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Phosphatidylinositol 3-kinase type 2α (PI3KC2α) and related class II PI3K isoforms are of increasing biomedical interest because of their crucial roles in endocytic membrane dynamics, cell division and signaling, angiogenesis, and platelet morphology and function. Herein we report the development and characterization of PhosphatidylInositol Three-kinase Class twO INhibitors (PITCOINs), potent and highly selective small-molecule inhibitors of PI3KC2α catalytic activity. PITCOIN compounds exhibit strong selectivity toward PI3KC2α due to their unique mode of interaction with the ATP-binding site of the enzyme. We demonstrate that acute inhibition of PI3KC2α-mediated synthesis of phosphatidylinositol 3-phosphates by PITCOINs impairs endocytic membrane dynamics and membrane remodeling during platelet-dependent thrombus formation. PITCOINs are potent and selective cell-permeable inhibitors of PI3KC2α function with potential biomedical applications ranging from thrombosis to diabetes and cancer.

Arabidopsis PLDζ1 and PLDζ2 localize to the trans-Golgi network and to compartments including the trans-Golgi network, multi-vesicular bodies, and the tonoplast, respectively, depending on their N-terminal regions containing PX-PH domains. Phospholipase D (PLD) is involved in dynamic cellular processes, including membrane trafficking, cytoskeletal reorganization, and signal transduction for gene expression, through the production of phosphatidic acid in membrane compartments specific to each process. Although PLD plays crucial roles in various plant phenomena, the underlying processes involving PLD for each phenomenon remain largely elusive, partly because the subcellular localization of PLD remains obscure. In this study, we performed comparative subcellular localization analyses of the Arabidopsis thaliana PX-PH-PLDs PLDζ1 and PLDζ2. In mature lateral root cap cells, own promoter-driven fluorescence protein fusions of PLDζ1 localized to the entire trans-Golgi network (TGN) while that of PLDζ2 localized to punctate structures including part of the TGN and multi-vesicular bodies as well as the tonoplast. These localization patterns were reproduced using N-terminal partial proteins, which contain PX-PH domains. An inducibly overexpressed fluorescence protein fusion of the PLDζ2 partial protein first localized to punctate structures, and then accumulated predominantly on the tonoplast. Further domain dissection analysis revealed that the N-terminal moiety preceding the PX-PH domain of PLDζ2 was required for the tonoplast-predominant accumulation. These findings suggest that PLDζ1 and PLDζ2 play partially overlapping but nonetheless distinctive roles in post-Golgi compartments along the membrane trafficking pathway from the TGN to the tonoplast.

Purpose The MERCURY study aimed to evaluate the effects on visual acuity and psychological symptoms, and safety, of ranibizumab and subsequent treatment in patients with diabetic macular oedema (DME) and impaired visual acuity (VA). We report data from the prespecified 12-month interim analysis. Methods This was a 24-month, phase 4, open-label, single-arm, prospective, observational study conducted at 20 specialised retinal centres in Japan. Participants were 209 patients with DME and impaired VA, not previously treated with either intravitreal or systemic anti-vascular endothelial growth factor (anti-VEGF) agents, who initiated ranibizumab 0.5 mg per investigator discretion. Following ranibizumab administration, patients were treated per routine clinical practice. Other treatments were allowed. The main outcome measure was the mean change in best-corrected VA (BCVA) in logarithmic minimum angle of resolution (logMAR) from baseline to month 12. An exploratory objective was to assess patients’ psychological status using the Hospital Anxiety and Depression Scale (HADS). Results The mean ± standard deviation BCVA at baseline was 0.43 ± 0.39 logMAR. The mean number of injections of ranibizumab and anti-VEGF agents from baseline to month 11 was 3.2 ± 2.0 and 3.6 ± 2.4, respectively. The BCVA change from baseline to 12 months was − 0.08 ± 0.34 logMAR (p = 0.011), showing a significant improvement; the HADS-anxiety score also decreased significantly (p = 0.001) and the depression score decreased numerically (p = 0.080). Conclusion MERCURY study data confirm the effectiveness of real-world treatment initiated with ranibizumab in Japanese patients with DME. In addition, treatment was able to positively influence anxiety via VA improvement.

Phosphatidylinositol-3-phosphate (PtdIns3P) is essential for generating autophagosomes and regulating endocytic trafficking. Recently, we have shown that the activities of human PIK3C3/VPS34-containing complexes I and II, which synthesize PtdIns3P, are greatly affected by three membrane physicochemical parameters: lipid unsaturation, membrane curvature, and negative charge. Both complexes are more active on membranes composed of unsaturated lipids than saturated lipids, and high membrane curvature can compensate for the negative effect of high lipid saturation. Negatively charged phosphatidylserine (PS) activates the complexes, as well as PIK3C3/VPS34 alone. The kinase activity of complex I depends critically on the ATG14 BATS domain, whereas complex II relies on the BECN1 BARA domain. Our findings highlight the importance of the membrane character as sensed by the unique membrane binding motifs/domain of the complexes for regulating PIK3C3/VPS34 activity.

Autophagy is a process through which intracellular cargoes are catabolised inside lysosomes. It involves the formation of autophagosomes initiated by the serine/threonine kinase ULK and class III PI3 kinase VPS34 complexes. Here, unbiased phosphoproteomics screens in mouse embryonic fibroblasts deleted for Ulk1/2 reveal that ULK loss significantly alters the phosphoproteome, with novel high confidence substrates identified including VPS34 complex member VPS15 and AMPK complex subunit PRKAG2. We identify six ULK‐dependent phosphorylation sites on VPS15, mutation of which reduces autophagosome formation in cells and VPS34 activity in vitro. Mutation of serine 861, the major VPS15 phosphosite, decreases both autophagy initiation and autophagic flux. Analysis of VPS15 knockout cells reveals two novel ULK‐dependent phenotypes downstream of VPS15 removal that can be partially recapitulated by chronic VPS34 inhibition, starvation‐independent accumulation of ULK substrates and kinase activity‐regulated recruitment of autophagy proteins to ubiquitin‐positive structures., ULK‐dependent phosphorylation of the VPS34 complex component VPS15 regulates starvation‐induced autophagy in mammalian cells.

