bluuebunny/crossref_metadata_2025 huggingface dataset:
Neurons in a Subregion of the Medial Parabrachial Nucleus (mPBN) Attenuate the Gain of the Hering‐Breuer (H‐B) Reflex
Authors: Zuperku Edward J. , Stucke Astrid G. , Tomlinson Jack , Krolikowski John G. , Hopp Francis A. , Stuth Eckehard A.
Published: June 2021

This study provides new evidence showing that a subregion of the mPBN, which plays a key role in the control of breathing frequency (1), also attenuates the H‐B expiratory facilitatory reflex. Experiments were performed in decerebrate, vagotomized dogs during isocapnic hyperoxia. Phase durations TI and TE were obtained from the phrenic neurogram(PNG). A small concentric bipolar electrode was used to microstimulate the mPBN subregion. Lung inflations and/or weak electrical stimulation of a desheathed cervical vagus nerve were used to activate the slowly adapting pulmonary stretch receptors (PSRs) to produce the H‐B reflex. Pulse‐train PSR stimulation at various frequencies (Fs) were delivered during the E‐phase of test cycles separated by several non‐stimulated control cycles. During every other PSR test cycle, pulse‐train stimulation of the pontine subregion at a predetermined frequency was concurrently delivered to determine its effect on the PSR‐induced increase in TE (e.g., fig A). The effect of the pontine stimulus in the absence of the PSR input was also measured. Plots of TE vs. PSR step Fs have the shape of an increasing exponential function. The effect of the pontine input was a proportional reduction in TE at each of the PSR step Fs, and was shown as an attenuated curve in the TE‐Fs plots (fig B). The relationship between the reduction in the H‐B reflex and the pontine stimulus frequency was linear over the range of 0 to 20 Hz, with a maximum attenuation of the H‐B reflex to ~20% of control (no pontine stimulus) produced by pontine stimulus frequencies in the 20–40 Hz range. These findings suggest that as inputs from the pontine subregion increase breathing frequency, this region concurrently attenuates the H‐B expiratory facilitatory reflex. This mechanism counteracts the PSR‐induced increase in TE, which reduces breathing frequency.

Support or Funding Information

Supported by VA grant 2 I01BX000721‐07

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Veins Are Essential for Arteries: Immune Cells Implication
Authors: Champin Tristan , Grenier Céline , Munier Mathilde , Henrion Daniel , Loufrani Laurent
Published: June 2021

Shear stress produces arterial diameter expansion associated with an hypertrophy induced by inflammation, macrophages, neutrophils or lymphocytes cells. Furthermore, arteries and veins are often associated through perivascular fat, allowing transfer of immune cells between both possible.

So, we investigated the vein implication on immune cells recruitment for arterial remodeling of 12 weeks C57BL6/J mice. We produced various ligation models in mesenteric arteries or common carotids to produce High Flow Arteries (HFA). For each model, mesenteric or jugular veins associated with the HFA are ligatured or not.

In resistance arteries, the absence of the vein associated with HFA, prevents HFA remodeling (HFA Ctrl: 320μm±27 vs HFA without vein: 218μm±19). Also, in conductance arteries, jugular ligation associated with HF carotid is able to prevents remodeling. qRT‐PCRs experiment have shown an increase of VCAM1 expression in veins during high‐flow mediated arteries remodeling. Vein ligation increase oxidative stress production. ROS generate a SIRT1 transcriptional activation leading to a decrease of VCAM1 expression. Immunostaining analysis, show that neutrophils and macrophages migrate from vein to artery. Immune cells are early recruited in vein but diapedesis modification is modulated by VCAM1 expression on vein endothelium.

To conclude, we demonstrated for the first time that artery and vein interact each other by perivascular fat to generate mesenteric and conductance arteries remodeling.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Type 2 Diabetic Coronary Vascular Smooth Muscle Cells Exhibit Decreased Stiffness and Decreased Adhesion
Authors: McCallinhart Patricia , Sun Zhe , Meininger Gerald A. , Trask Aaron J.
Published: June 2021

We previously reported that type 2 diabetic (T2D) coronary resistance microvessels (CRMs) undergo early inward hypertrophic remodeling with reduced vessel stiffness that is associated with a proliferative VSMC phenotype. We also previously revealed that there is no difference in stiffness of decellularized CRMs from diabetic (db/db) and normal (Db/db) mice, suggesting that the decreased stiffness of the intact diabetic CRM is not due to alterations in the extracellular matrix (ECM) of those microvessels. Given that diabetic CRMs are less stiff and this decreased stiffness does not appear to be due differences in the mechanical strength of the surrounding ECM, the aim of the current study was to determine whether a reduction in cellular mechanical stiffness between Db/db and db/db CRM vascular smooth muscle cells (VSMCs) may be responsible. VSMCs were isolated from 16 week old Db/db and db/db mice and passage 1 cells were used for all experiments (n=6–7 for all groups). Using an atomic force microscope with probes coated with fibronectin (FN), a nano‐indentation protocol was used to measure cellular stiffness and adhesion to FN. Elastic modulus, a measurement of cellular stiffness, was reduced in diabetic coronary VSMCs compared to normal (Db/db: 6.74 ± 0.40 kPa vs. db/db: 4.96 ± 0.35 kPa, p = 0.004). By comparison, there were no significant differences in elastic modulus between normal and diabetic aortic VMSCs (Db/db: 5.39 ± 0.79 kPa vs. db/db: 5.18 ± 0.87 kPa, p = 0.861). Additionally, diabetic coronary VSMCs had reduced adhesive force to fibronectin compared to normal coronary VSMCs (Db/db: 54.78 ± 5.49 vs. db/db: 39.8 ± 3.43 pN, p = 0.03). In a separate experiment, qPCR was performed on mRNA from normal and diabetic CRM VSMCs. Alpha‐smooth muscle actin trended lower in db/db CRM VSMCs (p=0.07), indicating a de‐differentiated phenotype. Cell cycle regulator, p53, had significantly reduced mRNA expression in the diabetic coronary VSMCs over control VSMCs (Db/db 1.00±0.06 vs. db/db 0.63±0.08, p<0.01). These data show that reductions in cellular stiffness and adhesion to the ECM of diabetic CRM VSMCs may underlie ex vivo whole microvessel mechanical properties to account for reduced CRM stiffness. The changes in diabetic coronary VSMC stiffness and adhesion may be the result of VMSC phenotypic switching to a more de‐differentiated/proliferative phenotype.

