bluuebunny/crossref_metadata_2025 huggingface dataset:
Post‐menopausal Cerebral Microvascular Alterations in a Mouse Model of Alzheimer’s Disease
Authors: Blackwell Jade , Louis Emma , Uhlorn Josh , Brooks Heddwen L. , Pires Paulo W.
Published: May 2022

Cognitive decline, as seen in Alzheimer’s disease (AD), is a growing public health concern and is linked to decreased cerebral blood flow, particularly in women after menopause. Impaired cerebrovascular function precedes the onset of AD, possibly due to reduced endothelial function in parenchymal arterioles, the bottlenecks of the cerebral microcirculation, although exact mechanisms by menopause remain elusive. The goal of this study was to determine whether menopause impairs endothelial function in parenchymal arterioles of wild‐type (WT) and 5x‐FAD mouse model of AD. Menopause was induced by the 4‐vinylcyclohexene diepoxide (VCD) model, which induces gradual ovarian failure. Cerebral parenchymal arterioles were isolated and studied by pressure myography. Data are means ± SEM. In WT mice, menopause caused a significant increase in myogenic tone (myogenic tone: 24.73 ± 1.2 vs. 34 ± 2.6%, vehicle vs VCD, n = 5 / 8; p<0.05), as well as a small but significant reduction in resting lumen diameter (32.68 ± 1.3 vs. 28.40 ± 2.1 µm, vehicle vs VCD, n = 5 / 8; p<0.05). We then tested endothelial function, focusing on K+ channels, namely small and intermediate Ca2+‐activated K+ channels (SKCa / IKCa) and inwardly rectifying K+ channels (KIR2). Menopause did not affect arteriolar dilation to a cumulative concentration‐response curve of the SKCa/ IKCa activator NS‐309 (at 1 µM, vasodilation: 23.49 ± 11.5 vs. 17.80 ± 1.7%, vehicle vs VCD, n = 3 / 6). Similarly, KIR2 function was unchanged (vasodilation: 16.06 ± 6.6 vs. 16.21 ± 2.9%, vehicle vs VCD, n = 4 / 8). In the 5x‐FAD mice, menopause did not affect myogenic tone (myogenic tone: 23.49 ± 2.2 vs. 28 3.2%, vehicle vs VCD, n = 10 / 7), although there was a significant decrease in resting lumen diameter (39.16 ± 2.6 vs. 27.79 ± 2.0 µm, vehicle vs VCD, n = 10 / 7 p<0.05). Arterioles from menopausal 5x‐FAD mice showed a significantly blunted response to NS‐309 (at 1 µM, vasodilation: 14.96 ± 2.9 vs. 9.42 ± 1.4%, vehicle vs VCD, n = 7 / 4, p<0.05), without differences in KIR2‐induced dilation (vasodilation: 16.02 ± 1.3 vs. 13.69 ± 2.3%, vehicle vs VCD n = 9 / 6). In conclusion, we found that in WT mice, menopause leads to changes in myogenic tone without affecting K+‐dependent dilation. However, in 5x‐FAD mice, menopause impairs endothelial SKCa/IKCa channels independent of KIR2 function or myogenic tone. These findings identify new targets modulated by menopause in the cerebral microvasculature of WT and AD mice, which may contribute to cognitive dysfunction.
Sialylation of CD11b/CD18 regulates Neutrophil Transepithelial Migration and Neutrophil Inflammatory function in the Intestine
Authors: Brazil Jennifer , Azcutia Veronica , Kelm Matthias , Fink Dylan , Cummings Richard , Nusrat Asma , Parkos Charles
Published: May 2022

Neutrophils (PMNs) are the first immune responders to infection/inflammation playing critical roles in clearing invading microbes and promoting tissue repair. However, dysregulated trafficking of PMNs across mucosal surfaces is a pathological hallmark of diseases characterized by persistent or intermittent bursts of inflammation including inflammatory bowel disease (IBD). The critical final step in PMN trafficking into mucosal lined organs (including the gut) involves transepithelial migration (TEpM). The glycoprotein CD11b/CD18 is the predominant β2 integrin mediating PMN TEpM into the intestinal lumen. Furthermore, CD11b/CD18 also regulates key PMN inflammatory effector functions that are implicated in IBD pathogenesis including superoxide release. We have previously demonstrated that specific GlcNAc and Lewis X glycan structures on CD11b/CD18 can be targeted to reduce PMN intestinal trafficking. Recent mass spectrometry analyses revealed the presence of specific sialylated glycan structures on PMN CD11b. However the role of sialylation in regulating PMN CD11b/CD18 function is not well understood. Using lectins recognizing the most common linkages of sialic acid we show that sialidase mediated removal of α2‐3 sialic acid from CD11b/CD18 occurs during PMN migration across intestinal epithelia. Furthermore, specific inhibition of sialidase activity was found to reduce PMN intestinal migration in vitro and in vivo. In addition to limiting PMN intestinal trafficking, sialidase inhibition also reduced key PMN effector functions including degranulation and superoxide release. Finally, we demonstrate that sialidase inhibition impedes PMN CD11b activation and blocks downstream β2 integrin signaling mediated by Spleen tyrosine kinase (Syk). Therefore α2‐3 sialylated glycans on CD11b/CD18 represent novel targets for ameliorating PMN mediated intestinal tissue damage and reducing mucosal inflammation in IBD.
Synergistic Contribution of Epac1‐/‐ and Epac2‐/‐ for Urinary Concentrating Ability
Authors: Stavniichuk Anna , Pyrshev Kyrylo , Tomilin Viktor , Zaika Oleg , Mei Fang , Cheng Xiaodong , Pochynyuk Oleh
Published: May 2022