VPS34 phosphorylates phosphatidylinositol to produce PtdIns3P and is the progenitor of the phosphoinositide 3-kinase (PI3K) family. VPS34 has a simpler domain organization than class I PI3Ks, which belies the complexity of its quaternary organization, with the enzyme always functioning within larger assemblies. PtdIns3P recruits specific recognition modules that are common in protein-sorting pathways, such as autophagy and endocytic sorting. It is best characterized in two heterotetramers, complexes I and II. Complex I is composed of VPS34, VPS15, Beclin 1, and autophagy-related gene (ATG)14L, whereas complex II replaces ATG14L with UVRAG. Because VPS34 can form a component of several distinct complexes, it enables independent regulation of various pathways that are controlled by PtdIns3P. Complexes I and II are critical for early events in autophagy and endocytic sorting, respectively. Autophagy has a complex association with cancer. In early stages, it inhibits tumorigenesis, but in later stages, it acts as a survival factor for tumors. Recently, various disease-associated somatic mutations were found in genes encoding complex I and II subunits. Lipid kinase activities of the complexes are also influenced by posttranslational modifications (PTMs). Mapping PTMs and somatic mutations on three-dimensional models of the complexes suggests mechanisms for how these affect VPS34 activity.

The lipid phosphatidylinositol-3-phosphate (PI3P) is a regulator of two fundamental but distinct cellular processes, endocytosis and autophagy, so its generation needs to be under precise temporal and spatial control. PI3P is generated by two complexes that both contain the lipid kinase VPS34: complex II on endosomes (VPS34/VPS15/Beclin 1/UVRAG), and complex I on autophagosomes (VPS34/VPS15/Beclin 1/ATG14L). The endosomal GTPase Rab5 binds complex II, but the mechanism of VPS34 activation by Rab5 has remained elusive, and no GTPase is known to bind complex I. Here we show that Rab5a–GTP recruits endocytic complex II to membranes and activates it by binding between the VPS34 C2 and VPS15 WD40 domains. Electron cryotomography of complex II on Rab5a-decorated vesicles shows that the VPS34 kinase domain is released from inhibition by VPS15 and hovers over the lipid bilayer, poised for catalysis. We also show that the GTPase Rab1a, which is known to be involved in autophagy, recruits and activates the autophagy-specific complex I, but not complex II. Both Rabs bind to the same VPS34 interface but in a manner unique for each. These findings reveal how VPS34 complexes are activated on membranes by specific Rab GTPases and how they are recruited to unique cellular locations., The phosphatidylinositol-3-phosphate (PI3P) is generated by the lipid kinase VPS34, in the context of VPS34 complex I on autophagosomes or complex II on endosomes. Biochemical and structural analyses provide insights into the mechanism of both VPS34 complexes recruitment to and activation on membranes by specific Rab GTPases.

Macroautophagy/autophagy is triggered by various starvation and stress conditions. The phospholipid phosphatidylinositol-3-phosphate (PtdIns3P) is essential for the formation of the autophagosome both in yeast and mammals. The class III phosphatidylinositol 3-kinase, PIK3C3C in humans or Vps34 in yeast, produces PtdIns3P by phosphorylating the 3ʹ-OH position of phosphatidylinositol (PtdIns). In order to synthesize PtdIns3P for the initiation of autophagy, PIK3C3/Vps34 has a heterotetrameric core, the PIK3C3 complex I (hereafter complex I) composed of PIK3C3/Vps34, PIK3R4/Vps15, BECN1/Vps30, and ATG14/Atg14. A fifth component of complex I, NRBF2 in mammals and Atg38 in yeast, was found and has been characterized in the past decade. The field has been expanding from cell and structural biology to mouse model and cohort studies. Here I will summarize the structures and models of complex I binding NRBF2/Atg38, its intracellular roles, and its involvement in health and disease. Along with this expansion of the field, different conclusions have been drawn in several topics. I will clarify what has and has not been agreed, and what is to be clarified in the future.

The ZIPANGU study assessed the efficacy and safety of ranibizumab as a one loading dose + pro re nata (one + PRN) regimen with/without focal/grid laser among treatment-naïve patients suffering from macular edema (ME) following branch retinal vein occlusion (BRVO). ZIPANGU was a phase IV, prospective, randomized, open-label, active-controlled, 12-month, two-arm, multicenter study. Treatment-naïve patients with visual impairment (19–73 letters) caused by ME, defined as central subfield thickness (CSFT) > 300 µm, due to BRVO were randomly assigned to ranibizumab monotherapy (n = 29) or combination therapy (ranibizumab + focal/grid short-pulse laser, n = 30). The primary endpoint was the number of ranibizumab injections. Secondary endpoints were mean changes in best-corrected visual acuity (BCVA) and CSFT, and safety. There were no statistically significant differences in the mean number of ranibizumab injections between monotherapy (4.3 injections) vs. combination (4.1 injections) therapy, or in CSFT. BCVA improvement in the monotherapy arm (22.0 letters) was better than the combination therapy arm (15.0 letters) (p = 0.035). Overall, both regimens appeared to be safe and well tolerated. One + PRN ranibizumab is safe and efficacious in treatment-naïve patients with ME secondary to BRVO. A conjunctive laser treatment did not lead to better functional outcomes or fewer ranibizumab injections.

