Edoxaban suppresses the progression of atrial fibrosis and atrial fibrillation in a canine congestive heart failure model.

Coagulation factor Xa activates the protease-activated receptor 2 (PAR2) and causes tissue fibrosis; however, the effects of Xa inhibitor edoxaban on atrial fibrosis and atrial fibrillation (AF) have not been investigated. We examined the effect of edoxaban on the progression of atrial fibrosis in a canine congestive heart failure (CHF) model. Beagle dogs were assigned to sham, placebo, and edoxaban groups (n = 6/group). Dogs of the placebo or edoxaban groups received 19 days of medication with daily oral placebo or edoxaban, respectively, followed by 14 days of ventricular tachypacing. Dogs of the sham group had no medication or pacing. Ventricular tachypacing prolonged AF duration in dogs of the placebo group (159 ± 41 s, p < 0.01 vs. sham); however, this effect was suppressed by edoxaban treatment. Compared with the sham group, tachypacing alone also significantly increased the atrial fibrotic area (2.9 ± 0.1% vs. 7.8 ± 0.4%, p < 0.01), PAR2 expression (1.0 ± 0.1 vs. 1.8 ± 0.3, p < 0.05), and atrial fibronectin expression (1.0 ± 0.2 vs. 2.0 ± 0.2, p < 0.01). These responses were suppressed by edoxaban treatment (area 5.9 ± 0.4%, p < 0.01; PAR2 1.1 ± 0.1, p < 0.05; fibronectin 1.2 ± 0.2, p < 0.05 vs. placebo). Edoxaban showed suppressive effects on atrial remodeling, AF progression, and excessive expressions of PAR2 and fibronectin in a canine CHF model. The suppression of the Xa/PAR2 pathway might be a potential pharmacological target of edoxaban.

A mutant HCN4 channel in a family with bradycardia, left bundle branch block, and left ventricular noncompaction.

We found that a female infant presenting with left bundle branch block and left ventricular noncompaction carries uninvestigated gene mutations HCN4(G811E), SCN5A(L1988R), DMD(S2384Y), and EMD(R203H). Here, we explored the possible pathogenicity of HCN4(G811E), which results in a G811E substitution in hyperpolarization-activated cyclic nucleotide-gated channel 4, the main subunit of the cardiac pacemaker channel. Voltage-clamp measurements in a heterologous expression system of HEK293T cells showed that HCN4(G811E) slightly reduced whole-cell HCN4 channel conductance, whereas it did not affect the gating kinetics, unitary conductance, or cAMP-dependent modulation of voltage-dependence. Immunocytochemistry and immunoblot analysis showed that the G811E mutation did not impair the membrane trafficking of the channel subunit in the heterologous expression system. These findings indicate that HCN4(G811E) may not be a monogenic factor to cause the cardiac disorders.

Incipient progressive supranuclear palsy is more common than expected and may comprise clinicopathological subtypes: a forensic autopsy series.

We investigated 998 serial Japanese forensic autopsy cases (0-101 years old, mean age 61.7 ± 21.9), with no case selection, using immunohistochemistry to detect cases with progressive supranuclear palsy (PSP). Twenty-nine cases (mean age 82.3 ± 7.2 years, 11 males, 18 females) fulfilled the National Institute of Neuronal Disorders and Stroke (NINDS)-PSP pathological criteria (2.9% of all cases, 4.6% of cases over 60). All had neuronal and glial inclusions in the basal ganglia and brainstem. However, 13 cases had low tau pathology and were categorized as atypical PSP. In addition to PSP pathology, multiple types of astrocytic inclusions and comorbid proteinopathies, particularly a high prevalence of argyrophilic grain disease, were found. All cases had not been diagnosed with PSP and had preserved daily functioning prior to death. However, 14 (48.3%), 11 (37.9%), and 16 (55.2%) cases showed signs of dementia, depressive state, and gait disturbance, respectively. Sixteen accidental death cases (55.2%), including from falls and getting lost, and 11 suicide cases (37.9%) appear to have a relationship with incipient PSP pathology. Cluster analysis using the distribution and amount of 4-repeat-tau pathology classified the cases into three subgroups: Group 1 (10 cases) had typical PSP pathology and seven cases (70.0%) had dementia as the most frequent symptom; Group 2 (7 cases) had significantly higher frequency of gait disorder (6 cases, 85.7%), and less neocortical tau pathology than Group 1; Group 3 (12 cases) had relatively mild PSP pathology and high argyrophilic grain burdens. Granular-shaped astrocytes were the dominant astrocytic inclusion in all cases. We conclude that in forensic cases incipient PSP occurs with a higher prevalence than expected. If these findings can be extrapolated to other population-based cohorts, PSP may be more common than previously thought.

