bluuebunny/crossref_metadata_2025 huggingface dataset:
Intestinal tight junction CLDN2 gene is a direct target of the vitamin D receptor
Authors: Zhang Yong‐guo , Wu Shaoping , Lu Rong , Zhou David , Zhou Jingsong , Petrof Elaine , Claud Erika , Sun Jun
Published: June 2021

The breakdown of intestinal barrier is a common manifestation of GI disorders. Claudin‐2 is a tight junction protein that mediates paracellular water transport in leaky epithelium. Recent evidence suggests that vitamin D and vitamin D receptor (VDR) may regulate functions of tight junction proteins. However, it is unknown how Claudin‐2 is regulated by VDR signaling in normal and inflamed intestine. Using whole body VDR‐/‐ mice, intestinal epithelial VDR conditional knockout (VDRΔIEC) mice, human samples, and cultured human intestinal epithelial cells (IEC), we performed a series of molecular and biochemical experiments in vivo and in vitro. We provide evidence that the CLDN2 gene is a direct target of the transcription factor VDR. VDR‐inducing Claudin‐2 promoter activity required the Cdx binding site on the promoter of the CLDN2 gene. We further identify a functional vitamin D response element in the Claudin‐2 promoter. In vivo, VDR deletion in intestinal epithelial cells (IEC) led to decreased Claudin‐2 at both mRNA and protein levels. Functionally, we found a robust increase of Claudin‐2 in inflammatory IECs which lacked VDR regulation and allowed the inflammatory cytokines to take over. Furthermore, in inflamed intestine of ulcerative colitis patients, VDR expression is low and Claudin‐2 is enhanced. A lack of intestinal VDR regulation leads to dysfunction of Claudin‐2 in inflammatory responses. This study reveals a complex role and novel mechanism for intestinal VDR by regulation of epithelial barriers and inflammation.
Identification of an Occludin Regulatory Motif that Confers Dynamics to Epithelial Tight Junctions (TJs) and Adherens Junctions (AJs)
Authors: Manda B , Gangwar R. , Mir H. , Rao R.
Published: June 2021

Occludin is a transmembrane protein of epithelial TJs, the function of which remains unclear. Previous studies in Caco‐2 cells showed that the C‐terminal domain of occludin harbors a conserved phosphorylation hotspot, which we designated as 'Occludin Regulatory Motif' (ORM). To determine the functional significance of ORM, we cloned a stable, occludin‐deficient MDCK cell line (MDCKOKD) by shRNA transfection, and introduced GFP‐tagged human occludin deletion mutant (OclDM: Δ388‐415; lacking ORM) or wild type occludin (OclWT) and cloned stable cell lines. Confocal imaging of live cells, immunofluorescence staining and co‐immunoprecipitation analyses revealed a co‐localization of OclDM with ZO‐1 at the intercellular junctions similar to OclWT. Post seeding barrier development was faster in MDCKOKD‐OclDM and MDCKOKD‐Vec cells compared to those in MDCKOKD‐OclWT and native MDCK cells. FRAP analysis demonstrated that deletion of ORM significantly reduced the mobile fraction of occludin and elevated the proportion of detergent‐insoluble fraction of occludin. Incubation of MDCKOKD‐OclWT cell monolayers in low calcium medium (LCM) resulted in redistribution of OclWT from the junctions, but LCM had no effect on the distribution of OclDM. LCM reduced barrier function and induced ZO‐1 redistribution in MDCKOKD‐OclWT and native MDCK cells, but not in MDCKOKD‐VecorMDCKOKD‐OclDM cells, indicating the role of ORM in TJ dynamics. Depletion of occludin or lack of ORM also attenuated LCM‐mediated redistribution of E‐cadherin and β‐catenin and reorganization of actin cytoskeleton, indicating the role of ORM in AJs. In summary, results indicate a potential role for ORM in conferring dynamics to epithelial apical junctional complexes.
Western diet induces nonalcoholic fatty liver disease and impairs liver mitochondrial metabolism in adult mice
Authors: Pacheco Patricia , dos Santos Vivian , Neves Fabiana , Bernardo Amelia , Vieira Anatalia , Miranda Glauciane , Malafaia Tayanne , Guedes Roberta , Moura Anibal , Sichieri Rosely , de Souza Erica
Published: June 2021

The aim of this study was to investigate the liver mitochondrial metabolism of obese adult mice fed with Western diet (high fat diet rich in saturated fat). 21 days old male mice were divided in two groups: fed with control diet (CD) or Western diet (HFD), for 112 days. At 133 days of life the animals were sacrificed for evaluation of biometric parameters, fasting glucose, liver histology, liver mitochondrial function and proteins involved in the signaling cascade of ghrelin and insulin by Western Blotting (GHSR 1a, IRβ, PI3K, pAKT/AKT, CPT1, UCP2 and α‐tubulin). Animals subjected to HFD showed increased body weight (p <0.0001), visceral fat (p <0.0001), liver weight (p <0.0001) and hyperglycemia (p <0.0133) compared to CD group. The liver of HFD group presented damaged parenchymal cells with accumulation of lipid droplets and alterations in protein content compared to CD: reduced level of GHSR1a (p <0.0282) and pAKT/AKT ratio (p <0.0053), increased content of PI3K (p <0.0149), IRβ (p <0.0539), CPT1 (p <0.0417) and decreased content of UCP2 (p <0.0196). Through High resolution respirometry we observed a reduction in carbohydrate oxidation (p <0.0481) and increased fatty acid oxidation (p <0.0008) in the liver of HFD group. We concluded that Western diet alters energetic metabolism and impairs ghrelin signaling which favors the development of nonalcoholic fatty liver disease. Supported by FAPERJ and CNPq.
eNOS Deficiency Increases Susceptibility to Western Diet Induced Hepatic Mitochondrial Dysfunction
Authors: Sheldon Ryan , Linden Melissa , Meers Grace , Morris E. , Laughlin M. , Rector R.
Published: June 2021

