Cannabidiol Inhibits Tau Aggregation In Vitro

A hallmark of Alzheimer’s disease (AD) is the accumulation of tau protein in the brain. Compelling evidence indicates that the presence of tau aggregates causes irreversible neuronal destruction, eventually leading to synaptic loss. So far, the inhibition of tau aggregation has been recognized as one of the most effective therapeutic strategies. Cannabidiol (CBD), a major component found in Cannabis sativa L., has antioxidant activities as well as numerous neuroprotective features. Therefore, we hypothesize that CBD may serve as a potent substance to hamper tau aggregation in AD. In this study, we aim to investigate the CBD effect on the aggregation of recombinant human tau protein 1N/4R isoform using biochemical methods in vitro and in silico. Using Thioflavin T (ThT) assay, circular dichroism (CD), atomic force microscopy (AFM), we demonstrated that CBD can suppress tau fibrils formation. Moreover, by quenching assay, docking and job’s plot, we further demonstrate that one molecule of CBD interacts with one molecule of tau protein through a spontaneous binding. Experiments performed by quenching assay, docking and Thioflavin T assay further established that the main forces are hydrogenvan der Waals and some non-negligible hydrophobic forces, affecting the lag phase of tau protein kinetics. Taken together, this study provides new insights about a natural substance, CBD, for tau therapy which may offer new hope for the treatment of AD.

Multistep substrate binding and engagement by the AAA+ ClpXP protease

Escherichia coliClpXP is one of the most thoroughly studied AAA+ proteases, but relatively little is known about the reactions that allow it to bind and then engage specific protein substrates before the adenosine triphosphate (ATP)-fueled mechanical unfolding and translocation steps that lead to processive degradation. Here, we employ a fluorescence-quenching assay to study the binding of ssrA-tagged substrates to ClpXP. Polyphasic stopped-flow association and dissociation kinetics support the existence of at least three distinct substrate-bound complexes. These kinetic data fit well to a model in which ClpXP and substrate form an initial recognition complex followed by an intermediate complex and then, an engaged complex that is competent for substrate unfolding. The initial association and dissociation steps do not require ATP hydrolysis, but subsequent forward and reverse kinetic steps are accelerated by faster ATP hydrolysis. Our results, together with recent cryo-EM structures of ClpXP bound to substrates, support a model in which the ssrA degron initially binds in the top portion of the axial channel of the ClpX hexamer and then is translocated deeper into the channel in steps that eventually pull the native portion of the substrate against the channel opening. Reversible initial substrate binding allows ClpXP to check potential substrates for degrons, potentially increasing specificity. Subsequent substrate engagement steps allow ClpXP to grip a wide variety of sequences to ensure efficient unfolding and translocation of almost any native substrate.

Detection of target collagen peptides with single amino acid mutation using two fluorescent peptide probes

We have herein for the first time reported the development of a fluorescent self-quenching assay to detect target collagen peptides with a single amino acid mutation.

Anti-Biofilm and Anti-Quorum Sensing Properties of Ethanol and Aqueous Extracts of Orthosiphon Stamineus and Andrographis Paniculata Leaves and Stems

Introduction: Quorum sensing controls a wide spectrum of processes and phenotypic behaviours including biofilm formation that helps in the survival and virulence of bacteria in hosts. Thus, anti-quorum sensing is suggested to combat bacterial infections. This study aimed to evaluate the anti-biofilm forming and anti-quorum sensing activities which may contribute to the anti-bacterial effect of ethanol and aqueous extracts of Orthosiphon stamineus and Andrographis paniculata leaves and stems. Materials and method: General biofilm assay protocol with modification was used for the biofilm assay to test the effect of different concentration (2 mg/ml and 3 mg/ml) of extracts on the biofilm formation by Pseudomonas aeruginosa. Optical density of crystal violet/acetic acid solution was measured at 595 nm and the percentage of biofilm inhibition was calculated. In quorum quenching assay, visualization of reporter strain AI1-QQ.1 growth will be used for the detection of bio-molecules interfering with acyl homoserine lactone (AHL). Results: Extracts with the highest ability to inhibit biofilm formation were ethanol extract of O.stamineus stem and A.paniculata leaves with 77.84% at extracts concentration of 3 mg/ml. Aqueous extract of O.stamineus leaves showed the least potential with 13.41% inhibition. However, aqueous extract of A.paniculata showed zero inhibition at 3 mg/ml and two extracts (aqueous extract of O.stamineus and A.paniculata leaves) gave negative value at concentration of 3 mg/ml and 2 mg/ml, respectively. Quorum quenching assay has yet to be done. Conclusion: It could be concluded that most of the extracts possess anti-biofilm property.