MicroRNA-93 may control vascular endothelial growth factor A in circulating peripheral blood mononuclear cells in acute Kawasaki disease.

BACKGROUND: Kawasaki disease (KD) is the most common systemic vasculitis syndrome primarily affecting medium-sized arteries, particularly the coronary arteries. Though KD may be associated with immunological problems, the involvement of microRNAs (miRs) has not been fully described. METHODS: We enrolled 23 KD patients and 12 controls. We performed miR and mRNA microarray analysis of peripheral blood mononuclear cells (PBMCs) isolated from acute KD patients and controls. Continuously, we measured specific miRs, mRNA and the expression of proteins by using reverse-transcriptase PCR (RT-PCR) and enzyme-linked immunosorbent assay (ELISA). RESULTS: We identified strikingly high levels of miR-182 and miR-296-5p during the acute febrile phase, and of miR-93, miR-145-5p, miR-145-3p, and miR-150-3p in the defervescence stage, especially in refractory KD patients. The expression of vascular endothelial growth factor A (VEGFA) mRNA, previously reported to be controlled by miR-93, was significantly elevated during the febrile phase and normalized upon treatment, negatively correlating with the expression of miR-93. Further, plasma levels of VEGF-A correlated with PBMC VEGFA mRNA expression. CONCLUSION: Several miRs are highly specific to the acute phase of KD, and may participate in regulating the expression of genes in pathways associated with KD. In particular, miR-93 may participate in regulating expression of VEGF-A and contribute to the pathogenesis of arteritis in acute KD.

Effect of irbesartan on development of atrial fibrosis and atrial fibrillation in a canine atrial tachycardia model with left ventricular dysfunction, association with p53.

Effects of an angiotensin II receptor blocker, irbesartan (IRB), on the development of atrial fibrosis and atrial fibrillation (AF) were assessed in a canine model of atrial tachycardia remodeling (ATR) with left ventricular dysfunction, together with its possible association with involvement of p53. Atrial tachypacing (400 bpm for 4 weeks) was used to induce ATR in beagles treated with placebo (ATR-dogs, n = 6) or irbesartan (IRB-dogs, n = 5). Non-paced sham dogs served as control (Control-dogs, n = 4). ATR- and IRB-dogs developed tachycardia-induced left ventricular dysfunction. Atrial effective refractory period (AERP) shortened (83 ± 5 ms, p < 0.05), inter-atrial conduction time prolonged (72 ± 2 ms, p < 0.05), and AF duration increased (29 ± 5 s, p < 0.05 vs. baseline) after 4 weeks in ATR-dogs. ATR-dogs also had a larger area of atrial fibrous tissue (5.2 ± 0.5 %, p < 0.05 vs. Control). All these changes, except for AERP, were attenuated in IRB-dogs (92 ± 3 ms, 56 ± 3 ms, 9 ± 5 s, and 2.5 ± 0.7 %, respectively; p < 0.05 vs. ATR for each). In ATR-dogs, p53 expression in the left atrium decreased by 42 % compared with Control-dogs (p < 0.05); however, it was highly expressed in IRB-dogs (+89 % vs. ATR). Transforming growth factor (TGF)-ß1 expression was enhanced in ATR-dogs (p < 0.05 vs. Control) but reduced in IRB-dogs (p < 0.05 vs. ATR). Irbesartan suppresses atrial fibrosis and AF development in a canine ATR model with left ventricular dysfunction in association with p53.

