bluuebunny/crossref_metadata_2025_split huggingface dataset:
Analysis of the physical interaction between α1G T‐type calcium channel and endothelial nitric oxide synthase (NOS3) in pulmonary microvascular endothelial cells
Authors: Sellak Hassan , Chen Hairu , Alexeyev Mikhail , Wu Songwei 
Published: November 2023

We have observed that, in pulmonary microvascular endothelial cells (PMVECs), activation of the site‐specific α1G T‐type Ca2+ channel (α1G) provides a cytosolic Ca2+ source necessary for endothelial nitric oxide synthase (NOS3) activation. Furthermore, the interaction between α1G and NOS3 in PMVECs has been demonstrated by in vitro Förster resonance energy transfer studies, as well as by immunoprecipitation assays. The present work was undertaken to analyze the molecular basis of the α1G‐NOS3 interaction. We constructed retroviral expression vectors harboring different deletion mutants of α1G that encompasses its cytoplasmic domains including N‐terminus (α1G‐Nter, 1–80), loop I–II (396–744), loop II–III (966–1,251), loop III–IV (1,517–1,578), and C‐terminus (α1G‐Cter, 1,823–2,254). Additionally, Rat NOS3 was cloned in retroviral expression vector and five (5) deletion mutants were generated and similarly cloned, i.e., NOS3‐F1 (1–730), NOS3‐F2 (1–450), NOS3‐F3 (400–730), NOS3‐F4 (700–1,202), and NOS3‐F5 (850–1,202). PMVECs were retrovirally transduced with full‐length α1G alone, full‐length α1G combined with full‐length NOS3, or full‐length α1G combined with different NOS3 deletion mutants. Immunoprecipitation assays using protein extracts from these transduced cells showed that fragments NOS3‐F1 (1–730) and NOS3‐F5 (850–1,202) interact with full‐length α1G. In a reverse approach, PMVECs were retrovirally transduced with full‐length NOS3 alone or in combination with each α1G cytoplasmic domains. Immunoprecipitation and immunoblot assays were performed and results revealed that α1G loop I–II, loop II–III, and α1G C‐terminus interact with NOS3. Altogether, these findings demonstrated the existence of constitutive physical interaction between α1G T‐type Ca2+ channel and NOS3, and identified the critical domains that mediate α1G‐NOS3 interaction. Thus, α1G‐NOS3 association may form a signaling complex that governs the inflammatory response of pulmonary microvascular endothelium.Support or Funding InformationSupported by HL‐066299.
Protective role of ergothioneine from tobacco smoke‐induced oxidative stress in vitro and in vivo
Authors: Ehrhardt Carsten , Selo Mohammed Ali , Clerkin Caoimhe Geraldine , Talbot Brian Noel , Walsh John Jarlath , Nakamichi Noritaka , Kato Yukio , Lewis Joshua B , Reynolds Paul R , Nickel Sabrina 
Published: November 2023

Tobacco smoke is a major contributing factor for chronic obstructive pulmonary disease (COPD) and exposure leads to oxidative stress, which contributes to alveolar wall destruction, mucus hyper secretion, inflammation and defective tissue repair.Ergothioneine (ET), an amino acid transported across cell membranes by the OCTN1 (SLC22A4) transporter, has been shown to exhibit antioxidant capacities. The objective of this study is to investigate the role of OCTN1‐transported ET on lung epithelial cells subjected to tobacco smoke‐induced oxidative stress.For in vitro studies, human NCI‐H441 cells were cultured for at least 5 days in the presence or absence ET and then exposed to cigarette smoke extract (CSE). Reactive oxygen species were measured using H2DCFDA, enzymes involved in oxidative stress defence (e.g. thioredoxin, sulfiredoxin 1). In addition markers of xenobiotic defense (e.g. MRP1, BCRP, and PXR) were studied by qPCR and immunoblot. Moreover, octn1 knock‐out mice were exposed for 3 and 6 months to second hand smoke. The total cell numbers in broncho‐alveolar lavage fluid (BALF) were counted and smoke induced changes in lung morphology were assessed in hematoxylin‐eosin (HE) stained lung sections.q‐PCR and immunoblot revealed elevated expression levels of sulfiredoxin 1 and thioredoxin as well as PXR after ET treatment of NCI‐H441 cells. Moreover, lower levels of oxidative stress were observed in cells, which were cultured in the presence of ET prior to CSE exposure. When exposed to room air, KO mice showed no differences in HE staining or cell numbers in BALF compared to wild type (WT) animals. However, total cell numbers in BALF as well as increased alveolar damage and immune cell infiltration were observed in KO animals compared to WT mice when exposed to second hand smoke.These data suggest that ET can protect lung epithelial cells from oxidative damage and consequently, variants of OCTN1 might play a role in the pathogenesis of tobacco smoke‐induced COPD by regulating ET transport.Support or Funding InformationThis work has been financially supported by a Clinical Investigator Awards from the Flight Attendant Medical Research Institute (FAMRI CIA 130016). M.A.S. is the recipient of a PhD bursary from the Iraqi Ministry of Higher Education and Scientific Research (MOHESR) and C.G.C. is supported by a Trinity College Research Studentship Award.
Alveolar Acid Injury Causes Endothelial Mitochondrial Depolarization
Authors: Hough Rebecca F , Gusarova Galina A , Islam Mohammad N , Bhattacharya Jahar 
Published: November 2023

