Methacholine dose-response curves illustrate pharmacologic bronchoprotection against methacholine-induced airway hyperresponsiveness and can be used to quantitate changes in airway sensitivity (position), reactivity (slope), and maximal responsiveness following drug administration. Our objective was to determine the influence of single-dose glycopyrronium (long-acting muscarinic antagonist) and indacaterol (ultra-long acting β2 agonist), as monotherapy and in combination, on the methacholine dose-response curve of mild asthmatics and to compare these findings with a non-asthmatic control curve.

PINK1 deficiency causes the autosomal recessive PARK6 variant of Parkinson's disease. PINK1 activates ubiquitin by phosphorylation and cooperates with the downstream ubiquitin ligase PARKIN, to exert quality control and control autophagic degradation of mitochondria and of misfolded proteins in all cell types.

Some patients with chronic abdominal pain suffer from an anterior cutaneous nerve entrapment syndrome (ACNES). This somewhat illusive syndrome is thought to be caused by the entrapment of end branches of the intercostal nerves residing in the abdominal wall. If ACNES is suspected, a local injection of an anesthetic agent may offer relief. If pain is recurrent following multiple-injection therapy, an anterior neurectomy entailing removal of the entrapped nerve endings may be considered. After 1 year, a 70% success rate has been reported. Research on minimally invasive alternative treatments is scarce. Pulsed radiofrequency (PRF) treatment is a relatively new treatment for chronic pain syndromes. An electromagnetic field is applied around the nerve in the hope of leading to pain relief. This randomized controlled trial compares the effect of PRF treatment and neurectomy in patients with ACNES.

Adverse effects in diabetic mothers offspring (DMO) are a major concern of increasing incidence. Among these, chronic central complications in DMO remain poorly understood, and in extreme cases, diabetes can essentially function as a gestational brain insult. Nevertheless, therapeutic alternatives for DMO are limited.

Background While the patient and public involvement (PPI) evidence base has expanded over the past decade, the quality of reporting within papers is often inconsistent, limiting our understanding of how it works, in what context, for whom, and why.Objective To develop international consensus on the key items to report to enhance the quality, transparency, and consistency of the PPI evidence base. To collaboratively involve patients as research partners at all stages in the development of GRIPP2.Methods The EQUATOR method for developing reporting guidelines was used. The original GRIPP (Guidance for Reporting Involvement of Patients and the Public) checklist was revised, based on updated systematic review evidence. A three round Delphi survey was used to develop consensus on items to be included in the guideline. A subsequent face-to-face meeting produced agreement on items not reaching consensus during the Delphi process.Results 143 participants agreed to participate in round one, with an 86% (123/143) response for round two and a 78% (112/143) response for round three. The Delphi survey identified the need for long form (LF) and short form (SF) versions. GRIPP2-LF includes 34 items on aims, definitions, concepts and theory, methods, stages and nature of involvement, context, capture or measurement of impact, outcomes, economic assessment, and reflections and is suitable for studies where the main focus is PPI. GRIPP2-SF includes five items on aims, methods, results, outcomes, and critical perspective and is suitable for studies where PPI is a secondary focus.Conclusions GRIPP2-LF and GRIPP2-SF represent the first international evidence based, consensus informed guidance for reporting patient and public involvement in research. Both versions of GRIPP2 aim to improve the quality, transparency, and consistency of the international PPI evidence base, to ensure PPI practice is based on the best evidence. In order to encourage its wide dissemination this article is freely accessible on The BMJ and Research Involvement and Engagement journal websites.

Stroke is a costly and debilitating disease that disproportionately affects blacks. Despite the efficacy of statins, evidence suggests racial disparities may exist in statin prescribing.

Endocytosed cell surface membrane proteins rely on recycling pathways for their return to the plasma membrane. Although endosome-to-plasma membrane recycling is critical for many cellular processes, much of the required machinery is unknown. We discovered that yeast has a recycling route from endosomes to the cell surface that functions efficiently after inactivation of the sec7-1 allele of Sec7, which controls transit through the Golgi. A genetic screen based on an engineered synthetic reporter that exclusively follows this pathway revealed that recycling was subject to metabolic control through the Rag GTPases Gtr1 and Gtr2, which work downstream of the exchange factor Vam6. Gtr1 and Gtr2 control the recycling pathway independently of TORC1 regulation through the Gtr1 interactor Ltv1. We further show that the early-endosome recycling route and its control though the Vam6>Gtr1/Gtr2>Ltv1 pathway plays a physiological role in regulating the abundance of amino acid transporters at the cell surface.