Epac is a relatively new direct downstream effector of cAMP. Two known Epac isoforms, Epac1 and Epac2 are abundantly expressed in the renal epithelial cells of the proximal tubule and the collecting duct. We recently demonstrated that Epac1‐/‐ and Epac2‐/‐ mice develop polyuria largely due to decreased NHE‐3 expression in the proximal tubule. In contrast, AQP2‐dependent water reabsorption in the collecting duct was unaffected. In this study, we utilized double Epac knockout mice to determine whether both isoforms contribute complementarily to the urinary production and concentrating ability. We found that Epac1&2‐/‐ produced significantly higher 24h urinary volume and lower osmolarities compared to the values in single Epac knockouts. These differences were further exacerbated in response to water deprivation test suggesting a defect in the collecting duct. Consistently, Epac1&2 blockade with ESI‐09 inhibited AQP2 translocation to the apical plasma membrane in CD cells in response to AVP. At the same time, inhibition of a single Epac isoform fails to do so. Overall, our results demonstrate that concomitant Epac1‐/‐ and Epac2‐/‐deletion compromises reabsorption in both PT and CD, thus leading to a much greater in urine volume and decrease urinary concentrating ability than the single isoform Epac knockout.
Olfactory Receptor78 Participates in Carotid Body‐Dependent Sympathetic Activation and Hypertension in Murine Models of Obstructive Sleep Apnea
Authors: Peng Ying‐Jie , Su Xiaoyu , Wang Benjamin , Mathews Timothy , Nanduri Jayasri , Prabhakar Nanduri R.
Published: May 2022

Olfactory receptor (Olfr) 78 participates in carotid body (CB) activation by acute hypoxia. We examined the role of Olfr78 in CB‐dependent sympathetic activation and hypertension in: 1) mice treated with chronic intermittent hypoxia (CIH) patterned after blood O2 profiles during obstructive sleep apnea (OSA) and 2) heme oxygenase‐2 (HO‐2) null mice, which exhibit OSA. CIH treated wild type (WT) mice showed: enhanced CB sensory nerve response to hypoxia and sensory long‐term facilitation (sLTF), hypertension, activation of the sympathetic nervous system as evidenced by spectral analysis of inter‐beat interval as well as elevated plasma nor‐epinephrine (NE) levels. All these responses were absent in Olfr78 null mice. HO‐2 null mice showed spontaneous apneas with an apnea index (AI) of 58±1.2 apneas/hour compared to AI of 8±0.8 apneas/hour in WT mice. CBs of HO‐2 null mice showed enhanced CB response to hypoxia and sLTF as well as elevated plasma NE levels and hypertension. The magnitude of hypertension correlated to AI in HO‐2 null mice (P<0.01). In striking contrast, HO‐2/Olfr78 double null mice showed stable breathing with an AI of 12±1.3 apneas/hour. HO‐2/Olfr78 double null mice showed absence of augmented CB response to hypoxia, sLTF, elevated plasma NE levels and hypertension. These results demonstrate that Olfr78 participates in CB activation and ensuing sympathetic nerve excitation and hypertension in two mouse models of OSA.
Suppression of MT1 and Melatonin Treatment Improves Liver Phenotypes in Mdr2‐/‐ mice
Authors: Ceci Ludovica , Wu Nan , Carpino Guido , Chen Lixian , Zhou Tianhao , Kennedy Lindsey , Kyritsi Konstantina , Francis Heather , Franchitto Antonio , Onori Paolo , Gaudio Eugenio , Glaser Shannon , Alpini Gianfranco
Published: May 2022

Background & Aim

Primary Sclerosing Cholangitis (PSC) is a cholestatic liver disease characterized by hepatic fibrosis and portal inflammation. Melatonin is synthesized by arylalkylamine N‐acetyltransferase (AANAT) in the pineal gland, as well as extrapineal sites such as cholangiocytes. We previously found that: (i) the MT1 receptor is primarily expressed in cholangiocytes with low and absent expression in hepatic stellate cells and hepatocytes, respectively; (ii) melatonin reduces biliary proliferation via MT1 receptor signaling; and (iii) melatonin treatment for 1 wk decreases biliary proliferation and liver fibrosis in bile duct ligated rats by downregulation of MT1 and clock genes (PER1, CRY1, CLOCK and BMAL1). We aimed to evaluate the beneficial effects of long‐term melatonin treatment and MT1 signaling on biliary phenotypes, liver fibrosis and portal inflammation in Mdr2‐/‐ mice (a model of PSC).

Methods

Male FVB/NJ and Mdr2‐/‐ mice had access ad libitum to drinking water with/without melatonin for 3 months. We evaluated: (i) ductular reaction (DR) by immunohistochemistry (IHC) for CK19, (ii) liver fibrosis by Sirius Red staining and (iii) portal inflammation by IHC for F4/80. MT1, AANAT, and clock genes immunoreactivity was evaluated by immunofluorescence (IF) co‐stained with CK19 and qPCR in isolated cholangiocytes. Male C3H‐Hej (WT for MT1‐/‐), FVB/NJ (WT for Mdr2‐/‐), MT1‐/‐, Mdr2‐/‐ mice and MT1‐/‐/Mdr2‐/‐ (DKO) mice were euthanized at 12 wk of age. We analyzed: (i) MT1 expression by IF and mRNA expression in total liver by qPCR to validate our model; (ii) liver damage by H&E, DR by IHC for CK19 and liver fibrosis by Sirius Red staining and (iii) portal inflammation by IHC for F4/80 and expression of the inflammatory markers by qPCR in total liver.

Results

Long‐term melatonin treatment reduces DR, liver fibrosis and portal inflammation in Mdr2‐/‐mice. Prolonged administration of melatonin in Mdr2‐/‐mice improves liver phenotype by decreasing the immunoreactivity of MT1, AANAT and clock genes in Mdr2‐/‐ mice, which suggests the chronobiotic action of melatonin on biliary circadian rhythm. DKO mice have no MT1 expression and display ameliorated liver phenotype compared to Mdr2‐/‐ mice.

