the rats allowed to breathe spontaneously. The portal vein and mesenteric artery were exposed via a midline laparotomy and fitted with transit time flow probes. After stabilization, the blood pressure, heart rate, portal venous flow (PF) and mesenteric flow (MF) responses to pericardial injection of BK or vehicle were recorded.

Pericardial injection of saline vehicle had little effect (MAP: 2±2 mm Hg; HR: 13±6 bpm; PF: −5±1%; MF: −3±1%). In contrast, pericardial injection of BK (10 ug/kg) increased MAP (16±3 mm Hg) and HR (28±5 bpm). After an initial transient decrease, PF (10±2%) and MF (5±2%) were increased during the plateau phase of the MAP response (60–120 seconds).

We interpret these data to indicate that activation of bradykinin‐sensitive pericardial afferents increases portal venous flow. This finding is consistent with the view that activation of the cardiac sympathetic afferent reflex elicits a venoconstrictor response to increase venous return from the splanchnic compartment.

Support or Funding Information

Supported by NIH R01 HL136741‐03, and the Basic Biomedical Sciences program of the Sanford School of Medicine.
Making Functional Predictions Using Local Spatial Arrangements in the Haloacid Dehalogenase Superfamily
Authors: Ruffner Lydia Armentha , Pina Manuel , Beuning Penny J. , Ondrechen Mary Jo
Published: April 2020

Beginning with the sequencing of the human genome in the 1990s, major efforts have been underway to expand significantly upon our current working knowledge of proteins. Between the years 2000–2015, the Protein Structure Initiative (PSI) solved 15,000+ protein structures, but most of them have unknown or uncertain biochemical function or have an incorrect assigned putative function. With all these structures, methodology must be developed to functionally annotate these proteins in order to identify potential applications, such as bioremediation and understanding cellular processes. Enzymes in the Haloacid Dehalogenase Superfamily (HADSF) possess a wide range of functions, including phosphatases important in cell membrane biosynthesis and dehalogenases that possesses the ability to detoxify and degrade halogenated compounds. The Ondrechen Research Group (ORG) at Northeastern University has successfully developed methodology that is being used to predict biochemical function for Structural Genomics (SG) proteins in the HADSF. These methods are Partial Order Optimum Likelihood (POOL) and Structurally Aligned Local Sites of Activity (SALSA). POOL is a machine learning method that uses the electrostatic features and metrics from THEoreticalMicroscope Anomalous TItration Curve Shapes (THEMATICS), ligand binding pocket and geometric features from ConCavity, and the evolutionary scores and phylogenetic trees from INformation‐theoretic TREe traversal for Protein functional site IDentification (INTREPID) to make the predictions about which residues are catalytically active or otherwise important for protein function. SALSA uses the functional residue predictions for SG proteins obtained from POOL and aligns them with consensus signatures from known enzyme subfamilies according to the local spatial arrangement of predicted residues at the active site. So far for the HAD superfamily, using SALSA we have made predictions for 20 SG proteins: one dehalogenase, eight sugar phosphatases, three NagD‐like phosphatases, four P‐Type ATPases, and four soluble epoxide hydrolases. These predictions will be experimentally validated by biochemical assays to establish the function of each protein and to verify our computational approach to protein function prediction. The ability to predict computationally the biochemical function of protein structures of unknown or uncertain function adds tremendous value to genomics data.

Support or Funding Information

This project is funded by NSF CHE‐1305655, CHE‐1905214, and CHE‐1757078.
Generation of a Gelatin‐Based, Vancomycin‐Eluting Hemostatic Agent for the Prevention of Surgical Site Infections
Authors: Fralish Zachary , Shelby Shameka J
Published: April 2020