Lung inflammation and edema, the characteristic features of Acute Lung Injury (ALI), result from lung microvascular barrier deterioration. Since ALI associates with loss of mitochondrial function, we evaluated endothelial mitochondrial responses to alveolar acid instillation. We established isolated, blood‐perfused mouse lungs at constant pulmonary artery, left atrial and alveolar pressures (n=4). To view alveoli and capillaries by confocal microscopy, we micropunctured alveoli to load the cytosolic fluorescent dyes, calcein green (CG) and calcein red (CR), respectively. We loaded capillaries with the mitochondrial potentiometric dye, trimethylrhodamine ethyl ester (TMRE). To establish acid‐induced alveolar injury, we microinjected alveoli with concentrated hydrochloric acid (HCl). HCl decreased CG by 75±4% from baseline (p<0.05, mean±SE), indicating that alveolar acid instillation injured the epithelial plasma membrane. Concomitantly, TMRE, but not CR fluorescence decreased (p<0.05), indicating that despite lack of membrane injury endothelial mitochondria were depolarized. Alveolar PBS injection was without effect. Since loss of calcein dye is an indicator of plasma membrane injury, we interpret alveolar contact with concentrated acid caused membrane injury to epithelium, but not endothelium. Nevertheless, endothelial mitochondria were depolarized and possibly dysfunctional after alveolar acid injection. We suggest, acid‐induced alveolar injury abrogates endothelial bioenergetics, leading to the microvascular deterioration underlying ALI (HL105323, HL57556).Support or Funding InformationHL105323, HL57556
Copper manipulation of the transcriptional activity of hypoxia‐inducible factor‐1
Authors: Zhang Wenjing , Wu Zhijuan , Liu Xiaojuan , Li Pengfei , Fu Haiying , Kang Y. James 
Published: November 2023

Hypoxia‐inducible factor 1 (HIF‐1) controls the expression of more than 100 genes involved in multiple cellular events, however, it is unknown how the selectivity of the expression of a certain group of genes under certain circumstances is manipulated. We previously observed that copper (Cu) is required for the expression of some HIF‐1 controlled genes (such as BCL2/adenovirus E1B 19 kDa protein‐interacting protein 3 (BNIP3) and vascular endothelial growth factor (VEGF)), but not others (such as insulin‐like growth factor 2, IGF‐2). The present study was undertaken to understand the mechanism by which Cu manipulates the transcriptional activity of HIF‐1. Human umbilical vein endothelial cells (HUVECs) were treated with CoCl2 to induce HIF‐1α accumulation and with a Cu chelator tetraethelenepentamine (TEPA) to reduce the availability of Cu in cells. Chromatin immunoprecipitation assay (ChIP) was used to detect the binding of HIF‐1 transcription complex to hypoxia‐responsive elements (HREs) located in the promoters of Cu‐dependent genes (BNIP3 and VEGF) or Cu‐independent genes (IGF‐2) under normal or Cu‐reduced conditions. Nuclear extracts from normal and Cu‐reduced HUVECs were subjected to immunoprecipitation assay (IP) using anti‐HIF‐1α antibody and to immobilized metal affinity chromatography (IMAC) using immobilized columns loaded with Cu, followed by label free LC‐MS/MS to identify Cu binding proteins that may participate in the formation of the HIF‐1 transcription complex. Treatment with TEPA reduced Cu concentrations in the nuclei but did not change the protein level of HIF‐1α. The sequences of HRE (−550/−451) of BNIP3 and HRE (−355/−347) of IGF‐2 were defined as HIF‐1 binding sites by ChIP‐PCR assay. Cu depletion reduced HIF‐1 transcription complex binding to the HRE of BNIP3 significantly, but did not affect that of IGF‐2, suggesting that Cu manipulates the HIF‐1 transcriptional activity through affecting HIF‐1 transcription complex binding to HREs. LC‐MS/MS identified several Cu binding proteins, which will be further analyzed for their function in the regulation of HIF‐1 transcription complex binding to different HRE sequences. The results thus demonstrate that Cu may represent a means for selective manipulation of the transcriptional activity of HIF‐1.Support or Funding InformationSupported in part by National Science Foundation of China, grant # 81230004 and 81300107
New Theory of Holistic Integrative Physiology and Medicine I: Structural Frame of Life Control
Authors: Sun Xing‐Guo 
Published: November 2023