Conclusion

We demonstrated that chronic melatonin treatment improves liver histology and restores the biliary circadian rhythm by interaction with MT1. Suppression of MT1 receptor in Mdr2‐/‐mice ameliorates biliary/liver phenotypes through changes in clock genes and AANAT. Restoration of the circadian rhythm by modulation of melatonin/MT1 signaling may be key for the management of cholangiopathies.
Soluble VE‐cadherin disrupts endothelial barrier function via VE‐PTP/RhoA signalling
Authors: Knop Juna‐Lisa , Burkard Natalie , Flemming Sven , Schlegel Nicolas
Published: May 2022

Increased levels of soluble VE‐cadherin fragments (sVE‐cadherin) have previously been linked to inflammation‐induced loss of endothelial barrier function. We hypothesized that sVE‐cadherin independent of pro‐inflammatory stimuli may be critically involved in the context of endothelial barrier dysfunction.

Recombinant human sVE‐cadherin (extracellular domains EC1‐5) was generated and verified by sequencing and immunoblotting. Application of sVE‐cadherin on human dermal microvascular endothelial cells (HDMECs) dose‐dependently induced loss of endothelial barrier function as revealed by measurements of transendothelial electrical resistance (TER) and flux of 70 kDa FITC‐dextran across HDMEC monolayers. In an in vivo rat model i.v. application of sVE‐cadherin resulted in loss of endothelial barrier function and reduced microcirculatory flow, which was comparable to the conditions when bacterial Lipopolysaccharide (LPS) was applied in vivo. Immunofluorescence staining and western blot analysis revealed that the functional effects of sVE‐cadherin were paralleled by decreased expression of VE‐cadherin and reduction of α‐/ β‐/ γ‐/ δ‐catenin and tight‐junction associated protein ZO‐1 at the cell borders. VE‐protein tyrosine phosphatase (VE‐PTP) which is associated with VE‐cadherin under resting conditions was removed from the cell borders following application of sVE‐cadherin to HDMEC. These changes were accompanied by intercellular gap formation and increased stress fibers in the actin cytoskeleton. On a mechanistic level, sVE‐cadherin did not induce cell death but increased RhoA activity and reduced extracellular VE‐cadherin interaction as revealed by atomic force measurements. Rho‐kinase inhibitor Y27632 blocked all sVE‐cadherin‐induced effects on endothelial barrier function. Furthermore, VE‐PTP inhibitor AKB9778 attenuated both RhoA activation and all sVE‐cadherin‐induced effects.

In summary, sVE‐cadherin disrupts endothelial barrier function by dismantling the VE‐cadherin complex at the cell border via VE‐PTP‐dependent RhoA activation. This points to a novel pathophysiological role of sVE‐cadherin in the context of endothelial barrier dysfunction in inflammation.
Cerebral Ischemic Reperfusion Increases Autotaxin Expression Elevating Brain Endothelial Permeability
Authors: Bhattarai Susmita , Sharma Sudha , Ara Hosne , Subedi Utsab , Li Chun , Sun Grace , Murov Milena , Bhuiyan Md. Shenuarin , Sun Hong , Miriyala Sumitra , Panchatcharam Manikandan
Published: May 2022

Introduction

Ischemic reperfusion during stroke treatment increases the pathological progression of stroke. Reperfusion with r‐TPA and mechanical thrombectomy applied to stroke patients increases the risk of intracerebral hemorrhage. In addition, various molecular signals initiated during ischemic reperfusion exacerbate the disease due to increased endothelial permeability. However, a detailed molecular therapeutic regimen with r‐TPA and mechanical thrombectomy could limit the detrimental effects and increase the survival rate. Lysophosphatidic acid (LPA) is a bioactive phospholipid regulated in physiological and pathological conditions. LPA is produced by the enzyme autotaxin (ATX). ATX and LPA have been observed to deteriorate physiological status in various diseases such as myocardial infarction, Alzheimer’s disease, liver fibrosis, neuropathic pain etc. In cerebral ischemic‐reperfusion, the ATX‐LPA axis could initiate the endothelium disruption.

Hypothesis

Increased ATX expression in endothelium disrupts the endothelial barrier by increasing LPA during ischemic‐reperfusion.

Methods

Transient middle cerebral artery occlusion with 90 minutes of ischemia and 24 hours of reperfusion (I/R) was used as a stroke model in mice. LPA was analyzed with mass spectrometry and immunohistochemistry. AR‐2 probe fluorescence assay was used to measure ATX activity. Evans Blue Permeability in vivo was measured with an infrared imager. For the in‐vitro study, oxygen and glucose deprivation and reperfusion (OGDR) were carried out in mouse brain microvascular endothelial cells (MBMEC).

Results

ATX enzymatic activity is prominently raised (P<0.01) in I/R mice compared to sham mice. Increased ATX activity coheres with the Evans Blue permeability in the mice brain. Simultaneously LPA levels are elevated (P<0.01) in I/R mice. LPA was increased in brain tissue along the vasculature, indicating an unfavorable role of ATX in maintaining endothelial integrity. LPA receptors expression was regulated following stroke with upregulation of LPAR1, LPAR2, LPAR5, and LPAR6 following I/R. Permeability measured in MBMEC showed that LPA treatment increased the permeability in a time and concentration‐dependent manner. ATX expression was increased (P<0.05) in MBMEC with OGDR suggesting ATX regulation in endothelial cells following ischemic‐reperfusion. LPA produced during the process can disrupt the blood‐brain barrier, increasing cerebral permeability.

