Ultrastructural analysis of whole glomeruli using array tomography.

The renal glomerulus produces primary urine from blood plasma by ultrafiltration. The ultrastructure of the glomerulus is closely related to filtration function and disease development. The ultrastructure of glomeruli has mainly been evaluated using transmission electron microscopy; however, the volume that can be observed using transmission electron microscopy is extremely limited relative to the total volume of the glomerulus. Consequently, observing structures that exist in only one location in each glomerulus, such as the vascular pole, and evaluating low-density or localized lesions are challenging tasks. Array tomography (AT) is a technique used to analyze the ultrastructure of tissues and cells via scanning electron microscopy of serial sections. In this study, we present an AT workflow that is optimized for observing complete serial sections of the whole glomerulus, and we share several analytical examples that use the optimized AT workflow, demonstrating the usefulness of this approach. Overall, this AT workflow can be a powerful tool for structural and pathological evaluation of the glomerulus. This workflow is also expected to provide new insights into the ultrastructure of the glomerulus and its constituent cells.

The TGN/EE SNARE protein SYP61 and the ubiquitin ligase ATL31 cooperatively regulate plant responses to carbon/nitrogen conditions in Arabidopsis.

Ubiquitination is a post-translational modification involving the reversible attachment of the small protein ubiquitin to a target protein. Ubiquitination is involved in numerous cellular processes, including the membrane trafficking of cargo proteins. However, the ubiquitination of the trafficking machinery components and their involvement in environmental responses are not well understood. Here, we report that the Arabidopsis thaliana trans-Golgi network/early endosome localized SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) protein SYP61 interacts with the transmembrane ubiquitin ligase ATL31, a key regulator of resistance to disrupted carbon (C)/nitrogen/(N)-nutrient conditions. SYP61 is a key component of membrane trafficking in Arabidopsis. The subcellular localization of ATL31 was disrupted in knockdown mutants of SYP61, and the insensitivity of ATL31-overexpressing plants to high C/low N-stress was repressed in these mutants, suggesting that SYP61 and ATL31 cooperatively function in plant responses to nutrient stress. SYP61 is ubiquitinated in plants, and its ubiquitination level is upregulated under low C/high N-nutrient conditions. These findings provide important insights into the ubiquitin signaling and membrane trafficking machinery in plants.

Deubiquitinating enzymes UBP12 and UBP13 stabilize the brassinosteroid receptor BRI1.

Protein ubiquitination is a dynamic and reversible post-translational modification that controls diverse cellular processes in eukaryotes. Ubiquitin-dependent internalization, recycling, and degradation are important mechanisms that regulate the activity and the abundance of plasma membrane (PM)-localized proteins. In plants, although several ubiquitin ligases are implicated in these processes, no deubiquitinating enzymes (DUBs), have been identified that directly remove ubiquitin from membrane proteins and limit their vacuolar degradation. Here, we discover two DUB proteins, UBP12 and UBP13, that directly target the PM-localized brassinosteroid (BR) receptor BR INSENSITIVE1 (BRI1) in Arabidopsis. BRI1 protein abundance is decreased in the ubp12i/ubp13 double mutant that displayed severe growth defects and reduced sensitivity to BRs. UBP13 directly interacts with and effectively removes K63-linked polyubiquitin chains from BRI1, thereby negatively modulating its vacuolar targeting and degradation. Our study reveals that UBP12 and UBP13 play crucial roles in governing BRI1 abundance and BR signaling activity to regulate plant growth.

Low nitrogen conditions accelerate flowering by modulating the phosphorylation state of FLOWERING BHLH 4 in Arabidopsis.