The ZIPANGU study assessed the efficacy and safety of ranibizumab as a one loading dose + pro re nata (one + PRN) regimen with/without focal/grid laser among treatment-naïve patients suffering from macular edema (ME) following branch retinal vein occlusion (BRVO). ZIPANGU was a phase IV, prospective, randomized, open-label, active-controlled, 12-month, two-arm, multicenter study. Treatment-naïve patients with visual impairment (19-73 letters) caused by ME, defined as central subfield thickness (CSFT) 300 µm, due to BRVO were randomly assigned to ranibizumab monotherapy (n = 29) or combination therapy (ranibizumab + focal/grid short-pulse laser, n = 30). The primary endpoint was the number of ranibizumab injections. Secondary endpoints were mean changes in best-corrected visual acuity (BCVA) and CSFT, and safety. There were no statistically significant differences in the mean number of ranibizumab injections between monotherapy (4.3 injections) vs. combination (4.1 injections) therapy, or in CSFT. BCVA improvement in the monotherapy arm (22.0 letters) was better than the combination therapy arm (15.0 letters) (p = 0.035). Overall, both regimens appeared to be safe and well tolerated. One + PRN ranibizumab is safe and efficacious in treatment-naïve patients with ME secondary to BRVO. A conjunctive laser treatment did not lead to better functional outcomes or fewer ranibizumab injections.

VPS34 complex II (VPS34CII) is a 386-kDa assembly of the lipid kinase subunit VPS34 and three regulatory subunits that altogether function as a prototypical class III phosphatidylinositol-3-kinase (PI3K). When the active VPS34CII complex is docked to the cytoplasmic surface of endosomal membranes, it phosphorylates its substrate lipid (phosphatidylinositol, PI) to generate the essential signaling lipid phosphatidylinositol-3-phosphate (PI3P). In turn, PI3P recruits an array of signaling proteins containing PI3P-specific targeting domains (including FYVE, PX, and PROPPINS) to the membrane surface, where they initiate key cell processes. In endocytosis and early endosome development, net VPS34CII-catalyzed PI3P production is greatly amplified by Rab5A, a small G protein of the Ras GTPase superfamily. Moreover, VPS34CII and Rab5A are each strongly linked to multiple human diseases. Thus, a molecular understanding of the mechanism by which Rab5A activates lipid kinase activity will have broad impacts in both signaling biology and medicine. Two general mechanistic models have been proposed for small G protein activation of PI3K lipid kinases. 1) In the membrane recruitment mechanism, G protein association increases the density of active kinase on the membrane. And 2) in the allosteric activation mechanism, G protein allosterically triggers an increase in the specific activity (turnover rate) of the membrane-bound kinase molecule. This study employs an in vitro single-molecule approach to elucidate the mechanism of GTP-Rab5A-associated VPS34CII kinase activation in a reconstituted GTP-Rab5A-VPS34CII-PI3P-PX signaling pathway on a target membrane surface. The findings reveal that both membrane recruitment and allosteric mechanisms make important contributions to the large increase in VPS34CII kinase activity and PI3P production triggered by membrane-anchored GTP-Rab5A. Notably, under near-physiological conditions in the absence of other activators, membrane-anchored GTP-Rab5A provides strong, virtually binary on-off switching and is required for VPS34CII membrane binding and PI3P production.

The lipid kinase VPS34 orchestrates diverse processes, including autophagy, endocytic sorting, phagocytosis, anabolic responses and cell division. VPS34 forms various complexes that help adapt it to specific pathways, with complexes I and II being the most prominent ones. We found that physicochemical properties of membranes strongly modulate VPS34 activity. Greater unsaturation of both substrate and non-substrate lipids, negative charge and curvature activate VPS34 complexes, adapting them to their cellular compartments. Hydrogen/deuterium exchange mass spectrometry (HDX-MS) of complexes I and II on membranes elucidated structural determinants that enable them to bind membranes. Among these are the Barkor/ATG14L autophagosome targeting sequence (BATS), which makes autophagy-specific complex I more active than the endocytic complex II, and the Beclin1 BARA domain. Interestingly, even though Beclin1 BARA is common to both complexes, its membrane-interacting loops are critical for complex II, but have only a minor role for complex I.

Introduction: Direct oral anticoagulants are widely used in patients with nonvalvular atrial fibrillation (AF) to reduce the risk of stroke and systemic embolism, however, there is not enough real-world data of their effectiveness and safety in patients with worsening renal function (WRF). Xarelto post-authorization safety and effectiveness study in Japanese patients with atrial fibrillation (XAPASS) is a prospective observational post-marketing surveillance study mandated by the Japanese authority. It aims to examine safety and effectiveness of rivaroxaban in everyday clinical practice. This analysis investigated one year outcomes among patients with WRF and stable renal function (SRF) in XAPASS. Methods: One year follow-up data of 9578 patients enrolled in XAPASS were analyzed to evaluate baseline characteristics and safety/effectiveness profile among patients with WRF and SRF. WRF was defined as a decrease of more than 20% from enrollment creatinine clearance measurement at any time point during the study. SRF was defined as the absence of WRF at any time. Results: We identified 1229 patients (12.8%) with WRF and 6280 patients (65.6%) with SRF among 9578 patients. WRF patients were significantly older, and had significantly higher mean CHA 2 DS 2 -VASc score and modified HAS-BLED score compared to SRF patients. Prevalence of hypertension, congestive heart failure, ischemic stroke/transient ischemic attack, and myocardial infarction was higher in WRF patients. There was no difference in rates of major bleeding (hazard ratio (HR) 1.20; 95% confidence interval (CI) 0.75-1.90; p=0.45) or the composite endpoint of stroke, systemic embolism or myocardial infarction (HR 1.06; 95% CI 0.65-1.71; p=0.82) between patients with WRF and SRF. WRF patients experienced a higher incidence of transfusion of 2 units or more (0.46 versus 0.14 events per 100 patient-years; HR 3.19; 95% CI 1.04-9.74; p=0.03) versus SRF patients. Rates of other major bleeding subgroups defined according to ISTH criteria were similar between patients with WRF and SRF. Conclusions: WRF during rivaroxaban treatment did not significantly increase the rates of major bleeding or thromboembolic events, although it was associated with a higher incidence of transfusion of 2 units or more.

