Myosins are a multimember family of motor proteins with diverse functions in eukaryotic cells. African trypanosomes possess only two candidate myosins and thus represent a useful system for functional analysis of these motors. One of these candidates is an unusual class I myosin (TbMyo1) that is expressed at similar levels but organized differently during the life cycle of Trypanosoma brucei. This myosin localizes to the polarized endocytic pathway in bloodstream forms of the parasite. This organization is actin dependent. Knock down of TbMyo1 results in a significant reduction in endocytic activity, a cessation in cell division and eventually cell death. A striking morphological feature in these cells is an enlargement of the flagellar pocket, which is consistent with an imbalance in traffic to and from the surface. In contrast TbMyo1 is distributed throughout procyclic forms of the tsetse vector and a loss of ∼90% of the protein has no obvious effects on growth or morphology. These results reveal a life cycle stage specific requirement for this myosin in essential endocytic traffic and represent the first description of the involvement of a motor protein in vesicle traffic in these parasites.
Polymorphonuclear neutrophils (PMN) are critical for first line innate immune defence against Staphylococcus aureus . Mature circulating PMN maintain a short half-life ending in constitutive apoptotic cell death. This makes them unlikely candidates as a bacterial intracellular niche. However, there is significant evidence to suggest that S. aureus can survive intracellularly within PMN and this contributes to persistence and dissemination during infection. The precise mechanism by which S. aureus parasitizes these cells remains to be established. Herein we propose a novel mechanism by which S. aureus subverts both autophagy and apoptosis in PMN in order to maintain an intracellular survival niche during infection. Intracellular survival of S. aureus within primary human PMN was associated with an accumulation of the autophagic flux markers LC3-II and p62, while inhibition of the autophagy pathway led to a significant reduction in intracellular survival of bacteria. This intracellular survival of S. aureus was coupled with a delay in neutrophil apoptosis as well as increased expression of several anti-apoptotic factors. Importantly, blocking autophagy in infected PMN partially restored levels of apoptosis to that of uninfected PMN, suggesting a connection between the autophagic and apoptotic pathways during intracellular survival. These results provide a novel mechanism for S. aureus intracellular survival and suggest that S. aureus may be subverting crosstalk between the autophagic and apoptosis pathways in order to maintain an intracellular niche within human PMN.
Spherulitic assemblies have applications as carriers for drug delivery and as targeting vectors. Presently, the driving force behind the formation of spherulites comprising small drug molecules is not fully understood. Herein, the impact of different substrate types on spherulitic crystallization of salbutamol sulfate (SS) was investigated. Freshly cleaved mica, silicon (111) wafer (SiW), uncoated borosilicate glass (UG), silane coated glass (CG) and stainless steel (MS) were used as substrates. It was demonstrated that the spherulite growth can be controlled via the substrate selection. Spherulite formation was inhibited on hydrophilic substrates, such as mica, possibly due to stronger intermolecular interactions between SS and the substrate than SS-SS interactions. Contact angle measurements established that mica possessed the lowest contact angle (15.1±0.5°) and the values of this parameter increased in the order of UC˂SiW˂CG˂MS. Substrate roughness also played a key role in controlling spherulite formation. SiW, UC and CG had isotropic surfaces with low average roughness, facilitating spherulite formation. Overall, this work demonstrates that it is possible to successfully produce SS spherulites using a single step process at room temperature. Furthermore, the formation of SS spherulites can be tuned by the hydrophobicity of the substrate, an approach that could be applied to assembling spherulites of other small organic molecules. Presently, the driving force behind the formation of spherulites comprising small drug molecules is not fully understood. Herein, the impact of different substrate types on spherulitic crystallization of salbutamol sulfate (SS) was investigated. Freshly cleaved mica, silicon (111) wafer (SiW), uncoated borosilicate glass (UG), silane coated glass (CG) and stainless steel (MS) were used as substrates. It was demonstrated that the spherulite growth can be controlled via the substrate selection. Spherulite formation was inhibited on hydrophilic substrates, such as mica, possibly due to stronger intermolecular interactions between SS and the substrate than SS-SS interactions. Contact angle measurements established that mica possessed the lowest contact angle (15.1±0.5°) and the values of this parameter increased in the order of UC˂SiW˂CG˂MS. Substrate roughness also played a key role in controlling spherulite formation. SiW, UC and CG had isotropic surfaces with low average roughness, facilitating spherulite formation. Overall, this work demonstrates that it is possible to successfully produce SS spherulites using a single step process at room temperature. Furthermore, the formation of SS spherulites can be tuned by the hydrophobicity of the substrate, an approach that could be applied to assembling spherulites of other small organic molecules.
Gold-Copper core shell nanowires have been electrodeposited and their electrochemical and Raman properties probed. First, hollow copper nanotubes, 3.2 ± 0.1 μm long, with a uniform diameter of 70 ± 22 nm, were electrodeposited within the pores of a track etched polycarbonate membrane filter. Second, gold was then electrodeposited within these copper cylinders to yield the gold-copper core-shell nanowires. Nanowires, functionalised with probe strand DNA, that is complementary to that of the pathogen Staph. Aureus, only on their ends, can be immobilised onto an electrode surface in a DNA sandwich assay. Significantly, the charge associated with the selective oxidation of the copper shell depends linearly on the target DNA concentration from 1 nM to 100 μM.
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