Conclusion

After a stroke, enhanced LPA production by ATX can disrupt the blood‐brain barrier, leading to brain edema and neural cell damage followed by irreversible brain injury. Inhibition of the ATX‐LPA axis prospects to be a better therapeutic avenue for cerebral ischemic reperfusion.
Harness RNA‐binding protein HuR to boost Anti‐PD‐1 Immunotherapy in immunologically cold tumors
Authors: Zhang QI , Yang Zhe , Wu Xiaoqing , Wei Lanjing , Lan Lan , Xu Liang
Published: May 2022

Cancer immunotherapy, especially those targeting the immune checkpoint PD‐1 and PD‐L1 has emerged as a revolutionizing treatment modality. Immune checkpoint inhibitors (ICIs)‐mediated anti‐tumor response depends on T cell capability of recognizing and killing tumor cells. ‘Hot’ tumors, which are characterized by the T cell infiltration, show a better response rate to immune checkpoint blockade (ICB) treatment. In contrast, ‘Cold’ tumors have a particularly low objective response rate (ORR) to ICB. In consideration of the durable and impressive responses observed in ‘hot’ tumors, it will be valuable to make an endeavor to turn immunologically cold tumors into hot tumors. The RNA‐binding protein HuR is a member of the embryonic lethal abnormal vision (ELAV) family that is overexpressed in a variety of cancers and promotes tumorigenesis by interacting with a subset of oncogenic mRNAs.

In this study, we show the direct binding of HuR protein to PD‐L1 mRNA in multiple types of cells by Ribonucleoprotein immunoprecipitation (RNP‐IP) and further validated by RNA pulldown assay. We also show that PD‐L1 mRNA decay is greatly expedited in HuR‐knockout cells, which underlines the stabilization function of HuR on PD‐L1 transcripts. Immunoblotting results demonstrate that PD‐L1 protein expression level is decreased in HuR‐knockout cells while up‐regulated in HuR‐proficient cells in comparison with control. These data together clarify HuR is engaged in PD‐L1 post‐transcription regulation.

Based on this mechanism, we develop a combination strategy using HuR inhibitors and anti‐PD1 antibody to overcome the limitation by regulating the tumor immune microenvironment.

KH‐200 is a compound that disrupts the transportation of HuR from nuclear to cytoplasm. To test the combination strategy in vivo, we established the EMT6 syngeneic breast cancer model. After 4‐week treatment, the combination group shows great effects in inhibiting tumor growth and prolonging survival. The similar combination effect was further observed in Lewis lung syngeneic lung cancer model.

In conclusion, we preliminarily explored the role of HuR in tumor immune evasion. The method using HuR inhibitors as a combination in immunotherapy shows an effective in immunologically cold tumor treatment and may have reference significance for clinical cancer therapy.
Differential Roles of SWI/SNF Complexes in Metal Regulation
Authors: Carulli Nick
Published: May 2022

SWI/SNF complexes play a major role in chromatin remodeling in mammalian cells. Mammalian DNA is compacted into chromatin by nucleosomes and other factors. The level of compaction is regulated largely by SWI/SNF complexes. These complexes remodel nucleosomes by displacing or evicting them, or by exchanging histones. By controlling which areas of the chromatin are available for transcription, SWI/SNF complexes have a large impact on gene expression. There are three main classes of SWI/SNF complexes: Brg/Brm‐associated factor (BAF), polybromo‐associated BAF (PBAF), and non‐canonical BAF (ncBAF). We hypothesized that these distinct classes of SWI/SNF complexes may have different roles, and respond to environmental and cellular signals in a specific manner. Preliminary data from our lab showed that the PBAF, and potentially the BAF complexes, may play a role in stress, homeostasis, and metal regulation in proliferating and differentiating myoblasts. We used cultured C2C12 myoblasts partially depleted of a representative subunit of each SWI/SNF complex, as these cells have an intrinsic high demand for transition metals for proper growth, differentiation, and function. Knock‐down myoblasts were treated with sublethal concentrations of different metals, and we analyzed the correlation of each SWI/SNF complex with the expression of the metal protective transcription factor (Mtf1) and metal responsive metallothionein‐1 (Mt1) genes. Our work provides the first mechanistic evidence of the contribution of specific chromatin remodelers to the expression of the main metal protective transcription factor and effector genes necessary for surviving metal toxicity.
The Nova Hydroculture Project: Bringing a Community of Science Technology, Engineering, Arts, and Math (STEAM) Learners Together to Grow a Unique Garden
Authors: Schmitt Lavin Emily , Andrews Varun , Bell Espélancia , Bui Kenneth , Celestin Aleessa , Do Vincent , Joshi Neha , Lavin Alexander , Manikkuttiyil Carol , Nutalapati Shashank , Poothurail Alina , Roy Caroline , Vadlamudi Sanjana , McQuaid Shawn , Parsons Michele , Raja Aarti
Published: May 2022

The Nova Hydroculture Project (NHP) is a collaboration between the Nova Southeastern University (NSU) American Society for Biochemistry & Molecular Biology (ASBMB) and Sigma Xi chapters as well as Science Alive and the Nova Public Schools (K‐12) in Broward County, FL. Science Alive is a 501(c)(3) non‐profit organization (faculty‐sponsored and student‐run program) providing opportunities for teams of learners from all academic levels to explore STEAM (science, technology, engineering, arts, and mathematics) beyond textbooks through hands‐on, interactive discovery. By providing service learning opportunities for college students to lead in the local school community, we fill a need for the development of fun and educational science‐related activities in schools with traditionally underserved populations. For the NHP we are collaborating with schools that have the Title 1 designation meaning that at least 40% of the student population comes from low‐income families. Many of our college volunteers are the first in their families to attend college, as would be the case for many of the K‐12 students that we serve. Therefore, we are in an ideal environment to positively impact students that are “just like us”. The NHP involves building hydroculture gardens with food plants (such as tomatoes, peppers, and berries). Showing NHP participants how to grow vegetables and fruits in a small space with reduced amounts of water can teach alternate methods to grow fresh produce, especially in communities where traditional agricultural practices may not be possible. These techniques are also being used on the International Space Station (ISS) in their Vegetable Production System (Veggie). The first peppers from the ISS Veggie were harvested and consumed on the ISS in early November 2021, which was exciting for our teams to discuss. The NHP also provides experiential learning opportunities for young students to apply science and engineering concepts to garden‐building while working in teams with their mentors. The NHP teams document the growth and water quality of the gardens, take photos, and make science journal entries throughout the Project. Not only are hydroculture gardens fantastic opportunities to learn about science, math, technology, art, and engineering, but they are also living, breathing entities that require ongoing care and commitment, thus fostering a collaborative and interactive experience for everyone involved. The NHP brings together diverse teams of K‐12 students, parents, teachers, NSU students, and faculty as we work together and learn from each other in our diverse community.
Presence of an Abnormal Obturator Artery Nutrient Branch: Cadaveric Images of a Rare Anatomical Variation
Authors: Bosse Benjamin L. , Palacios Victoria J. , Dutcher Dustin , Etter Emily , Cobine Caroline A. , Lim Peter , Moritz Gillian L.
Published: May 2022

