Dynamic movement of the Golgi unit and its glycosylation enzyme zones.

Knowledge on the distribution and dynamics of glycosylation enzymes in the Golgi is essential for better understanding this modification. Here, using a combination of CRISPR/Cas9 knockin technology and super-resolution microscopy, we show that the Golgi complex is assembled by a number of small 'Golgi units' that have 1-3 µm in diameter. Each Golgi unit contains small domains of glycosylation enzymes which we call 'zones'. The zones of N- and O-glycosylation enzymes are colocalised. However, they are less colocalised with the zones of a glycosaminoglycan synthesizing enzyme. Golgi units change shapes dynamically and the zones of glycosylation enzymes rapidly move near the rim of the unit. Photobleaching analysis indicates that a glycosaminoglycan synthesizing enzyme moves between units. Depletion of giantin dissociates units and prevents the movement of glycosaminoglycan synthesizing enzymes, which leads to insufficient glycosaminoglycan synthesis. Thus, we show the structure-function relationship of the Golgi and its implications in human pathogenesis.

Bony Mallet Thumb Treated With the Ishiguro Extension Block Technique: A Case Report.

A bony mallet thumb is an extremely rare injury. An 82-year-old man fell from a standing height and injured his right thumb. Imaging examinations revealed a rare intra-articular fracture at the dorsal side of the base of the distal phalanx of the thumb called the bony mallet thumb. Conservative treatment was adopted initially; however, surgery was deemed necessary because of the redislocation of the bone fragment. Thus, the Ishiguro extension block technique was used, and three months later, satisfactory thumb function was achieved. The Ishiguro technique is a relatively simple procedure often performed for bony mallet fingers. The current case indicated that it can also be used to treat cases of bony mallet thumbs successfully.

Therapeutic Effects of Dual Dopaminergic Modulation With l-DOPA and Chlorpromazine in Patients With Idiopathic Cervical Dystonia.

Objectives: Imbalanced activities between dopamine D1 and D2 signals in striatal striosome-matrix system have been proposed as a cause of dystonia symptoms. The aim of this study was to assess the therapeutic effects of dual dopaminergic modulation (DDM) with l-DOPA and chlorpromazine (CPZ) in patients with idiopathic cervical dystonia (CD). Methods: We enrolled 21 patients with CD who responded poorly to botulinum toxin treatment. The severities of CD motor symptoms and CD-associated pain were determined using the Toronto Western Spasmodic Torticollis Rating Scale and the visual analog scale, respectively. Results: In patients with CD (n = 7), oral administration of l-DOPA combined with CPZ significantly attenuated both CD motor symptoms and CD-associated pain in a dose-related manner. By contrast, there was no improvement of CD symptoms in patients (n = 7) who ingested l-DOPA alone nor in those (n = 7) who ingested CPZ alone. Discussion: DDM with l-DOPA and CPZ may be an effective tool to treat dystonia symptoms in patients with botulinum toxin-resistant idiopathic CD. Our results may also indicate that CD dystonia symptoms could be attenuated through DDM inducing an increase in striosomal D1-signaling. Classification of Evidence: This study provides Class III evidence that treatment of botulinum toxin-resistant idiopathic cervical dystonia with l-DOPA and chlorpromazine is superior to either one alone.

PIGB maintains nuclear lamina organization in skeletal muscle of Drosophila.

The nuclear lamina (NL) plays various roles and participates in nuclear integrity, chromatin organization, and transcriptional regulation. Lamin proteins, the main components of the NL, form a homogeneous meshwork structure under the nuclear envelope. Lamins are essential, but it is unknown whether their homogeneous distribution is important for nuclear function. Here, we found that PIGB, an enzyme involved in glycosylphosphatidylinositol (GPI) synthesis, is responsible for the homogeneous lamin meshwork in Drosophila. Loss of PIGB resulted in heterogeneous distributions of B-type lamin and lamin-binding proteins in larval muscles. These phenotypes were rescued by expression of PIGB lacking GPI synthesis activity. The PIGB mutant exhibited changes in lamina-associated domains that are large heterochromatic genomic regions in the NL, reduction of nuclear stiffness, and deformation of muscle fibers. These results suggest that PIGB maintains the homogeneous meshwork of the NL, which may be essential for chromatin distribution and nuclear mechanical properties.

