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Fish air breathing is crucial for the transition of vertebrates from water to land. So far, the genes involved in fish air breathing have not been well identified. Here, we performed gene enrichment analysis of positively selected genes (PSGs) in loach (Misgurnus anguillicaudatus, an air-breathing fish) in comparison to Triplophysa tibetana (a non-air-breathing fish), haplotype-resolved genome assembly of the loach, and gene evolutionary analysis of air-breathing and non-air-breathing fishes and found that the PSG mex3a originated from ancient air-breathing fish species. Deletion of Mex3a impaired loach air-breathing capacity by inhibiting angiogenesis through its interaction with T-box transcription factor 20. Mex3a overexpression significantly promoted angiogenesis. Structural analysis and point mutation revealed the critical role of the 201st amino acid in loach Mex3a for angiogenesis. Our findings innovatively indicate that the ancient mex3a is a fish air-breathing gene, which holds significance for understanding fish air breathing and provides a valuable resource for cultivating hypoxia-tolerant fish varieties., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

The mitochondrial matrix peptidase CLPP is crucial during cell stress. Its loss causes Perrault syndrome type 3 (PRLTS3) with infertility, neurodegeneration, and a growth deficit. Its target proteins are disaggregated by CLPX, which also regulates heme biosynthesis via unfolding ALAS enzymes, providing access for pyridoxal-5'-phosphate (PLP). Despite efforts in diverse organisms with multiple techniques, CLPXP substrates remain controversial. Here, avoiding recombinant overexpression, we employed complexomics in mitochondria from three mouse tissues to identify endogenous targets. A CLPP absence caused the accumulation and dispersion of CLPX-VWA8 as AAA+ unfoldases, and of PLPBP. Similar changes and CLPX-VWA8 co-migration were evident for mitoribosomal central protuberance clusters, translation factors like GFM1-HARS2, the RNA granule components LRPPRC-SLIRP, and enzymes OAT-ALDH18A1. Mitochondrially translated proteins in testes showed reductions to <30% for MTCO1-3, the mis-assembly of the complex IV supercomplex, and accumulated metal-binding assembly factors COX15-SFXN4. Indeed, heavy metal levels were increased for iron, molybdenum, cobalt, and manganese. RT-qPCR showed compensatory downregulation only for Clpx mRNA; most accumulated proteins appeared transcriptionally upregulated. Immunoblots validated VWA8, MRPL38, MRPL18, GFM1, and OAT accumulation. Co-immunoprecipitation confirmed CLPX binding to MRPL38, GFM1, and OAT, so excess CLPX and PLP may affect their activity. Our data mechanistically elucidate the mitochondrial translation fidelity deficits which underlie progressive hearing impairment in PRLTS3.

Air-breathing has evolved independently serval times with a variety of air-breathing organs (ABOs) in fish. The physiology of the air-breathing in bimodal respiration fish has been well understood, while studies on molecular mechanisms of the character are very limited. In the present study, we first determined the gill indexes of 110 fish species including 25 and 85 kinds of bimodal respiration fishes and non-air-breathing fishes, respectively. Then combined with histological observations of gills and ABOs/non-ABOs in three bimodal respiration fishes and two non-air breathing fishes, we found that the bimodal respiration fish was always of a degeneration gill and a well-vascularized ABO. Meanwhile, a comparative transcriptome analysis of posterior intestines, namely a well vascularized ABO in Misgurnus anguillicaudatus and a non-ABO in Leptobotia elongata, was performed to expound molecular variations of the air-breathing character. A total of 5,003 orthologous genes were identified. Among them, 1,189 orthologous genes were differentially expressed, which were enriched in 14 KEGG pathways. More specially, the expressions of hemoglobin genes and various HIF/VEGF signaling pathway genes were obviously upregulated in the ABO of M. anguillicaudatus. Moreover, we found that HIF-1α, VEGFAa, and MAP2K1 were co-expressed dramatically higher in ABOs of bimodal respiration fishes than those of non-ABOs of non-air-breathing fishes. These results indicated that the HIF/VEGF pathway played an important role in ABO angiogenesis/formation to promote fish to do aerial respiration. This study will contribute to our understanding of molecular mechanisms of air-breathing in fish., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Evidence for a cerebellar role during cardiopulmonary challenges has long been established, but studies of cerebellar involvement in eupneic breathing have been inconclusive. Here we investigated temporal aspects of eupneic respiration in the Atoh1-En1/2 mouse model of cerebellar neuropathology. Atoh1-En1/2 conditional knockout mice have conditional loss of the developmental patterning genes Engrailed1 and 2 in excitatory cerebellar nuclear neurons, which leads to loss of a subset of medial and intermediate excitatory cerebellar nuclear neurons. A sample of three Atoh1-derived extracerebellar nuclei showed no cell loss in the conditional knockout compared to control mice. We measured eupneic respiration in mutant animals and control littermates using whole-body unrestrained plethysmography and compared the average respiratory rate, coefficient of variation, and the CV2, a measure of intrinsic rhythmicity. Linear regression analyses revealed that Atoh1-En1/2 conditional knockouts have decreased overall variability (p = 0.021; b = -0.045) and increased intrinsic rhythmicity compared to their control littermates (p < 0.001; b = -0.037), but we found no effect of genotype on average respiratory rate (p = 0.064). Analysis also revealed modestly decreased respiratory rates (p = 0.025; b = -0.82), increased coefficient of variation (p = 0.0036; b = 0.060), and increased CV2 in female animals, independent of genotype (p = 0.024; b = 0.026). These results suggest a cerebellar involvement in eupneic breathing by controlling rhythmicity. We argue that the cerebellar involvement in controlling the CV2 of respiration is indicative of an involvement of coordinating respiration with other orofacial rhythms, such as swallowing., (© 2021 The Authors. Genes, Brain and Behavior published by International Behavioural and Neural Genetics Society and John Wiley & Sons Ltd.)

Rett syndrome (RTT) is a neurodevelopmental disorder caused mostly by mutations in the MECP2 gene. RTT patients show periodical hypoventilation attacks. The breathing disorder contributing to the high incidence of sudden death is thought to be due to depressed central inspiratory (I) activity via unknown cellular processes. Demonstration of such processes may lead to targets for pharmacological control of the RTT-type hypoventilation. We performed in vivo recordings from medullary respiratory neurons on the RTT rat model. To our surprise, both I and expiratory (E) neurons in the ventral respiratory column (VRC) increased their firing activity in Mecp2-null rats with severe hypoventilation. These I neurons including E-I phase-spanning and other I neurons remained active during apneas. Consistent with enhanced central I drive, ectopic phrenic discharges during expiration as well as apnea were observed in the Mecp2-null rats. Considering the increased I neuronal firing and ectopic phrenic activity, the RTT-type hypoventilation does not seem to be caused by depression in central I activity, neither reduced medullary I premotor output. This as well as excessive E neuronal firing as shown in our previous studies suggests inadequate synaptic inhibition for phase transition. We found that the abnormal respiratory neuronal firing, ectopic phrenic discharge as well as RTT-type hypoventilation all can be corrected by enhancing GABAergic inhibition. More strikingly, Mecp2-null rats reaching humane endpoints with severe hypoventilation can be rescued by GABAergic augmentation. Thus, defective GABAergic inhibition among respiratory neurons is likely to play a role in the RTT-type hypoventilation, which can be effectively controlled with pharmacological agents., (© 2021 Wiley Periodicals LLC.)

Viral-mediated in vivo gene delivery methods currently dominate among therapeutic strategies within the clinical and experimental settings, albeit with well documented limitations arising from immunologic constraints. In this study, we demonstrate the utility of nonviral hepatotropic in vivo gene delivery of unpackaged expression constructs, including one encoding fibroblast growth factor 21 (FGF21). FGF21 is an important hepatokine whose expression positively correlates with therapeutic outcomes across various animal models of obesity. Our data demonstrate that FGF21 expression can be restored into the livers of immunocompetent FGF21 knockout mice for at least 2 weeks after a single injection with an FGF21 expression plasmid. In wild-type C57BL6/J mice, in vivo transfection with an FGF21-expressing plasmid induced weight loss, decreased adiposity, and activated thermogenesis in white fat within 2 weeks. Furthermore, in vivo FGF21 gene delivery protected C57BL6/J mice against diet-induced obesity by decreasing adiposity and increasing uncoupling protein 1-dependent thermogenesis in brown fat and by boosting respiratory capacity in subcutaneous and perigonadal white fat. Together, the data illustrate a facile and effective methodology for delivering prolonged protein expression specifically to the liver. We contend that this method will find utility in basic science research as a practical means to enhance in vivo studies characterizing liver protein function. We further believe our data provide a rationale for further exploring the potential clinical utility of nonviral gene therapy in mouse models of disease. SIGNIFICANCE STATEMENT: This study presents a valuable method for nonviral gene delivery in mice that improves upon existing techniques. The data provide a rationale for further exploring the potential clinical utility of nonviral gene therapy in mouse models of disease and will likely enhance in vivo studies characterizing liver protein function., (Copyright © 2021 by The Author(s).)

