bluuebunny/crossref_metadata_2025 huggingface dataset:
The Ventilatory Response to Hypercapnia in vivo Requires Serotoninergic Afferents to the Retrotrapezoid Nucleus
Authors: Leirão Isabela P. , Colombari Débora S.A. , da Silva Glauber S.F. , Zoccal Daniel B.
Published: April 2020

Chemosensory cells located in the brainstem are responsible to monitor the arterial levels of CO2/pH and provide excitatory inputs to the respiratory network to evoke a reflex increase in ventilation in conditions of hypercapnia. Among the candidates responsible for central chemoreception are the Raphe serotoninergic neurons. However, there is scarce functional evidence about the role of specific serotoninergic projections to the respiratory network and other chemosensitive sites. Herein, we explored the hypothesis that the hypercapnic ventilatory response in vivo requires, at least in part, the stimulation of the Raphe serotoninergic neurons that project to the retrotrapezoid nucleus (RTN) ‐ an important respiratory and CO2/pH chemosensitive region of the ventrolateral medulla. To reach this goal, pulmonary ventilation was evaluated under baseline conditions and during the exposure to hypercapnia (7% CO2) in unanesthetized juvenile Holtzman rats (60–90 g) that received bilateral microinjections of the toxin anti‐SERT‐SAP (0.1 mM) in the RTN to selectively lesion serotoninergic neurons that send projections to this region. After 15 days of anti‐SERT‐SAP microinjections in the RTN (n=7), baseline respiratory frequency (Rf) and tidal volume (VT) were similar to control rats that received vehicle microinjections (n= 9). On the other hand, the ablation of RTN‐projecting serotoninergic neurons markedly attenuated the CO2‐induced response of increase in the Rf (P=0.001), but not in the VT. Our preliminary analyses suggest that the impaired tachypneic response to hypercapnia induced by anti‐SERT‐SAP microinjections in the RTN was associated with reduced number of serotoninergic neurons in the Raphe Obscurus (Rob) and Magnus (RMg), but not in the Raphe Pallidus. Overall, our data support the idea that interactions between the serotoninergic neurons of the Rob/RMg and the RTN neurons are necessary for the processing of the hypercapnic ventilatory response in unanesthetized conditions.

Support or Funding Information

Supported by FAPESP and CNPq
Glutamate strengthening of GABAergic inhibition in the hypothalamic PVN: Dependence on EAAT3‐mediated Glu uptake into local GABA terminals
Authors: Yamaguchi Junya , Andrade Mary Ann , Truong Tamara , Toney Glenn M.
Published: April 2020

In unstressed animals, pre‐sympathetic neurons (PSNs) of the hypothalamic paraventricular nucleus (PVN) are held in a state of near discharge quiescence by the dominance of GABAergic inhibition. In brain slice studies we found that the amplitude of PSN GABAergic inhibitory postsynaptic currents (IPSCs) is increased by elevated ECF L‐glutamate (Glu). This Glu‐GABA strengthening (GGS) phenomenon, originally described in the hippocampus, can be elicited in PVN PSNs both by exposure to bath Glu (100 μM) or high K+ (+10 mM) for 10 min. PVN GGS is (1) mediated by a presynaptic mechanism that increases unitary quantal size (more GABA molecules/vesicle), (2) is unaffected by inhibition of glial GLT1‐mediated Glu uptake, and (3) is prevented by global inhibition of Glu uptake. Collectively, these findings indicate that PVN GGS depends on Glu uptake by the neuronal Glu transporter EAAT3. Importantly, bath Glu elicits a delayed GABA‐AR‐mediated suppression of PSN firing, indicating that the Glu‐induced increase of IPSC amplitude (i.e., GGS) is sufficient to regulate PSN discharge quiescence. Moreover, in vivo studies indicate that PVN Glu enhances the increase of renal SNA caused by local blockade of GABA‐AR, consistent with elevated Glu increasing functional GABAergic inhibitory tone in the PVN. Here we performed PVN immunohistochemistry and found EAAT3 localized diffusely within somatodendritic compartments and densely in puncta resembling synaptic terminals. EAAT3+ puncta co‐localized with GAD65 punctate staining, consistent with EAAT3 expression on local GABAergic synaptic terminals. To test dependence of GGS on EAAT3 uptake of Glu specifically by PVN GABA terminals, we used a CRISPR/Cas9 gene editing strategy in which spCas9 was expressed in a Cre‐dependent manner amongst vGAT+ (GABAergic) neurons. To knockdown (KD) EAAT3 specifically amongst GABAergic inputs to PVN, AAV1‐CAG‐EAAT3:sgRNA‐GFP was nanoinjected (400 nl) into the surrounding peri‐nuclear zone (PNZ). Western blot analysis of tissue punches (Cntrl: n=10, CRISPR EAAT3: n=12) revealed a nearly 40% loss of EAAT3 in PVN. Given that EAAT3 amongst non‐GABAergic (i.e., glutamatergic) PVN neurons were not targeted by our CRISPR/Cas9 strategy, loss of EAAT3 specifically amongst PVN‐projecting GABAergic PNZ neurons would likely have been much greater than ~40%. Crucially, strengthening of PNZ evoked GABAergic IPSCs by bath Glu was effectively abolished (before: −238 ± 10 pA, after: −226 ± 12 pA; n=3) by CRISPR EAAT3 KD amongst PNZ GABAergic neurons. Results indicate that EAAT3‐mediated Glu uptake into PVN GABA terminals actively defends and restores PSN quiescence following acute glutamatergic excitation.