ObjectiveThe modern systemic physiology, based on each functional system alone, has significant limitations. Human being is an organic set of all functions. We plan to discuss the life control involving all systems.MethodsWe did establish the raw functional structure of new theory to explain the control and regulation of life.ResultsAfter birth, independent human being's life is depended upon two core functional axis. The first one is respiration‐circulation‐metabolism, and the second one is digestion and absorption‐circulation‐metabolism. The main function of them is to maintain the homeostasis for cells’ metabolic activities in the body. The first functional axis is dominantly to keep balance between supply and demand of gases (O2, and CO2 etc.). The second is to keep balance between supply and demand of materials (glucose, fatty acid, amino acid and lactic etc.) in the body. Use circulation as a central coupling, we constructed a main control circles for life. The circulation‐metabolism is shared parts by both axis. After birth, we start our independent life. Traditionally, breathing is the only vital sign, so that we focused on the breathing control (be discussed in others abstracts) linking with all others.The structural frame of life controlThe cortex is the mind over control for almost functions. Under cortex, main breathing control circle consists of peripheral chemical/pressure receptors in arterial line (aorta arch and carotid body), afferent nerves, central integrative center (medulla and central chemical receptor), efferent nerves, N‐M connection, muscles (alternative constriction and relaxation), diaphragm and chest wall, lung and airway, pulmonary capillary, pulmonary vein, left atrium, left ventricle, and artery. The signal from receptors to muscles is transferred electronically, which dominantly neuron‐control; but signal from lung to receptors at artery is transferred by blood flow, which dominantly heart‐control. The secondary circle of breathing control is the circulation. It consists of artery, capillary (all cells metabolic activities: resting, exercise and sleeping. GI uptake, liver metabolism and kidney elimination), vein, right atrium, right ventricle, pulmonary artery, pulmonary capillary, pulmonary vein, left atrium, left ventricle, and artery. The pulmonary capillary, pulmonary vein, left atrium, left ventricle, and artery are the shared parts in both main and side circles. And the autonomic (sympathetic and parasympathetic) nerves, from central integrative center, control the heart and blood vessel for circulatory regulation.ConclusionWe can explain that the life control through the integration of all functions of respiration, circulation, nerve, digestion, absorption and elimination, metabolism, exercise, sleeping, etc. together.Support or Funding InformationCNSF‐Medical Science Program (81470204); NCCVD Fuwai Hospital Start Program (2012‐YJR02)
Inhibitory control of active expiration by the Kölliker‐Fuse in rats
Authors: Jenkin Sarah E. M. , Milsom William K , Zoccal Daniel B 
Published: November 2023

