Replacing the 238th aspartic acid with an arginine impaired the oligomerization activity and inflammation-inducing property of pyolysin

<p><i>Trueperella pyogenes</i> (<i>T. pyogenes</i>) is an important opportunistic pathogen. Pyolysin (PLO) importantly contributes to the pathogenicity of <i>T. pyogenes</i>. However, the relationship between the structure and function and the virulence of PLO is not well documented. In the current study, recombinant PLO (rPLO) and three rPLO mutants were prepared. rPLO D238R, a mutant with the 238th aspartic acid replaced with an arginine, showed impairment in oligomerization activity on cholesterol-containing liposome and pore-forming activity on sheep red blood cell membrane. Further study employing the prepared mutants confirmed that the pore-forming activity of PLO is essential for inducing excessive inflammation responses in mice by upregulating the expression levels of IL-1β, TNF-α, and IL-6. By contrast, rPLO P499F, another mutant with impaired cell membrane binding capacity, elicited an inflammation response that was dependent on pathogen-associated molecular pattern (PAMP) activity, given that the mutant significantly upregulated the expression of IL-10 in macrophages and in mice, whereas rPLO did not. Results indicated that domain 1 of the PLO molecule plays an important role in maintaining pore-forming activity. Moreover, the PLO pore-forming activity and not PAMP activity is responsible for the inflammation-inducing effect of PLO. The results of this study provided new information for research field on the structure, function, and virulence of PLO.</p> <p><b>Abbreviations</b>: <i>T. pyogenes: Trueperella pyogenes</i>; PLO: Pyolysin; rPLO: recombinant PLO; PAMP: pathogen-associated molecular pattern; CDCs: cholesterol-dependent cytolysins; PLY: pneumolysin; NLRP3: NLR family pyrin domain containing protein 3; PRRs: pattern recognition receptors; Asp: aspartic acid; TLR4: Toll-like receptor 4; Arg: arginine; Asn: asparagine; IPTG: Isopropyl-β-d-thiogalactoside; PBS: phosphate-buffered saline; sRBCs: sheep red blood cells; TEM: Transmission electron microscopy; RBCM: red blood cell membrane; SDS-PAGE: sodium dodecyl sulfate–polyacrylamide gel electrophoresis; NC membrane: nitrocellulose membrane; SDS-AGE: dodecyl sulfate agarose gel electrophoresis; MDBK cells: Madin–Darby bovine kidney cells; RPMI-1640 medium: Roswell Park Memorial Institute-1640 medium; FBS: fetal bovine serum; BMDMs: bone marrow-derived macrophages; TNF-α: tumor necrosis factor α; IL-1β: interleukin-1β; IFN-γ: interferon-γ; TGF-β: transforming growth factor-β; ELISA: enzyme-linked immunosorbent assay</p

Datasheet1_Association between red cell distribution width-to-albumin ratio and prognostic outcomes in pediatric intensive care unit patients: a retrospective cohort study.pdf

ObjectiveThis study aimed to assess the association between Red Cell Distribution Width-to-Albumin Ratio (RAR) and the clinical outcomes in Pediatric Intensive Care Unit (PICU) patients.DesignThis is a retrospective cohort study.MethodsWe conducted a retrospective cohort study based on the Pediatric Intensive Care database. The primary outcome was the 28-day mortality rate. Secondary outcomes included the 90-day mortality rate, in-hospital mortality rate, and length of hospital stay. We explored the relationship between RAR and the prognosis of patients in the PICU using multivariate regression and subgroup analysis.ResultsA total of 7,075 participants were included in this study. The mean age of the participants was 3.4 ± 3.8 years. Kaplan–Meier survival curves demonstrated that patients with a higher RAR had a higher mortality rate. After adjusting for potential confounding factors, we found that for each unit increase in RAR, the 28-day mortality rate increased by 6% (HR = 1.06, 95% CI: 1.01–1.11, P = 0.015). The high-RAR group (RAR ≥ 4.0) had a significantly increased 28-day mortality rate compared to the low-RAR group (RAR ≤ 3.36) (HR = 1.7, 95% CI: 1.23–2.37, P ConclusionOur study suggests a significant association between RAR and adverse outcomes in PICU patients. A higher RAR is associated with higher 28-day, 90-day, and in-hospital mortality rates.</p

Areal data from summer 2004 and winter 2006 cruises.

