Epithelial tubes of the correct size and shape are vital for the function of the lungs, kidneys, and vascular system, yet little is known about epithelial tube size regulation. Mutations in the Drosophila gene sinuous have previously been shown to cause tracheal tubes to be elongated and have diameter increases. Our genetic analysis using a sinuous null mutation suggests that sinuous functions in the same pathway as the septate junction genes neurexin and scribble, but that nervana 2, convoluted, varicose, and cystic have functions not shared by sinuous. Our molecular analyses reveal that sinuous encodes a claudin that localizes to septate junctions and is required for septate junction organization and paracellular barrier function. These results provide important evidence that the paracellular barriers formed by arthropod septate junctions and vertebrate tight junctions have a common molecular basis despite their otherwise different molecular compositions, morphologies, and subcellular localizations.

Epithelial tubes are the functional units of many organs, but little is known about how tube sizes are established. Using the Drosophila tracheal system as a model, we previously showed that mutations in varicose (vari) cause tubes to become elongated without increasing cell number. Here we show vari is required for accumulation of the tracheal size-control proteins Vermiform and Serpentine in the tracheal lumen. We also show that vari is an essential septate junction (SJ) gene encoding a membrane associated guanylate kinase (MAGUK). In vivo analyses of domains important for MAGUK scaffolding functions demonstrate that while the Vari HOOK domain is essential, the L27 domain is dispensable. Phylogenetic analyses reveal that Vari helps define a new MAGUK subgroup that includes mammalian PALS2. Importantly, both Vari and PALS2 are basolateral, and the interaction of Vari with the cell-adhesion protein Neurexin IV parallels the interaction of PALS2 and another cell-adhesion protein, Necl-2. Vari therefore bolsters the similarity between Drosophila and vertebrate epithelial basolateral regions, which had previously been limited to the common basolateral localization of Scrib, Dlg and Lgl, proteins required for epithelial polarization at the beginning of embryogenesis. However, by contrast to Scrib, Dlg and Lgl, Vari is not required for cell polarity but rather is part of a cell-adhesion complex. Thus, Vari fundamentally extends the similarity of Drosophila and vertebrate basolateral regions from sharing only polarity complexes to sharing both polarity and cell-adhesion complexes.

Although hydroxyapatite (HAp) has been doped with dozens of different ions, the quest for an ion imparting a combination of properties conducive to bone healing is still ongoing. Because of its protean potency and the similarity in size and shape to the phosphate tetrahedron, selenite ion presents a natural ionic substitute in HAp. The incorporation of selenite into synthetic HAp using two different methods – co-precipitation and ion-exchange sorption - was studied for its effect on crystal properties and on a triad of biological responses: antibacterial, anticancer and osteoinductive. Co-precipitation yielded HAp with higher selenite contents than sorption and the stoichiometry of HAp richest in selenite was represented as Ca9.75(PO4)5.75(SeO3)0.25(OH)1.75. Crystallinity of HAp decreased in direct proportion with the amount of selenite incorporated. Because of their lower selenite content, HAp powders prepared by ion-exchange exhibited a consistently higher crystallinity compared to the co-precipitated ones. Annealing partially recovered the crystallinity, yet the difference in crystallinity between powders prepared by co-precipitation and by ion-exchange remained, suggesting that the amorphization is mainly due to structural incorporation of selenite, not its effect on the crystal growth kinetics. The addition of selenite changed the morphology of HAp nanoparticles from acicular to rounded and affected the crystal lattice parameters in different ways depending on whether the powders were annealed or not. As for the annealed powders, the incorporation of selenite contracted the lattice in both a and c crystallographic directions. In the agar diffusion assay, the effectiveness of HAp was more dependent on the presence or absence of selenite in it than on its concentration and was highest against E. coli and S. aureus, moderately high against S. enteritidis and ineffective against P. aeruginosa. In liquid inoculation tests, on the other hand, the antibacterial activity of HAp was directly proportional to the amount of selenite contained in it. The viability of K7M2 osteosarcoma cells decreased in direct proportion with the amount of selenite in HAp and was significantly different from the untreated control and from pure HAp at contents equal to or higher than 1.9 wt.%. In contrast, no reduction was observed in the viability of primary fibroblasts treated with HAp incorporating different amounts of selenite ions, suggesting their potentially selective anticancer activity: lethal for the cancer cells and harmless for the healthy cells. Finally, mRNA expression of bone gamma-carboxyglutamate protein (BGLAP3) was higher in differentiated MC3T3-E1 osteoblastic cells treated with selenite-incorporated HAp particles than in cells treated with pure HAp. The osteoinductive effect was due to an overall higher metabolic activity of cells treated with the particles and not due to increased proliferation. In such a way, a triad of antibacterial, osteoinductive and anticancer activities was attributed to selenite-incorporated ...