Pathological features of preclinical or early clinical stages of corticobasal degeneration: a comparison with advanced cases.

AIMS: The manner in which pathological lesions of corticobasal degeneration (CBD) progress remains poorly understood. Because the pathology of early disease stages may be fundamental for elucidating a border between clinical and preclinical states of CBD, the present study aimed to detect preclinical or early clinical CBD cases by examining a series of forensic autopsy cases. METHODS: A series of 887 brains from medicolegal autopsies was examined. Immunohistochemistry for tau (AT8, 3, and 4-repeat-tau) and Gallyas-Braak was applied for diagnosis. Neuropathological diagnosis of CBD followed criteria updated in 2002 by a working group. RESULTS: Three autopsy cases (0.34%) were identified that fulfilled CBD pathological criteria. Two cases were preclinical or very early clinical cases without brain atrophy; the other case had exhibited a 5-year history of advanced frontotemporal dementia. Significant microscopic differences between the subclinical and clinical cases included occurrence of neuronal loss with spongiosis and gliosis, as well as a difference in degree of tau pathology in the superficial layer of the neocortical areas and white matter. Anatomical hierarchy of tau pathology in the brain was not evident, but asymmetric neocortical tau pathology that might influence the clinical phenotype was found in preclinical and early clinical cases. CONCLUSIONS: The results revealed the pathological features of subclinical and early clinical CBD cases. Comparison with clinical CBD cases showed that neuronal loss, cortical atrophy and volume reduction of white matter may be involved in the occurrence of clinical symptoms of CBD. Additionally, immunohistochemistry is essential for detecting preclinical CBD cases, regardless of case selection.

The Susceptibilities of Human Ether-à-Go-Go-Related Gene Channel with the G487R Mutation to Arrhythmogenic Factors.

The human ether-à-go-go-related gene (hERG) channel mediates the rapid delayed rectifier potassium current (IKr) responsible for shaping the repolarization phase of cardiac action potentials. hERG mutation may cause hERG channel malfunction, leading to long QT syndrome and other arrhythmic disorders. Elucidation of the genotype-phenotype relationships of individual hERG mutations is key to the development of treatment for such arrhythmic disorders. We previously identified hERG(G487R), a missense mutant with a glycine-to-arginine substitution at position 487. In the absence of arrhythmogenic factors, hERG(G487R) subunit-containing channels show normal surface expression and gating kinetics. However, it remains unknown whether the mutation exacerbates hERG channel malfunction induced by arrhythmogenic factors. Here we used a voltage-clamp technique to compare the effects of the major arrythmogenic factors on wild-type hERG [hERG(WT)] and hERG(G487R) channel currents (IhERG) in HEK-293T cells. The extent of IhERG blockade by the antiarrhythmic drug dofetilide or E4031 was not different between these channels. On the other hand, the extracellular K(+) concentration ([K(+)]ex)-dependent changes in the rates of recovery from inactivation and deactivation of IhERG were rather less obvious for hERG(G487R) channel than for hERG(WT) channel. These findings suggest that the inheritance of hERG(G487R) does not increase the risk of arrhythmic disorders induced by antiarrhythmic drugs or hypokalemia.

A590T mutation in KCNQ1 C-terminal helix D decreases IKs channel trafficking and function but not Yotiao interaction.

KCNQ1 encodes the α subunit of the voltage-gated channel that mediates the cardiac slow delayed rectifier K(+) current (IKs). Here, we report a KCNQ1 allele encoding an A590T mutation [KCNQ1(A590T)] found in a 39-year-old female with a mild QT prolongation. A590 is located in the C-terminal α helical region of KCNQ1 that mediates subunit tetramerization, membrane trafficking, and interaction with Yotiao. This interaction is known to be required for the proper modulation of IKs by cAMP. Since previous studies reported that mutations in the vicinity of A590 impair IKs channel surface expression and function, we examined whether and how the A590T mutation affects the IKs channel. Electrophysiological measurements in HEK-293T cells showed that the A590T mutation caused a reduction in IKs density and a right-shift of the current-voltage relation of channel activation. Immunocytochemical and immunoblot analyses showed the reduced cell surface expression of KCNQ1(A590T) subunit and its rescue by coexpression of the wild-type KCNQ1 [KCNQ1(WT)] subunit. Moreover, KCNQ1(A590T) subunit interacted with Yotiao and had a cAMP-responsiveness comparable to that of KCNQ1(WT) subunit. These findings indicate that the A590 of KCNQ1 subunit plays important roles in the maintenance of channel surface expression and function via a novel mechanism independent of interaction with Yotiao.