Nitrogen (N) is an essential nutrient that affects multiple plant developmental processes, including flowering. As flowering requires resources to develop sink tissues for reproduction, nutrient availability is tightly linked to this process. Low N levels accelerate floral transition; however, the molecular mechanisms underlying this response are not well understood. Here, we identify the FLOWERING BHLH 4 (FBH4) transcription factor as a key regulator of N-responsive flowering in Arabidopsis Low N-induced early flowering is compromised in fbh quadruple mutants. We found that FBH4 is a highly phosphorylated protein and that FBH4 phosphorylation levels decrease under low N conditions. In addition, decreased phosphorylation promotes FBH4 nuclear localization and transcriptional activation of the direct target CONSTANS (CO) and downstream florigen FLOWERING LOCUS T (FT) genes. Moreover, we demonstrate that the evolutionarily conserved cellular fuel sensor SNF1-RELATED KINASE 1 (SnRK1), whose kinase activity is down-regulated under low N conditions, directly phosphorylates FBH4. SnRK1 negatively regulates CO and FT transcript levels under high N conditions. Together, these results reveal a mechanism by which N levels may fine-tune FBH4 nuclear localization by adjusting the phosphorylation state to modulate flowering time. In addition to its role in flowering regulation, we also showed that FBH4 was involved in low N-induced up-regulation of nutrient recycling and remobilization-related gene expression. Thus, our findings provide insight into N-responsive growth phase transitions and optimization of plant fitness under nutrient-limited conditions.

TUNEL-positive structures in activated microglia and SQSTM1/p62-positive structures in activated astrocytes in the neurodegenerative brain of a CLN10 mouse model.

Neuronal ceroid lipofuscinosis is a group of pediatric neurodegenerative diseases. One of their causative genes, CLN10/CtsD, encodes cathepsin D, a major lysosomal protease. Central nervous system (CNS)-specific CtsD-deficient mice exhibit a neurodegenerative disease phenotype with accumulation of ceroid lipofuscins, granular osmiophilic deposits, and SQSTM1/p62. We focused on activated astrocytes and microglia in this neurodegenerative mouse brain, since there are few studies on the relationship between these accumulators and lysosomes in these glial cells. Activated microglia and astrocytes in this mouse thalamus at p24 were increased by approximately 2.5- and 4.6-fold compared with the control, while neurons were decreased by approximately half. Granular osmiophilic deposits were detected in microglial cell bodies and extended their processes in the thalamus. LAMP1-positive lysosomes, but not SQSTM1/p62 aggregates, accumulated in microglia of this mouse thalamus, whereas both lysosomes and SQSTM1/p62 aggregates accumulated in its astrocytes. TUNEL-positive signals were observed mainly in microglia, but few were observed in neurons and astrocytes. These signals were fragmented DNA from degenerated neurons engulfed by microglia or in the lysosomes of microglia. Abnormal autophagic vacuoles also accumulated in the lysosomes of microglia. Granular osmiophilic deposit-like structures localized to LAMP1-positive lysosomes in CtsD-deficient astrocytes. SQSTM1/p62-positive but LAMP1-negative membranous structures also accumulated in the astrocytes and were less condensed than typical granular osmiophilic deposits. These results suggest that CtsD deficiency leads to intracellular abnormalities in activated microglia and astrocytes in addition to neuronal degeneration.

Homeostatic p62 levels and inclusion body formation in CHCHD2 knockout mice.

Inactivation of constitutive autophagy results in the formation of cytoplasmic inclusions in neurones, but the relationship between impaired autophagy and Lewy bodies (LBs) remains unknown. α-Synuclein and p62, components of LBs, are the defining characteristic of Parkinson's disease (PD). Until now, we have analyzed mice models and demonstrated p62 aggregates derived from an autophagic defect might serve as 'seeds' and can potentially be a cause of LB formation. P62 may be the key molecule for aggregate formation. To understand the mechanisms of LBs, we analyzed p62 homeostasis and inclusion formation using PD model mice. In PARK22-linked PD, intrinsically disordered mutant CHCHD2 initiates Lewy pathology. To determine the function of CHCHD2 for inclusions formation, we generated Chchd2-knockout (KO) mice and characterized the age-related pathological and motor phenotypes. Chchd2 KO mice exhibited p62 inclusion formation and dopaminergic neuronal loss in an age-dependent manner. These changes were associated with a reduction in mitochondria complex activity and abrogation of inner mitochondria structure. In particular, the OPA1 proteins, which regulate fusion of mitochondrial inner membranes, were immature in the mitochondria of CHCHD2-deficient mice. CHCHD2 regulates mitochondrial morphology and p62 homeostasis by controlling the level of OPA1. Our findings highlight the unexpected role of the homeostatic level of p62, which is regulated by a non-autophagic system, in controlling intracellular inclusion body formation, and indicate that the pathologic processes associated with the mitochondrial proteolytic system are crucial for loss of DA neurones.