Leigh syndrome is a severe mitochondrial neurodegenerative disease with no effective treatment. In the Ndufs4 -/- mouse model of Leigh syndrome, continuously breathing 11% O 2 (hypoxia) prevents neurodegeneration and leads to a dramatic extension (~5-fold) in lifespan. We investigated the effect of hypoxia on the brain metabolism of Ndufs4 -/- mice by studying blood gas tensions and metabolite levels in simultaneously sampled arterial and cerebral internal jugular venous (IJV) blood. Relatively healthy Ndufs4 -/- and wildtype (WT) mice breathing air until postnatal age ~38 d were compared to Ndufs4 -/- and WT mice breathing air until ~38 days old followed by 4-weeks of breathing 11% O 2 . Compared to WT control mice, Ndufs4 -/- mice breathing air have reduced brain O 2 consumption as evidenced by an elevated partial pressure of O 2 in IJV blood (P ijv O 2 ) despite a normal PO 2 in arterial blood, and higher lactate/pyruvate (L/P) ratios in IJV plasma revealed by metabolic profiling. In Ndufs4 -/- mice, hypoxia treatment normalized the cerebral venous P ijv O 2 and L/P ratios, and decreased levels of nicotinate in IJV plasma. Brain concentrations of nicotinamide adenine dinucleotide (NAD + ) were lower in Ndufs4 -/- mice breathing air than in WT mice, but preserved at WT levels with hypoxia treatment. Although mild hypoxia (17% O 2 ) has been shown to be an ineffective therapy for Ndufs4 -/- mice, we find that when combined with nicotinic acid supplementation it provides a modest improvement in neurodegeneration and lifespan. Therapies targeting both brain hyperoxia and NAD + deficiency may hold promise for treating Leigh syndrome., Competing Interests: Declaration of Competing Interest V.K.M. and W.M.Z. are co-inventors on a patent application submitted by Massachusetts General Hospital on the use of hypoxia as a therapy. V.K.M. owns equity stake in Raze Therapeutics and is a paid advisor for Janssen Pharmaceuticals and 5 AM Ventures. R.S. holds equity in BlueBird Bio., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Hepatocellular carcinoma (HCC) is the second leading cause of cancer-related death worldwide. Currently, recent risk stratification has only focused on liver function and tumor characteristics. Thus, the purpose of this study was to develop a prognostic model based on genes involved in aerobic respiration. Matched tumor and normal tissues from TCGA and ICGC cohorts were analyzed to identify 15 overlapping differential expressed genes. Cox univariate analysis of the 15 genes in the TCGA cohort revealed they were all associated with disease-specific survival (DSS) in HCC patients. Using LASSO estimation and the optimal value for penalization coefficient lambda 12 genes were selected for the prognostic model, and then HCC patients in the TCGA cohort were dichotomized into low-risk and high-risk groups. Univariate and multivariate Cox analysis demonstrated patients in low-risk group had better survival. Validation of the risk score model with the ICGC cohort produces results consistent with those of the TCGA cohort. In conclusion, this study developed and validated a prognostic model of HCC through a comprehensive analysis of genes involved in aerobic respiration. This model may help develop personalized treatments for patients with HCC.

Vagus nerve stimulation has shown many benefits for disease therapies but current approaches involve imprecise electrical stimulation that gives rise to off-target effects, while the functionally relevant pathways remain poorly understood. One method to overcome these limitations is the use of optogenetic techniques, which facilitate targeted neural communication with light-sensitive actuators (opsins) and can be targeted to organs of interest based on the location of viral delivery. Here, we tested whether retrograde adeno-associated virus (rAAV2-retro) injected in the heart can be used to selectively express opsins in vagus nerve fibers controlling cardiac function. Furthermore, we investigated whether perturbations in cardiac function could be achieved with photostimulation at the cervical vagus nerve. Viral injection in the heart resulted in robust, primarily afferent, opsin reporter expression in the vagus nerve, nodose ganglion, and brainstem. Photostimulation using both one-photon stimulation and two-photon holography with a GRIN-lens incorporated nerve cuff, was tested on the pilot-cohort of injected mice. Changes in heart rate, surface electrocardiogram, and respiratory responses were observed in response to both one- and two-photon photostimulation. The results demonstrate feasibility of retrograde labeling for organ targeted optical neuromodulation.

Brainstem networks that control regular tidal breathing depend on excitatory drive, including from tonically active, CO 2 /H + -sensitive neurons of the retrotrapezoid nucleus (RTN). Here, we examine intrinsic ionic mechanisms underlying the metronomic firing activity characteristic of RTN neurons. In mouse brainstem slices, large-amplitude membrane potential oscillations are evident in synaptically isolated RTN neurons after blocking action potentials. The voltage-dependent oscillations are abolished by sodium replacement; blocking calcium channels (primarily L-type); chelating intracellular Ca 2+ ; and inhibiting TRPM4, a Ca 2+ -dependent cationic channel. Likewise, oscillation voltage waveform currents are sensitive to calcium and TRPM4 channel blockers. Extracellular acidification and serotonin (5-HT) evoke membrane depolarization that augments TRPM4-dependent oscillatory activity and action potential discharge. Finally, inhibition of TRPM4 channels in the RTN of anesthetized mice reduces central respiratory output. These data implicate TRPM4 in a subthreshold oscillation that supports the pacemaker-like firing of RTN neurons required for basal, CO 2 -stimulated, and state-dependent breathing., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Cilia and centrosomes of eukaryotic cells play important roles in cell movement, fluid transport, extracellular sensing, and chromosome division. The physiological functions of cilia and centrosomes are generated by their dynamics, motions, and forces controlled by the physical, chemical, and biological environments. How an individual cilium achieves its beat pattern and induces fluid flow is governed by its ultrastructure as well as the coordination of associated molecular motors. Thus, a bottom-up understanding of the physiological functions of cilia and centrosomes from the molecular to tissue levels is required. Correlations between the structure and motion can be understood in terms of mechanics. This review first focuses on cilia and centrosomes at the molecular level, introducing their ultrastructure. We then shift to the organelle level and introduce the kinematics and mechanics of cilia and centrosomes. Next, at the tissue level, we introduce nodal ciliary dynamics and nodal flow, which play crucial roles in the organogenetic process of left-right asymmetry. We also introduce respiratory ciliary dynamics and mucous flow, which are critical for protecting the epithelium from drying and exposure to harmful particles and viruses, i.e., respiratory clearance function. Finally, we discuss the future research directions in this field., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

The bighead catfish (Clarias macrocephalus) and channel catfish (Ictalurus punctatus) are freshwater species in the Siluriformes order. C. macrocephalus has both gills and modified gill structures serving as an air-breathing organ (ABO), while I. punctatus does not possess such an organ, and cannot breathe in air, providing an excellent model for studying the molecular basis of ABO development in teleost fish. To investigate the critical time window for the development of air-breathing function, seven development stages were selected based on hypoxia challenge results, and RNA-seq was performed upon C. macrocephalus to compare with the non-air-breathing I. punctatus. Five-hundred million reads were generated and 25,239 expressed genes were annotated in C. macrocephalus. Among those, 8675 genes were differentially expressed across developmental stages. Comparative genomic analysis identified 1458 C. macrocephalus specific genes, which were absent in I. punctatus. Gene network and protein-protein interaction analyses identified 26 key hub genes involved in the air-breathing function. Three top candidate genes, mb, ngb, hbae, are mainly associated with oxygen carrying, oxygen binding, and heme binding activities. Our study provides a rich data set for exploring the genomic basis of air-breathing function in C. macrocephalus and offers insights into the adaption to hypoxic environments.