Support or Funding Information

Support: NIH R00 HL116769 and S10 OD023438 and Nationwide Children's Hospital to AJT

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Microvascular Endothelial Function across the Cancer Survivorship Continuum
Authors: Banister Heather R. , Sutterfield Shelbi L. , Caldwell Jacob T. , Post Hunter K. , Lovoy Garrett M. , Ade Carl J.
Published: June 2021

Background

Chemotherapy for the treatment of various cancers has previously been shown to have direct cardiotoxic effects. However, changes in endothelial function due to chemotherapy‐induced toxicity in cancer survivors during the years following treatment remains poorly understood. Therefore, the purpose of this study was to evaluate microvascular endothelial‐dependent vasodilation across the cancer survivorship continuum as well as in healthy controls. We hypothesized that early cancer survivors would yield a decreased percent change in skin blood flow and cutaneous vascular conductance when compared to late survivors and controls.

Methods

To date, thirteen female cancer survivors and thirteen healthy controls (cancer free with no history of chemotherapy) participated in this study. Of the cancer survivors nine were defined as early survivors (within 5 years of chemotherapy treatment) and four as late survivors (> 5 years post chemotherapy treatment). Skin blood flow (SkBF) was measured via integrated laser Doppler flowmetry. Mean arterial pressure (MAP) was measured and used to calculate cutaneous vascular conductance (CVC = SkBF/MAP *100). Endothelium‐dependent cutaneous microvascular function was determined as the percent change in SkBF and CVC in response to iontophoresis of 2% acetylcholine (ACh).

Results

There were no differences in age between groups (early vs. late: P=0.8; late vs. control: P=0.7; early vs. control: P=0.8). The hyperemic responses for SkBF (early: 1081 ± 718% Δ; late: 1458 ± 653% Δ; P=0.4) and CVC (early: 1060 ± 694% Δ; late: 1499± 751% Δ; P=0.5) were not significantly different between early and late cancer survivors. Similarly, the SkBF (early: 1081 ± 718% Δ; control: 1554 ± 655% Δ; P=0.3) and CVC (early: 1060 ± 694% Δ; control: 1472 ± 626% Δ; P=0.1) were not significantly different in the early cancer survivors compared to controls. There was no significant difference in SkBF (late: 1458 ± 653% Δ; control: 1554 ± 655% Δ; P=0.8) and CVC (late: 1499± 751% Δ; control: 1472 ± 626% Δ; P=0.9) between late cancer survivors and controls.

Conclusion

Endothelium‐dependent cutaneous microvascular function is not different between early cancer survivors compared to those >5 years post‐chemotherapy and healthy controls. These results suggest that microvascular endothelial function is restored in the early years following chemotherapy and is maintained in later years of survivorship. Additional work is required to determine if endothelial dysfunction occurs in other important vascular beds.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Glucose‐Derived Microparticles Induce Adhesion Molecule Surface Expression on Endothelial Cells
Authors: Bammert Tyler D. , Hijmans Jamie G. , Stockelman Kelly A. , Brewster Lillian M. , Greiner Jared J. , DeSouza Christopher A.
Published: June 2021

