A Blood-Brain Barrier (BBB) Disrupter Is Also a Potent α-Synuclein (α-syn) Aggregation Inhibitor: A NOVEL DUAL MECHANISM OF MANNITOL FOR THE TREATMENT OF PARKINSON DISEASE (PD)*

The development of disease-modifying therapy for Parkinson disease has been a main drug development challenge, including the need to deliver the therapeutic agents to the brain. Here, we examined the ability of mannitol to interfere with the aggregation process of α-synuclein in vitro and in vivo in addition to its blood-brain barrier-disrupting properties. Using in vitro studies, we demonstrated the effect of mannitol on α-synuclein aggregation. Although low concentration of mannitol inhibited the formation of fibrils, high concentration significantly decreased the formation of tetramers and high molecular weight oligomers and shifted the secondary structure of α-synuclein from α-helical to a different structure, suggesting alternative potential pathways for aggregation. When administered to a Parkinson Drosophila model, mannitol dramatically corrected its behavioral defects and reduced the amount of α-synuclein aggregates in the brains of treated flies. In the mThy1-human α-synuclein transgenic mouse model, a decrease in α-synuclein accumulation was detected in several brain regions following treatment, suggesting that mannitol promotes α-synuclein clearance in the cell bodies. It appears that mannitol has a general neuroprotective effect in the transgenic treated mice, which includes the dopaminergic system. We therefore suggest mannitol as a basis for a dual mechanism therapeutic agent for the treatment of Parkinson disease.     

High Incidence of Tuberculosis,Low Sensitivity of Current Diagnostic Scheme and Prolonged Culture Positivity in Four Colombian Prisons. A Cohort Study

Objective

To determine the incidence of pulmonary tuberculosis (TB) in inmates, factors associated with TB, and the time to sputum smear and culture conversion during TB treatment.

Methods

Prospective cohort study. All prisoners with respiratory symptoms (RS) of any duration were evaluated. After participants signed consent forms, we collected three spontaneous sputum samples on consecutive days. We performed auramine-rhodamine staining, culturing with the thin-layer agar method, Löwestein-Jensen medium and MGIT, susceptibility testing for first-line drugs; and HIV testing. TB cases were followed, and the times to smear and culture conversion to negative were evaluated.

Results

Of 9,507 prisoners held in four prisons between April/30/2010 and April/30/2012, among them 4,463 were screened, 1,305 were evaluated for TB because of the lower RS of any duration, and 72 were diagnosed with TB. The annual incidence was 505 cases/100,000 prisoners. Among TB cases, the median age was 30 years, 25% had <15 days of cough, 12.5% had a history of prior TB, and 40.3% had prior contact with a TB case. TB-HIV coinfection was diagnosed in three cases. History of prior TB, contact with a TB case, and being underweight were risk factors associated with TB. Overweight was a protective factor. Almost a quarter of TB cases were detected only by culture; three cases were isoniazid resistant, and two resistant to streptomycin. The median times to culture conversion was 59 days, and smear conversion was 33.

Conclusions

The TB incidence in prisons is 20 times higher than in the general Colombian population. TB should be considered in inmates with lower RS of any duration. Our data demonstrate that patients receiving adequate anti-TB treatment remain infectious for prolonged periods. These findings suggest that current recommendations regarding isolation of prisoners with TB should be reconsidered, and suggest the need for mycobacterial cultures during follow-up.     

Peptide-based hydrogel nanoparticles as effective drug delivery agents

Peptide-based hydrogel nanoparticles represent a promising alternative to current drug delivery approaches. We have previously demonstrated that the Fmoc-FF aromatic dipeptide building block can self-assemble in aqueous solutions to form nano-scaled ordered hydrogels of remarkable mechanical rigidity. Here, we present a scalable process for the assembly of this peptide into hydrogel nanoparticles (HNPs) aimed to be utilized as potential drug delivery carriers. Fmoc-FF based HNPs were formulated via modified inverse-emulsion method using vitamin E-TPGS as an emulsion stabilizer and high speed homogenization. The formed HNPs exhibited two distinguishable populations with an average size of 21.5 ± 1.3 and 225.9 ± 0.8 nm. Gold nanoparticles were encapsulated within the hydrogel nanoparticles as contrast agents to monitor the formation of the assemblies and their ultrastructural properties. Next, we demonstrated a robust experimental procedure developed and optimized for the formulation, purification, storage and handling procedures of HNPs. Encapsulation of doxorubicin (Dox) and 5-flourouracil (5-Fu) within the HNPs matrix showed release kinetics of the drugs depending on their chemical structure, molecular weight and hydrophobicity. The results clearly indicate that Fmoc-FF based hydrogel nanoparticles have the potential to be used as encapsulation and delivery system of various drugs and bioactive molecules.     

