This paper systematically investigated and reported for the first time the identification and quantification of co-eluting impurities as low as 0.05 area% by PDA with i-PDeA II deconvolution software in the LabSolutions Chromatographic Data System (CDS) using an integrated multivariate curve resolution-alternating least squares (MCR-ALS) algorithm with a bidirectional exponentially modified Gaussian (BEMG) model function. The algorithm was able to consistently identify 0.
View Article and Find Full Text PDFBackground: cell envelope-associated proteins play a relevant role in infection mechanisms. They can contribute to the antibiotic resistance of the bacterial cells and be involved in the interaction with host cells. Thus, studies contributing to elucidating these key molecular elements are of great importance to find alternative therapeutics.
View Article and Find Full Text PDFEnvironmental bacteria are most often endowed with native surface-attachment programs that frequently conflict with efforts to engineer biofilms and synthetic communities with given tridimensional architectures. In this work, we report the editing of the genome of KT2440 for stripping the cells of most outer-facing structures of the bacterial envelope that mediate motion, binding to surfaces, and biofilm formation. To this end, 23 segments of the chromosome encoding a suite of such functions were deleted, resulting in the surface-naked strain EM371, the physical properties of which changed dramatically in respect to the wild type counterpart.
View Article and Find Full Text PDFThe Pseudomonas aeruginosa PA3685 locus encodes a conserved protein that shares 49% sequence identity with Escherichia coli YeaZ, which was recently reported as involved in the biosynthesis of threonylcarbamoyl adenosine (t(6)A), a universal modified tRNA nucleoside. Many YeaZ orthologues were reported as "essential for life" among various bacterial species, suggesting a critical role for both these proteins and for the t(6)A biosynthetic pathway. We provide here evidences that PA3685 protein (PaYeaZ) is essential.
View Article and Find Full Text PDFThe evaluation of weight average molecular weight (Mw) and molecular weight distribution represents one of the most controversial aspects concerning the characterization of low molecular weight heparins (LMWHs). As the most commonly used method for the measurement of such parameters is high performance size exclusion chromatography (HP-SEC), the soundness of results mainly depends on the appropriate calibration of the chromatographic columns used. With the aim of meeting the requirement of proper Mw standards for LMWHs, in the present work the determination of molecular weight parameters (Mw and Mn) by HP-SEC combined with a triple detector array (TDA) was performed.
View Article and Find Full Text PDFBackground: Antibiotics in current use target a surprisingly small number of cellular functions: cell wall, DNA, RNA, and protein biosynthesis. Targeting of novel essential pathways is expected to play an important role in the discovery of new antibacterial agents against bacterial pathogens, such as Pseudomonas aeruginosa, that are difficult to control because of their ability to develop resistance, often multiple, to all current classes of clinical antibiotics.
Results: We aimed to identify novel essential genes in P.
We report on specific magneto-capturing followed by Multidimensional Protein Identification Technology (MudPIT) for the analysis of surface-exposed proteins of intact cells of the bacterial opportunistic pathogen Pseudomonas aeruginosa. The magneto-separation of cell envelope fragments from the soluble cytoplasmic fraction allowed the MudPIT identification of the captured and neighboring proteins. Remarkably, we identified 63 proteins captured directly by nanoparticles and 67 proteins embedded in the cell envelope fragments.
View Article and Find Full Text PDFThe Gram-negative bacterium Pseudomonas aeruginosa is an important opportunistic pathogen in compromised individuals, such as patients with cystic fibrosis, severe burns or impaired immunity. In this work we aimed to screen novel essential genes of P. aeruginosa by shotgun antisense identification, a technique that was developed a decade ago for the Gram-positive bacterium Staphylococcus aureus and was under-used in Gram-negative bacteria for a considerable period of time.
View Article and Find Full Text PDFAs part of a more extensive investigation on structural features of different low-molecular-weight heparins (LMWHs) that can affect their biological activities, Enoxaparin, Tinzaparin and Dalteparin were characterised with regards to the distribution of different chain length oligosaccharides as determined by size-exclusion (SE) chromatography, as well as their structure as defined by 2D-NMR spectra (HSQC). The three LMWHs were also fractionated into high affinity (HA) and no affinity (NA) pools with regards to their ability to bind antithrombin (AT). The HA fractions were further subfractionated and characterised.
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