Prospective randomized comparative study of the effect of pemafibrate add-on or double statin dose on small dense low-density lipoprotein-cholesterol in patients with type 2 diabetes and hypertriglyceridemia on statin therapy.

AIMS/INTRODUCTION: Small dense low-density lipoprotein (sdLDL) is a more potent atherogenic lipoprotein than LDL. As sdLDL-cholesterol (C) levels are determined by triglyceride and LDL-C levels, pemafibrate and statins can reduce sdLDL-C levels. However, it remains unclear whether adding pemafibrate or increasing statin doses would more effectively reduce sdLDL-C levels in patients receiving statin therapy. MATERIALS AND METHODS: A total of 97 patients with type 2 diabetes and hypertriglyceridemia who were treated with statins were randomly assigned to the pemafibrate 0.2 mg/day addition or statin dose doubled, and followed for 12 weeks. sdLDL-C was measured by our established homogenous assay. RESULTS: The percentage and absolute reductions of sdLDL-C levels were significantly greater in the pemafibrate add-on group than the statin doubling group (-32.8 vs -8.1%; -16 vs -3 mg/dL, respectively). Triglyceride levels were reduced only in the pemafibrate add-on group (-44%), and LDL-C levels were reduced only in the statin doubling group (-8%), whereas levels of non-high-density lipoprotein-C and apolipoprotein B were similarly decreased (7-9%) in both groups. The absolute reductions of sdLDL-C levels were closely associated with decreased triglyceride, LDL-C, non-high-density lipoprotein-C and apolipoprotein B. In the subgroup analysis, the effect of pemafibrate add-on on sdLDL-C reductions was observed irrespective of baseline lipid parameters or statin type. No serious adverse effects were observed in both groups. CONCLUSIONS: In patients with type 2 diabetes and hypertriglyceridemia, the addition of pemafibrate to a statin is superior to doubling a statin in reducing sdLDL-C without increasing adverse effects.

Cranial geometry in patients with dystonia and Parkinson's disease.

Abnormal skull shape has been reported in brain disorders. However, no studies have investigated cranial geometry in neurodegenerative disorders. This study aimed to evaluate the cranial geometry of patients with dystonia or Parkinson's disease (PD). Cranial computed tomography images of 36 patients each with idiopathic dystonia (IDYS), PD, and chronic subdural hematoma (CSDH) were analyzed. Those with IDYS had a significantly higher occipital index (OI) than those with CSDH (p = 0.014). When cephalic index (CI) was divided into the normal and abnormal groups, there was a significant difference between those with IDYS and CSDH (p = 0.000, α = 0.017) and between PD and CSDH (p = 0.031, α = 0.033). The age of onset was significantly correlated with the CI of IDYS (τ = - 0.282, p = 0.016). The Burke-Fahn-Marsden Dystonia Rating Scale motor score (BFMDRS-M) showed a significant correlation with OI in IDYS (τ = 0.372, p = 0.002). The cranial geometry of patients with IDYS was significantly different from that of patients with CSDH. There was a significant correlation between age of onset and CI, as well as between BFMDRS-M and OI, suggesting that short heads in the growth phase and skull balance might be related to the genesis of dystonia and its effect on motor symptoms.

An uncommon midfoot injury, naviculocuneiform and calcaneocuboid fracture dislocation: a case report.