T cell-dependent germinal center (GC) responses require coordinated interactions of T cells with two antigen-presenting cell (APC) populations, B cells and dendritic cells (DCs), in the presence of B7- and CD40-dependent co-stimulatory pathways. Contrary to the prevailing paradigm, we found unique cellular requirements for B7 and CD40 expression in primary GC responses to vaccine immunization with protein antigen and adjuvant: B7 was required on DCs but was not required on B cells, whereas CD40 was required on B cells but not on DCs in the generation of antigen-specific follicular helper T cells, antigen-specific GC B cells, and high-affinity class-switched antibody production. There was, in fact, no requirement for coexpression of B7 and CD40 on the same cell in these responses. Our findings support a substantially revised model for co-stimulatory function in the primary GC response, with crucial and distinct contributions of B7- and CD40-dependent pathways expressed by different APC populations and with important implications for understanding how to optimize vaccine responses or limit autoimmunity.

Really interesting new gene (RING)-finger protein 52 (RNF52), an E3 ubiquitin ligase, is found in eukaryotes from yeast to humans. Human RNF52 is known as breast cancer type 1 susceptibility protein (BRCA1)-associated protein 2 (BRAP or BRAP2). The central catalytic domain of BRAP comprises four subdomains: nucleotide-binding α/β plait (NBP), really interesting new gene (RING) zinc finger, ubiquitin-specific protease (UBP)-like zinc finger (ZfUBP), and coiled-coil (CC). This domain architecture is conserved in RNF52 orthologs; however, the domain's function in the ubiquitin system has not been delineated. In the present study, we discovered that the RNF52 domain, comprising NBP-RING-ZfUBP-CC, binds to ubiquitin chains (oligo-ubiquitin) but not to the ubiquitin monomers, and can utilize various ubiquitin chains for ubiquitylation and auto-ubiquitylation. The RNF52 domain preferentially bound to M1- and K63-linked di-ubiquitin chains, weakly to K27-linked chains, but not to K6-, K11-, or K48-linked chains. The binding preferences of the RNF52 domain for ubiquitin-linkage types corresponded to ubiquitin usage in the ubiquitylation reaction, except for K11-, K29-, and K33-linked chains. Additionally, the RNF52 domain directly ligated the intact M1-linked, tri-, and tetra-ubiquitin chains and recognized the structural alterations caused by the phosphomimetic mutation of these ubiquitin chains. Full-length BRAP had nearly the same specificity for the ubiquitin-chain types as the RNF52 domain alone. Mass spectrometry analysis of oligomeric ubiquitylation products, mediated by the RNF52 domain, revealed that the ubiquitin-linkage types and auto-ubiquitylation sites depend on the length of ubiquitin chains. Here, we propose a model for the oligomeric ubiquitylation process, controlled by the RNF52 domain, which is not a sequential assembly process involving monomers.

One proposed mechanism of cellular aging is the gradual loss of certain cellular components that are insufficiently renewed. In an earlier study, multidrug resistance transporters (MDRs) were postulated to be such aging determinants during the yeast replicative life span (RLS). Aged MDR proteins were asymmetrically retained by the aging mother cell and did not diffuse freely into the bud, whereas newly synthesized MDR proteins were thought to be deposited mostly in the bud before cytokinesis. In this study, we further demonstrate the proposed age asymmetry of MDR proteins in dividing yeast cells and investigate the mechanism that controls diffusive properties of MDR proteins to maintain this asymmetry. We found that long-chain sphingolipids, but not the septin/endoplasmic reticulum-based membrane diffusion barrier, are important for restricting MDR diffusion. Depletion of sphingolipids or shortening of their long acyl chains resulted in an increase in the lateral mobility of MDR proteins, causing aged MDR protein in the mother cell to enter the bud. We used a mathematical model to understand the effect of diminished MDR age asymmetry on yeast cell aging, the result of which was qualitatively consistent with the observed RLS shortening in sphingolipid mutants.