Nitric oxide produced by endothelial nitric oxide synthase (eNOS) can influence mitochondrial biogenesis and electron transport chain activity. Hepatic mitochondrial dysfunction is a hallmark feature of nonalcoholic fatty liver disease (NAFLD), yet the underlying mechanisms remain elusive. Here, we tested the hypothesis that eNOS plays an important role in sustaining hepatic mitochondrial function in NAFLD. Wild‐type (WT) and eNOS knockout (eNOS‐/‐) mice received either control (CON; 10% fat, 3.5% sucrose) or a western diet high in fat (45%), sucrose (17%), and cholesterol (1%; HFC) for 18‐weeks. HFC diet feeding induced histological NAFLD and increased weight gain and % body fat in both genotypes compared to CON diet. Complete hepatic mitochondrial palmitate oxidation to CO2 was elevated in both eNOS‐/‐ groups compared with WT mice(p<0.05), while incomplete acid‐soluble metabolite production was elevated only in eNOS‐/‐‐CON compared to other groups. In addition, maximal FCCP‐uncoupled mitochondrial respiration tended to be elevated in eNOS‐/‐‐CON compared to WT‐CON (+24%; p=0.08) but was significantly reduced with HFC feeding in eNOS‐/‐ mice (‐37% vs eNOS‐/‐‐CON; p<0.05). Finally, HFC feeding dramatically reduced hepatic mRNA expression of the putative control for mitochondrial biogenesis, PGC‐1α, in WT (‐33%) and eNOS‐/‐ (‐58%) compared with WT‐CON (p<0.05). These results support the hypothesis that eNOS limits susceptibility to western diet‐induced hepatic mitochondrial dysfunction and highlights a potential role for eNOS in hepatic mitochondrial biogenesis. Supported by:HL036088 (MHL) and VHA‐CDA2 1299‐03(RSR).
Dorsal Medulla Inspiratory Neurons Contribute to Increased Inspiratory Drive during Swallow
Authors: Pitts Teresa , Shen Tabitha , Varechova Silvia , Demoulin Bruno , Guannan Zhou , Tsai Hsiu‐Wen , Musslewhite M. , Rose Melanie , Poliacek Ivan , Morris Kendall , Davenport Paul , Bolser Donald
Published: June 2021

Schluckatmung, or swallow breath is believed to be an evolutionarily preserved inspiratory drive from deglutition to eupnea. This inspiratory motor drive creates a net negative trans‐esophageal pressure that promotes bolus movement across the upper esophageal sphincter. A multi‐channel electrode array was used to record dorsal respiratory group neurons in anesthetized, spontaneously breathing cats. Swallowing was induced by injection of water into the oropharynx. Electromyograms (EMG) were recorded from pharyngeal, laryngeal, and chest wall muscles, and intra‐esophageal pressure was recorded from the mid‐thoracic esophagus. A total of 104 inspiratory (I) and expiratory (E) phasic neurons were recorded in 6 animals during breathing and swallow. Approximately 90% of I neurons increased their firing frequency during swallow, and approximately 80% of E neurons decreased their firing frequency during swallow. The increased firing rate of I neurons was associated with costal diaphragm and parasternal EMG bursts along with a negative esophageal pressure during swallow. Previous work has shown low activity of I neurons during swallow in the ventral respiratory column. We conclude that increased dorsal medullary inspiratory neuron activity contributes to enhanced inspiratory drive during swallow. Supported by HL 111215(K99/R00) & HL 103415
Ventral Respiratory Column Expiratory Neuron Activity is Altered during Fictive Swallow in the Cat
Authors: Zhou Guannan , Tsai Hsiu‐Wen , Horton Kofi‐Kermit , Morris Kendall , Gestreau Christian , Nuding Sarah , Segers Lauren , Lindsey Bruce , Bolser Donald , Davenport Paul
Published: June 2021

Swallow and breathing are both fundamental functions for mammals. Anatomically, breathing and swallow share a common passageway, the pharynx. To coordinate with swallow and prevent aspiration, inspiration is inhibited, while expiration is prolonged. The swallow central pattern generator (sCPG) and the rCPG share common neural components. We hypothesize that the respiratory neuronal network in the VRC, has neurons that will be retasked during swallow to participate in production of the swallow breathing pattern. We stimulated the superior laryngeal nerve (SLN) of paralyzed, decerebrated, ventilated cats to elicit swallow. Extracellular neuronal activity was recorded with microelectrode arrays during fictive swallow and breathing. SLN elicited fictive swallow inhibited phrenic nerve activity and remodeled activity of expiratory neurons (E neurons) in the VRC. During fictive swallows, the activity of a subpopulation of expiratory decrementing (E‐Dec) neurons was suppressed, and a subpopulation of E‐Dec neurons was facilitated. Similar responses have been seen in different subgroups of expiratory augmenting (E‐Aug) neurons during fictive swallows. These results suggest that the VRC E neurons underwent reconfiguration to generate a coordinated swallow breathing pattern. The various response categories suggest that the E neurons located in the VRC are functionally heterogenous.
Specific Role of Membrane Progesterone Receptors α and β on Ventilation during Sleep in Female Mice.
Authors: Boukari Ryma , Rossignol Orlane , Marcouiller François , Bairam Aida , Joseph Vincent
Published: June 2021