The cerebral cortex computes through the canonical microcircuit that connects six stacked layers; however, how cortical processing streams operate in vivo, particularly in the higher association cortex, remains elusive. By developing a novel MRI-assisted procedure that reliably localizes recorded single neurons at resolution of six individual layers in monkey temporal cortex, we show that transformation of representations from a cued object to a to-be-recalled object occurs at the infragranular layer in a visual cued-recall task. This cue-to-target conversion started in layer 5 and was followed by layer 6. Finally, a subset of layer 6 neurons exclusively encoding the sought target became phase-locked to surrounding field potentials at theta frequency, suggesting that this coordinated cell assembly implements cortical long-distance outputs of the recalled target. Thus, this study proposes a link from local computation spanning laminar modules of the temporal cortex to the brain-wide network for memory retrieval in primates.

The phosphatidylinositol 3-kinase Vps34 is part of several protein complexes. The structural organization of heterotetrameric complexes is starting to emerge, but little is known about organization of additional accessory subunits that interact with these assemblies. Combining hydrogen-deuterium exchange mass spectrometry (HDX-MS), X-ray crystallography and electron microscopy (EM), we have characterized Atg38 and its human ortholog NRBF2, accessory components of complex I consisting of Vps15-Vps34-Vps30/Atg6-Atg14 (yeast) and PIK3R4/VPS15-PIK3C3/VPS34-BECN1/Beclin 1-ATG14 (human). HDX-MS shows that Atg38 binds the Vps30-Atg14 subcomplex of complex I, using mainly its N-terminal MIT domain and bridges the coiled-coil I regions of Atg14 and Vps30 in the base of complex I. The Atg38 C-terminal domain is important for localization to the phagophore assembly site (PAS) and homodimerization. Our 2.2 Å resolution crystal structure of the Atg38 C-terminal homodimerization domain shows 2 segments of α-helices assembling into a mushroom-like asymmetric homodimer with a 4-helix cap and a parallel coiled-coil stalk. One Atg38 homodimer engages a single complex I. This is in sharp contrast to human NRBF2, which also forms a homodimer, but this homodimer can bridge 2 complex I assemblies.

Background Although the efficacy and safety of the factor Xa inhibitor rivaroxaban for the prevention of stroke and systemic embolism in patients with non-valvular atrial fibrillation (NVAF) were shown in global and Japanese phase III clinical trials, safety and effectiveness data from unselected patients in everyday clinical practice are limited. The objective of the XAPASS (Xarelto Post-Authorization Safety & Effectiveness Study in Japanese Patients with Atrial Fibrillation) is to investigate the safety and effectiveness of rivaroxaban in Japanese real-world clinical practice. Methods The XAPASS is a prospective, single-arm, real-world observational study mandated by the Japanese authority as post-marketing surveillance. In total, 11,308 patients with NVAF who began treatment with rivaroxaban were enrolled from April 2012 to June 2014, and 9578 patients were analyzed to examine the one-year outcomes. Results The mean treatment duration was 300 ± 119 days. The patients’ age was 73.2 ± 9.8 years, and their CHADS2 score was 2.2 ± 1.3. Any bleeding and major bleeding occurred in 602 patients (7.6 events per 100 patient-years) and 143 patients (1.8 events per 100 patient-years), respectively. Stroke/non-central nervous system systemic embolism/myocardial infarction was observed in 144 patients (1.8 events per 100 patient-years). Conclusions Real-world outcomes of the XAPASS showed incidence rates of major bleeding and thromboembolic events, suggesting that rivaroxaban is safe and effective in Japanese daily clinical practice (Clinicaltrials.gov: NCT01582737 ).

Background The phase III Japanese Rivaroxaban Once‐Daily Oral Direct Factor Xa Inhibition Compared with Vitamin K Antagonism for Prevention of Stroke and Embolism Trial in Atrial Fibrillation (J‐ROCKET AF) showed that the rivaroxaban group had a lower event rate of intracranial bleeding than the warfarin group and that rivaroxaban was noninferior to warfarin for the principal safety outcome. However, safety and effectiveness data from unselected patients with AF in everyday clinical practice in Japan are lacking. Methods The Xarelto Post‐Authorization Safety & Effectiveness Study in Japanese Patients with Atrial Fibrillation (XAPASS) is a real‐world, prospective, single‐arm, observational study mandated by the Japanese authority as postmarketing surveillance. XAPASS involves patients with nonvalvular AF prescribed rivaroxaban. The principal safety outcome is a composite of major and nonmajor bleeding events, and the primary effectiveness outcome is the incidence of ischemic stroke, hemorrhagic stroke, noncentral nervous system systemic embolism, and myocardial infarction. Results In total, 11 308 patients were enrolled from April 2012 to June 2014. Their age was 73.1 ± 9.9 years, and their CHADS 2 score was 2.2 ± 1.3. Female patients, patients aged ≥75 years, patients with a body weight of ≤50 kg, and patients with a creatinine clearance of

The cerebellum consists of the cerebellar cortex and the cerebellar nuclei. Although the basic neuronal circuitry of the cerebellar cortex is uniform everywhere, anatomical data demonstrate that the input and output relationships of the cortex are spatially segregated between different cortical areas, which suggests that there are functional distinctions between these different areas. Perturbation of cerebellar cortical functions in a spatially restricted fashion is thus essential for investigating the distinctions among different cortical areas. In the cerebellar cortex, Purkinje cells are the sole output neurons that send information to downstream cerebellar and vestibular nuclei. Therefore, selective manipulation of Purkinje cell activities, without disturbing other neuronal types and passing fibers within the cortex, is a direct approach to spatially restrict the effects of perturbations. Although this type of approach has for many years been technically difficult, recent advances in optogenetics now enable selective activation or inhibition of Purkinje cell activities, with high temporal resolution. Here we discuss the effectiveness of using Purkinje cell-specific optogenetic approaches to elucidate the functions of local cerebellar cortex regions. We also discuss what improvements to current methods are necessary for future investigations of cerebellar functions to provide further advances.

