The importance of patient perspectives in pulmonary hypertension.

The assessment of objective measurement of cardiopulmonary status has helped us achieve better clinical outcomes for patients and develop new therapies through to the point of market access; however, patient surveys indicate that more can be done to improve holistic care and patient engagement. In this multidisciplinary review, we examine how clinical teams can acknowledge and embrace the individual patient's perspective, and thus improve the care for individual patients suffering from pulmonary hypertension by cultivating the importance and relevance of health-related quality of life in direct clinical care. At the individual level, patients should be provided with access to accredited specialist centres which provide a multidisciplinary approach where there is a culture focused on narrative medicine, quality of life, shared decision making and timely access to palliative care, and where there is participation in education. On a larger scale, we call for the development, expansion and promotion of patient associations to support patients and carers, lobby for access to best care and treatments, and provide input into the development of clinical trials and registries, focusing on the patients' perspective.

Differences of Recovery from Rocuronium-induced Deep Paralysis in Response to Small Doses of Sugammadex between Elderly and Nonelderly Patients.

WHAT WE ALREADY KNOW ABOUT THIS TOPIC: WHAT THIS ARTICLE TELLS US THAT IS NEW: BACKGROUND:: Complete recovery from rocuronium-induced muscle paralysis with sugammadex is reported to be delayed in elderly patients. The authors tested a hypothesis that recovery from deep neuromuscular block with low-dose sugammadex is slower (primary hypothesis) and incidence of recurarization is higher (secondary hypothesis) in elderly patients than in nonelderly patients. METHODS: In anesthetized elderly (n = 20; 76.9 ± 5.0 yr of age) and nonelderly patients (n = 20; 53.7 ± 12.8 yr of age) under deep paralysis with rocuronium, change of train-of-four ratio per minute (primary outcome variable) was measured with an acceleromyograph neuromuscular monitor during spontaneous recovery from rocuronium-induced muscle paralysis (0.6 mg/kg) and after infusion of low-dose sugammadex (50 µg · kg · min). Recurarization was defined as the negative change of train-of-four ratio. RESULTS: Spontaneous train-of-four ratio recovery rate was significantly slower in the elderly group (median [25th percentile, 75th percentile]: 1.89 [1.22, 2.90] %/min) than in the nonelderly group (3.45 [1.96, 4.25] %/min, P = 0.024). Train-of-four ratio change rate in response to low-dose sugammadex was significantly slower in elderly (0.55 [-0.29, 1.54] %/min) than in the nonelderly group (1.68 [0.73, 3.13] %/min, P = 0.024). Incidence of recurarization was significantly higher in the elderly group than in the nonelderly group (35% vs. 5%, P = 0.044). Multiple linear regression analyses indicate that slower spontaneous train-of-four ratio recovery rate and impaired renal function are two major contributing factors that decrease train-of-four ratio change rate in response to low-dose sugammadex. CONCLUSIONS: Elderly patients are at greater risk for recurarization and residual muscle paralysis when low-dose sugammadex is administered.

Neural activity selects myosin IIB and VI with a specific time window in distinct dynamin isoform-mediated synaptic vesicle reuse pathways.

Presynaptic nerve terminals must maintain stable neurotransmissions via synaptic vesicle (SV) resupply despite encountering wide fluctuations in the number and frequency of incoming action potentials (APs). However, the molecular mechanism linking variation in neural activity to SV resupply is unknown. Myosins II and VI are actin-based cytoskeletal motors that drive dendritic actin dynamics and membrane transport, respectively, at brain synapses. Here we combined genetic knockdown or molecular dysfunction and direct physiological measurement of fast synaptic transmission from paired rat superior cervical ganglion neurons in culture to show that myosins IIB and VI work individually in SV reuse pathways, having distinct dependency and time constants with physiological AP frequency. Myosin VI resupplied the readily releasable pool (RRP) with slow kinetics independently of firing rates but acted quickly within 50 ms after AP. Under high-frequency AP firing, myosin IIB resupplied the RRP with fast kinetics in a slower time window of 200 ms. Knockdown of both myosin and dynamin isoforms by mixed siRNA microinjection revealed that myosin IIB-mediated SV resupply follows amphiphysin/dynamin-1-mediated endocytosis, while myosin VI-mediated SV resupply follows dynamin-3-mediated endocytosis. Collectively, our findings show how distinct myosin isoforms work as vesicle motors in appropriate SV reuse pathways associated with specific firing patterns.

Folic acid supplementation dysregulates gene expression in lymphoblastoid cells--implications in nutrition.