A 57-year-old man fell from a height of 6 m and injured his right foot. Imaging studies showed an uncommon injury; naviculocuneiform and calcaneocuboid joint fracture dislocations. He underwent a temporary fixation with Kirschner wires (K-wires), and the injured foot was immobilized with a below-knee splint. Weight-bearing was started gradually. The K-wires were removed at 8 weeks. Full weight-bearing was achieved 14 weeks after the operation. At 19 weeks, he returned to his carpentry job. At 1 year, he had no marked limitation of daily activities. Early recognition of these injuries is required to prevent persistent foot pain and long-term dysfunction.

Brain abscess in a patient with generalized dystonia after deep brain stimulation: illustrative case.

BACKGROUND: Infections related to deep brain stimulation (DBS) devices are not rare, but abscess formation in brain parenchyma is extremely rare. OBSERVATIONS: A 50-year-old man with generalized dystonia had undergone DBS of bilateral globus pallidus internus. The authors attempted to remove the bilateral DBS system due to repeated device infections caused by metal allergies. However, the intracranial lead had to be left in place, because the lead was strongly adherent to brain parenchyma. Five years later, magnetic resonance imaging showed ring-like enhancement localized around the tip of the intracranial lead, suggesting brain abscess. In response to the symptoms, the remaining left intracranial electrode was removed. Brain abscesses require several months of treatment with appropriate antibiotics, but good outcomes can be achieved with appropriate treatment. LESSONS: Brain abscess is a rare complication of DBS. In the present case, the infection spread from the subcutaneous infected foci to the intracranial area through the lead, resulting in the formation of a brain abscess. Removing as much of the device as possible from the body is therefore important, even if adhesions with brain parenchyma or other tissues are present, because of the risk of serious complications, as seen in this case.

Circadian Rhythm of Subspecies of Low-Density Lipoprotein-Cholesterol and High-Density Lipoprotein-Cholesterol in Healthy Subjects and Patients with Type 2 Diabetes.

AIMS: We established automated assay kits for quantifying small dense low-density lipoprotein (sdLDL)-cholesterol (C), LDL-triglyceride (TG), and high-density lipoprotein (HDL)3-C, and apolipoprotein (apo)E-rich HDL-C, and these have been recognized as sensitive biomarkers for predicting coronary artery disease. We investigated the circadian rhythms of these novel lipids to determine if fasting is required to determine basal levels. METHODS: Forty-eight inpatients with type 2 diabetes and 19 healthy volunteers were studied. Blood samples were collected at seven time points, which were obtained after an overnight fast, before and 2 h after each meal, and before the next breakfast. sdLDL-C, LDL-TG, remnant-like particle (RLP)-C, TG-rich lipoprotein (TRL-C), HDL3-C, and apoE-rich HDL-C were measured by the homogeneous methods. NonHDL-C, large buoyant (lb)LDL-C and HDL2-C were calculated by subtracting sdLDL-C from LDL-C or HDL3-C from HDL-C, respectively. RESULTS: Serum TG levels were significantly increased after meals in both healthy participants and patients with diabetes. RLP-C and TRL-C were also increased postprandially. LDL-TG, LDL-C, nonHDL-C, HDL2,3-C, and apoE-rich HDL-C did not exhibit significant fluctuation during the day in healthy participants and patients with diabetes. sdLDL-C was slightly increased postprandially in subjects with diabetes (1-2 mg/dl, 3%-9%), though its increase was not significant compared to the baseline (fasting) level. Significant postprandial reduction was observed with LDL-C and lbLDL-C. There was no influence of statin therapy or oral anti-diabetes drugs on the circadian rhythm of LDL-C subspecies. CONCLUSIONS: Subtle postprandial increase in sdLDL-C is considered a negligible level in general clinical practice. Fasting is not mandatory to measure basal concentrations of LDL and HDL subspecies.

Emergence of the super antenna effect in mixed crystals of ytterbium and lutetium complexes showing near-infrared luminescence.