Breathing is critically dependent on synchronized activity generated by neural respiratory centres within the pons and medulla. When respiratory drive is elevated, active expiration (AE) is observed, typically expressed by abdominal muscle activity at the end of the expiratory phase (late‐E). It is currently unknown what role the dorsolateral pons, specifically the Kolliker‐Fuse (KF), plays in generating the AE breathing pattern. Using the decerebrated in situ working heart‐brainstem preparation of juvenile male Holtzman rats (60–70 g), motor output from the vagus, phrenic and abdominal nerves were recorded during baseline conditions. The preparations were then exposed to hypercapnic conditions (8% CO2) to recruit AE. Hypercapnia resulted in a significant increase in the amplitude of abdominal late‐E activity (Δ: 118±30%, P<0.05), with little change to the other recorded parameters. During hypercapnia, the pharmacological inhibition of the KF, with bilateral microinjections of (50–75 nL) isoguvacine (10 mM), caused a significant decrease in breathing frequency (Δ: −5±2 cpm, P<0.05) due to increased inspiratory time (0.86±0.1 vs 1.37±0.1 s, P<0.05). Interestingly, while there was no change to the overall expiratory time, the duration (1.86±0.21 vs 1.47±0.13 s, P<0.05) and amplitude (3.6±0.4 vs 0.7±0.2 μV, P<0.05) of post inspiratory (post‐I) vagal activity decreased while the onset of the abdominal late‐E bursts occurred significantly earlier (1.3±0.1 vs 2.4±0.2 s, P<0.05). No changes were observed in the abdominal late‐E burst amplitude after KF inhibition. These findings suggest that the KF plays a relevant inhibitory role in coordinating the transition between post‐I activity and AE during hypercapnia.Support or Funding InformationFinancial support: NSERC, FAPESP (2013/17251‐6) and NIH/NCCIH (1R01AT008632‐01).
Acute Lung Injury Alters AMPA Receptor Subunit Density in the Dorsal Vagal Complex
Authors: Litvin David Gregory , Dick Thomas E , Jacono Frank J 
Published: November 2023

Ventilatory pattern is a neurally integrated output that is altered by lung injury. Work in our lab has provided evidence that acute lung injury (ALI) promotes changes in ventilatory pattern, which persist in situ and are associated with synaptic plasticity at 2nd order nucleus tractus solitarii (nTS) neurons. Therefore, in the current study, we sought to identify evidence that brainstem autonomic circuits were undergoing changes in glutamatergic transmission following ALI. We hypothesized that ALI could be promoting changes in brainstem α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid (AMPA) receptor subunits. To test this hypothesis, we intratrachially instilled bleomycin (Bleo) in postnatal (P) 11 Sprague Dawley rats and sacrificed them at P18 for fluorescent immunostaining of the AMPA‐R subunits GluR1, GluR2, and GluR4, followed by confocal microscopy and densitometric analysis. AMPA‐R subunit immunoreactivity was measured in the nTS, dorsal motor nucleus of the vagus (DMNX), and hypoglossal nucleus (XII). Significant increases in GluR2 immunoreactive density were observed in the nTS of Bleo (0.185±0.036AU n=5) versus PBS (0.108±0.035AU p=0.02, n=5) rats. There were no significant differences in GluR1 (p=0.31) or GluR4 (p=0.70) immunoreactive density in the nTS. In the DMNX, there were significant increases in GluR1 (Bleo: 0.071±0.017AU vs. PBS: 0.0496±0.01AU, p=0.047) and GluR2 (Bleo: 0.057±0.016AU vs. PBS: 0.028±0.0134AU, p=0.03) immunoreactivity. There was a significant decrease in GluR4 density in the DMNX (Bleo: 0.238±0.023AU vs. PBS: 0.295±0.025AU, p=0.02). Finally, there were no significant changes in GluR1 (p=0.209), GluR2 (p=0.275), and GluR4 (p=0.103) in the XII. Taken together, these results show that bleo induced lung injury promotes changes in the density of AMPA‐R subunits in the dorsal vagal complex. These findings suggest that altered glutamatergic transmission in the dorsal vagal complex may contribute to synaptic plasticity and ventilatory pattern changes in the setting of lung injury.Support or Funding InformationVA Research Service Award Number I01BX000873 and NIH T32HL007913
The background sodium channel NALCN regulates basal excitability of chemosensitive retrotrapezoid nucleus neurons and stimulation of breathing by CO2
Authors: Shi Yingtang , Abe Chikara , Holloway Benjamin , Shu Shaofang , Kumar Natasha , Weaver Janelle , Sen Josh , Perez‐Reyes Edward , Stornetta Ruth , Guyenet Patrice , Bayliss Douglas 
Published: November 2023