Identification and characterization of a novel genetic mutation with prolonged QT syndrome in an unexplained postoperative death.

INTRODUCTION: The human ether-à-go-go-related gene (hERG) encodes the α-subunit of a cardiac potassium channel. Various mutations of hERG, including missense mutations, have been reported to cause long QT syndrome (LQTS) and severe arrhythmic disorders such as sudden cardiac death. We identified a novel hERG frameshift mutation (hERG(ΔAT)) in the S5-pore region from a LQTS patient who died suddenly and analyzed its genetic profile and the molecular and electrophysiological behaviors of the protein product to assess the pathogenicity of hERG(ΔAT). METHODS AND RESULTS: We performed direct sequencing of hERG and evaluated its transcript level by using a whole blood sample from the patient. We performed immunoblotting, immunocytochemistry, and patch-clamp recordings of HEK-293 T cells transfected with hERG(ΔAT), wild-type hERG (hERG(WT)), or both. The patient demonstrated an AT deletion (c.1735_1736del) in hERG and a decrease in hERG mRNA transcripts. HEK-293 T cells showed lower production and cell surface expression of hERG(ΔAT) compared with hERG(WT) protein. In addition, the hERG(∆AT) protein failed to form functional channels, while the activation kinetics of functional channels, presumably consisting of hERG(WT) subunits, were unaffected. CONCLUSION: The ΔAT mutation may decrease the number of functional hERG channels by impairing the posttranscriptional and posttranslational processing of the mutant product. This decrease may partly explain the cardiac symptoms of the patient who was heterozygous for hERG(ΔAT).

Glycine/Serine polymorphism at position 38 influences KCNE1 subunit's modulatory actions on rapid and slow delayed rectifier K+ currents.

BACKGROUND: KCNE1 encodes a modulator of KCNH2 and KCNQ1 delayed rectifier K(+) current channels. KCNE1 mutations might cause long QT syndrome (LQTS) by impairing KCNE1 subunit's modulatory actions on these channels. There are major and minor polymorphismic KCNE1 variants whose 38(th) amino acids are glycine and serine [KCNE1(38G) and KCNE1(38S) subunits], respectively. Despite its frequent occurrence, the influence of this polymorphism on the K(+) channels' function is unclear. METHODS AND RESULTS: Patch-clamp recordings were obtained from human embryonic kidney -293T cells. KCNH2 channel current density in KCNE1(38S)-transfected cells was smaller than that in KCNE1(38G)-transfected cells by 34%. The voltage-sensitivity of the KCNQ1 channel current in KCNE1(38S)-transfected cells was lowered compared to that in KCNE1(38G)-transfected cells, with a +13mV shift in the half-maximal activation voltage. KCNH2 channel current density or KCNQ1 channel voltage-sensitivity was not different between KCNE1(38G)-transfected cells and cells transfected with both KCNE1(38G) and KCNE1(38S). Moreover, the KCNH2 channel current in KCNE1(38S)-transfected cells was more susceptible to E4031, a QT prolonging drug and a condition with hypokalemia, than that in KCNE1(38G)-transfected cells. CONCLUSIONS: Homozygous inheritance of KCNE1(38S) might cause a mild reduction of the delayed rectifier K(+) currents and might thereby increase an arrhythmogenic potential particularly in the presence of QT prolonging factors. By contrast, heterozygous inheritance of KCNE1(38G) and KCNE1(38S) might not affect the K(+) currents significantly. (Circ J 2014; 78: 610-618).

