GABAB receptors induce phasic release from medial habenula terminals through activity-dependent recruitment of release-ready vesicles.

GABAB receptor (GBR) activation inhibits neurotransmitter release in axon terminals in the brain, except in medial habenula (MHb) terminals, which show robust potentiation. However, mechanisms underlying this enigmatic potentiation remain elusive. Here, we report that GBR activation on MHb terminals induces an activity-dependent transition from a facilitating, tonic to a depressing, phasic neurotransmitter release mode. This transition is accompanied by a 4.1-fold increase in readily releasable vesicle pool (RRP) size and a 3.5-fold increase of docked synaptic vesicles (SVs) at the presynaptic active zone (AZ). Strikingly, the depressing phasic release exhibits looser coupling distance than the tonic release. Furthermore, the tonic and phasic release are selectively affected by deletion of synaptoporin (SPO) and Ca2+-dependent activator protein for secretion 2 (CAPS2), respectively. SPO modulates augmentation, the short-term plasticity associated with tonic release, and CAPS2 retains the increased RRP for initial responses in phasic response trains. The cytosolic protein CAPS2 showed a SV-associated distribution similar to the vesicular transmembrane protein SPO, and they were colocalized in the same terminals. We developed the "Flash and Freeze-fracture" method, and revealed the release of SPO-associated vesicles in both tonic and phasic modes and activity-dependent recruitment of CAPS2 to the AZ during phasic release, which lasted several minutes. Overall, these results indicate that GBR activation translocates CAPS2 to the AZ along with the fusion of CAPS2-associated SVs, contributing to persistency of the RRP increase. Thus, we identified structural and molecular mechanisms underlying tonic and phasic neurotransmitter release and their transition by GBR activation in MHb terminals.

[Creating a Predictive Model of the Contrast Enhancement for Coronary CT Angiography by Using Statistical Analysis and Machine Learning].

The purpose of this study was to calculate statistically significant patient data and test bolus (TB) parameters in order to predict the contrast enhancement of main bolus (eMB) in coronary computed tomography (CT) angiography, and to create a predictive model of eMB with the calculated parameters by machine learning. A total of 126 patients underwent coronary CT angiography. Contrast material was administered at a fixed injection rate and volume. The peak enhancement (PE) and the time needed to reach peak (TP) of the TB were calculated for each patient. The dependency of MB contrast attenuation on these parameters was evaluated. Significant correlations were obtained among PE, TP, and the patient body surface area (BSA) with the eMB. The coefficient of determination of the linear regression model to estimate eMB by machine learning using the above three variables was 0.70 for the training data and 0.55 for the test data. For comparison, the coefficient of determination of the model using only BSA was 0.55 for the training data and 0.36 for the test data; the accuracy of the model created during this time was confirmed.

Variations in Ca2+ Influx Can Alter Chelator-Based Estimates of Ca2+ Channel-Synaptic Vesicle Coupling Distance.

The timing and probability of synaptic vesicle fusion from presynaptic terminals is governed by the distance between voltage-gated Ca2+ channels (VGCCs) and Ca2+ sensors for exocytosis. This VGCC-sensor coupling distance can be determined from the fractional block of vesicular release by exogenous Ca2+ chelators, which depends on biophysical factors that have not been thoroughly explored. Using numerical simulations of Ca2+ reaction and diffusion, as well as vesicular release, we examined the contributions of conductance, density, and open duration of VGCCs, and the influence of endogenous Ca2+ buffers on the inhibition of exocytosis by EGTA. We found that estimates of coupling distance are critically influenced by the duration and amplitude of Ca2+ influx at active zones, but relatively insensitive to variations of mobile endogenous buffer. High concentrations of EGTA strongly inhibit vesicular release in close proximity (20-30 nm) to VGCCs if the flux duration is brief, but have little influence for longer flux durations that saturate the Ca2+ sensor. Therefore, the diversity in presynaptic action potential duration is sufficient to alter EGTA inhibition, resulting in errors potentially as large as 300% if Ca2+ entry durations are not considered when estimating VGCC-sensor coupling distances.SIGNIFICANT STATEMENT The coupling distance between voltage-gated Ca2+ channels and Ca2+ sensors for exocytosis critically determines the timing and probability of neurotransmitter release. Perfusion of presynaptic terminals with the exogenous Ca2+ chelator EGTA has been widely used for both qualitative and quantitative estimates of this distance. However, other presynaptic terminal parameters such as the amplitude and duration of Ca2+ entry can also influence EGTA inhibition of exocytosis, thus confounding conclusions based on EGTA alone. Here, we performed reaction-diffusion simulations of Ca2+-driven synaptic vesicle fusion, which delineate the critical parameters influencing an accurate prediction of coupling distance. Our study provides guidelines for characterizing and understanding how variability in coupling distance across chemical synapses could be estimated accurately.