The target of rapamycin (Tor) is a Ser/Thr protein kinase that regulates a range of anabolic and catabolic processes. Tor is present in two complexes, TORC1 and TORC2, in which the Tor–Lst8 heterodimer forms a common sub-complex. We have determined the cryo-electron microscopy (EM) structure of Tor bound to Lst8. Two Tor–Lst8 heterodimers assemble further into a dyad-symmetry dimer mediated by Tor–Tor interactions. The first 1,300 residues of Tor form a HEAT repeat-containing α-solenoid with four distinct segments: a highly curved 800-residue N-terminal 'spiral', followed by a 400-residue low-curvature 'bridge' and an extended ‘railing' running along the bridge leading to the 'cap' that links to FAT region. This complex topology was verified by domain insertions and offers a new interpretation of the mTORC1 structure. The spiral of one TOR interacts with the bridge of another, which together form a joint platform for the Regulatory Associated Protein of TOR (RAPTOR) regulatory subunit., The target of rapamycin (Tor) is a Ser/Thr protein kinase that regulates a wide range of anabolic and catabolic processes. Here the authors describe a sub-nanometer cryo-EM structure of a yeast Tor–Lst8 complex and propose an overall topology that differs from that previously suggested for mTORC1.

The optogenetic approach to primate brain circuitry has unparalleled potential for uncovering genetically and temporally resolved neuronal mechanisms of higher brain functions. In order to optogenetically investigate the large and complex primate brain, an optical-/electrical probe, or "optrode", must be inserted deeply, which requires the optrode to be not only long and stiff, but also sharp and smooth to reduce possible tissue damage. This study presents a tungsten microelectrode-based optrode that encloses optical fibers within its insulation glass. Optical fibers and a tungsten wire were tightly bound to each other and integrally coated with a smooth, thin layer of glass. This design satisfied the structural requirements for use in deep brain structures. The performance of the optrode was then examined in the thalamus of the rat and macaque monkeys which were injected with lentiviral vectors carrying the channelrhodopsin-2-enhanced yellow fluorescent protein (ChR2-EYFP) transgene. With fluorescence measurements via the optical fiber, ChR2-EYFP expression was detected clearly in vivo, which was confirmed by histological analysis in the rat. With photostimulation and extracellular recording, photo-responsive single-unit activities were isolated in the monkeys. The depth distribution of these units and the peak of the EYFP fluorescence profile overlapped consistently with each other. Thus, by developing a new probe, optogenetic methodology was successfully applied to a primate subcortical structure. This smooth glass-coated optrode is a promising tool for chronic in vivo experiments with various research targets including deep brain structures in behaving monkeys.

Although the parallel visual pathways are a fundamental basis of visual processing, our knowledge of their molecular properties is still limited. Here, we uncovered a parvocellular-specific molecule in the dorsal lateral geniculate nucleus (dLGN) of higher mammals. We found that FoxP2 transcription factor was specifically expressed in X cells of the adult ferret dLGN. Interestingly, FoxP2 was also specifically expressed in parvocellular layers 3-6 of the dLGN of adult old world monkeys, providing new evidence for a homology between X cells in the ferret dLGN and parvocellular cells in the monkey dLGN. Furthermore, this expression pattern was established as early as gestation day 140 in the embryonic monkey dLGN, suggesting that parvocellular specification has already occurred when the cytoarchitectonic dLGN layers are formed. Our results should help in gaining a fundamental understanding of the development, evolution, and function of the parallel visual pathways, which are especially prominent in higher mammals.

Precise localization of single-neuron activity has elucidated functional architectures of the primate cerebral cortex, related to vertically stacked layers and horizontally aligned columns. The traditional “gold standard” method for localizing recorded neuron is histological examination of electrolytic lesion marks at recording sites. Although this method can localize recorded neurons with fine neuroanatomy, the necessity for postmortem analysis prohibits its use in long-term chronic experiments. To localize recorded single-neuron positions in vivo, we introduced MRI-detectable elgiloy deposit marks, which can be created by electrolysis of an elgiloy microelectrode tip and visualized on highly contrasted magnetic resonance (MR) images. Histological analysis validated that the deposit mark centers could be localized relative to neuroanatomy in vivo with single-voxel accuracy, at an in-plane resolution of 200 μm. To demonstrate practical applications of the technique, we recorded single-neuron activity from a monkey performing a cognitive task and localized it in vivo using deposit marks (deposition: 2 μA for 3 min; scanning: fast-spin-echo sequence with 0.15 × 0.15 × 0.8 mm3 resolution, 120/4,500 ms of echo-time/repetition-time and 8 echo-train-length), as is usually performed with conventional postmortem methods using electrolytic lesion marks. Two localization procedures were demonstrated: 1) deposit marks within a microelectrode track were used to reconstruct a dozen recorded neuron positions along the track directly on MR images; 2) combination with X-ray imaging allowed estimation of hundreds of neuron positions on MR images. This new in vivo method is feasible for chronic experiments with nonhuman primates, enabling analysis of the functional architecture of the cerebral cortex underlying cognitive processes.