Support or Funding Information

NIH HL088052 (GMT)
Therapeutic Inhibition of Sphingosine Kinase SphK2 lowers Blood Pressure Levels in an Experimental Model of Hypertension by Reducing CD4+ T‐cell Responses
Authors: Don-Doncow Nicholas , Vanherle Lotte , Matthes Frank , Petzka Henning , Meissner Anja
Published: April 2020

Hypertension is a major cause of premature death in the world and the primary contributor to target organ damage. Although many therapeutic strategies to combat hypertension and its comorbidities exist, none of the current anti‐hypertension medications target the immune system, which evolved as key player in the pathogenesis of hypertension. During hypertension, the activation of the immune system links to alterations in sphingosine‐1‐phosphate (S1P) signaling and specifically, to sphingosine kinase 2 (SphK2)‐mediated immune cell chemotaxis. Our previous findings revealed a protection from the development of hypertension in SphK2 knockout mice. In this study, we explored the efficacy of pharmacological SphK2 inhibition to lowering elevated blood pressure and immune activation during angiotensin II‐induced hypertension in mice.

In a murine model of hypertension where we treated mice with the SphK2 inhibitor ABC294640 or saline for two weeks, we assessed BP levels using tail cuff plethysmography, immune cell populations using flow cytometry, and circulating S1P levels using mass spectrometry. Using decision trees as a method of supervised machine learning, we set out to identify a sequential set of rules on measurement outcomes, which predict a successful reduction of BP.

Pharmacological SphK2 inhibition significantly lowered BP levels, without affecting plasma S1P levels. Notwithstanding its incapacity to lower S1P plasma levels, pharmacological SphK2 inhibition drastically affected T‐cell populations in the blood as evidenced by significantly reduced overall circulating CD4+ T‐cells compared to placebo‐treated hypertensive mice. Besides dampening T‐cell responses typically associated to hypertension (i.e., TH17 and TH1 responses), pharmacological SphK2 inhibition resulted in marked increases of the IL4+TH2 subset. Interestingly, the rules learned by a decision tree identify CD4+ T‐cells as critical modulators for BP responses. Thus, therapeutic administration of CD4 blocking antibody diminished the number of circulating CD4+ T‐cells and significantly lowered elevated blood pressure levels.

Our results provide profound evidence that the SphK2‐S1P signaling axis regulates systemic immune cell homeostasis and T‐cell phenotype changes important to blood pressure regulation. Taken together, our work identified first evidence for SphK2 inhibitors as successful immune‐based therapy in hypertension.

Support or Funding Information

This research was supported by the Swedish Research Council (VR 2017‐01243), the Ake Wibergs Stiftelse (M17‐0031), and the Knut & Alice Wallenberg Foundation.
Analysis of Assembly of Complex I and Characterization Of Clinically Identified Mutations In Membrane Subunits ND2, ND4, and ND5, Using an E. coli Model System
Authors: Zhang Fang , Vik Steven
Published: April 2020

NADH: ubiquinone oxidoreductase, also known as Complex I, is the first and largest enzyme of the respiratory chain, and has a central role in cellular energy production. We use Complex I from E. coil as a model system, which contains 13 subunits named A‐N. Subunits A, H, J, K, L, M and N, constitute the membrane arm, while the rest of the subunits, B, CD, E, F, G, and I, make up the peripheral arm. The assembly pathway of the bacterial Complex I is uncertain. The assembly pathway of subsets of the membrane subunits has been analyzed by native gel electrophoresis and by co‐immunoprecipitation. A gentle detergent, dodecyl maltoside, was used to extract complexes from cellular membranes. Expression of all subunits leads to a large complex in native gels that appears to be Complex I. Expression of subsets of the membrane subunits leads to formation of sub‐complexes M‐N, J‐K‐M‐N, and A‐H‐J. Subunit L only formed complexes when all subunits were expressed. This question was further addressed by expressing the subunits from 2 different plasmids, simultaneously, or sequentially. In addition several clinically identified mutations in subunits L, M and N (ND5, ND4, and ND2, respectively) were constructed, and analysis by western blotting, native gel electrophoresis, and NADH oxidase activity is underway. In conclusion, E. coli Complex I is an effective model system for studies of assembly and analysis of human mutations.

Support or Funding Information

This work was supported by a NIH grant R15 GM 126507.
Small but greedy: a minimal rDNA array outcompetes the genome for replication initiation
Authors: Kwan Elizabeth X. , Alvino Gina M. , Queitsch Christine , Brewer Bonita J. , Raghuraman M. K.
Published: April 2020

Ribosomal DNA (rDNA) copy number varies widely among individuals in many species, but the phenotypic consequences of such copy number fluctuations remain relatively unexplored. Initiation of DNA replication has been detected within the rDNA, presumably required for maintenance of large rDNA arrays. Previous work in S. cerevisiae demonstrated that the rDNA locus—containing an origin of replication in each repeat—can be a significant competitor for replication resources, suggesting that rDNA copy number variation could affect timely completion of genome‐wide replication. To test the hypothesis that reduction in rDNA copy number (and thus rDNA replication origins) reduces competition from the rDNA locus and thereby improves non‐rDNA genome replication, we engineered an S. cerevisiae strain to have a short rDNA array of 35 copies, a minimal array that still maintains wild‐type level ribosome function. We compared this strain to a matched wild‐type strain with 180 rDNA copies. Contrary to our expectations, we found that the 35‐copy rDNA strain displayed classic replication defects: decreased plasmid maintenance, delayed completion of chromosomal replication, and increased sensitivity to replication stress agonists. The 35‐copy rDNA strain also exhibited delayed non‐rDNA origin firing and slower replication progression genome‐wide, reminiscent of mutants with hyperactive rDNA replication. We found that shrinking the rDNA locus dramatically alters rDNA replication time. In wild type cells, the majority of rDNA replicates in mid/late S‐phase of the cell cycle. When reduced to 35 copies, the rDNA locus becomes one of the earliest and most efficiently replicating regions of the genome, starting and completing replication 15 minutes ahead of the normally late‐replicating wild‐type rDNA locus. We therefore advocate that reduction of rDNA copy number alters genome replication through competition with an early‐replicating rDNA locus. Our results indicate that precocious rDNA replication, not total number of rDNA initiations, compromises replication of the genome and suggest that the conserved late‐replicating state of nucleoli may promote genome replication. Thus reductions in rDNA copy number may confer replication stress rather than replication relief, supporting an active role for rDNA copy number variation in determining stability of the genome.
Perinatal nicotine exposure alters miR‐181a‐mediated signaling leading to gender‐dependent development of cardiac ischemia‐sensitive phenotype in adult male offspring
Authors: Jian Jie , Zhang Peng , Li Yong , Liu Bailin , Xiao Daliao
Published: April 2020