Surgical site infections (SSIs) following major surgeries are a growing concern in the healthcare industry. These infections lead to lengthened hospital stays, additional surgical procedures, prolonged antibiotic use, and increased patient morbidity. Vancomycin is often the last line of defense against strains of staphylococcal and streptococcal bacteria that are resistant to beta‐lactam antibiotics including Methicillin‐Resistant Staphylococcus Aureus (MRSA). Upon the event of SSIs, typical treatment includes the intravenous delivery of vancomycin which leads to lengthened hospital stays and increased patient expenditures. Current prevention methods include oral administration of antibiotics before surgery, coating of antiseptic agents on incision sites and operating utensils, and systemic administration of antibiotics during surgery. With widespread delivery of antibiotics, there is an increased likelihood of further expansion of antimicrobial resistance. The development of a localized antibiotic‐eluting hemostatic agent as a preventative measure will likely improve the efficiency and efficacy of antimicrobial prophylaxis and subsequently reduce the occurrence of SSIs. Therefore, the current investigation identified a methodology to produce a vancomycin‐linked hemostatic agent through liquid‐phase peptide bond formation. Type B gelatin was incubated with vancomycin and the cross‐linking agent 1‐ethyl‐3‐(‐3‐dimethylaminopropyl) carbodiimide (EDC) to allow for internal cross‐linking of gelatin and conjugation of gelatin with vancomycin. Concentrations of gelatin, EDC, and vancomycin were optimized through release profile analysis. Analysis of eluted fractions by UV‐Vis spectrometry indicated that approximately 30% of loaded vancomycin was released over a two week period. Further analysis by infrared spectroscopy and UV‐Vis spectrometry confirmed that vancomycin released from the loaded hemostatic agent maintained its structural integrity following the cross‐linking procedure. The method described here allows for the production of a vancomycin‐eluting hemostatic agent for the prevention of SSIs. Incorporation of this technology into post‐operative procedures may expedite surgical protocols and limit cost burdens and mortality rates associated with SSIs. On‐going work will analyze the efficacy and toxicity of these agents within biological systems.

Support or Funding Information

Florida Southern College Department of Chemistry, Biochemistry, & Physics

Berend Research Funds
Pyridoxal 5 Phosphate Decarboxylates Angiotensin II: Putative Mechanism for Generation of Angiotensin A
Authors: Stoyell-Conti Filipe Fernandes , Chabbra Alesa , Perry Richard , Speth Robert C.
Published: April 2020

In 2007, a new renin‐angiotensin system (RAS) peptide, Angiotensin A (Ang A; Ala‐Arg‐Val‐Tyr‐Ile‐His‐Pro‐Phe) was discovered, which differs from Angiotensin II (Ang II; Asp‐Arg‐Val‐Tyr‐Ile‐His‐Pro‐Phe) by having alanine (Ala) in position one instead of aspartic acid (Jankowski et al., 2007). Previous studies have shown that Ang A has a higher affinity for angiotensin II receptor type 2 (AT2R) compared to Ang II. It is important to note that AT2R counter‐regulates the adverse effects of angiotensin II receptor type 1 (AT1R). In addition, data from our lab shows that Ang A is ~17‐fold less potent than Ang II at AT1R. Together these observations suggest that Ang A may be less harmful to the cardiovascular system compared to Ang II. Alamandine (Ala‐Arg‐Val‐Tyr‐Ile‐His‐Pro) can be derived from Ang A through enzymatic hydrolysis by angiotensin‐converting enzyme 2 (ACE2) and other carboxypeptidases. Alamandine may also be produced from Ang‐(1‐7) by decarboxylation of the Asp1 residue (Lautner et al., 2013). Alamandine is reported to be ~1000‐fold more potent than Ang‐(1‐7) at counteracting the AT1R mediated adverse effects of Ang II by activating Mas and MrgD receptors (Tetzner et al., 2018). Pyridoxal 5 Phosphate (P5P), the active form of vitamin B6, can promote non‐enzymatic reactions such as decarboxylation and transamination. We hypothesized that P5P could decarboxylate Asp of Ang II to form Ang A. To test this hypothesis, we incubated a mixture containing Ang II (1 mM), P5P (3 mM), and 3‐(N‐morpholino)propanesulfonic acid, MOPS) buffer (100 mM) at pH 7.4 and a temperature of 37 °C for 24 hours. High performance liquid chromatography‐mass spectrometry (HPLC‐MS) analysis of the reaction mixture produced ion signals at m/z 335 and m/z 502 corresponding to [Ang II – CO2 + 3H]3+ and [Ang II – CO2 + 2H]2+, respectively. The mass spectra and retention times for these ions are similar to an Ang A standard. These preliminary results suggests that P5P converts Ang II to Ang A via decarboxylation of Asp. Further studies are underway to confirm the site of decarboxylation.
An Invitation to Modeling
Authors: Dahlquist Kam D.
Published: April 2020

