Immediate implant loading following computer-guided surgery

AbstractPurposeThe aim of this study was to develop and apply a new method for easy intraoperative adjustment of a provisional fixed full-arch restoration, in order to allow immediate implant loading following computer-guided surgery, regardless of any implant positioning errors compared to the virtual planning.MethodsIn accordance with the NobelGuide™ protocol, a provisional restoration for immediate loading of six maxillary implants was prepared prior to surgery. Because small shifts between the planned and the actual implant positions were to be expected, the provisional restoration was not fabricated directly on temporary cylinders as a conventional one-piece superstructure, but was divided into two portions: six custom made abutments and a long span fixed restoration which were left unconnected. After implantation, the custom abutments were attached to the six implants to be immediately loaded, and the superstructure was cemented simultaneously to all abutments using dual cure resin cement. After the excess cement was cleaned and polished, the superstructure was then reseated. Passive fit was achieved between implants and the superstructure.ConclusionThe superstructure described in this article can be easily seated and adjusted to accommodate any possible shifts in implant positioning occurring during computer-guided surgery. Through this method uneventful immediate implant loading can be achieved in a reasonable operative time

Tuberous Sclerosis 2 Gene Is Expressed at High Levels in Specific Types of Neurons in the Mouse Brain

Tuberous sclerosis (TSC) is an autosomal dominant disorder characterized by mental retardation, epilepsy and hamartomatous growth in many tissues. The gene (TSC2) encoding a tumor suppressor protein whose mutations cause TSC, has been demonstrated to be expressed at high levels in the adult and developing brain, raising the question of whether or not the TSC2 gene product has unique roles in differentiation related to cytoskeletal interactions within the central nervous system, in addition to a tumor suppressor function. To determine the expression of TSC2 in functionally distinct neuron types of the mouse brain, we carried out in situ hybridization with digoxigenin-labeled riboprobes for the detection of TSC2 mRNA. High levels of the TSC2 gene were in neurons of the pyramidal and dentate granular layer in the hippocampus, cerebellar Purkinje cells, neurons of the piriform cortex, motor neurons in the medulla and interneurons in the striatum, while intermediate levels were in cortical neurons, striatal neurons, septal neurons, thalamic neurons and neurons in the substantia nigra compacta. Thus, the high expression of the TSC2 gene has restricted distribution in specific neuronal types which are characterized by well-developed dendrites and rich in use-dependent long-term changes in synaptic efficacy. These results suggest that the function of the TSC2 gene product may be involved on a cellular basis in neuronal plasticity and relevant to mental retardation observed in TSC patients

Evaluation of the Pichia pastoris expression system for the production of GPCRs for structural analysis

<p>Abstract</p> <p>Background</p> <p>Various protein expression systems, such as <it>Escherichia coli </it>(<it>E. coli</it>), <it>Saccharomyces cerevisiae </it>(<it>S. cerevisiae</it>), <it>Pichia pastoris </it>(<it>P. pastoris</it>), insect cells and mammalian cell lines, have been developed for the synthesis of G protein-coupled receptors (GPCRs) for structural studies. Recently, the crystal structures of four recombinant human GPCRs, namely β₂adrenergic receptor, adenosine A_2areceptor, CXCR4 and dopamine D3 receptor, were successfully determined using an insect cell expression system. GPCRs expressed in insect cells are believed to undergo mammalian-like posttranscriptional modifications and have similar functional properties than in mammals. Crystal structures of GPCRs have not yet been solved using yeast expression systems. In the present study, <it>P. pastoris </it>and insect cell expression systems for the human muscarinic acetylcholine receptor M2 subtype (CHRM2) were developed and the quantity and quality of CHRM2 synthesized by both expression systems were compared for the application in structural studies.</p> <p>Results</p> <p>The ideal conditions for the expression of CHRM2 in <it>P. pastoris </it>were 60 hr at 20°C in a buffer of pH 7.0. The specific activity of the expressed CHRM2 was 28.9 pmol/mg of membrane protein as determined by binding assays using [³H]-quinuclidinyl benzilate (QNB). Although the specific activity of the protein produced by <it>P. pastoris </it>was lower than that of Sf9 insect cells, CHRM2 yield in <it>P. pastoris </it>was 2-fold higher than in Sf9 insect cells because <it>P. pastoris </it>was cultured at high cell density. The dissociation constant (Kd) for QNB in <it>P. pastoris </it>was 101.14 ± 15.07 pM, which was similar to that in Sf9 insect cells (86.23 ± 8.57 pM). There were no differences in the binding affinity of CHRM2 for QNB between <it>P. pastoris </it>and Sf9 insect cells.</p> <p>Conclusion</p> <p>Compared to insect cells, <it>P. pastoris </it>is easier to handle, can be grown at lower cost, and can be expressed quicker at a large scale. Yeast, <it>P. pastoris</it>, and insect cells are all effective expression systems for GPCRs. The results of the present study strongly suggested that protein expression in <it>P. pastoris </it>can be applied to the structural and biochemical studies of GPCRs.</p

Identification of a novel SEREX antigen family, ECSA, in esophageal squamous cell carcinoma