Clinical interest in endothelial‐derived microparticles (EMPs) has increased due to their role in the pathogenesis of vascular disease. Hyperglycemic conditions, such as diabetes, are known to stimulate EMP release; however, the effects of these glucose‐derived microparticles on endothelial health and function are not fully understood. The aim of this study was to determine, in vitro, the effects of glucose‐induced EMPs on endothelial cell expression of the following adhesion molecules: E‐selectin, intercellular cell adhesion molecule‐1 (ICAM‐1), vascular cell adhesion molecule‐1 (VCAM‐1) and platelet cell adhesion molecule‐1 (PECAM‐1). Human umbilical vein endothelial cells (HUVECs) were cultured (3rd passage) and plated in 6‐well plates at a density of 5.0 × 105 cells/condition. HUVECs were incubated with media containing either 25 mM D‐glucose (concentration representing a hyperglycemic state) or 5 mM D‐glucose (normoglycemic condition) for 48 hr to generate EMPs. EMPs derived from both conditions were pelleted by centrifugation and resuspended in culture media. Characterization and concentration of EMPs was determined by flow cytometry using VE‐Cadherin (CD‐144‐PE). To determine cell adhesion molecule expression, HUVECs (3 × 106 cells/condition) were treated with either high glucose‐derived EMPs (hgEMPs) or normal glucose‐derived EMPs (ngEMPs) for 24 hr and surface expression of E‐selectin (CD62E‐PE), ICAM‐1 (CD54‐FITC), VCAM‐1 (CD106‐APC) and PECAM‐1 (CD31‐BV) was assessed by flow cytometry and reported as mean fluorescent intensity (MFI). The high glucose condition induced markedly higher (~260%; P<0.05) EMP release compared with normal glucose (136±38 vs. 37±13 MP/μL). Surface expression of E‐selectin (2614±132 vs. 2010±204 MFI), ICAM‐1 (2110±81 vs. 1688±152 MFI), and PECAM‐1 (4237±395 vs. 2525±269 MFI) on endothelial cells were significantly higher in the hgEMP treated compared with ngEMP treated cells. VCAM‐1 expression was not significantly different between cells treated with hgEMP (3741±680 MFI) or ngEMPs (2340±546 MFI). These results demonstrate that high glucose‐derived EMPs markedly increase endothelial cell expression of E‐selectin, ICAM‐1 and PECAM‐1. Elevations in endothelial expression of these cell adhesion molecules has been shown to be associated with both the development and progression of atherosclerosis. Microparticle‐induced cell adhesion molecule expression provides potential novel mechanistic insight regarding the heightened and accelerated risk of atherosclerotic vascular disease associated with hyperglycemia.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Evidence for Circadian Control of Endothelial Function in Mice on a High Fat Diet
Authors: Pati Paramita , Allan John Miller , Colson Jackson , Young Martin , Bailey Shannon , Pollock David M. , Gamble Karen , Pollock Jennifer S.
Published: June 2021

Both human and animal studies demonstrate that a chronic high fat diet (HFD) leads to endothelial dysfunction. Timing of feeding and fasting is known to influence risk for cardiometabolic disease. Ad libitum chronic HFD disrupts the normal circadian rhythm in metabolism and peripheral molecular clock gene rhythm in mice. However, specific circadian‐dependent mechanisms affecting endothelial function during chronic HFD are poorly understood. Thus, we hypothesized that ad libitum chronic HFD mediates endothelial dysfunction via metabolic arrhythmia and clock gene dysfunction. To test this hypothesis, we designed experiments to measure vascular function in ad libitum and restricted feeding (RF), defined as food availability only during the active phase (ZT 12 to 0 or 7 PM to 7 AM). C57BL/6J 8 week old male mice were provided HFD for 20 weeks ad libitum and separate mice were exposed to RF from weeks 20 to 22 of chronic HFD. Endothelial‐dependent relaxation to acetylcholine in aorta (wire myography) was 1.58 ±0.34 mN in mice provided ad libitum HFD. HFD+RF significantly improved endothelial‐dependent relaxation to 5.12±1.07 mN (n=5–6, p<0.01). In contrast, aortic endothelial‐independent relaxation to the exogenous NO donor, sodium nitroprusside, was similar in ad libitum HFD and HFD+RF mice. In additional experiments, male and female Period2:Luciferase (Per2:Luc) reporter mice were given a normal diet (ND) or HFD for 20 weeks ad libitum and separate groups were subjected to RF from weeks 20 to 22 of ND or HFD. Food consumption and body weight between groups did not differ with 2 weeks of RF. Chronic HFD induced a significantly shortened circadian period of Per2 in aorta (n=4, p=0.0054) compared to ND, and 2 weeks of RF restored the aortic Per2 circadian period in mice on HFD (n=3–4, p=0.0417). Circadian period was unaffected by RF in mice on ND. Interestingly, Per2 rhythm in the perivascular adipose tissue was unaffected by HFD. These findings suggest circadian control of endothelial function during chronic HFD and that HFD induces clock gene arrhythmia specifically disrupting vascular peripheral clocks.

Support or Funding Information

This research was supported by the NIH T32 DK007545 and the UAB School of Medicine AMC21 Multi‐Investigator Grant “Circadian Disruption and Susceptibility to Target Organ Damage.”

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Nighttime sodium intake is associated with cardiometabolic risk and insulin resistance in night shift nurses
Authors: Speed Joshua S. , Molzov Hylton E. , Johnson Russell L. , Becker Bryan K. , Pollock David M. , Gamble Karen L.
Published: June 2021

Night shift work and salt intake are each independent risk factors for cardiovascular disease and possibly insulin resistance. Rhythms in renal sodium excretory function are observed regardless of the time of intake suggesting that salt ingested during the typical inactive period is retained until the following active period. Thus, we hypothesized that eating more salt at night contributes to cardiometabolic disease risk in night shift workers. Day and night shift nurses were followed for 8 days total (5 days off work and 3 days on work) keeping strict food diaries. Nutrient intake was estimated using the Nutrition Data System for Research. Fasting blood draws were taken on day 9. Interestingly, 24‐hour Na+ intake was similar between day and night shift workers throughout the observation period. Both day and night shift workers had similar Na+ intake patterns during off days. As expected, night shift workers consumed significantly more Na+ during the night while day shift workers consumed significantly more Na+ during the day on workdays. The major finding of the study is that greater work‐night Na+ intake was a significant predictor of elevated homeostatic model assessment ‐ insulin resistance (HOMA‐IR), a measure of insulin resistance, for night‐shift nurses only, even after controlling for the effect of work‐night caloric intake (F(1,5)Δ = 16.4, p < 0.05; b = 0.84). In addition, nighttime Na+ intake accounted for 71% of the variance in HOMA‐IR in night shift nurses. Finally, a significant positive correlation was observed between nighttime Na+ intake and fasting total cholesterol (p<0.05, r2=0.57) and triglycerides (p<0.05, r2=0.66) in night shift nurses. These data suggest that lowering nighttime salt intake in night shift workers may improve cardiometabolic risk and insulin resistance in this population with a high prevalence of cardiovascular disease.