For over a decade, folic acid (FA) supplementation has been widely prescribed to pregnant women to prevent neural tube closure defects in newborns. Although neural tube closure occurs within the first trimester, high doses of FA are given throughout pregnancy, the physiological consequences of which are unknown. FA can cause epigenetic modification of the cytosine residues in the CpG dinucleotide, thereby affecting gene expression. Dysregulation of crucial gene expression during gestational development may have lifelong adverse effects or lead to neurodevelopmental defects, such as autism. We have investigated the effect of FA supplementation on gene expression in lymphoblastoid cells by whole-genome expression microarrays. The results showed that high FA caused dysregulation by ≥ four-fold up or down to more than 1000 genes, including many imprinted genes. The aberrant expression of three genes (FMR1, GPR37L1, TSSK3) was confirmed by Western blot analyses. The level of altered gene expression changed in an FA concentration-dependent manner. We found significant dysregulation in gene expression at concentrations as low as 15 ng/ml, a level that is lower than what has been achieved in the blood through FA fortification guidelines. We found evidence of aberrant promoter methylation in the CpG island of the TSSK3 gene. Excessive FA supplementation may require careful monitoring in women who are planning for, or are in the early stages of pregnancy. Aberrant expression of genes during early brain development may have an impact on behavioural characteristics.

The current status of bereavement follow-up in Japanese emergency departments: A cross-sectional nationwide survey.

INTRODUCTION: The aim of this study was to investigate the current status of bereavement follow-up in Japanese emergency departments. METHODS: This study employed a cross-sectional design and conducted a nationwide survey of all emergency departments in Japan. Self-reported questionnaires were sent to the nurse leaders of each emergency department. RESULTS: Of 289 nurse leaders approached, 145 (50.2%) responded. Only 17.9% emergency departments provided bereavement follow-up strategies, and the most frequent strategy was referral to a specialist for psychological treatment. Most nurse leaders perceived that bereavement follow-up is necessary, and the greatest need of the bereaved as perceived by the nurse leaders was explanation of the patient's death. However, 60% of the nurse leaders perceived bereavement follow-up to be necessary but difficult, and the major challenges in bereavement follow-up were lack of time, knowledge, and skill. CONCLUSION: In contemporary Japan, the prevalence of bereavement follow-up strategies offered by emergency departments was low, and although most nurse leaders perceived follow-up as necessary, it could not be provided because of limitations in human resources and staff training.

Dyrk1A binds to multiple endocytic proteins required for formation of clathrin-coated vesicles.

In spite of a nuclear targeting sequence, a substantial amount of dual-specificity tyrosine phosphorylation-regulated kinase (Dyrk1A) is located within the cytoplasm of neurons. Analysis of fractionated rat brains revealed that the majority of Dyrk1A was in the postnuclear precipitate. The kinase in this fraction was resistant to high salt and Triton X-100 extraction at pH 6.5. Hypothesizing that Dyrk1A binds tightly with cell constituents, we searched for Dyrk1A binding proteins in the Triton X-100-insoluble fraction extracted with urea and fractionated by column chromatography. An overlay assay using the recombinant kinase revealed that multiple proteins are capable of binding to Dyrk1A. Among them, we identified clathrin heavy chain and dynamin 1 as potential candidates. An overlay assay using purified and partially purified proteins showed the binding of Dyrk1A with both proteins. Under native conditions, Dyrk1A precipitated with newly formed clathrin cages and with dynamin via the GST-amphiphysin SH3 domain. We also identified another endocytic protein, endophilin 1, as an additional Dyrk1A binding protein. We then tested whether the clathrin-coated vesicle (CCV)-associated proteins could be phosphorylated by Dyrk1A. Multiple proteins apparently distinctive from the known substrates were phosphorylated in the brain CCV. Our findings suggest a role for Dyrk1A in controlling synaptic vesicle recycling processes.

[GERD and sleep disorder].

The prevalence of gastroesophageal reflux disease (GERD) is increasing in Japan. Symptoms of GERD negatively affect their quality of life and sleep. There are several reasons for sleep disorder with GERD as follows. Nocturnal GERD symptoms sometimes directly avoid sleeping. Sleep apnea syndrome and GERD are sometimes concomitant. The both are sharing similar risk factor such as obesity and cause sleep disorder. When untypical symptoms of GERD are not diagnosed, patients are severely anxious about their physical condition. Then they feel stressful and sometimes get secondary depressive state including sleep disorder. We had better take care about patients' psychosocial factors and treat symptoms of GERD and sleep disorder together with holistic approach.