Comparison of three different approaches to very high-power short-duration ablation using the QDOT-MICRO catheter.

BACKGROUND/OBJECTIVES: The QDOT-MICRO™ catheter allows very high-power and short-duration (vHPSD) ablation. This study aimed to investigate lesion characteristics using different ablation settings. METHODS: Radiofrequency applications (90 W/4 s, temperature-control mode with 55°C or 60°C target) were performed in excised porcine myocardium using three different approaches: single (SA), double nonrepetitive (DNRA), and double repetitive applications (DRA). Applications were performed with an interval of 1 min for DNRA, and without interval for DRA. RESULTS: A total of 480 lesions were analyzed. Lesion depth and volume were largest for DRA followed by DNRA and SA regardless of catheter direction (depth: 3.8 vs. 3.3 vs. 2.6 mm, p < .001 for all comparisons; volume: 176.6 vs. 145.1 vs. 97.0 mm3 , p < .001 for all comparisons). Surface area was significantly larger for DRA than for SA (45.1 vs. 38.3 mm2 , p < .001) and larger for DNRA than for SA (44.5 vs. 38.3 mm2 , p < .001), but was similar between DRA and DNRA (45.1 vs. 44.5 mm2 , p = .54). Steam-pops more frequently occurred for DRA than for SA (15.6% vs. 4.4%, p = .004) and DNRA (15.6% vs. 6.9%, p = .061), but the incidence was similar between SA and DNRA (4.4% vs. 6.9%, p = 1). Although surface area and lesion volume were larger in lesions with steam-pops than without steam-pops (46.5 vs. 38.1 mm2 , p = .018 and 128.3 vs. 96.8 mm3 , p = .068, respectively), lesions were not deeper (pop(+): 2.5 mm vs. pop(-): 2.6 mm, p = .75). CONCLUSIONS: DNRA produces larger lesions than SA without increasing the risk of steam-pops. DRA produces the largest lesions among the three groups, but with an increased risk of steam-pops. Even with steam-pops, lesions do not become deeper in vHPSD ablation.

Comparison of lesion characteristics using temperature-flow-controlled versus conventional power-controlled ablation with fixed ablation index.

INTRODUCTION: The QDOT-MicroTM catheter is a novel irrigated contact force (CF) sensing catheter which benefits from thermocouples for temperature monitoring, allowing temperature-flow-controlled (TFC) ablation. We compared lesion metrics at fixed ablation index (AI) value during TFC-ablation and conventional power-controlled (PC)-ablation. METHODS: A total of 480 RF-applications were performed on ex-vivo swine myocardium with predefined AI targets (400/550) or until steam-pop occurred, using the QDOT-MicroTM (TFC-ablation) and Thermocool SmartTouch SFTM (PC-ablation). RESULTS: Both TFC-ablation and PC-ablation produced similar lesions in volume (218 ± 116 vs. 212 ± 107 mm3 , p = .65); however, lesions using TFC-ablation were larger in surface area (41.3 ± 8.8 vs. 34.8 ± 8.0 mm2 , p < .001) and shallower in depth (4.0 ± 1.0 vs. 4.2 ± 1.1 mm, p = .044). Average power tended to be lower in TFC-alation (34.2 ± 8.6 vs. 36.9 ± 9.2, p = .005) compared to PC-ablation due to automatic regulation of temperature and irrigation-flow. Although steam-pops were less frequent in TFC-ablation (24% vs. 15%, p = .021), they were particularly observed in low-CF (10 g) and high-power ablation (50 W) in both PC-ablation (n = 24/240, 10.0%) and TFC-ablation (n = 23/240, 9.6%). Multivariate analysis revealed that high-power, low-CF, long application time, perpendicular catheter orientation, and PC-ablation were risk factors for steam-pops. Furthermore, activation of automatic regulation of temperature and irrigation-flow was independently associated with high-CF and long application time while ablation power had no significant relationship. CONCLUSIONS: With a fixed target AI, TFC-ablation reduced the risk of steam-pops, producing similar lesions in volume, but with different metrics in this ex-vivo study. However, lower CF and higher power in fixed-AI ablation may increase the risk of steam-pops.

Impact of baseline pool impedance on lesion metrics and steam pops in catheter ablation.

