Investigation of Protein - Protein Interactions in Clathrin-Mediated Membrane Transport

Seitdem entdeckt wurde, dass die Rezyklierung synaptischer Vesikel (SV) kinetisch in zwei unterschiedlichen Phasen erfolgt, werden die zugehörigen molekularen Mechanismen kontrovers diskutiert. Diese Studie hatte zum Ziel, die molekularen Details der Rezyklierung SV in kleinen, zentralen Synapsen aufzuklären. Wir haben Methoden zur Inhibition der Clathrin-vermittelten Endozytose in primären, hippokampalen Neuronen entwickelt und deren Effekte auf die Endozytose SV durch Anwendung von FM1-43 Membranfarbstoffen und SynaptopHluorin analysiert. Im Zuge dieser Studien haben wir gefunden, dass der Großteil synaptischer Vesikel AP-2/Clathrin- und Dynamin-abhängig internalisiert wird. Schnelle und langsame Komponenten der SV Endozytose waren gleichermaßen betroffen.Während der grundlegende Mechanismus der Clathrin-vermittelten Endozytose generell gut verstanden ist, blieb bisher ungeklärt, wie spezifische SV Proteine von der Endozytose-Maschinerie erkannt werden. Genetische und biochemische Studien deuten darauf hin, dass das SV Membranprotein Synaptotagmin an der Kopplung von Exo- und Endozytose beteiligt ist. Wir haben Stonin 2 als einen Synaptotagmin-spezifischen endozytotischen Sortierungsadaptor identifiziert, der durch direkte Interaktion mit Synaptotagmin und AP-2 als Bindeglied zwischen Synaptotagmin und der Clathrin-Maschinerie fungiert. Sowohl die Wechselwirkung zwischen Synaptotagmin und Stonin 2 als auch die endozytotische Funktion von Stonin 2 sind abhängig von den Aminosäure-Resten KYE783-785, die in der µ-homologen Domäne (µHD) von Stonin 2 lokalisiert sind. Ferner implizieren unsere Daten einen synergistischen Effekt zwischen den beiden Synaptotagmin C2 Domänen im Hinblick auf Stonin 2 Bindung. Wenn die C2 Domänen allerdings einzeln angeboten werden, interagiert Stonin 2 hauptsächlich mit der C2A Domäne. Die C2B Domäne, wie auch die früher beschriebene direkte Interaktion zwischen AP-2 und Synaptotagmin, sind nicht notwendig für die AP-2/Stonin 2-abhängige Synaptotagmin Internalisierung in Fibroblasten. Wir vermuten, dass Stonin 2 als Linker zwischen Synaptotagmin und AP-2/Clathrin fungiert, indem es direkt mit den C2 Domänen assoziiert.Weiterhin ist es uns gelungen GIT1 (G protein-coupled receptor kinase interacting protein 1) als neuen Stonin 2 Interaktionspartner zu identifizieren. Stonin 2 bindet direkt an die ARF-GAP (ADP ribosylation factor GTPase activating protein) Domäne von GIT1. Das könnte bedeuten, dass Stonin 2 eine regulierende Funktion auf die enzymatische Aktivität von GIT1 ausübt. GIT1 ist an zahlreichen zellulären Vorgängen beteiligt, wie beispielsweise an Umformungen des Zytoskeletts, die zur Zell-Motilität beitragen, an endosomalem Vesikeltransport und an der Synapsenbildung. Eine funktionelle Relevanz der Stonin 2 GIT1 Interaktion in einem oder mehreren dieser Prozesse muss noch nachgewiesen werden

Molecular basis of synaptic vesicle cargo recognition by the endocytic sorting adaptor stonin 2

Journal ArticleSynaptic transmission depends on clathrin-mediated recycling of synaptic vesicles (SVs). How select SV proteins are targeted for internalization has remained elusive. Stonins are evolutionarily conserved adaptors dedicated to endocytic sorting of the SV protein synaptotagmin. Our data identify the molecular determinants for recognition of synaptotagmin by stonin 2 or its Caenorhabditis elegans orthologue UNC-41B

Hybrid inorganic-organic fluorescent silica nanoparticles—influence of dye binding modes on dye leaching