We recently showed that deletion of nuclear progesterone receptors disrupts breathing during sleep. Here we tested the hypotheses that deletion of membrane progesterone receptors (mPR) α and β in the medulla‐brain stem regions decreases ventilation and increases breathing instabilities during sleep. Female mice were chronically instrumented and small interfering RNA (siRNA) against mPRαor mPRβ (or a control solution) were infused in the fourth ventricle for 2 weeks. Ventilation (Ve) and CO2 production (VCO2) were continuously recorded (whole body plethysmography) during 4 h (9:00‐13:00) in normoxia. Respiratory frequency, tidal volume, minute ventilation and the frequency of spontaneous and post‐sigh apneas were determined during sleep, defined as periods of stable breathing pattern lasting at least 10 minutes. Compared to control, deletion of mPRα decreased Ve and Ve/VCO2 during sleep, but did not affect apnea or sigh frequency. The deletion of mPRβ had no effect on Ve but decreased VCO2, increased Ve/VCO2 and increased the frequency of spontaneous and post‐sigh apneas. Specific deletions of mPRα and mPRβ have been verified by immunohistochemistry on serial brainstem slices. Our results show that mPRα and β play specific role on respiratory control during sleep, and suggest that these receptors mediate some of the respiratory effects of progesterone. Founded by CIHR (MOP‐102715).
Cortical Activation Patterns in Response to Respiratory Transient Occlusions
Authors: Chan Pei‐Ying , Ju Ya‐Jhih , Wu Yi‐Jhen
Published: June 2021

Cortical awareness of respiratory stimulation can be tested with the transient respiratory occlusions. This mechanism has been investigated by the respiratory‐related evoked potential method with scalp surface electrodes. The above evidence suggests the corresponding cortical areas of neuronal activation in response to inspiratory interruptions. The electrophysiological method provides excellent temporal evidence but little spatial information with regards to the cortical areas activated with respiratory sensory gating. Therefore, the purpose of this study was to systematically investigate the effect of inspiratory occlusions on respiratory sensation. We hypothesized that the stimulation paradigm would result in cortical activation patterns in the thalamus, somatosensory, and parietal cortex.

We tested this hypothesis in a group of healthy adult participants. During the experimental recordings, one run of 40 inspiratory interruptions was presented to the participants. Inspiratory interruptions of 150‐msec were provided every 2 to 4 breaths. During each scanner run, the individual was instructed to keep breathing in until the end of their regular inspiratory phase even after they detect the inspiratory interruption.

Our preliminary data showed that single inspiratory occlusions elicited brain activation including the inferior parietal gyrus, parietal and central opercular gyrus. The finding is consistent with our hypothesis and more participants are needed in order to confirm the areas of interests activated by the inspiratory occlusion paradigm.
Role of Mucin 5ac in Murine Airways
Authors: Cho Hye‐Youn , Miller‐DeGraff Laura , Park Soojung , Lao Huei‐Chen , Langenbach Robert , Kleeberger Steven
Published: June 2021

Mucin is a highly glycosylated protein in epithelial lining of respiratory, gastrointestinal, and reproductive tracts. To determine the role of Muc5ac, the major secreted airway mucin, in respiratory pathogenesis, Muc5ac‐deficient mice (Muc5ac‐/‐), generated by deletion of exons 21‐31 encoding the glycosylation region, and wild type controls (Muc5ac+/+) were examined in models of respiratory syncytial virus (RSV) disease (intranasal infection, 106 plaque forming unit RSV/mouse) and pulmonary fibrosis (intratracheal instillation, 0.5 U bleomycin/Kg body weight). Phenotypes were assessed by histopathologic and bronchoalveolar lavage analyses. Reduced levels of intraepithelial mucus from nasal to bronchial airways, atrophic maxilloturbinates, and enriched nasal glands were manifest in Muc5ac‐/‐, compared to Muc5ac+/+. In Muc5ac‐/‐, RSV caused more severe nasal airway injuries including subepithelial congestion and gland secretion and compensatory increase of mucus secretion than in Muc5ac+/+ at 1‐5 d post‐infection. Bronchoepithelial injury and hyperplasia/hyperproliferation as well as pulmonary perivascular‐peribronchiolar edema and smooth muscle thickening were more evident in Muc5ac‐/‐ relative to Muc5ac+/+ at 1‐5 d post‐infection. Serum IgE level was also significantly higher in Muc5ac‐/‐ than in Muc5ac+/+ at 5 d after infection. Muc5ac‐/‐ exhibited significantly more severe pulmonary inflammation than Muc5ac+/+ at 7‐14 d post‐bleomycin. Overall, lack of epithelial barrier component Muc5ac impaired the airway defense against virus and fibrogen, and caused compensatory regulation of other secretory proteins. Supported by the NIEHS Intramural Program.
The Effects of Norbinaltorphimine Preexposure on THC‐induced Place and Taste Avoidance in Sprague‐Dawley Rats.
Authors: Flax Shaun , Wakeford Alison , Rice Kenner , Riley Anthony
Published: June 2021

Studying the mechanisms that modulate cannabinoid abuse potential is important, given that it is the most widely used drug of abuse in the United States. Although a drug's abuse potential is thought to be mediated by the balance between its rewarding and aversive effects, little is known about the aversive effects of Δ9‐tetrahydrocannabinol (THC), the main psychoactive constituent of cannabis, and how these effects might vary with specific experiential and subject factors. The aversive effects of THC have been studied in CB57/BL mice using the place conditioning design, and these studies have argued that activity at the kappa opioid receptor (KOR) mediates the aversive effects of THC. Specifically, both genetic ablation and pharmacological antagonism of the KOR system blocks THC‐induced place avoidance. Given reported differences between mice and rats in other behavioral preparations, it is not known if this effect is evident in other species. To address this, the present experiment assessed the effects of the KOR antagonist, norbinaltorphimine (norBNI), on THC‐induced place and taste avoidance in Sprague‐Dawley rats. Specifically, rats were injected with 15 mg/kg of norBNI 24h prior to the pairing of a distinct side of a place conditioning chamber (Experiment 1) or a novel taste (Experiment 2) with one of three doses of THC (0.56, 1.0 and 3.2 mg/kg). Independent of the specific assay, norBNI had no significant effect on the aversive effects of THC. These data suggest that the specific mediation of THC's aversive effects by KOR activity may be species dependent.
Investigation of Sex Differences in Neurotensin following Nicotine Self‐Administration
Authors: Pittenger Steven , Smith Misty , Hoonakker Amanda , Hanson Glen , Fleckenstein Annette , Bevins Rick
Published: June 2021

