Autophagy Modulation and Its Implications on Glioblastoma Treatment.

Autophagy is a vital cellular process that functions to degrade and recycle damaged organelles into basic metabolites. This allows a cell to adapt to a diverse range of challenging conditions. Autophagy assists in maintaining homeostasis, and it is tightly regulated by the cell. The disruption of autophagy has been associated with many diseases, such as neurodegenerative disorders and cancer. This review will center its discussion on providing an in-depth analysis of the current molecular understanding of autophagy and its relevance to brain tumors. We will delve into the current literature regarding the role of autophagy in glioma pathogenesis by exploring the major pathways of JAK2/STAT3 and PI3K/AKT/mTOR and summarizing the current therapeutic interventions and strategies for glioma treatment. These treatments will be evaluated on their potential for autophagy induction and the challenges associated with their utilization. By understanding the mechanism of autophagy, clinical applications for future therapeutics in treating gliomas can be better targeted.

Identification of the trade-offs/synergies between rural landscape services in a spatially explicit way for sustainable rural development.

Rural problems are becoming increasingly prominent in sustainable development in China. Landscape services (LSs) are an emerging concept associated with ecosystem services and play an important role in promoting sustainable development. However, a comprehensive framework of rural LS and empirical research are still lacking. Here, we proposed a rural LSs system based on its multiple functions of "ecological maintenance, agricultural production and human living", including 9 LS types. Combined with the impact of landscape pattern on LSs, we optimized the assessment method of LS, and the LS capabilities were assessed at the village scale in Hangzhou city. Furthermore, we identified the spatial differentiation of rural LSs capabilities and trade-offs/synergies along urban-rural and terrain gradients. The results showed that the LSs capabilities of the rural landscape were greatly affected by the urban radiation and terrain, and we found that the turning point of LSs capabilities was 35 km from the urban fringe and the terrain gradient T5. The LSs in most villages (77%) revealed trade-offs, and the villages with LS synergies largely occurred in the middle and eastern regions of the study area. This study could improve our current understanding of LSs in regard to sustainable rural development, and identifying the trade-offs/synergies of LSs in a spatially explicit way can provide suggestions for the differentiated management of rural landscape sustainability.

The Intracranial Aneurysm Gene THSD1 Connects Endosome Dynamics to Nascent Focal Adhesion Assembly.

BACKGROUND/AIMS: We recently discovered that harmful variants in THSD1 (Thrombospondin type-1 domain-containing protein 1) likely cause intracranial aneurysm and subarachnoid hemorrhage in a subset of both familial and sporadic patients with supporting evidence from two vertebrate models. The current study seeks to elucidate how THSD1 and patient-identified variants function molecularly in focal adhesions. METHODS: Co-immunostaining and co-immunoprecipitation were performed to define THSD1 subcellular localization and interacting partners. Transient expression of patient-identified THSD1 protein variants and siRNA-mediated loss-of-function THSD1 were used to interrogate gene function in focal adhesion and cell attachment to collagen I in comparison to controls. RESULTS: THSD1 is a novel nascent adhesion protein that co-localizes with several known markers such as FAK, talin, and vinculin, but not with mature adhesion marker zyxin. Furthermore, THSD1 forms a multimeric protein complex with FAK/talin/vinculin, wherein THSD1 promotes talin binding to FAK but not to vinculin, a key step in nascent adhesion assembly. Accordingly, THSD1 promotes mature adhesion formation and cell attachment, while its rare variants identified in aneurysm patients show compromised ability. Interestingly, THSD1 also localizes at different stages of endosomes. Clathrin-mediated but not caveolae-mediated endocytosis pathway is involved in THSD1 intracellular trafficking, which positively regulates THSD1-induced focal adhesion assembly, in contrast to the traditional role of endosomes in termination of integrin signals. CONCLUSIONS: The data suggest that THSD1 functions at the interface between endosome dynamics and nascent focal adhesion assembly that is impaired by THSD1 rare variants identified from intracranial aneurysm patients.

THSD1 (Thrombospondin Type 1 Domain Containing Protein 1) Mutation in the Pathogenesis of Intracranial Aneurysm and Subarachnoid Hemorrhage.