We developed a bicistronic HIV1-derived lentiviral vector system co-expressing green fluorescent protein (AcGFP1) and wheat germ agglutinin (WGA) mediated by picornaviral 2A peptide. This system was first applied to the analysis of the rat cerebellar efferent pathways. When the lentiviral vector was injected into a specific lobule, the local Purkinje cell population (first-order neurons) was efficiently infected and co-expressed both AcGFP1 and WGA protein. In the second-order neurons in the cerebellar and vestibular nuclei, WGA but not AcGFP1 protein was differentially detected, demonstrating that the presence of AcGFP1 protein enables discrimination of first-order neurons from second-order neurons. Furthermore, WGA protein was detected in the contralateral ventrolateral thalamic nucleus (third-order nucleus). This system also successfully labeled rat cortical pathways from the primary somatosensory cortex and monkey cerebellar efferent pathways. Thus, this bicistronic lentiviral vector system is a useful tool for differential transsynaptic tracing of neural pathways originating from local brain regions.

The integral membrane protein Atg9 is delivered to the autophagosome in yeast and mammalian cells. We find that Atg9 does not originate from mitochondria and cannot reach the autophagosome directly from the ER. Instead, pairwise combinations of mutations in Golgi-endosomal traffic components cause defects in Atg9 delivery during starvation., While many of the proteins required for autophagy have been identified, the source of the membrane of the autophagosome is still unresolved with the endoplasmic reticulum (ER), endosomes, and mitochondria all having been evoked. The integral membrane protein Atg9 is delivered to the autophagosome during starvation and in the related cytoplasm-to-vacuole (Cvt) pathway that occurs constitutively in yeast. We have examined the requirements for delivery of Atg9-containing membrane to the yeast autophagosome. Atg9 does not appear to originate from mitochondria, and Atg9 cannot reach the forming autophagosome directly from the ER or early Golgi. Components of traffic between Golgi and endosomes are known to be required for the Cvt pathway but do not appear required for autophagy in starved cells. However, we find that pairwise combinations of mutations in Golgi-endosomal traffic components apparently only required for the Cvt pathway can cause profound defects in Atg9 delivery and autophagy in starved cells. Thus it appears that membrane that contains Atg9 is delivered to the autophagosome from the Golgi-endosomal system rather than from the ER or mitochondria. This is underestimated by examination of single mutants, providing a possible explanation for discrepancies between yeast and mammalian studies on Atg9 localization and autophagosome formation.

Opening up Vps34 protein complexes During intracellular membrane trafficking, large protein complexes regulate and adapt the activity of signal transducer enzymes such as the class III phosphatidylinositol 3-kinase Vps34. These large enzyme complexes are present in all eukaryotic cells, having widespread importance in neurodegeneration, aging, and cancer; however, a structural understanding has been lacking. Rostislavleva et al. provide atomic-resolution insights into the structures of the Vps34-containing protein complexes required for autophagy, endocytic sorting, and cytokinesis. The V-shaped complexes can undergo opening motions, which allows them to adapt to and phosphorylate membranes. Science , this issue p. 10.1126/science.aac7365

Pathway-specific gene delivery is requisite for understanding complex neuronal systems in which neurons that project to different target regions are locally intermingled. However, conventional genetic tools cannot achieve simultaneous, independent gene delivery into multiple target cells with high efficiency and low cross-reactivity. In this study, we systematically screened all receptor-envelope pairs resulting from the combination of four avian sarcoma leukosis virus (ASLV) envelopes (EnvA, EnvB, EnvC, and EnvE) and five engineered avian-derived receptors (TVA950, TVB(S3), TVC, TVB(T), and DR-46TVB) in vitro. Four of the 20 pairs exhibited both high infection rates (TVA-EnvA, 99.6%; TVB(S3)-EnvB, 97.7%; TVC-EnvC, 98.2%; and DR-46TVB-EnvE, 98.8%) and low cross-reactivity (2.5%). Next, we tested these four receptor-envelope pairs in vivo in a pathway-specific gene-transfer method. Neurons projecting into a limited somatosensory area were labeled with each receptor by retrograde gene transfer. Three of the four pairs exhibited selective transduction into thalamocortical neurons expressing the paired receptor (98%), with no observed cross-reaction. Finally, by expressing three receptor types in a single animal, we achieved pathway-specific, differential fluorescent labeling of three thalamic neuronal populations, each projecting into different somatosensory areas. Thus, we identified three orthogonal pairs from the list of ASLV subgroups and established a new vector system that provides a simultaneous, independent, and highly specific genetic tool for transferring genes into multiple target cells in vivo. Our approach is broadly applicable to pathway-specific labeling and functional analysis of diverse neuronal systems.

The localization of microelectrode recording sites in the layers of primate cerebral cortex permits the analysis of relationships between recorded neuronal activities and underlying anatomical connections. We present a magnetic resonance imaging method for precise in vivo localization of cortical recording sites. In this method, the susceptibility-induced effect thickens the appearance of the microelectrode and enhances the detectability of the microelectrode tip, which usually occupies less than a few percent of the volume of an image voxel. In a phantom study, the optimized susceptibility-induced effect allowed tip detection with single-voxel accuracy (in-plane resolution, 50 mum). We applied this method to recording microelectrodes inserted into the brains of macaque monkeys, and localized the microelectrode tip at an in-plane resolution of 150 mum within the cortex of 2-3 mm in thickness. Subsequent histological analyses validated the single-voxel accuracy of the in vivo tip localization. This method opens up a way to investigate information flow during cognitive processes in the brain.

A novel re-hybridization protocol for pathology archive sections that uses microwave-assisted fluorescence in situ hybridization (FISH) is described. Stripping the probe from the pathology archive sections with HCl and re-hybridizing with the next probe by intermittent microwave irradiation generated clear signals without background noise. Repeated stripping and hybridization with numerous bacterial artificial chromosome (BAC)-derived probes would identify the profile of genome-wide changes in small lesions on sections.