Green tea extracts interfere with the stress-protective activity of PrP and the formation of PrP

A hallmark in prion diseases is the conformational transition of the cellular prion protein (PrP(C)) into a pathogenic conformation, designated scrapie prion protein (PrP(Sc)), which is the essential constituent of infectious prions. Here, we show that epigallocatechin gallate (EGCG) and gallocatechin gallate, the main polyphenols in green tea, induce the transition of mature PrP(C) into a detergent-insoluble conformation distinct from PrP(Sc). The PrP conformer induced by EGCG was rapidly internalized from the plasma membrane and degraded in lysosomal compartments. Isothermal titration calorimetry studies revealed that EGCG directly interacts with PrP leading to the destabilizing of the native conformation and the formation of random coil structures. This activity was dependent on the gallate side chain and the three hydroxyl groups of the trihydroxyphenyl side chain. In scrapie-infected cells EGCG treatment was beneficial; formation of PrP(Sc) ceased. However, in uninfected cells EGCG interfered with the stress-protective activity of PrP(C). As a consequence, EGCG-treated cells showed enhanced vulnerability to stress conditions. Our study emphasizes the important role of PrP(C) to protect cells from stress and indicate efficient intracellular pathways to degrade non-native conformations of PrP(C).     

Polyphenol-induced dissociation of various amyloid fibrils results in a methionine-independent formation of ROS

Fibrillization of amyloid polypeptides is accompanied by formation of reactive oxygen species (ROS), which, in turn, is assumed to further promote amyloid-related pathologies. Different polyphenols, all of which are established antioxidants, cause dissociation of amyloid fibrils. This study addresses the latter, poorly understood process. Specifically, we have investigated the dissociation of Abeta(42) fibrils by six different polyphenols, using electron microscopy and spectrofluorometric analysis. Simultanously, we have monitored the production of ROS using electron spin resonance (ESR) and the commercially available peroxide assay kit. Using the same methods we found that curcumin, one of the most potent destabilizing agents of Abeta(42), induced dissociation of fibrils of other amyloid polypeptides [Abeta(40), Abeta(42)Nle35, islet amyloid polypeptide and a fragment of alpha-synuclein]. When the solution contained traces of transition metal, all the dissociation reactions were accompanied by ROS formation, independent of the presence of a methionine residue. Kinetic studies show that the formation of ROS lags behind dissociation, indicating that if casual relationship exists between these two processes, then ROS formation may be considered a consequence and not a cause of dissociation. These findings open new avenues in amyloid research that will be required to gain further understanding of our results and of their implications.     

The fruit fly PUB: a phagostimulation unit bioassay system to quantitatively measure ingestion of baits by individual flies

Abstract:  A bioassay to investigate quantitative phagostimulation and ingestion physiology of baits on individual fruit flies is presented. The study was undertaken using two fruit fly species: the Mediterranean fruit fly ( Ceratitis capitata ), a cosmopolitan insect pest, and the Ethiopian fruit fly ( Dacus ciliatus ), a quarantine insect in Israel. Our model bait suspension included spinosad as the toxic agent, and 1% yeast hydrolysate with 10% sucrose as phagostimulant. A preliminary toxicology study showed that the two fruit flies are highly sensitive to low concentrations of spinosad baited with this phagostimulant. The maximum concentration needed to kill 90% of the female flies was 4.2 and 8.5 p.p.m. for C. capitata and D. ciliatus , respectively. The bioassay was able to detect the ingestion of low volumes (e.g. 1  μ l) of tested solutions. The bioassay was also able to detect differences in intake of different concentrations of spinosad solutions and relate ingestion to fruit fly mortality. Additionally, the bioassay was sensitive enough to highlight differences in intake related to the physiological status of the fruit fly and fly species. The bioassay can also be used to follow ingestion kinetics of baits. We expect that this bioassay will contribute in the exploration of more efficient bait systems for fruit flies.