BACKGROUND AND PURPOSE: A ruptured intracranial aneurysm (IA) is the leading cause of a subarachnoid hemorrhage. This study seeks to define a specific gene whose mutation leads to disease. METHODS: More than 500 IA probands and 100 affected families were enrolled and clinically characterized. Whole exome sequencing was performed on a large family, revealing a segregating THSD1 (thrombospondin type 1 domain containing protein 1) mutation. THSD1 was sequenced in other probands and controls. Thsd1 loss-of-function studies in zebrafish and mice were used for in vivo analyses and functional studies performed using an in vitro endothelial cell model. RESULTS: A nonsense mutation in THSD1 was identified that segregated with the 9 affected (3 suffered subarachnoid hemorrhage and 6 had unruptured IA) and was absent in 13 unaffected family members (LOD score 4.69). Targeted THSD1 sequencing identified mutations in 8 of 507 unrelated IA probands, including 3 who had suffered subarachnoid hemorrhage (1.6% [95% confidence interval, 0.8%-3.1%]). These THSD1 mutations/rare variants were highly enriched in our IA patient cohort relative to 89 040 chromosomes in Exome Aggregation Consortium (ExAC) database (P<0.0001). In zebrafish and mice, Thsd1 loss-of-function caused cerebral bleeding (which localized to the subarachnoid space in mice) and increased mortality. Mechanistically, THSD1 loss impaired endothelial cell focal adhesion to the basement membrane. These adhesion defects could be rescued by expression of wild-type THSD1 but not THSD1 mutants identified in IA patients. CONCLUSIONS: This report identifies THSD1 mutations in familial and sporadic IA patients and shows that THSD1 loss results in cerebral bleeding in 2 animal models. This finding provides new insight into IA and subarachnoid hemorrhage pathogenesis and provides new understanding of THSD1 function, which includes endothelial cell to extracellular matrix adhesion.

Molecular Insights of SARS-CoV-2 Infection and Molecular Treatments.

The coronavirus disease emerged in December 2019 (COVID-19) is caused by Severe Acute Respiratory Syndrome-related coronavirus 2 (SARS-CoV-2). Its rapid global spread has brought an international health emergency and urgent responses for seeking efficient prevention and therapeutic treatment. This has led to imperative needs for illustration of the molecular pathogenesis of SARS-CoV-2, identification of molecular targets or receptors, and development of antiviral drugs, antibodies, and vaccines. In this study, we investigated the current research progress in combating SARS-CoV-2 infection. Based on the published research findings, we first elucidated, at the molecular level, SARS-CoV-2 viral structures, potential viral host-cell-invasion, pathogenic mechanisms, main virus-induced immune responses, and emerging SARS-CoV-2 variants. We then focused on the main virus- and host-based potential targets and summarized and categorized effective inhibitory molecules based on drug development strategies for COVID-19 that can guide efforts for the identification of new drugs and treatment for this problematic disease. Current research and development of antibodies and vaccines were also introduced and discussed. We concluded that the main virus entry route- SARS-CoV-2 spike protein interaction with ACE2 receptors played a key role in guiding the development of therapeutic treatments against COVID-19. Four main strategies may be considered in developing molecular therapeutics, and drug repurposing is likely to be an easy, fast and low-cost approach in such a short period of time with urgent need of antiviral drugs. Additionally, the quick development of antibody and vaccine candidates has yielded promising results, but the wide-scale deployment of safe and effective COVID-19 vaccines remains paramount in solving the pandemic crisis. As new variants of the virus emerge, the efficacy of these vaccines and treatments must be closely evaluated. Finally, we discussed the possible challenges of developing molecular therapeutics for COVID-19 and suggested some potential future efforts. Despite the limited availability of literature, our attempt in this work to provide a relatively comprehensive overview of current SARS-CoV-2 studies can be helpful for quickly acquiring the key information of COVID-19 and further promoting this important research to control and diminish the pandemic.

A systematic coupling analysis framework and multi-stage interaction mechanism between urban land use efficiency and ecological carrying capacity.