Malignant Hyperthermia during Thoracoscopic Pulmorrhaphy in a 70-Year-Old Man.

Malignant hyperthermia (MH) is a rare but potentially fatal complication that may develop under general anesthesia (GA) and is rarely reported in elderly patients. We encountered a case of mild-onset MH in a 70-year-old patient who was receiving an elective thoracoscopic pulmorrhaphy and had a history of several GA procedures. Anesthesia was induced with propofol, fentanyl, and rocuronium and maintained with sevoflurane and remifentanil. His body temperature (BT) was 37.9°C after induction. During the procedure, the end-tidal CO2 (ETCO2) increased steadily to 47-50 mmHg, presumably in response to the single lung ventilation. At the end, BT was 38.1°C and ETCO2 was 47 mmHg under spontaneous breathing. After extubation, the patient wheezed on inspiration and expiration, and his trachea was reintubated. Sixty minutes after surgery, BT increased to 40.5°C and the arterial blood gas analysis showed severe metabolic acidosis. Based on these findings, MH was suspected and a bolus dose of dantrolene was administered. He responded to the dantrolene, and no complications or recurrence of MH was observed postoperatively. In this patient, the initial signs of MH were so subtle that making the diagnosis of MH was difficult. A high degree of suspicion is necessary to prevent a fulminant MH crisis.

Phosphorylation by Dyrk1A of clathrin coated vesicle-associated proteins: identification of the substrate proteins and the effects of phosphorylation.

Dyrk1A phosphorylated multiple proteins in the clathrin-coated vesicle (CCV) preparations obtained from rat brains. Mass spectrometric analysis identified MAP1A, MAP2, AP180, and α- and ß-adaptins as the phosphorylated proteins in the CCVs. Each protein was subsequently confirmed by [(32)P]-labeling and immunological methods. The Dyrk1A-mediated phosphorylation released the majority of MAP1A and MAP2 and enhanced the release of AP180 and adaptin subunits from the CCVs. Furthermore, Dyrk1A displaced adaptor proteins physically from CCVs in a kinase-concentration dependent manner. The clathrin heavy chain release rate, in contrast, was not affected by Dyrk1A. Surprisingly, the Dyrk1A-mediated phosphorylation of α- and ß-adaptins led to dissociation of the AP2 complex, and released only ß-adaptin from the CCVs. AP180 was phosphorylated by Dyrk1A also in the membrane-free fractions, but α- and ß-adaptins were not. Dyrk1A was detected in the isolated CCVs and was co-localized with clathrin in neurons from mouse brain sections and from primary cultured rat hippocampus. Previously, we proposed that Dyrk1A inhibits the onset of clathrin-mediated endocytosis in neurons by phosphorylating dynamin 1, amphiphysin 1, and synaptojanin 1. Current results suggest that besides the inhibition, Dyrk1A promotes the uncoating process of endocytosed CCVs.

Activation of DNA methyltransferase 1 by EBV latent membrane protein 2A leads to promoter hypermethylation of PTEN gene in gastric carcinoma.

CpG island promoter methylation of tumor suppressor genes is one of the most characteristic abnormalities in EBV-associated gastric carcinoma (GC). Aberrant promoter methylation and expression loss of PTEN were evaluated in cancer tissues of GC by methylation-specific PCR and immunohistochemistry, respectively, showing that both abnormalities occurred concurrently in EBV-associated GC. PTEN abnormalities were reiterated in GC cell lines MKN-1 and MKN-7 infected with recombinant EBV, and DNA methyltransferase 1 (DNMT1) was commonly overexpressed in both cell lines. Stable and transient transfection systems in MKN-1 similarly showed that viral latent membrane protein 2A (LMP2A) up-regulated DNMT1, leading to an increase in methylation of the PTEN promoter. Importantly, the level of phosphorylated signal transducer and activator of transcription 3 (pSTAT3) increased in the nuclei of LMP2A-expressing GC cells, and knockdown of STAT3 counteracted LMP2A-mediated DNMT1 overexpression. Immunohistochemistry for both pSTAT3 and DNMT1 showed diffuse labeling in the nuclei of the cancer cells in GC tissues, especially in EBV-associated GC. Taken together, LMP2A induces the phosphorylation of STAT3, which activates DNMT1 transcription and causes PTEN expression loss through CpG island methylation of the PTEN promoter in EBV-associated GC. LMP2A plays an essential role in the epigenetic abnormalities in host stomach cells and in the development and maintenance of EBV-associated cancer.