Research has revealed sex differences in nicotine addiction vulnerability. Women, on average, progress to dependence more rapidly, attempt to quit less frequently, remain abstinent for shorter durations during quit attempts, and have a higher relapse rate to smoking. Recent research suggests a role of the endogenous neuropeptide neurotensin (NT) in nicotine addiction via modulation of dopamine. Experimenter‐administered nicotine alters NT levels. However, possible variation of NT levels between the sexes following nicotine self‐administration (SA) needs to be determined. In a series of studies, age‐matched male and female Sprague‐Dawley rats were surgically implanted with indwelling jugular catheters. Half of the rats were then trained to self‐administer nicotine (Nic‐Male; Nic‐Female); the other half received saline infusions yoked to a nicotine‐administering rat (Sal‐Male; Sal‐Female) during daily sessions (1‐h; 21 days). Brains were extracted 3 hours after the last SA session. NT was assessed in the nucleus accumbens core (NAc) and shell (NAs), substantia nigra (SN), globus palladus (GP), anterior (AC) and posterior caudate (PC), frontal cortex (FR), ventral tegmental area (VTA) and habenula (Hb). We found a reliable NT increase in the Nic‐Males compared to the Sal‐Males in the AC, PC, GP, and FR. Potential sex differences in the PC (Nic‐Male NT > Nic‐Female NT) are currently under additional validation. Research supported by DA034389 and DA031883.
Activation of Voltage‐Gated Sodium Channels with Brevetoxin‐2 (PbTx‐2) Increases Dendritic Spine Density in Murine Organotypic Hippocampal Slice Culture
Authors: Gomez Dina , Murray Thomas
Published: June 2021

The N‐methyl‐D‐aspartate receptor (NMDAR) is essential for activity‐dependent neurite outgrowth, dendritic arborization and spine formation. We previously demonstrated that the voltage gated sodium channel activator, PbTx‐2, augments NMDAR function in dissociated cultures of cerebrocortical neurons. Here we assessed the effect of PbTx‐2 on spine dynamics in an organotypic hippocampal slice culture that better retains neuronal cytoarchitecture and synaptic circuits. This system also allowed us to ask whether the effects of PbTx‐2 on cerebrocortical neurons generalized to hippocampal neurons. The effects of PbTx‐2 on spine dynamics were compared to those of NMDA. Organotypic hippocampal slice culture (450 μm thick sections) was prepared from postnatal day 5‐6 mouse pups.. Hippocampal slices were placed on a membrane at the interface between air and culture medium. The slices were treated with vehicle, PbTx‐2 or NMDA 18 hours after plating. Slices were diolistically or biolistically labeled, fixed and mounted. A Leica SP8 microscope was used to generate Z‐stack confocal images and Imaris‐XT software was used create 3D‐reconstructed images of dendrites to analyze dendritic spine dynamics. Our results demonstrated that PbTx‐2 treatment produced significant increases in spine density. The fold increment in spine density produced by PbTx‐2 was similar to that observed with NMDA. The use of the organotypic slice culture confirmed the impact of a voltage‐gated sodium channel activator on spinogenesis, and demonstrated that this effect generalizes to hippocampal neurons. Sodium channel activators may represent a novel pharmacological strategy to promote neuronal structural plasticity. NIH‐RO1N5053398‐12
Single‐Cell Calcium Imaging of RFP Labeled Interneurons in the Neocortex of Tbr1‐Deficient Neonatal mice
Authors: Easton Curtis , Moody William , Kalume Franck , Hevner Robert
Published: June 2021

Cortical development involves initial structuring of network features genetically programmed signaling pathways. Later, ion channel activity helps refine neuronal connections. In this study we examine propagating waves of activity in a mouse genetically engineered to lack the transcription factor T‐box, brain 1 (Tbr1), which is involved in the differentiation of cortical excitatory neurons and cortical patterning. Using calcium imaging, we find that wave activity appears at normal frequencies. Additionally we examine the distribution of cortical inhibitory neurons in Tbr1 deficient cortex using red‐fluorescent‐protein (RFP), and find that these neurons are displaced similarly to what is observed in reeler mice. Sensitivity of waves to blockade of GABA signaling is preserved in Tbr1 deficient mice. Finally, we show using dual green and red‐channel imaging of calcium indicator dye and RFP fluorescence, respectively, that inhibitory interneurons participate in spontaneous calcium signaling at normal frequencies in Tbr1 deficient mice. We identify two distinct types of spontaneous calcium signals: one that is consistent with wave activity, and another that is restricted to single cells and not correlated with neighboring cells, and sensitive to blockers of L‐type calcium channels. These results provide evidence that wave activity is a robust property of developing networks, and establish new methods of recording calcium signals from genetically labeled cell populations. We identify distinct calcium signals in immature interneurons that may serve separate purposes in development.
Novel Peptides that Block the Interaction between Wnt and Frizzled
Authors: Hermans Kevin , Fernández‐Llamazares Ana , Dailly Nicolas , Blankesteijn W Matthijs , Timmerman Peter
Published: June 2021

Introduction

Wnt/Frizzled signaling is involved in many developmental processes but is mostly silent in healthy adult organs. However, a reactivation of this signaling pathway is generally observed during pathological conditions, which makes it a promising therapeutic target. The recently presented crystal structure of Xenopus Wnt8 in complex with the cysteine rich domain (CRD) of Frizzled8 demonstrates that Wnt has a shape resembling the outline of a hand with a thumb and index finger grasping the CRD at two opposing sites. The thumb contains a palmitic acid lipid at the tip that engages a groove on the CRD whereas the index finger forms a strong hydrophobic contact with a groove on the opposite side of the CRD. Here we investigated the antagonistic properties of peptides that resemble fragments of Wnts that interact with the CRD of Frizzled receptors on canonical Wnt/Frizzled signaling in cell‐based assays.