INTRODUCTION: Little is known about the impact of blood-pool local impedance (LI) on lesion characteristics and the incidence of steam pops. METHODS: Radiofrequency applications at a range of powers (30, 40, and 50 W), contact forces (CF) (5, 15, and 25 g), and durations (15, 30, 45, and 120 s) using perpendicular/parallel catheter orientation were performed in 40 excised porcine preparations, using a catheter capable of monitoring LI (StablePoint©, Boston Scientific). To simulate the variability in blood-pool impedance, the saline-pool LI was modulated by calibrating saline concentrations. Lesion characteristics were compared under three values of saline-pool LI: 120, 160, and 200 Ω. RESULTS: Of 648 lesions created, steam pops occurred in 175 (27.0%). When power, CF, time, and catheter orientation were adjusted, ablation at a saline-pool impedance of 160 or 200 Ω more than doubled the risk of steam pops compared with a saline-pool impedance of 120 Ω (Odds ratio = 2.31; p = .0002). Lesions in a saline-pool impedance of 120 Ω were significantly larger in surface area (50 [38-62], 45 [34-56], and 41 [34-60] mm2 for 120, 160, and 200 Ω, p < .05), but shallower in depth (4.0 [3-5], 4.4 [3.2-5.3], and 4.5 [3.8-5.5] mmfor 120, 160, and 200 Ω, respectively, p < .05) compared with the other two settings. The correlation between the absolute LI-drop and lesion size weakened as the saline-pool LI became higher (e.g., 120 Ω group (r2 = .30, r2 = .18, and r2 = .16, respectively for 120, 160, and 200 Ω), but the usage of %LI-drop (= absolute LI-drop/initial LI) instead of absolute LI-drop may minimize this effect. CONCLUSIONS: In an experimental model, baseline saline-pool impedance significantly affects the lesion metrics and the risk of steam pops.

Superiority of the Combination of Input and Output Parameters to the Single Parameter for Lesion Size Estimation.

BACKGROUND: For lesion size prediction, each input parameter, including ablation energy (AE), and output parameter, such as impedance, is individually used. We hypothesize that using both parameters simultaneously may be more optimal.Methods and Results: Radiofrequency applications at a range of power (30-50 W), contact force (10 g and 20 g), duration (10-60 s), and catheter orientation with normal saline (NS)- or half-normal saline (HNS)-irrigation were performed in excised porcine hearts. The correlations, with lesion size of AE, absolute impedance drop (∆Imp-drop), relative impedance drop (%Imp-drop), and AE*%Imp-drop were examined. Lesion size was analyzed in 283 of 288 lesions (NS-irrigation, n=142; HNS-irrigation, n=141) without steam pops. AE*%Imp-drop consistently showed the strongest correlations with lesion maximum depth (NS-irrigation, ρ=0.91; HNS-irrigation, ρ=0.94), surface area (NS-irrigation, ρ=0.87; HNS-irrigation, ρ=0.86), and volume (NS-irrigation, ρ=0.94; HNS-irrigation, ρ=0.94) compared with the other parameters. Moreover, compared with AE alone, AE*%Imp-drop significantly improved the strength of correlation with lesion maximum depth (AE vs. AE*%Imp-drop, ρ=0.83 vs. 0.91, P<0.01), surface area (ρ=0.73 vs. 0.87, P<0.01), and volume (ρ=0.84 vs. 0.94, P<0.01) with NS-irrigation. This tendency was also observed with HNS-irrigation. Parallel catheter orientation showed a better correlation with lesion depth and volume using ∆Imp-drop, %Imp-drop, and AE*%Imp-drop than perpendicular orientation. CONCLUSIONS: The combination of input and output parameters is more optimal than each single parameter for lesion prediction.

Safety verification of a novel irrigation catheter with flexible tip of laser-cut kerfs and contact force sensor.