Dual-specificity tyrosine phosphorylation-regulated kinase 1A does not require tyrosine phosphorylation for activity in vitro.

The dual-specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A) gene is localized in human chromosome 21, and its overexpression has been associated with the learning and memory deficits of Down syndrome. DYRK1A contains a Y319XY321 motif shared by all members of the DYRK protein kinase family. Residue Y321 in the motif is phosphorylated in DYRK1A prepared from Escherichia coli and from eukaryotic cells. It has been proposed that the YXY motif is an equivalent of the TXY motif, the activation loop, of mitogen-activated protein kinase and that phosphorylation at the motif is required for DYRK activity. In this study, the role of tyrosine phosphorylation in the activity of DYRK1A was investigated in detail. Wild-type DYRK1A with a reduced level of phosphotyrosine (pY) was prepared by treating E. coli-produced DYRK1A with two different protein tyrosine phosphatases. The resulting pY-depleted DYRK1A could not regain pY during autophosphorylation but was as active as the untreated control. These findings were further supported by the observation that DYRK1A retained significant enzymatic activity when both tyrosine residues in the YXY motif were replaced with either histidine or glutamine. Together, we conclude that tyrosine phosphorylation and tyrosine residues in the YXY motif are not directly involved in DYRK1A enzymatic activity in vitro.

MNB/DYRK1A phosphorylation regulates the interactions of synaptojanin 1 with endocytic accessory proteins.

MNB/DYRK1A is a proline-directed serine/threonine kinase implicated in Down syndrome (DS). In an earlier screening, two proteins from adult rat brain, one 100kDa and the other 140 kDa, were found to be prominently phosphorylated by the kinase. The 100-kDa protein was previously characterized as an isoform of dynamin 1. In this study, we identified the 140-kDa protein as synaptojanin 1 (SJ1). MNB/DYRK1A phosphorylates SJ1 at multiple sites and produces complex behaviors in binding to amphiphysin 1 and intersectin 1 (ITSN1). However, the phosphorylation has little effect on the phosphatidylinositol phosphatase activity of SJ1. These results suggest that MNB/DYRK1A is involved in regulating the recruitment activity but not the phosphatase activity of SJ1. Our findings may be especially important in the etiology of DS because MNB/DYRK1A, SJ1, and ITSN1 are all located at or near the region of human chromosome 21, which is postulated to be involved in the disease.

Kinetic properties of a MNB/DYRK1A mutant suitable for the elucidation of biochemical pathways.

Minibrain kinase/dual-specificity tyrosine phosphorylation regulated kinase 1A (MNB/DYRK1A) is a proline/arginine-directed serine/threonine kinase implicated in the learning deficits of Down syndrome. Epigallocatechin-3-gallate (EGCG), the major tea polyphenolic compound, is a potent MNB/DYRK1A inhibitor. In this study, we investigated the mechanism of EGCG inhibition of MNB/DYRK1A using a combination of genetic and biochemical approaches. In the testing system using MNB/DYRK1A-promoted Gli 1-dependent transcription as the readout, NIH3T3 cells expressing EGCG resistant MNB/DYRK1A mutant R21 were found to acquire EGCG resistance for a wide range of drug concentrations. Mutant R21 harbors a single K465R substitution, which produces a 3-fold gain in the EGCG resistance in vitro. However, the gain in the EGCG resistance alone cannot fully interpret the effectiveness of mutant R21 in suppressing EGCG in cultured cells. Kinetic analysis suggests that EGCG functions as a noncompetitive inhibitor against ATP. Interestingly, the K465R mutation changes the mode of EGCG inhibition on MNB/DYRK1A so that it becomes a competitive inhibitor against ATP. This competitive mode of EGCG inhibition coupled with high intracellular ATP concentrations and an elevated EGCG resistance are likely to be the basis for the resistant property of mutant R21 in cultured cells. The K465R mutation apparently transforms the intramolecular interactions required for MNB/DYRK1A catalysis. This mutant would also be valuable for the elucidation of the mechanisms of MNB/DYRK1A-catalyzed reaction.

Phosphorylation of amphiphysin I by minibrain kinase/dual-specificity tyrosine phosphorylation-regulated kinase, a kinase implicated in Down syndrome.

