The role of α-synuclein (α-syn) pathology in Parkinson's disease (PD) is well established; however, effective therapies remain elusive. Two mechanisms central to PD neurodegeneration are the intracellular aggregation of misfolded α-syn and the uptake of α-syn aggregates into neurons. Cationic arginine-rich peptides (CARPs) are an emerging class of molecule with multiple neuroprotective mechanisms of action, including protein stabilisation. This study characterised both intracellular α-syn aggregation and α-syn uptake in cortical neurons in vitro. Thereafter, this study examined the therapeutic potential of the neuroprotective CARP, R18D (18-mer of D-arginine), to prevent the aforementioned PD pathogenic processes through a cell-free thioflavin-T (ThT) assay and in cortical neurons. To induce intracellular α-syn aggregation, rat primary cortical neurons were exposed to α-syn seed (0.14 μM) for 2 h to allow uptake of the protein, followed by R18D treatment (0.0625, 0.125, 0.25, 0.5 μM), and a subsequent measurement of α-syn aggregates 48 h later using a homogenous time-resolved fluorescence (HTRF) assay. To assess neuronal uptake, α-syn seeds were covalently labelled with an Alexa-Fluor 488 fluorescent tag, pre-incubated with R18D (0.125, 0.25, 0.5 μM), and then exposed to cortical neurons for 24 h and assessed via confocal microscopy. It was demonstrated that R18D significantly reduced both intracellular α-syn aggregation and α-syn seed uptake in neurons by 37.8% and 77.7%, respectively. Also, R18D reduced the aggregation of α-syn monomers in the cell-free assay. These findings highlight the therapeutic potential of R18D to inhibit key α-syn pathological processes and PD progression.
We examined the efficacy of R18 in a transient MCAO model and compared its effectiveness to the well-characterized neuroprotective NA-1 peptide. R18 and NA-1 peptides were administered intravenously (30, 100, 300, 1000nmol/kg), 60min after the onset of 90min of MCAO. Infarct volume, cerebral swelling and functional outcomes (neurological score, adhesive tape and rota-rod) were measured 24h after MCAO. R18 reduced total infarct volume by 35.1% (p=0.008), 24.8% (p=0.059), 12.2% and 9.6% for the respective 1000 to 30nmol/kg doses, while the corresponding doses of NA-1 reduced lesion volume by 26.1% (p=0.047), 16.6%, 16.5% and 7%, respectively. R18 also reduced hemisphere swelling by between 46.1% (1000 and 300nmol/kg; p=0.009) and 24.4% (100nmol/kg; p=0.066), while NA-1 reduced swelling by 25.7% (1000nmol/kg; p=0.054). In addition, several R18 and NA-1 treatment groups displayed a significant improvement in at least one parameter of the adhesive tape test. These results confirm the neuroprotective properties of R18, and suggest that the peptide is a more effective neuroprotective agent than NA-1. This provides strong justification for the continuing development of R18 as a neuroprotective treatment for stroke.
The competitive interactions of genetically distinct isolates of Giardia duodenalis with different growth rates were studied in vitro. Electrophoretic analysis of mixed cultures showed that competition between 2 cloned isolates occurs under normal in vitro culture conditions, with faster-growing isolates outcompeting those with slower growth rates. The addition of sublethal concentrations of metronidazole to clonal mixtures in vitro prevented the competitive exclusion, which was seen in normal culture. This apparently occurred because the drug reduced the growth rate of the faster-growing but not the slower-growing clone.
There is considerable experimental evidence that hypothermia is neuroprotective and can reduce the severity of brain damage after global or focal cerebral ischaemia. However, despite successful clinical trials for cardiac arrest and perinatal hypoxia-ischaemia and a number of trials demonstrating the safety of moderate and mild hypothermia in stroke, there are still no established guidelines for its use clinically. Based upon a review of the experimental studies we discuss the clinical implications for the use of hypothermia as an adjunctive therapy in global cerebral ischaemia and stroke and make some suggestions for its use in these situations.
Using a novel method for cloning Giardia duodenalis from cultures and fecal samples, 47 clones from 7 isolates were established in vitro. Average colony-forming efficiency in established cultures was 43.2% compared to 11.2% when cloning directly from excystation. The highest success rate of cloning was found with the Portland (P1, ATCC No. 30888) isolate, with a colony-forming efficiency of 92.7%. Cloned and parent populations were compared over a range of 13 enzymes using starch gel electrophoresis. No genetic difference was found between any of the clones and the parent isolates.
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