Since urban land use efficiency (ULUE) bridges urbanization and economic efficiency while ecological carrying capacity (ECC) is the basic natural endowments support, the coupling coordination degree (CCD) between ULUE and ECC represents a combination of resource-intensive and environment-friendly, which can serve as an effective tool to evaluate sustainable development. We first quantified ULUE and ECC by super-efficiency DEA, DPSIR framework, and entropy-TOPSIS from a coupling perspective, attempting to compensate for the lack of clarity regarding urban sustainability constraint factors in the holistic perspective. On this basis, we formulate an integrated coupling coordination analysis framework comprising temporal and spatial characteristics, disorder diagnosis and interaction mechanism to synthesize the current scattered research directions into a logically clear framework and serve as a guide for future research on coupling. Moreover, to extend the macroscopic mechanism to a microscopic level at a theoretical level and facilitate more effective and sustainable urban management practices, this paper highlights a detailed multi-stage coupling mechanism corresponding to different stages of urban development, deriving an urban sustainable development spiral upward model. The results indicated that the CCD between ULUE and ECC exhibits a significant clustering pattern accompanied by a spatial spillover effect, which was closely related to economic development level and natural resource endowment. Besides, the disorder factor in the eastern Jilin province was ULUE while the western was ECC. Furthermore, the ULUE will take precedence over ECC breaking the old balance, in which technological innovation is the internal driving factor. These findings also illustrate the analysis framework and coupling mechanism mentioned in this paper can act as a nexus between interdisciplinary perspectives to enhance our understanding of changing social-ecological systems, thus serving urban sustainable development.

Combined treatment with niclosamide and camptothecin enhances anticancer effect in U87 MG human glioblastoma cells.

Glioblastoma multiforme (GBM) is one of the deadliest cancers of the brain. Its ability to infiltrate healthy brain tissues renders it difficult to remove surgically. Furthermore, it exhibits high rates of radio- and chemoresistance, making the survival rates of patients with GBM poor. Therefore, novel effective therapies for GBM remain urgently in demand. Niclosamide is an anti-helminthic drug and recently it has been receiving attention due to its reported anticancer effects in cancer models, including GBM. Furthermore, camptothecin (CPT) is a naturally-occurring alkaloid and has been previously reported to be a potential chemotherapeutic agent by targeting the nuclear topoisomerase I. In the present study, the possible combined chemotherapeutic effects of niclosamide and CPT on the human glioblastoma cell line U87 MG was investigated by MTT assay and western blot analysis. Niclosamide exhibited synergistic activities with CPT to suppress the proliferation of U87 MG cells. Additionally, niclosamide suppressed cell proliferation and induced cell death mainly by triggering ER stress and autophagy, whilst CPT induced cell apoptosis mainly through p53-mediated mitochondrial dysfunction and activation of the MAPK (ERK/JNK) pathways. Overall, these findings suggest that co-administration of niclosamide and CPT may provide a novel therapeutic treatment strategy for GBM.

The NMDA receptor NR1 subunit is critically involved in the regulation of NMDA receptor activity by C-terminal Src kinase (Csk).

Previous studies have shown that Csk plays critical roles in the regulation of neural development, differentiation and glutamate-mediated synaptic plasticity. It has been found that Csk associates with the NR2A and 2B subunits of N-methyl-D-aspartate receptors (NMDARs) in a Src activity-dependent manner and serves as an intrinsic mechanism to provide a "brake" on the induction of long-term synaptic potentiation (LTP) mediated by NMDARs. In contrast to the NR2A and 2B subunits, no apparent tyrosine phosphorylation is found in the NR1 subunit of NMDARs. Here, we report that Csk can also associate with the NR1 subunit in a Src activity-dependent manner. The truncation of the NR1 subunit C-tail which contains only one tyrosine (Y837) significantly reduced the Csk association with the NR1-1a/NR2A receptor complex. Furthermore, we found that either the truncation of NR2A C-tail at aa 857 or the mutation of Y837 in the NR1-1a subunit to phenylalanine blocked the inhibition of NR1-1a/NR2A receptors induced by intracellular application of Csk. Thus, both the NR1 and NR2 subunits are required for the regulation of NMDAR activity by Csk.

Heavy metal contamination risk assessment and correlation analysis of heavy metal contents in soil and crops.