Carbonic anhydrases (CAs) are essential metalloenzymes in nature, catalyzing the carbon dioxide reversible hydration into bicarbonate and proton. In humans, breathing and many other critical physiological processes depend on this enzymatic activity. The CA superfamily function and inhibition in pathogenic bacteria has recently been the object of significant advances, being demonstrated to affect microbial survival/virulence. Targeting bacterial CAs may thus be a valid alternative to expand the pharmacological arsenal against the emergence of widespread antibiotic resistance. Here, we report an extensive study on the inhibition profile of the recently discovered ι-CA class present in some bacteria, including Burkholderia territorii , namely BteCAι, using substituted benzene-sulfonamides and clinically licensed sulfonamide-, sulfamate- and sulfamide-type drugs. The BteCAι inhibition profile showed: (i) several benzene-sulfonamides with an inhibition constant lower than 100 nM; (ii) a different behavior with respect to other α, β and γ-CAs; (iii) clinically used drugs having a micromolar affinity. This prototype study contributes to the initial recognition of compounds which efficiently and selectively inhibit a bacterial member of the ι-CA class, for which such a selective inhibition with respect to other protein isoforms present in the host is highly desired and may contribute to the development of novel antimicrobials.

Iron-sulfur (Fe/S) proteins are present in virtually all living organisms and are involved in numerous cellular processes such as respiration, photosynthesis, metabolic reactions, nitrogen fixation, radical biochemistry, protein synthesis, antiviral defense, and genome maintenance. Their versatile functions may go back to the proposed role of their Fe/S cofactors in the origin of life as efficient catalysts and electron carriers. More than two decades ago, it was discovered that the in vivo synthesis of cellular Fe/S clusters and their integration into polypeptide chains requires assistance by complex proteinaceous machineries, despite the fact that Fe/S proteins can be assembled chemically in vitro. In prokaryotes, three Fe/S protein biogenesis systems are known; ISC, SUF, and the more specialized NIF. The former two systems have been transferred by endosymbiosis from bacteria to mitochondria and plastids, respectively, of eukaryotes. In their cytosol, eukaryotes use the CIA machinery for the biogenesis of cytosolic and nuclear Fe/S proteins. Despite the structural diversity of the protein constituents of these four machineries, general mechanistic concepts underlie the complex process of Fe/S protein biogenesis. This review provides a comprehensive and comparative overview of the various known biogenesis systems in Biology, and summarizes their common or diverging molecular mechanisms, thereby illustrating both the conservation and diverse adaptions of these four machineries during evolution and under different lifestyles. Knowledge of these fundamental biochemical pathways is not only of basic scientific interest, but is important for the understanding of human 'Fe/S diseases' and can be used in biotechnology., (Copyright © 2020 The Author(s). Published by Elsevier B.V. All rights reserved.)

Light/dark cycle affects the physiology of vertebrates and hypothalamic orexin neurons (ORX) are involved in this function. The breathing pattern of the green iguana changes from continuous to episodic across the light/dark phases. Since the stimulatory actions of ORX on breathing are most important during arousal, we hypothesized that ORX regulates changes of breathing pattern in iguanas. Thus, we: (1) Localized ORX neurons with immunohistochemistry; (2) Quantified cyclic changes in plasma orexin-A levels by ELISA; (3) Compared breathing pattern at rest and during hypoxia and hypercarbia; (4) Evaluated the participation of the ORX receptors in ventilation with intracerebroventricular microinjections of ORX antagonists during light and dark phases. We show that the ORX neurons of I. iguana are located in the periventricular hypothalamic nucleus. Orexin-A peaks during the light/active phase and breathing parallels these cyclic changes: ventilation is higher during the light phase than during the dark phase. However, inactivation of ORX-receptors does not affect the breathing pattern. Iguanas increase ventilation during hypoxia only during the light phase. Conversely, CO 2 promotes post-hypercarbic hyperpnea during both phases. We conclude that ORXs potentiate the post-hypercarbic (but not the hypoxic)-drive to breathe and are not involved in light/dark changes in the breathing pattern.

The Krebs cycle enzyme fumarase is a dual-targeted protein that is located in the mitochondria and cytoplasm of eukaryotic cells. Besides being involved in the TCA cycle and primary metabolism, fumarase is a tumour suppressor that aids DNA repair in human cells. Using mass spectrometry, we identified modifications in peptides of cytosolic yeast fumarase, some of which were absent when the cells were exposed to DNA damage (using the homing endonuclease system or hydroxyurea). We show that DNA damage increased the enzymatic activity of fumarase, which we hypothesized to be affected by post-translational modifications. Succinylation and ubiquitination of fumarase at lysines 78 and 79, phosphorylation at threonine 122, serine 124 and threonine 126 as well as deamidation at arginine 239 were found to be functionally relevant. Upon homology analysis, these residues were also found to be evolutionally conserved. Serine 128, on the other hand, is not evolutionary conserved and the Fum1S128D phosphorylation mimic was able to aid DNA repair. Our molecular model is that the above modifications inhibit the enzymatic activity of cytosolic fumarase under conditions of no DNA damage induction and when there is less need for the enzyme. Upon genotoxic stress, some fumarase modifications are removed and some enzymes are degraded while unmodified proteins are synthesized. This report is the first to demonstrate how post-translational modifications influence the catalytic and DNA repair functions of fumarase in the cell., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Breathing is coupled to metabolism. Leptin, a peptide mainly secreted in proportion to adipose tissue mass, increases energy expenditure with a parallel increase in breathing. We demonstrate that optogenetic activation of LepRb neurons in the nucleus of the solitary tract (NTS) mimics the respiratory stimulation after systemic leptin administration. We show that leptin activates the sodium leak channel (NALCN), thereby depolarizing a subset of glutamatergic (VGluT2) LepRb NTS neurons expressing galanin. Mice with selective deletion of NALCN in LepRb neurons have increased breathing irregularity and central apneas. On a high-fat diet, these mice gain weight with an associated depression of minute ventilation and tidal volume, which are not detected in control littermates. Anatomical mapping reveals LepRb NTS-originating glutamatergic axon terminals in a brainstem inspiratory premotor region (rVRG) and dorsomedial hypothalamus. These findings directly link a defined subset of NTS LepRb cells to the matching of ventilation to energy balance., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

The neural control system underlying breathing is sexually dimorphic with males being more vulnerable to dysfunction. Microglia also display sex differences, and their role in the architecture of brainstem respiratory rhythm circuitry and modulation of cervical spinal cord respiratory plasticity is becoming better appreciated. To further understand the molecular underpinnings of these sex differences, we performed RNA sequencing of immunomagnetically isolated microglia from brainstem and cervical spinal cord of adult male and female rats. We used various bioinformatics tools (Gene Ontology, Kyoto Encyclopedia of Genes and Genomes, Reactome, STRING, MAGICTRICKS) to functionally categorize identified gene sets, as well as to pinpoint common transcriptional gene drivers that may be responsible for the observed transcriptomic differences. We found few sex differences in the microglial transcriptomes derived from the brainstem, but several hundred genes were differentially expressed by sex in cervical spinal microglia. Comparing brainstem and spinal microglia within and between sexes, we found that the major factor guiding transcriptomic differences was central nervous system (CNS) location rather than sex. We further identified key transcriptional drivers that may be responsible for the transcriptomic differences observed between sexes and CNS regions; enhancer of zeste homolog 2 emerged as the predominant driver of the differentially downregulated genes. We suggest that functional gene alterations identified in metabolism, transcription, and intercellular communication underlie critical microglial heterogeneity and sex differences in CNS regions that contribute to respiratory disorders categorized by dysfunction in neural control. These data will also serve as an important resource data base to advance our understanding of innate immune cell contributions to sex differences and the field of respiratory neural control. SIGNIFICANCE STATEMENT: The contributions of central nervous system (CNS) innate immune cells to sexually dimorphic differences in the neural circuitry controlling breathing are poorly understood. We identify key transcriptomic differences, and their transcriptional drivers, in microglia derived from the brainstem and the C3-C6 cervical spinal cord of healthy adult male and female rats. Gene alterations identified in metabolism, gene transcription, and intercellular communication likely underlie critical microglial heterogeneity and sex differences in these key CNS regions that contribute to the neural control of breathing., (Copyright © 2020 by The American Society for Pharmacology and Experimental Therapeutics.)

