A proposed framework to evaluate the quality and reliability of targeted metabolomics assays from the UK Consortium on Metabolic Phenotyping (MAP/UK).

Targeted metabolite assays that measure tens or hundreds of pre-selected metabolites, typically using liquid chromatography-mass spectrometry, are increasingly being developed and applied to metabolic phenotyping studies. These are used both as standalone phenotyping methods and for the validation of putative metabolic biomarkers obtained from untargeted metabolomics studies. However, there are no widely accepted standards in the scientific community for ensuring reliability of the development and validation of targeted metabolite assays (referred to here as 'targeted metabolomics'). Most current practices attempt to adopt, with modifications, the strict guidance provided by drug regulatory authorities for analytical methods designed largely for measuring drugs and other xenobiotic analytes. Here, the regulatory guidance provided by the European Medicines Agency, US Food and Drug Administration and International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use are summarized. In this Perspective, we have adapted these guidelines and propose a less onerous 'tiered' approach to evaluate the reliability of a wide range of metabolomics analyses, addressing the need for community-accepted, harmonized guidelines for tiers other than full validation. This 'fit-for-purpose' tiered approach comprises four levels-discovery, screening, qualification and validation-and is discussed in the context of a range of targeted and untargeted metabolomics assays. Issues arising with targeted multiplexed metabolomics assays, and how these might be addressed, are considered. Furthermore, guidance is provided to assist the community with selecting the appropriate degree of reliability for a series of well-defined applications of metabolomics.

Mycobacterium komaniense sp. nov., a rapidly growing non-tuberculous Mycobacterium species detected in South Africa.

Some species of non-tuberculous mycobacteria (NTM) have been reported to be opportunistic pathogens of animals and humans. Recently there has been an upsurge in the number of cases of NTM infections, such that some NTM species are now recognized as pathogens of humans and animals. From a veterinary point of view, the major significance of NTM is the cross-reactive immune response they elicit against Mycobacterium bovis antigens, leading to misdiagnosis of bovine tuberculosis. Four NTM isolates were detected from a bovine nasal swab, soil and water, during an NTM survey in South Africa. These were all found using 16S rRNA gene sequence analysis to be closely related to Mycobacterium moriokaense. The isolates were further characterised by sequence analysis of the partial fragments of hsp65, rpoB and sodA. The genome of the type strain was also elucidated. Gene (16S rRNA, hsp65, rpoB and sodA) and protein sequence data analysis of 6 kDa early secretory antigenic target (ESAT 6) and 10 kDa culture filtrate protein (CFP-10) revealed that these isolates belong to a unique Mycobacterium species. Differences in phenotypic and biochemical traits between the isolates and closely related species further supported that these isolates belong to novel Mycobacterium species. We proposed the name Mycobacterium komaniense sp. nov. for this new species. The type strain is GPK 1020T (=CIP 110823T=ATCC BAA-2758).

Functionalization of PLGA Nanoparticles with 1,3-ß-glucan Enhances the Intracellular Pharmacokinetics of Rifampicin in Macrophages.

PURPOSE: Mycobacterium tuberculosis which causes tuberculosis, is primarily resident within macrophages. 1,3-ß-glucan has been proposed as a ligand to target drug loaded nanoparticles (NPs) to macrophages. In this study we characterized the intracellular pharmacokinetics of the anti-tubercular drug rifampicin delivered by 1,3-ß-glucan functionalized PLGA NPs (Glu-PLGA). We hypothesized that Glu-PLGA NPs would be taken up at a faster rate than PLGA NPs, and consequently deliver higher amounts of rifampicin into the macrophages. METHODS: Carbodiimide chemistry was employed to conjugate 1,3-ß-glucan and rhodamine to PLGA. Rifampicin loaded PLGA and Glu-PLGA NPs as well as rhodamine functionalized PLGA and Glu-PLGA NPs were synthesized using an emulsion solvent evaporation technique. Intracellular pharmacokinetics of rifampicin and NPs were evaluated in THP-1 derived macrophages. A pharmacokinetic model was developed to describe uptake, and modelling was performed using ADAPT 5 software. RESULTS: The NPs increased the rate of uptake of rifampicin by a factor of 17 and 62 in case of PLGA and Glu-PLGA, respectively. Expulsion of NPs from the macrophages was also observed, which was 3 fold greater for Glu-PLGA NPs than for PLGA NPs. However, the ratio of uptake to expulsion was similar for both NPs. After 24 h, the amount of rifampicin delivered by the PLGA and Glu-PLGA NPs was similar. The NPs resulted in at least a 10-fold increase in the uptake of rifampicin. CONCLUSIONS: Functionalization of PLGA NPs with 1,3-ß-glucan resulted in faster uptake of rifampicin into macrophages. These NPs may be useful to achieve rapid intracellular eradication of Mycobacterium tuberculosis.

Mycobacterium malmesburyense sp. nov., a non-tuberculous species of the genus Mycobacterium revealed by multiple gene sequence characterization.