Methods

Peptides resembling the thumb and index finger of Wnt3a and Wnt5a were synthesized and tested in HEK293 and 3T3 cells expressing the TOPFlash reporter. Canonical Wnt signaling was induced by addition of Wnt3a conditioned medium.

Results

Peptide fragments of both thumb and index finger demonstrated inhibition of Wnt3a induced signaling in micromolar concentrations. Combined administration of both fragments demonstrated an additive inhibition. Furthermore, we demonstrated that palmitoylation, tertiary structure and length of the peptide are critical for antagonistic activity.

Conclusion

Fragments of Wnt that interact with Frizzled CRDs can serve as new antagonists for Wnt/Frizzled signaling.

Supported by the Dutch Heart Foundation (2010B196) and Cyttron (FES0908)
Teratogenic Effects Of Some Commonly Used Medicinal Plants On The Cardiovascular System
Authors: Afolayan Gbenga , Afolayan Olasunmbo , Awodele Olufunsho , Agbaje Esther , Kumar Suresh , Klingler Rebekah , Svoboda Kurt , Carvan Michael
Published: June 2021

The use of medicinal plants by pregnant women has not been extensively validated and screened for their influence on offspring. The aim of this study was to determine the medicinal plants used, prevalence of their use among pregnant women in Lagos state, Nigeria, and to investigate the effect of these plants on cardiovascular system development.
An Ethnobothanical survey was carried out among traditional herbal practitioners and pregnant women attending antenatal clinics in Lagos State public hospitals using structured questionnaires. Transgenic zebrafish embryos were treated with various concentrations (0.08mg/ml – 1.28mg/ml) of plant extracts between 4 – 72 hours post‐fertilization to determine the effects on the cardiovascular system.

The survey showed that 44.2% of the pregnant women used herbal medicine and 50% of these women had used Enantia chlorantha which was among the recommended plants for use by herb sellers. Pericardial edema and the presence of a tubular heart were observed in the 1.28mg/ml group. There was also, a significant dose dependent decrease in the blood flow rate and a disruption in the morphology of the intersegmental vessels some of which failed to develop.

These research findings suggest that Enantia chlorantha possibly has an effect on angiogenesis. And it, offers a basis for further investigation of the molecular mechanism by which this plant might be responsible for these effects.
Medication Use by U.S. Crewmembers on the International Space Station
Authors: Wotring V
Published: June 2021

This study examined medication use during long‐duration spaceflight. Medication records from 24 crewmembers on 20 missions (> 30 days duration) were examined for trends in usage rates, efficacy, indication, as well as adverse event qualities, frequencies and severities. No controls were possible in this observational, retrospective analysis of available data; comparisons are made to similar studies of individuals on short‐duration spaceflights and submarine deployments. The most frequently used medications were for sleep problems, pain, congestion or allergy. Medication use during spaceflight missions was similar to what is seen in adult ambulatory medicine; one notable exception is that usage of sleep aids was about 10 times higher in spaceflight. There were also two apparent treatment failures in cases of skin rash, raising questions about the efficacy or suitability of the treatments used. Many spaceflight‐related medication uses were linked to extravehicular‐activities and operationally‐driven schedule changes. The data suggest that sleep and skin rash merit additional study prior to longer space exploration missions. It also seems likely that alterations in schedule‐shifting or extravehicular activity suits would reduce the need for many medication uses, preserving resources as well as improving crew quality of life.
Inhibitory effects of Leishmanial sphingolipid on Endotoxin induced macrophage activation and sepsis associated inflammatory injury
Authors: Das Subhadip , Das Saha Krishna
Published: June 2021

Sepsis is the reflection of systemic immune response that manifests in the sequential inflammatory process in presence of infection with excessive production of inflammatory mediators and causes severe tissue injuries. Total lipid of attenuated Leishmania donovani establish as a bioactive. Moreover, sphingolipids moderate various cellular processes. So, this finding encourages us to evaluate inflammatory response in system using the microbial sphingolipid.

In this study, we have used bacterial endotoxin stimulated macrophages to evaluate the efficacy of leishmanial sphingolipid (LSPL) towards the amelioration of inflammatory mediators (TNF‐α, IL‐1β, IL‐6, IL‐17, IL‐10) and transcriptional factors with associated TLR4‐CD14 expression. In vivo system, endotoxin induced murine sepsis model was used in focusing on multiple organ injury (Lungs, Liver, and Kidney) and status of different cytokine‐chemokine(s) storm. Due to the effect of LSPL, endotoxin induced inflammatory cytokine‐chemokine(s) levels were significantly reduced in serum and other major organs with the reduction of vascular permeability factors and suppressed the expression of cell adhesion molecule in septic mice evident by immunochemical approaches.