As part of an working group at the National Institute for Mathematical and Biological Synthesis (NIMBioS), I joined an an interdisciplinary group of biologists, mathematicians, mathematical biologists, and education researchers who came together to address the challenges of teaching modeling. This working group provided the opportunity over two years to explore the education research on modeling, share how modeling is applied and taught in our various disciplines, and examine our individual teaching experiences for best practices. In the process, members of the group became more thoughtful in our practice and have developed more granular, nuanced, and inclusive definitions of models and modeling in a broad sense, and mathematical modeling in particular (Eaton et al., 2019; doi: 10.1080/10511970.2018.1489318). This has led to insight about how we might improve our approach to teaching with models in biology courses (Dahlquist et al., 2017; doi: 10.7287/peerj.preprints.3215v1). Models and the process of modeling are fundamental to the discipline of biology, and therefore should be incorporated into undergraduate biology courses. Models are ubiquitous in biology, including representations of the process of science itself. We advocate for a model of the process of science that highlights parallel tracks of mathematical and experimental modeling investigations. With this recognition, we suggest ways in which instructors can call students’ attention to biological models more explicitly by using modeling language, facilitating metacognition about the use of models, and employing model‐based reasoning. We then provide guidance on how to begin to engage students in the process of modeling, encouraging instructors to scaffold a progression to mathematical modeling. I will discuss how I use this framework in a lower division sophomore‐level Cell Function course and upper division courses in Molecular Biology, Bioinformatics, and Biomathematical modeling. We suggest that by making even a small shift in the way models and modeling are discussed in the classroom, students gain understanding of key biological concepts, practice realistic scientific inquiry, and build quantitative and communication skills.

Support or Funding Information

This work was conducted as part of the “Unpacking the Black Box: Teaching Quantitative Biology” Working Group at the National Institute for Mathematical and Biological Synthesis (NIMBioS), sponsored by the National Science Foundation through NSF Award #DBI‐1300426, with additional support from The University of Tennessee, Knoxville. Working group members included: Melissa L. Aikens, Joseph T. Dauer, Samuel S. Donovan, Carrie Diaz Eaton, Hannah Callender Highlander, Kristin P. Jenkins, John R. Jungck, M. Drew LaMar, Glenn Ledder, Robert L. Mayes, and Richard C. Schugart.
Genetic ablation of bone marrow beta‐adrenergic receptors alters miRNA‐transcriptome networks for microglia activation and inflammation in the paraventricular nucleus of the hypothalamus
Authors: Martyniuk Chris J. , Martinez Ruben , Kostyniuk Dan , Mennigan Jan , Zubcevic Jasenka
Published: April 2020

Hypertension (HTN) is a preventable condition with complex etiology. Neurogenic origins of HTN such as aberrant signaling in the paraventricular nucleus (PVN) of the hypothalamus remain a high priority for therapeutic interventions, as most anti‐HTN treatments remain ineffective in a significant portion of patients. Discerning the molecular mechanisms underlying neurogenic HTN is critical for understanding how an overactive sympathetic drive contributes to high blood pressure. We generated a novel bone marrow‐specific adrenergic beta 1 and beta 2 knockout mouse chimera (AdrB1.B2 KO mouse) to study how sympathetic drive to the bone, or lack thereof, affects the molecular responses in the PVN. These mice have previously been characterized by dampened systemic immune responses and decreased blood pressure during the active period. Here, loss of sympathetic drive in the AdrB1.B2 KO chimera lead to an overwhelming suppression of transcriptional networks in the PVN that included leukocyte cell adhesion and migration as well as lymphocyte activation and adhesion, and T cell‐ activation and recruitment. Furthermore, transcriptome networks associated with astroglia, microglial, and neuronal function were suppressed in the PVN of AdrB1.B2 KO chimera. Transcriptional networks related to IL‐17a signaling and the renin‐angiotensin system related genes were also suppressed in the PVN of the AdrB1.B2 KO chimera. Using computational predictive tools, we identified a number of miRNAs expected to regulate global transcriptome responses in the PVN. These included miR‐27b3p, miR‐150, miR‐223, and miR‐326, some of which have been implicated in HTN. In addition, using qPCR, we observed a downregulation in the relative expression of miR‐150, miR‐205, miR‐223, miR‐375, miR‐499a, and miR‐27b3p in the PVN of AdrB1.B2 KO chimera, thus in part confirming the results of computational predictive tools. This study identifies novel molecular mechanisms involved in neural‐immune interactions that underlie neurogenic HTN and elucidate new pathways for therapeutic intervention.