Ataxic phenotype with altered CaV3.1 channel property in a mouse model for spinocerebellar ataxia 42.

Spinocerebellar ataxia 42 (SCA42) is a neurodegenerative disorder recently shown to be caused by c.5144G > A (p.Arg1715His) mutation in CACNA1G, which encodes the T-type voltage-gated calcium channel CaV3.1. Here, we describe a large Japanese family with SCA42. Postmortem pathological examination revealed severe cerebellar degeneration with prominent Purkinje cell loss without ubiquitin accumulation in an SCA42 patient. To determine whether this mutation causes ataxic symptoms and neurodegeneration, we generated knock-in mice harboring c.5168G > A (p.Arg1723His) mutation in Cacna1g, corresponding to the mutation identified in the SCA42 family. Both heterozygous and homozygous mutants developed an ataxic phenotype from the age of 11-20 weeks and showed Purkinje cell loss at 50 weeks old. Degenerative change of Purkinje cells and atrophic thinning of the molecular layer were conspicuous in homozygous knock-in mice. Electrophysiological analysis of Purkinje cells using acute cerebellar slices from young mice showed that the point mutation altered the voltage dependence of CaV3.1 channel activation and reduced the rebound action potentials after hyperpolarization, although it did not significantly affect the basic properties of synaptic transmission onto Purkinje cells. Finally, we revealed that the resonance of membrane potential of neurons in the inferior olivary nucleus was decreased in knock-in mice, which indicates that p.Arg1723His CaV3.1 mutation affects climbing fiber signaling to Purkinje cells. Altogether, our study shows not only that a point mutation in CACNA1G causes an ataxic phenotype and Purkinje cell degeneration in a mouse model, but also that the electrophysiological abnormalities at an early stage of SCA42 precede Purkinje cell loss.

Ca2+ channel to synaptic vesicle distance accounts for the readily releasable pool kinetics at a functionally mature auditory synapse.

Precise regulation of synaptic vesicle (SV) release at the calyx of Held is critical for auditory processing. At the prehearing calyx of Held, synchronous and asynchronous release is mediated by fast and slow releasing SVs within the readily releasable pool (RRP). However, the posthearing calyx has dramatically different release properties. Whether developmental alterations in RRP properties contribute to the accelerated release time course found in posthearing calyces is not known. To study these questions, we performed paired patch-clamp recordings, deconvolution analysis, and numerical simulations of buffered Ca(2+) diffusion and SV release in postnatal day (P) 16-19 mouse calyces, as their release properties resemble mature calyces of Held. We found the P16-P19 calyx RRP consists of two pools: a fast pool (τ ≤ 0.9 ms) and slow pool (τ ∼4 ms), in which release kinetics and relative composition of the two pools were unaffected by 5 mm EGTA. Simulations of SV release from the RRP revealed that two populations of SVs were necessary to reproduce the experimental release rates: (1) SVs located close (∼5-25 nm) and (2) more distal (25-100 nm) to VGCC clusters. This positional coupling was confirmed by experiments showing 20 mm EGTA preferentially blocked distally coupled SVs. Lowering external [Ca(2+)] to in vivo levels reduced only the fraction SVs released from the fast pool. Therefore, we conclude that a dominant parameter regulating the mature calyx RRP release kinetics is the distance between SVs and VGCC clusters.

[Pixel values of [15O]H2O2 PET images with OSEM algorithm depending on numbers of subset and iteration times: comparative assessment to FBP].

To investigate a potential application of ordered subsets expectation maximization (OSEM) algorithm for clinical [15O]H2O PET studies, region of interest (ROI) measurements were performed on both images with OSEM and filtered back projection (FBP). Forty OSEM images were reconstructed with variable combinations of numbers of the subset (1-40) and iteration times (2-12). PET scans were acquired using a PET/CT scanner (Discovery ST Elite, GE), and 3T-MRI images were obtained for fusion images. The mean values were measured on the frontal cortical regions in the middle cerebral artery distribution. Differences of the values between the OSEM and FBP were evaluated as %Error. Relationship between ROI mean values and the iteration times was investigated on the OSEM images. The smallest %Error 0.4% was measured in the combination of the subset number 10 and iteration times 8 [10, 8], and in that of [28, 2].The mean values were stable with iteration number 8 or more. OSEM image with [28, 2] was reconstructed in a shorter time (2.5 min) than that with [10, 8] (6 min). OSEM image with [28, 2] was superior to that with [10, 8] in the qualitative evaluation. The mean values on OSEM images with [28, 2] were comparable with those on FBP images with little artifacts and higher spatial resolution. OSEM with optimal parameter setting seemed applicable for both quantitative and qualitative [15O]H2O PET studies.