The predominant types of dendritic cells (DC) in the skin and mucosa are Langerhans cells (LC) and interstitial dermal DC (iDDC). LC and iDDC process cutaneous antigens and migrate out of the skin and mucosa to the draining lymph nodes to present antigens to T and B cells. Because of the strategic location of LC and iDDC and the ability of these cells to capture and process pathogens, we hypothesized that they could be infected with human herpesvirus 8 (HHV-8) (Kaposi's sarcoma [KS]-associated herpesvirus) and have an important role in the development of KS. We have previously shown that HHV-8 enters monocyte-derived dendritic cells (MDDC) through DC-SIGN, resulting in nonproductive infection. Here we show that LC and iDDC generated from pluripotent cord blood CD34+ cell precursors support productive infection with HHV-8. Anti-DC-SIGN monoclonal antibody (MAb) inhibited HHV-8 infection of iDDC, as shown by low expression levels of viral proteins and DNA. In contrast, blocking of both langerin and the receptor protein tyrosine kinase ephrin A2 was required to inhibit HHV-8 infection of LC. Infection with HHV-8 did not alter the cell surface expression of langerin on LC but downregulated the expression of DC-SIGN on iDDC, as we previously reported for MDDC. HHV-8-infected LC and iDDC had a reduced ability to stimulate allogeneic CD4+ T cells in the mixed-lymphocyte reaction. These results indicate that HHV-8 can target both LC and iDDC for productive infection via different receptors and alter their function, supporting their potential role in HHV-8 pathogenesis and KS.IMPORTANCE Here we show that HHV-8, a DNA tumor virus that causes Kaposi's sarcoma, infects three types of dendritic cells: monocyte-derived dendritic cells, Langerhans cells, and interstitial dermal dendritic cells. We show that different receptors are used by this virus to infect these cells. DC-SIGN is a major receptor for infection of both monocyte-derived dendritic cells and interstitial dermal dendritic cells, yet the virus fully replicates only in the latter. HHV-8 uses langerin and the ephrin A2 receptor to infect Langerhans cells, which support full HHV-8 lytic replication. This infection of Langerhans cells and interstitial dermal dendritic cells results in an impaired ability to stimulate CD4+ helper T cell responses. Taken together, our data show that HHV-8 utilizes alternate receptors to differentially infect and replicate in these tissue-resident DC and support the hypothesis that these cells play an important role in HHV-8 infection and pathogenesis.

Calorie restriction (CR) may extend longevity by modulating the mechanisms involved in aging. Different hypotheses have been proposed for its main mode of action. We quantified hepatic transcripts of male C57BL/6 mice exposed to graded levels of CR (0% to 40% CR) for three months, and evaluated the responses relative to these various hypotheses. Of the four main signaling pathways implied to be linked to the impact of CR on lifespan (insulin/insulin like growth factor 1 (IGF-1), nuclear factor-kappa beta (NF-ĸB), mechanistic target of rapamycin (mTOR) and sirtuins (SIRTs)), all the pathways except SIRT were altered in a manner consistent with increased lifespan. However, the expression levels of SIRT4 and SIRT7 were decreased with increasing levels of CR. Changes consistent with altered fuel utilization under CR may reduce reactive oxygen species production, which was paralleled by reduced protection. Downregulated major urinary protein (MUP) transcription suggested reduced reproductive investment. Graded CR had a positive effect on autophagy and xenobiotic metabolism, and was protective with respect to cancer signaling. CR had no significant effect on fibroblast growth factor-21 (FGF21) transcription but affected transcription in the hydrogen sulfide production pathway. Responses to CR were consistent with several different hypotheses, and the benefits of CR on lifespan likely reflect the combined impact on multiple aging related processes.

Multiple hereditary exostoses (MHE) is characterized by the development of numerous benign bony tumors (osteochondromas). Although it has been well established that MHE is caused by mutations in EXT1 and EXT2, which encode glycosyltransferase essential for heparan sulfate (HS) biosynthesis, the cellular origin and molecular mechanisms of MHE remain elusive. Here, we show that in Ext1 mutant mice, osteochondromas develop from mesenchymal stem cell-like progenitor cells residing in the perichondrium, and we show that enhanced BMP signaling in these cells is the primary signaling defect that leads to osteochondromagenesis. We demonstrate that progenitor cells in the perichondrium, including those in the groove of Ranvier, highly express HS and that Ext1 ablation targeted to the perichondrium results in the development of osteochondromas. Ext1-deficient perichondrial progenitor cells show enhanced BMP signaling and increased chondrogenic differentiation both in vitro and in vivo. Consistent with the functional role for enhanced BMP signaling in osteochondromagenesis, administration of the small molecule BMP inhibitor LDN-193189 suppresses osteochondroma formation in two MHE mouse models. Together, our results demonstrate a role for enhanced perichondrial BMP signaling in osteochondromagenesis in mice, and they suggest the possibility of pharmacological treatment of MHE with BMP inhibitors.