The synthesis of luminescent molecular crystalline materials requires a good understanding of the luminescence properties of crystals in which many molecules are densely packed. Previously, we studied the near-infrared (NIR) luminescence of a trivalent ytterbium (Yb(iii)) complex with a Schiff base ligand, tris[2-(5-methylsalicylideneimino)ethyl]amine (H3L). Herein, we extended our study on the Yb complex (YbL) to enhance and understand its solid-state luminescence via mixed crystallization with the lutetium complex (LuL). We prepared (YbL) x (LuL)1-x mixed crystals (x = 0.01, 0.05, 0.1, 0.2, 0.3, 0.5, and 0.7) and studied their NIR luminescence properties. The NIR luminescence intensity per Yb(iii) ion for (YbL)0.01(LuL)0.99 was determined to be two orders of magnitude larger than that for YbL. The excitation spectral shape of (YbL)0.01(LuL)0.99 was different from the absorption spectral shape of YbL but similar to that of LuL. We attribute this observation to the emergence of an intermolecular energy-migration path. In the mixed crystals, LuL molecules acted as a light-harvesting super antenna for Yb(iii) luminescence. Decay measurements of the NIR luminescence for (YbL) x (LuL)1-x with x > 0.2 showed mono-exponential decay, while (YbL) x (LuL)1-x with x < 0.1 showed a grow-in component, which reflected the lifetime of the intermediate state for energy migration. The decay lifetime values tended to increase with decreasing x, suggesting that Yb(iii) isolation resulted in a reduction in concentration quenching. We propose that the luminescence enhancement in the highly Yb-diluted conditions was mainly caused by an increase in the super antenna effect.

Identification of essential intron sequences that enhance gene expression independently of splicing in the yeast Saccharomyces cerevisiae.

Gene expression in eukaryotes is enhanced by the presence of introns in a process known as intron-mediated enhancement (IME), but its mechanism remains unclear. In Saccharomyces cerevisiae, sequences at the 5'-splice sites (SS) and branch point sites (BPS) are highly conserved compared with other higher eukaryotes. Here, the minimum intron sequence essential for IME was investigated using various short introns and a yeast codon-optimized luciferase gene as an IME model. Mutations at the 5'-SS conserved sequence and branch point in the QCR10 intron caused splicing deficiency with either a complete loss or a marked decrease in IME. By contrast, however, the 3'-AG to tG mutant was spliced and retained IME function. Moreover, heterologous introns, which did not show IME in S. cerevisiae, gained splicing competency and IME ability by substitutions to the S. cerevisiae-type 5'-SS and BPS sequences. Intriguingly, several deletion mutants between the 5'-SS and BPS in introns exhibited high levels of IME despite a loss in splicing competency. In most cases, further deletions or substitutions did not recover splicing competency and were found to decrease IME. However, a 16-nt variant consisting of the conserved 5'-SS and BPS sequences and 3'-CAG showed an IME level comparable with that of the wild-type intron. These results indicate that IME can be independent of splicing in S. cerevisiae while intron sequences at the 5'-SS and BPS play an essential role in IME.

Hrd1-dependent Degradation of the Unassembled PIGK Subunit of the GPI Transamidase Complex.

Glycosylphosphatidylinositol (GPI)-anchored proteins are post-transcriptionally modified with GPI and anchored to the plasma membrane. GPI is attached to nascent proteins in the endoplasmic reticulum by the GPI transamidase complex, which consists of PIGT, PIGK, GPAA1, PIGU, and PIGS. Of these, PIGK is a catalytic subunit that is unstable without PIGT. This study investigated the pathway by which unassembled PIGK not incorporated into the complex is degraded. We showed that unassembled PIGK was degraded via the proteasome-dependent pathway and that Hrd1 (also known as SYVN1), a ubiquitin ligase involved in the endoplasmic reticulum-associated degradation pathway, was responsible for degradation of unassembled PIGK.Key words: Glycosylphosphatidylinositol, GPI transamidase complex, protein stability, transamidation, ERAD.

Metagenomic Shotgun Sequencing Analysis of Canalicular Concretions in Lacrimal Canaliculitis Cases.