These findings indicate that LSPL may prove to be a potential anti‐inflammatory agent as protection against bacterial sepsis with associated organ injury.
Assessment of Active Metabolites of Levobunolol and Betaxolol in Rat, Rabbit and Human Ocular and Liver S9 Fractions
Authors: Bushee Jennifer , Argikar Upendra , Dunne Christine
Published: June 2021

Little is known about ocular metabolism and formation of pharmacologically active metabolites for drugs dosed topically to the eye. We devised a simple in vitro approach coupled with high resolution mass spectrometry to measure the rate and extent of ocular metabolism for comparison with liver. Levobunolol and betaxolol are antagonists of β‐adrenergic receptors and have been clinically utilized for treatment of primary open‐angle glaucoma and/or ocular hypertension. Levobunolol has been reported to be metabolized to an equipotent active metabolite, dihydrolevobunolol, in plasma and urine of mice, rat, dog, and human. Hydroxybetaxolol, an active metabolite of betaxolol, has been reported in the liver. We aimed to understand whether the eye has functional metabolic activity and to understand the extent of formation of two historically reported in vivo active metabolites, one each for levobunolol and betaxolol, across rat (R), rabbit (Rbt) and human (H) ocular S9 (OS9) fractions and liver S9 (LS9) fractions. Based on high resolution mass spectrometry M11 was identified as the active dihydroxylevobunolol while M2 was identified as the active hydroxybetaxolol. The in vitro data shows that betaxolol's M2 was formed in the eye across preclinical species and human. M2 pharmacological activity for antagonism toward β1‐ or β2‐receptors has not been assessed to date. This is the first report of hydroxybetaxolol being formed in the eye. Results from levobunolol and betaxolol metabolism studies point toward species differences in conversion of levobunolol and betaxolol to their respective active metabolites and strongly indicate that LS9 fractions are a poor surrogate for assessment of active metabolites formed in the eye.
Role of Calcium Sensitivity of Troponin C in the Development of Dilated Cardiomyopathy
Authors: Tikunova Svetlana , Singh Sonal , Fan Qiying , Hernandez Adriana , Portillo Jesus , McConnell Bradley
Published: June 2021

Dilated cardiomyopathy (DCM) is a disease of cardiac muscle characterized by left ventricular dilation and systolic dysfunction. In 30‐40% of all cases, DCM is believed to be caused by mutations of cytoskeletal and sarcomeric proteins. The objective of this project was to determine whether decreasing the calcium sensitivity of the regulatory N‐domain of cardiac troponin C (cTnC) is sufficient to trigger the development of DCM. In order to achieve the desired objective, we knocked‐in a calcium desensitizing D73NcTnC substitution into the cardiac and slow skeletal muscle of mice.

The heterozygous knock‐in mice on C57BL/6J background appeared phenotypically normal and were fertile. Western blotting demonstrated that levels of cTnC, cTnI, or phosphorylation of cTnI by protein kinase A were not affected by the D73NcTnC mutation. At four, eight and twelve weeks, knock‐in mice exhibited increased heart weight‐to‐body weight ratios. Echocardiography showed that at eight weeks, knock‐in mice exhibited increased left ventricular dimensions with thinner walls. Echocardiography also showed that contractility parameters such as ejection fraction and fractional shortening were dramatically reduced in knock‐in mice, while heart rate was not affected. Electrocardiography demonstrated that knock‐in mice exhibited prolonged QRS and QTc intervals. Small but significant increase in fibrotic lesions was observed at twelve weeks in the ventricles of knock‐in mice. Death of the heterozygous knock‐in mice occurred between six and nineteen weeks. Thus, knock‐in mice were able to closely recapitulate the phenotypes of human patients with DCM.
Effect of Small Chemicals like N‐Methyl Pyrrolidone (NMP) on the Orchestration of Bone Remodeling by Osteocytes
Authors: Siegenthaler Barbara , Ghayor Chafik , Weber Franz
Published: June 2021

Osteoporosis is a bone disease that is characterized by gradually decreasing bone mass and hence increasing fracture risk with age. This bone mass is regulated by a tightly controlled balance between osteoblasts and osteoclasts orchestrated by osteocytes. Mediators of this orchestration are nitric oxide, RANKL, prostaglandin, and Sclerostin. The latter is expressed by mature osteocytes and has been shown to compete with Wnt signaling, thereby negatively regulating bone formation.

Recently our lab identified N‐Methyl Pyrrolidone (NMP) as a potent agent for bone regeneration on the level of osteoblasts and osteoclasts. The aim of this study was to characterize the effect of NMP and NMP‐like small molecules on osteocytes.

We used the mouse osteocytic cell line IDG‐SW3 and the rat mature osteoblast cell line UMR‐106 to investigate the effect of small the chemicals on the expression levels of Sclerostin. Chemical treatment was performed in different concentrations over multiple weeks. Cell viability was measured to investigate for toxic chemical concentrations and Alkaline Phosphatase activity assays were performed to check the cellular potential of calcium deposition. Furthermore, total RNA was isolated and real time PCR performed to assess for gene expression alterations with the different treatments.

Our preliminary results indicate the NMP and NMLs are capable to reduce sclerostin expression and thereby could possibly diminish the competitive binding to the LRP5/6 receptors and facilitate bone formation. Further experiments are needed to investigate the effect of NMP and NMLs on bone cells to understand the molecular and cellular basis of their possible anti‐osteoporotic potential.
Role of PGE2/EP2 Receptor Signaling on The Effects of Neonatal Hyperoxia on Airway Smooth Muscle
Authors: Sopi Ramadan , Mladenov Mitko , Dreshaj Ismail , Neziri Burim , Mehmeti Mirsad , Bislimi Kemajl , Jakupaj Muharrem
Published: June 2021

It was shown that neonatal hyperoxic‐exposure associates with airway hyperresponsiveness and impaired nonadrenergic noncholinergic relaxation. It was demonstrated that reduced relaxant responses were associated with an impairment of prostaglandin E2 (PGE2) release from airway smooth muscle (ASM). In this study we tested the hypothesis that supplementation of PGE2 or EP2 agonists to ASM restore relaxant responses. Tracheal cylinders were obtained from 12 days rat pups exposed to hyperoxia or room air for seven days. These cylinders were used to study contractile and relaxant responses. In hyperoxic tracheal preparations the contractile responses to bethanechol were enhanced as compared to room air controls. These enhanced contractile responses were attenuated by pre‐incubation of tissues with PGE2 (1µM). Similar effect was showed by using an EP2 agonist. This effect of PGE2 and EP2 agonist was reversed by an EP2 antagonist. PGE2 and EP2 induced dose response relaxations of ASM, which were reduced by an EP2 antagonist.