Silica nanoparticles with embedded fluorescent dyes represent an important class of markers for example in biological imaging. We systematically studied the various incorporation mechanisms of fluorescent xanthene dyes in 30–40 nm silica nanoparticles. An important parameter was the interaction of the dye with the matrix material, either by weak electrostatic or strong covalent interactions, which also has implications on the stability of fluorescence and brightness of the dyes. Factors that can influence leaching of dyes such as the position of the dyes in particles and the intensity of the particle-dye interaction were investigated by using the solvatochromic effect of xanthene dyes and by stationary fluorescence anisotropy measurements. We compared uranine and rhodamine B, which were physically embedded, with modified fluorescein isothiocyanate and rhodamine B isothiocyanate, which were covalently bound to the silica matrix within a usual Stöber synthesis. Systematic leaching studies of time spans up to 4 days revealed that covalent bonding of dyes like fluorescein isothiocyanate or rhodamine B isothiocyanate is necessary for fluorescence stability, since dyes bound by physical interaction tend to leach out of porous silica networks. Coverage of silica particles with hydrophobic protection layers of alkyltrialkoxysilanes or hydrophilic polyethylene glycol (PEG) groups resulted in a better retention of physisorbed dyes and provides the possibility to adapt the particles to the polarity of the medium. Best results were archived with PEG groups, but even small trimethylsilyl (TMS) groups already reduce leaching

Changes of radiopacity around implants of different lengths: Five-year follow-up data of a randomized clinical trial

OBJECTIVE To follow-up the radiographic changes in peri-implant bone of short (6 mm, test group) and long (10 mm, control group) single-unit implants five years after loading. MATERIALS AND METHODS Forty-three implants of the test and 44 implants of the control group could be reassessed from 96 originally included implants. Standardized areas of interest (AOI) were defined in the peri-implant bone at pre-defined locations at mid-length on both sides of the implants, and at the apex. An arbitrary mean grey scale value (GSV) was calculated for the AOI after brightness calibration of the radiographs. Changes for GSV were calculated and tested for possible inter- and intra-group differences using the Mann-Whitney and Wilcoxon tests. RESULTS The calculated intra-group differences between baseline and 5 years in the test group accounted for 2.4 ± 19.6 (i.e. slight brightening) and -6.2 ± 20.2 for the control group (i.e. slight shading), which resulted in a statistically significant difference in GSV change (p < .05). Crown-to-implant ratio was the only parameter showing an effect on GSV change (p = .001). CONCLUSIONS Assessing conventional radiographs, longer implants showed a slightly stronger change of radiopacity of the peri-implant bone (slight loss of density) than short ones (slightly enhanced density) after five years of loading

Survival and complication rates of two dental implant systems supporting fixed restorations: 10-year data of a randomized controlled clinical study

OBJECTIVES To compare clinical, radiographic, biological and technical long-term outcomes of two types of dental implants over a period of 10 years. MATERIALS AND METHODS Ninety-eight implants were placed in 64 patients, randomly allocated to one of two manufacturers (AST and STM). All implants were loaded with fixed restorations. Outcome measures were assessed at implant insertion (T$_{i}$), at baseline examination (T$_{L}$), at 1, 3, 5, 8 and 10 (T$_{10}$) years. Data analysis included survival, bone level changes, complications and clinical measures. RESULTS Re-examination was performed in 43 patients (23 AST and 20 STM) at 10 years. The implant level analysis was based on 37 (AST) and 32 (STM) implants. Survival rates of 100% were obtained for both groups. The median changes of the marginal bone levels between baseline and T$_{10}$ (the primary endpoint) amounted to a loss of 0.07 mm for group AST and a gain of 0.37 mm for group STM (intergroup p = 0.008). Technical complications occurred in 27.0% of the implants in group AST and in 15.6% in group STM. The prevalence of peri-implant mucositis was 29.7% (AST) and 50.1% (STM). The prevalence of peri-implantitis amounted to 0% (AST) and 6.3% (STM). CONCLUSIONS Irrespective of the implant system used, the survival rates after 10 years were high. Minimal bone level changes were observed, statistically significant but clinically negligible in favor of STM. Technical complications were more frequently encountered in group AST, while group STM had a higher prevalence of peri-implant mucositis

Histologic analyses of flapless ridge preservation in sockets with buccal dehiscence defects using two alloplastic bone graft substitutes