<p>Abstract</p> <p>Background</p> <p>Diagnosis of esophageal squamous cell carcinoma (SCC) may improve with early diagnosis. Currently it is difficult to diagnose SCC in the early stage because there is a limited number of tumor markers available.</p> <p>Results</p> <p>Fifty-two esophageal SCC SEREX antigens were identified by SEREX (serological identification of antigens by recombinant cDNA expression cloning) using a cDNA phage library and sera of patients with esophageal SCC. Sequence analysis revealed that three of these antigens were similar in amino acid sequences, and they were designated as ECSA (esophageal carcinoma SEREX antigen)-1, -2 and -3. The ECSA family was also similar to an EST clone, hepatocellular carcinoma-associated antigen 25a (HCA25a). Serum antibody levels to ECSA-1, -2 and -3 were significantly higher in patients with esophageal SCC than in healthy donors. Based on the conserved amino acid sequences, three peptides were synthesized and used for enzyme-linked immunosorbent assays (ELISA). The serum antibody levels against one of these peptides were significantly higher in patients with esophageal SCC. This peptide sequence was also conserved in FAM119A, GOSR1 and BBS5, suggesting that these are also ECSA family members. Reverse transcription followed by quantitative PCR analysis showed that the mRNA expression levels of ECSA-1, -2 and -3 and FAM119A but not of HCA25a, GOSR1 and BBS5 were frequently elevated in esophageal SCC tissues.</p> <p>Conclusions</p> <p>We have identified a new gene family designated ECSA. Serum antibodies against the conserved domain of the ECSA family may be a promising tumor marker for esophageal SCC.</p

Comparing serial X-ray crystallography and microcrystal electron diffraction (MicroED) as methods for routine structure determination from small macromolecular crystals.

Innovative new crystallographic methods are facilitating structural studies from ever smaller crystals of biological macromolecules. In particular, serial X-ray crystallography and microcrystal electron diffraction (MicroED) have emerged as useful methods for obtaining structural information from crystals on the nanometre to micrometre scale. Despite the utility of these methods, their implementation can often be difficult, as they present many challenges that are not encountered in traditional macromolecular crystallography experiments. Here, XFEL serial crystallography experiments and MicroED experiments using batch-grown microcrystals of the enzyme cyclophilin A are described. The results provide a roadmap for researchers hoping to design macromolecular microcrystallography experiments, and they highlight the strengths and weaknesses of the two methods. Specifically, we focus on how the different physical conditions imposed by the sample-preparation and delivery methods required for each type of experiment affect the crystal structure of the enzyme

Structure of the dopamine D2 receptor in complex with the antipsychotic drug spiperone

統合失調症に関わるドパミン受容体の構造解明 --副作用を抑えた薬の迅速な探索・設計が可能に--. 京都大学プレスリリース. 2020-12-24.In addition to the serotonin 5-HT2A receptor (5-HT2AR), the dopamine D2 receptor (D2R) is a key therapeutic target of antipsychotics for the treatment of schizophrenia. The inactive state structures of D2R have been described in complex with the inverse agonists risperidone (D2Rris) and haloperidol (D2Rhal). Here we describe the structure of human D2R in complex with spiperone (D2Rspi). In D2Rspi, the conformation of the extracellular loop (ECL) 2, which composes the ligand-binding pocket, was substantially different from those in D2Rris and D2Rhal, demonstrating that ECL2 in D2R is highly dynamic. Moreover, D2Rspi exhibited an extended binding pocket to accommodate spiperone’s phenyl ring, which probably contributes to the selectivity of spiperone to D2R and 5-HT2AR. Together with D2Rris and D2Rhal, the structural information of D2Rspi should be of value for designing novel antipsychotics with improved safety and efficacy

Targeted deletion of the C-terminus of the mouse adenomatous polyposis coli tumor suppressor results in neurologic phenotypes related to schizophrenia

Background: Loss of adenomatous polyposis coli (APC) gene function results in constitutive activation of the canonical Wnt pathway and represents the main initiating and rate-limiting event in colorectal tumorigenesis. APC is likely to participate in a wide spectrum of biological functions via its different functional domains and is abundantly expressed in the brain as well as in peripheral tissues. However, the neuronal function of APC is poorly understood. To investigate the functional role of Apc in the central nervous system, we analyzed the neurological phenotypes of Apc 1638T/1638T mice, which carry a targeted deletion of the 3′ terminal third of Apc that does not affect Wnt signaling. Results: A series of behavioral tests revealed a working memory deficit, increased locomotor activity, reduced anxiety-related behavior, and mildly decreased social interaction in Apc 1638T/1638T mice. Apc 1638T/1638T mice showed abnormal morphology of the dendritic spines and impaired long-term potentiation of synaptic transmission in the hippocampal CA1 region. Moreover, Apc 1638T/1638T mice showed abnormal dopamine and serotonin distribution in the brain. Some of these behavioral and neuronal phenotypes are related to symptoms and endophenotypes of schizophrenia. Conclusions: Our results demonstrate that the C-terminus of the Apc tumor suppressor plays a critical role in cognitive and neuropsychiatric functioning. This finding suggests a potential functional link between the C-terminus of APC and pathologies of the central nervous system