BACKGROUND AND AIMS: The safety evaluation of TactiFlex, a novel contact-force sensing catheter with a flexible 4-mm tip irrigated through laser-cut kerfs, has been ongoing. This study aimed to verify the safety of this type of catheter. METHODS: Study 1: Radiofrequency (RF) applications at a range of powers (30-50 W), contact forces (10-20 g), and durations (10-60 s) using perpendicular/parallel catheter orientation with half-normal (HNS) or normal saline irrigation were compared between TactiFlex (4-mm tip) and TactiCath (3.5-mm tip) with temperature-controlled mode in excised porcine hearts. Study 2: The relation between RF applications using TactiFlex and the incidence of steam-pops in the real clinical cases were examined. RESULTS: Study-1: 576 RF lesions were examined. TactiFlex demonstrated a significantly lower risk of steam-pops (5[1.7%] vs. 59[20.5%], p < .0001). Compared to 3.5-mm-tip catheter (TactiCath), 4-mm-tip catheter (TactiFlex) produced smaller lesion volume at perpendicular (193[98-554]mm3 vs. 263[139-436]mm3 , p < .0001), but relatively similar lesion volume at parallel contact (243[105-443]mm3 vs. 278[180-440]mm3 , p = .06). HNS-irrigation tended to increase the lesion volume in both catheters and to increase the incidence of steam-pops with TactiCath, but not with TactiFlex. The cut-off value of %impedance-drop ( = absolute impedance-drop/initial impedance) of 20% predicted steam-pops with a sensitivity = 100% and specificity = 89.6% in TactiFlex. Study-2: 5496 RF applications in 84 patients (51AFs/8ATs/3AVNRTs/4AVRTs/17PVCs/4VTs) using TactiFlex were analyzed. Four steam-pops (0.07%) in three patients with pericardial effusion were observed (%impedance-drop = 24%/26%/29%/35%, respectively). The cut-off value of %impedance-drop = 20%, derived from ex-vivo study, showed sensitivity = 100% and specificity = 90.1% in detecting steam-pops. CONCLUSION: TactiFlex reduced the risk of steam-pops than TactiCath. %impedance-drop ≤ 20% may be reasonable for safely use with a sufficient safety margin. For 4-mm-tip catheter, parallel-contact may be recommended for larger lesion creation.

Deubiquitinating enzymes UBP12 and UBP13 regulate carbon/nitrogen-nutrient stress responses by interacting with the membrane-localized ubiquitin ligase ATL31 in Arabidopsis.

Ubiquitination is an important post-translational modification that regulates multiple cellular activities in plants including environmental stress responses. In addition to activity of ubiquitin ligases, the activity of deubiquitinating enzymes (DUBs) is critical for modulating the optimal ubiquitination status of target proteins in response to environmental stimuli. However, while several ubiquitin ligases have been isolated to date, little is known about the DUBs involved in plant stress responses. Here, we report that two DUBs, UBP12 and UBP13, function in response to disrupted carbon (C)/nitrogen (N)-nutrient stress conditions in Arabidopsis. Knockdown of UBP12 and UBP13 expression resulted in hypersensitivity to high C/low N-nutrient stress conditions, whereas overexpression of UBP13 reduced the sensitivity. Additionally, UBP13 physically interacted with and deubiquitinated the ubiquitin ligase ATL31, a key regulator of plant resistance to high C/low N-nutrient stress conditions. Genetic analysis showed that the loss of ATL31 and its homolog ATL6 suppressed the high C/low N-hyposensitivity of UBP13-overexpressing plants, suggesting that ATL31 is epistatic to UBP12 and UBP13. Taken together, our results suggest that the DUBs UBP12 and UBP13 function together with the ubiquitin ligase ATL31 to mediate C/N-nutrient stress responses in plants.

Pulmonary vein narrowing after visually guided laser balloon ablation: Occurrence and clinical correlates.