Heavy metal pollution is a notable threat to agricultural production. Soil heavy metal pollution can cause potential ecological risk (ERI), and crop heavy metal pollution can cause human health risk (HRI). However, most previous studies partially focused on heavy metal pollution in soil or crop but often neglected the relationship between them. Actually, soil heavy metal can pollute crops to some extent, while not all heavy metal pollution in crops comes from soil. The inner relationship of pollution risk in soil-crop system is worth attention. In this study, we selected Ningbo as the study region and used sample data to assess both soil and crop heavy metal risks, in order to explore the differences between heavy metal contamination risks in soil and crops as well as the relationships between heavy metal contents in soil and crops. Our results showed that Hg was the most polluted heavy metal in soil, which led to the highest ecological risk in Jiangbei (Comprehensive ERI = 567) with the maximum ERI of Hg (430). However, As in crops contributed the most to health risk and caused the highest health risk in Fenghua (HRI = 10) with the largest contribution of 64.5%. Such differences of pollution risk assessment indicated that the contents of the same heavy metal were inconsistent in soil and crops. Our results further showed that the heavy metals in soil had the greatest influence on Zn in crops. Pb and Cr in soil had synergistic effects on the crop absorption of Zn, whereas As, Hg and Cu played antagonistic roles in the crop absorption of Zn. Our study confirms that heavy metals in soil would variously influence heavy metals in crops and the interaction of heavy metals is very important for pollution risk control, which have been largely ignored yet.

Zinc-chelating postsynaptic density-95 N-terminus impairs its palmitoyl modification.

Chemical synaptic transmission represents the most sophisticated dynamic process and is highly regulated with optimized neurotransmitter balance. Imbalanced transmitters can lead to transmission impairments, for example, intracellular zinc accumulation is a hallmark of degenerating neurons. However, the underlying mechanisms remain elusive. Postsynaptic density protein-95 (PSD-95) is a primary postsynaptic membrane-associated protein and the major scaffolding component in the excitatory postsynaptic densities, which performs substantial functions in synaptic development and maturation. Its membrane association induced by palmitoylation contributes largely to its regulatory functions at postsynaptic sites. Unlike other structural domains in PSD-95, the N-terminal region (PSD-95NT) is flexible and interacts with various targets, which modulates its palmitoylation of two cysteines (C3/C5) and glutamate receptor distributions in postsynaptic densities. PSD-95NT contains a putative zinc-binding motif (C2H2) with undiscovered functions. This study is the first effort to investigate the interaction between Zn2+ and PSD-95NT. The NMR titration of 15 N-labeled PSD-95NT by ZnCl2 was performed and demonstrated Zn2+ binds to PSD-95NT with a binding affinity (Kd ) in the micromolar range. The zinc binding was confirmed by fluorescence and mutagenesis assays, indicating two cysteines and two histidines (H24, H28) are critical residues for the binding. These results suggested the concentration-dependent zinc binding is likely to influence PSD-95 palmitoylation since the binding site overlaps the palmitoylation sites, which was verified by the mimic PSD-95 palmitoyl modification and intact cell palmitoylation assays. This study reveals zinc as a novel modulator for PSD-95 postsynaptic membrane association by chelating its N-terminal region, indicative of its importance in postsynaptic signaling.

Characterization of neuronal Src kinase purified from a bacterial expression system.