BACKGROUND Lung cancers are resistant to conventional chemotherapeutic interventions such as paclitaxel. Notch signaling is crucial in the chemoresistance of lung cancer cells. The Notch inhibitor gamma-secretase inhibitor (GSI) inhibits the Notch signaling pathway. MATERIAL AND METHODS Here, we evaluated how Notch-3 inhibition by GSI can enhance the sensitivity of lung cancer cells to paclitaxel. To study how Notch-3-specific inhibition affects non-small cell lung cancer (NSCLC), we compared the cell viability, apoptosis, and colony formation of A549 and H1299 cells treated with Notch-3 siRNA and GSI. RESULTS The expression levels of Notch-3 or Notch intracellular domain 3 (NICD3) and apoptosis-related proteins were measured and compared between different groups. Notch-3 was significantly overexpressed in both cell lines, and Notch-3 expression was elevated after paclitaxel treatment, indicating activation of the Notch signaling pathway. Inhibition of the Notch signaling pathway by GSI and Notch-3 siRNA reduced cell proliferation and induced apoptosis in A549 and H1299 cells, thereby boosting sensitivity of the cell lines to paclitaxel. Concomitant treatment with paclitaxel and GSI or siRNA downregulated Bcl-2 expression and upregulated Bax expression levels. CONCLUSIONS These results indicate a synergistic effect of Notch-3-specific inhibition and paclitaxel through alteration of the intrinsic apoptosis pathway, which was involved in Notch-3-induced chemoresistance in NSCLC cells, and GSI inhibited Notch-3-induced chemoresistance in a concentration-dependent manner. This approach that combines Notch-3-specific inhibition and paclitaxel would be likely to apply in NSCLC.

Succinyl Coenzyme A synthetase (SCS) is a key mitochondrial enzyme. Defected SCS ADP-forming β subunit (SCS A-β) is linked to lethal infantile Leigh or leigh-like syndrome. However, the impacts of SCS A-β deficiency on mitochondria specifically in neurons have not yet been comprehensively investigated. Here, by down-regulating the expression levels of SCS A-β in cultured mouse neurons, we have found that SCS A-β deficiency induces severe mitochondrial dysfunction including lowered oxidative phosphorylation (OXPHOS) efficiency, increased mitochondrial superoxide production, and mtDNA depletion as well as aberrations of mitochondrial fusion and fission proteins, which eventually leads to neuronal stress. Our data also suggest that the deregulation of mitochondrial nucleoside diphosphate kinase (NDPK) together with defects in mitochondrial transcription factors including mitochondrial DNA pol γ and Twinkle contribute to SCS A-β deficiency-mediated mtDNA instability. Furthermore, we have found that SCS A-β deficiency has detrimental influence on neuronal mitochondrial dynamics. Put together, the results have furnished our knowledge on the pathogenesis of SCS A-β deficiency-related mitochondrial diseases and revealed the vital role of SCS A-β in maintaining neuronal mitochondrial quality control and neuronal physiology.

Skeletal muscle is composed of heterogeneous populations of myofibers that are classified as slow- and fast-twitch fibers. The muscle fiber-type is regulated in a coordinated fashion by multiple genes, including transcriptional factors and microRNAs (miRNAs). However, players involved in this regulation are not fully elucidated. One of the members of the Vestigial-like factors, Vgll2, is thought to play a pivotal role in TEA domain (TEAD) transcription factor-mediated muscle-specific gene expression because of its restricted expression in skeletal muscles of adult mice. Here, we generated Vgll2 null mice and investigated Vgll2 function in adult skeletal muscles. These mice presented an increased number of fast-twitch type IIb fibers and exhibited a down-regulation of slow type I myosin heavy chain (MyHC) gene, Myh7, which resulted in exercise intolerance. In accordance with the decrease in Myh7, down-regulation of miR-208b, encoded within Myh7 gene and up-regulation of targets of miR-208b, Sox6, Sp3, and Purβ, were observed in Vgll2 deficient mice. Moreover, we detected the physical interaction between Vgll2 and TEAD1/4 in neonatal skeletal muscles. These results suggest that Vgll2 may be both directly and indirectly involved in the programing of slow muscle fibers through the formation of the Vgll2-TEAD complex.