Characterization of a novel mutant KCNQ1 channel subunit lacking a large part of the C-terminal domain.

A mutation of KCNQ1 gene encoding the alpha subunit of the channel mediating the slow delayed rectifier K(+) current in cardiomyocytes may cause severe arrhythmic disorders. We identified KCNQ1(Y461X), a novel mutant gene encoding KCNQ1 subunit whose C-terminal domain is truncated at tyrosine 461 from a man with a mild QT interval prolongation. We made whole-cell voltage-clamp recordings from HEK-293T cells transfected with either of wild-type KCNQ1 [KCNQ1(WT)], KCNQ1(Y461X), or their mixture plus KCNE1 auxiliary subunit gene. The KCNQ1(Y461X)-transfected cells showed no delayed rectifying current. The cells transfected with both KCNQ1(WT) and KCNQ1(Y461X) showed the delayed rectifying current that is thought to be mediated largely by homomeric channel consisting of KCNQ1(WT) subunit because its voltage-dependence of activation, activation rate, and deactivation rate were similar to the current in the KCNQ1(WT)-transfected cells. The immunoblots of HEK-293T cell-derived lysates showed that KCNQ1(Y461X) subunit cannot form channel tetramers by itself or with KCNQ1(WT) subunit. Moreover, immunocytochemical analysis in HEK-293T cells showed that the surface expression level of KCNQ1(Y461X) subunit was very low with or without KCNQ1(WT) subunit. These findings suggest that the massive loss of the C-terminal domain of KCNQ1 subunit impairs the assembly, trafficking, and function of the mutant subunit-containing channels, whereas the mutant subunit does not interfere with the functional expression of the homomeric wild-type channel. Therefore, the homozygous but not heterozygous inheritance of KCNQ1(Y461X) might cause major arrhythmic disorders. This study provides a new insight into the structure-function relation of KCNQ1 channel and treatments of cardiac channelopathies.

A novel missense mutation causing a G487R substitution in the S2-S3 loop of human ether-à-go-go-related gene channel.

INTRODUCTION: Mutations of human ether-à-go-go-related gene (hERG), which encodes a cardiac K(+) channel responsible for the acceleration of the repolarizing phase of an action potential and the prevention of premature action potential regeneration, often cause severe arrhythmic disorders. We found a novel missense mutation of hERG that results in a G487R substitution in the S2-S3 loop of the channel subunit [hERG(G487R)] from a family and determined whether this mutant gene could induce an abnormality in channel function. METHODS AND RESULTS: We made whole-cell voltage-clamp recordings from HEK-293T cells transfected with wild-type hERG [hERG(WT)], hERG(G487R), or both. We measured hERG channel-mediated current as the "tail" of a depolarization-elicited current. The current density of the tail current and its voltage- and time-dependences were not different among all the cell groups. The time-courses of deactivation, inactivation, and recovery from inactivation and their voltage-dependences were not different among all the cell groups. Furthermore, we performed immunocytochemical analysis using an anti-hERG subunit antibody. The ratio of the immunoreactivity of the plasma membrane to that of the cytoplasm was not different between cells transfected with hERG(WT), hERG(G487R), or both. CONCLUSION: hERG(G487R) can produce functional channels with normal gating kinetics and cell-surface expression efficiency with or without the aid of hERG(WT). Therefore, neither the heterozygous nor homozygous inheritance of hERG(G487R) is thought to cause severe cardiac disorders. hERG(G487R) would be a candidate for a rare variant or polymorphism of hERG with an amino acid substitution in the unusual region of the channel subunit.

A functional neuron maturation device provides convenient application on microelectrode array for neural network measurement.