We conclude that PGE2 induces relaxation of airways via EP2 receptors, and the use of EP2 agonists may serve as a therapeutic approach to overcome the adverse effects of neonatal hyperoxia on relaxant responses of ASM.

Support by MEST
Loss of PPARγ Activates ERK 1/2‐NF‐κB‐Nox4‐H2O2 Signaling Axis to Promote Human Pulmonary Artery Smooth Muscle Cell Proliferation
Authors: Bijli Kaiser , Kleinhenz Jennifer , Murphy Tamara , Kang Bum‐Yong , Adesina Sherry , Sutliff Roy , Hart Charles
Published: June 2021

Hypoxia stimulates pulmonary hypertension (PH) in part by increasing the proliferation of pulmonary vascular wall cells. Recent evidence suggests that hypoxia stimulates sustained nuclear factor‐kappaB (NF‐κB) activation, increased NADPH oxidase4 (Nox4) expression, and downregulation of peroxisome proliferator‐activated receptor‐gamma (PPARγ) to cause human pulmonary artery smooth muscle cell (HPASMC) proliferation. To further understand the role of reduced PPARγ in PH pathobiology, siRNA was employed to reduce PPARγ levels in HPASMC in vitro under normoxic conditions. PPARγ protein levels were reduced to levels comparable to those observed under hypoxic conditions. Knockdown of PPARγ for 24‐72 hours activated mitogen‐activated protein kinase, ERK 1/2, and NF‐κB. Inhibition of ERK 1/2 prevented NF‐κB activation caused by PPARγ knockdown indicating that ERK 1/2 lies upstream of NF‐κB activation. Knockdown of PPARγ for 72 hours increased NF‐κB‐dependent Nox4 protein expression and H2O2 production. Degradation of H2O2 using PEG‐catalase or inhibition of NF‐κB or Nox4 attenuated HPASMC proliferation caused by PPARγ knockdown. In contrast, PPARγ overexpression reduced ERK 1/2 and NF‐κB activation, Nox4 expression, and proliferation in HPASMC. Collectively, these findings provide novel evidence that PPARγ plays a central role in the regulation of the ERK 1/2‐NF‐κB‐Nox4‐H2O2 signaling axis in HPASMC and that reductions in PPARγ caused by pathophysiological stimuli such as prolonged hypoxia exposure are sufficient to promote the proliferation of pulmonary vascular smooth muscle cells observed in PH pathobiology.
Hypercapnia potentiates the action of μ‐opioid receptor agonists on synaptic transmission in the preBötzinger complex
Authors: Garcia Alfredo , Anderson Tatiana , Ramirez Jan
Published: June 2021

Acute respiratory depression is a leading cause of mortality in individuals who overdose on opiates. Moreover, the magnitude of respiratory depression is variable and unpredictable, making the use of opiates particularly dangerous. Opiates can blunt the hypercapnic respiratory response, yet the precise mechanisms underlying this phenomenon are unclear. The objective of this study was to determine how hypercapnia influences the action of the μ‐opioid receptor agonist, DAMGO, on rhythmogenesis and synaptic transmission within the preBötC. Electrophysiological recordings were made from rhythmic transverse brainstem slices (mice, P6‐P12) during the following conditions: (1) normocapnia (90% O2,5% CO2; 25mM NaHCO3); (2) hypercapnic acidosis (HA: 90% O2, 10% CO2; 25mM NaHCO3). In normocapnia, 25nM DAMGO suppressed the frequency of rhythmogenesis by 24 ± 11%. HA exaggerated the effect of 25nM DAMGO reducing frequency by 66 ± 10%. Similarly, HA augmented the effects of morphine (7.5nM) on the preBötC. Recordings of evoked excitatory postsynaptic potentials shows that HA exaggerates the suppression of glutamatergic drive to preBötC neurons. Moreover, during normocapnia 25nM DAMGO fully suppressed rhythmogenesis in the presence of 20μM CPP, a NMDA receptor antagonist. These data indicate that HA augments the effects of μ‐opioid agonists on glutamatergic synaptic transmission in the preBötC.
Serotonin Receptor Expression on Astrocytes Near Spinal Phrenic Motor Circuitry
Authors: Gonzalez‐Rothi Elisa , Armstrong Gregory , Cerreta Anthony , Mitchell Gordon , Reier Paul , Fuller David
Published: June 2021

Spinal 5‐HT2 and 5‐HT7 receptor activation both trigger respiratory motor plasticity expressed as long‐lasting phrenic motor facilitation, although via distinct cellular mechanisms. Although most studies of serotonin‐dependent spinal respiratory plasticity have focused on motoneurons, 5‐HT receptors are also expressed in glia. Although little is known concerning the function of glia in spinal respiratory plasticity, glia play important roles in in other models of neuroplasticity. Here we present preliminary results concerning 5‐HT receptor expression on GFAP‐positive astrocytes near spinal phrenic motor circuitry. Using immunofluorescence and pseudorabies virus as a retrograde transynaptic tracer from the diaphragm of adult rats, we identified phrenic motoneurons, pre‐phrenic cervical interneurons and nearby GFAP‐positive astrocytes. Initial results indicate 5‐HT2A and 5‐HT7 receptor co‐localization in GFAP positive cells in the vicinity of phrenic‐associated neurons in the cervical gray matter, raising the possibility that spinal astrocytes play an important role in 5‐HT‐dependent phrenic motor plasticity.
A significant portion of Dbx1 neurons in the intermediate reticular formation are XII inspiratory premotoneurons
Authors: Revill A , Kottick A , Akins V , Vann N , Gray P , Del Negro C , Funk G
Published: June 2021