Back ground

Cigarette smoking/nicotine exposure during pregnancy is one of the major perinatal insults resulting in an increased risk of cardiovascular diseases in offspring. However, the fetal nicotine exposure‐induced programming of adult cardiovascular dysfunction remains largely elusive. The present study tested the hypothesis that perinatal nicotine exposure caused gender‐dependent increase in heart vulnerability to ischemia‐reperfusion injury and cardiac dysfunction in adult male rat offspring through up‐regulation of miRNA‐181a (miR‐181a)‐mediated signaling pathway.

Methods

Nicotine or saline was administered to pregnant rats via subcutaneous osmotic minipumps from gestational day 4 until postnatal day 10. The adult (~ 3 month old) offspring were treated with miR‐181a antisense (LNA‐miR‐181a) (10 mg/kg × 2 every 5 day, i.p.) or saline. After 10 days of treatment, the rats underwent the cardiac ischemia/reperfusion (I/R) procedure and echocardiography analysis, and the left ventricle (LV) tissues were isolated for molecular biological studies.

Results

Perinatal nicotine exposure enhanced I/R‐induced cardiac infraction and dysfunction in adult male but not in female offspring. The miR‐181a levels in LV tissues were significantly higher in the nicotine‐treated group than the saline control group. In addition, nicotine exposure decreased global DNA methylation levels but enhanced AT1 and AT2 receptor protein expression, and ROS production in LV tissues in the male offspring. Furthermore, the nicotine exposure up‐regulated autophagy‐related proteins (Atg‐5, beclin‐1, LC3 II) expressions as compared to controls in the male offspring. The most importance, treatment with LNA‐miR‐181a reversed nicotine‐mediated I/R‐induced cardiac infarction and dysfunction in male offspring, which was associated with an elimination of the nicotine’s effects on miR‐181a expression, DNA methylation, AT1R/AT2R protein, ROS, and autophagy‐related proteins expressions in the male offspring.

Conclusion

Our data demonstrated that perinatal nicotine exposure could cause a gender‐dependent fetal programing of adult heart ischemia‐sensitive phenotype. Our findings further suggest that miR‐181a plays a casual role in nicotine‐mediated up‐regulation of oxidative stress, renin‐angiotensin system (RAS) and autophagic flux, which may contribute to the development of ischemia‐sensitive phenotype in adult male rats.

Support or Funding Information

Supported in part by the NIH grants: R01HL135623, R03DA041492, and R01HD088039; TRDRP grants: 29IR0437 and T30FT0936
SLO and Steady: Role of Streptolysin O during Inflammasome Activation in Group A Streptococcus‐infected THP‐1 Macrophages
Authors: Alcantara Cheldon Ann T. , Okumura Cheryl Y.M.
Published: April 2020

Group A Streptococcus (GAS; Streptococcus pyogenes) is a Gram‐positive bacterium that infects millions of people globally each year, with a number of cases developing into serious infections such as septicemia, necrotizing fasciitis, and streptococcal toxic shock syndrome. Upon phagocytosis of GAS, intracellular detection of the bacteria triggers a localized inflammatory response by activation of the NLRP3 inflammasome, a cytosolic multimeric protein complex that produces pro‐inflammatory cytokines. A two‐signal system is required for NLRP3 activation which 1) induces transcription of the oligomer components and pro‐IL‐1β, and 2) detects cellular damage to elicit cleavage of pro‐IL‐1β to mature IL‐1β and subsequent extracellular release. Although several GAS proteins have been reported to trigger NLRP3 inflammasome activation, the mechanisms by which bacteria cause inflammation are generally unknown. We have shown that proteins up to 40kDa can escape GAS‐infected phagosomes via Streptolysin O (SLO)‐induced pores, which suggests that either a host phagolysosomal protein or a secreted bacterial protein is detected by cytosolic pathogen recognition receptors (PRRs) to trigger NLRP3‐induced inflammation. To test this, we first infected THP‐1 macrophages with WT or heat‐killed (HK) GAS to establish if a secreted protein activates the inflammasome by monitoring IL‐1β secretion using enzyme‐linked immunosorbent assay (ELISA). We found that HK GAS did not stimulate IL‐ Iβ release, indicating that secretion of bacterial components are necessary for inflammasome activation. To determine essential proteins for GAS‐induced inflammation, we infected THP‐1 macrophages with various secretory protein knockout strains and found that only strains lacking SLO displayed significantly decreased IL‐1β release, confirming that pore formation is necessary for inflammasome activation. Using recombinant SLO (rSLO), we found that the protein itself does not stimulate IL‐1β release. However, when rSLO is coupled with HK GAS, IL‐1β release occurs, indicating that host phagolysosomal proteins may be the trigger for inflammasome activation. The information gained through our investigation provides insight into how GAS and possibly other pathogens or agents that damage the phagosome activate the inflammasome, which may contribute to the development of more effective therapies for infections that cause life‐threatening inflammation.