Lacrimal canaliculitis is a rare infection of the lacrimal canaliculi with canalicular concretions formed by aggregation of organisms. Metagenomic shotgun sequencing analysis using next-generation sequencing has been used to detect pathogens directly from clinical samples. Using this technology, we report cases of successful pathogen detection of canalicular concretions in lacrimal canaliculitis cases. We investigated patients with primary lacrimal canaliculitis examined in the eye clinics of four hospitals from February 2015 to July 2017. Eighteen canalicular concretion specimens collected from 18 eyes of 17 patients were analyzed by shotgun metagenomics sequencing using the MiSeq platform (Illumina). Taxonomic classification was performed using the GenBank NT database. The canalicular concretion diversity was characterized using the Shannon diversity index. This study included 18 eyes (17 patients, 77.1 ± 6.1 years): 82.4% were women with lacrimal canaliculitis; canalicular concretions were obtained from 12 eyes using lacrimal endoscopy and six eyes using canaliculotomy with curettage. Sequencing analysis detected bacteria in all samples (Shannon diversity index, 0.05-1.47). The following genera of anaerobic bacteria (>1% abundance) were identified: Actinomyces spp. in 15 eyes, Propionibacterium spp., Parvimonas spp. in 11 eyes, Prevotella spp. in 9 eyes, Fusobacterium spp. in 6 eyes, Selenomonas spp. in 5 eyes, Aggregatibacter spp. in 3 eyes, facultative and aerobic bacteria such as Streptococcus spp. in 13 eyes, Campylobacter spp. in 6 eyes, and Haemophilus spp. in 3 eyes. The most common combinations were Actinomyces spp. and Streptococcus spp. and Parvinomonas spp. and Streptococcus spp., found in 10 cases. Pathogens were identified successfully using metagenomic shotgun sequencing analysis in patients with canalicular concretions. Canalicular concretions are polymicrobial with anaerobic and facultative, aerobic bacteria.

SPPL3-dependent downregulation of the synthesis of (neo)lacto-series glycosphingolipid is required for the staining of cell surface CD59.

CD59 is a small glycoprotein modified with a glycophosphatidylinositol (GPI) anchor that prevents the formation of the membrane attack complex, thereby protecting host cells from lysis. A previous study identified that cell surface CD59 staining required the intramembrane protease signal peptide peptidase-like 3 (SPPL3). However, the effect of SPPL3 on the staining of CD59 remains unknown. This study shows that SPPL3 is essential for the surface labeling of CD59 but not of major GPI-anchored proteins. Surface CD59 staining requires the intramembrane protease activity of SPPL3 and SPPL3-mediated suppression of the (neo)lacto-series glycosphingolipids (nsGSLs)-but not N-glycan-synthesis pathway. The abundance of nsGSLs may affect complement-dependent cytotoxicity by altering the abundance or accessibility of cell surface CD59.

Long-Term Follow-Up of 12 Patients Treated with Bilateral Pallidal Stimulation for Tardive Dystonia.

Tardive dystonia (TD) is a side effect of prolonged dopamine receptor antagonist intake. TD can be a chronic disabling movement disorder despite medical treatment. We previously demonstrated successful outcomes in six patients with TD using deep brain stimulation (DBS); however, more patients are needed to better understand the efficacy of DBS for treating TD. We assessed the outcomes of 12 patients with TD who underwent globus pallidus internus (GPi) DBS by extending the follow-up period of previously reported patients and enrolling six additional patients. All patients were refractory to pharmacotherapy and were referred for surgical intervention by movement disorder neurologists. In all patients, DBS electrodes were implanted bilaterally within the GPi under general anesthesia. The mean ages at TD onset and surgery were 39.2 ± 12.3 years and 44.6 ± 12.3 years, respectively. The Burke-Fahn-Marsden Dystonia Rating Scale (BFMDRS) performed the preoperative and postoperative evaluations. The average BFMDRS improvement rate at 1 month postoperatively was 75.6 ± 27.6% (p < 0.001). Ten patients were assessed in the long term (78.0 ± 50.4 months after surgery), and the long-term BFMDRS improvement was 78.0 ± 20.4%. Two patients responded poorly to DBS. Both had a longer duration from TD onset to surgery and older age at surgery. A cognitive and psychiatric decline was observed in the oldest patients, while no such decline ware observed in the younger patients. In most patients with TD, GPi-DBS could be a beneficial therapeutic option for long-term relief of TD.