<p>All parameters for the summer cruise have been integrated over the entire euphotic layer (1% incident PAR at surface; it was similar to the mixed layer depth or deeper). The winter PP has been integrated over the euphotic layer while other parameters have been integrated over the mixed layer depth (epipelagic) and from there down to 750 m (mesopelagic layer). Averages and standard deviation in parenthesis.</p><p>PP = primary production; BCP = bacterial carbon production; Summer excess DOC = summer dissolved organic carbon values minus average winter constant value (36.5±2.8 µM C s.d.); BCD = bacterial carbon demand (BCP/bacterial growth efficiency); N.D. = not determined.</p>*<p>BCD calculated using a bacterial growth efficiency derived by the curve in ref. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0006941#pone.0006941-Rivkin1" target="_blank">[20]</a> (∼36% in summer and 39% in winter).</p>**<p>BCD calculated using bacterial growth efficiency of 13% in summer and 6.2% in winter (averaging all data for summer and only HNLC for winter from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0006941#pone.0006941-Obernosterer1" target="_blank">[12]</a>).</p>§<p>no s.d. reported because the value is derived from a single depth profile.</p>°<p>DOC data for winter are not reported, since they are considered as constant refractory DOC values, and have been used to determine summer excess DOC.</p

List of GenBank accession number and closest relative to 16S rDNA archaeal sequences obtained from DGGE.

*<p>phylotypes found in both seasons. Ref. = References of the closest relative.</p

Depth profile of bacterial parameters in summer 2004 and winter 2006.

<p>Winter data are from 32 stations from 5 to 400 m; at 6 stations samples were also taken from 750 m. Summer: blue circles = ACC water; red circles = shelf water; green circles = mixed water; empty square = Winter. (Panel A) Bacterial abundance; (panel B) BCP = bacterial carbon production; BCP calculated from ³H-Leucine incorporation, employing a conversion factor of 3.1 kg C per mol of Leu <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0006941#pone.0006941-Simon1" target="_blank">[17]</a>; (panel C) μ = bacterial growth rate. Cell-specific growth rate calculations assumed 20 fg C per cell <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0006941#pone.0006941-Lee1" target="_blank">[18]</a>.</p

List of GenBank accession number and closest relative to 16S rDNA bacterial sequences obtained from DGGE.

*<p>phylotypes found in both seasons. Ref. = References of the closest relative.</p

DGGE fingerprint of <i>Archaea</i> amplicons from Summer 2004 (A) and Winter 2006 (B) samples.

<p>The numbered bands have been excised for sequencing.</p

List and parameters of stations sampled for bacterial community analysis.

*<p>positive temperatures in summer at surface, in winter at depth.</p

Depth profile of chemotrophic carbon production in winter 2006.

<p>DIC = dissolved inorganic carbon.</p

Depth distribution of organic matter pools during summer and winter cruises.

<p>Summer 2004: blue circles = ACC water; red circles = shelf water; green circles = mixed water; empty square = Winter 2006. (Panel A) DOC = Dissolved organic carbon. The data presented are measurements of total organic carbon, but since POC represents a negligible contribution to total organic carbon (POC represented only between 7% and 2% of the total pool) we can consider the analysis as DOC values. The shaded area covers the range of winter concentration. For a comparison, a range of variations of DOC from the FRUELA cruise study area in summer (Gerlache Strait, Bransfield Strait and Bellinghausen Strait) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0006941#pone.0006941-Doval1" target="_blank">[22]</a> has been reported (black triangles). (Panel B) POC = Particulate organic carbon.</p