Nine halogenated anilines, consisting of di- and tri-substituted fluoro-, chloro-, and bromoanilines, were subjected to analysis of their cytochrome P450 (CYP) 2E1/2A6-selective inhibitory effects on metabolic activation of dimetylnitrosamine (DMN) in human liver microsomes. Inhibitory activities (IC50) of these anilines on human recombinant CYP2E1 ranged from 8.0 to 549 μM. However, most of these anilines showed no remarkable inhibition of CYP1A2, while their IC50 values on CYP2A6 ranged from 2.9 to 232 μM. The CYP2E1 selectivity of these anilines in terms of the ratio of the IC50 values of these anilines on CYP2A6 to those on CYP2E1, ranged from 0.1- to 5.2-fold. Their CYP2E1 selectivity decreased in the following order: 3,4,5-trifluoroaniline (3,4,5-triF-A) > 3,5-diF-A >> 3,5-dichloroaniline (3,5-diCl-A) > 3,4-diCl-A > 2,6-diCl-A > 2,3,4-trichloroaniline (2,3,4-triCl-A) > 2,4,6-triCl-A > 2,6-dibromoaniline (2,6-diBr-A) > 3,4,5-triCl-A. The inhibitory effects of these anilines on metabolic activation of DMN were analyzed using human liver microsomes. The IC50 values of these anilines on demethylation metabolism of DMN correlated with those of these anilines on CYP2E1. These results suggest that these halogenated anilines may be useful as indicators of CYP-selectivity involved in metabolic activation of nitrosamines.

GLABRA2 (GL2)/ATHB-10 encodes a homeodomain protein that belongs to the homeodomain-leucine zipper family. Mutant studies have revealed that this gene is involved in trichome, root-hair and seed-coat development. We used reverse genetics to investigate the role of GL2 in trichome development. A transgene consisting of a GL2-coding fragment preceded by the cauliflower mosaic virus 35S promoter (35S::GL2) did not complement defects in the gl2-1 mutant. In the wild-type genetic background, 35S::GL2 caused gl2-mutant-like and scarcely viable phenotypes, suggesting that ectopic overexpression of GL2 interrupts endogenous GL2 function in trichome development and is toxic to plants. On the other hand, another GL2 transgene containing the GL2 promoter (pGL2::GL2) complemented the gl2-1 mutation. Entopically additive expression of GL2 by introduction of pGL2::GL2 in the wild-type genetic background noticably increased the number of trichomes and induced production of adjacent trichomes. Consistent with this result, gl2-1/+ heterozygous leaves, whose GL2 expression was expected to decrease, had fewer trichomes than +/+ leaves. These results indicate that GL2 quantitatively regulates the frequency of trichome initiation and is involved in determining trichome spacing.

Lentiviral vectors deliver transgenes efficiently to a wide range of neuronal cell types in the mammalian central nervous system. To drive gene expression, internal promoters are essential; however, the in vivo properties of promoters, such as their cell type specificity and gene expression activity, are not well known, especially in the nonhuman primate brain. Here, the properties of five ubiquitous promoters (murine stem cell virus [MSCV], cytomegalovirus [CMV], CMV early enhancer/chicken β-actin [CAG], human elongation factor-1α [EF-1α], and Rous sarcoma virus [RSV]) and two cell type-specific promoters (rat synapsin I and mouse α-calcium/calmodulin-dependent protein kinase II [CaMKIIα]) in rat and monkey motor cortices in vivo were characterized. Vesicular stomatitis virus G (VSV-G)-pseudotyped lentiviral vectors expressing enhanced green fluorescent protein (EGFP) under the control of the various promoters were prepared and injected into rat and monkey motor cortices. Immunohistochemical analysis revealed that all of the VSV-G-pseudotyped lentiviral vectors had strong endogenous neuronal tropisms in rat and monkey brains. Among the seven promoters, the CMV promoter showed modest expression in glial cells (9.4%) of the rat brain, whereas the five ubiquitous promoters (MSCV, CMV, CAG, EF-1α, and RSV) showed expression in glial cells (7.0-14.7%) in the monkey brain. Cell type-specific synapsin I and CaMKIIα promoters showed excitatory neuron-specific expression in the monkey brain (synapsin I, 99.7%; CaMKIIα, 100.0%), but their specificities for excitatory neurons were significantly lower in the rat brain (synapsin I, 94.6%; CaMKIIα, 93.7%). These findings could be useful in basic and clinical neuroscience research for the design of vectors that efficiently deliver and express transgenes into rat and monkey brains.

We present two patients with multiple microhamartomas of the biliary tract (von Meyenburg complexes) with multiple liver cysts with special reference to their diagnostic imaging and laparoscopic findings. Case 1 was a 76-year-old male and case 2 was 51-year-old male, who was later found to have adenocarcinoma of the common bile duct as a complication. The characteristic findings of ultrasonography (US) were markedly heterogeneous and there was a severe hyperechoic pattern in the liver with small cystic lesions. Computed tomography (CT) demonstrated multiple low attenuation focal defects scattered throughout both lobes which showed no enhancement following the application of intravenous contrast medium. Laparoscopically, multiple whitish nodular or stellate-shaped lesions, which were slightly depressed, and multiple small dark greenish cystic lesions of various sizes were scattered on the smooth surface of both lobes. Some dark greenish cystic lesions and whitish lesions were adjoining. A histological study of the whitish nodular lesions revealed a collection of proliferated and irregularly dilated bile ducts and ductules in a dense fibrous stroma. These findings are compatible with microhamartomas. These features are characteristic of this disease, and support the close association between liver cysts and microhamartomas. Our hypothesis is that a polycystic liver may result from cystic dilatation of microhamartomas.

Rubella was accompanied by hepatic dysfunction in a 28-year-old male. Serum aminotransferase levels were moderately elevated and LDH markedly increased, especially LDH isoenzyme 5, whereas total bilirubin and ALP remained almost normal. GOT, GPT and LDH levels were completely normalized by the 21st hospital day. Paired antibody titers of viruses which may cause hepatitis, other than rubella, were of no significance. Laparoscopy showed enlarged, red liver. Histologic and electron microscopic findings of the liver were consistent with acute hepatitis. Hepatic damage with rubella is rare, and it is possible that the hepatic dysfunction seen in adult rubella may be mediated by an immunopathologic mechanism.