Non-tuberculous mycobacteria (NTM) are ubiquitous in the environment, and an increasing number of NTM species have been isolated and characterized from both humans and animals, highlighting the zoonotic potential of these bacteria. Host exposure to NTM may impact on cross-reactive immune responsiveness, which may affect diagnosis of bovine tuberculosis and may also play a role in the variability of the efficacy of Mycobacterium bovis BCG vaccination against tuberculosis. In this study we characterized 10 NTM isolates originating from water, soil, nasal swabs of cattle and African buffalo as well as bovine tissue samples. These isolates were previously identified during an NTM survey and were all found, using 16S rRNA gene sequence analysis to be closely related to Mycobacterium moriokaense. A polyphasic approach that included phenotypic characterization, antibiotic susceptibility profiling, mycolic acid profiling and phylogenetic analysis of four gene loci, 16S rRNA, hsp65, sodA and rpoB, was employed to characterize these isolates. Sequence data analysis of the four gene loci revealed that these isolates belong to a unique species of the genus Mycobacterium. This evidence was further supported by several differences in phenotypic characteristics between the isolates and the closely related species. We propose the name Mycobacterium malmesburyense sp. nov. for this novel species. The type strain is WCM 7299T (=ATCC BAA-2759T=CIP 110822T).

Micronization, characterization and in-vitro dissolution of shellac from PGSS supercritical CO2 technique.

The purpose of this investigation was to determine whether shellac, a naturally occurring material with enteric properties, could be processed in supercritical CO2 (sc-CO2) using the particles from gas saturated solution (PGSS) process and how process parameters affect the physico-chemical properties of shellac. In-situ attenuated total reflection fourier transform infra-red (ATR-FTIR) spectroscopy showed that CO2 dissolves in shellac with solubility reaching a maximum of 13% (w/w) at 300 bar pressure and 40 °C and maximum swelling of 28%. The solubility of sc-CO2 in shellac allowed for the formation of porous shellac structures of which the average pore diameter and pore density could be controlled by adjustment of operating pressure and temperature. In addition, it was possible to produce shellac microparticles ranging in average diameter from 180 to 300 µm. It was also shown that processing shellac in sc-CO2 resulted in accelerated esterification reactions, potentially limiting the extent of post-processing "ageing" and thus greater stability. Due to additional hydrolysis reactions enhanced by the presence of sc-CO2, the solubility of shellac at pH 7.5 was increased by between 4 and 7 times, while dissolution rates were also increased. It was also shown that the in-vitro dissolution profiles of shellac could be modified by slight adjustment in operating temperatures.

Curdlan-Conjugated PLGA Nanoparticles Possess Macrophage Stimulant Activity and Drug Delivery Capabilities.

PURPOSE: There is significant interest in the application of nanoparticles to deliver immunostimulatory signals to cells. We hypothesized that curdlan (immune stimulating polymer) could be conjugated to PLGA and nanoparticles from this copolymer would possess immunostimulatory activity, be non-cytotoxic and function as an effective sustained drug release system. METHODS: Carbodiimide chemistry was employed to conjugate curdlan to PLGA. The conjugate (C-PLGA) was characterized using (1)H and (13)C NMR, FTIR, DSC and TGA. Nanoparticles were synthesized using an emulsion-solvent evaporation technique. Immunostimulatory activity was characterized in THP-1 derived macrophages. MTT assay and real-time impedance measurements were used to characterize polymer and nanoparticle toxicity and uptake in macrophages. Drug delivery capability was assessed across Caco-2 cells using rifampicin as a model drug. RESULTS: Spectral characterization confirmed successful synthesis of C-PLGA. C-PLGA nanoparticles enhanced phosphorylated ERK production in macrophages indicating cell stimulation. Nanoparticles provided slow release of rifampicin across Caco-2 cells. Polymers but not nanoparticles altered the adhesion profiles of the macrophages. Impedance measurements suggested Ca(2+) dependent uptake of nanoparticles by the macrophages. CONCLUSIONS: PLGA nanoparticles with macrophage stimulating and sustained drug delivery capabilities have been prepared. These nanoparticles can be used to stimulate macrophages and concurrently deliver drug in infectious disease therapy.

Permeation of PLGA nanoparticles across different in vitro models.

Many drug delivery systems have indicated improvement in delivery of various drug molecules and among these biodegradable and biocompatible polymers such as poly(D,L-lactide-co-glycolide) (PLGA) have been shown to enhance intracellular uptake of drug candidates when formulated as nanoparticles. PLGA nanoparticles were prepared by means of a double emulsion solvent evaporation technique and evaluated in terms of size, encapsulation efficiency, surface charge, isoniazid release and in vitro transport. The nanoparticles have an average size of 237 nm and were previously shown to be distributed in several tissues after oral administration without triggering an immune response. This study focussed on the in vitro permeation of the PLGA nanoparticles across different membranes and showed that although Rhodamine 6G-labelled nanoparticles are efficiently delivered across the intestinal epithelium, its epithelial permeability changes when a drug such as isoniazid is encapsulated. Future studies should focus on ways to optimise PLGA nanoparticle delivery when a drug such as isoniazid is encapsulated for instance by coating with polymers such as polyethylene glycol.