Support or Funding Information

Supported by AHA grant 14SDG18300010 to JZ and NIH R21AT010192 Award AWD05242 P0104932 to CJM and JZ.
Evaluation of Common Crosslinking Agents, Glutaraldehyde and EDC, for the Magnetic Isolation of Phagosomes from Cultured Retinal Pigment Epithelial Cells
Authors: Norberg Ashley , Shelby Shameka J.
Published: April 2020

Daily exposure of photoreceptors to light leads to damage and subsequent shedding of the photoreceptor outer segments (OS). The retinal pigment epithelium (RPE) is responsible for internalization, processing, and recycling of the shed photoreceptor OS. This phenomena, termed RPE phagocytosis, proceeds through a relatively unknown mechanism. To study RPE phagocytosis, previous studies labeled OS with magnetic nanoparticles through a glutaraldehyde cross‐linker to allow for magnetic isolation of phagosomes from the RPE. While isolation of phagosomes from the glutaraldehyde cross‐linking method was successful, the addition of this linker may impact the phagocytic ingestion pathway. Comparatively, 1‐ethyl‐3‐(3‐dimethylaminopropyl) carbodiimide (EDC), a zero‐length cross‐linker, may be used to create a direct cross‐link between the carboxylic acids of the OS protein and the amines of prepared magnetite particles. As such, the purpose of this study was to compare the outcomes of linking OS to nanomagnetites via glutaraldehyde and EDC. To prepare the nanomagnetites, Fe3O4 particles coated with primary amine groups were centrifuged to separate nanoparticles from larger particles. The nanomagnetites were then linked to purified bovine OS with either glutaraldehyde or EDC for 3 hours or 15 minutes, respectively. Linked OS were then fed to cultured RPE cells for 4 hours. Cells were washed and then gently lysed using dounce homogenization to obtain intact phagosomes. Lysed cells were subjected to a magnetic field to separate phagosomes containing magnetite‐labeled OS from the remainder of the lysate. Immunoblot analysis of the purified phagosomes confirmed the presence of rhodopsin, a major component of OS, in both methods. In addition, isolated phagosomes from both methods were shown to contain proteins typically known to be associated with phagosomes including Rab 5, EEA1, and Annexin V. These results suggest that linkage of nanomagnetites to OS with either glutaraldehyde or EDC can serve as efficient methods to accomplish purification of phagosomes from the RPE. Due to the readily tunable linkage conditions, ease of use, and the formation of a direct peptide bond between the magnetite and OS, linkage of nanomagnetites to OS with EDC can serve as a more effective method for purification of phagosomes from RPE cells. Analysis of RPE phagosomes isolated by magnetic selection will contribute to the further elucidation of the RPE phagocytic process.

Support or Funding Information

Florida Southern College Department of Chemistry, Biochemistry, & Physics
Endothelial Permeability Following Skeletal Muscle Injury in Males Versus Females Ages 4 weeks to 1 Year.
Authors: Tusavitz Sabrina , Lee Hannah , Carpenter Apeil
Published: April 2020

Capillary permeability following skeletal muscle injury is necessary for the entry of blood cells and plasma factors necessary for healing. However, it is unclear how aging and sex differences affect capillary permeability after muscle injury.

PURPOSE

To compare endothelial permeability 48 hours post skeletal muscle injury between male and female mice at 1, 3, 6 and 12s month of ag between male and female mice.

METHODS

Barium chloride (BaCl2) was injected into the left tibialis anterior(TA) to create a chemical injury and sodium chloride (NaCl) was injected into the right leg as a control. Evans Blue (EB) dye was injected through the lateral tail vein at 48 hours post‐injury. The mouse was sacrificed 30 min following EB injection and both TA’s were removed, weighed and soaked in formamide to remove extravasated EB from the muscles. Absorbance of extravasated EB was measured. To determine the effect of testosterone and estradiol on permeability, castrated and ovariectomized mice at 12 weeks of age were tested for permeability using Evan’s Blue dye injection 48 hours after injury.

