bluuebunny/crossref_metadata_2025 huggingface dataset:
A novel cross talk between mechanosensitive ion channel TRPV4 and VEGFR2 in human endothelial cells
Authors: Kanugula Anantha Koteswararao , Adapala Ravi Kumar , Cappelli Holly C , Paruchrui Sailaja , Thodeti Charles K
Published: November 2023

Angiogenesis is critical for cardiovascular function, however, insufficient or excessive angiogenesis has been implicated in various diseases. Although both soluble (VEGF) and mechanical factors regulate angiogenesis, most of the studies on angiogenesis are focused on targeting soluble signaling. We have recently shown that mechanosensitive ion channel TRPV4 (transient receptor potential vanilloid 4) negatively regulates angiogenesis via modulation of Rho/Rho kinase‐dependent endothelial mechanosensing. We also found that pharmacological activation of TRPV4 normalized abnormal tumor vasculature and improved cancer therapy. However, it is not known if there is a cross‐talk exists between TRPV4 and VEGF signaling. To explore this cross‐talk, we knocked down TRPV4 in human endothelial cells and measured VEGFR2 expression, localization and phosphorylation. We found a significant increase in VEGFR2 phosphorylation at Y951 (kinase insert domain) and Y1175 (c‐terminal domain) in TRPV4 siRNA knocked down cells. Further, we found that knockdown of TRPV4 increased VEGFR2 expression with concomitant increase in angiogenic transcriptional factor GATA2. Importantly, immunofluorescence analysis revealed reduced accumulation of VEGFR2 in perinuclear Golgi compartment suggesting increased trafficking of VEGFR2. Taken together, our results suggest that TRPV4 mechanotransduction regulates VEGFR2 expression and trafficking and identifies a novel cross‐talk between mechanical (TRPV4) and soluble (VEGF) signaling in human endothelial cells.

Support or Funding Information

NIH‐NHLBI (R01), NIH‐NCI (R15), AHA (Grant‐in‐Aid), NIH‐NHLBI (R15)
Allostery in Nuclear Hormone Receptor Transactivation
Authors: Fernandez Elias John
Published: November 2023

Nuclear hormone receptors (NR) relay cellular signals through distinct multiprotein assemblies. Hormonal signals produce structural changes within NRs that determine the composition of the interacting proteins. NRs are characteristically modular proteins. At the N terminus is the DNA‐binding domain (DBD). The ligand‐binding domain (LBD) contains a dimerization interface that associates with the retinoid X receptor (RXR) and a C‐terminal ligand‐dependent transactivation domain (AF2). Agonist binding results in conformational changes associated with a transcriptionally active state where the AF2 domain rearranges along the receptor surface creating a docking site for coactivator proteins such as the steroid receptor coactivator 1 (SRC1). Using the constitutive androstane (CAR) and thyroid hormone receptors (TR) as typical NR systems, we propose models for allostery within NRs that address the following questions: (i) What role do ligands play in the stoichiometric assembly of the NR:coregulator complex? (ii) How do discrete sites regulate the activity of these receptors? (iii) How do the DBD and LBD communicate to regulate coactivator and DNA recognition? With biophysical and cell‐based transcription assays (PMID:25053412; 22474364; 19561066) we report (1) that agonist ligands can potentiate transactivation through both coactivator‐binding sites on CAR:RXR, which distinctly bind two SRC1 molecules; (2) on the structural basis for negative cooperativity between the TR and RXR ligand‐binding pockets in TR:RXR. We also propose a novel ‘frustrated fit’ allosteric mechanism (PMID: 25658131 & 27110634) and (3) on quantitative evidence of direct, DNA‐dependent, communication between the DBD and LBD.
Cardiovascular Protective vs. Anti‐Cancer Properties: Novel Actions of the AT2R Agonist, NP‐6A4
Authors: Belenchia Anthony M , Beauparlant Paige , Mahmood Abuzar , Bajwa Jamal , Zhang Qiong , Khare Sharad , Pulakat Lakshmi
Published: November 2023

Angiotensin II receptor AT2R is a member of the anti‐inflammatory branch of Renin‐Angiotensin System that exerts vasodilative, cardioreparative, and neuroprotective effects. AT2R agonist therapy represents a potential approach to treating cardiac repair following ischemia. However, low level expression of AT2R encoded by the Agtr2 gene in adult tissues present challenges in using AT2R activation as a therapy for cardiovascular protection. AT2R agonists such as CGP42112A (a peptide agonist) or C21 (a non‐peptide agonist) are not reported to increase Agtr2 expression. We previously reported that NP‐6A4, a novel peptide agonist of the AT2R (from Novopyxis Inc), could improve cell survival and viability of serum‐starved mouse cardiomyocyte HL‐1 cells and primary human coronary artery vascular smooth muscle cells (hCAVSMC). Here we report that treatment of hCAVSMC with NP‐6A4 (1μM) for 48 hours resulted in significant increases in Agtr2 expression (up to 4 fold; p<0.05). To determine whether NP‐6A4 could increase Agtr2 expression in non‐vascular cells, we investigated the effect of NP‐6A4 on human dopaminergic neuronal cell line SH‐SY5Y and the human hepatocellular carcinoma cell line, HepG2. SH‐SY5Y cells are an in vitro model of neuronal function and differentiation. NP‐6A4 treatment (48 hours) increased Agtr2 expression in SH‐SY5Y cells (2‐fold; p<0.05). Similarly NP‐6A4 treatment of HepG2 cells (48 hours) resulted in a 3.2‐fold increase in Agtr2 expression (p<0.05). Interestingly, while NP‐6A4 improved neurite outgrowth of SH‐SY5Y cells, it suppressed cell growth of HepG2 cells. HepG2 cell growth and viability was determined by xCELLigence real‐time cell analyzer (RTCA), which provides a single cell index (CI) value based on cell size, proliferation, and adhesion. HepG2 cells treated with NP‐6A4 showed a significant reduction in CI compared to untreated cells (p<0.05). Moreover, NP‐6A4 treatment entirely repressed proliferation of HepG2 cells, as determined by MTS assay (p<0.05). To further determine whether NP‐6A4 could inhibit growth of other cancer cell lines, we investigated the effect of NP‐6A4 on the human breast adenocarcinoma cell line MCF‐7. Treatment of MCF‐7 cells with NP‐6A4 (48 hours) significantly reduced both their CI (determined by RTCA) and cell proliferation (determined by MTS assay) compared to untreated cells (p<0.05). These data demonstrate that in addition to the well‐documented cardioprotective effects of AT2R agonists, NP‐6A4 is capable of inhibiting carcinoma cell growth—highlighting its unique capabilities as an onco‐cardiovascular therapeutic.
Myoendothelial junctions in in vitro model of human pulmonary microvessels
Authors: Bhatta Anil , Pagaduan Jayson , Gracias David , Hartley Lewis H.
Published: November 2023