INTRODUCTION: There are limited data focusing on pulmonary vein (PV) narrowing following ablation using a visually guided laser balloon (VGLB). We sought to assess the frequency and predictors of PV narrowing after VGLB ablation. METHODS AND RESULTS: Patients with paroxysmal atrial fibrillation treated with VGLB were screened. Study participants underwent contrast-enhanced computed tomography (CT) scanning before and 6 months after the procedure. We defined 25% to 49%, 50% to 74%, and 75% to 100% reduction in PV cross-sectional area as mild, moderate, and severe narrowing, respectively. Of 146 PVs in 38 patients analyzed, severe narrowing developed in two right superior and one right inferior PV. Moderate or severe narrowing occurred in 40 veins (27% of all PVs, 50% of the right superior, 22% of the right inferior, 21% of the left superior, and 14% of the left inferior PV). In PVs with moderate-severe narrowing, the baseline orifice area was significantly larger (4.1 [interquartile range, 3.2-4.8] vs 2.5 [1.9-3.3] cm2 , P < .0001), the narrowest region of stenosis was significantly more distal into the vessel (1.9 [0.7-2.9] vs 0 [0-1.7] mm from the orifice, P = .0006) and the total amount of energy delivered per vein was significantly greater (5190 ± 970 vs 4626 ± 1573 J, P = .018) than in PVs with mild or no significant narrowing. The baseline orifice area independently predicted moderate-severe narrowing in multivariate analysis (odds ratio, 1.8 [1.3-2.5] per 1 cm2 increase, P = .0003). No patient exhibited any signs or symptoms of PV stenosis. CONCLUSIONS: Baseline PV orifice area, ablating distally inside the veins, and total amount of laser energy are associated with PV narrowing after VGLB ablation.

Clinical outcome of pulmonary vein isolation alone ablation strategy using VISITAG SURPOINT in nonparoxysmal atrial fibrillation.

BACKGROUND: Recent studies have shown the improvement in long-term effectiveness with standardized pulmonary vein isolation (PVI) aimed at creating durable and contiguous lesions with VISITAG SURPOINT (VS) in paroxysmal atrial fibrillation (PAF). OBJECTIVE: We aimed to assess efficacy of PVI alone strategy using VS in non-PAF patients and evaluate factors associated with corresponding clinical outcomes. METHODS: Consecutive patients who underwent PVI for persistent/long-standing persistent AF between May 2017 to July 2019 were studied retrospectively. PVI was performed with 30-50 W guided by VS (posterior target: 400-500, anterior target: 500). Left atrial voltage maps were created during atrial pacing after PVI. RESULTS: A total of 140 patients (119 males, age 62 ± 10 years, long-standing persistent AF: 35) were included and followed for median of 454 days. No adverse events were reported in any patients during periprocedural and follow-up period of up to 28 months. Kaplan-Meier analysis estimated that freedom from atrial tachycardia or AF (AT/AF) without antiarrhythmics at 1-year was 70%. Radiofrequency delivery with higher power was associated with increased first-pass isolation rate, but not with freedom from AT/AF. In multivariate analysis, long-standing persistent AF and % low-voltage zone (%LVZ) were independent predictors of clinical outcome. The best cut-off value of %LVZ for predicting AT/AF recurrence was 3.24%. Freedom from AT/AF was 88% in patients with persistent AF and %LVZ < 3.24%, while 27% in those with long-standing persistent AF and %LVZ ≥ 3.24%. CONCLUSIONS: PVI alone using VS was associated with excellent 1-year success in patients with persistent AF and %LVZ < 3.24%, but was insufficient in those with long-standing persistent AF and/or %LVZ ≥ 3.24%.

Long-Term Performance of Right Ventricular Implantable Cardioverter-Defibrillator Leads in Arrhythmogenic Right Ventricular Cardiomyopathy and Hypertrophic Cardiomyopathy.

Patients with arrhythmogenic right ventricular cardiomyopathy (ARVC) and hypertrophic cardiomyopathy (HCM) implanted with implantable cardioverter-defibrillators (ICDs) may show a large decrease in R-wave amplitude during long-term follow-up. However, it is unclear whether this decrease is higher in these patients than in those without structural heart disease. This study investigated ICD-lead intracardiac parameters over a long duration in patients with ARVC and HCM and compared these parameters with those of a control group. We included 50 patients (mean age, 55.2 ± 17.2 years; 26% female) with ICD leads in the right ventricular apex, and compared 7 ARVC and 14 HCM patients with 29 control patients without structural heart disease. ICD-lead parameters, including R-wave amplitude, pacing threshold, and impedance during follow-up, were compared. The difference in these parameters between the time of implantation and year 5 were also compared. There were no significant differences in R-wave amplitude at implantation among the 3 groups. The change in R-wave amplitude between the time of implantation and year 5 was significantly greater in the ARVC group (-3.3 ± 5.4 mV, P = 0.012) in comparison to the control group (1.3 ± 2.8 mV); the HCM group showed no significant difference (-0.4 ± 2.3 mV, P = 0.06). Thus, in the ARVC group, R-wave amplitude at year 5 was significantly lower than that in the control group (5.7 ± 4.8 mV versus 12.5 ± 4.5 mV, P = 0.001). In ARVC patients with ICDs, ventricular sensing is likely to deteriorate during long-term follow-up; however, in HCM patients, sensing may not deteriorate.