Multimorbidity of Allergic Diseases Is Associated With Functional Gastrointestinal Disorders in a Young Japanese Population.

Background/Aims: Although certain allergic diseases have been reported to be associated with the prevalence of functional dyspepsia (FD) and irritable bowel syndrome (IBS), it is unclear whether the presence of multiple allergic diseases further increases the prevalence of FD and IBS. The aim of this study is to determine this issue in young people. Methods: A cohort of 8923 Japanese university students was enrolled and diagnoses of FD and IBS were confirmed using Rome III criteria. Allergic disorders diagnosed at medical institutions were obtained by means of a self-administered questionnaire. Results: The prevalence of FD, IBS, and their overlap was found to be 1.9%, 6.5%, and 1.1%, respectively. Pollen allergy was independently positively correlated with FD, IBS, and overlap of FD and IBS. Allergic rhinitis was positively linked to IBS. Drug allergy was positively associated with FD. The presence of multiple allergic diseases was positively correlated with FD and IBS (FD: adjusted OR for 2 allergic diseases: 1.95 [95% CI, 1.24-2.98], P for trend = 0.003; and IBS: adjusted OR for 1 allergic disease: 1.40 [95% CI, 1.15-1.69], 2 allergic diseases 1.47 [95% CI, 1.12-1.91], and 3 or more allergic diseases: 2.22 [95% CI, 1.45-3.28], P for trend = 0.001). Additionally, the concomitant existence of multiple allergic diseases was also demonstrated to have a trend that correlated with the overlap of FD and IBS (P for trend = 0.018). Conclusion: Allergic disease multimorbidity is positively correlated with the prevalence of FD and IBS in a young population.

Activation mechanism of melB tyrosinase from Aspergillus oryzae by acidic treatment.

The pro form of recombinant tyrosinase from Aspergillus oryzae (melB) shows no catalytic activity, but acid treatment (around pH 3.5) of protyrosinase activates it to induce tyrosinase activity. Circular dichroism spectra, gel filtration analysis, and colorimetric assay have indicated that acid treatment around pH 3.5 induced the disruption of the conformation of the C-terminal domain covering the enzyme active site. These structural changes induced by the acid treatment may open the entrance to the enzyme active site for substrate incorporation. To compare the mechanism of hydroxylation by the acid-treated tyrosinase with that by trypsin-treated tyrosinase, a detailed steady-state kinetic analysis of the phenolase activity was performed by monitoring the O(2)-consumption rate using a Clark-type oxygen electrode. The results clearly show that the phenolase activity (phenol hydroxylation) of the activated tyrosinase involves an electrophilic aromatic substitution mechanism as in the case of mushroom tyrosinase (Yamazaki and Itoh in J. Am. Chem. Soc. 125:13034-13035, 2003) and activated hemocyanin with urea (Morioka et al. in J. Am. Chem. Soc. 128:6788-6789, 2006).

Multifunctions of MelB, a fungal tyrosinase from Aspergillus oryzae.

The pro form of melB tyrosinase from the melB gene of Aspergillus oryzae was over-produced from E. coli and formed a homodimer that exhibited the spectral features of met-tyrosinase. In the presence of NH(2)OH (reductant), the proenzyme bound dioxygen to give a stable (µ-η(2):η(2) -peroxo)dicopper(II) species (oxy form), thus indicating that the pro form tyrosinase can function as an oxygen carrier or storage protein like hemocyanin. The pro form tyrosinase itself showed no catalytic activity toward external substrates, but proteolytic digestion with trypsin activated it to induce tyrosinase activity. Mass spectroscopy analyses, mutagenesis experiments, and colorimetry assays have demonstrated that the tryptic digestion induced cleavage of the C-terminal domain (Glu458-Ala616), although the dimeric structure of the enzyme was retained. The structural changes induced by proteolytic digestion might open the entrance to the enzyme active site for substrate incorporation.

Serum Biomarkers in a Radiological Pattern of Non-Fibrotic Hypersensitivity Pneumonitis: Implications for Mechanistic Difference and Differential Diagnosis.