Pulmonary hypertension (PAH) is a cardiovascular disease associated with augmented vasoconstriction, inflammation, an imbalance of vasoactive mediators, and vascular remodeling. Understanding pathophysiological mechanisms that cause PAH is key to designing effective therapies and reducing the health care cost burden associated with this disease. Intercellular communication within the blood vessel wall plays an important role in pulmonary resistance vessel function. Myoendothelial junctions (MEJ), have been cited as key elements in vascular physiology and disease, but less is known about the role of MEJ in the development and progression of pulmonary hypertension. Using a well‐established vascular cell co‐culture (VCCC) model, we have successfully reproduced a model of MEJ using human pulmonary microvascular endothelial cells (HPMEC) and pulmonary artery smooth muscle cells (HPASMC). Lattice Light Sheet Microscopy was used to visualize cellular projections from HPMEC through the porous Transwell® membranes onto the HPASMC. Immunoblotting was used to verify segregation of HPASMC from HPMEC by expression of the cell‐specific markers SM‐a‐actin and VE‐cadherin, respectively, and VE‐cadherin was also expressed in the MEJ fraction. We hypothesized that hypoxia would diminish NO signaling through MEJ. We measured a marked reduction in cyclic GMP levels in HPMEC that were selectively harvested from VCCC after exposure to hypoxic conditions (1% oxygen) for 24h. We anticipate similar reductions in NO and cGMP in the MEJ and SMC fractions from hypoxic VCCC. This VCCC system with functional MEJ may be used as an in vitro model of the human pulmonary microcirculation, in order to test specific aspects of pulmonary hypertension pathobiology and therapy.
SMAD3 contributes to lung vascular remodeling in pulmonary arterial hypertension via MRTF disinhibition
Authors: Zabini Diana , Granton Elise , Bonnet Sandra Breuils , Bonnet Sebastién , Morrell Nicholas , Connelly Kim , Provencher Steeve , Ghanim Bahil , Klepetko Walter , Olschewski Andrea , Kapus Andras , Kuebler Wolfgang
Published: November 2023

Introduction

Vascular remodeling in pulmonary arterial hypertension (PAH) results from smooth muscle cell hypertrophy and proliferation of vascular cells. Loss of bone morphogenetic protein receptor 2 (BMPR‐II) signaling and increased signaling via transforming growth factor β (TGF‐β) and its downstream mediators SMAD2/3 has been proposed to drive lung vascular remodeling; yet, proteomic analyses indicate a loss of SMAD3 in PAH.

Objective

We thus proposed that SMAD3 may be dysregulated in PAH, and that loss of SMAD3 may present a master switch in the disease process. Specifically we hypothesized that SMAD3 loss may promote lung vascular remodeling via disinhibition of its interaction partner myocardin‐related transcription factor (MRTF) which drives muscle protein expression.

Methods and Results

SMAD3 was downregulated in lungs of PAH patients, and in pulmonary arteries of three PAH animal models (monocrotaline or sugen/hypoxia rats, and BMPR2+/R899X mice with dysfunctional BMPR‐II). TGF‐β treatment replicated the loss of SMAD3 in pulmonary artery smooth muscle (huPASMCs) and endothelial (huPAECs) cells. SMAD3 silencing increased proliferation and migration in huPASMCs and huPAECs. Co‐immunoprecipitation revealed reduced interaction of MRTF with SMAD3 in TGF‐β treated huPASMCs and pulmonary arteries of PAH animal models. In huPASMC, loss of SMAD3 or BMPR‐II increased smooth muscle actin expression, which was attenuated by MRTF inhibition. Conversely, SMAD3 overexpression prevented actin stress fiber formation in BMPR2 silenced huPASMC. MRTF inhibition attenuated PAH and lung vascular remodeling in sugen/hypoxia rats.

Conclusion

Loss of SMAD3 presents a novel central pathomechanism in PAH that promotes both vascular cell proliferation and ‐ via MRTF disinhibition ‐ hypertrophy of huPASMC, thereby reconciling the parallel induction of a synthetic and contractile huPASMC phenotype. While basal MRTF signaling has been shown to maintain BMP‐regulated vascular homeostasis in healthy lungs, disinhibition of MRTF following functional loss of BMPR‐II and subsequent downregulation of SMAD3 drives lung vascular remodeling in PAH.