Reduced tonic and inspiratory drive to XII motoneurons (MNs) that innervate the genioglossus muscle (GG, a tongue protruder) is implicated in obstructive sleep apnea. Reduced excitability of glutamatergic inspiratory XII premotoneurons (preMNs) during sleep may contribute. Little is known about XII inspiratory preMNs due to their sparse distribution in the intermediate reticular formation (IRt). We hypothesized that ipsilaterally‐projecting XII inspiratory preMNs derive from Dbx1 progenitors, a transcription factor expressed in the IRt. Rhythmic, transverse medullary slices (550 μm) were produced from Dbx1ERCreT2; R26tdTomato neonatal mice (postnatal day 0‐5). Approximately 20% of the Dbx1 IRt neurons received inspiratory drive. In total we identified 29 inspiratory‐modulated Dbx1 IRt neurons via whole‐cell recording. 10 of 24 neurons were antidromically activated from the ipsilateral XII nucleus. Anatomical reconstruction of biocytin‐filled neurons revealed 10 commissural axons and 4 ipsilateral axons projecting to the XII nucleus. Using in situ hybridization, 57.3% (range 54.0‐58.9%) of Dbx1 IRt neurons were glutamatergic. Laser ablation of individual Dbx1 IRt neurons caused a significant (36 ± 3.6%) drop in ipsilateral, but not contralateral, XII inspiratory burst amplitude, and had no effect on inspiratory burst period. Thus, a subpopulation of Dbx1‐derived neurons in the IRt are XII inspiratory preMNs that contribute significantly to XII inspiratory output. This discovery will aid study of XII inspiratory preMNs. Funding: CIHR, NSERC, WCHRI, AIHS, CFI, NIH.
The evolution of air‐breathing is the adaptive response to high environmental temperatures
Authors: Giomi Folco , Fusi Marco , Pörtner Hans‐Otto
Published: June 2021

The transition to air breathing is the key innovation during evolution of terrestrial animal life from aquatic ancestors in a huge variety of taxa. This transition has happened numerous times and independently in all animal phyla. Especially in fishes and crabs, the proximate factors of this physiological innovation were poorly understood. A previous paradigm saw hypoxia in estuarine and fresh waters as an initial driver of the transition from water‐ to air‐breathing but was not able to explain the geographical distribution of extant genera of amphibious fish and crabs, a long standing puzzle in evolutionary biology. Here we provide evidence that high temperature extremes act as a key and universal driver for this transition, and that thermal extremes can explain the present phylogenetic knowledge and distribution of amphibious and terrestrial crabs. Our results demonstrate that breathing air rather than water saves energy and extends aerobic performance to a wider thermal niche. These findings provide a mechanistic explanation of the recurrent and repeated evolution of air‐breathing in response to the effects of temperature demonstrating that the emergence of this novel respiratory mode has a direct benefit for improving thermal tolerance by enhancing the oxygen provision.
Mitochondrial‐Derived Vesicle Formation in Cardiac Mitochondrial Quality Control
Authors: Cadete Virgilio , Deschênes Sonia , Cuillerier Alexanne , Picard Martin , McBride Heidi , Burelle Yan
Published: June 2021

Mitochondrial quality control (QC) is critical for maintenance of efficient cardiac function. Recently, a novel mechanism of mitochondrial QC, acting upstream of autophagy, has been uncovered – mitochondrial derived vesicle (MDV) formation. Our aim was to demonstrate MDV formation in a cardiac model and determine its relevance in mitochondrial pathophysiology. In H9c2 cardioblasts, common mitochondrial stressors (antimycin‐A and H2O2) resulted in a significant increase in MDV formation after only 60 min of exposure. The chemotherapeutic doxorubicin (DOX) induced maximal MDV formation after 30 min of treatment, with the levels of MDVs returning to baseline at 24h. In vivo MDV formation and response to stress was analyzed in mice subjected to Sham injection or acute DOX treatment. Despite the absence of echocardiographic differences between groups, mitochondrial functional analysis revealed decreased mitochondrial respiratory capacity, increased ROS production, and susceptibility to PTP opening in the DOX‐treated group. Morphological analysis by TEM revealed a substantial increase in the number of MDVs in the heart of DOX‐treated animals. Our results thus demonstrate that in a cardiac cell model MDV formation is a fast‐response mechanism of mitochondrial QC stimulated by mitochondrial and cellular stressors. Moreover, in addition to the previous reports of in vitro MDV formation, this study demonstrates the existence and responsiveness of MDV formation in cardiac mitochondrial QC.
The Mitochondrial Permeability Transition Pore Regulator Cyclophilin D Exhibits Tissue‐Specific Control of Metabolic Homeostasis
Authors: Laker Rhianna , Taddeo Evan , Zhang Mei , Hoehn Kyle , Yan Zhen
Published: June 2021

The mitochondrial permeability transition pore (mPTP) is a key regulator of mitochondrial health, and the mPTP has been implicated in the pathogenesis of metabolic disease. Cyclophilin D (CypD) is a critical regulator that directly binds to mPTP constituents to facilitate pore opening. Mice lacking CypD in all tissues are protected from diet‐induced glucose intolerance; however, the tissue‐specific function of CypD and mPTP have not been ascertained. To determine the functional role of CypD in metabolic tissues that control glucose homeostasis, we generated liver‐specific and muscle‐specific CypD knockout mice (LKO and MKO, respectively; n=9‐11/gp). These mice were fed either normal chow (NC) or 45% high fat diet (HFD) and evaluated for metabolic phenotypes. We observed that MKO mice were statistically identical to floxed controls with respect to body weight gain or glucose tolerance when fed either diet. However, LKO mice fed HFD developed greater hepatic steatosis, adipose mass, glucose intolerance and pyruvate intolerance compared with flox control mice. These findings demonstrate that CypD has tissue‐specific roles in the context of metabolic physiology, and the loss of CypD in liver is sufficient to exacerbate whole‐body metabolic dysfunction with high fat feeding.

Supported by NIH grant R01AR050429