No abstract

Cheap and simple to make, calcium phosphate (CP), thanks to its unusual functional pleiotropy, belongs to the new wave of abundant and naturally accessible nanomaterials applicable as a means to various technological ends. It is used in a number of industries, including the biomedical, but its intrinsic antibacterial activity in the nanoparticle form has not been sufficiently explored to date. In this study, we report on this intrinsic antibacterial effect exhibited by two distinct CP phases: an amorphous CP (ACP) and hydroxyapatite (HAp). The effect is prominent against a number of regular bacterial species, including Staphylococcus aureus, Staphylococcus epidermis, Enterococcus faecalis, Escherichia coli, and Pseudomonas aeruginosa, but also their multidrug-resistant (MDR) analogues. Although ACP and HAp displayed similar levels of activity against Gram-negative organisms, ACP proved to be more effective against the Gram-positive ones, with respect to which HAp was mostly inert, yet this trend became reversed for the MDR strains. In addition to the intrinsic antimicrobial effect of CP nanoparticles, we have also observed a synergistic effect between the nanoparticles and certain antibiotics. Both forms of CP were engaged in a synergistic relationship with a variety of concomitantly delivered antibiotics, including ampicillin, kanamycin, oxacillin, vancomycin, minocycline, erythromycin, linezolid, and clindamycin, and enabled even antibiotics completely ineffective against particular bacterial strains to significantly suppress their growth. This relationship was complex; depending on a particular CP phase, bacterial strain and antibiotic, the antibacterial activity (i) intensified proportionally to the nanoparticle concentration, (ii) plateaued immediately after the introduction of nanoparticles in minute amounts, or (iii) exhibited concentration-dependent minima due to stress-induced biofilm formation. These findings present grounds for the further optimization of CP properties and maximization of this intriguing effect, which could in the long run make this material comparable in activity to the inorganics of choice for this application, including silver, copper, or zinc oxide, while retaining its superb safety profile and positive eukaryotic versus prokaryotic cell selectivity.

We report new w À fluorescent balancers scorable from stage 13 through adulthood that bear a nuclearlocalized yellow fluorescent protein marker directly driven by dfd and GMR enhancer elements. The utility of this marker is enhanced by identification of an anti-GFP/yellow fluorescent protein (YFP) serum that is compatible with heat fixation.

Osteomyelitis, an infectious disease predominantly tied to poor sanitary conditions in underdeveloped regions of the world, is in need of inexpensive, easily in situ synthesizable and administrable materials for its treatment. The results of this study stem from the attempt to create one such affordable and minimally invasive therapeutic platform in the form of a self-setting, injectable cement with a tunable drug release profile, composed of only nanoparticulate hydroxyapatite, the synthetic version of the bone mineral. Cements comprised two separately synthesized hydroxyapatite powders, one of which, HAP2, was precipitated abruptly, retaining the amorphous nature longer, and the other one of which, HAP1, was precipitated at a slower rate, more rapidly transitioning to the crystalline structure. Cements were made with four different weight ratios of the two hydroxyapatite components: 100/ 0, 85/15, 50/50, and 0/100 with respect to HAP1 and HAP2. Both the setting and the release rates measured on two different antibiotics, vancomycin and ciprofloxacin, were controlled using the weight ratio of the two hydroxyapatite components. Various inorganic powder properties were formerly used to control drug release, but here we demonstrate for the first time that the kinetics of the mechanism of formation of a solid compound can be controlled to produce tunable drug release profiles. Specifically, it was found that the longer the precursor calcium phosphate component of the cement retains the amorphous nature of the primary precipitate, the more active it was in terms of speeding up the diffusional release of the adsorbed drug. The setting rate was, in contrast, inversely proportional to the release rate and to the content of this active hydroxyapatite component, HAP2. The empirical release profiles were fitted to a set of equations that could be used to tune the release rate to the therapeutic occasion. All of the cements loaded with vancomycin or ciprofloxacin inhibited the growth of Gram-positive Staphylococcus aureus and Gram-negative Escherichia coli and Pseudomonas aeruginosa in both agar diffusion assays and broth dilution tests with intensities either comparable to the antibiotic per se, as in the case of ciprofloxacin, or even larger than the antibiotic alone, as in the case of vancomycin. Interestingly, even the pure cements exhibited an antibacterial effect ranging from moderate to strong, while demonstrating high levels of biocompatibility with osteoclastic RAW264.7 cells and only slightly affecting the viability of the osteoblastic MC3T3-E1 cells, in direct proportion with the amount of the more active hydroxyapatite component in the cements. This antibacterial effect was especially noticeable against Gram-negative bacteria, where the growth inhibition by the cements was comparable to or even stronger than that of the pure antibiotics. The antibiofilm assay against P. aeruginosa biofilms reiterated the antibiotic effectiveness of pure, antibiotic-free cements. That the carrier per se, composed of a nontoxic, ...

Previous studies have demonstrated that Pseudomonas putida strains are not only capable of growth on a wide range of organic substrates, but also chemotactic towards many of these compounds. However, in most cases the specific chemoreceptors that are involved have not been identified. The complete genome sequences of P. putida strains F1 and KT2440 revealed that each strain is predicted to encode 27 methyl-accepting chemotaxis proteins (MCPs) or MCP-like proteins, 25 of which are shared by both strains. It was expected that orthologous MCPs in closely related strains of the same species would be functionally equivalent. However, deletion of the gene encoding the P. putida F1 orthologue (locus tag Pput_4520, designated mcfS) of McpS, a known receptor for organic acids in P. putida KT2440, did not result in an obvious chemotaxis phenotype. Therefore, we constructed individual markerless MCP gene deletion mutants in P. putida F1 and screened for defective sensory responses to succinate, malate, fumarate and citrate. This screen resulted in the identification of a receptor, McfQ (locus tag Pput_4894), which responds to citrate and fumarate. An additional receptor, McfR (locus tag Pput_0339), which detects succinate, malate and fumarate, was found by individually expressing each of the 18 genes encoding canonical MCPs from strain F1 in a KT2440 mcpS-deletion mutant. Expression of mcfS in the same mcpS deletion mutant demonstrated that, like McfR, McfS responds to succinate, malate, citrate and fumarate. Therefore, at least three receptors, McfR, McfS, and McfQ, work in concert to detect organic acids in P. putida F1.