Support or Funding Information

This work was supported by the Erwin‐Schroedinger Fellowship of the Austrian FWF Foundation to DZ, and grants‐in‐aid from the Canadian Institutes of Health Research (CIHR) and the Heart & Stroke Foundation of Canada (HSFC) to WMK
Rapid High‐Fat Diet‐Induced Glucose Intolerance is Not Associated with Hepatic Interleukin‐6 Resistance
Authors: Townsend Logan K. , Trottier Sarah K. , Peppler Will T. , Wright David C.
Published: November 2023

Background

High‐fat diet (HFD)‐induced hepatic insulin resistance is associated with attenuated hepatic interleukin‐6 (IL‐6) action. This is important as ablation of hepatic IL‐6 signalling worsens systemic glucose tolerance. Hepatic IL‐6 resistance may result from increased content or activity of Src homology phosphatase 2 (SHP2) in the liver, a phosphatase which dephosphorylates STAT3, a downstream IL‐6 signaling intermediate.

Objective

Determine if the initial development of glucose intolerance with a high fat diet is associated with the onset of liver IL‐6 resistance.

Hypothesis

HFD will rapidly induce hepatic IL‐6 resistance, in association with impairments in whole body glucose homeostasis and increased liver SHP2 content and activity.

Methods

C57bl/6 mice (10 per group) had ad libitum access to standard chow or HFD (60% kcal from fat) for 3 d. On the 4th d, mice were fasted for 6 h before an intraperitoneal (IP) glucose tolerance test. The following day half of the mice in each group were injected IP with IL‐6 (3 ηg kg−1 body weight) or sterile saline. Tissues were harvested 15 min post‐injection. STAT3 phosphorylation and SHP2 activity were assessed via Western blotting.

Results

Mice fed a HFD were glucose intolerant (P<0.001). This was not associated with attenuated hepatic STAT‐3 phosphorylation following IL‐6 injection. Nor was hepatic SHP2 content or activity increased in HFD mice.

Conclusions

HFD rapidly causes systemic glucose intolerance but this is not the result of blunted hepatic IL‐6 signalling.

Support or Funding Information

Ontario Graduate Scholarship and Dairy Farmers of Ontario Doctoral Research Assistantship (LKT) and Natural Sciences and Engineering Research Council and Canada Research Chair (DCW)
Lipopolysaccharide Induced Activation of VRAC (LRRC8) in Nodose Neurons Is Dependent on TLR4 Receptor Mediated ROS Generation
Authors: Wang Runping , Lu Yongjun , Cicha Michael Z , Chapleau Mark W , Abboud Francois M
Published: November 2023

The Volume‐Regulated Anion Channel and its essential subunit Leucine Rich Repeats Containing protein 8a (VRAC/LRRC8) is a widely expressed Cl− channel critical for cell volume regulation. This channel is activated by osmotic gradient and ionic strength (Voss et al., 2014, Qiu et al., 2014, Syeda et al., 2016). Using patch‐clamp techniques, we reported that channel activation is also induced in the majority of vagal afferent neurons isolated from mouse (C57BL/6) nodose ganglia by hypoosmolarity and by extracellular low pH (Wang et al., 2017). We have also observed that this current is activated by lipopolysaccharide (LPS) at 10μg/ml. Here we tested the hypothesis that the response to LPS is dependent on toll‐like receptor 4 (TLR4) and is mediated by intracellular reactive oxygen species (ROS). The current was reduced from 26.7±5.4 pA/pF (n=5) in control peptide‐treated neurons to 6.4±2.1 pA/pF (n=6, p<0.05) in neurons treated with the TLR4 inhibitor peptide VIPER. Application of LPS at a lower dose (100ng/ml) increased intracellular neuronal ROS measured by dihydroethidium within several minutes. The cell permeable hydrogen peroxide scavenger PEG‐catalase decreased the LPS‐induced current from 10.2±2.5 pA/pF (n=7) to 2.3±1.2 pA/pF (n=5, p<0.05). We conclude that LPS induces activation of the VRAC/LRRC8 channel in nodose neurons via TLR4 receptor‐mediated ROS generation. The results indicate a diverse functional role of this channel beyond volume homeostasis with potential implications in high fat diet‐ or LPS‐induced decrease in satiety signaling in intestinal vagal afferents.

Support or Funding Information

HL14388
Bisphenol‐A Alters Human Neutrophil Function
Authors: Balistrieri Angela , Insel Paul A , Corriden Ross
Published: November 2023

Bisphenol‐A (BPA), a synthetic organic compound, is frequently present in plastic‐lined cans, water bottles, toys, and teeth sutures. Previous studies have shown that BPA can produce adverse health effects that include defects in reproductive function and altered prenatal/childhood development. However, little is known regarding the effects of BPA on immune function. In this study, we assessed the effect of BPA on human polymorphonuclear neutrophils (PMNs), a critical component of the innate immune system's defense against pathogens. Using the fluorescent reactive oxygen species (ROS) dye DCFDH, we found that BPA induces a concentration‐dependent increase in ROS generation by PMNs. The membrane‐permeable calcium chelator BAPTA‐AM and/or removal of extracellular calcium inhibited BPA‐induced ROS production, indicating that the process is calcium dependent. Pre‐incubation of PMNs with chelerythrine chloride, a protein kinase C (PKC) antagonist, also inhibited BPA‐induced ROS production, implying that this action of BPA is PKC‐dependent. Transwell chemotaxis assays revealed that BPA exposure reduces the chemotactic capacity of PMNs in a gradient of the bacterial cell wall component f‐Met‐Leu‐Phe, a potent chemoattractant. Our findings reveal that BPA alters the in vitro function of PMNs, including ROS production and chemotaxis, and raise the possibility of altered innate immunity in vivo, especially in those with compromised immune function and who can be exposed to BPA in a wide variety of products.
Mitochondrial Toxicity in Breakdown Products of the Lampricide 3‐trifluoromethyl‐4‐nitrophenol (TFM)
Authors: Dunn David A
Published: November 2023