Support or Funding Information

This research was supported by NIH grant K99HL127178 to JSS, the UAB Center for Clinical and Translational Science (CCTS) Grant Number ULITR000165 to KLG, the UAB Department of Vision Sciences, the UAB Comprehensive Diabetes Center, and the UAB Department of Psychiatry Office of Clinical Research

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Drowning a frog respiratory oscillator in a wash of network excitability
Authors: Wilson Richard JA. , Baghdadwala Mufaddal
Published: June 2021

To be part of a respiratory rhythm generating circuit, a brainstem nuclei must (1) have phasic activity timed to the rhythm (correlation), (2) be able to speed the rhythm when excited (sufficiency), and (3) be able to slow the rhythm or terminate it when inhibited (necessity). A discrete brainstem nucleus passing these tests is currently considered a critical site for respiratory rhythm generation (an oscillator) if the nuclei continues to be rhythmic when physically isolated.

Compelling and accumulating evidence in support of oscillators has been found in many vertebrates including lampreys, bullfrogs, turtles, birds, mice and rats. In bullfrogs and rodents, three putative oscillators have been discovered: the lung power stroke, lung priming and buccal oscillators in frogs; and in mammals the para Facial Respiratory Group (pFRG), Post‐Inspiratory Complex (PICO), and the PreBötzinger Complex (PreBötC). As the buccal oscillator in frogs and the PreBötC in mammals are located in the same brainstem rhombomere, r7, they may be homologous. Identification of multiple ‐‐ possibly homomogous ‐‐ oscillators in different species has led to the Oscillator Hypothesis which, in its strongest form, posits that respiratory rhythm is produced solely by brainstem oscillators and phase of motor bursts is determine by their interaction.

However, almost complete (90%) destruction of the putative PreBötC in goats over several days was insufficient to terminate breathing. One explanations for this observation is a network architecture in which respiratory oscillators are no more than isolatable units in a much larger rhythmogenic network. To test if this is the case in frogs, we (a) located the frog buccal oscillator using necessity and sufficiency tests in an isolated hemisected brainstem preparation; (b) increased the excitability of the hemisection which increases the frequency and amplitude of the buccal motor pattern; and (c) again tested for necessity. We found that with network excitation, the necessity of the buccal oscillator disappeared, replaced by distributed rhythmogenesis in the surrounding premotor network.

These data suggest that the Oscillator Hypothesis may need to be revised in frogs and possibly other vertebrates; the existence of the buccal oscillator as a discrete site of rhythm generation appears to be state dependent and may only be demonstrable in some conditions.

Support or Funding Information

This work was supported by NSERC

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Viral‐genetic Mapping of Neural Circuit Organization of Nociceptive Neurons in the Thalamus
Authors: Umorin Mikhail
Published: June 2021

There is a critical need to determine the location, connections and neurochemical identities of neurons participating in the transmission and modulation of pain in the CNS so that these circuits can be precisely targeted for pain intervention. Without such knowledge the development of effective, low side‐effect pain treatments will be difficult. In this talk I will report our progress using recent technological advancements to determine detailed neural activation patterns and synaptic circuit organization of neurons related to pain sensation and modulation in the thalamus. We have identified and visualized thalamic nociceptive neurons activated by chronic pain, using a strategy of genetic capture of active neurons on the basis of activity dependent c‐fos gene expression. Specifically, pain‐activated neurons are captured using Fos‐CreER mice with tamoxifen‐dependent recombinase CreERT2 under the promoter of the immediate early gene c‐fos. These mice are crossed with Cre reporter Ai9 mice to express tdTomato fluorescent proteins. A noxious stimulus is evoked by injection of complete Freund's adjuvant (CFA) into the hind paw; only neurons that are functionally active within a limited time window of 4‐OHT administration are genetically labeled. Then we build on this technical development to combine the Fos‐CreER based strategy with new viral tracing of circuit connections to nociceptive neurons in the ventral posterolateral (VPL) nucleus. Genetically modified rabies is used to trace global direct synaptic connections to pain activated neurons in VPL. These studies allow us to map molecular and neurochemical identities (e.g., excitatory versus inhibitory) of whole brain‐wide presynaptic neurons that are directly connected to thalamic nociceptive neurons. We foresee that this new research can address the critical barrier in the field and will advance our understanding of thalamic nociceptive neuron circuits and pain modulating processes.