Support or Funding Information

American Heart Association (17GRNT3410851), Occidental College Undergraduate Research Center
Systems thinking in biochemistry: Assessing students’ understanding of metabolic pathway dynamics and regulation
Authors: Manley Andrew , Villafane Sachel M. , Lewis Jennifer E. , Loertscher Jennifer , Minderhout Vicky
Published: April 2020

Systems thinking requires consideration of dynamic processes, how parts of a whole interact through causal relationships, and how such systems change over time, often in response to environmental stimuli. A systems thinking approach is essential to understanding the complexities of metabolic pathways, yet few tools exist to probe students’ proficiency in this area. We have developed and used an assessment instrument to measure undergraduate students’ understanding of biochemical pathway dynamics and regulation in a metabolic context. A community of biochemistry educators was involved in an iterative process of designing and testing the instrument and accompanying rubric. The assessment instrument was embedded on final exams in an introductory biochemistry course and student responses were collected over several terms. Analysis of student responses showed that most students were able to interpret visual conventions in an unfamiliar metabolic pathway. Furthermore, many students could make basic predictions about how the system would be expected to respond to changes. However, fewer students generated nuanced responses that accounted for both microscopic changes at the protein level and macroscopic changes in pathway product outputs. Interviews revealed that students struggled to process the volume of complex information provided in prompts and the metabolic pathway diagram. This suggests that supporting information processing skills may be an important part of teaching systems thinking and metabolic logic.

Support or Funding Information

NSF‐DUE‐1224868
Selective Phosphodiesterase 1 Inhibitor LY1 Reduces Blood Pressure in Spontaneously Hypertensive and Dahl Salt Sensitive Rats
Authors: Khedr Sherif , Dey Asim , Terashvili Maia , Porras Leah , Meredith Tamika , Willard Francis , Wiley Michael , Atkinson Simon , Kowala Mark , Staruschenko Alexander , Rekhter Mark
Published: April 2020

Regulation of the peripheral vascular resistance via modulating the vessel diameter has been considered as a main determinant of the arterial blood pressure. Phosphodiesterase enzymes (PDE1‐11) hydrolyse cyclic nucleotides, which are key players controlling the vessel diameter and thus peripheral resistance, aiming at achieving and maintaining a homeostatic balance between their production and destruction. On the contrary, pathological shift in the balance to words the destruction leads to depletion of the cyclic nucleotides and dysregulation of the blood pressure. Here, we have tested and reported the effects of a novel selective PDE1 inhibitor (LY1) on the cardiovascular system. In vitro experiments have been conducted to determine the IC50 of LY1 using phosphodiesterase radiometric enzyme assay. We used rat mesenteric artery to conduct myogrphy study to determine the effect of the drug on the vessel diameter. Also, short as well as long term telemetric studies were done on normal Sprague Dawley (SD), spontaneously hypertensive (SHR), and Dahl salt sensitive (SS) rats to document the in vivo efficacy of the compound. LY1 inhibited all three isoforms of PDE1 in nanomolar concentration (1.74 nM, 2.44 nM and 1.20 nM for PDE1A, PDE1B and PDE1C, respectively), while micromolar concentrations were needed to induce effective inhibition for other PDEs. The myography study revealed a potent vasodilatory effect of the drug, which was confirmed in vivo by an increase in the blood flow in the rat ear arteriole reflected by the rise in the temperature. LY1 proved a high efficacy in lowering the blood pressure in two animal models for hypertension i.e. SHR and SS rats. 3 mg/kg of LY1 twice daily through oral gavage could efficiently reduce the blood pressure in both SHR and SS rats approximately 40 mmHg compared to the untreated control group. Therefore, LY1 induced an efficient vasodilation that was accompanied by a significant reduction of blood pressure in different hypertensive rat models. Thus, PDE1 appears to be a tempting therapeutic target for treatment of resistant hypertension.
Relaxin‐3 receptor (RXFP3) mediated modulation of central respiratory activity
Authors: Furuya Werner Issao , Dhingra Rishi R. , Gundlach Andrew L. , Hossain Mohammed Akhter , Dutschmann Mathias
Published: April 2020

The neuropeptide, relaxin‐3, is expressed by pontine nucleus incertus (NI) neurons. Relaxin‐3 and synthetic agonist peptides modulate arousal and cognitive processes via activation of the
rela
xin‐
family
peptide 3 receptor (RXFP3). We recently demonstrated that a double‐chain RXFP3 peptidomimetic (RXFP3‐A2) in the nucleus of the solitary tract (NTS) triggered a mild stimulation of respiration and augmented the chemoreceptor reflex in an in situ perfused brainstem preparation (
Furuya et al., 2020). In the present study, we assessed the central respiratory effects of systemic application and local microinjection into the NTS, Kölliker‐Fuse nucleus (KF) or NI of a single chain RXFP3 peptidomimetic (B18) in the perfused brainstem preparation.