Several studies have highlighted the inconstant branching pattern of pelvic arteries, with a recent focus on the obturator artery (OA). The OA has an inconstant origin from the internal iliac artery, external iliac artery, or inferior epigastric artery. Within the pelvis, the OA can give off muscular branches and nutrient vessels to the ilium and pubis. While categorizations of the origin of the OA are well established, limited literature exists on the muscular and nutrient branches arising from this vessel. Furthermore, few resources employ cadaveric images that depict branches arising from the obturator arteries. In the cadaveric case presented here, we examined 34 hemisected pelves and identified one individual with a substantial atypical vessel branching from the obturator artery unilaterally to supply the arcuate line of the ilium. Case studies such as this aim to highlight variations of the OA and nutrient arteries to aid physicians, anatomical instructors, and students alike in the proper identification of such variants and to better understand their prevalence. To our knowledge, this aberrancy is not described in previous studies, despite its size and course which make it particularly vulnerable during intrapelvic surgeries such as pelvic lymph node dissection or in procedures requiring arterial embolization of the obturator artery.
Cholinergic signaling impairs cardiomyocyte cohesion
Authors: Yeruva Sunil , Körber Lars , Hiermaier Matthias , Egu Desalegn , Kempf Ellen , waschke Jens
Published: May 2022

Cardiac autonomic nervous system (ANS) dysregulation is a hallmark of cardiovascular diseases such as heart failure, arrhythmias and myocardial infarction. To understand the role of ANS in cardiomyocyte cohesion, we have previously identified that adrenergic signaling enhances cardiomyocyte cohesion via PKA‐mediated plakoglobin phosphorylation at serine 665, which we termed as positive adhesiotropy. In this study, we investigated the role of parasympathetic nervous system in cardiomyocyte cohesion, using carbachol (CCH) as a cholinergic agonist that binds to the muscarinic receptors. Dissociation assays performed in HL‐1 cells, grown in norepinephrine (NE) containing medium for baseline adrenergic stimulation, and in murine cardiac slice cultures from wild type (wt) and plakoglobin (Pg) knockout (Jup‐/‐) mice revealed an impaired cardiomyocyte cohesion after CCH treatment. Electron microscopy revealed that CCH reduced intercalated disc plaque thickness in both wt and Jup‐/‐mice. Immunostaining and atomic force microscopy in HL‐1 cells demonstrated that CCH reduced desmoglein 2 (Dsg2) localization and binding at cell borders. Immunoprecipitation showed no alterations in the interaction of Dsg2, desmoplakin, plakophilin 2 and Pg after CCH treatment. Nevertheless, knockdown of any of the above genes abrogated the loss of cardiomyocyte cohesion in response to CCH. In HL‐1 cells, CCH inhibited ERK phosphorylation but not Pg phosphorylation at serine 665 induced by forskolin/rolipram (FR) combination used for adrenergic stimulation. In addition, CCH activated the AKT/GSK‐3β axis in the presence of NE. In conclusion, our results demonstrate that cholinergic signaling antagonizes the positive effect of adrenergic signaling on cardiomyocyte cohesion and thus causes negative adhesiotropy, which is independent of Pg phosphorylation.
Hepatic Arterial Patterns and Variations: Using CT Data Visualization Software to Decrease Procedural Complications Due to Hepatic Arterial Variations
Authors: Krueger Christopher , Withnell Charles B.
Published: May 2022

Intro

Three‐dimensional (3D) models have recently been used in pre‐operative planning in rare, complex surgeries in many disciplines. The goal of this study was to determine the efficacy of using 3D models in common procedures that involve increased risk of injury to hepatic arteries in the biliary region. This would allow surgeons to plan around individual variation and prevent intra‐operative complications in these regions. This study focuses on the feasibility of converting computed tomography (CT) scans into 3D models to identify anatomical variation and compares the results to the published rates of anatomical variation for the structures considered.

Methods

A total of 27 (n=27) CT scans of healthy individuals were obtained from the Cancer Imaging Archive and digitally segmented with Dragonfly and the 3D Slicer software using the semi‐automated grow from seeds tool. The results were then manually reviewed and edited for accuracy. The liver, gall bladder, and spleen were segmented as well as the associated hepatic arterial vasculature: celiac trunk, splenic artery, left gastric artery, common hepatic artery, gastroduodenal artery, proper hepatic artery, right and left hepatic arteries, and the superior mesenteric artery. The segmented scans were then converted to a 3D model where they were classified using the CRL classification system which has a 99.8% classification rate.

Results

All 3D models were classified into a CRL classification type. Nineteen (n=19) models were classified as Type 1, two (n=2) models as Type 2, one (n=1) model as Type 3, one (n=1) model as Type 4, two (n=2) models as Type 5, one (n=1) model as Type 9, and one (n=1) model did not fit under the classification system and remained unclassified. Previously published data was used to calculate the statistical expected value of each CRL type. A chi‐square analysis was performed to determine if the dataset fit normal patterns of variation. The results of the statistical test were statistically different χ2=0.0008 than previously published patterns of anatomical variation due to the rare unclassifiable variant.