Neuronal Src (n-Src) is an alternative isoform of Src kinase containing a 6-amino acid insert in the SH3 domain that is highly expressed in neurons of the central nervous system (CNS). To investigate the function of n-Src, wild-type n-Src, constitutively active n-Src in which the C-tail tyrosine 535 was mutated to phenylalanine (n-Src/Y535F) and inactive n-Src in which the lysine 303 was mutated to arginine in addition to the mutation of Y535F (n-Src/K303R/Y535F), were expressed and purified from Escherichia coli BL21(DE3) cells. We found that all three types of n-Src constructs expressed at very high yields (∼500 mg/L) at 37°C, but formed inclusion bodies. In the presence of 8M urea these proteins could be solubilized, purified under denaturing conditions, and subsequently refolded in the presence of arginine (0.5M). These Src proteins were enzymatically active except for the n-Src/K303R/Y535F mutant. n-Src proteins expressed at 18°C were soluble, albeit at lower yields (∼10-20 mg/L). The lowest yields were for n-Src/Y535F (∼10 mg/L) and the highest for n-Src/K303R/Y535F (∼20 mg/L). We characterized the purified n-Src proteins expressed at 18°C. We found that altering n-Src enzyme activity either pharmacologically (e.g., application of ATP or a Src inhibitor) or genetically (mutation of Y535 or K303) was consistently associated with changes in n-Src stability: an increase in n-Src activity was coupled with a decrease in n-Src stability and vice versa. These findings, therefore, indicate that n-Src activity and stability are interdependent. Finally, the successful production of functionally active n-Src in this study indicates that the bacterial expression system may be a useful protein source in future investigations of n-Src regulation and function.

Measuring the Urban Particulate Matter Island Effect with Rapid Urban Expansion.

Rapid urbanization has posed numerous negative impacts on the environment, including fine particulate matter (PM2.5) pollution. However, quantitative investigations of the PM2.5 concentration trends over an urban-rural gradient at the local level are still lacking. The urban particulate matter island (UPI) effect, representing the phenomenon that high particle concentrations in urban areas are gradually attenuated to surrounding areas, was adopted and modified in this paper to study the Hangzhou Bay area from 2000 to 2015. We found the following: (1) every urban area in the Hangzhou Bay area experienced rapid expansion, especially during 2000-2005; (2) more than half of the urban areas suffered UPI problems, and these urban areas had relatively high and stable UPI intensity (UPII) values, although the UPI footprint (UPIFP) values decreased with urban expansion; and (3) urban areas could be divided into three categories: plain areas, hilly areas and the junction of plains and hills, and the probability of the UPI effect varied significantly for different categories. This paper can compensate for the lack of research on the UPI effect at the local level and provide scientific evidence for air pollution control during urban agglomeration planning.

Injectable thermosensitive alginate/ß-tricalcium phosphate/aspirin hydrogels for bone augmentation.

In this study, an injectable and thermo-sensitive alginate/ß-tricalcium phosphate hydrogel (TSAH/ß-TCP) was prepared for aspirin release to a bone defect. Aspirin was dissolved into a mixture of poly(N-isopropylacrylamide) (PNIPAAm), an aminated alginate-g-PNIPAAm co-polymer, and ß-TCP powders. Scanning electron microscopy showed that TSAH/ß-TCP had an interconnected porous microstructure with a porosity of 86.78%. The cross-linked hydrogel released approximately 40% of the aspirin in the first 3 days and then slowly released the remainder. At a low concentration (≤100 µg/mL), aspirin did not promote cell proliferation, but enhanced the alkaline phosphatase activity, and osteocalcin (OCN) and collagen I expression of human bone marrow-derived mesenchymal stem cells. The TSAH/ß-TCP/aspirin hydrogel was injected into the periosteum of the rat cranial bone, and its in vivo bone-forming ability was evaluated at 12 weeks. A bone morphogenetic protein 2 (BMP-2)-loaded TSAH/ß-TCP hydrogel was injected as a control. Micro-computed tomography showed that the percentage of mineralized tissue in the TSAH/ß-TCP/BMP-2 and TSAH/ß-TCP/aspirin groups were similar and significantly higher than that in the TSAH/ß-TCP group. Immunohistochemical staining showed considerable expression of OCN, especially in the TSAH/ß-TCP/BMP-2 and TSAH/ß-TCP/aspirin groups. These results suggest that the injectable TSAH/ß-TCP/aspirin hydrogel has great potential for bone regeneration. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 1739-1751, 2018.

Hippocampal protein kinase D1 is necessary for DHPG-induced learning and memory impairments in rats.

