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%;

Neurology Problem Based Learning with Limited Neurologist Resources is Feasible

Objective: To determine if a single neurologist can lead neurology PBL for an entire medical school class and assess the effect of a neurologist assisting in facilitating during neurology module problem-based learning on student perception of knowledge gained. Background: Problem-based learning (PBL) groups are widely becoming integrated into medical school curriculum. During each module, clinicians from the specialty area of study help conduct PBL and facilitate students through cases. For example, during the neurology module, neurologist facilitate cases pertaining to their study. At the University of Texas Rio Grande Valley (UTRGV), there is a shortage of neurologists and it is not possible to have one neurologist lead multiple groups at once. Neurology PBL groups are therefore facilitated by non-physicians. The neurologist serves a few functions: 1) to create a written facilitator guide, 2) to open PBL to the entire class before splitting into small groups, 3) to make rounds during PBL sessions to ensure groups are on track and answer any questions, and 4) to serve as lead teacher during wrap-up of the PBL. Design/Methods: Using module surveys administered by UTRGV, we will compare student perception of PBL. Perceptions will be quantified by how they rated PBL. Data from previous modules will be used to compare surveys from the current neurology module. Results: Feedback from last year’s PBL session showed that 68.00% of students agree that neurology PBL was a useful way to learn about basic science and its application to patient care and clinical practice. Preliminary feedback from this year’s class indicates greater satisfaction with PBL than last year, where there was no neurologist involved, and formal feedback is in progress. Conclusions: Neurology PBL for an entire medical school class with a single neurologist is feasible, and appears effective based on preliminary feedback. We are awaiting formal feedback

The Role of Zinc in PSD-95 Palmitoyl Modification and NMDAR surface expression.

NMDA receptor in an excitatory ligand-gated ion channel present in the neurons of the central nervous system. Zinc and calcium ions are an important part of synaptic transmission and disruption of these can be seen in neurological diseases. PSD95 is a major protein involved in the stability of NMDAR at the synapse. This protein can be palmitoylated and de-palmitoylated, a process that is thought to be regulated by Zinc. The exact mechanism of how Zinc is involved in the stability (palmitoylation) of the PSD95 and indirectly, the NMDA receptor, is unclear and will be investigated in our research. Our hypothesis is that Zinc de-palmitoylates PSD95 leads to the surface reduction of the NMDR. We will be using HEK293 (human embryonic kidney 293 cells) and transfect them to express NMDAR and PSD95. PSD95 palmitoylation by zinc will be assayed using acyl-biotinylation exchange (ABE) and parallelly NMDA receptor expression will be assayed using biotinylation assay and immunochemistry

The Role of Zinc in PSD-95 Palmitoyl Modification

Postsynaptic density-95 (PSD-95) is a membrane-associated guanylate kinase that mediates localization of receptors in the excitatory postsynaptic density. It has been reported that PSD-95 mediates postsynaptic localization of NMDA receptors and anchors postsynaptic AMPAR receptors mainly through its postsynaptic membrane targeting by its N-terminal palmitoylation. Recent studies have shown that Ca2+/calmodulin blocks palmitoylation of PSD-95 by binding at the N-terminus of PSD-95, which promotes dissociation of PSD-95 from the postsynaptic membrane and causes loss of surface AMPARs in cultured neurons. Another metal ion zinc is found in various areas of the brain. As an endogenous neuromodulator, zinc plays a role in synaptic transmission and is important in the maintenance of postsynaptic density stability. However, whether or not Zn2+ interacts with PSD-95 and regulates PSD-95 modification remain unknown. This study was carried out in human embryonic kidney 293 (HEK-293) cells. Cells were transfected with PSD-95 plasmids. After incubation for 48 hours, the cells were stimulated with 0.1 mM ZnCl2 for 5 min. And then the cells were harvested and the palmitoylation of PSD-95 was assessed using acyl-biotinyl exchange (ABE) method and Western blot. PBS was used in the control group in place of the ZnCl2. Our data showed that zinc stimulation decreased PSD-95 palmitoylation by 40%. The potential effect of the Zn2+ -induced depalmitoylation of PSD-95 will be further studied, such as PSD-95 postsynaptic stability and PSD-95 postsynaptic localization. And more work needs to be done to unveil the mechanism underlying the impact of the depalmitoylation of PSD-95 on the postsynaptic localization of NMDARs and AMPRs in response to zinc stimulation