RESULTS

Permeability 48 hours post‐injection was significantly elevated in the BaCl2 injected muscles of mice aged 4 weeks compared to NaCl Male: 6.53±0.1.25 vs. 16.68±8.712 ngEB/mgTA, p<0.001; Female: 2.435±1.56 vs. 9.411±2.799, p<0.01). Permeability 48 hours post‐injection was significantly elevated in the BaCl2 injected muscles of mice aged 12 weeks compared to NaCl Female: 3.02 + 2.43 vs. 16.425 ± 6.63, p<0.0001). Permeability 48 hours post‐injection was significantly elevated in the BaCl2 injected muscles of mice aged 6 months compared to NaCl Male: 2.2743 + .0.932 vs. 8.37 ± 81.66 ngEB/mgTA, p<0.01; Female: 1.593 ±.0.991 vs. 6.3 ± 4.846, p<0.001). Permeability 48 hours post‐injection was significantly elevated in the BaCl2 injected muscles of mice aged 12 months compared to NaCl Male: 1.55 ± 0.32 vs. 4.371 ± 0.299 ngEB/mgTA, p<0.01; Female: 2.78 + 0.79 vs.. 12.02 ± 2.84, p<0.0001). Following castration, permeability in male mice at 12 weeks was not significantly different than non‐castrated mice (5.64 ± 1.033; 7.505 ± 2.131vs 4.927 ± 0.549 vs. 7.505 ± 2.131, respectively). Following ovariectomy, permeability in female mice at 12 weeks was not significantly different than non‐ovariectomized mice (3.02 + 2.43 vs. 16.53 ± 6.63; 4.927 ± 0.549 vs. 9.152 ± 2.96, respectively)

CONCLUSION

Both age and gender contribute to permeability 48 hrs post‐skeletal muscle injury. This affect does not appear to be dependent on testosterone or estradiol.
Activation of nicotinic acetylcholine receptors induces potentiation and synchronization within in vitro hippocampal networks
Authors: Djemil Sarra , Chen Xin , Lee Jisoo , Rauf Mikael , Pak Daniel T.S. , Dzakpasu Rhonda
Published: April 2020

Nicotinic acetylcholine receptors (nAChRs) are known to play a role in cognitive functions of the hippocampus, such as memory consolidation. Given that they conduct Ca2+ and are capable of regulating the release of glutamate and γ‐aminobutyric acid (GABA) within the hippocampus, thereby shifting the excitatory‐inhibitory ratio, we hypothesized that the activation of nAChRs will result in the potentiation of hippocampal networks and alter synchronization. We used nicotine as a tool to investigate the impact of activation of nAChRs on neuronal network dynamics in primary embryonic rat hippocampal cultures prepared from timed‐pregnant Sprague‐Dawley rats. We perturbed cultured hippocampal networks with increasing concentrations of bath‐applied nicotine and performed network extracellular recordings of action potentials using a microelectrode array (MEA). We found that nicotine modulated network dynamics in a concentration‐dependent manner; it enhanced firing of action potentials as well as facilitated bursting activity. In addition, we used pharmacological agents to determine the contributions of discrete nAChR subtypes to the observed network dynamics. We found that β4‐containing nAChRs are necessary for the observed increases in spiking, bursting and synchrony, while the activation of α7 nAChRs augments nicotine‐mediated network potentiation but is not necessary for its manifestation. We also observed that antagonists of N‐methyl‐D‐aspartate receptors (NMDARs) and group I metabotropic glutamate receptors (mGluRs) partially blocked the effects of nicotine. Furthermore, nicotine exposure promoted autophosphorylation of Ca2+/calmodulin‐dependent kinase II (CaMKII) and serine 831 phosphorylation of the α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid receptor (AMPAR) subunit GluA1. These results suggest that nicotinic receptors induce potentiation and synchronization of hippocampal networks and glutamatergic synaptic transmission. Findings from this work highlight the impact of cholinergic signaling in generating network‐wide potentiation in the form of enhanced spiking and bursting dynamics that coincide with molecular correlates of memory such as increased phosphorylation of CaMKII and GluA1.