Conclusion

The study showed that it is feasible to use pre‐operative CT scans to reconstruct 3D models that can be used to identify variant anatomy pre‐operatively to assist in the planning of complex surgeries. The 3D slicer software did not require a computer with substantial hardware requirements and is able to be used on a personal computer. This means it is not necessary to purchase special equipment to implement in the hospital and could potentially allow physicians to explain procedures to patients in clinics outside of hospitals. In order to expand on the capabilities of pre‐operative 3D models, it is necessary to investigate more automated methods to accurately segment the appropriate anatomy.
Application of a Second‐Order Control‐Systems Model to Investigate Effects of Impaired Capillary Signalling in Skeletal Muscle on Regulation of Capillary Blood Flow Velocity and Tissue Oxygenation
Authors: Afas Keith C. , Goldman Daniel
Published: May 2022

Microcirculation is the main region of the vasculature where oxygen (O2) is transferred directly from blood to metabolically active tissue. Tissue O2 partial pressure (PO2) in the microcirculation has been historically infeasible to measure, but critical as an indicator of capillary and surrounding tissue health. Thus, theoretical modelling of capillary‐tissue O2 transport is crucial as a complement to in vivo experiments in exploring structure‐function relationships in microvascular networks (Ellis et al, Microcirc 2012, 19:5). To properly complement experiments which stimulate O2 flow‐dependent regulation systems, modelling of microcirculatory O2 transport requires incorporation of knowledge on regulatory mechanisms.

Capillary networks modulate their blood velocity to recruit sufficient O2 delivery in many local O2 conditions; this has led to the conclusion that endothelium signals upstream to arterioles in an O2‐dependent manner (Ghonaim et al, Microcirc 2021, e12699). Recently, a second‐order control systems model was developed to investigate the interaction between capillary‐tissue O2 transport and O2‐dependent blood flow regulation under an externally applied O2 stimulus. This utilized elements from previous arteriolar diameter alteration models in response to skeletal muscle O2 gradients, as well as elements from a recently developed continuous‐capillary O2 transport model (Afas et al, Math Biosci 2021, 333:108535).

The control systems model aimed to predict modulations in blood velocity through endothelial signalling which homogenized outlet capillary PO2 in skeletal muscle. The model demonstrated that variations in O2 transport parameters such as capillary network density and tissue O2 consumption rate had varying effects on the averaged blood velocity in a small skeletal muscle segment perfused by a capillary network. In addition, the externally supplied PO2 had an effect on both blood velocity and the tissue‐capillary O2 balance. While the tissue‐capillary PO2 balance and velocity adaptation were investigated, the endothelial signalling parameters were not reported (Afas & Goldman, Vasc. Bio. 2021, conference).

The present study investigates implications of capillary network metric variations on the endothelial signal communicated upstream to modulate capillary blood flow in response to various muscle surface PO2 levels. Constraints imposed by pathogenic impairment of endothelial signalling will be investigated and interpreted in the context of capillary flow regulation.
N6‐Methyladenosine (m6A) Modifies Regenerative Transcripts in the Intestinal Epithelium
Authors: Danan Charles H. , Hayer Katharina E. , Simon Lauren A. , Weitzman Matthew D. , Hamilton Kathryn E.
Published: May 2022

Background

The intestinal epithelium forms a critical barrier between foreign antigens in the intestinal lumen and immune cells in the underlying mucosa. This barrier is compromised in inflammatory bowel disease (IBD), resulting in inflammation and epithelial cell death. Improved understanding of intestinal epithelial regeneration would identify novel therapeutic targets to enhance epithelial barrier repair in diseases such as IBD. After injury, tissues such as the blood, skeletal muscle, and central nervous system activate a regenerative program that is partially regulated by N6‐methyladeonsine (m6A) modification of RNA. However, few data exist regarding the role of m6A within the intestinal epithelium. The goal of this study was to profile m6A‐modification transcriptome‐wide in the regenerating intestinal epithelium. We hypothesize that after injury, m6A promotes regeneration within the intestinal epithelium by modifying transcripts important in epithelial repair.

Methods

Wildtype mice were given dextran sodium sulfate (DSS) in drinking water to induce intestinal inflammation and epithelial cell loss. Control mice were provided untreated water. DSS was followed by a two‐week washout period to allow for regenerating epithelium to appear adjacent to areas of epithelial ulceration. After DSS washout, regenerating intestinal epithelium was isolated by flow cytometry, RNA was extracted, and m6A‐modified transcripts were immunoprecipitated and sequenced (m6A‐seq).

Results

m6A‐seq yielded ~5500 total m6A‐modified sites within the regenerating intestinal epithelium. This corresponded to 314 m6A‐modified mRNAs that were unique to the regenerating as compared to the homeostatic epithelium. These unique transcripts were enriched for pathways involved in RNA transcription and processing, cell‐cell junctions, and Hippo pathway signaling, all of which have previously been implicated in intestinal epithelial regeneration.

Conclusions

. Our data support the hypothesis that m6A modifies many transcripts with known roles in intestinal epithelial regeneration. Ongoing studies are evaluating how m6A modification contributes to regulation of these transcripts and whether m6A identifies transcripts with previously unknown roles in regeneration. We are also evaluating whether m6A is required for optimal regeneration following epithelial injury.
NUCLEAR PARATHYROID HORMONE‐RELATED PROTEIN IS NECESSARY FOR PITUITARY SOMATOTROPHS AND GROWTH HORMONE PRODUCTION Waleed J. Hashmi1, Noriko Kantake2, Shiu Yuan2,3, Ibiaghani Max Harry2,3, Nathan K. Hoggard1, Alex H. Fishbach2, Ramiro E. Toribio4, Thomas J. Rosol2,3, Department of Biomedical Sciences, Translation Biomedical Sciences Program, Ohio University, Athens, 45701, OH1,Department of Biomedical Sciences, Heritage College of Osteopathic Medicine, Ohio University2, Department of Biological Sciences, Molecular and Cellular Biology Program, Ohio University, Athens, 45701, OH3, Department of Veterinary Clinical Sciences, Ohio State University, Columbus, Ohio 432104
Authors: Hashmi Waleed J. , Kantake Noriko , Yuan Shiyu , Max Harry Ibiagbani , Hoggard Nathan K. , Fischbach Alex H. , Toribio Ramiro E. , Rosol Thomas J.
Published: May 2022