Support or Funding Information

Start‐up funds from Texas A&M University

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Expanding the Horizon of Recombination Repair in Mycobacteria: Identification and Characterization of Novel Proteins
Authors: Singh Amandeep
Published: June 2021

In prokaryotes, repair by homologous recombination provides a major means to reinstate the genetic information lost in DNA damage. Recent research on recombination repair in mycobacteria has led to significant revision of the understanding of this system. A comparative analysis of the system, carried out in 43 mycobacterial genomes, lead to identification of novel proteins. To substantiate the predictions, biochemical and structural investigations on probable roles of two proteins, namely, SSBb and RecGwed, were carried out. The structure of M. smegmatis SSBb (MsSSBb), was determined at 2.5 □ resolution. Solution studies indicate that the canonical SSB (MsSSBa) is more stable towards thermal and chemical denaturation than MsSSBb. Also, MsSSBa has a two‐fold higher DNA binding affinity than MsSSBb. A direct physical interaction between MsSSBb and MsRecA was established, suggesting a role during recombination. The expression levels of ssbB gene increased by approximately two‐ and sevenfold in UV and hypoxic stress, respectively, while concurrently the levels of ssbA expression declined. These results indicate a role of MsSSBb in recombination repair during stress. MsRecGwed binds a variety of branched DNA structures in vitro, while it does not bind ss‐ or dsDNA. The up‐regulation of recGwed during UV damage, and down‐regulation during H2O2/MMS stress, in M. smegmatis indicated a possible role in post‐replicative recombination events that proceed though branched DNA intermediates. Taken together, this work augments the understanding of the repertoire of proteins known to be involved in DNA repair pathways in mycobacteria.

Support or Funding Information

Department of Biotechnology, India

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
REVERSAL OF PERIPHERAL AND CNS MEDIATED A1 ADENOSINE RECEPTOR HYPOTENSION
Authors: Laughlin Bernard W , Baily Isaac , Tagaban Sierra , Drew Kelly
Published: June 2021

Introduction

A1 adenosine receptor (A1AR) agonists such as N6‐cyclohexyladenosine (CHA) are known for their neuroprotective and anti‐convulsant properties and for modulation of thermogenesis. We have demonstrated a neuroprotective, thermolytic effect following systemic administration of CHA combined with the peripherally acting adenosine receptor antagonist, 8‐(p‐sulfophenyl)theophylline (8‐SPT). However, A1AR‐mediated cardiovascular side effects limit the translation of this drug class, and clear understanding of the effects of this drug combination on hypotension and bradycardia are lacking. CHA delivered into the CNS increases para‐sympathetic influence on the heart to produce bradycardia similar to what is seen during onset of hibernation. Systemic administration of A1AR agonist produces bradycardia via direct negative chronotropic and inotropic effects. Evidence also supports other mechanisms of A1AR agonist modulation of blood pressure including indirect activation of peripheral β2 adrenergic receptors and central H1 receptors.

Hypothesis

We test the hypothesis that CHA lowers blood pressure via increased para‐sympathetic tone to the heart and direct effects on the heart.

Methods

Rats were implanted with telemetry transmitters to monitor Tb, heart rate (HR), mean arterial pressure (MAP) and administered drugs IP as follows, CHA (1.0mg/kg), 8‐SPT (25.0mg/kg), Atropine (1.0mg/kg), diphenhydramine (4.0mg/kg), or pro‐pranolol (4.0mg/kg), 15min prior to CHA. Cage surface temperature was initially cooled to 4°C and adjusted to maintain Tb at 32°C.

Results

8‐SPT with atropine reversed hypotension. 8‐SPT alone increased rate of resolution in MAP to baseline value. Pre‐treatment with propranolol, atropine, or diphenhydramine alone did not influence MAP. Diphenhydramine with 8‐SPT reduced the magnitude of hypotension (− 24% average one hour baseline change after CHA) compared to pre‐treatment with 8‐SPT (− 36%) alone.

Conclusions

Our results suggest that A1AR induced hypotension is mediated through increased parasympathetic tone to the heart in addition to direct effect on the heart. Histamine receptor activation may also play a role, although the resolution of MAP seen may have been due to the anticholinergic effect of diphenhydramine instead of H1 receptor antagonism. Reversing hypotension necessitates addressing both mechanism with atropine and 8‐SPT

Support or Funding Information

NIH U54GM115371, NIH R15 NS070779, NIH: RL5GM118900, TL4 GM 18992, 1UL1GN118991

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Calmodulin Binds to and Inhibits H‐Ras Activation of PI3K: A Single Molecule Study
Authors: Buckles Thomas , Ziemba Brian , Masson Glenn , Williams Roger , Falke Joseph
Published: June 2021

The lipid‐anchored GTPase Ras is well known for its role in oncogenesis, with around 25% of human tumors showing mutations in a Ras family member. One of the best characterized Ras effectors is the highly oncogenic lipid kinase phosphatidylinositol 3‐kinase (PI3K). PI3K phosphorylates the constitutive plasma membrane lipid PIP2, yielding the vital second messenger lipid PIP3. This PIP3 signal regulates many cell processes including migration and growth. Calmodulin also activates PI3K by tightly binding MARCKS, a PIP2 sequestering protein, freeing the lipid. The enhanced free PIP2 population serves as additional substrate for PI3K, increasing its activity. The findings presented here reveal that surprisingly, Ras activation of PI3K is blocked by the presence of calmodulin, with important implications for PIP3 signaling. We are observing these mechanisms using single molecule TIRF microscopy to probe the interactions and activities of single proteins on a supported target membrane surface. Preliminary evidence monitoring PI3K binding and lipid kinase activity at the single molecule level reveals that biologically relevant concentrations of calmodulin strongly inhibit Ras activation of PI3K. These results imply a new and additional level of control for an important and oncogenic signaling pathway.