Systemic application of B18 (2 μM) triggered a dose‐dependent increase in respiratory rate by 22 ± 8%. At this concentration of B18, the NaCN‐evoked (0.1% w/v, 100 μl, bolus injection) tachycardia of the arterial chemoreceptor reflex was augmented by 95 ± 14% compared to control (p<0.001, n=4). Local microinjections into the NTS also increased respiratory frequency (28 ± 5%, p<0.05, n=6) and enhanced the NaCN‐evoked tachycardia by 59%. Microinjections into the KF only triggered a mild increase in respiratory frequency (18 ± 7%, p<0.05, n=6) but had no effect on the NaCN‐evoked chemoreceptor reflex. Finally, microinjections into the NI (n=6) had no effect on either stationary breathing activity, or on the chemoreceptor reflex. We conclude that relaxin‐3 neurons target RXFP3 in respiratory control areas and acts as a general respiratory stimulant, causing mild increases in respiratory frequency. Importantly, RXFP3 stimulation significantly enhanced the respiratory response of arterial chemoreceptor reflex, implicating a major neuromodulatory role in this specific reflex pathway.
Development of a Novel Approach for Real‐Time Two‐Photon Imaging of the Rat Hypothalamus In Vivo
Authors: Brown Colin H. , Roy Ranjan K. , Hamm Jordan P. , Stern Javier E.
Published: April 2020

Approaches affording high spatiotemporal resolution with low tissue damage have recently enabled imaging of bidirectional communication among neurons, glial cells and the local microvasculature in living rodents under physiological and disease conditions. However, this approach has been largely limited to superficial dorsal brain areas, omitting hypothalamic centres in which neuro‐glial‐vascular interactions could be critically important for hypothalamic function. Hence, the objective of this study was to develop a preparation to enable in vivo, real‐time imaging of interactions among these cell types in the hypothalamus. As proof of principle, we first focussed on neurovascular interactions. Briefly, urethane‐anaesthetised male and female transgenic rats expressing GFP driven by the vasopressin (VP) promoter (VP‐GFP rats) were placed supine in a stereotaxic frame. The left femoral vein was catheterized and a modification of transpharyngeal surgery developed for in vivo electrophysiological recordings was used to expose the surface of the right hypothalamus and optic chiasm. The vasculature was labelled with rhodamine (70 kDa, 0.2 ml of 20 mg/ml IV). Rats were placed under a two‐photon microscope and imaged at 860–920 nm with a 16X objective to obtain in vivo images of somata, dendrites and axons of individual VP‐GFP neurons within the hypothalamic supraoptic (SON) and suprachiasmatic (SCN) nuclei. Pial and intraparenchymal vessels, including penetrating arterioles and capillary network within the SON and SCN, were imaged after labelling with rhodamine. To monitor neurovascular coupling in the SON, 2 M NaCl was infused (1 ml over 30 min, IV) to activate VP neurons, which significantly reduced vessel diameter (mean peak response −15.7 ± 4.1%, between 15 and 20 min after infusion onset, 13 vessels from 3 rats, P < 0.01), and this vasoconstriction was prevented by topical application of a V1a‐receptor antagonist (V225, 100 μM, 7.5 ± 3.4%, 12 vessels from 2 rats, P = 0.70). To the best of our knowledge, this is the first in vivo imaging of activity‐dependent hypothalamic neurovascular interactions. This approach could be adapted to visualise other hypothalamic areas within ~500 μm of the surface of the brain (e.g. VP‐GFP neurons in the SCN were also imaged), and used in combination with opto/chemogenetics to precisely activate discrete cell populations.
Long Term Effects of Low Dose Radiation Exposure During Late Gestation on Cardiac Metabolism and Oxidative Stress
Authors: Nemec-Bakk Ashley , Niccoli Sarah , Davidson Caitlund , Boreham Douglas , Lees Simon , Khaper Neelam
Published: April 2020

An adverse in utero environment can result in fetal programming that can manifest as diseases later in life. Ionizing radiation (IR) is known to cause detrimental fetal programming especially during organogenesis. High doses of radiation cause severe cellular damage that can lead to mortality, however, the physiological effects of low dose radiation are not fully understood. This study examines the effect of low to non‐lethal radiation exposure during late gestation on cardiac metabolism and oxidative stress in adult mice. Pregnant C57BL/6J mice were exposed to 50, 300, or 1000mGy of gamma radiation or were sham irradiated on gestational day 15. At 4 months of age, 18FDG uptake was observed using PET imaging as an indicator of cardiac glucose uptake. Western blot was used to determine changes in 4‐hydroxynonenal adduct formation, catalase, superoxide dismutase (SOD), and glutathione peroxidase (GPx) protein expression and catalase, SOD and GPx enzyme activity was measured. Both male and female irradiated pups had lower body weights when compared to the sham group. Female pups receiving 1000mGy in utero, demonstrated a significant increase in 18FDG uptake and increased glycogen storage compared to sham group. However, female pups receiving 50mGy IR did not show a significant difference in 18FDG uptake or glycogen storage when compared to sham. Male pups did not exhibit increased 18FDG uptake, however at 50 and 300mGy IR there was decreased glycogen storage compared to sham. The reduced/oxidized glutathione redox ratio, a marker of oxidative stress, was significantly decreased in the female 300mGy and 1000mGy groups compared to sham but there was no change in males. We conclude that non‐lethal doses of radiation during late gestation can alter glucose uptake and storage as glycogen as well as oxidative status in female off‐spring. These data provide evidence that non‐lethal doses of radiation during the third trimester alter cardiac metabolism later in life and sex may have a role in fetal programming.