Sea lamprey (Petromyzon marinus) is an invasive species in the Great Lakes basin. Lamprey control is partially accomplished via 3‐trifluoromethyl‐4‐nitrophenol (TFM) application in Great Lakes tributaries to eradicate lamprey larvae. TFM toxicity occurs via dissipating the proton gradient across the inner mitochondrial membrane and thus uncoupling oxidative phosphorylation. Following deployment of TFM in tributaries, it is converted into a reduced form and various organic and inorganic compounds. Despite its widespread use as a lampricide, physiological/toxicological effects of TFM degradation products are understudied. This study tested the hypothesis that TFM breakdown products display mitochondrial toxicity. Respiration defects were detected in rainbow trout (Oncorhynchus mykiss) liver mitochondria in the presence of TFM breakdown products. These results suggest that further studies are warranted into the potential toxic effects of TFM degradation products.
Incidental Discovery of Supernumerary Breast Tissue in Dissection Course: Case Report and Review of Literature
Authors: da Rocha Andrea Oxley , Bonatto‐Costa João , Pedron Julia , De Campos Deivis , Tomaz Gabriela Rocha , Bodi Mohamed Anaas , Pacini Gabriel Sartori
Published: November 2023

Accessory mammary tissue is rare in the clinical practice, and may be present as polythelia or polymastia. Polythelia (one or more supernumerary nipples) is a congenital condition, being detectable during childhood. Polymastia (more than two mammary glands) may not be evident until puberty. During the seventh gestational week, the embryologic mammary line (“milk line”) is represented by two thickened strips of ectoderm, which extend from the axilla to the inguinal region, and only a small portion of this line might persist. In case it doesn't disappear, an accessory breast may develop anywhere along the streak. The objective of this work is to report the finding of accessory breasts in the lower thoracic wall of a body belonging to the Body Donation Program at UFCSPA. The body in question was female, Caucasian, 84 years old, with no history of breast cancer. During dissection, two accessory breasts were identified in the lower thoracic wall, below the chest muscles, equidistant from each other, the right one measuring 3,0 × 2,7cm, and the left measuring 2,5 × 2,5cm. Samples for histological analysis were extracted and using hematoxylin and eosin staining. Breast tissue was identified, confirming the hypothesis of polymastia. Accessory breast tissue is an uncommon finding in women (2–6%) as well as in men (1–3%). Usually it develops along the course of the embryologic mammary line, and may be solitary or multiple, arising on one or both sides of the line. They are usually asymptomatic, not demanding medical intervention. However, in cases of development in females, the surgical resection of the accessory breast may be necessary, once there is greater symptomatology due to the influence of hormonal variations. There are reports of neoplasic proliferation in this tissue (0,3%). Therefore, this finding becomes important in the medical practice, since it should be remembered as a possible source of malignant neoplasm, and in the differential diagnosis of other soft‐tissue tumors.
Chemical and Histological Analysis of Leptomeningeal Plaques
Authors: Coovert Daniel , Millspaugh‐Storms Brittany , Fitch Richard
Published: November 2023

Study Objective

This study was carried out to investigate the composition of arachnoidal plaques discovered during routine cadaver dissection during gross anatomy courses at Indiana State University.

Background

Leptomeningeal calcified plaques are uncommon formations that have been periodically described in dissection or in autopsy for over 100 years (1,2). These typically benign leptomeningeal plaques are usually asymptomatic, and are distinct from those associated with the chronic proliferative disorder Arachnoiditis ossificans (AO). The etiological cause of arachnoidal plaques and AO are unknown, but iatrogenic, traumatic, metastatic, and infective factors have been associated with these disorders (2).

Methods

Leptomeningeal plaques identified during routine cadaver dissection were excised and examined by SDS‐PAGE, histological stains, and electron microscopy. Proteins present in the inclusion were resolved on a 10% gel, and were detected using silver staining. Presence of surface protein was detected using amidoblack. Ultrastructral analysis and elemental composition was carried out using a JOEL 7008F Field Emission Scanning Electron Microscope at the Integrated Nanosystems Development Institute (INDI) at Purdue University Indianapolis.

Results

Amidoblack staining indicated the plaques are covered by a thin protein layer derived from the arachnoid. Electron microscopy confirmed the surface is composed of a fibrous network, presumably collagen fibers derived from the arachnoid. These results indicate the plaque formation process initiates within the arachnoid rather than aggregating on the luminal surface of the arachnoid and growing into the subarachnoid space. Elemental analysis indicates the plaque is highly biased toward carbon with little nitrogen and lacks calcium or phosphorus. This suggests the plaque is highly enriched with lipid. Protein analysis indicated the protein composition was similar to the composition of the arachnoid.

Conclusions

Leptomeningeal plaques are lipid rich inclusions that form within the arachnoid layer.
Hypothalamic vasopressinergic and orexinergic pathways converge with midbrain aminergic inputs on LHb intrinsic GABAergic system regulated by neurosteroid signaling: implications for contingency‐dependent motivated behavior
Authors: Zhang Limei , Hernandez Vito S. , Eiden Lee E.
Published: November 2023