BACKGROUND: Understanding molecular mechanisms underlying the induction of learning and memory impairments remains a challenge. Recent investigations have shown that the activation of group I mGluRs (mGluR1 and mGluR5) in cultured hippocampal neurons by application of (S)-3,5-Dihydroxyphenylglycine (DHPG) causes the regulated internalization of N-methyl-D-aspartate receptors (NMDARs), which subsequently activates protein kinase D1 (PKD1). Through phosphorylating the C-terminals of the NMDAR GluN2 subunits, PKD1 down-regulates the activity of remaining (non-internalized) surface NMDARs. The knockdown of PKD1 does not affect the DHPG-induced inhibition of AMPA receptor-mediated miniature excitatory post-synaptic currents (mEPSCs) but prevents the DHPG-induced inhibition of NMDAR-mediated mEPSCs in vitro. Thus, we investigated the in vivo effects of bilateral infusions of DHPG into the hippocampal CA1 area of rats in the Morris water maze (MWM) and the novel object discrimination (NOD) tests. METHODS: A total of 300 adult male Sprague Dawley rats (250-280 g) were used for behavioral tests. One hundred ninety four were used in MWM test and the other 106 rats in the NOD test. Following one week of habituation to the vivarium, rats were bilaterally implanted under deep anesthesia with cannulas aimed at the CA1 area of the hippocampus (CA1 coordinates in mm from Bregma: AP -3.14; lateral +/-2; DV -3.0). Through implanted cannulas artificial cerebrospinal fluid (ACSF), the group1 mGluR antagonist 6-Methyl-2-(phenylethynyl)pyridine (MPEP), the dynamin-dependent internalization inhibitor Dynasore, or the PKD1 inhibitor CID755673 were infused into the bilateral hippocampal CA1 areas (2 µL per side, over 5 min). The effects of these infusions and the effects of PKD1 knockdown were examined in MWM or NOD test. RESULTS: DHPG infusion increased the latency to reach the platform in the MWM test and reduced the preference for the novel object in the NOD task. We found that the DHPG effects were dose-dependent and could be maintained for up to 2 days. Notably, these effects could be prevented by pre-infusion of the group1 mGluR antagonist MPEP, the dynamin-dependent internalization inhibitor Dynasore, the PKD1 inhibitor CID755673, or by PKD1 knockdown in the hippocampal CA1 area. CONCLUSION: Altogether, these findings provide direct evidence that PKD1-mediated signaling may play a critical role in the induction of learning and memory impairments by DHPG infusion into the hippocampal CA1 area.

[Four year's clinical evaluation of glass fiber reinforced resin-bonded fixed partial denture as a periodontal splint to replace lost anterior teeth].

OBJECTIVE: To evaluate the clinical performance of glass fiber reinforced resin-bonded fixed partial denture(GFR-RBFPD) as a periodontal splint on abutment teeth with reduced periodontal support. METHODS: Thirty fixed-fixed GFR-RBFPD were delivered to restore anterior partial edentulous dentitions. The adjacent abutment teeth had severely reduced periodontal support and were not indicated for full crown retained FPD. The success rate and functional survival rate had been recorded and the periodontal condition had been evaluated for 4 years. The results were statistically analyzed with single factor variance analysis and chi square test(α=0.05). RESULTS: In the first, second, and third year following restoration, one connector fractured in each year and they were repaired with adhesive resin and the pontics were kept in place for function. In the third and fourth years after restoration, there was one pontic displacement with fracture of connectors. The total survive rate was 83%(25/30) and the functional survival rate was 93% (28/30) at the end of the fourth year. The main reason of failure was fracture of connector. About 22%(13/60) of the adjacent teeth showed marginal bone height decrease, while the other 78% (47/60) increased with statistic significant difference from one year after the restoration to the end of the observation term(P<0.05). The periodontal condition of the adjacent teeth was improved after the restoration. CONCLUSIONS: The four years clinical evaluation indicates that the GFR-RBFPD may be used as fixed prostheses to replace lost one to three anterior teeth with reduced periodontal support of abutment teeth.

Four-Year Clinical Evaluation of GFRC-RBFPDs as Periodontal Splints to Replace Lost Anterior Teeth.