Objective: To propose alternative learning strategies for glossopharyngeal breathing in patients with Duchenne muscular dystrophy (DMD) and healthy men., Design: A feasibility study with small case series., Subjects: Five boys with DMD and 7 male physical therapists as healthy controls who had not learned glossopharyngeal breathing., Methods: Participants were instructed in a glossopharyngeal breathing protocol, including induction methods comprising sucking motions and phonation with inhalation. The protocol consisted of 1-6 sessions (10-15 min each; total 60 min). Criteria for glossopharyngeal breathing mastery were vital capacity with glossopharyngeal insufflation (VCGI)/VC ratio > 1.10 for the DMD group and > 1.05 for the Healthy group. Feasibility outcomes were time required for mastering glossopharyngeal breathing, self-reported outcomes, adverse events and drop-outs., Results: All participants learned glossopharyngeal breathing within the allocated 60 min. Mean VCGI/VC ratio was 1.31 for the DMD group and 1.09 for the Healthy group. No adverse events or drop-outs were encountered during the protocol. In most cases, self-reported outcomes showed that motivation increased and difficulty decreased., Conclusion: Induction methods for sucking motions and phonation with inhalation for glossopharyngeal breathing learning are feasible. This paper proposes alternative strategies for glossopharyngeal breathing learning in boys with DMD and their instructors.

Arterial tortuosity syndrome (ATS) is a recessively inherited connective tissue disorder, mainly characterized by tortuosity and aneurysm formation of the major arteries. ATS is caused by loss-of-function mutations in SLC2A10, encoding the facilitative glucose transporter GLUT10. Former studies implicated GLUT10 in the transport of dehydroascorbic acid, the oxidized form of ascorbic acid (AA). Mouse models carrying homozygous Slc2a10 missense mutations did not recapitulate the human phenotype. Since mice, in contrast to humans, are able to intracellularly synthesize AA, we generated a novel ATS mouse model, deficient for Slc2a10 as well as Gulo, which encodes for L-gulonolactone oxidase, an enzyme catalyzing the final step in AA biosynthesis in mouse. Gulo;Slc2a10 double knock-out mice showed mild phenotypic anomalies, which were absent in single knock-out controls. While Gulo;Slc2a10 double knock-out mice did not fully phenocopy human ATS, histological and immunocytochemical analysis revealed compromised extracellular matrix formation. Transforming growth factor beta signaling remained unaltered, while mitochondrial function was compromised in smooth muscle cells derived from Gulo;Slc2a10 double knock-out mice. Altogether, our data add evidence that ATS is an ascorbate compartmentalization disorder, but additional factors underlying the observed phenotype in humans remain to be determined., (© The Author(s) 2020. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Small noncoding RNAs (sRNAs) from mRNA 3' UTRs seem to present a previously unrecognized layer of bacterial post-transcriptional control whereby mRNAs influence each other's expression, independently of transcriptional control. Studies in Escherichia coli and Salmonella enterica showed that such sRNAs are natural products of RNase E-mediated mRNA decay and associate with major RNA-binding proteins (RBPs) such as Hfq and ProQ. If so, there must be additional sRNAs from mRNAs that accumulate only under specific physiological conditions. We test this prediction by characterizing candidate NarS that represents the 3' UTR of nitrate transporter NarK whose gene is silent during standard aerobic growth. We find that NarS acts by Hfq-dependent base pairing to repress the synthesis of the nitrite transporter, NirC, resulting in mRNA cross-regulation of nitrate and nitrite transporter genes. Interestingly, the NarS-mediated repression selectively targets the nirC cistron of the long nirBDC-cysG operon, an observation that we rationalize as a mechanism to protect the bacterial cytoplasm from excessive nitrite toxicity during anaerobic respiration with abundant nitrate. Our successful functional assignment of a 3' UTR sRNA from a non-standard growth condition supports the notion that mRNA crossregulation is more pervasive than currently appreciated., (© The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The precise pattern of motor neuron (MN) activation is essential for the execution of motor actions; however, the molecular mechanisms that give rise to specific patterns of MN activity are largely unknown. Phrenic MNs integrate multiple inputs to mediate inspiratory activity during breathing and are constrained to fire in a pattern that drives efficient diaphragm contraction. We show that Hox5 transcription factors shape phrenic MN output by connecting phrenic MNs to inhibitory premotor neurons. Hox5 genes establish phrenic MN organization and dendritic topography through the regulation of phrenic-specific cell adhesion programs. In the absence of Hox5 genes, phrenic MN firing becomes asynchronous and erratic due to loss of phrenic MN inhibition. Strikingly, mice lacking Hox5 genes in MNs exhibit abnormal respiratory behavior throughout their lifetime. Our findings support a model where MN-intrinsic transcriptional programs shape the pattern of motor output by orchestrating distinct aspects of MN connectivity., Competing Interests: AV, KG, CD, MM, JC, LJ, NZ, LL, PP No competing interests declared, (© 2020, Vagnozzi et al.)

Brachycephalic syndrome (BS) is a pathophysiological disorder caused by excessive soft tissue within the upper airways of short-nosed dog breeds, causing obstruction of the nasal, pharyngeal and laryngeal lumen, resulting in severe respiratory distress. As the prevalence of BS appears to be high among some of the affected breeds, there is an urgent need for breeding efforts to improve the health status of those dogs. In the present study, we evaluated correlations between morphometric and other phenotypic characteristics and BS in a population of 69 French bulldogs from Denmark to identify parameters that could serve as a basis for breeding against BS. Furthermore, the genetic variation was monitored to determine whether it would be possible to breed based on these characteristics without simultaneously causing a critical reduction in genetic variation. Six phenotypic characteristics were correlated with the Brachycephalic Syndrome Functional (BSF) score. Among the morphometric risk factors, nostril stenosis (NS) and neck girth (NG) had the highest impact on the BSF score, accounting for 32% and 4% of the variation, respectively. The genetic variation in the population was comparable to other pure breeds, i.e. estimated and observed heterozygosity was 0.60 and the average inbreeding coefficient was 0.01. If only dogs with Grades 1 and 2 NS (no or only mild NS) were selected for breeding the mean BSF score would be reduced significantly. However, it would result in the exclusion of 81% of the population for breeding and this is not prudent. Excluding only dogs with severe stenosis (Grade 4) would exclude 50% of the population without any adverse impact on genetic variation within the population. Although exclusion of dogs with Grade 4 would result in an apparent reduction in the mean BSF score, this reduction is not significant. As NS accounts for 32% of the variation in BSF score, a possible long term strategy to reduce the prevalence of the BS in French bulldogs would seem to be a selection scheme that first excluded dogs with the most severe NS from breeding, gradually moving towards selecting dogs with lower NS grades. According to our findings there is no viable short term solution for reducing the prevalence of BS in the French bulldog population., Competing Interests: The authors have declared that no competing interests exist.

The prevalence of delivery through cesarean-section (C-section) has been increasing worldwide. Although different modes of delivery, such as vaginal birth and C-section, are associated with incidence of some diseases in humans, little is known about how delivery stimuli affect short- and long-term brain function. Phenotypic analyses of Atp1a2 homozygous knockout (Atp1a2 -/- ) neonates showed that the mode of delivery affected neural phenotypes; Atp1a2 -/- mice born by vaginal delivery started spontaneous breathing, while Atp1a2 -/- mice born by C-section showed a complete absence of breathing followed by their death. This life or death phenotype prompted us to examine several aspects of the neonatal brain following C-section or vaginal delivery. We found significantly different levels of several monoamines and transporters/channel proteins and a different c-Fos expression pattern. Furthermore, these mice showed different behaviors in adulthood. Our results suggest that birth mode impacts neurotransmission and functional network formation in the neonatal brain., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Dravet syndrome (DS) is a form of epilepsy with a high incidence of sudden unexpected death in epilepsy (SUDEP). Respiratory failure is a leading cause of SUDEP, and DS patients' frequently exhibit disordered breathing. Despite this, mechanisms underlying respiratory dysfunction in DS are unknown. We found that mice expressing a DS-associated Scn1a missense mutation (A1783V) conditionally in inhibitory neurons ( Slc32a1 cre/+ ::Scn1a A1783V fl/+ ; defined as Scn1a ΔE26 ) exhibit spontaneous seizures, die prematurely and present a respiratory phenotype including hypoventilation, apnea, and a diminished ventilatory response to CO 2 . At the cellular level in the retrotrapezoid nucleus (RTN), we found inhibitory neurons expressing the Scn1a A1783V variant are less excitable, whereas glutamatergic chemosensitive RTN neurons, which are a key source of the CO 2 /H + -dependent drive to breathe, are hyper-excitable in slices from Scn1a ΔE26 mice. These results show loss of Scn1a function can disrupt respiratory control at the cellular and whole animal levels., Competing Interests: FK, CC, JL, XC, DM No competing interests declared, (© 2019, Kuo et al.)