We have recently reported the existence of a vasopressinergic input from the hypothalamic paraventricular nucleus (PVN) to the medial division of the lateral habenula (LHbM), reporting to a subpopulation of putative GABAergic interneurons there. Stimulation of this pathway influences escape behaviors in the rat. Here, we identify additional hunger‐related orexinergic and reward‐related midbrain aminergic (dopaminergic and serotoninergic) inputs converging on this habenular cell group, and uniquely expressing the estrogen receptor ERalpha. The origins of each of the projection types were determined by fluorogold retrograde tracing. Orexinergic, dopaminergic and serotonergic projections originate in lateral hypothalamus (LH), ventral tegmental area (VTA) and substantia nigra (SN), and dorsal raphe (DR), respectively. They share, with the vasopressinergic inputs from the PVN, sex‐steroid responsivity, i.e. they express androgen receptors (ARs) and aromatase, the latter visualizable in axon terminals within the habenula. The distinct LHbM cell population onto which these inputs converge are likely to be functionally GABAergic, as determined by in situ hybridization histochemistry identifying the expression of gad1, gad2, Slc6A1, Slc6A11, Slc32A1 within them. This cell group also expresses mRNA encoding receptors for serotonin, dopamine, vasopressin, and orexin. Of physiological importance, AR and aromatase expression in the input neurons from PVN, LH, VTA/SN and DR are dependent on both estrogen levels, in female rats, and history of sexual activity, in male rats. Castration‐induced behavioral changes in passive vs active coping strategies before a predator were also observed. We postulate that all four pathways studied here contribute to the hormonal/homeostatic control of behavioral motivation in a coordinated or convergent fashion, through the use of estrogen as a co‐transmitter impingent on LHbM neurons. These findings are relevant within a broader physiological context, reflecting the influence of hunger and thirst circuitry on contingency‐dependent motivated behavior.

Support or Funding Information

Partially supported by: DGAPA‐UNAM‐PAPIIT‐IN216214, CONACYT‐CB‐176919 & CB‐238744 to LZ and NIMH‐IRP‐1ZIAMH002386 to LEE. LZ is a Fulbright visiting scholar, also supported by PASPA‐DGAPA‐UNAM fellowships for her sabbatical research stay hosted by LEE of SMN‐NIMH‐NIH‐USA.
Quantum Dynamics Neuronal Stimulation by Sigma Photonics Quantum Energy
Authors: Einstein George P , Anaushvili Avtavandil P , Tulp Orien L
Published: November 2023

It is well known that the human brain generates waves (oscillation). For example, it is known that brain generates electromagnetic waves, and this phenomenon is used in electro encephalography. In a given model of a human brain, the two hemispheres are considered as two independent generators of oscillation processes. The mathematical description of the specified wave model of a brain has been determined (Anuashvili). The mathematical description of the specified wave model of a brainwill be presented. For the oscillatory processes occurring in the hemispheres, we shall present the mathematical formulae we have developed. The domination of one of hemispheres can be determined by the size of oscillitory displacement of the source of the oscillations during an exposition or proportional value ‐ difference of amplitudes of oscillatory processes occurring in hemispheres. Thus, in wave model of a brain two parameters are allocated:

Domination of one of hemispheres,

Coordination (coherence) of oscillatory processes in hemispheres.

Thus, this research determned the mathematical formulae to describe hemispheric electromagnetic wave functions and may contribute to the clinical diagnostic profile of the individuals under study under a variety of clinical conditions.

Support or Funding Information

Institutional Support of the Einstein Medical Institute and the College of Medicine, University of Science Arts and Technology, Montserrat, British SWest Indies.
NMDA receptor 2B Subunit and BK Channel May Affect Mechanical Allodynia by Regulating the Activity of Enkephalinergic Neurons in the Spinal Dorsal Horn
Authors: Tanaka Shiho , Kato Eiko , Kofuji Takefumi , Fukushima Teruyuki , Hori Yuuichi
Published: November 2023

Enkephalin (ENK)‐containing neurons in the spinal superficial dorsal horn (SDH) play an important role in regulating the transmission of nociceptive information. Using BAC transgenic mice, in which ENK‐containing neurons can be identified by green fluorescent protein expressed under the control of preproenkephalin gene promoter, we have observed that ENK‐containing neurons in the SDH express the NMDA receptor 2B (NR2B) subunit and large conductance calcium‐activated potassium (BK) channels. In experiments, we investigated the effects on nociceptive behavior of intrathecally administrating NR2B subunit antagonist and BK channel antagonist in peripheral nerve‐injured mice.

The experiments were performed in 6‐ to 8‐week‐old male ICR mice. Partial sciatic nerve ligation was performed as described previously (PSL, Seltzer model). To assess the development of mechanical allodynia, we measured the frequency of withdrawal responses to 10 repetitive stimuli with von Frey filaments. Behavioral assessments were performed beginning 3 days prior to the ligation surgery. On post‐surgical day 7, effects on nociceptive behavior of the BK channel antagonist, charybdotoxin (CTX), and the NR2B antagonist, ifenprodil (IFN), were analyzed. Additionally, in order to clarify whether the actions of CTX and IFN are mediated by an alteration in the release of enkephalin, we investigated the effect of the selective δ‐opioid receptor antagonist naltrindole (NTI).

CTX (1 pmol/10 μL per mouse), IFN (50 nmol/10 μL per mouse) and saline as a control (10 μL per mouse) was intrathecally injected according to the modified method originally described by Hylden. All behavioral experiments were performed in double‐blind fashion.

PSL significantly increased the occurrence of withdrawal reflex to von Frey stimuli being indicative of the development of mechanical allodynia. Intrathecal CTX significantly reduced the occurrence of withdrawal reflex as compared to saline group. Additionally, intraperitoneal administration of NTI at the dose of 5 mg/kg body weight attenuated the effect on nociceptive behavior of CTX. Intrathecal IFN significantly reduced the occurrence of withdrawal reflex. This effect was also blocked by intraperitoneal NTI.

These behavioral observations might suggest that NMDA receptors and BK channels inhibit the activity of enkephalin‐containing neurons in the spinal dorsal horn, which is speculated to be involved in peripheral nerve injury‐induced chronic pain state.