Characterization of Anti-Cancer properties of Fungal Metabolite Ophiobolin A

Background: Ophiobolin A (Oph A) is a secondary metabolite and a phytotoxin produced by the pathogenic fungi Cochliobolus heterostrophus that causes “southern corn leaf blight” disease in maize via modulation of the calcium binding protein calmodulin. Numerous studies have found antiproliferative effects of Ophiobolin A against a variety of cells including bacteria and various cancers including melanoma, glioma and leukemia. Recent studies have shown that OphA induces paraptosis-like cell death in glioblastoma multiforme (GBM) cells via vacuolization of the cytoplasm and enlargement of the mitochondria and endoplasmic reticulum. Notably, unlike apoptosis, paraptosis cell death lacks DNA fragmentation and activation of caspases, creating a possible mechanism for targeted treatment of malignant brain tissue of GBM patients who have GBM cells that are highly resistant to pro-apoptotic treatments. This study aimed to further characterize the effects of this promising anti-cancer agent on glioblastoma (U118 and U87), breast cancer (MCF7 and T47D), neuroblastoma cells (SH-SY5Y) and rat pheochromocytoma cells (PC12). SH-SY5Y and rat PC12 commonly serve as two popular neuron models to test drug toxicity and viability. Methods: The effects of Ophiobolin A were studied in six cancer cell lines from various tissue types including glioblastoma, breast cancer and rat pheochromocytoma cells: U87, MCF-7, T47D, U118, SH-SY5Y and PC12. Over 4 weeks, cell lines were recovered and cultured until adequate confluence was reached with subculturing and medium refreshing. Once adequate growth and attachment was confirmed with microscopy, cells were seeded in 6-well plates from 100k to 600k and treated with either DMSO or 1 µM of Ophiobolin A. Cell morphology was monitored using inverted microscope at 1 hour, 3 hours, and 6 hours following Ophiobolin A application. Cell survivability was measured using Countess at 6 hours post drug treatment. Results: Similar to previous studies, OphA was found to induce cell apoptosis and decrease cell numbers in glioblastoma cell lines, U87 and U118. As expected, similar results were observed in the breast cancer line MCF7. The impact of Oph A on cell morphology changes demonstrated a time-dependent manner in both glioblastoma cell lines, showing elongated neuronal bodies and cell processes. In MCF7 cells, we observed increased vacuolization after drug treatment at 1 hour, 3 hours, and 6 hours. Cell survival rates were significantly reduced in all tested cancer cells in compared to the control groups. Conclusions: Ophiobolin A has been shown to induce cell apoptosis in glioblastoma cells and breast cancer cells. The effect of OphA on healthy cells and the mechanisms underlying the OphA-induced cell apoptosis will be studied

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

Anti-cancer effect of Cissus quadrangularis on human glioblastoma cells

Objectives Glioblastoma multiforme (GBM) is a common and fatal brain tumour in the central nervous system with a poor survival rate and a median survival time of 15 months only. The standard treatment is aggressive surgical resection followed by radiotherapy and chemotherapy. However, effective drugs available in chemotherapy are limited. This study was designed to evaluate, for the first time, the potential therapeutic effect of Cissus quadrangularis (CQ) in human glioblastoma cells and to investigate its possible mechanisms of action. Methods In this study, we examined the anticancer activity of CQ in human glioblastoma U87 MG cells by cell viability assay, cell migration assay, immunofluorescence staining and Western blot. Results Our results demonstrated that CQ treatment induced U87 cytotoxicity, cell cycle arrest and cell death. The cytotoxicity of CQ mediates ER stress, autophagy and mitochondrial apoptosis by suppressing pro-survival signalling pathways (extracellular signal-regulated kinase and signal transducer and activator of transcription 3 pathways). Conclusions The findings of this study imply that CQ is a promising anti-cancer candidate for the treatment of GBM. Highlights The anticancer effect of Cissus quadrangularis (CQ) was studied in human glioblastoma U87 MG cells. It was demonstrated that CQ treatment induced cytotoxicity, cell cycle arrest and cell death in U87 MG cells. CQ may become a potential chemotherapy component for the treatment of glioblastoma multiforme

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 (SAH). 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 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 (thrombospondin type-1 domain-containing protein 1) was identified that segregated with the 9 affected (3 suffered SAH; 6 had unruptured IA) and 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 SAH (1.6% [95% CI, 0.8%–3.1%]). These THSD1 mutations/rare variants were highly enriched in our IA patient cohort relative to 89,040 chromosomes in ExAC database (p\u3c0.0001). 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 two animal models. This finding provides new insight into IA and SAH pathogenesis and provides new understanding of THSD1 function, which includes endothelial cell to extracellular matrix adhesion