Upwelling of global deep waters to the sea surface in the Southern Ocean closes the global overturning circulation and is fundamentally important for oceanic uptake of carbon and heat, nutrient resupply for sustaining oceanic biological production, and the melt rate of ice shelves. However, the exact pathways and role of topography in Southern Ocean upwelling remain largely unknown. Here we show detailed upwelling pathways in three dimensions, using hydrographic observations and particle tracking in high-resolution models. The analysis reveals that the northern-sourced deep waters enter the Antarctic Circumpolar Current via southward flow along the boundaries of the three ocean basins, before spiraling southeastward and upward through the Antarctic Circumpolar Current. Upwelling is greatly enhanced at five major topographic features, associated with vigorous mesoscale eddy activity. Deep water reaches the upper ocean predominantly south of the Antarctic Circumpolar Current, with a spatially nonuniform distribution. The timescale for half of the deep water to upwell from 30° S to the mixed layer is ~60-90 years.Deep waters of the Atlantic, Pacific and Indian Oceans upwell in the Southern Oceanbut the exact pathways are not fully characterized. Here the authors present a three dimensional view showing a spiralling southward path, with enhanced upwelling by eddy-transport at topographic hotspots.

People vary considerably in moral reasoning. According to Kohlberg's theory, individuals who reach the highest level of post-conventional moral reasoning judge moral issues based on deeper principles and shared ideals rather than self-interest or adherence to laws and rules. Recent research has suggested the involvement of the brain's frontostriatal reward system in moral judgments and prosocial behaviors. However, it remains unknown whether moral reasoning level is associated with differences in reward system function. Here, we combined arterial spin labeling perfusion and blood oxygen level-dependent functional magnetic resonance imaging and measured frontostriatal reward system activity both at rest and during a sequential risky decision making task in a sample of 64 participants at different levels of moral reasoning. Compared to individuals at the pre-conventional and conventional level of moral reasoning, post-conventional individuals showed increased resting cerebral blood flow in the ventral striatum and ventromedial prefrontal cortex. Cerebral blood flow in these brain regions correlated with the degree of post-conventional thinking across groups. Post-conventional individuals also showed greater task-induced activation in the ventral striatum during risky decision making. These findings suggest that high-level post-conventional moral reasoning is associated with increased activity in the brain's frontostriatal system, regardless of task-dependent or task-independent states.

This study assesses a novel bile solubility test and MALDI-TOF for the differentiation of Streptococcus pneumoniae from other mitis group streptococci, including differentiation of S. pneumoniae from Streptococcus pseudopneumoniae. Eighty-four species verified mitis group isolates were subjected to our bile solubility test (which measures and calculates the differences of absorbance in the test tube containing 10% sodium deoxycholate versus a blank control tube, after incubation for 10 minutes at 36 °C using a spectrophotometer) and MALDI-TOF MS (both the standard result output and by visual spectra evaluation). Applying a calculated optimal cut-off absorbance-value of 2.1, differentiated S. pneumoniae from all but one other mitis group streptococci (one S. mitis isolate generated an OD-value above 2.1). MALDI-TOF score value identification identified correctly 46 S. pneumoniae and 4 S. pseudopneumoniae but misidentified 16 other mitis group strains. Visual spectra evaluation correctly identified all S. pneumoniae and S. pseudopneumoniae strains but misidentified 13 other mitis group strains. The bile solubility test based on spectrophotometric reading described in this study can differentiate S. pneumoniae from other Streptococcus species. Combining the bile solubility test and the MALDI-TOF spectra results provide a correct identification of all S. pneumoniae and S. pseudopneumoniae isolates.

What are the neural mechanisms underlying beauty based on objective parameters and beauty based on subjective social construction? This study scanned participants with fMRI while they performed aesthetic judgments on concrete pictographs and abstract oracle bone scripts. Behavioral results showed both pictographs and oracle bone scripts were judged to be more beautiful when they referred to beautiful objects and positive social meanings, respectively. Imaging results revealed regions associated with perceptual, cognitive, emotional and reward processing were commonly activated both in beautiful judgments of pictographs and oracle bone scripts. Moreover, stronger activations of orbitofrontal cortex (OFC) and motor-related areas were found in beautiful judgments of pictographs, whereas beautiful judgments of oracle bone scripts were associated with putamen activity, implying stronger aesthetic experience and embodied approaching for beauty were elicited by the pictographs. In contrast, only visual processing areas were activated in the judgments of ugly pictographs and negative oracle bone scripts. Results provide evidence that the sense of beauty is triggered by two processes: one based on the objective parameters of stimuli (embodied natural beauty) and the other based on the subjective social construction (social endowed beauty).