Support or Funding Information

This work was supported by Dokkyo Medical University Endowment Fund.
Investigating the Role of the DYW Deaminase in RNA Processing
Authors: Aldana‐Mendoza Jonathan A , Hayes Michael lloyd
Published: November 2023

RNA editing is the process by which ribonucleic acid (RNA) molecules are enzymatically modified post synthesis on specific nucleosides. In land plants it is mostly comprised of cytidine (C) to uridine (U) transformations linked to proteins with the DYW deaminase domain within the pentatricopeptide repeat (PPR) protein family. The DWY deaminase domain is likely the catalytic RNA editing domain because of its conserved glutamate residue of the HXE motif, required in other known catalyzed cytidine deaminases. PPR proteins have been shown to play important roles in transcript maturation. The molecular specificity of RNA‐binding proteins is essential for RNA regulation. Understanding RNA recognition also allows re‐coding of RNA‐binding proteins that may be employed to modulate RNA regulation in vivo. We plan on investigating the binding of PPRs to natural RNA transcripts by using transition state analogs (TSA's) which could inhibit RNA editing in vitro. Using different synthetic RNA's we can identify the requirements for RNA editing activity. Using chloroplast extracts in vitro RNA editing efficiency can be compared to determine the necessary cis‐‐elements. In order to discover the associated PPR specificity factor. This would also provide insight into the degenerate recognition code of RNA editing.

Support or Funding Information

NIH Grant # GM 49001
Emersion Profiles and Recovery: Quantifying Physiological Responses in Mussels
Authors: Dilly Geoff Fowler , Brazier Garrick David , Christensen Katherine Mary , Moran Randy William
Published: November 2023

Rocky intertidal zones are in a constant state of flux. Sessile intertidal organisms must tolerate a suite of dynamic abiotic conditions: temperature, wave action, pH, hypoxia, and most dramatically, emersion. The duration of emersion also changes daily depending on the tides; additionally, air temperatures shift with diurnal cycles, seasonal differences, and weather conditions. Thus, the West Coast mussel, Mytilus californianus (M.c.), a key indicator species of intertidal community health, must tolerate rapid shifts between a wide variety of submerged and emerged conditional regimes. Previous literature on M.c. stress responses to conditional shifts have focused primarily on respirometric rates during immersion experiments and/or protein turnover with relation to emersion. Our full‐factorial study combines these approaches to explore the respirometric responses of M.c. to emersion at a range of environmentally relevant thermal regimes (air: 7, 18, and 35°C; water: 14 and 24°C), emersion durations (4 and 8 hour), and recovery times (0 and 30 minute) using a custom‐designed respirometry system.

To conduct in vivo respirometry experiments, we built a new customizable isolation system named the Chambers for Organismal Response to Environmental Stressors (CORES) to interface with a NeoFox spectrophotometer (Ocean Optics, Dunedin, Fl). This low‐cost, novel system allows for continual non‐invasive measurements of dissolved oxygen on a variety of marine invertebrates. Mytilus californianus were collected from Ventura, California (34.272° N, −119.287° W) and acclimated at temperature in lab aquaria. After 2‐hour respirometric trials, gills were excised and divided evenly for dry‐weight respirometric calculations and RNA extractions. We quantify key gene expression stress responses using RT‐qPCR with HSP70, HSC71 (thermal), catalase (oxidative), phosphofructokinase (metabolic), and caspase (apoptosis) on a CFX96 RT‐PCR System (Bio Rad, Hercules, CA).

Calculated respiration rates were positively correlated with water temperature and emersion duration, indicating that Mytilus californianus needed to regenerate their depleted aerobic capacity after exposure to these stresses. Additionally, our data suggest M.c. maintained at 24°C experience a stress‐induced state of torpor following 8‐hour periods of emersion at boundary air temperatures (7 and 35°C). A differential response in respiration rate was observed at the median air temperature of 18°C, indicating that air temperatures at the boundaries of environmental conditions increase the time of recovery to homeostasis after mussels are re‐submerged.

Support or Funding Information

This work is supported by CSU Channel Islands and an NSF I‐Corps CSUPERB sub‐award.
Development of a Patient‐Based Computational Modeling Framework for Analyzing the Mechanisms of Doxorubicin Cardiotoxicity
Authors: Lewalle Alexandre , Land Sander , Niederer Steven
Published: November 2023

Doxorubicin, a potent anticancer drug, has well known dose‐dependent cardiotoxic side effects. In particular, doxorubicin exposure disrupts protein expression and impacts multiple biochemical pathways through the generation of reactive oxide species and oxidative stress, which in turn leads to cardiomyopathy and congestive heart failure. Specific effects of doxorubicin exposure at the biochemical, cellular, and tissue levels, have been extensively studied. However, a systematic quantitative assessment of the relative significance of these effects on the separate subsystems of the heart, in the perspective of overall cardiac function, remains to be done. Gaining such an understanding of the mechanisms underlying heart failure is essential for the development of diagnoses and treatments.

Computational modeling provides a unique platform for exploring cardiac function in silico and gaining insight into the interplay between its separate mechanistic contributions, within the context of doxorubicin cardiotoxicity. We therefore developed a multi‐scale computational modeling framework to simulate the heart contraction cycle. The model was designed to be sufficiently detailed to include the main known aspects of cardiac muscle behavior, while retaining enough simplicity to enable its parameterization using clinical and experimental data. This approach provides a “virtual workbench” for simulating cardiac function with maximal objectivity and faithfulness with regard to measurements derived from patients.

The model parameters were constrained using available data (echocardiography, cuff pressure, biopsy‐based proteomics analyses) derived from either doxorubicin‐treated or “control” cohorts. The flexibility of the model allows the determination of the sensitivities of the main cardiac phenotypes to specific parameters, in both cohorts. We thus aimed to identify the predominant pathway for the onset of doxorubicin cardiotoxicity, as characterized by a substantial lowering of the left‐ventricular ejection fraction (LVEF).

Our results suggest that a structural remodeling of the heart (e.g., a widening of the left‐ventricular diameter) contributes partially to the decrease in LVEF. Additional necessary contributions must include changes to the passive or active contractile properties of the heart tissue. By using available proteomics analyses derived from patient biopsies, we aim to discern quantitatively between these contributions, resulting from cumulative doxorubicin exposure.