CO2/H+‐sensitive neurons of the retrotrapezoid nucleus (RTN) are a nexus for integration of multiple respiratory‐related inputs in service of homeostatic regulation of the rate and depth of breathing, blood gases, and arterial pH. The spike firing activity of RTN neurons controls respiratory output. Using a transgenic mouse line in which RTN chemosensory neurons are marked with GFP, we identified two populations of RTN neurons ‐‐ Type I and Type II ‐‐ with different firing activities evident over distinct pH ranges. The higher baseline firing rate in Type II cells is associated with greater inward current at −60 mV and larger NMDG‐sensitive, TTX‐resistant current. We hypothesized that NALCN, a “leak” Na+ channel might underlie this RTN current. Single cell RT‐PCR (scPCR) and in situ hybridization showed that NALCN is expressed in nearly all RTN neurons (>90%), and quantitative scPCR revealed that NALCN expression is highest in Type II cells. Using viral‐mediated shRNA knockdown of NALCN, we found that NALCN depletion favors Type I properties in vitro and blunts ventilatory responses to CO2 in vivo. Therefore, NALCN plays an important role in establishing baseline excitability of RTN neurons and contributes to control of breathing by these cells.Support or Funding InformationNIH (HL074011, PGG; HL108609, DAB)
Characteristics of Breathing Rate Control by Neurons in the Pontine Medial Parabrachial Region (PBR)
Authors: Zuperku Edward J , Stucke Astrid G , Hopp Francis A , Stuth Eckehard A 
Published: November 2023

A subregion within the medial PBR plays a key role in the control of expiratory duration (TE). Drug‐induced activation of PBR neuronal activity decreases TE, and neuronal inhibition increases TE. AMPA‐induced pontine‐mediated tachypnea is associated with increased pre‐I neuronal activity in the preBotzinger (preBC) region. This study used bipolar microstimulation to activate the PBR neurons to more precisely control the input pattern to the preBC/BC region and examine peri‐stimulus time histograms (PSTHs) for short‐time scale correlations in neuronal activity suggestive of direct synaptic connections. Multichannel recordings of preBC/BC neurons were performed in decerebrate, vagotomized dogs. The phrenic neurogram (PNG) was used to assess phase‐timing responses. PBR step frequency stimulus patterns markedly decreased TE in particular when applied during the E‐phase. The E off‐switch threshold decreased with time into the E‐phase. In preBC/BC neurons of various subtypes, PSTHs showed short duration evoked excitation, longer duration inhibition or both, suggesting fast EPSP‐ and slow IPSP‐like synaptic inputs. These results were also reflected in cycle‐triggered histograms. The data suggest that the control of TE from the PBR is mediated by inhibition of E‐neurons and excitation of pre‐I neurons.Support or Funding InformationMerit ReviewAward # 2 I01 BX000721‐05 from the United States Dept. of Veterans Affairs, Biomedical Laboratory Research and Development Program. Edward J. Zuperku, Ph.D. (PI)
Long‐range gene regulation in the context of chromatin domains
Authors: Dekker Job 
Published: November 2023

The 3D organization of the genome is critical for gene regulation, genome stability and faithful transmission of chromosomes to daughter cells. We apply Chromosome Conformation Capture‐based technologies to determine how cells fold their chromosomes and to discover the mechanisms that drive the spatial organization of genomes. I will discuss our recent proposal that in interphase nuclei mammalian chromosomes are hierarchically organized (Gibcus and Dekker, 2013). First, at the nuclear level, chromosomes are compartmentalized into large multi‐Mb domains that are either active and open or inactive and closed. These compartments themselves are composed of smaller sub‐Mb Topologically Associating domains (TADs) (Dixon et al. 2012, Nora et al. 2012). Finally, long‐range gene regulation occurs within TADs through long‐range looping interactions between genes and regulatory elements.TAD organization, and their role in gene regulation have important implications in cancer cells. I will highlight two new studies from my laboratory that reveal different roles of TADs in chromosome‐wide gene regulation along the dosage compensated X‐chromosomes. First, in C. elegans we find that a Condensin complex mediates TAD formation and dosage compensation. Second, in mouse the dosage compensated X chromosome folds in a unique way with global loss of TADs, except around loci that escapee inactivation.