Support or Funding Information

This work was funded by the Natural Sciences and Engineering Research Council of Canada (NSERC) and by our industry partner, Bruce Power.
Epigenetic Mechanisms Involved in Anti‐Inflammatory Actions of a Diabetes Regulated lncRNA DRAIR in Monocytes
Authors: Reddy Marpadga A. , Amaram Vishnu , Das Sadhan , Tanwar Vinay Singh , Ganguly Rituparna , Wang Mei , Lanting Linda , Zhang Lingxiao , Wu Xiwei , Devaraj Sridevi , Natarajan Rama
Published: April 2020

Type 2 diabetes (T2D) is associated with monocyte activation and chronic inflammation implicated in accelerated cardiovascular disease. Evidence shows that epigenetic mechanisms and long non‐coding RNAs (lncRNA)s can regulate inflammatory phenotype of monocytes, but their role in T2D induced monocyte dysfunction is unclear. Here, we describe the regulation and functions of a novel lncRNA
Diabetes
Regulated
anti‐
inflammatory
RNA (DRAIR) in monocytes. Using RNA‐seq we identified downregulation of several anti‐inflammatory genes and lncRNAs including DRAIR in peripheral blood CD14+ monocytes isolated from subjects with T2D versus healthy controls. Further studies showed that high glucose and palmitic acid significantly downregulated DRAIR expression in CD14+ monocytes and THP1 human monocytes. Conversely, DRAIR was upregulated by anti‐inflammatory cytokines and by PMA (inducer of macrophage differentiation). ChIP assays showed that KLF4 transcription factor binds at KLF4 binding sites in the DRAIR promoter and KLF4 overexpression upregulates DRAIR expression in monocytes. Loss and gain of function approaches revealed that DRAIR regulates anti‐inflammatory phenotype in monocytes and macrophages. Mouse orthologous Drair was also similarly downregulated in macrophages from T2D db/db mice and its knockdown with LNA‐GapmeRs increased expression of pro‐inflammatory genes. Furthermore, we found that DRAIR is a chromatin associated lncRNA. Chromatin isolation by RNA purification and RNA‐immunoprecipitation revealed DRAIR interaction with chromatin near target genes and with a histone methyl transferase G9a. Mechanistically, DRAIR overexpression inhibited G9a recruitment and enrichment of the repressive histone modification H3K9me2 at anti‐inflammatory genes. Together, these results demonstrate that DRAIR can mediate anti‐inflammatory phenotype in monocytes/macrophages via epigenetic mechanisms, and its downregulation in diabetes might contribute to mechanisms promoting chronic inflammation associated with diabetes complications.

Support or Funding Information

This research work was funded by National Institutes of Health grant NIH R01 DK 065073.
Effects of 2‐APB, a Stim‐Activated TRPC‐ORAI Channels Blocker, on the Vascular Remodeling and Pulmonary Hypertension Induced by Chronic Intermittent Hypoxia
Authors: Castillo-Galán Sebastian Andrés , Arenas Germán , Krause Bernardo , Iturriaga Rodrigo
Published: April 2020

Obstructive sleep apnea (OSA), a breathing disorder characterized by episodes of chronic intermittent hypoxia (CIH), is an independent risk for systemic hypertension. Furthermore, 20–60% of OSA patients develop moderate pulmonary hypertension (PH). Rats exposed to CIH developed pulmonary vascular remodeling and PH, but the mechanisms underlaying these responses are not well known. Stim‐activated TRPC‐ORAI channel (STOC), cationic calciumpermeable channels, are overexpressed in the lung and plays a key role in the development of pulmonary vascular remodeling and PH in animals exposed to sustained hypoxia. We previously demonstrated that STOC are overexpressed in lung of rats exposed to CIH. However, whether these channels are involved in the pulmonary vascular remodeling and PH induced by CIH have not been addressed. Thus, we studied the effects of treatment of 2‐aminoethyldiphenylborinate (2‐APB), a STOC blocker, in rats exposed to CIH. Male Sprague‐Dawley rats (~200g) were exposed for 28 days to CIH (5% O2, 12 times/h, for 8h). At 14 days of CIH, rats under anesthesia were implanted with osmotic pumps containing 2‐APB (10 mg/kg/day) or its vehicle, and then rats were exposed to CIH for 14 more days. At the end of CIH exposure, the right ventricular systolic pressure (RVSP) was measure under anesthesia with urethane/α‐cloralose (800 mg/kg, i.p). Animals were then euthanized and lungs dissected to determine vascular remodeling in arteries of 200–300 μm, and the mRNA levels of the STOC forming subunits TRPC1, TRPC4, TRPC6, ORAI 1 and ORAI 2 by qPCR. 2‐APB infusion prevented the increase of the RVSP (36.5 ± 1.1 mmHg vs. 24.7 ± 1.6 mmHg, 28d‐CIH vs. 2‐APB respectively), and the pulmonary vascular remodeling (66.3 ± 9.4 % vs 48.2 ± 3.5 %, CIH, 28d‐CIH vs. 2‐APB, respectively). In addition, 2‐APB treatment reverted the increase in the mRNA levels of TRPC1, TRPC4 and TRPC6, but induced an overexpression of ORAI 1, while ORAI 2 remained unchanged. Present results suggest that STOC plays a key role in the developing of the vascular remodeling and pulmonary hypertension induced by CIH.

Support or Funding Information

SUPPORTED BY FONDECYT 1150040 and 1180341
Mice with Bile acid‐CoA: amino acid N‐acyltransferase deletion are Protected from Obesity
Authors: Alrehaili Bandar D. , Lee Mikang , Takahashi Shogo , Gonzalez Frank J. , Lee Yoon-Kwang
Published: April 2020

Background

Bile acid‐CoA: amino acid N‐acyltransferase (BAAT) is the enzyme which is responsible for bile acid (BA) conjugation with glycine and taurine in the final step of bile acid synthesis in humans. More than 98% of BA conjugation occurs in the liver. BAAT deficiency affects lipid homeostasis in the whole body. BA conjugation is essential to improve fat solubility and counteract passive diffusion in intestine by reducing their pKa values. Thus, the absence of conjugated BAs in the duodenum will affect the absorption of dietary fat and lipid soluble vitamins.

