Pseudomonas aeruginosa is an opportunistic human pathogen and has been established as a model organism to study bacterial biofilm formation. At least three exopolysaccharides (alginate, Psl, and Pel) contribute to the formation of biofilms in this organism. Here mutants deficient in the production of one or more of these polysaccharides were generated to investigate how these polymers interactively contribute to biofilm formation. Confocal laser scanning microscopy of biofilms formed in flow chambers showed that mutants deficient in alginate biosynthesis developed biofilms with a decreased proportion of viable cells than alginate-producing strains, indicating a role of alginate in viability of cells in biofilms. Alginate-deficient mutants showed enhanced extracellular DNA (eDNA)-containing surface structures impacting the biofilm architecture. PAO1 ⌬pslA ⌬alg8 overproduced Pel, and eDNA showing meshwork-like structures presumably based on an interaction between both polymers were observed. The formation of characteristic mushroom-like structures required both Psl and alginate, whereas Pel appeared to play a role in biofilm cell density and/or the compactness of the biofilm. Mutants producing only alginate, i.e., mutants deficient in both Psl and Pel production, lost their ability to form biofilms. A lack of Psl enhanced the production of Pel, and the absence of Pel enhanced the production of alginate. The function of Psl in attachment was independent of alginate and Pel. A 30% decrease in Psl promoter activity in the alginate-overproducing MucA-negative mutant PDO300 suggested inverse regulation of both biosynthesis operons. Overall, this study demonstrated that the various exopolysaccharides and eDNA interactively contribute to the biofilm architecture of P. aeruginosa.

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Background Tranexamic acid reduces surgical bleeding and reduces death due to bleeding in patients with trauma. Meta-analyses of small trials show that tranexamic acid might decrease deaths from gastrointestinal bleeding. We aimed to assess the effects of tranexamic acid in patients with gastrointestinal bleeding. Methods We did an international, multicentre, randomised, placebo-controlled trial in 164 hospitals in 15 countries. Patients were enrolled if the responsible clinician was uncertain whether to use tranexamic acid, were aged above the minimum age considered an adult in their country (either aged 16 years and older or aged 18 years and older), and had significant (defined as at risk of bleeding to death) upper or lower gastrointestinal bleeding. Patients were randomly assigned by selection of a numbered treatment pack from a box containing eight packs that were identical apart from the pack number. Patients received either a loading dose of 1 g tranexamic acid, which was added to 100 mL infusion bag of 0•9% sodium chloride and infused by slow intravenous injection over 10 min, followed by a maintenance dose of 3 g tranexamic acid added to 1 L of any isotonic intravenous solution and infused at 125 mg/h for 24 h, or placebo (sodium chloride 0•9%). Patients, caregivers, and those assessing outcomes were masked to allocation. The primary outcome was death due to bleeding within 5 days of randomisation; analysis excluded patients who received neither dose of the allocated treatment and those for whom outcome data on death were unavailable. This trial was registered with Current Controlled Trials, ISRCTN11225767, and ClinicalTrials.gov, NCT01658124.

Alginates are polysaccharides with many industrial and medical uses, from food additives to encapsulation agents in the emerging transplantation technologies. Alginate is composed of variable proportions of β-D-mannuronic acid and α-Lguluronic acid linked by 1-4 glycosidic bonds. Traditionally, commercial alginate has been produced by farmed brown seaweeds, but this alginate suffers from heterogeneity in composition and quality partly due to environmental variation. Two bacterial genera, Pseudomonas and Azotobacter, are also capable of producing alginate as an exopolysaccharide. These bacterial alginate producers can provide the means to produce alginates with defined monomer composition and possibly through genetic and protein engineering may allow for the production of 'tailor made' bacterial alginates. The paper discusses the mechanisms behind alginate production in bacteria and how they may be used in the commercial production of alginates.