Identification of Arabidopsis CCR4-NOT Complexes with Pumilio RNA-Binding Proteins, APUM5 and APUM2.

CCR4/CAF1 are widely conserved deadenylases in eukaryotes. They form a large complex that includes NOT1 as a scaffold protein and various NOT proteins that are core components of multiple levels of gene expression control. The CCR4-NOT complex also contains several RNA-binding proteins as accessory proteins, which are required for target recognition by CCR4/CAF1 deadenylases. AtCCR4a/b, orthologs of human CCR4 in Arabidopsis, have various physiological effects. AtCCR4 isoforms are likely to have specific target mRNAs related to each physiological effect; however, AtCCR4 does not have RNA-binding capability. Therefore, identifying factors that interact with AtCCR4a/b is indispensable to understand its function as a regulator of gene expression, as well as the target mRNA recognition mechanism. Here, we identified putative components of the AtCCR4-NOT complex using co-immunoprecipitation in combination with mass spectrometry using FLAG-tagged AtCCR4b and subsequent verification with a yeast two-hybrid assay. Interestingly, four of 11 AtCAF1 isoforms interacted with both AtCCR4b and AtNOT1, whereas two isoforms interacted only with AtNOT1 in yeast two-hybrid assays. These results imply that Arabidopsis has multiple CCR4-NOT complexes with various combinations of deadenylases. We also revealed that the RNA-binding protein Arabidopsis Pumilio 5 and 2 interacted with AtCCR4a/b in the cytoplasm with a few foci.

Nitrate Reductase Modulation in Response to Changes in C/N Balance and Nitrogen Source in Arabidopsis.

Environmental cues modulate the balance of carbon (C) and nitrogen (N) which are essential elements for plant metabolism and growth. In Arabidopsis, photochemical efficiency of PSII, phosphorylation status and localization of many enzymes, and the level of total soluble sugars were affected by an unbalanced C/N ratio. Since differences in C/N affect these parameters, here we checked whether different sources of N have different effects when a high C/N ratio is imposed. NO3- and NH4+ were separately provided in C/N medium. We investigated the effects on photochemical efficiency of PSII, the level of total soluble sugars and nitrate reductase activity under stressful C/N conditions compared with control conditions. We found that treated plants accumulated more total soluble sugars when compared with control. Photochemical efficiency of PSII did not show significant differences between the two sources of nitrogen after 24 h. The actual nitrate reductase activity was the result of a combination of activity, activation state and protein level. This activity constantly decreased starting from time zero in control conditions; in contrast, the actual nitrate reductase activity showed a peak at 2 h after treatment with NO3-, and at 30 min with NH4+. This, according to the level of total soluble sugars, can be explained by the existence of a cross-talk between the sugars in excess and low nitrate in the medium that blocks the activity of nitrate reductase in stressful sugar conditions until the plant is adapted to the stress.

Purkinje Cells Are More Vulnerable to the Specific Depletion of Cathepsin D Than to That of Atg7.

Neurologic phenotypes of cathepsin D (CTSD)-deficient mice, a murine model of neuronal ceroid lipofuscinoses, indicate the importance of CTSD for the maintenance of metabolism in central nervous system neurons. To further understand the role of CTSD in central nervous system neurons, we generated mice with a CTSD deficiency specifically in the Purkinje cells (PCs) (CTSDFlox/Flox;GRID2-Cre) and compared their phenotypes with those of PC-selective Atg7-deficient (Atg7Flox/Flox;GRID2-Cre) mice. In both strains of mice, PCs underwent degeneration, but the CTSD-deficient PCs disappeared more rapidly than their Atg7-deficient counterparts. When CTSD-deficient PCs died, the neuronal cell bodies became shrunken, filled with autophagosomes and autolysosomes, and had nuclei with dispersed small chromatin fragments. The dying Atg7-deficient PCs also showed similar ultrastructures, indicating that the neuronal cell death of CTSD- and Atg7-deficient PCs was distinct from apoptosis. Immunohistochemical observations showed the formation of calbindin-positive axonal spheroids and the swelling of vesicular GABA transporter-positive presynaptic terminals that were more pronounced in Atg7-deficient PCs than in CTSD-deficient PCs. An accumulation of tubular vesicles may have derived from the smooth endoplasmic reticulum; nascent autophagosome-like structures with double membranes was a common feature in the swollen axons of these PCs. These results suggested that PCs were more vulnerable to CTSD deficiency in lysosomes than to autophagy impairment, and this vulnerability does not depend on the severity of axonal swelling.