Support or Funding Information

National Science Foundation (PHY‐1205919 and IOS‐ 1755033), National Institutes of Health (RF1 AG056603‐01) and the Georgetown‐MedStar CERSI Scholars program.
Development of Hypertension and Cardiac Hypertrophy is associated with Endothelial Cell Dysfunction in Npr1 Gene‐disrupted Mice
Authors: Das Subhankar , Noland Whitney , Periasamy Ramu , Krazit Stephen T. , Pandey Kailash N.
Published: April 2020

Atrial and brain natriuretic peptides (ANP and BNP) are cardiac hormones that elicit natriuretic, diuretic, vasorelaxant, and antiproliferative responses, all of which contribute to lowering blood pressure and blood volume. ANP and BNP bind to guanylyl cyclase ‐A/natriuretic peptide receptor ‐A (GC‐A/NPRA), which is considered to be the major natriuretic peptide receptor that synthesizes the intracellular second messenger cGMP. The objective of the present study was to examine the effect of Npr1 (coding for NPRA) gene‐disruption on the process of endothelial cell dysfunction in the development of hypertension and cardiac hypertrophy. We used six‐month old mice having different copies of Npr1 gene (0‐copy, 1‐copy, and 2‐copy). The blood pressure and echocardiographic measurements were performed in each group. Blood was collected by direct cardiac puncture and cardiac tissues were harvested and snap frozen in liquid nitrogen. The 0‐copy (Npr1−/−) mice showed 35–45 mmHg higher systolic blood pressure (SBP) as compared to 2‐copy (Npr1+/+) mice. Echocardiographic measurements of Npr1 gene‐disrupted 1‐copy (Npr1+/−) and 0‐copy mice showed significant increased LVEDD (27% in 0‐copy and 10% in 1‐copy mice), intraventricular septal wall thickness (IVSTDD, 30% in 0‐copy and 15% in 1‐copy mice), and reduced fractional shortening by 24% in 0‐copy and 12% in 1‐copy mice as compared with control mice. There was also a significant increase in plasma levels of plasminogen activator inhibitor‐1 (PAI‐1; 2.2‐fold), soluble intracellular adhesion molecule (sICAM; 1.8‐fold), soluble vascular cell adhesion molecule (sVCAM; 30%). In the present study, increased plasma levels of sICAM, sVCAM, and PAI‐1 suggested the involvement of endothelial cell dysfunction in producing cardiac hypertrophy in Npr1 null mutant mice. Our results implicate that ANP/NPRA signaling promotes inhibitory effect on the agents, which cause endothelial dysfunction in the heart leading to cardiac hypertrophy and cardiac remodeling.

Support or Funding Information

This work was supported by NIH grant (HL062147).
Histone 3 Lysine 9 Methylating Enzyme G9a Regulates Transcriptional Expression of Nuclear Receptors and Cytochrome P450 Genes in HepaRG Cells
Authors: Pande Parimal , Zhong Xiao-bo , Ku Warren
Published: April 2020

Histone modification transcriptionally regulate gene expression. Cytochrome P450 genes are studied extensively in terms of regulation of expression through histone modification. Histone 3 lysine 4 (H3K4) and Histone 3 lysine 27 (H3K27) methylation are studied extensively in regulation of P450 gene expression. Another histone modification Histone 3 lysine 9 methylation, is well known for transcriptional repression of genes but not studies well in regulation of nuclear receptors and cytochrome P450 gene expression. In this study, the role of histone methyl transferase G9a, which is responsible for mono and di‐methylation of histone 3 lysine 9 residue, was studied in regulation of drug metabolizing enzymes and nuclear receptors. We treated HepaRG cell with small interfering RNA to selectively knockdown EHMT2 mRNA and using adenovirus, G9a was overexpressed to study the effect on nuclear receptors CAR (NR1I3), PXR (NR1I2), AHR and heterodimer partner RXRα along with the selected drug metabolizing cytochrome P450 genes. In pathological conditions such as steatosis, the expression of nuclear receptors and cytochrome P450 genes is affected along with G9a in a mice model treated with high fat diet. In this study, we treated HepaRG cells with fatty acids to induce steatosis. In conclusion we observed that loss of G9a shown to decrease mRNA expression of nuclear receptors and cytochrome P450 genes whereas overexpression of G9a increased expression of nuclear receptors CAR (NR1I3), PXR (NR1I2)and selected drug metabolizing enzymes CYP1A2, CYP2B6, CYP2C9, CYP3A4 etc. In fatty acid treated cells overexpression of G9a was observed to prevent fatty acid mediated down regulation of nuclear receptor CAR (NR1I3) and drug metabolizing enzyme genes CYP2C19, CYP2C8 and CYP3A4.