Support or Funding Information

Funding for this work was provided by the National Institutes of Health ( R01 GM063235 to J.J.F.), the Medical Research Council ( MC U105184308 to R.L.W.), the AstraZeneca/LMB Blue Skies Initiative ( MC A024‐5PF9G to R.L.W.), and St. Catharine's College (G.R.M.).

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The Psycho ‐ Neuro – Immuno Axis of Cancer
Authors: Zillow Anna L , Tulp Orien L , Einstein George P
Published: June 2021

The bio‐psycho‐social model of medicine suggests that the mind affects the brain, and which affects the body. In this manner we suggest a psycho‐neuro‐immuno axis of disease that may be found in the relationship between emotional stress and cancer.

Psychological tension causes a dysregulation of the hormonal stress response resulting in an increase in the circulating levels of the stress hormone cortisol. The physiological mechanism follows that emotional stress causes the brain's hypothalamus to secrete corticotropin releasing hormone. This hormone increases the amount of adrenocorticotropin hormone flowing from the anterior pituitary gland, which ultimately magnifies the amount of cortisol released from the adrenal gland.

Cortisol increase has been shown to rapidly decrease the marginalization and exit of the immunological cells known as neutrophils from the blood into tissues(1). Marginalization is an early‐phase inflammatory response during which cells move from the axial circulation in the center of the blood flow to the periphery of the cross‐sectional lumen. Therefore, despite the increased number of neutrophils, the decreased tissue deployment may have the effect of blunting the immune response. This proposed immune‐incompetence in and of itself may predispose the body to cancer via the upregulation of inflammatory markers and the simultaneous down‐regulation of anti‐inflammatory cytokines. Specifically, the multiplication of pro‐inflammatory cytokines, such as IL‐6 and tumor necrosis factor (2) are likely candidates for involvement in the carcinogenic process.

Moreover, the mere abundance of neutrophils, marginalized or not, may be correlated with increased cancer risk (3). Of course, such findings don't verify a direct causal relationship between emotional stress and cancer, since other mediating variables might influence both variables. However, if with future studies, the prospective nature of the data prove consistent and the relationship holds even when other standard prognostic variables are controlled may suggest, we would consider this more convincing evidence.

Septon et al assert interestingly that “associations of cortisol profiles with psychological stress suggest that rhythm dysregulation may be a marker or mediator of psychosocial effects on cancer progression” (4). Accordingly, we propose that this statement is instructive in our making the assertion that our Psycho ‐ Neuro – Immuno axis operates on a feedback loop. That is to say psychology affects neurology affects immunology and that immunological pathology can cause even more psychological stress – thereby starting the cycle all over again.

Support or Funding Information

Supportde by Institutional Resources of the University of Science Arts & Technology, Montserrat, and the Einstein Medical Institute.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The Role of Platelet Syntaxins in Endocytosis
Authors: Tichacek Laura , Whiteheart Sidney W.
Published: June 2021

In healthy patients, platelets serve as the first line of defense in forming a hemostatic plug when damage to the vasculature is detected. Hemostasis is a balancing act, however, where hyperactive platelets can cause unwanted blood clots which may lead to heart attack or stroke, while hypoactive platelets can cause excessive bleeding. For this reason, examining the proteins critical to platelet function and understanding the bleeding phenotype when these proteins are knocked out can provide potential drug targets and help explain and diagnose platelet disorders. In addition to their role in hemostasis, platelets act as vascular “sentries,” patrolling the vasculature for damage and endocytosing small molecules and viral particles they encounter [1]. While the exact purpose of platelet endocytosis of viral particles is unknown, studies have shown that AIDS patients have increased chances of experiencing a thrombotic event [2], which implies that the uptake of the HIV virus by platelets could be implicated in this disparity, perhaps by altering platelet function. Platelets contain SNARE (soluble N‐ethylmaleimide sensitive fusion protein attachment protein receptor) proteins which are known to control trafficking in most cells [3] but this trafficking role has not been established in platelets, making them an attractive target of study. To this end, we created Syntaxin 2, Syntaxin 4 (two t‐SNARE proteins) and double knock out mice to study the endocytic capabilities of their platelets. Platelets from the Syntaxin 4 and double knock out mice showed a defect in the uptake of total fibrinogen while Syntaxin 4 knockouts did not show a defect in uptake of transferrin.