BACKGROUND: Microelectrode array (MEA) systems are valuable for in vitro assessment of neurotoxicity and drug efficiency. However, several difficulties such as protracted functional maturation and high experimental costs hinder the use of MEA analysis requiring human induced pluripotent stem cells (hiPSCs). Neural network functional parameters are also needed for in vitro to in vivo extrapolation. METHODS: In the present study, we produced a cost effective nanofiber culture platform, the SCAD device, for long-term culture of hiPSC-derived neurons and primary peripheral neurons. The notable advantage of SCAD device is convenient application on multiple MEA systems for neuron functional analysis. RESULTS: We showed that the SCAD device could promote functional maturation of cultured hiPSC-derived neurons, and neurons responded appropriately to convulsant agents. Furthermore, we successfully analyzed parameters for in vitro to in vivo extrapolation, i.e., low-frequency components and synaptic propagation velocity of the signal, potentially reflecting neural network functions from neurons cultured on SCAD device. Finally, we measured the axonal conduction velocity of peripheral neurons. CONCLUSIONS: Neurons cultured on SCAD devices might constitute a reliable in vitro platform to investigate neuron functions, drug efficacy and toxicity, and neuropathological mechanisms by MEA.

Identification of antigen-specific B cells by concurrent monitoring of intracellular Ca2+ mobilization and antigen binding with microwell array chip system equipped with a CCD imager.

B cells are very heterogeneous, consisting of more than 10(9) B-cell clones with distinct specificities for antigens in each individual. To identify single B cells with antigen specificity, we have been developing cell microarray technology using microwell array chips whose microwells each capture a single B cell. Using microwell array chips, we detected antigen-specific B cells by monitoring antigen-induced intracellular Ca2+ mobilization with a CCD scanner (MAC-CCD system) or the binding of fluorescence-labeled antigen to cells with a confocal laser scanner. We retrieved target cells from the chip, cloned immunoglobulin genes, and produced antigen-specific antibodies. However, these methods present some difficulties: the former technique could not detect cells whose frequency was less than 0.05% and the latter one took a long time to identify the objective cells although it could detect cells at a frequency of 0.01%. Here, we have combined the advantages of these two methods. Monitoring antigen-induced intracellular Ca2+ mobilizations and the binding of fluorescence-labeled antigens simultaneously with a MAC-CCD system enabled us to detect rapidly, antigen-specific B cells whose frequency was less than 0.01% with high efficiency. Our system provides a superior screening system for antigen-specific B cells and extends the horizons of multiparameter single-cell analysis in heterogeneous cell populations.

Lack of modulatory effect of the SCN5A R1193Q polymorphism on cardiac fast Na+ current at body temperature.

SCN5A encodes the main subunit of the NaV1.5 channel, which mediates the fast Na+ current responsible for generating cardiac action potentials. The single nucleotide polymorphism SCN5A(R1193Q), which results in an amino acid replacement in the subunit, is common in East Asia. SCN5A(R1193Q) is often identified in patients with type 3 long QT syndrome and Brugada syndrome. However, its linkage to arrhythmic disorders is under debate. Previous electrophysiological studies performed at room temperature inconsistently reported the gain- or loss-of-function effect of SCN5A(R1193Q) on the NaV1.5 channel. More recently, it was theoretically predicted that SCN5A(R1193Q) would exert a loss-of-function effect at body temperature. Here, we experimentally assessed whether SCN5A(R1193Q) modulates the NaV1.5 channel at various temperatures including normal and febrile body temperatures. We compared voltage-gated Na+ currents in SCN5A(R1193Q)-transfected and wild-type SCN5A-transfected HEK293T cells using a whole-cell voltage-clamp technique. First, we made comparisons at constant temperatures of 25°C, 36.5°C, and 38°C, and found no difference in the conductance density, voltage dependence of gating, or time dependence of gating. This suggested that SCN5A(R1193Q) does not modulate the NaV1.5 channel regardless of temperature. Second, we made comparisons while varying the temperature from 38°C to 26°C in 3 min, and again observed no difference in the time course of the amplitude or time dependence of gating during the temperature change. This also indicated that SCN5A(R1193Q) does not modulate the NaV1.5 channel in response to an acute body temperature change. Therefore, SCN5A(R1193Q) may not be a monogenic factor that triggers arrhythmic disorders.