Small GTPase Rab1B is associated with ATG9A vesicles and regulates autophagosome formation.

ATG9 is a membrane protein that is essential for autophagy and is considered to be directly involved in the early steps of autophagosome formation. Yeast Atg9 is mainly localized to small vesicles (Atg9 vesicles), whereas mammalian ATG9A is reportedly localized to the trans-Golgi network, the endosomal compartment, and other unidentified membrane structures. To dissect the ATG9A-containing membranes, we examined the subcellular localization of ATG9A and performed immunoisolation of those membranes. ATG9A-green fluorescent protein in human culture cells was observed as numerous puncta that move rapidly throughout the cytoplasm. We isolated these cytoplasmic membranes and found that they were small vesicles that resemble the yeast Atg9 vesicle. One of the proteins obtained via proteomic analyses of the mammalian ATG9A vesicle was Rab1, a small GTPase that is essential in endoplasmic reticulum-to-Golgi vesicle trafficking. Knockdown studies of Rab1B showed a suppression of autophagy. In these Rab1B-depleted cells, ATG9A accumulated in intermediate membrane structures at autophagosome formation sites. These results indicate that Rab1B is involved in regulating the proper development of autophagosomes.-Kakuta, S., Yamaguchi, J., Suzuki, C., Sasaki, M., Kazuno, S., Uchiyama, Y. Small GTPase Rab1B is associated with ATG9A vesicles and regulates autophagosome formation.

Atrial fibrillation type matters: greater infarct volume and worse neurological defects seen in acute cardiogenic cerebral embolism due to persistent or permanent rather than paroxysmal atrial fibrillation.

Aims: Some studies have shown that the type of atrial fibrillation (AF), whether paroxysmal AF (PAF) or persistent or permanent AF (PeAF), affects the incidence of ischaemic stroke. This study sought to determine the relationship between the AF pattern and the severity and brain volume of infarction in an AF population including transient ischaemic attack (TIA) patients. Methods and results: This was a retrospective observational study. We studied 161 consecutive patients who were admitted to our stroke care unit with cardiogenic embolism or TIA related to non-valvular AF (age 79 ± 9.5, 78 females, and 87 PAF patients). We evaluated the differences in severity and infarct volume between the types of AF. Additionally, we divided the patients into three groups according to severe stroke (n = 38), TIA (n = 28), and those who were neither (stroke, n = 95) for the assessment of the predictors of severe stroke and TIA. Persistent or permanent atrial fibrillation patients with acute cardiogenic stroke or TIA had worse peak National Institute of Health Stroke Scale (NIHSS) scores [PAF median 4 (range 3-14), PeAF 17 (5.8-25); P < 0.0001] and worse NIHSS scores at discharge [PAF 2.0 (1-7), PeAF 11 (3-22); P < 0.0001]. Their infarct brain volume assessed by computed tomography or magnetic resonance imaging was also larger [PAF 4.4 (1.1-32) mL, PeAF 64 (6.9-170) mL; P < 0.0001]. Multivariate analysis of severe stroke vs. non-severe stroke patients showed that having PeAF was the only independent predictor of severe stroke [odds ratio (OR) 4.27, 95% confidence interval (CI) 1.91-10.2; P = 0.0003]. Comparison of TIA vs. non-TIA patients showed that PeAF (OR 0.120, 95% CI 0.0230-0.444; P = 0.0008) and anticoagulant use (OR 8.24, 95% CI 2.15-40.8; P = 0.0018) were independent predictors of TIA. Conclusion: Cardiogenic emboli due to non-valvular PeAF are associated with a worse acute clinical course and greater volume of infarction than those due to PAF.