PURPOSE: The aim of this study was to evaluate the clinical performance of glass fiber-reinforced composite-based resin-bonded fixed partial dentures (GFRC-RBFPDs) as periodontal splints for periodontal support-reduced anterior partially edentulous dentition and for replacing lost teeth. MATERIALS AND METHODS: A total of 39 subjects were enrolled who required fixed restorations for lost mandibular anterior teeth where the adjacent teeth offered severely reduced periodontal support. GFRC-RBFPDs were cemented to replace the lost teeth and to stabilize the adjacent teeth. The survival rates were recorded, and the periodontal condition (bone height, bleeding index, and probing depth) was evaluated at 1, 2, 3, and 4 years after the restorations. The results were statistically analyzed with single-factor variance analysis and chi-square tests (α = .05). RESULTS: The complete survival rate was 89.7%, and the functional survival rate was 92.3% at the fourth year. The main reason for failure was fracture of the connector of the GFRC-RBFPDs. In 21.7% of adjacent teeth, the bone height decreased; in the other 78.3%, it increased from 1 year after the restoration to the end of the observation period and the heights were statistically different from the initial values. The periodontal condition of the adjacent teeth was improved after the restoration. CONCLUSIONS: This 4-year clinical evaluation indicated that GFRC-RBFPDs may be useful as fixed prostheses to replace one to three lost anterior teeth with damaged periodontal support in adjacent teeth.

Minimally traumatic alveolar ridge augmentation with a tunnel injectable thermo-sensitive alginate scaffold.

UNLABELLED: Injectable bone substitutes and techniques have been developed for use in minimally invasive procedures for bone augmentation. OBJECTIVE: To develop a novel injectable thermo-sensitive alginate hydrogel (TSAH) as a scaffold to induce bone regeneration, using a minimally invasive tunnelling technique. MATERIAL AND METHODS: An injectable TSAH was prepared from a copolymer solution of 8.0 wt% Poly(N-isopropylacrylamide) (PNIPAAm) and 8.0 wt% AAlg-g-PNIPAAm. In vitro properties of the material, such as its microstructure and the sustained release of recombinant human bone morphogenetic protein-2 (rhBMP-2), were investigated. Then, with the subperiosteal tunnelling technique, this material, carrying rhBMP-2, was injected under the labial periosteum of the maxillary anterior alveolar ridge in a rabbit model. New bone formation was evaluated by means of X-ray, micro-computed tomography (micro-CT), fluorescence labelling, histological study, and immunohistochemistry study. RESULTS: The material exhibited good injectability and thermo-irreversible properties. SEM showed an interconnected porous microstructure of the TSAH. The result of ALP activity indicated sustained delivery of BMP-2 from the TSAH from days 3 to 15. In a rabbit model, both TSAH and TSAH/rhBMP-2 induced alveolar ridge augmentation. The percentage of mineralised tissue in the TSAH/rhBMP-2 group (41.6 ± 3.79%) was significantly higher than in the TSAH group (31.3 ± 7.21%; p < 0.05). The density of the regenerating tissue was higher in the TSAH/rhBMP-2 group than in the other groups (TSAH group, positive control, blank control; p < 0.05). CONCLUSIONS: The TSAH provided convenient handling properties for clinical application. To some extent, TSAH could induce ridge augmentation and mineral deposition, which can be enhanced when combined with rhBMP-2 for a minimally invasive tunnelling injection.

Regulated internalization of NMDA receptors drives PKD1-mediated suppression of the activity of residual cell-surface NMDA receptors.

BACKGROUND: Constitutive and regulated internalization of cell surface proteins has been extensively investigated. The regulated internalization has been characterized as a principal mechanism for removing cell-surface receptors from the plasma membrane, and signaling to downstream targets of receptors. However, so far it is still not known whether the functional properties of remaining (non-internalized) receptor/channels may be regulated by internalization of the same class of receptor/channels. The N-methyl-D-aspartate receptor (NMDAR) is a principal subtype of glutamate-gated ion channel and plays key roles in neuronal plasticity and memory functions. NMDARs are well-known to undergo two types of regulated internalization - homologous and heterologous, which can be induced by high NMDA/glycine and DHPG, respectively. In the present work, we investigated effects of regulated NMDAR internalization on the activity of residual cell-surface NMDARs and neuronal functions. RESULTS: In electrophysiological experiments we discovered that the regulated internalization of NMDARs not only reduced the number of cell surface NMDARs but also caused an inhibition of the activity of remaining (non-internalized) surface NMDARs. In biochemical experiments we identified that this functional inhibition of remaining surface NMDARs was mediated by increased serine phosphorylation of surface NMDARs, resulting from the activation of protein kinase D1 (PKD1). Knockdown of PKD1 did not affect NMDAR internalization but prevented the phosphorylation and inhibition of remaining surface NMDARs and NMDAR-mediated synaptic functions. CONCLUSION: These data demonstrate a novel concept that regulated internalization of cell surface NMDARs not only reduces the number of NMDARs on the cell surface but also causes an inhibition of the activity of remaining surface NMDARs through intracellular signaling pathway(s). Furthermore, modulating the activity of remaining surface receptors may be an effective approach for treating receptor internalization-induced changes in neuronal functions of the CNS.