Background: Spinal muscular atrophy (SMA) is a neuromuscular disorder classified into four types based on the age of onset of the disease. Early onset is correlated with a higher mortality rate, mainly due to respiratory complications. Valproic acid (VPA) is a histone deacetylase (HDAC) inhibitor that has shown positive results on SMA both in experimental and cohort studies., Objectives: This systematic review and meta-analysis aimed to investigate the efficacy and safety of VPA in patients with SMA., Methods: Eleven databases were systematically searched on 30 May 2017 for clinical trials that reported the efficacy and safety of VPA in SMA patients. The primary outcome was the efficacy of VPA in terms of gross motor function and expression of both full-length spinal motor neuron (SMN) gene (FL-SMN) and exon 7-lacking SMN. The secondary outcome was the safety of VPA in terms of reported adverse effects. The protocol was registered at PROSPERO (CRD42017067203)., Results: Five of the ten included studies were used in the meta-analysis (n = 126). The overall effect estimate, comparing pre- and post-VPA treatment, regardless of carnitine co-administration and design of the studies, showed significant improvement in gross motor function (standard mean difference [SMD] = 0.302, 95% confidence interval [CI] 0.048-0.556, P = 0.02) using the Hammersmith Functional Motor Scale (HFMS), Modified Hammersmith Functional Motor Scale (MHFMS), and MHFMS-Extend, with no significant heterogeneity. Similarly, in non-randomized controlled studies, the results indicated that there was a significant improvement detected (SMD = 0.335, 95% CI 0.041-0.628, P = 0.025), with no significant heterogeneity. Meanwhile, our results suggest that there was no significant improvement in treatment with co-administered carnitine (SMD = 0.28, 95% CI - 0.02 to 0.581, P = 0.067). No significant differences were found between pre- and post-VPA treatment co-administered with carnitine, in terms of the change in FL-SMN and exon 7-lacking SMN. Qualitative synthesis showed that other motor functions were not improved, while respiratory function test results were contradictory. Regarding the safety of the treatment, a double-blind, randomized, placebo-controlled trial reported no statistically significant differences for adverse events (AEs) between groups. Moreover, most of the included studies reported no serious AEs related to VPA use, although weight gain, gastrointestinal symptoms and respiratory symptoms were notable problems., Conclusions: Our study suggests that VPA treatment results in an improvement in gross motor functions for SMA patients, but not in other assessments of motor function or, possibly, in respiratory function. Furthermore, VPA appears to be a relatively safe drug, although treatment may be associated with a wide range of AEs (including body weight increase, fatigue, fever, flu-like symptoms, irritability, and pain). Double-blind, randomized, controlled trials are required to confirm these findings.

Protein misfolding in the endoplasmic reticulum (ER) is accompanied by adaptive cellular responses to promote cell survival. We now show that activation of mitochondrial respiration is a critical component of an adaptive ER stress response, requiring the unfolded protein response (UPR) sensor Ire1, and also calcium signaling via calcineurin. In yeast and mammalian cells lacking Ire1 or calcineurin, respiratory activation is impaired in response to ER stress; accumulation of mitochondrial reactive oxygen species (ROS) triggers cell death as abrogation of ROS by antioxidants or loss of the electron transport chain (in yeast) can rescue cells from death. Significantly, cells are rescued from ER stress-induced death by mitochondrial uncoupling by CCCP to increase O 2 consumption (and increase the efficiency of electron transfer). Remarkably, genetic and pharmacologic strategies to promote mitochondrial biogenesis and increase O 2 consumption also alleviate ER stress-mediated ROS and death in yeast and mammalian cells. Moreover, in a yeast genetic screen, three mitochondrial proteins Mrx9, Mrm1, and Aim19 that increase mitochondrial biogenesis were identified as high copy suppressors of ER stress-mediated cell death. Our results show that enhanced mitochondrial biogenesis, linked to improved efficiency of the electron transport chain, is a powerful strategy to block ROS accumulation and promote cell survival during ER stress in eukaryotic cells.

Iron is an essential element for plants as well as other organisms, functioning in various cellular processes, including respiration, chlorophyll biosynthesis, and photosynthesis. Plants take up iron from soil in which iron solubility is extremely low especially under aerobic conditions at high-pH range. Therefore, plants have evolved efficient iron-uptake mechanisms. Because iron is prone to being precipitated and excess ionic iron is cytotoxic, plants also have sophisticated internal iron-transport mechanisms. These transport mechanisms comprise iron chelators including nicotianamine, mugineic acid family phytosiderophores and citrate, and various types of transporters of these chelators, iron-chelate complexes, or free iron ions. To maintain iron homeostasis, plants have developed mechanisms for regulating gene expression in response to iron availability. Expression of various genes involved in iron uptake and translocation is induced under iron deficiency by transcription factor networks and is negatively regulated by the ubiquitin ligase HRZ/BTS. This response is deduced to be mediated by cellular iron sensing as well as long-distance iron signaling. The ubiquitin ligase HRZ/BTS is a candidate intracellular iron sensor because it binds to iron and zinc, and its activity is affected by iron availability. The iron-excess response of plants is thought to be partially independent of the iron-deficiency response. In this review, we summarize and discuss extant knowledge of plant iron transport and its regulation., (Copyright © 2018. Published by Elsevier Inc.)

Introduction: Murine studies have shown that apolipoprotein E modulates pulmonary function during development, aging, and allergen-induced airway disease. It is not known whether the polymorphic human APOE gene influences pulmonary function., Objectives: We assessed whether an association exists between the polymorphic human APOE ε2, ε3, and ε4 alleles and pulmonary function among participants in the Long Life Family Study., Methods: Data from 4,468 Caucasian subjects who had genotyping performed for the APOE ε2, ε3, and ε4 alleles were analyzed, with and without stratification by sex. Statistical models were fitted considering the effects of the ε2 allele, defined as ε2/2 or ε2/3 genotypes, and the ε4 allele, defined as ε3/4 or ε4/4 genotypes, which were compared to the ε3/3 genotype., Results: The mean FEV1/FVC ratio (the forced expiratory volume in one second divided by the forced vital capacity) was lower among women with the ε4 allele as compared to women with the ε3/3 genotype or the ε2 allele. Carriage of the APOE ε4 allele was associated with FEV1/FVC, which implied lower values. Further analysis showed that the association primarily reflected women without lung disease who were older than 70 years. The association was not mediated by lipid levels, smoking status, body mass index, or cardiovascular disease., Conclusions: This study for the first time identifies that the APOE gene is associated with modified lung physiology in women. This suggests that a link may exist between the APOE ε4 allele, female sex, and a reduction in the FEV1/FVC ratio in older individuals., Competing Interests: The authors have declared that no competing interests exist.

While barotrauma, decompression sickness, and drowning-related injuries are common morbidities associated with diving and decompression from depth, it remains unclear what impact rapid decompression has on mitochondrial function. In vitro diving simulation was performed with human dermal fibroblast cells subjected to control, air, nitrogen, and oxygen dive conditions. With the exception of the gas mixture, all other related variables, including absolute pressure exposure, dive and decompression rates, and temperature, were held constant. High-resolution respirometry was used to examine key respiratory states. Mitochondrial dynamic function, including net movement, number, and rates of fusion/fission events, was obtained from fluorescence microscopy imaging. Effects of the dive conditions on cell cytoskeleton were assessed by imaging both actin and microtubules. Maximum respiration was lower in fibroblasts in the air group than in the control and nitrogen groups. The oxygen group had overall lower respiration when compared with all other groups. All groups demonstrated lower mitochondrial motility when compared with the control group. Rates of fusion and fission events were the same between all groups. There were visible differences in cell morphology consistent with the actin staining; however, there were no appreciable changes to the microtubules. This is the first study to directly assess mitochondrial respiration and dynamics in a cell model of decompression. Both hyperbaric oxygen and air dive conditions produce deleterious effects on overall mitochondrial health in fibroblasts.

The emergence of systems neuroscience tools requires parallel generation of objective analytical workflows for experimental neuropathology. We developed an objective analytical workflow that we used to determine how specific autonomic neural lineages change during postnatal development. While a wealth of knowledge exists regarding postnatal alterations in respiratory neural function, how these neural circuits change and develop in the weeks following birth remains less clear. In this study, we developed our workflow by combining genetic mouse modeling and quantitative immunofluorescent confocal microscopy and used this to examine the postnatal development of neural circuits derived from the transcription factors NKX2.2 and OLIG3 into three medullary respiratory nuclei. Our automated FIJI-based image analysis workflow rapidly and objectively quantified synaptic puncta in user-defined anatomic regions. Using our objective workflow, we found that the density and estimated total number of Nkx2.2-derived afferents into the pre-Bötzinger Complex significantly decreased with postnatal age during the first three weeks of postnatal life. These data indicate that Nkx2.2-derived structures differentially influence pre-Bötzinger Complex respiratory oscillations at different stages of postnatal development., (© 2018 Wiley Periodicals, Inc.)