Parathyroid hormone‐related protein (PTHrP) is an important regulator of bone growth, energy metabolism, and lung development. PTHrP is expressed by the pituitary endocrine cells. The pituitary gland plays an important role in homeostasis, energy metabolism, and development. The first phylogenetic identification of PTHrP in animals has shown that it originally developed as a secretory pituitary hormone in fishes. PTHrP is both a secreted and nuclear protein. A novel knock‐in mouse (PTHrP∆/∆) with a lack of the nuclear localization sequence (NLS) of PTHrP was developed. The mice fail to grow and die 4 to 5 days after birth. We hypothesized that the nuclear localization sequence (NLS) of PTHrP was necessary to many tissues for normal development and physiological processes. Histological analysis was performed to study the morphological structure of the pituitaries in these mice. Compared to the wildtype and heterozygote mice(PTHrP+/∆), the pituitary pars distalis from 1‐day‐old PTHrP∆/∆ mice was hypocellular, with a decreased density of pituitary epithelial cells throughout the gland (hypoplasia of the pituitary pars distalis). Occasional pituitary epithelial cells were apoptotic with shrunken pyknotic or fragmented nuclei, with increased cytoplasmic eosinophilia. The pituitary pars intermedia, pars nervosa, and rathke pouch epithelium was unaffected. Immunohistochemistry using GH primary antibody showed decreased GH secretion in somatotrophs which may contribute to early senescence in the mice. Immunoreactivity to GH was strongest in the pituitary epithelial cells within lateral areas of the pars distalis in PTHrP+/∆ mice. The GH immunoreactive cells in the pituitary epithelial cells of PTHrP∆/∆ mice was low compared to PTHrP+/∆ mice. The GH is expressed at low levels in the pituitaries from the PTHrP∆/∆ mice compared to PTHrP+/∆mice as verified by PCR. Understanding the mechanisms and functions of nuclear PTHrP in the pituitary provides the platform for improved medical therapies for endocrine diseases as well as novel insights into the impact of the PTHrP on processes such as growth and metabolism.
Using Whole Genome Sequencing to Identify Mutations in E. coliLeading to Cefsulodin Resistance
Authors: Hays Jade M. , Dinkel Brittney A. , Weiss David S.
Published: May 2022

Peptidoglycan (PG) is the key component of the cell wall and a hallmark of bacteria. It forms a net‐like structure that surrounds the bacterial cell, giving the organism its characteristic shape and protecting it from lysis due to turgor pressure. PG is synthesized by Class A and Class B Penicillin‐Binding Protein (aPBP, bPBP). Cefsulodin is a beta‐lactam antibiotic that inactivates aPBPs. Even though the bPBPs are still active, they are not sufficient to support proper peptidoglycan synthesis, so E. coli growing in the presence of cefsulodin will lyse and die. We identified cefsulodin resistant mutants by plating E. coli onto media containing cefsulodin and waiting for (rare) colonies to grow. Characterization of seven CefR mutants confirmed increased resistance to cefsulodin. Whole genome sequencing revealed one CefR mutant had a mutation in ihf‐α(a DNA binding protein), and another had a mutation in yidC (inserts proteins into the membrane). The remaining five CefR mutants had point mutations in mrcB, which encodes the class A PBP called PBP1b, a target of cefsulodin. Biochemical assays confirmed two PBP1b mutants had reduced binding to cefsulodin. Mapping the mutations onto a published crystal structure of PBP1b revealed the mutations are near the active site. Thus, selection for antibiotic resistance identified amino acids that affect binding of cefsulodin to its target PBP1b. This information may help in developing new antibiotics to avoid resistance.
The Role of C‐Terminus and Nuclear Localization Sequence of Parathyroid Hormone‐Related Protein (PTHrP) in Pancreatic Islet Morphology and Glucose Homeostasis
Authors: Max‐Harry Ibiagbani , Kantake Noriko , Toribio Ramiro , Nunemaker Craig , Rosol Thomas
Published: May 2022

Parathyroid Hormone‐related protein (PTHrP) is an important polyhormone with multiple functions in development and cell regulation. PTHrP is expressed by pancreatic beta cells which are responsible for insulin secretion. Previous studies have focused on the ability of N‐terminal PTHrP to stimulate proliferation in beta cells. We have developed a knock‐in mouse model lacking the C‐terminal and nuclear localization sequence of PTHrP, retaining only N‐terminal PTHrP. These mice die at ~day 5, are severely stunted in growth (weigh 54% less than control mice), have chondrodystrophy, hypoinsulinemia, hypoglycemia (~40‐60mg/dL), hypotriglyceridemia, and lack body fat. Percentage stomach weight (including ingested milk) measurements in relation to body weight were consistent among all mouse genotypes indicating that inanition was not the cause of death. To characterize the pancreatic islets in these mice, islets (~10‐20) were isolated from 2‐5 day‐old‐mice using collagenase digestion and a glucose‐stimulated intracellular calcium assay was conducted at 0, 4, 8, 12, 16 and 20mM glucose. Results showed higher intracellular calcium response in knock‐in islets compared to control islets and increased insulin secretion after overnight incubation. Data from immunofluorescence also showed less glucagon staining in knock‐in islets compared to control islets. This study revealed that the C‐terminus and nuclear localization sequence of PTHrP are crucial to life, including regulation of glucose homeostasis and islet response to glucose metabolism. It also revealed that these portions of PTHrP may be important for the developmental stage of islets and differentiation into alpha or beta cells.
Tracking and Inhibiting Atypical Vascular Inflammation
Authors: Grimsey Neil J. , Lane Elizabeth L. , Ghosh Sneha , Yoder Megan , Lane Payton , Burton Jeremy C.
Published: May 2022

