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Experiment 1. Vehicle-treated animals received DMSO only. After 6 weeks, the mice were euthanized and perfused with PBS, and brains were harvested and frozen in liquid nitrogen.
Experiment 2. To study the efficacy of gingipain inhibitors to decrease P. The mice received gingipain inhibitors or moxifloxacin for 5 weeks days 36 to Vehicle-treated animals received PBS or pluronic only. A group of mock-infected and P. After 10 weeks, P.
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Experiment 3. Vehicle-treated animals received PBS only. Results from both assays were corrected for protein content and normalized to the mock group, and the means of both assays were analyzed as a combined dataset.
The amplified product was identified by electrophoresis in a 1. Images were taken on an Olympus BX61 microscope with a Zyla 5. Fluorescence intensity was quantified on a PerkinElmer Envision Plate reader. The primers were based on single copy of P. The number of copies of the P. Anti-GAD67 antibody, clone 1G The area of the hilus was defined as the area between the blades of the dentate gyrus connected by a straight line on the open side.
The number of cells on every 40th section through the hippocampus was counted. The results are presented as the number of cells per volume of tissue. The supernatant was discarded. The supernatant containing protein lysate was collected. Gel was run at 75 V for 10 min and then at V for 1.
Oral subgingival plaque and saliva samples were obtained from five human subjects with periodontal disease under an IRB-approved clinical protocol.
An unstimulated saliva sample about 1 ml was obtained by collection into a sterile ml falcon tube following a 2-min water rinse. Samples were collected at a consistent time of day to avoid diurnal effects and were kept cold during and following collection. The sampling sites were gently air-dried and isolated with cotton pellets to avoid saliva contamination.
The paper points were inserted in the pockets for 30 s until resistance was felt. Paper points were held with pliers, removed from the site, and placed into prelabeled 1. Each plaque sample was processed in this manner separately but combined for analysis. The primers and methods were the same as those used for saliva above.
On the day of the experiment, the primary culture was diluted to obtain OD of 0. Then, the bacteria were collected by centrifuging at rpm for 10 min and washed.
Clinical Studies – RESCEU
The resultant pellet was resuspended using 10 ml of defined medium to assess growth. The defined medium consists of the following: salt base supplement [ Vehicle cultures were treated with 0. Resistance was assessed by incubation of 16 serial passages of P. Because COR does not completely inhibit P. Resistance was assessed in two standard methods, with the final data reported as an average of both methods. Cultures were first prepared with drug and moxifloxacin in a range of doses passaging each time for 17 passages and monitoring MIC with each passage.
In a separate study, drug concentrations were gradually increased between passages. Data were analyzed with GraphPad Prism version 7. Outliers were not removed from data presented in Fig. To determine whether the data were normally distributed, we performed a Shapiro-Wilk test. If P values were below 0. Table S4. Demographic information of patients with CP who donated saliva and subgingival plaque samples.
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Science Advances 23 Jan : eaau Table of Contents. All rights reserved. Stephen S. Cortexyme, Inc.
AD diagnosis correlates with gingipain load in brain Tissue microarrays TMAs containing sex- and age-matched brain tissue cores from the middle temporal gyrus MTG of both AD patients and neurologically normal individuals were used for immunohistochemical IHC studies tables S1 and S2. Download high-res image Open in new tab Download Powerpoint. RgpB colocalizes with neurons, astrocytes, and pathology in AD hippocampus In AD, the hippocampus is one of the first brain areas to be damaged.
Detection of Kgp in AD cerebral cortex AD is also associated with atrophy of the gray matter of the cerebral cortex.
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Identification of the P. Tau is fragmented by gingipains Because we identified colocalization of gingipain with tau tangles in AD brain Fig. Small-molecule gingipain inhibitors are neuroprotective To determine whether gingipains are toxic to neurons in vitro, we exposed differentiated SH-SY5Y cells to either RgpB or Kgp for 24 hours.
Oral infection of mice with P. Oral administration of a Kgp inhibitor effectively treats P. COR treatment shows dose-dependent effects on brain P. Sequence analysis of P. Effects of P. Small-molecule gingipain inhibitor characterization Structure-based design was used to develop a library of gingipain inhibitors, which were tested on purified Kgp and RgpB to assess potency and determine inhibition constants.
Effect of gingipain inhibitors on P. Stereotactic injection of gingipains in mouse hippocampus A 7-day study was designed to detect gingipain-induced hippocampal neurodegeneration with Fluoro-Jade C FJC , a fluorescent stain that has been shown to exhibit maximum staining of degenerating neurons 1 week after a neurotoxic insult Growth of P.
Endpoint PCR analysis of P. Preparation of P. COR activity probe labeling of Kgp P. Collection and processing of human saliva and subgingival plaque samples Oral subgingival plaque and saliva samples were obtained from five human subjects with periodontal disease under an IRB-approved clinical protocol.
Determination of Kgp-dependent growth of P. Assessment of in vitro resistance of P. Statistical analysis Data were analyzed with GraphPad Prism version 7. CAB analysis of non-AD neurological disease brain microarrays. Sequencing of P. Table S1. Table S2.
Table S3. Tau fragments identified by MS after gingipain exposure. Wyss-Coray , J.
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Rogers , Inflammation in Alzheimer disease—A brief review of the basic science and clinical literature. Cold Spring Harb. Kaushal , R. Dye , P.