Support or Funding Information

This study is supported and funded as part of the European Commission HeCaTos Project (Hepatic and Cardiax Toxicity Systems modeling), Proposal 602156‐2.
MicroRNA Profile of Excised Cutaneous Squamous Cell Carcinoma Tissues Comparative to Adjacent Normal Tissue
Authors: Pulford Christopher S. , Loomis Ellise M. , Montgomery McKale R. , Uppalapati Chandana K. , Pascual Agnes S. , Hull Elizabeth E. , Leyva Kathryn J.
Published: November 2023

Cutaneous Squamous Cell Carcinoma (cSCC) is the second most common form of skin cancer commonly arising from sun‐induced mutations. As most cases of cSCC are treated by tumor excision, research aimed at understanding factors influencing the metastasis, and possible alternative treatments, of cSCC is limited. Cancer metastasis is a multistep process, including epithelial‐to‐mesenchymal transition (EMT). During EMT, epithelial cells undergo a dedifferentiation process whereby they transition to a highly mobile and invasive mesenchymal cell type with metastatic capacities. Although the factors influencing EMT are varied, one important mediator of intercellular communication shown to be involved in oncogenesis is miRNA. Our work focuses on miRNAs, which are small non‐coding RNA species that may be one epigenetic factor contributing to the progression of cSCC. In this study, we compared the expression pattern of seven miRNAs (miR‐21, ‐31, ‐34a, ‐125b, ‐199a, ‐204, and ‐1246) between fresh primary tumor samples and fresh adjacent normal tissue (ANT) collected from patients diagnosed with either cSCC, SCC in situ, or invasive SCC. Total RNA was isolated from each of the samples and qPCR on all samples was performed to determine the expression level of each miRNA relative to control. Of the seven miRNA tested, two were significantly upregulated (miR‐21 and miR‐31) in the patient SCC samples compared to ANT. As many of these miRNAs are predicted to target genes involved in EMT in other cancers, we have begun examining the expression of known EMT markers via qPCR and/or Western blot from RNA and protein isolated from both SCC and ANT tissues. Preliminary results indicate that mRNA expression of TGFβ1 is significantly increased and mRNA expression of E‐cadherin is significantly reduced in cSCC relative to normal tissue, indicative of a progression of EMT in the cancerous tissue. Other EMT markers are being examined as well. Further work investigating the function of these differentially‐expressed miRNAs will help to begin elucidating the role miRNAs are playing in the progression of cSCC.

Support or Funding Information

Biomedical Sciences Program and MWU Intramural Funds
Effects of Ketogenic Diet in Young Adult and Old Rats
Authors: Duplisea Michael J. J. , Wilson Esther K. , Cheung Eric C.K. , Parker Brian A. , Carr Sheryl T. , Ashby Conrad K. , Bikman Benjamin T. , Thomson David M.
Published: November 2023

Ketogenic diets (KD) have been promoted extensively for weight loss and health. However, their effects in aged skeletal muscle are not well‐defined. In this study, we tested the effect of a ketogenic diet in young adult (YA; 5 month old) and old (O; 28 month old) male Fisher 344 rats (n = 4–6/group). Rats were assigned to either standard chow (STD; Evigo Teklad Rodent Diet, 8604; 32% protein, 14% fat, 54% carbohydrate) or ketogenic diet (KETO; Envigo Teklad Custom Diet, TD.10911; 22.4% protein, 77.1% fat, 0.5% carbohydrate) for 4 weeks, and weighed weekly. KETO rats were pair fed isocalorically with STD chow rats within each age group with the YA rats eating 0.132 kcal/g body weigh/day, and O rats eating 0.109 kcal/g body weight/day. After 24 or 25 days glucose tolerance testing was performed by fasting the rats for 6 hours followed by an injection of 1 mg glucose/g BW. Blood glucose concentration was measured from tail blood immediately before and 15, 30, 60 and 120 minutes after the glucose injection. Ketones were also measured from tail blood immediately before the glucose injection. After the 4‐week treatment period, rats were euthanized and gastrocnemius muscle was collected for determination of mitochondrial respiration and AMP‐activated protein kinase (AMPK; an important stimulator of glucose and fatty acid metabolism) phosphorylation. We found that KETO feeding increased blood ketone levels, albeit non‐significantly (p=0.065), in YA but not O rats. KETO resulted in significant weight loss within 2 weeks in both YA and O rats. Glucose tolerance was impaired in O‐STD vs. YA‐STD rats, while KETO impaired glucose tolerance in YA, but not O rats. KETO increased mitochondrial respiration in YA, but not O rats. AMPK phosphorylation was decreased by KETO vs. STD diet in YA, but not O rats. We conclude that a KETO diet induces a significant drop in body weight and enhanced mitochondrial respiration, but impaired glucose tolerance in YA rats, suggesting a shift from glycolytic to oxidative metabolism. Somewhat surprisingly, these KETO‐induced changes in YA rats are associated with decreased AMPK activation. O rats are resistant to all of these effects of KETO diet except for weight loss, suggesting impaired metabolic plasticity with old age. Further study will be required to elucidate the exact molecular pathways underlying these shifts.