Support or Funding Information

This project work was funded by Boehringer Ingelheim Pharmaceuticals
The Effects of Diet‐Induced Obesity in Male Sprague Dawley Rats on Blood Glucose, Ketones and Markers of Mental and Physical fatigue
Authors: Kaviani Sepideh , Niepoetter Paige , Viernow Coral , Ruholl Hannah , Gopalan Chaya
Published: April 2020

Objective

Previous studies on the pathology of obesity and obesity‐induced metabolic disorders utilized genetically modified obese rat models. To better mimic the onset and the physiology of obesity in humans, the use of diet‐induced obese rat models is lacking. Therefore, the purpose of this study is to induce obesity in Sprague Dawley rats by administering a high fat (HF) diet and to further investigate the effects of obesity on blood glucose and ketone concentrations as well as locomotor and mental activities as indicators of fatigue.

Methods

Fifty‐four 7‐week‐old male Sprague Dawley rats were randomized into either a high‐fat diet (HFD) group (n=28) or a standard diet (SD) group (n=26). All rats were housed in individual cages under controlled laboratory conditions and had access to food and water ad libitum. The HFD group received a HF diet (60% fat, 20% CHO, and 20% Pr) for six weeks, while the SD group received a standard diet (17% fat, 56% CHO, 27% Pr). Body weight (BW) was measured weekly and blood glucose and ketones were measured twice a week for 6 weeks. The open field test (OF) was conducted daily for 8 days in week 5, followed by Novel object recognition (NOR) testing at 4 separate days during week 6.

Results

Both groups gained weight from week 1 to week 6 (HFD: 223.0±16.2g vs. 387.7±23.6g, p<0.001; at week 1 vs. week 6, respectively, SD: 215.0±15.9g vs. 361.5±23.6g, p<0.001; at week 1 vs. week 6, respectively). However, the average within‐subject BW gain was greater in the HFD compared to the SD (168.1±21.6g vs 130.1±16.9g, p<0.001; HFD vs. SD, respectively). Although the average glucose levels were not different between the two groups throughout the study, the HFD showed higher fluctuations of blood glucose levels and at week 4 they had higher glucose levels than the SD (104.1±11.2mg/dL vs. 96.6±6.9mg/dL, p=0.005; for HFD vs. SD, respectively). Ketone levels were not different between the two groups. Although there were no overall differences in OF and NOR test results in HFD vs. SD, the difference between the recognition time of the familiar vs. the novel object in the HFD tended to be smaller than that of the SD at all 4 days of NOR testing (p=ns).

Conclusions

The HF diet results in significant weight gain consistent with obesity in the majority of rats within a 6‐week period of receiving the diet. Overall, diet‐induced obesity was not found to have adverse effects on glucose and ketone levels. However, it might negatively affect memory and lead to mental fatigue in rats.
Isopropyl Alcohol and Methylene Chloride as Alternatives for pre‐plastination Dehydration and Defatting processes
Authors: Lopez-McCormick Juan Sebastian , Pedraza Juan Daniel , Rueda-Esteban Roberto Javier
Published: April 2020

Plastination as a technique creates durable, long lasting, realistic specimens with a high value in anatomy teaching and research. While the original protocol using Acetone has shown great results, the use of this substance is restricted in some countries due to socioeconomical or governmental limitations. The aim of this work is to develop a protocol in which acetone has been replaced as a dehydration and defatting medium.

Three groups of specimens were procured. Each of them had a sample of abdominal organs with varying fragility (kidney‐liver‐small intestine), skeletal muscle and brain tissue. Group 1 and 2 (G1–G2) were previously fixated with formaldehyde 4%, while group 3 (G3) was preserved in a glycerin‐based formaldehyde free solution. A conventional plastination technique was carried out including fixation, dehydration, defatting, forced impregnation and curing.

All groups were fixated with formaldehyde 10%, although G3 required cleansing with a 50% ethylic alcohol (EA) solution to remove the glycerin. G1 was dehydrated and defatted with EA. G2 & G3 were dehydrated with isopropyl alcohol (IA), and defatted with methylene chloride (MC). All groups were impregnated in low temperatures with 1% S3–S10 Biodur silicone‐cross linker mixture. Finally, curing was divided in two steps, passive curing for 6 months and S6 cross linking. The specimens were analyzed based on tissue retraction, color degradation, morphological alterations and final specimen pliability.