Minibrain kinase/dual-specificity tyrosine phosphorylation-regulated kinase (Mnb/Dyrk1A) is a proline-directed serine/threonine kinase encoded in the Down syndrome critical region of human chromosome 21. This kinase has been shown to phosphorylate dynamin 1 and synaptojanin 1. Here we report that amphiphysin I (Amph I) is also a Mnb/Dyrk1A substrate. This kinase phosphorylated native Amph I in rodent brains and recombinant human Amph I expressed in Escherichia coli. Serine 293 (Ser-293) was identified as the major site, whereas serine 295 and threonine 310 were found as minor kinase sites. In cultured cells, recombinant Amph I was phosphorylated at Ser-293 by endogenous kinase(s). Because mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) has been suggested to phosphorylate Amph I at Ser-293, our efforts addressed whether Ser-293 is phosphorylated in vivo by MAPK/ERK or by Mnb/Dyrk1A. Overnight serum-withdrawal inactivated MAPK/ERK; nonetheless, Ser-293 was phosphorylated in Chinese hamster ovary and SY5Y cells. Epigallocatechin-3-gallate, a potent Mnb/Dyrk1A inhibitor in vitro, apparently reduced the phosphorylation at Ser-293, whereas PD98059, a potent MAPK/ERK inhibitor, did not. High frequency stimulation of mouse hippocampal slices reduced the phosphorylation at Ser-293, albeit in the midst of MAPK/ERK activation. The endophilin binding in vitro was inhibited by phosphorylating Amph I with Mnb/Dyrk1A. However, phosphorylation at Ser-293 did not appear to alter cellular distribution patterns of the protein. Our results suggest that Mnb/Dyrk1A, not MAPK/ERK, is responsible for in vivo phosphorylation of Amph I at Ser-293 and that phosphorylation changes the recruitment of endophilin at the endocytic sites.

Localization of myosin II and V isoforms in cultured rat sympathetic neurones and their potential involvement in presynaptic function.

While vesicle transport is one of the principal functions of myosin motors in neurones, the role played by specific myosin subtypes in discrete vesicle trafficking is poorly understood. We conducted electrophysiological and morphological experiments to determine whether myosin isoforms II and V might be involved in the transport of small synaptic vesicles in presynaptic nerve terminals of a model cholinergic synapse. Electron microscopy revealed the presence of normal synaptic architecture and synaptic vesicle density in presynaptic terminals of cultured superior cervical ganglion neurones (SCGNs) from myosin Va null rats (dilute-opisthotonus, dop). Similarly, electrophysiological analyses of synaptic transmission and synaptic vesicle cycling at paired SCGN synapses failed to uncover any significant differences in synaptic development and function between normal and dop rats. Immunocytochemistry and in situ localization of green fluorescent protein (GFP)-fusion proteins in wild-type synapses revealed that myosins IIB and Va were distributed throughout the cell soma and processes of SCGNs, while myosins IIA and Vb were not detected in SCGNs. Myosin Va was conspicuously absent in presynaptic nerve terminals, but myosin IIB alone was found to be expressed. Furthermore, synaptic transmission was inhibited by introduction of myosin IIB heavy chain fragments into presynaptic terminals of SCGNs. Together these results suggest that only myosin IIB isoform participates in vesicle trafficking in presynaptic nerve terminals of cultured SCGNs.

Mnb/Dyrk1A phosphorylation regulates the interaction of dynamin 1 with SH3 domain-containing proteins.

Mnb/Dyrk1A is a proline-directed serine/threonine kinase implicated in Down's syndrome. Mnb/Dyrk1A was shown to phosphorylate dynamin 1 and alter its interactions with several SH3 domain-containing endocytic accessory proteins. To determine the mechanism of regulation, we mapped the Mnb/Dyrk1A phosphorylation sites in dynamin 1. Using a combination of deletion mutants and synthetic peptides, three potential Mnb/Dyrk1A phosphorylation sites (S778, S795, and S857) were first identified. Phosphorylation at S795 and S857 was confirmed in full-length dynamin 1, and S857 was subsequently determined to be the major Mnb/Dyrk1A phosphorylation site in vitro. Phosphorylation at S857 was demonstrated to be the basis for altering the binding of dynamin 1 to amphiphysin 1 and Grb 2 by site-directed mutants mimicking phosphorylation. Furthermore, S857 of dynamin 1 is phosphorylated by the endogenous kinase in brain extracts and in PC12 cells. In PC12 cells, the state of S857 phosphorylation is dependent on membrane potentials. These results suggest that S857 phosphorylation is a physiological event, which regulates the binding of dynamin 1 to SH3 domain-containing proteins. Since S857 is unique to dynamin 1xa isoforms, Mnb/Dyrk1A regulation of dynamin 1 is expected to be specific to these spliced variants.