-Nanotechnology has opened a new horizon of research in various fields including applied physics, chemistry, electronics, optics, robotics, biotechnology and medicine. In the biomedical field, nanomaterials have shown remarkable potential as theranostic agents. Materials which are considered inert are often used in nanomedicine owning to their nontoxic profile. At nanoscale, these inert materials have shown unique properties that differ from bulk and dissolved counterparts. In the case of metals, this unique behavior not only imparts paramount advantages but also confers toxicity due to their unwanted interaction with different cellular processes. In the literature, the toxicity of nanoparticles made from inert materials has been investigated and many of these have revealed toxic potential under specific conditions. The surge to understand underlying mechanism of toxicity has increased and different means have been employed to overcome toxicity problems associated with these agents. In this review, we have focused nanoparticles of three inert metallic materials i.e. gold, silver and iron as these are regarded as biologically inert in the bulk and dissolved form. These materials have gained wider research interest and studies indicating the toxicity of these materials are also emerging. Oxidative stress, physical binding and interference with intracellular signaling are the major role player in nanotoxicity and their predominance is highly dependent upon size, surface coating and administered dose of nanoparticles. Current strategies to overcome toxicity have also been reviewed in the light of recent literature. The authors also suggested that uniform testing standards and well-designed studies are needed to evaluate nanotoxicity of these materials that are otherwise considered as inert.

Pseudomonas aeruginosa produces three exopolysaccharides, Psl, Pel, and alginate, that play vital roles in biofilm formation. Pel is a glucose-rich, cellulose-like exopolysaccharide. The essential Pel biosynthesis proteins are encoded by seven genes, pelA to pelG. Bioinformatics analysis suggests that PelF is a cytosolic glycosyltransferase. Here, experimental evidence was provided to support this PelF function. A UDP-glucose dehydrogenase-based assay was developed to quantify UDP-glucose. UDP-glucose was proposed as the substrate for PelF. The isogenic pelF deletion mutant accumulated 1.8 times more UDP-glucose in its cytosol than the wild type. This suggested that PelF, which was found localized in the cystosol, uses UDP-glucose as substrate. Additionally, in vitro experiments confirmed that PelF uses UDP-glucose as substrate. To analyze the functional roles of conserved residues in PelF, site-directed mutagenesis was performed. The presence of the EX 7 E motif is characteristic for various glycosyltransferase families, and in PelF, E405/E413 are the conserved residues in this motif. Replacement of E405 with A resulted in a reduction of PelF activity to 30.35% ؎ 3.15% (mean ؎ standard deviation) of the wild-type level, whereas replacement of the second E, E413, with A did not produce a significant change in the activity of PelF. Moreover, replacement of both E residues did not result in a loss of PelF function, but replacement of the conserved R325 or K330 with A resulted in a complete loss of PelF activity. Overall, our data show that PelF is a soluble glycosyltransferase that uses UDP-glucose as the substrate for Pel synthesis and that conserved residues R325 and K330 are important for the activity of PelF.

BackgroundNewcastle disease (ND) is one of the most deadly diseases of poultry around the globe. The disease is endemic in Pakistan and recurrent outbreaks are being reported regularly in wild captive, rural and commercial poultry flocks. Though, efforts have been made to characterize the causative agent in some of parts of the country, the genetic nature of strains circulating throughout Pakistan is currently lacking.Material and methodsTo ascertain the genetics of NDV, 452 blood samples were collected from 113 flocks, originating from all the provinces of Pakistan, showing high mortality (30–80%). The samples represented domesticated poultry (broiler, layer and rural) as well as wild captive birds (pigeons, turkeys, pheasants and peacock). Samples were screened with real-time PCR for both matrix and fusion genes (1792 bp), positive samples were subjected to amplification of full fusion gene and subsequent sequencing and phylogenetic analysis.ResultsThe deduced amino acid sequence of the fusion protein cleavage site indicated the presence of motif (112RK/RQRR↓F117) typical for velogenic strains of NDV. Phylogenetic analysis of hypervariable region of the fusion gene indicated that all the isolates belong to lineage 5 of NDV except isolates collected from Khyber Pakhtunkhwa (KPK) province. A higher resolution of the phylogenetic analysis of lineage 5 showed the distribution of Pakistani NDV strains to 5b. However, the isolates from KPK belonged to lineage 4c; the first report of such lineage from this province.ConclusionsTaken together, data indicated the prevalence of multiple lineages of NDV in different poultry population including wild captive birds. Such understanding is crucial to underpin the nature of circulating strains of NDV, their potential for interspecies transmission and disease diagnosis and control strategies.

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