G1 showed maximum tissue shrinkage, morphological alteration and color degradation acquiring a brownish tone. G2 & G3 suffered less tissue retraction, color degradation was minimal, morphological alteration was not observed. Specimen pliability remained the same across the three groups, specific tissues such as skeletal muscle, blood vessels and intestine were more pliable.

Lesser tissue retraction and increased color preservation were obtained with the use of IA and MC for dehydration and defatting, compared with exclusive use of EA for both steps. Specimens previously preserved with glycerin‐based formaldehyde free solutions where successfully plastinated with this technique.

While this study serves as a proof of concept that IA and MC are viable substitutes for Acetone in the dehydration and defatting steps, the lack of objective measurements and a control group with acetone are strong limitations of this study. Future studies with a higher number of standardized specimens and controls are needed to determine the impact of the proposed replacement in the plastination protocol.
Effect of Prostate Cancer and Endurance Exercise Training on Aerobic Capacity
Authors: Baumfalk Dryden R. , Colburn Trenton D. , Horn Andrew G. , Kunkel Olivia N. , Weber Ramona E. , Musch Timothy I. , Behnke Bradley J.
Published: April 2020

Background

Prostate cancer is the most commonly diagnosed non‐skin cancer in men accounting for 20% of all diagnoses. Exercise intolerance is common in cancer patients, in particular, in those undergoing adjuvant therapies (i.e., androgen deprivation therapy) that leads to reductions in quality of life. Recent evidence suggests prostate cancer, independent of treatment, hastens fatigue (i.e., reduces time to exhaustion) which is associated with atrophic effects on whole heart and left ventricular (LV) mass. However, moderate‐intensity endurance training mitigates cardiac mass loss in prostate tumor‐bearing rats. Using a pre‐clinical orthotopic model of prostate cancer, we tested the hypothesis that prostate cancer would reduce aerobic capacity, assessed as maximal oxygen consumption (V̇o2max). We also investigated whether moderate‐intensity endurance training would mitigate any loss of aerobic capacity associated with prostate cancer.

Methods

Dunning R‐3327 MatLyLu prostate adenocarcinoma cells (2×105) in 0.1ml of physiological saline solution were injected in the ventral lobe of 4–5‐month‐old male RNU (NIH nude) rat (n=10). These animals were randomized into 2 groups, Tumor Bearing Exercise (TBEX, n=6) or Tumor Bearing Sedentary (TBS, n=4). After ~5 days of recovery, TBEX animals began progressive exercise training on a motorized treadmill at 25 m/min with a 5% incline for 10 minutes a day progressing to 60 min/day for a ~5 week period. Pre‐injection (Pre) and post‐exercise training (Post) animals performed V̇o2max testing at least twice to verify attainment of maximal value (i.e validation test), with a minimum of 24 h of recovery between bouts to ensure reproducibility.

Results

There were no significant differences (all p>0.05) in tumor mass between groups (TBEX 6.4±1.1; TBS 7.6±1.2 g), body mass (TBEX 322±15; TBS 361±16 g), or Pre‐injection V̇o2max (TBEX 82.2±2.1; TBS 78.5±2.5 ml·min−1·kg−1). Heart to body mass ratio was lower in TBS group compared to TBEX (2.9±0.1 vs. 3.3±0.1 mg/g, p<0.05). Following training, TBEX had increases in V̇o2max (6.4±1.3 ml·min−1·kg−1, p<0.05) whereas TBS had a decrease in aerobic capacity
(
−
5.6±2. ml·min−1·kg−1, p<0.05).

Conclusion

This study suggests that prostate cancer, independent of treatment significantly diminishes maximal aerobic capacity. Further, moderate‐ intensity exercise training not only mitigates this cancer‐associated loss, but effectively improves aerobic capacity vs. values obtained prior to cancer development. Given prostate cancer patients often present fatigue and loss of quality of life, moderate‐ intensity exercise training may be useful to improve both facets via maintaining or improving aerobic capacity.

Support or Funding Information

NIH HL137156‐01A1ACS RSG‐14‐150‐01‐CCE