Support or Funding Information

This work is supported by grants from the National Institutes of Health, National Heart, Lung, and Blood Institute (HL56652 and HL138179), and a Veterans Affairs Merit Award to SWW.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Serine Rich Splicing Factor 2 (SRSF2) Flips for RNA Binding
Authors: Arnholt Mark , Bohn M. , Cina M. , Downing A. , Flanagan M. , Hautala L. , Jaeke A. , Kennedy C. , Lane B. , O'Bryon A. , Pepin J. , Rohloff E. , Rrahmani F. , Schmitt S. , Spudich J. , Staus S. , Tadlock K. , Tomashek M. , Witt E. , McNally Mark
Published: June 2021

Mistakes in alternative splicing of RNA cause diseases such as acute myeloid leukemia. Serine Rich Splicing Factor 2 (SRSF2) is a splicing factor that controls alternative splicing by promoting exon inclusion, so it is not surprising that mutations to SRFS2 are linked to cancers. SR proteins harbor an RNA recognition motif (RRM) at the N‐terminus that binds to mRNA. SRSF2 has the unusual ability to bind to both pyrimidine and purine rich RNA sequences by flipping two C or G nucleotides in the mRNA into anti or syn orientations. The RRM specifically recognizes only C2, C3, and G5. Arg61 forms a hydrogen bond to C3, Phe59 hydrogen bonds to C2, and Lys17 is involved in flipping C2 or G2 into syn or anti conformations. Tyr92 binds with C2 and forms a hydrogen bond with C3. The mutation Pro95His binds better to UCCAGU and has been linked to cancer. The Hartford Union High School SMART (Students Modeling A Research Topic) Team modeled SRSF2 using 3D printing technology to highlight structural characteristics involved in RNA binding. Additional research on SRSF2 mutations and sequence binding has the potential to find both causes of and treatments for diseases like cancer.

Support or Funding Information

The MSOE Center for BioMolecular Modeling would like to acknowledge and thank the National Institutes of Health Science Education Partnership Award (NIH‐SEPA 1R25OD010505‐01) and the National Institutes of Health Clinical and Translational Science Award (NIH‐CTSA UL1RR031973) for their support in funding the 2017‐2018 SMART Team Team program.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Co‐Transcriptional Folding of a Riboswitch Controls the Fate of the Transcriptional Machinery
Authors: Walter Nils G.
Published: June 2021

Folding of nascent transcripts can be modulated by properties of the RNA polymerase (RNAP) that carries out the transcription process. For example, the site‐specific pausing of the RNAP and co‐transcriptional RNA‐protein interactions have been shown in several cases to be important for co‐transcriptional RNA folding. In bacteria, riboswitches are genetic elements frequently found in the 5′ untranslated regions of mRNAs, where they respond to cellular metabolites to regulate gene expression either at the level of transcription or translation. Since they change conformation “on‐the‐fly”, riboswitches are tractable models for studying the role of the transcription process in guiding the folding of nascent transcripts.

The preQ1 riboswitch from Bacillus subtilis regulates gene expression by a transcriptional mechanism in which the binding of its ligand, the queuosine precursor preQ1, causes a conformational change that leads to the formation of a terminator hairpin. Previous studies have deciphered the structure and dynamics of the isolated riboswitch aptamer, but less is known about the impact of the transcriptional machinery on riboswitch folding and vice versa. We have identified a strong pause by RNAP during riboswitch transcription that is stabilized by a previously characterized consensus pause sequence and the ligand‐free conformation of the riboswitch. By using single molecule Förster resonance energy transfer (smFRET) analysis of the riboswitch in a paused elongation complex, we show that RNAP has a profound impact on RNA dynamics, stabilizing a tightly folded structure at this specific pause site. Molecular modeling and biochemical assays further support a specific functional interaction between the nascent transcript and the exit channel of RNAP through which ligand binding to the riboswitch unpauses the polymerase. We are currently using cryo‐electron microscopy of the paused elongation complex to further pinpoint the specific structural rearrangements in both riboswitch and RNAP that are induced first by pausing, then by ligand binding. Finally, we are evaluating the impact of transcription factors on pausing and riboswitch folding. Our study provides significant insights into the mechanisms by which small RNA structures can regulate the function of the macromolecular transcription machinery, and vice versa.

Support or Funding Information

NIH R01 grants GM062357, GM118524 and GM122803

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Differential Response of mTOR and ERK Signaling in Human Skeletal Muscle Following Resistance Exercise and Acetaminophen Consumption
Authors: D'Lugos Andrew C , Patel Shivam H , Fry Christopher S , Carroll Chad C , Dickinson Jared M
Published: June 2021

Previous data have shown that cyclooxygenase (COX) inhibiting drugs may interact with the cellular mechanisms governing the adaptive responses of skeletal muscle to exercise. However, findings from acute exercise trials of attenuated growth responses with COX inhibitors do not translate to chronic exercise training, where COX inhibitors may augment muscle adaptation. The purpose of this study was to determine the extent to which prior consumption of acetaminophen (APAP) alters skeletal muscle signaling of the mammalian target of rapamycin (mTORC1) and ERK pathways in response to RE. In a double‐blinded, counter‐balanced, crossover design, healthy young men (n=8; 25 ± 1 yr, BMI: 26 ± 2 kg·m−2) performed two trials of unilateral knee extension (8 sets, 10 reps, 70% 1RM). For 24h prior to each trial, subjects ingested either APAP (1000mg/6h) or placebo (PLA) (final dose consumed immediately post RE). Muscle biopsies (vastus lateralis) were collected at rest and at 1 and 3h postexercise during each trial. Western blot was utilized to assess mTORC1 and ERK signaling proteins and immunohistochemistry was utilized to assess localization of mTOR to the lysosome (LAMP2). Localization of mTOR to the lysosome (LAMP2) increased in myosin heavy chain (MHC) II fibers 3h postexercise only in PLA (P<0.05). At 1h p‐rpS6Ser240/244 was increased in both groups, but to a greater extent in PLA (P<0.05). At 3h p‐S6K1Thr389 was elevated only in PLA, whereas p‐S6K1Thr421/424 was elevated only in APAP (P<0.05). Similarly, p‐ERK1/2Thr202/Tyr204 was increased to a greater extent in APAP at 3h (P<0.05). Consumption of APAP prior to resistance exercise blunts the translocation of mTORC1 to the lysosome and the phosphorylation of S6K1 at Thr389. Conversely, prior consumption of APAP augments ERK signaling following resistance exercise, leading to the phosphorylation of S6K1 at Thr421/Ser424. These data support a model in which ERK signaling may provide a compensatory response to inhibition of mTORC1 signaling. These findings also further highlight the intricate interaction between COX‐inhibiting drugs and the cellular processes regulating skeletal muscle adaptation to exercise.