OBJECTIVES To investigate whether one of two synthetic bone substitute materials used for ridge preservation in the extraction sockets with buccal dehiscence defects was superior regarding new bone formation and ridge preservation and to compare it to sites left for spontaneous healing. MATERIALS AND METHODS In sixteen dogs, P3 and P4 were hemi-sectioned and the respective distal roots were extracted. Following the preparation of a mucoperiosteal flap without vertical releasing incisions, 50% of the buccal bone was carefully removed. The extraction sites were randomly assigned either to a ridge preservation procedure (alloplastic bone substitute material (two test groups)) or to spontaneous healing (control group). Descriptive histology and histomorphometric analyses were performed at healing times of 4, 8, and 16 weeks. In case of homogeneous variances, the results were analyzed by one-way ANOVA, followed by Tukey's post-hoc test. If inhomogeneous, the data was analyzed using Welch-type ANOVA, followed by the Games-Howell post-hoc test. RESULTS The use of bone substitute material led to significantly greater horizontal dimensions amounting to 3.3 mm (SD = 0.67; test 1) and 3.5 mm (SD = 0.72; test 2) compared to spontaneous healing (1.7 mm, SD = 0.23) at 16 weeks of healing (p  0.05). The final ridge profile was more favorable after ridge preservation (p < 0.001) as demonstrated by a loss of 28.8% (spontaneous healing) and an increase in both test groups at 16 weeks (test 1 = 60.5% and test 2 = 31.2%). CONCLUSIONS The use of alloplastic materials rendered greater horizontal dimensions and a more favorable maintenance of the ridge profile. CLINICAL RELEVANCE Alloplastic bone substitute materials can successfully be used for ridge preservation procedures

Early implant placement with or without alveolar ridge preservation in single tooth gaps renders similar esthetic, clinical and patient-reported outcome measures: One-year results of a randomized clinical trial

OBJECTIVES To test whether early implant placement with alveolar ridge preservation (ARP) results in different esthetic, clinical and patient-reported outcome measures (PROMs) compared with early implant placement without ARP. MATERIAL AND METHODS Seventy-five patients requiring single tooth extraction in the anterior maxilla were recruited. Following tooth extraction, the patients were randomly allocated to three groups: (a) ARP using demineralized bovine bone mineral containing 10% collagen (DBBM-C) covered by a collagen matrix (CM) (n = 25), (b) ARP using DBBM-C covered with a palatal graft (PG) (n = 25) and (c) spontaneous healing (control) (n = 25). Eight weeks after tooth extraction, a CBCT was taken and early implant placement was performed in all patients. Esthetic, clinical and PROMs were evaluated one year post-loading. RESULTS A total of 70 patients were available for re-examination at one year post-loading. The median mid-facial mucosal margin change amounted to -0.02 mm (IQR -0.27-0.46) in the CM group, -0.13 mm (IQR -0.44-0.25) in the PG group and -0.14 mm (IQR -0.29-0.07) in the control group, with no significant differences between the groups. Mean PES scores amounted to 7.0 ± 1.4 in the CM group, 7.1 ± 1.5 in the PG group and 7.3 ± 1.7 in the control group without significant differences between the groups. Plaque, bleeding on probing and probing depth did not differ between treatment groups. PROMs in general revealed no significant differences between the groups. CONCLUSION Early implant placement with ARP using either a collagen matrix or a palatal graft rendered similar esthetic, clinical and PROMs to early implant placement without ARP. When a failing tooth can be replaced with an implant within 2 months after tooth extraction, the added value of ARP might be clinically negligible

Risk Characteristics of Peri-Implant Infections: A Retrospective Evaluation in a University Consultation Setting

Peri-implantitis is a common biological complication in dentistry. The aim of the present study was to retrospectively analyze risk characteristics in a group of patients referred to a university-based consultation for peri-implantitis. In all, 190 initial cases from 2010 to 2019 were evaluated and descriptively summarized. The evaluation included various parameters such as periodontitis, smoking and oral hygiene status, implant position, type of prosthetic restoration and retention, mucosal quality, and further anamnestic and clinical findings related to the potential risk of developing peri-implantitis. Peri-implantitis was diagnosed in 83% of the cases, with peri-implant mucositis alone in 16% of cases; furthermore, 38% of the patients were diagnosed with active/instable periodontitis, while 14% had stable periodontitis. Residual cement was considered as a potential co-factor of peri-implant inflammation in 43% of cases. Suboptimal implant positioning was found in 19% of patients. Peri-implantitis or peri-implant mucositis was present in about one-third of patients in the absence of smoking and periodontitis factors. Of note, 6% showed no identifiable risk factors. Factors related to an increased risk of peri-implantitis should be taken into consideration when planning implant treatment. Adequate prosthetic implant position, restoration, and cleanability remain important for long-term success

Gray’s revised Reinforcement Sensitivity Theory in relation to Attention-Deficit/Hyperactivity and Tourette-like behaviors in the general population

Attention-Deficit/Hyperactivity Disorder (ADHD) and Tourette Syndrome (TS) present as distinct conditions clinically; however, they show comorbidity and inhibitory control deficits have been proposed to underlie both. The role of reinforcement sensitivity in ADHD has been studied previously, but no study has addressed this in relation to TS-like behaviors in the general population. The present study examined these associations within the remit of the revised Reinforcement Sensitivity Theory (rRST). One hundred and thirty-eight participants completed psychometric measures of the rRST, and self-report checklists for ADHD- and TS-like behaviors