Numerous physiological and morphological adaptations were achieved during the transition to lungless respiration that accompanied evolutionary lung loss in plethodontid salamanders, including those that enable efficient gas exchange across extrapulmonary tissue. However, the molecular basis of these adaptations is unknown. Here, we show that lungless salamanders express in the larval integument and the adult buccopharynx-principal sites of respiratory gas exchange in these species-a novel paralogue of the gene surfactant-associated protein C ( SFTPC ), which is a critical component of pulmonary surfactant expressed exclusively in the lung in other vertebrates. The paralogous gene appears to be found only in salamanders, but, similar to SFTPC , in lunged salamanders it is expressed only in the lung. This heterotopic gene expression, combined with predictions from structural modelling and respiratory tissue ultrastructure, suggests that lungless salamanders may produce pulmonary surfactant-like secretions outside the lungs and that the novel paralogue of SFTPC might facilitate extrapulmonary respiration in the absence of lungs. Heterotopic expression of the SFTPC paralogue may have contributed to the remarkable evolutionary radiation of lungless salamanders, which account for more than two thirds of urodele species alive today., (© 2018 The Authors.)

Over the last decades, predictive microbiology has made significant advances in the mathematical description of microbial spoiler and pathogen dynamics in or on food products. Recently, the focus of predictive microbiology has shifted from a (semi-)empirical population-level approach towards mechanistic models including information about the intracellular metabolism in order to increase model accuracy and genericness. However, incorporation of this subpopulation-level information increases model complexity and, consequently, the required run time to simulate microbial cell and population dynamics. In this paper, results of metabolic flux balance analyses (FBA) with a genome-scale model are used to calibrate a low-complexity linear model describing the microbial growth and metabolite secretion rates of Escherichia coli as a function of the nutrient and oxygen uptake rate. Hence, the required information about the cellular metabolism (i.e., biomass growth and secretion of cell products) is selected and included in the linear model without incorporating the complete intracellular reaction network. However, the applied FBAs are only representative for microbial dynamics under specific extracellular conditions, viz., a neutral medium without weak acids at a temperature of 37℃. Deviations from these reference conditions lead to metabolic shifts and adjustments of the cellular nutrient uptake or maintenance requirements. This metabolic dependency on extracellular conditions has been taken into account in our low-complex metabolic model. In this way, a novel approach is developed to take the synergistic effects of temperature, pH, and undissociated acids on the cell metabolism into account. Consequently, the developed model is deployable as a tool to describe, predict and control E. coli dynamics in and on food products under various combinations of environmental conditions. To emphasize this point,three specific scenarios are elaborated: (i) aerobic respiration without production of weak acid extracellular metabolites, (ii) anaerobic fermentation with secretion of mixed acid fermentation products into the food environment, and (iii) respiro-fermentative metabolic regimes in between the behaviors at aerobic and anaerobic conditions., Competing Interests: The authors have declared that no competing interests exist.

Mevalonate kinase deficiency (MKD) an orphan drug rare disease affecting humans with different clinical presentations, is still lacking information about its pathogenesis; no animal or cell model mimicking the genetic defect, mutations at MVK gene, and its consequences on the mevalonate pathway is available. Trying to clarify the effects of MVK gene impairment on the mevalonate pathway we used a yeast model, the erg12-d mutant strain Saccharomyces cerevisiae (orthologous of MKV) retaining only 10% of mevalonate kinase (MK) activity, to describe the effects of reduced MK activity on the mevalonate pathway. Since shortage of isoprenoids has been described in MKD, we checked this observation using a physiologic approach: while normally growing on glucose, erg12-d showed growth deficiency in glycerol, a respirable carbon source, that was not rescued by supplementation with non-sterol isoprenoids, such as farnesol, geraniol nor geranylgeraniol, produced by the mevalonate pathway. Erg12-d whole genome expression analysis revealed specific downregulation of RSF2 gene encoding general transcription factor for respiratory genes, explaining the absence of growth on glycerol. Moreover, we observed the upregulation of genes involved in sulphur amino acids biosynthesis that coincided with the increasing in the amount of proteins containing sulfhydryl groups; upregulation of ubiquinone biosynthesis genes was also detected. Our findings demonstrated that the shortage of isoprenoids is not the main mechanism involved in the respiratory deficit and mitochondrial malfunctioning of MK-defective cells, while the scarcity of ubiquinone plays an important role, as already observed in MKD patients.

The magur catfish (Clarias magur) is a facultative air-breather and regularly encounters with various environmental changes along with exposure to various bacterial pathogens in its natural habitats. Occurrence of various biochemical adaptational strategies related to nitrogen metabolism in magur catfish is already known. The present investigation aimed at determining the possible induction of inducible nitric oxide synthase (inos) gene and stimulation of nitric oxide (NO) production in this catfish while challenging with lipopolysaccharide (LPS, a bacterial endotoxin) treatment, and also to determine the involvement of nuclear factor kappa B (NFkB) in induction of inos gene. Intra-peritoneal injection of LPS led to more production and accumulation of NO in different body tissues of magur catfish as a consequence of induction of iNOS activity. The induction of iNOS activity was associated with the induction of inos gene as evidenced by more expression of inos mRNA and more abundance of iNOS enzyme protein in different tissues of magur catfish with certain variations in zonal specific expression patterns. Similar observations related to more production of NO and induction of inos gene were also made when the isolated hepatocytes were treated with LPS in vitro condition. LPS treatment also led to activation of NFĸB in hepatic cells. However, in presence of a specific inhibitor of NFkB, the LPS-mediated induction of inos gene and extra production of NO were almost blocked, thereby suggesting that the induction of inos gene due to LPS treatment was mediated via the NFkB in magur catfish. It is hypothesized that the induction of iNOS activity, and more synthesis and accumulation of NO could serve as indicators to determine the pathophysiological conditions of the fish living in bacterial contaminated water bodies. Further, it can be contemplated that more synthesis of NO through iNOS enzyme probably serves as an important pharmacological tool against fish pathogen and also plays an important role in host defense mechanisms in this unique group of magur catfish., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Background: Chinese giant salamander (Andrias davidianus) undergoes a metamorphosis from aquatic larvae to terrestrial adults, with concomitant transfer of respiration from gills to lungs prior to metamorphosis. These two tissues, as well as skin, were sampled to identify the differentially expressed miRNAs., Results: High-coverage reference transcriptome was generated from combined gill, lung and skin tissues of metamorphosing juveniles, and lung tissue of adults: 86,282 unigenes with total length of approximately 77,275,634 bp and N50 of 1732 bp were obtained. Among these, 13,246 unigenes were assigned to 288 pathways. To determine the possible involvement of miRNAs in the respiratory transition, small RNA libraries were sequenced; 282 miRNAs were identified, 65 among which were known and 217 novel. Based on the hierarchical clustering analysis, the twelve studied samples were classified into three major clusters using differentially expressed miRNAs. We have validated ten differentially expressed miRNAs and some of their related target genes using qPCR. These results largely corroborated the results of transcriptomic and miRNA analyses. Finally, an miRNA-gene-network was constructed. Among them, two miRNAs with target genes related to oxygen sensing were differentially expressed between gill and lung tissues. Three miRNAs were differentially expressed between the lungs of larvae and lungs of adults., Conclusions: This study provides the first large-scale miRNA expression profile overview during the respiration transition from gills to lungs in Chinese giant salamander. Five differentially expressed miRNAs and their target genes were identified among skin, gill and lung tissues. These results suggest that miRNA profiles in respiratory tissues play an important role in the regulation of respiratory transition.

Augmentative and alternative communication (AAC) systems tend to rely on the interpretation of purposeful gestures for interaction. Existing AAC methods could be cumbersome and limit the solutions in terms of versatility. The study aims to interpret breathing patterns (BPs) to converse with the outside world by means of a unidirectional microphone and researches breathing-pattern interpretation (BPI) to encode messages in an interactive manner with minimal training. We present BP processing work with (1) output synthesized machine-spoken words (SMSW) along with single-channel Weiner filtering (WF) for signal de-noising, and (2) k -nearest neighbor ( k-NN ) classification of BPs associated with embedded dynamic time warping (DTW). An approved protocol to collect analogue modulated BP sets belonging to 4 distinct classes with 10 training BPs per class and 5 live BPs per class was implemented with 23 healthy subjects. An 86% accuracy of k-NN classification was obtained with decreasing error rates of 17%, 14%, and 11% for the live classifications of classes 2, 3, and 4, respectively. The results express a systematic reliability of 89% with increased familiarity. The outcomes from the current AAC setup recommend a durable engineering solution directly beneficial to the sufferers., Competing Interests: The authors declare no conflict of interest.