Hypersensitivity pneumonitis (HP) is a consequence of immune-mediated reactions caused by recurrent exposure to environmental agents. Recently, clinical practice guidelines for the diagnosis of HP were published and increased interest in HP. On the other hand, novel therapies have recently emerged for various diseases, and the management of drug-related pneumonitis (DRP) has become increasingly important. Among DRP, the HP pattern (DRP-HP) shows small, poorly defined centrilobular nodules with or without widespread areas of ground-glass opacity or lobular areas of decreased attenuation and vascularity. A similar radiological pattern of non-fibrotic HP can be induced, irrespective of inhalation (non-fibrotic HP) or intravenous administration (DRP-HP). However, their difference has not been well described, although the distribution of lesions in the lungs was slightly different between these two conditions. In this review, we focus on serum biomarkers of lung epithelial cells in order to investigate the difference between DRP-HP and non-fibrotic HP (common-HP). Serum levels of Krebs von den Lungen 6 (KL-6) might be relatively lower (occasionally normal) in DRP-HP than in common-HP, implying a mechanistic difference. KL-6 could be useful in discriminating between DRP and non-fibrotic HP (common type).

Post-translational His-Cys cross-linkage formation in tyrosinase induced by copper(II)-peroxo species.

Autocatalytic formation of His-Cys cross-linkage in the enzyme active site of tyrosinase from Aspergillus oryzae has been demonstrated to proceed by the treatment of apoenzyme with Cu(II) under aerobic conditions, where a (µ-η(2):η(2)-peroxo)dicopper(II) species has been suggested to be involved as a key reactive intermediate.

Drug-related pneumonitis with radiographic hypersensitivity pneumonitis pattern: Three case series.

Novel therapies have recently emerged for various diseases, and the management of drug-related pneumonitis (DRP) has become increasingly important. In particular, the hypersensitivity pneumonitis (HP) pattern of DRP has been increasingly recognized due to development of new therapeutic strategies, such as immunotherapy. However, literature describing detailed clinical cases is still lacking. Herein, we report three cases of DRP with typical HP radiographic pattern. These patients were treated with different drugs, namely nano albumin-bound (nab)-paclitaxel, everolimus, or nivolumab, but had common clinical features, including a good prognosis.

Mediastinal undifferentiated pleomorphic sarcoma with pleural effusion cytopathologically misdiagnosed as epithelial malignant pleural mesothelioma: An autopsy case report.

Undifferentiated pleomorphic sarcoma (UPS) is a new disease in the World Health Organization's classification of tumors of soft tissue and bone published in 2013. Primary mediastinal UPS is rare, especially with pleural effusion. Herein, we describe the pathological findings of pleural effusion followed by mediastinal UPS, which was initially misdiagnosed as epithelial malignant pleural mesothelioma (MPM). The cytopathological findings of the pleural effusion cell block often contribute to the diagnosis of various malignant tumors. However, these findings may lead to misdiagnosis of highly invasive mediastinal tumors such as UPS. A biopsy for primary mediastinal lesions should be performed because MPM rarely mimics mediastinal tumors with pleural effusion.

Disruption of the ether-a-go-go K+ channel gene BEC1/KCNH3 enhances cognitive function.

The K+ channel, one of the determinants for neuronal excitability, is genetically heterogeneous, and various K+ channel genes are expressed in the CNS. The therapeutic potential of K+ channel blockers for cognitive enhancement has been discussed, but the contribution each K+ channel gene makes to cognitive function remains obscure. BEC1 (KCNH3) is a member of the K+ channel superfamily that shows forebrain-preferential distribution. Here, we show the critical involvement of BEC1 in cognitive function. BEC1 knock-out mice performed behavioral tasks related to working memory, reference memory, and attention better than their wild-type littermates. Enhanced performance was also observed in heterozygous mutants. The knock-out mice had neither the seizures nor the motor dysfunction that are often observed in K+ channel-deficient mice. In contrast to when it is disrupted, overexpression of BEC1 in the forebrain caused the impaired performance of those tasks. It was also found that altering BEC1 expression could change hippocampal neuronal excitability and synaptic plasticity. The results indicate that BEC1 may represent the first K+ channel that contributes preferentially and bidirectionally to cognitive function.

[Optimization of contrast injection protocol for coronary CTA: adjustment of injection iodine volume on test bolus parameters and patients' sex and BSA].

