CcrZ is a recently discovered cell cycle regulator that connects DNA replication initiation with cell division in pneumococci and may have a similar function in related bacteria. CcrZ is also ...annotated as a putative kinase, suggesting that CcrZ homologs could represent a novel family of bacterial kinase-dependent cell cycle regulators. Here, we investigate the CcrZ homolog in Bacillus subtilis and show that cells lacking ccrZ are sensitive to a broad range of DNA damage. We demonstrate that increased expression of ccrZ results in over-initiation of DNA replication. In addition, increased expression of CcrZ activates the DNA damage response. Using sensitivity to DNA damage as a proxy, we show that the negative regulator for replication initiation (yabA) and ccrZ function in the same pathway. We show that CcrZ interacts with replication initiation proteins DnaA and DnaB, further suggesting that CcrZ is important for replication timing. To understand how CcrZ functions, we solved the crystal structure bound to AMP-PNP to 2.6 Å resolution. The CcrZ structure most closely resembles choline kinases, consisting of a bilobal structure with a cleft between the two lobes for binding ATP and substrate. Inspection of the structure reveals a major restructuring of the substrate-binding site of CcrZ relative to the choline-binding pocket of choline kinases, consistent with our inability to detect activity with choline for this protein. Instead, CcrZ shows activity on D-ribose and 2-deoxy-D-ribose, indicating adaptation of the choline kinase fold in CcrZ to phosphorylate a novel substrate. We show that integrity of the kinase active site is required for ATPase activity in vitro and for function in vivo. This work provides structural, biochemical, and functional insight into a newly identified, and conserved group of bacterial kinases that regulate DNA replication initiation.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Biofilms are organized multicellular communities encased in an extracellular polymeric substance (EPS). Biofilm-resident bacteria resist immunity and antimicrobials. The EPS provides structural ...stability and presents a barrier; however, a complete understanding of how EPS structure relates to biological function is lacking. This review focuses on the EPS of three Gram-negative pathogens: Pseudomonas aeruginosa, nontypeable Haemophilus influenzae, and Salmonella enterica serovar Typhi/Typhimurium. Although EPS proteins and polysaccharides are diverse, common constituents include extracellular DNA, DNABII (DNA binding and bending) proteins, pili, flagella, and outer membrane vesicles. The EPS biochemistry promotes recalcitrance and informs the design of therapies to reduce or eliminate biofilm burden.
Virtually all cells living in multicellular structures such as tissues and organs are encased in an extracellular matrix. One of the most important features of a biofilm is the extracellular ...polymeric substance that functions as a matrix, holding bacterial cells together. Yet very little is known about how the matrix forms or how matrix components encase bacteria during biofilm development. Pseudomonas aeruginosa forms environmentally and clinically relevant biofilms and is a paradigm organism for the study of biofilms. The extracellular polymeric substance of P. aeruginosa biofilms is an ill-defined mix of polysaccharides, nucleic acids, and proteins. Here, we directly visualize the product of the polysaccharide synthesis locus (Psl exopolysaccharide) at different stages of biofilm development. During attachment, Psl is anchored on the cell surface in a helical pattern. This promotes cell-cell interactions and assembly of a matrix, which holds bacteria in the biofilm and on the surface. Chemical dissociation of Psl from the bacterial surface disrupted the Psl matrix as well as the biofilm structure. During biofilm maturation, Psl accumulates on the periphery of 3-D-structured microcolonies, resulting in a Psl matrix-free cavity in the microcolony center. At the dispersion stage, swimming cells appear in this matrix cavity. Dead cells and extracellular DNA (eDNA) are also concentrated in the Psl matrix-free area. Deletion of genes that control cell death and autolysis affects the formation of the matrix cavity and microcolony dispersion. These data provide a mechanism for how P. aeruginosa builds a matrix and subsequently a cavity to free a portion of cells for seeding dispersal. Direct visualization reveals that Psl is a key scaffolding matrix component and opens up avenues for therapeutics of biofilm-related complications.
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Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
To update the 2006 American Society of Clinical Oncology guideline on the use of hematopoietic colony-stimulating factors (CSFs).
The American Society of Clinical Oncology convened an Update ...Committee and conducted a systematic review of randomized clinical trials, meta-analyses, and systematic reviews from October 2005 through September 2014. Guideline recommendations were based on the review of the evidence by the Update Committee.
Changes to previous recommendations include the addition of tbo-filgrastim and filgrastim-sndz, moderation of the recommendation regarding routine use of CSFs in older patients with diffuse aggressive lymphoma, and addition of recommendations against routine dose-dense chemotherapy in lymphoma and in favor of high-dose-intensity chemotherapy in urothelial cancer. The Update Committee did not address recommendations regarding use of CSFs in acute myeloid leukemia or myelodysplastic syndromes in adults.
Prophylactic use of CSFs to reduce the risk of febrile neutropenia is warranted when the risk of febrile neutropenia is approximately 20% or higher and no other equally effective and safe regimen that does not require CSFs is available. Primary prophylaxis is recommended for the prevention of febrile neutropenia in patients who are at high risk on the basis of age, medical history, disease characteristics, and myelotoxicity of the chemotherapy regimen. Dose-dense regimens that require CSFs should only be used within an appropriately designed clinical trial or if supported by convincing efficacy data. Current recommendations for the management of patients exposed to lethal doses of total-body radiotherapy, but not doses high enough to lead to certain death as a result of injury to other organs, include the prompt administration of CSFs.