MicroRNA-145-5p and microRNA-320a encapsulated in endothelial microparticles contribute to the progression of vasculitis in acute Kawasaki Disease.

Kawasaki Disease (KD) is an acute inflammatory disease that takes the form of systemic vasculitis. Endothelial microparticles (EMPs) have been recognized as an important transcellular delivery system. We hypothesized whether EMPs are involved in vasculitis in acute KD. Fifty patients with acute KD were enrolled, divided into two subgroups: those with coronary artery lesions (CAL) (n = 5) and those without CAL (NCAL) (n = 45). EMPs were measured using flow cytometry, and microRNA (miR) expression profiling was performed by microRNA array. The percentage of EMPs in acute KD was significantly higher than in controls (P < 0.0001). EMPs in patients with CAL rapidly increased after the initial treatment, and was significantly higher than those in NCAL (P < 0.001). In patients with CAL, we identified 2 specific miRs encapsulated in EMPs, hsa-miR-145-5p and hsa-miR-320a, which are predicted to affect monocyte function using in silico analysis, and were demonstrated to upregulate inflammatory cytokine mRNAs in THP-1 monocytes. In situ hybridization confirmed that hsa-miR-145-5p was preferentially expressed in CAL. EMPs may serve as a sensitive marker for the severity of vasculitis in acute KD. Moreover, these 2 specific miRs encapsulated in EMPs might be involved in inflammatory cytokine regulation and the pathogenesis of vasculitis in acute KD.

Ternary complex formation of pVHL, elongin B and elongin C visualized in living cells by a fluorescence resonance energy transfer-fluorescence lifetime imaging microscopy technique.

The tumor suppressor von Hippel-Lindau (VHL) gene product forms a complex with elongin B and elongin C, and acts as a recognition subunit of a ubiquitin E3 ligase. Interactions between components in the complex were investigated in living cells by fluorescence resonance energy transfer (FRET)-fluorescence lifetime imaging microscopy (FLIM). Elongin B-cerulean or cerulean-elongin B was coexpressed with elongin C-citrine or citrine-elongin C in CHO-K1 cells. FRET signals were examined by measuring a change in the fluorescence lifetime of donors and by monitoring a corresponding fluorescence rise of acceptors. Clear FRET signals between elongin B and elongin C were observed in all combinations, except for the combination of elongin B-cerulean and citrine-elongin C. Although similar experiments to examine interaction between pVHL30 and elongin C linked to cerulean or citrine were performed, FRET signals were rarely observed among all the combinations. However, the signal was greatly increased by coexpression of elongin B. These results, together with results of coimmunoprecipitation experiment using pVHL, elongin C and elongin B, suggest that a conformational change of elongin C and/or pVHL was induced by binding of elongin B. The conformational change of elongin C was investigated by measuring changes in the intramolecular FRET signal of elongin C linked to cerulean and citrine at its N- and C-terminus, respectively. A strong FRET signal was observed in the absence of elongin B, and this signal was modestly increased by coexpression of elongin B, demonstrating that a conformation change of elongin C was induced by the binding of elongin B.

An Autopsy Case of Sudden Unexpected Death of a Young Adult in a Hot Bath: Molecular Analysis Using Next-Generation DNA Sequencing.

We report a case of sudden unexpected death of a young woman who was found in a bathtub of hot water. The autopsy concluded that all possible causes of sudden loss of consciousness, except cardiac origin, could be excluded. However, the heart did not show any obvious pathological changes. We used next-generation DNA sequencing (NGS) to examine 73 genes and detected 3 rare, potentially pathogenic variants with minor allele frequencies ⩽1.0%. The pathogenicity of these variants was evaluated using 8 in silico predictive algorithms, and SCN5A_p.Gly289Ser, CACNB2_p.Ser502Leu, and MYH11_p.Lys1573Glu were detected as possible pathogenic variants. Inherited heart disease is a likely cause of sudden unexpected deaths of young people in hot baths, even before the clinical manifestation of the disease. In the future, molecular analysis by NGS may help to predict young to early middle-aged people who could be at risk of sudden arrhythmogenic fatality in hot baths.