The purpose of this study was to correlate test bolus (TB) parameters and patient information and cardiac function with main bolus (MB) contrast density for 64-slice computed tomography, and to evaluate MB contrast density on a fixed injection rate, in comparison with an injection rate adjusted with TB parameters and patient information. A total of 256 patients underwent coronary CT angiography. In 126 patients (group 1), contrast material was administered at a flow rate of 4 ml/sec. The peak enhancement, the time needed to reach the peak of the TB and cardiac function of the MB were calculated for each patient in this group. The dependency of MB contrast attenuation on these parameters was evaluated. Significant correlations were obtained between the peak density of the TB and the patient's body surface area (BSA) with the enhancement of MB. Furthermore, females showed a higher peak enhancement of MB than males. In view of the results of group 1, in the other 130 patients (group 2) injection protocols were computed by using regression analysis of each patient's attenuation response to a TB and patient sex and BSA. Compared with group 1, the variations of MB contrast density of group 2 were reduced. Optimized contrast injection protocols are useful to reduce variations of MB contrast density, by taking these TB parameters and patient sex and BSA into account.

EGTA Can Inhibit Vesicular Release in the Nanodomain of Single Ca2+ Channels.

The exogenous Ca2+ chelator EGTA (ethylene glycol tetraacetic acid) has been widely used to probe the coupling distance between Ca2+ channels and vesicular Ca2+ sensors for neurotransmitter release. Because of its slow forward rate for binding, EGTA is thought to not capture calcium ions in very proximity to a channel, whereas it does capture calcium ions at the remote distance. However, in this study, our reaction diffusion simulations (RDSs) of Ca2+ combined with a release calculation using vesicular sensor models indicate that a high concentration of EGTA decreases Ca2+ and vesicular release in the nanodomain of single channels. We found that a key determinant of the effect of EGTA on neurotransmitter release is the saturation of the vesicular sensor. When the sensor is saturated, the reduction in the Ca2+ concentration by EGTA is masked. By contrast, when the sensor is in a linear range, even a small reduction in Ca2+ by EGTA can decrease vesicular release. In proximity to a channel, the vesicular sensor is often saturated for a long voltage step, but not for a brief Ca2+ influx typically evoked by an action potential. Therefore, when EGTA is used as a diagnostic tool to probe the coupling distance, care must be taken regarding the presynaptic Ca2+ entry duration as well as the property of the vesicular Ca2+ sensor.

Underpinning heterogeneity in synaptic transmission by presynaptic ensembles of distinct morphological modules.

Synaptic heterogeneity is widely observed but its underpinnings remain elusive. We addressed this issue using mature calyx of Held synapses whose numbers of bouton-like swellings on stalks of the nerve terminals inversely correlate with release probability (Pr). We examined presynaptic Ca2+ currents and transients, topology of fluorescently tagged knock-in Ca2+ channels, and Ca2+ channel-synaptic vesicle (SV) coupling distance using Ca2+ chelator and inhibitor of septin cytomatrix in morphologically diverse synapses. We found that larger clusters of Ca2+ channels with tighter coupling distance to SVs elevate Pr in stalks, while smaller clusters with looser coupling distance lower Pr in swellings. Septin is a molecular determinant of the differences in coupling distance. Supported by numerical simulations, we propose that varying the ensemble of two morphological modules containing distinct Ca2+ channel-SV topographies diversifies Pr in the terminal, thereby establishing a morpho-functional continuum that expands the coding capacity within a single synapse population.

Two-component latency distributions indicate two-step vesicular release at simple glutamatergic synapses.

It is often assumed that only stably docked synaptic vesicles can fuse following presynaptic action potential stimulation. However, during action potential trains docking sites are increasingly depleted, raising the question of the source of synaptic vesicles during sustained release. We have recently developed methods to reliably measure release latencies during high frequency trains at single synapses between parallel fibers and molecular layer interneurons. The latency distribution exhibits a single fast component at train onset but contains both a fast and a slow component later in the train. The contribution of the slow component increases with stimulation frequency and with release probability and decreases when blocking the docking step with latrunculin. These results suggest that the slow component reflects sequential docking and release in immediate succession. The transition from fast to slow component, as well as a later transition to asynchronous release, appear as successive adaptations of the synapse to maintain fidelity at the expense of time accuracy.

Pulmonary Hemorrhaging as a Fatal Complication of IgA Vasculitis.

A 64-year-old man was admitted to our hospital for purpuric rash, joint pain, and a fever. He had earlier undergone a follow-up examination for interstitial lung disease. At the current visit, the diagnosis was immunoglobulin A (IgA) vasculitis, based on skin and renal biopsy findings. He developed sudden breathlessness and hemoptysis. Chest computed tomography revealed ground glass opacity in the right lower lung fields, suggesting pulmonary hemorrhaging associated with IgA vasculitis. Despite steroid and cyclophosphamide therapy, and plasma exchange, he died 52 days after admission. Early aggressive therapies may be recommended for old patients with IgA vasculitis who have an additional comorbidities.