Biodegradable 3D-printed polycaprolactone scaffolds for bone tissue engineering applications have been extensively studied as they can provide an attractive porous architecture mimicking natural ...bone, with tunable physical and mechanical properties enhancing positive cellular response. The main drawbacks of polycaprolactone-based scaffolds, limiting their applications in tissue engineering are: their hydrophobic nature, low bioactivity and poor mechanical properties compared to native bone tissue. To overcome these issues, the surface of scaffolds is usually modified and covered with a ceramic layer. However, a detailed description of the adhesion forces of ceramic particles to the polymer surface of the scaffolds is still lacking. Our present work is focused on obtaining PCL-based composite scaffolds to strengthen the architecture of the final product. In this manuscript, we report qualitative and quantitative evaluation of low temperature plasma modification followed by detailed studies of the adhesion forces between chemically attached ceramic layer and the surface of polycaprolactone-nanohydroxyapatite composite 3D-printed scaffolds. The results suggest modification-dependent alteration of the internal structure and morphology, as well as mechanical and physical scaffold properties recorded with atomic force microscopy. Moreover, changes in the material surface were followed by enhanced adhesion forces binding the ceramic layer to polymer-based scaffolds.
•Polycaprolactone-based bone tissue engineering scaffolds were fabricated and modified using low-temperature plasma.•Low temperature plasma modification of scaffolds altered their surface structure, roughness, stiffness and wettability.•The scaffolds were coated with hydroxyapatite and adhesion forces between the coating and polymeric surface was assesed.
Bacterial extracellular polysaccharides are a key constituent of the extracellular matrix material of biofilms. Pseudomonas aeruginosa is a model organism for biofilm studies and produces three ...extracellular polysaccharides that have been implicated in biofilm development, alginate, Psl and Pel. Significant work has been conducted on the roles of alginate and Psl in biofilm development, however we know little regarding Pel. In this study, we demonstrate that Pel can serve two functions in biofilms. Using a novel assay involving optical tweezers, we demonstrate that Pel is crucial for maintaining cell-to-cell interactions in a PA14 biofilm, serving as a primary structural scaffold for the community. Deletion of pelB resulted in a severe biofilm deficiency. Interestingly, this effect is strain-specific. Loss of Pel production in the laboratory strain PAO1 resulted in no difference in attachment or biofilm development; instead Psl proved to be the primary structural polysaccharide for biofilm maturity. Furthermore, we demonstrate that Pel plays a second role by enhancing resistance to aminoglycoside antibiotics. This protection occurs only in biofilm populations. We show that expression of the pel gene cluster and PelF protein levels are enhanced during biofilm growth compared to liquid cultures. Thus, we propose that Pel is capable of playing both a structural and a protective role in P. aeruginosa biofilms.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Correlating electromechanical and dielectric properties with nanometre-scale order is the defining challenge for the development of piezoelectric oxides. Current lead (Pb)-based relaxor ...ferroelectrics can serve as model systems with which to unravel these correlations, but the nature of the local order and its relation to material properties remains controversial. Here we employ recent advances in diffuse scattering instrumentation to investigate crystals that span the phase diagram of PbMg
Nb
O
-xPbTiO
(PMN-xPT) and identify four forms of local order. From the compositional dependence, we resolve the coupling of each form to the dielectric and electromechanical properties observed. We show that relaxor behaviour does not correlate simply with ferroic diffuse scattering; instead, it results from a competition between local antiferroelectric correlations, seeded by chemical short-range order, and local ferroic order. The ferroic diffuse scattering is strongest where piezoelectricity is maximal and displays previously unrecognized modulations caused by anion displacements. Our observations provide new guidelines for evaluating displacive models and hence the piezoelectric properties of environmentally friendly next-generation materials.
The transcription factor AmrZ regulates genes important for P. aeruginosa virulence, including type IV pili, extracellular polysaccharides, and the flagellum; however, the global effect of AmrZ on ...gene expression remains unknown, and therefore, AmrZ may directly regulate many additional genes that are crucial for infection. Compared to the wild type strain, a ΔamrZ mutant exhibits a rugose colony phenotype, which is commonly observed in variants that accumulate the intracellular second messenger cyclic diguanylate (c-di-GMP). Cyclic di-GMP is produced by diguanylate cyclases (DGC) and degraded by phosphodiesterases (PDE). We hypothesized that AmrZ limits the intracellular accumulation of c-di-GMP through transcriptional repression of gene(s) encoding a DGC. In support of this, we observed elevated c-di-GMP in the ΔamrZ mutant compared to the wild type strain. Consistent with other strains that accumulate c-di-GMP, when grown as a biofilm, the ΔamrZ mutant formed larger microcolonies than the wild-type strain. This enhanced biofilm formation was abrogated by expression of a PDE. To identify potential target DGCs, a ChIP-Seq was performed and identified regions of the genome that are bound by AmrZ. RNA-Seq experiments revealed the entire AmrZ regulon, and characterized AmrZ as an activator or repressor at each binding site. We identified an AmrZ-repressed DGC-encoding gene (PA4843) from this cohort, which we named AmrZ dependent cyclase A (adcA). PAO1 overexpressing adcA accumulates 29-fold more c-di-GMP than the wild type strain, confirming the cyclase activity of AdcA. In biofilm reactors, a ΔamrZ ΔadcA double mutant formed smaller microcolonies than the single ΔamrZ mutant, indicating adcA is responsible for the hyper biofilm phenotype of the ΔamrZ mutant. This study combined the techniques of ChIP-Seq and RNA-Seq to define the comprehensive regulon of a bifunctional transcriptional regulator. Moreover, we identified a c-di-GMP mediated mechanism for AmrZ regulation of biofilm formation and chronicity.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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