Cellular oxygen consumption and lactate production rates have been measured in both placental and myometrial cells to study obstetrics-related disease states such as preeclampsia. Platelet metabolic alterations indicate systemic bioenergetic changes that can be useful as disease biomarkers. We tested the hypothesis that platelet mitochondria display functional alterations in preeclampsia. Platelets were harvested from women in the third trimester of either a healthy, non-preeclamptic or preeclamptic pregnancy, and from healthy, non-pregnant women. Using Seahorse respirometry, we analyzed platelets for oxygen consumption (OCR) and extracellular acidification (ECAR) rates, indicators of mitochondrial electron transport and glucose metabolism, respectively. There was a 37% decrease in the maximal respiratory capacity measured in platelets from healthy, non-preeclamptic compared with preeclamptic pregnancy ( P <0.01); this relationship held true for other measurements of OCR, including basal respiration; ATP-linked respiration; respiratory control ratio (RCR); and spare respiratory capacity. RCR, a measure of mitochondrial efficiency, was significantly lower in healthy pregnant compared with non-pregnant women. In contrast with increased OCR, basal ECAR was significantly reduced in platelets from preeclamptic pregnancies compared with either normal pregnancies (-25%; P <0.05) or non-pregnant women (-22%; P <0.01). Secondary analysis of OCR revealed reduced basal and maximal platelet respiration in normal pregnancy prior to 34 weeks' estimated gestational age (EGA) compared with the non-pregnant state; these differences disappeared after 34 weeks. Taken together, findings suggest that in preeclampsia, there exists either a loss or early (before the third trimester) reversal of a normal biologic mechanism of platelet mitochondrial respiratory reduction associated with normal pregnancy., (© 2018 The Author(s).)

Introduction: Recently, a great deal of attention has been paid to the investigation of regulatory functions of microRNA. Currently, many different mechanisms involved in the pathogenesis of asthma are known, but the whole picture of pathogenesis has not yet been studied., Conclusions: MicroRNAs play an important role in the regulation of many cellular processes. Undoubtedly, these regulatory molecules are involved in the pathogenesis of asthma, and therefore can be potential targets for treatment., (Copyright © 2018 SEICAP. Published by Elsevier España, S.L.U. All rights reserved.)

High-resolution respirometry allows for the measurement of oxygen consumption of isolated mitochondria, cells and tissues. Beta cells play a critical role in the body by controlling blood glucose levels through insulin secretion in response to elevated glucose concentrations. Insulin secretion is controlled by glucose metabolism and mitochondrial respiration. Therefore, measuring intact beta cell respiration is essential to be able to improve beta cell function as a treatment for diabetes. Using intact 832/13 INS-1 derived beta cells we can measure the effect of increasing glucose concentration on cellular respiration. This protocol allows us to measure beta cell respiration in the presence or absence of various compounds, allowing one to determine the effect of given compounds on intact cell respiration. Here we demonstrate the effect of two naturally occurring compounds, monomeric epicatechin and curcumin, on beta cell respiration under the presence of low (2.5 mM) or high glucose (16.7 mM) conditions. This technique can be used to determine the effect of various compounds on intact beta cell respiration in the presence of differing glucose concentrations.

Bcs1p is a chaperone that is required for the incorporation of the Rieske subunit within complex III of the mitochondrial respiratory chain. Mutations in the human gene BCS1L (BCS1-like) are the most frequent nuclear mutations resulting in complex III-related pathologies. In yeast, the mimicking of some pathogenic mutations causes a respiratory deficiency. We have screened chemical libraries and found that two antibiotics, pentamidine and clarithromycin, can compensate two bcs1 point mutations in yeast, one of which is the equivalent of a mutation found in a human patient. As both antibiotics target the large mtrRNA of the mitoribosome, we focused our analysis on mitochondrial translation. We found that the absence of non-essential translation factors Rrf1 or Mif3, which act at the recycling/initiation steps, also compensates for the respiratory deficiency of yeast bcs1 mutations. At compensating concentrations, both antibiotics, as well as the absence of Rrf1, cause an imbalanced synthesis of respiratory subunits which impairs the assembly of the respiratory complexes and especially that of complex IV. Finally, we show that pentamidine also decreases the assembly of complex I in nematode mitochondria. It is well known that complexes III and IV exist within the mitochondrial inner membrane as supramolecular complexes III 2 /IV in yeast or I/III 2 /IV in higher eukaryotes. Therefore, we propose that the changes in mitochondrial translation caused by the drugs or by the absence of translation factors, can compensate for bcs1 mutations by modifying the equilibrium between illegitimate, and thus inactive, and active supercomplexes., (Copyright © 2017. Published by Elsevier B.V.)

Vertebrate hypoxia tolerance can emerge from modifications to the oxygen (O2) transport cascade, but whether there is adaptive variation to O2 binding at the terminus of this cascade, mitochondrial cytochrome c oxidase (COX), is not known. In order to address the hypothesis that hypoxia tolerance is associated with enhanced O2 binding by mitochondria we undertook a comparative analysis of COX O2 kinetics across species of intertidal sculpins (Cottidae, Actinopterygii) that vary in hypoxia tolerance. Our analysis revealed a significant relationship between hypoxia tolerance (critical O2 tension of O2 consumption rate; Pcrit), mitochondrial O2 binding affinity (O2 tension at which mitochondrial respiration was half maximal; P50), and COX O2-binding affinity (apparent Michaelis-Menten constant for O2 binding to COX; Km,app O2). The more hypoxia tolerant species had both a lower mitochondrial P50 and lower COX Km,app O2, facilitating the maintenance of mitochondrial function to a lower O2 tension than in hypoxia intolerant species. Additionally, hypoxia tolerant species had a lower overall COX Vmax but higher mitochondrial COX respiration rate when expressed relative to maximal electron transport system respiration rate. In silico analyses of the COX3 subunit postulated as the entry point for O2 into the COX protein catalytic core, points to variation in COX3 protein stability (estimated as free energy of unfolding) contributing to the variation in COX Km,app O2. We propose that interactions between COX3 and cardiolipin at four amino acid positions along the same alpha-helix forming the COX3 v-cleft represent likely determinants of interspecific differences in COX Km,app O2., (© The Author 2017. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Endocannabinoids (ECs) are bioactive lipid mediators acting on two distinct cannabinoid receptors (CB1 and CB2), which are ubiquitously expressed in many tissues including the respiratory system. Despite numerous experimental data showing that cannabinomimetics influence respiration, the role of endogenously produced ECs in respiratory control has not been verified yet. Pulse oximetry was used in the present study to directly measure changes in respiratory parameters during elevation of EC levels. The cannabinoid reuptake inhibitor AM-404 (10mgkg -1 , i.v.), but not its vehicle, induced a transient reduction of respiratory rate with a concomitant depression of arterial oxygen saturation and increase in breath distension in wild-type mice. In contrast, CB1 knock-out mice showed no alteration in any of these parameters upon administration of AM-404. Our results imply that the EC system has an important role in the physiological control of respiration by modulating the respiratory rate and consequently influencing arterial oxygen saturation. Furthermore, this mechanism is entirely dependent on CB1 receptors., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Despite the recent explosive rise in number of genetic markers for complex disease traits identified in genome-wide association studies, there is still a large gap between the known heritability of these traits and the part explained by these markers. To gauge whether this 'heritability gap' is closing, we first identified genome-wide significant SNPs from the literature and performed replication analyses for 32 highly relevant traits from five broad disease areas in 13 436 subjects of the Lifelines Cohort. Next, we calculated the variance explained by multi-SNP genetic risk scores (GRSs) for each trait, and compared it to their broad- and narrow-sense heritabilities captured by all common SNPs. The majority of all previously-associated SNPs (median=75%) were significantly associated with their respective traits. All GRSs were significant, with unweighted GRSs generally explaining less phenotypic variance than weighted GRSs, for which the explained variance was highest for height (15.5%) and varied between 0.02 and 6.7% for the other traits. Broad-sense common-SNP heritability estimates were significant for all traits, with the additive effect of common SNPs explaining 48.9% of the variance for height and between 5.6 and 39.2% for the other traits. Dominance effects were uniformly small (0-1.5%) and not significant. On average, the variance explained by the weighted GRSs accounted for only 10.7% of the common-SNP heritability of the 32 traits. These results indicate that GRSs may not yet be ready for accurate personalized prediction of complex disease traits limiting widespread adoption in clinical practice.