The regulation of N-methyl-D-aspartate receptors by Src kinase.

Src family kinases (SFKs) play critical roles in the regulation of many cellular functions by growth factors, G-protein-coupled receptors and ligand-gated ion channels. Recent data have shown that SFKs serve as a convergent point of multiple signaling pathways regulating N-methyl-d-aspartate (NMDA) receptors in the central nervous system. Multiple SFK molecules, such as Src and Fyn, closely associate with their substrate, NMDA receptors, via indirect and direct binding mechanisms. The NMDA receptor is associated with an SFK signaling complex consisting of SFKs; the SFK-activating phosphatase, protein tyrosine phosphatase α; and the SFK-inactivating kinase, C-terminal Src kinase. Early studies have demonstrated that intramolecular interactions with the SH2 or SH3 domain lock SFKs in a closed conformation. Disruption of the interdomain interactions can induce the activation of SFKs with multiple signaling pathways involved in regulation of this process. The enzyme activity of SFKs appears 'graded', exhibiting different levels coinciding with activation states. It has also been proposed that the SH2 and SH3 domains may stimulate catalytic activity of protein tyrosine kinases, such as Abl. Recently, it has been found that the enzyme activity of neuronal Src protein is associated with its stability, and that the SH2 and SH3 domain interactions may act not only to constrain the activation of neuronal Src, but also to regulate the enzyme activity of active neuronal Src. Collectively, these findings demonstrate novel mechanisms underlying the regulation of SFKs.

Minimally traumatic alveolar ridge augmentation with a tunnel injectable thermo-sensitive alginate scaffold

Injectable bone substitutes and techniques have been developed for use in minimally invasive procedures for bone augmentation. Objective : To develop a novel injectable thermo-sensitive alginate hydrogel (TSAH) as a scaffold to induce bone regeneration, using a minimally invasive tunnelling technique. Material and Methods : An injectable TSAH was prepared from a copolymer solution of 8.0 wt% Poly(N-isopropylacrylamide) (PNIPAAm) and 8.0 wt% AAlg-g-PNIPAAm. In vitro properties of the material, such as its microstructure and the sustained release of recombinant human bone morphogenetic protein-2 (rhBMP-2), were investigated. Then, with the subperiosteal tunnelling technique, this material, carrying rhBMP-2, was injected under the labial periosteum of the maxillary anterior alveolar ridge in a rabbit model. New bone formation was evaluated by means of X-ray, micro-computed tomography (micro-CT), fluorescence labelling, histological study, and immunohistochemistry study. Results : The material exhibited good injectability and thermo-irreversible properties. SEM showed an interconnected porous microstructure of the TSAH. The result of ALP activity indicated sustained delivery of BMP-2 from the TSAH from days 3 to 15. In a rabbit model, both TSAH and TSAH/rhBMP-2 induced alveolar ridge augmentation. The percentage of mineralised tissue in the TSAH/rhBMP-2 group (41.6±3.79%) was significantly higher than in the TSAH group (31.3±7.21%; p<0.05). The density of the regenerating tissue was higher in the TSAH/rhBMP-2 group than in the other groups (TSAH group, positive control, blank control; p<0.05). Conclusions : The TSAH provided convenient handling properties for clinical application. To some extent, TSAH could induce ridge augmentation and mineral deposition, which can be enhanced when combined with rhBMP-2 for a minimally invasive tunnelling injection. .