Role of the Drosophila YATA protein in the proper subcellular localization of COPI revealed by in vivo analysis.

yata mutants of Drosophila melanogaster exhibit phenotypes including progressive brain shrinkage, developmental abnormalities and shortened lifespan, whereas in mammals, null mutations of the yata ortholog Scyl1 result in motor neuron degeneration. yata mutation also causes defects in the anterograde intracellular trafficking of a subset of proteins including APPL, which is the Drosophila ortholog of mammalian APP, a causative molecule in Alzheimer's disease. SCYL1 binds and regulates the function of coat protein complex I (COPI) in secretory vesicles. Here, we reveal a role for the Drosophila YATA protein in the proper localization of COPI. Immunohistochemical analyses performed using confocal microscopy and structured illumination microscopy showed that YATA colocalizes with COPI and GM130, a cis-Golgi marker. Analyses using transgenically expressed YATA with a modified N-terminal sequence revealed that the N-terminal portion of YATA is required for the proper subcellular localization of YATA. Analysis using transgenically expressed YATA proteins in which the C-terminal sequence was modified revealed a function for the C-terminal portion of YATA in the subcellular localization of COPI. Notably, when YATA was mislocalized, it also caused the mislocalization of COPI, indicating that YATA plays a role in directing COPI to the proper subcellular site. Moreover, when both YATA and COPI were mislocalized, the staining pattern of GM130 revealed Golgi with abnormal elongated shapes. Thus, our in vivo data indicate that YATA plays a role in the proper subcellular localization of COPI.

Subunits of the GPI transamidase complex localize to the endoplasmic reticulum and nuclear envelope in Drosophila.

A total of 10-20% of plasma membrane proteins are anchored by glycosylphosphatidylinositol (GPI). GPI is attached to proteins by GPI transamidase (GPI-T), which contains five subunits named PIGK, PIGS, PIGT, PIGU, and GPAA1. We previously reported that PIGT localizes near the nucleus in Drosophila. However, localizations of the other four subunits remain unknown. Here, we show that a catalytic subunit of GPI-T, PIGK, mainly localizes to the endoplasmic reticulum (ER), while the other four subunits localize to the nuclear envelope (NE) and ER. The NE/ER localization ratio of PIGS differs between cell types and developmental stages. Our results suggest that GPI-T catalyzes GPI attachment in the ER and the other four subunits may have other unknown functions in the NE.

Short Radiative Lifetime and Non-Triplet Sensitization in Near-Infrared-Luminescent Yb(III) Complex with Tripodal Schiff Base.

We prepared Ln(III) (Ln=Eu, Gd, and Yb) complexes with a tripodal Schiff base, tris[2-(5-methylsalicylideneimino)ethyl]amine (H3 L) and studied their photophysical properties. Upon ligand excitation, YbL showed Yb(III)-centered luminescence in the near-infrared region. While the overall quantum yield (0.60(1)%) of YbL in acetonitrile was moderate among the reported values for Yb(III) complexes, its radiative lifetime (0.33(2) ms) was significantly shorter than those reported previously. We propose that the ligand-to-metal charge-transfer (LMCT) state mediated the sensitization in YbL. The emission and excitation spectra of EuL indicated the participation of the LMCT state in the sensitization. The radiative lifetime (0.84(7) ms) for EuL in the solid state was rather short compared to those of reported Eu(III) complexes. Our results show that the Yb(III) complex with the Schiff base ligand has two features: the short radiative lifetime and the non-triplet sensitization path.