Background: Genome-wide association studies have identified numerous genetic regions that influence cross-sectional lung function. Longitudinal decline in lung function also includes a heritable component but the genetic determinants have yet to be defined., Objectives: We aimed to determine whether regions associated with cross-sectional lung function were also associated with longitudinal decline and to seek novel variants which influence decline., Methods: We analysed genome-wide data from 4167 individuals from the Busselton Health Study cohort, who had undergone spirometry (12 695 observations across eight time points). A mixed model was fitted and weighted risk scores were calculated for the joint effect of 26 known regions on baseline and longitudinal changes in FEV 1 and FEV 1 /FVC. Potential additional regions of interest were identified and followed up in two independent cohorts., Results: The 26 regions previously associated with cross-sectional lung function jointly showed a strong effect on baseline lung function (p=4.44×10 -16 for FEV 1 /FVC) but no effect on longitudinal decline (p=0.160 for FEV 1 /FVC). This was replicated in an independent cohort. 39 additional regions of interest (48 variants) were identified; these associations were not replicated in two further cohorts., Conclusions: Previously identified genetic variants jointly have a strong effect on cross-sectional lung function in adults but little or no effect on the rate of decline of lung function. It is possible that they influence COPD risk through lung development. Although no genetic variants have yet been associated with lung function decline at stringent genome-wide significance, longitudinal change in lung function is heritable suggesting that there is scope for future discoveries., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/.)

Aims: Hyperoxic breathing might lead to redox imbalance and signaling changes that affect cerebral function. Paradoxically, hypoxic breathing is also believed to cause oxidative stress. Our aim is to dissect the cerebral tissue responses to altered O 2 fractions in breathed air by assessing the redox imbalance and the recruitment of the hypoxia signaling pathways., Results: Mice were exposed to mild hypoxia (10%O 2 ), normoxia (21%O 2 ) or mild hyperoxia (30%O 2 ) for 28 days, sacrificed and brain tissue excised and analyzed. Although one might expect linear responses to %O 2 , only few of the examined variables exhibited this pattern, including neuroprotective phospho- protein kinase B and the erythropoietin receptor. The major reactive oxygen species (ROS) source in brain, NADPH oxidase subunit 4 increased in hypoxia but not in hyperoxia, whereas neither affected nuclear factor (erythroid-derived 2)-like 2, a transcription factor that regulates the expression of antioxidant proteins. As a result of the delicate equilibrium between ROS generation and antioxidant defense, neuron apoptosis and cerebral tissue hydroperoxides increased in both 10%O 2 and 30%O 2 , as compared with 21%O 2 . Remarkably, the expression level of hypoxia-inducible factor (HIF)-2α (but not HIF-1α) was higher in both 10%O 2 and 30%O 2 with respect to 21%O 2 INNOVATION: Comparing the in vivo effects driven by mild hypoxia with those driven by mild hyperoxia helps addressing whether clinically relevant situations of O 2 excess and scarcity are toxic for the organism., Conclusion: Prolonged mild hyperoxia leads to persistent cerebral damage, comparable to that inferred by prolonged mild hypoxia. The underlying mechanism appears related to a model whereby the imbalance between ROS generation and anti-ROS defense is similar, but occurs at higher levels in hypoxia than in hyperoxia., (Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.)

Slow, controlled breathing has been used for centuries to promote mental calming, and it is used clinically to suppress excessive arousal such as panic attacks. However, the physiological and neural basis of the relationship between breathing and higher-order brain activity is unknown. We found a neuronal subpopulation in the mouse preBötzinger complex (preBötC), the primary breathing rhythm generator, which regulates the balance between calm and arousal behaviors. Conditional, bilateral genetic ablation of the ~175 Cdh9 / Dbx1 double-positive preBötC neurons in adult mice left breathing intact but increased calm behaviors and decreased time in aroused states. These neurons project to, synapse on, and positively regulate noradrenergic neurons in the locus coeruleus, a brain center implicated in attention, arousal, and panic that projects throughout the brain., (Copyright © 2017, American Association for the Advancement of Science.)

Sleep apnea, which is the periodic cessation of breathing during sleep, is a major health problem affecting over 10 million people in the United States and is associated with several sequelae, including hypertension and stroke. Clinical studies suggest that abnormal carotid body (CB) activity may be a driver of sleep apnea. Because gaseous molecules are important determinants of CB activity, aberrations in their signaling could lead to sleep apnea. Here, we report that mice deficient in heme oxygenase-2 (HO-2), which generates the gaseous molecule carbon monoxide (CO), exhibit sleep apnea characterized by high apnea and hypopnea indices during rapid eye movement (REM) sleep. Similar high apnea and hypopnea indices were also noted in prehypertensive spontaneously hypertensive (SH) rats, which are known to exhibit CB hyperactivity. We identified the gaseous molecule hydrogen sulfide (H 2 S) as the major effector molecule driving apneas. Genetic ablation of the H 2 S-synthesizing enzyme cystathionine-γ-lyase (CSE) normalized breathing in HO-2 -/- mice. Pharmacologic inhibition of CSE with l-propargyl glycine prevented apneas in both HO-2 -/- mice and SH rats. These observations demonstrate that dysregulated CO and H 2 S signaling in the CB leads to apneas and suggest that CSE inhibition may be a useful therapeutic intervention for preventing CB-driven sleep apnea., Competing Interests: The authors declare no conflict of interest.

Inspiratory accessory respiratory muscles (ARMs) enhance ventilation when demands are high, such as during exercise and/or pathological conditions. Despite progressive degeneration of phrenic motor neurons innervating the diaphragm, amyotrophic lateral sclerosis (ALS) patients and rodent models are able to maintain ventilation at early stages of disease. In order to assess the contribution of ARMs to respiratory compensation in ALS, we examined the activity of ARMs and ventilation throughout disease progression in SOD1 G93A ALS model mice at rest using a combination of electromyography and unrestrained whole body plethysmography. Increased ARM activity, accompanied by increased ventilation, is observed beginning at the onset of symptoms. However, ARM recruitment fails to occur at rest at late stages of disease, even though the same ARMs are used for other behaviors. Using a chemogenetic approach, we demonstrate that a glutamatergic class of neurons in the brainstem and spinal cord, the V2a class, is sufficient to drive increased ARM activity at rest in healthy mice. Additionally, we reveal pathology in the medial reticular formation of the brainstem of SOD1 G93A mice using immunohistochemistry and confocal imaging. Both spinal and brainstem V2a neurons degenerate in ALS model mice, accompanied by regional activation of astrocytes and microglia. These results establish inspiratory ARM recruitment as one of the compensatory mechanisms that maintains breathing at early stages of disease and indicate that V2a neuron degeneration may contribute to ARM failure at late stages of disease., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Genetic determinants of sleep-disordered breathing (SDB), a common set of disorders that contribute to significant cardiovascular and neuropsychiatric morbidity, are not clear. Overnight nocturnal oxygen saturation (SaO2) is a clinically relevant and easily measured indicator of SDB severity but its genetic contribution has never been studied. Our recent study suggests nocturnal SaO2 is heritable. We performed linkage analysis, association analysis and haplotype analysis of average nocturnal oxyhaemoglobin saturation in participants in the Cleveland Family Study (CFS), followed by gene-based association and additional tests in four independent samples. Linkage analysis identified a peak (LOD = 4.29) on chromosome 8p23. Follow-up association analysis identified two haplotypes in angiopoietin-2 (ANGPT2) that significantly contributed to the variation of SaO2 (P = 8 × 10-5) and accounted for a portion of the linkage evidence. Gene-based association analysis replicated the association of ANGPT2 and nocturnal SaO2. A rare missense SNP rs200291021 in ANGPT2 was associated with serum angiopoietin-2 level (P = 1.29 × 10-4), which was associated with SaO2 (P = 0.002). Our study provides the first evidence for the association of ANGPT2, a gene previously implicated in acute lung injury syndromes, with nocturnal SaO2, suggesting that this gene has a broad range of effects on gas exchange, including influencing oxygenation during sleep., (© The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)