Support or Funding Information

This research was supported by Brigham Young University Mentoring Environment Grants (MEG) and Gerontology Research Grants.
LKB1 Knockout in Skeletal Muscle Improves High Fat Diet‐Induced Insulin Tolerance, but not Glucose Tolerance
Authors: Chen Ting , Thomson David M.
Published: November 2023

Many studies have shown that a long‐term high fat diet can induce impaired glucose and insulin tolerance in mice. In skeletal muscle, LKB1 is a master kinase regulating the AMPK family of protein kinases, which are major regulators of metabolism. LKB1‐knockout in muscle has been shown to improve insulin sensitivity. In this study, we asked whether skeletal muscle specific LKB1‐knockout (KO) can protect mice from long‐term high fat diet‐induced impairment in glucose and insulin tolerance. Control (CON) and littermate LKB1‐KO (KO) mice were fed either high fat (HF) chow or standard (STD) chow for 14 weeks. Body weight and retroperitoneal fat weight increased similarly with HF diet feeding in both CON and KO mice, while the HF diet didn't affect heart and muscle weight. HF diet significantly (p≤0.05) worsened both glucose and insulin tolerance for both CON and KO mice, but the effect on insulin tolerance (but not glucose tolerance) was attenuated by 30.7% in KO vs. CON mice. Phosphorylation of AS160 (an important regulator of glucose transporter‐4 translocation and glucose uptake) at the Akt‐target site, Ser318, increased 64.7% with HF diet in the KO, but did not change in CON mice. Therefore, knocking out LKB1 in skeletal muscle can improve long‐term high fat diet‐induced insulin tolerance, and this may be through increased AS160 phosphorylation.

Support or Funding Information

This work was funded by Brigham Young University a Mentoring Environment Grant and a Graduate Research Fellowship Award.
Micro‐RNA 203 Regulates Myometrial Smooth Muscle Cell Expression of the Transient Receptor Vanilloid 4 Channel
Authors: Ying Lihua , Barnes Elizabeth A , Rodriguez Saidie , Alvira Cristina M , Cornfield David N
Published: November 2023

Background

We recently demonstrated that the expression and activity of the transient receptor vanilloid 4 (TRPV4) channel increase in myometrial smooth muscle cells (mSMC) during pregnancy. However, the molecular mechanisms that dynamically regulate TRPV4 expression and activity have not been identified. Accumulating evidence suggest that micro RNA molecules (miRNA) may influence the physiology of pregnancy by regulating the expression of molecules that affect implantation, uterine contraction, inflammation, and cervical remodeling. Although miR‐203 represses TRPV4 expression in chondrocytes, whether pregnancy associated changes in miR‐203 expression contribute to the heightened expression of TRPV4 in the pregnant myometrium remains unknown.

Objective

We hypothesized that with pregnancy, mSMC miR‐203 expression decreases as TRPV4 expression increases.

Methods

To test these hypotheses, miR‐203 and TRPV4 expression was determined by qPCR in primary mSMC isolated from non‐pregnant and pregnant (D18) mice. TRPV4 gene expression was also determined in human mSMC that were transfected with either non‐targeting control (NTC) or a miR‐203 mimetic. In separate studies, mSMC contraction was assessed in human mSMC transfected with either NTC or miR‐203 mimetic that were embedded in collagen gels.

Results

miR‐203 levels were almost 14‐fold higher in the non‐pregnant as compared to late pregnant mSMC (2.38±0.76 vs. 0.17±0.05, p=0.04). Treatment of human mSMC with the miR‐203 mimic increased miR‐203 expression by over 2000‐fold (p<0.0001), and decreased TRPV4 expression by approximately 90% (p=0.013). In addition, in the collagen gel assays, gels containing mSMC transfected with miR‐203 demonstrated an increase in area of 17% (p=0.016, versus control), consistent with relaxation.

Conclusions

These data demonstrate that in mSMC, miR‐203 suppresses TRPV4 gene expression. We speculate that reductions in miR‐203 expression during late gestation may allow for increased expression of TRPV4 near term, and represent a viable target to suppress uterine contractility, prolong pregnancy and prevent preterm delivery.

Support or Funding Information

Burroughs Wellcome Fund, Pretern Birth Intiative
Regulation of Glucose Metabolism by Contractile Activity in Human Skeletal Muscle Myotubes from Severely Obese Individuals
Authors: Park Sanghee , Houmard Joseph
Published: November 2023

Introduction

Exercise/ or contractile activity can enhance insulin sensitivity and oxidative capacity in skeletal muscle. However, the effects of contractile activity in skeletal muscle from severely obese individuals still remain unestablished.

Purpose

We investigated insulin‐stimulated glucose metabolism in primary skeletal muscle myotubes from severely obese individuals (BMI ≥ 40 kg/m2) in response to contractile activity induced by electrical pulse stimulation.

Method

Fully mature myotubes from lean (BMI: 23.1 ± 0.7 kg/m2) and obese (BMI: 44.9± 2.5 kg/m2) individuals were stimulated for 24‐hours by applying an electrical pulse at 11.5 V, 1 Hz and 2 ms. Following contraction, insulin was added to examine the combined effects of muscle contraction and insulin on anabolic signaling.

Result

In myotubes from the severely obese individuals, insulin‐stimulated glycogen synthesis and glucose oxidation were normalized to the levels seen in muscle from the lean subjects without electrical stimulation. However, responses to electrical stimulation were blunted in the severely obese vs. lean subjects in terms of the magnitude of the improvements in insulin‐stimulated glucose metabolism. In addition, electrical stimulation increased insulin‐stimulated lactate production in severely obese but not lean subjects. Electrical stimulation increased adenosine monophosphate (AMP) accumulation and AMP‐activated protein kinase (AMPK) signaling, but to a lesser degree in myotubes from the severely obese donors. Electrical stimulation provided an improvement in insulin signal transduction in lean but not severely obese individuals. Especially, sites on AS160 phosphorylation (T642, S588, S341) showed enhanced response to electrical stimulation in lean but no changes in severely obese individuals.

Conclusion

These data reveal that in the skeletal muscle of severely obese individuals, contractile activity can improve insulin‐stimulated glucose metabolism independent of insulin signaling. In addition, transient increase in AMPK signaling by accumulation of AMP for contractile activity appears to play a critical role in enhancing glucose metabolism in severely obese individual in response to electrical stimulation.

Support or Funding Information

DK56112