The Arabidopsis thaliana GLABRA2 (GL2) gene encodes a transcription factor involved in the cell differentiation of various epidermal tissues. During root hair pattern formation, GL2 suppresses root hair development in non-hair cells, acting as a node between the gene regulatory networks for cell fate determination and cell differentiation. Despite the importance of GL2 function, its molecular basis remains obscure because the GL2 target genes leading to the network for cell differentiation are unknown. We identified five basic helix-loop-helix (bHLH) transcription factor genes (ROOT HAIR DEFECTIVE6 [RHD6], RHD6-LIKE1 [RSL1], RSL2, Lj-RHL1-LIKE1 [LRL1], and LRL2) as GL2 direct targets using transcriptional and posttranslational induction systems. Chromatin immunoprecipitation analysis confirmed GL2 binding to upstream regions of these genes in planta. Reporter gene analyses showed that these genes are expressed in various stages of root hair development and are suppressed by GL2 in non-hair cells. GL2 promoter-driven GFP fusions of LRL1 and LRL2, but not those of the other bHLH proteins, conferred root hair development on non-hair cells. These results indicate that GL2 directly suppresses bHLH genes with diverse functions in root hair development.

During sensory deprivation, the barrel cortex undergoes expansion of a functional column representing spared inputs (spared column), into the neighboring deprived columns (representing deprived inputs) which are in turn shrunk. As a result, the neurons in a deprived column simultaneously increase and decrease their responses to spared and deprived inputs, respectively. Previous studies revealed that dendritic spines are remodeled during this barrel map plasticity. Because cofilin1, a predominant regulator of actin filament turnover, governs both the expansion and shrinkage of the dendritic spine structure in vitro, it hypothetically regulates both responses in barrel map plasticity. However, this hypothesis remains untested. Using lentiviral vectors, we knocked down cofilin1 locally within layer 2/3 neurons in a deprived column. Cofilin1-knocked-down neurons were optogenetically labeled using channelrhodopsin-2, and electrophysiological recordings were targeted to these knocked-down neurons. We showed that cofilin1 knockdown impaired response increases to spared inputs but preserved response decreases to deprived inputs, indicating that cofilin1 dependency is dissociated in these two types of barrel map plasticity. To explore the structural basis of this dissociation, we then analyzed spine densities on deprived column dendritic branches, which were supposed to receive dense horizontal transcolumnar projections from the spared column. We found that spine number increased in a cofilin1-dependent manner selectively in the distal part of the supragranular layer, where most of the transcolumnar projections existed. Our findings suggest that cofilin1-mediated actin dynamics regulate functional map plasticity in an input-specific manner through the dendritic spine remodeling that occurs in the horizontal transcolumnar circuits. These new mechanistic insights into transcolumnar plasticity in adult rats may have a general significance for understanding reorganization of neocortical circuits that have more sophisticated columnar organization than the rodent neocortex, such as the primate neocortex., In vivo measurement of the electrophysiology and shape of neurons reveals that cofilin1 is needed for remodeling dendritic spines in circuits that connect mouse whisker barrels, so aiding experience-dependent plasticity in the neocortex., Author Summary Plasticity in the adult neocortex is the basis of our learning and memory. However, its molecular mechanisms are still unclear. In the sensory barrel cortex of rodents, a well-characterized model for neocortical plasticity, neurons directly code for whisker displacement—neurons within a given barrel will fire when the whisker that that barrel represents is moved. Strikingly, the deprivation of all but a single whisker alters the original representations—cortical columns representing the deprived inputs shrink and that representing the spared inputs expands, intruding into the surrounding deprived columns. Because single-neuron-level structural changes are suggested to be involved in this plasticity, here we focused on cofilin1, a protein that is known to modulate the cytoskeleton and to regulate the structure of dendritic spines. We induced experience-dependent plasticity in the D1 column by sparing only the D1 whisker, and knocked down the expression of cofilin1 in the D2 column. Cofilin1 knockdown differentially affected plasticity, such that experience-dependent increases in spared input representation were impaired, whereas decreases in deprived input representation were intact. We then found that during these plastic changes, the density of dendritic spines increased in a cofilin1-dependent manner around the connections between the D1 and D2 columns. Cofilin1-dependent density increase was observed only in the most superficial part of the cortex but not in deeper parts, consistent with the distribution patterns of axons that transmit spared and deprived information, respectively. These results suggest that cofilin1 regulates neocortical functional plasticity through the remodeling of dendritic spines within circuits that connect columns.

Plant cells frequently undergo endoreduplication, a process in which chromosomal DNA is successively duplicated in the absence of mitosis. It has been proposed that endoreduplication is regulated at its entry by mitotic cyclin-dependent kinase activity. However, the regulatory mechanisms for its termination remain unclear, although plants tightly control the ploidy level in each cell type. In the process of searching for regulatory factors of endoreduplication, the promoter of an Arabidopsis thaliana cyclin A gene, CYCA2;3, was revealed to be active in developing trichomes during the termination period of endoreduplication as well as in proliferating tissues. Taking advantage of the situation that plants encode highly redundant cyclin A genes, we were able to perform functional dissection of CYCA2;3 using null mutant alleles. Null mutations of CYCA2;3 semidominantly promoted endocycles and increased the ploidy levels achieved in mature organs, but they did not significantly affect the proportion of cells that underwent endoreduplication. Consistent with this result, expression of the CYCA2;3–green fluorescent protein fusion protein restrained endocycles in a dose-dependent manner. Moreover, a mutation in the destruction box of CYCA2;3 stabilized the fusion protein in the nuclei and enhanced the restraint. We conclude that CYCA2;3 negatively regulates endocycles and acts as a key regulator of ploidy levels in Arabidopsis endoreduplication.