Aim

We aimed to investigate the physiological effects of BAAT deficiency on the mice in diet‐induced obesity using BAAT knockout (BAAT KO) mice.

Methods

Male BAAT KO mice and their littermate WT control mice were fed a WD for 15 weeks. LC‐MS were used to analyze BAs in the gallbladder. Body weight, EchoMRI analysis, CLAMS, gene expression, and plasma analysis were used to discern the severity of fat malabsorption that is associated with the deficiency of BAAT when mice were challenged with a WD.

Results

Total BA levels in serum, liver, and urine were induced in KO mice. Levels of unconjugated BAs were significantly higher and taurine conjugated but not glycine conjugated BAs were almost completely absent in the gallbladders of KO mice. Cyp7a1, Bsep, Abca1a, Abcc4, Papss2, and Sult2a1, genes involved in BA metabolism, were strongly upregulated in the livers of KO mice. Triglyceride levels in the liver exhibited a significant reduction while fecal total lipids levels were increased in KO mice compared with controls after WD challenge. CLAMS data displays an increased in the respiratory exchange ratio and food intake in KO mice when they were on WD. Their BW gains were also significantly impaired during gestation period.

Conclusion

BAAT KO mice fail to gain body weight either during gestation or when they consumed a WD due to the defect in fat absorption, which is caused by conjugated bile acid deficiency. BAATKO mice is a novel model to study bile acid and lipid metabolism.

Support or Funding Information

This work was supported by a bridge fund (Y.K.L) from NEOMED.
FSTL3 Promotes Tumor Cell Proliferation, Invasion, Survival and Associates with Poor Prognosis in Colorectal Cancer Patients
Authors: Fang Chih-Yeu , Hsiao Michael
Published: April 2020

Follistatin like‐3 (FSTL3) is a potent inhibitor that antagonizes TGF‐β family ligands including activin, myostatin and bone morphogenetic proteins. Though usually a secreted glycoprotein, nuclear FSTL3 expression had been noted in several cell types, suggesting the existence of specific intracellular functions of this protein. Colorectal cancer (CRC) is one of the leading malignant tumors worldwide. Its strong metastatic ability and chemoresistance tendency is a major obstacle in clinical treatment. In large cohort survival analyses, we have found that FSTL3 has a significant prognostic predictive power in colorectal cancer at both RNA and protein level. Immunohistochemistry staining of CRC specimens revealed that patients with high FSTL3 expression are significantly associated with poor survival. Our study further showed that knockdown of FSTL3 inhibited the migration and invasion of colon cancer cells and reduced tumorigenesis in vivo. Conversely, ectopic over‐expression of FSTL3 elicited the opposite effects in CRC cells. In addition, the overexpression of FSTL3 was found to confer the resistance to 5‐fluorouracil in CRC cells. mRNA profiling analyses suggested that FSTL3 drives CRC tumorigenesis through various carcinogenesis‐related pathways. Our results unveil the significance of FSTL3 in the oncogenesis of CRC, with implications for its potential use as a diagnostic or prognostic biomarker and also a candidate for therapeutic target in this disease.

Support or Funding Information

This research was supported by Academia Sinica [AS‐SUMMIT‐108].
Physiome: Encouraging Reproducible and FAIR Computational Modelling
Authors: Nickerson David Phillip , Lundengård Karin , Watts Jared , Porter Simon , Yu Tommy , Nielsen Poul , Hunter Peter
Published: April 2020

Computational modelling is a commonly used tool in the physiological sciences. For these models to be useful, it is essential that the model is reproducible and is available for other scientists to reuse. Despite development of standards and supporting software, widespread adoption of publishing reproducible and reusable computer models of biological systems is still lacking.

Physiome is a new journal committed to reproducibility and reusability of mathematical models of physiological processes. Every article published in Physiome is connected to a curated and permanent version of the model code with a persistent identifier. Through the Physiome paper, the code necessary to run the model is easily accessible by just clicking a link, to be reused as it is or as a module in a bigger model. It is also connected to a primary paper published in a domain‐appropriate journal, where the validation and scientific value of the model is evaluated and presented. A Physiome publication is a complement to your primary article that ensures reproducibility, reusability and discoverability of your model. The format encourages modularity that facilitates the combination of different models to develop the next level of systems understanding. And all the models are in one place, easy to Find, Access, Interoperable, and Reuse (FAIR).

Reproducibility and confirmation of results is crucial for useful science and should be one of the supporting pillars of good research. Yet, publication of it is rarely incentivised, often treated as a secondary result at best, which undermines the quality of our work. With the strict formulation of equations and easily shared code, one would expect mathematical models to be reproducible by default, but in fact less than 10% of the models published in scientific journals work when implemented by another group. Physiome will save valuable time and effort on trying to implement models from papers that lack information, or having your results lost because others cannot use them. In doing so, Physiome encourages model authors to make their work available in a reproducible and FAIR manner.

For more information visit:
https://journal.physiomeproject.org.

Support or Funding Information

We acknowledge the support of the International Union of Physiological Sciences, Digital Science, Figshare, Overleaf, and the University of Auckland.
MIF‐CD74 Signaling Protects against Endothelial Senescence in Chronic Obstructive Pulmonary Disease
Authors: Kim So-Jin , Wan Feng , Zhang Xuchen , Zhang Yi , Ifedigbo Emeka , Leng Lin , Bucala Richard , Sauler Maor , Lee Patty
Published: April 2020

RATIONALE

Chronic obstructive pulmonary disease (COPD) is characterized by gradual destruction of alveolar architecture and subsequent airflow obstruction. Advancing age and significant exposure to cigarette smoke (CS) are common risk factors, yet the pathologic manifestations of COPD are heterogenous and the biologic risk factors for COPD are unclear. MIF is an important regulator of innate immunity, cellular homeostasis and survival. We previously reported that circulating MIF was lower in smokers with COPD compared with smokers without COPD. Others have confirmed these findings and correlated decreased MIF protein with greater radiologic/functional evidence of emphysema in patients.