Support or Funding Information

Supported by intramural funds from ASU.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
rHbI‐S2H as a Potential Novel Hydrogen Sulfide Donor
Authors: Oliveras Alfredo Reyes , Arias Hazel Borges , López‐Garriga Juan
Published: June 2021

Hydrogen sulfide (H2S) is a well‐known poisonous gas whose toxic effects have been studied for many years. In recent studies, it was found that H2S is produced endogenously in humans through enzymatic pathways and has functioned as a vasodilator, anti‐inflammatory, an antioxidant and smooth muscle relaxant. As result, current studies of H2S are focused as a signaling molecule involved in various physiological processes like NO and CO. Also, H2S concentration in human physiology has been associated with diseases like hypertension, Alzheimer's, cancer, arthritis, diabetes, ulcerative colitis and cardiovascular diseases. Biochemical and physiological studies of H2S have been performed by using compounds that release or promote the production of H2S in biological samples for its therapeutic attribution known as H2S donors. Hemoglobin I (HbI) is a hemoprotein found in the clam Lucina pectinata that has the property of binding H2S in its heme group forming the HbI‐SH2 complex. For this reason, the viability of the HbI from L. pectinata was studied for the delivery of H2S in biological systems to obtain a reliable H‐2S donor. Kinetics studies were performed using a UVV is spectroscopy at physiological conditions with the following parameters; Krebs' buffer at pH 7.4 and 25°C and 37 °C for 24 hours. During the 24 hours, the Soret band showed a displacement from 426 nm (HbI‐SH2) to 407 nm (HbImet) indicating the release of H2S from the protein. The half‐life of the complex was approximately 6 hours. The dissociation constant (koff) 0.43×10−3 seg−1. This result shows the high affinity of the H2S for the protein regardless of physiological pH and buffer.

Support or Funding Information

This work was funded by the National Science Foundation STC award number 1231306. Research reported in this publication was supported by the National Institute Of General Medical Sciences of the National Institutes of Health under Award Number T34GM008419. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Potential Influence of Follistatin and Myostatin on Body Composition during the Yukon Arctic Ultra
Authors: Johannsen Michelle , Schalt Adriane , Kim Jimin , Chen Richard , Coker Melynda S. , Gunga Hanns‐Christian , Coker Robert H. , Steinach Mathias
Published: June 2021

Objective

The objective of this study was to determine the potential roles of follistatin and myostatin on body composition under conditions of prolonged physical activity, chronic cold exposure and remote isolation.

Methods

Participants traveling on foot in the 2017 Yukon Arctic Ultra 692‐km (430‐mile) were recruited for the study. Measurements and samples were obtained at pre‐event, mile 173 at Carmacks checkpoint 1 (C1), mile 239 at Pelly Crossing (C2) and post‐event. Body composition measurements were obtained using bioelectrical impedance analysis. Wrist worn accelerometer devices were utilized to provide an estimation of caloric expenditure and dietary recall provided an assessment of caloric intake. Blood serum samples were collected, centrifuged on‐site, frozen and later analyzed using enzyme linked immunosorbent assays to determine myostatin and follistatin concentrations. Results were analyzed using one‐way ANOVA, presented as means±SEM and considered significant at P<0.05.

Results

Ten participants (37±10 yr, body mass index: 24.4±2.6 kg/m2; 8 males, 2 females) were recruited, and five male participants completed the entire event in 260±9 hours. Caloric intake/expenditure was 4,244±471 kcal/day and 6,669±427 kcal/day, respectively, indicating a caloric deficit of 2,425±653 kcal/day. Total mass, body mass index and fat mass were reduced at each time point of the event. Fat free mass was unchanged throughout the event (Pre‐event: 60±3 kg, C1: 60±3 kg, C2: 60±3 kg, Post‐event: 59±1 kg). Follistatin was increased at C1 (1,715±310 pg/mL) in comparison to all other time points. Myostatin trended towards an increased level at C1 (20,494±5,531 pg/mL).

Conclusions

Despite a remarkable caloric deficit and extreme cold conditions, fat free mass was preserved in participants traveling on foot in the event. Transient alterations in follistatin and myostatin may occur during chronic exercise and may potentially influence the regulation of lean tissue retention under these conditions. Future studies will be directed at the role of nutrient strategies and sleep deprivation on cytokines and physiological resilience under these conditions.

Support or Funding Information

Research reported in this publication was primarily supported by the DLR grant 50WB1330. Additional support was also provided by the National Institute Of General Medical Sciences of the National Institutes of Health under Award Numbers UL1GM118991, TL4GM118992, or RL5GM118990. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.