Mitogen‐activated protein kinase (MAPK) p38 drives the onset and progression of many clinically challenging diseases. However, three decades of research have failed to yield clinically viable, selective MAPK p38 therapeutics. The extensive and ongoing studies have principally focused on therapeutics targeting the ATP‐pocket or active site of p38. These have failed in part due to the ubiquitous expression and dichotomous role of p38, leading to blockade of both physiological and pathological signaling pathways. A promising alternative strategy is to target either the substrates of p38 or the activators that drive p38‐induced pathological signaling. Our prior studies identified an atypical mechanism for G protein‐coupled receptor mediated activation of pathological p38 signaling during vascular inflammation. Several critical problems remain: firstly, how does atypical signaling drive inflammation, and secondly how can we stop pathological p38 activity? To further define how atypical p38 signaling induces inflammation we developed a fluorescent resonance energy transfer (FRET) biosensor platform to map atypical p38 signaling. This system revealed that GPCR induced p38 signaling is initiated via an endosomal axis. Blockade of receptor internalization shifts the spatial bias to that seen for MKK3/6‐dependent signaling with enhanced nuclear p38 activity. We predict that this endosomal axis drives pathological atypical p38 activity and the progression of vasculopathic disease progression. Critically, atypical p38 activity is initiated by a direct interaction between the adaptor protein TAB1 and two binding sites on p38. Blockade of the TAB1‐p38 interaction through mutation of select residues on TAB1 can disrupt the TAB1‐p38 interaction and prevent atypical p38 signaling. Modeling studies show that small molecule inhibitors that bind to the TAB1 binding motif on p38 can block this interaction. As an alternative strategy we focused on the TAB1 peptides that directly bind to p38. Using a phage‐display library of humanized nanobodies and a yeast two‐hybrid screen we have developed an array of 40 TAB1 blocking intrabodies (IntraT‐ab’s) as the first generation of selective atypical p38 inhibitors. We show that intraT‐ab’s bind TAB1, selectively blocking GPCR induced p38 (endosomal) signaling while leaving classical MKK3/6 dependent signaling intact. IntraT‐ab’s and our FRET platform provide the first opportunity to examine in detail the specific downstream mechanisms, targets and physiological consequences of atypical p38 signaling. Intracellular delivery/expression of intraT‐ab’s could also provide a therapeutic strategy for reducing p38 signaling in multiple diseases, including acute lung injury, retinopathies and neuroinflammation.
Prenatal E‐Cigarette Use Disrupts Blood‐Brain Barrier (BBB) Integrity and Induces Pro‐Inflammatory Cytokines in Postnatal Brain
Authors: Rahman Archie Sabrina , Sifat Ali Ehsan , Villalba Heidi , Sharma Sejal , Nozohouri Saeideh , Zhang Yong , Abbruscato Thomas
Published: May 2022

Purpose

Electronic cigarette (e‐Cig) is often considered as a safer alternative to smoking for pregnant women however, its potential toxic effect on neonatal health warrants further investigation. Toxic effects of prenatal tobacco smoking on postnatal health have been reported in several studies however, regarding thorough investigation of e‐Cig exposure during pregnancy is currently unavailable. Due to the staggering growth in e‐Cig usage among pregnant women (10%), it has become critical to explore the consequences of maternal e‐cig use on postnatal health outcomes. Hence, in this study, we have evaluated the consequences of prenatal e‐Cig use during pregnancy on postnatal blood‐brain barrier (BBB) integrity, proinflammatory cytokine levels and behavioral outcomes in offspring.

Method

Pregnant CD1 mice (E5) were exposed to e‐Cig vapor (2.4% nicotine) till postnatal day (PD) 7. Weight of the offspring was measured up to PD 90. Nicotine and cotinine concentration in plasma and brain were measured in PD 7 offspring by LC‐MS/MS. The expression level of structural elements of the BBB including, tight junction proteins (ZO‐1, claudin‐5, occludin), astrocyte (GFAP), pericyte (PDGFRβ) and basement membrane (Laminin α1, Laminin α4) were analyzed in offspring using western blot and immunohistochemistry at PD 7, PD 23, PD 45 and PD 90. The expression level of pro‐inflammatory cytokines (IL‐1α, IL‐1β, IL‐2, IL‐4, IL‐5, IL‐6, IL‐7, IL‐10, IL‐12, IL‐17α, KC, LIX, MIP‐2 and TNF‐α) in brain were measured at PD 7 and PD 90. To evaluate behavioral outcomes, open field test, novel object recognition test and morris water maze test were performed on adolescent and adult offspring.

Results

Reduced body weight was observed in e‐Cig exposed offspring at all time points till PD 90 compared to control (P <0.05). Plasma concentration of nicotine and cotinine in PD 7 offspring was 3.215 ng/mL and 3.373 ng/mL respectively whereas in brain, these two concentrations were 43.72 ng/mL and 1.123 ng/mL respectively. Significantly reduced expression of brain tight junction proteins and GFAP were observed in male and female offspring at PD 7, PD 23, PD 45 and PD 90 (P <0.05). Moreover, higher level of brain IL‐1α, IL‐6, IL‐7, IL‐10, IL‐17α, KC and TNF‐α was found in prenatally e‐cig exposed offspring at PD 7. However, only IL‐6 and IL‐17α were observed at higher levels in brain for prenatally e‐cig exposed adult offspring at PD 90 (P <0.05). Additionally, disrupted motor, learning and memory function were observed in prenatally e‐Cig exposed adolescent and adult offspring.

Conclusion

Our findings suggest that prenatal e‐Cig exposure induces potential neurotoxic effects on neonates by disrupting postnatal blood‐brain barrier integrity and inducing proinflammatory brain cytokines which may result in alteration in neurological, cerebrovascular, and behavioral outcomes.