METHODS

Gene expression microarray analysis was performed on lung tissue from individuals with/without COPD (n=262), and endothelial cell (Ec) CD74 expression was analyzed in single‐cell RNA‐seq (control=19, COPD=29). CD74 expression was determined by immunohistochemistry in human lung. CS extract (CSE) treated to HUVEC along with the following recombinant‐MIF (rMIF), MIF‐siRNA, neutralizing anti‐MIF monoclonal antibody (anti‐MIF mAb), MIF098 (MIF antagonist), or MIF20 (MIF agonist). U0126 (ERK inhibitor) was treated 1 hour prior to MIF20 treatment. MIF‐KO mice were treated with a control or MIF expressing lentiviral‐vector. Ec were isolated via MACS sorting. AKR/J mice were exposed to CS (4 months), and MIF20 was administered orally (3 mg/mouse) to mice every day for the final 2 months of CS exposure. Pressure‐Volume (PV) loops were measured by FlexiVent.

RESULTS

CD74 expression was increased in COPD patients especially in lung Ec. CSE increased p16 and p21 mRNA expression in HUVEC. This increase was attenuated by rMIF or MIF20, while augmented by anti‐MIF mAb or MIF98. CSE increased SA‐β‐gal activity and MIF20 attenuated it. The protective effect of MIF20 against CS‐induced Ec senescence was abolished by ERK signaling inhibitor. MIF‐KO mice showed an upshift of PV loop and increased Ec p21 expression, which were attenuated by restoration of MIF expression. CS induced upshift of PV loop, increased p16 and p21 expression, which were attenuated by MIF20.

CONCLUSIONS

The expression of CD74, the cognate receptor for MIF, was increased in COPD. Senescence was induced in Ec by CS, an important causative factor for COPD. MIF20, a novel small molecule MIF agonist, attenuated CS‐induced senescence via ERK signaling, which is known to support cell survival and proliferation. Furthermore, MIF20 prevented CS‐induced lung emphysema. These data suggest that therapeutic modulation of MIF targeting in lung Ec is feasible represents a novel strategy to protect against senescence‐induced COPD.

Support or Funding Information

M.S. is supported by NIH/NHLBI K08HL135402 and FAMRI YCSA 142017. P.J.L. is supported by NIH/NHLBI R01 HL138386, VAORD11858595, US Department of Defense PR150809, and FAMRI 150074.
Antibiotics in Apis mellifera Hemolymph
Authors: Brandt Liam J.
Published: April 2020

The western honey bee (Apis mellifera) is a large contributor to the food supply for humans. Over the past decades, there has been a decrease in bee colonies caused by multiple stress factors. A possible contributor to this decline is due to multiple infectious agents being introduced to the honey bees. In some hives, more than 90% of the dying bees contained a gram‐negative bacterium
[1]. Honey bees’ immunities against diseases are not fully understood, so to better understand their immunity a method was devised to test for useful proteins and antibodies in Apis mellifera’s hemolymph. Our research begins by extracting the bee’s hemolymph while they are in a comatose state and freezing it. Next, the hemolymph is used in an aggravate solution and injected into mice over a period of several months several months for their bodies to start producing the unknown antibodies. After the immunization process is completed, the mice are sacrificed by extracting their spleen where it is homogenized and the supernatant is extracted and incubated into a media. The hybridoma cells are duplicated into monoclonal colonies and frozen in liquid nitrogen for further testing or continuously grown for immediate testing. The supernatant from the hybridoma cells is taken for analysis where multiple analytical tests are performed to identify which supernatants contain antibodies. Through analytical testing, we hope to discover antibodies that will help researchers better understand honey bee immunity to combat in the decline of honey bee colonies.
Contributing annotations of predicted intrinsic protein disorder to the RCSB Protein Data Bank
Authors: Bui Jonathan T.
Published: April 2020

The Protein Data Bank (PDB) holds information about three‐dimensional (3D) structures of biological macromolecules. It’s a global resource used by industry, researchers, students and educators for various ends. To serve users with diverse needs, the RCSB PDB (accessible at rcsb.org) integrates additional information associated with each structure, among which are properties derived from sequence data. Predictions of highly dynamic disordered regions are available for PDB entries, calculated using JRONN, a Java port of RONN algorithm. The IUPred2A method for calculations of disordered protein regions is evaluated to replace the previously used JRONN method. Previously published benchmark studies show that IUPred2A is more accurate than JRONN. By pre‐computing disordered regions and serving this information with each protein sequence in the PDB, users gain additional reference information and adds context to solved protein structures without requiring users to derive property information on their own. Furthermore, the integration of disorder calculations can be combined with other reference data on the PDB. For example, many entries in the PDB are highly structured globular molecules, but the addition of predicted regions of disorder can reveal short unstructured regions that can be important for protein‐protein interactions. Calculations of intrinsically disordered regions in protein sequences contributes valuable reference information to the PDB and can serve diverse research needs.

Support or Funding Information

National Science Foundation (DBI‐1832184), the US Department of Energy (DE‐SC0019749), and the National Cancer Institute, National Institute of Allergy and Infectious Diseases, and National Institute of General Medical Sciences of the National Institute of Health under grant R01GM133198.