Effect of the induction of P<i>lasR</i>-bound proteins on P<i>lasR</i> promoter activity.

<p>(A) Histogram reporting P<i>lasR</i> maximal promoter activity (grey bars) and the corresponding cell density (white bars) measured in <i>P. aeruginosa</i> PAO1 P<i>lasR::lux</i> strains carrying the plasmids indicated below the graph, grown in LB supplemented with 0.1% (w/v) l-arabinose. (B) Graph reporting P<i>lasR</i> promoter activity (filled symbols) and cell density (open symbols) measured during the growth curve in <i>P. aeruginosa</i> PAO1 P<i>lasR::lux</i> carrying pHERD30T (triangles) or pR3699 (circles), grown in LB supplemented with 0.1% (w/v) l-arabinose. (C) Histogram reporting P<i>lasR</i> maximal promoter activity measured in <i>P. aeruginosa</i> PAO1 P<i>lasR::lux</i> strains carrying pHERD30T (grey bars) or pR3699 (white bars) grown in LB supplemented with different l-arabinose concentrations (%, w/v), indicated below the graph. In (A), (B) and (C) the average of three independent experiments is reported with standard deviations; in (A) and (C) statistical significance with respect to <i>P. aeruginosa</i> PAO1 P<i>lasR::lux</i> (pHERD30T) is indicated with one asterisk (<i>p</i> < 0.01).</p

P<i>lasR</i> activity and P<i>lasR</i>-affinity chromatography.

<p>(A) Growth curve of the <i>P. aeruginosa</i> PAO1 wild type strain carrying the pMP<i>lasR</i>::<i>lacZ</i> plasmid (dashed line) and corresponding P<i>lasR</i> promoter activity (solid line). The points of the growth curve at which the protein crude extracts for DNA-affinity chromatography were prepared are indicated by arrows. (B) SDS-PAGE analysis of proteins bound to the P<i>lasR</i> promoter region. Protein crude extracts were prepared from <i>P. aeruginosa</i> PAO1 cultures grown in LB broth to the indicated cell densities (A₆₀₀). Bands analysed by MALDI-TOF mass spectrometry are indicated by arrows, and the corresponding protein name is reported.</p

Effect of PA3699 induction on <i>P. aeruginosa</i> virulence factors production.

<p>Histogram reporting elastase, pyocyanin and proteases production measured in <i>P. aeruginosa</i> PAO1 carrying pHERD30T (grey bars) or pR3699 (white bars), grown in LB supplemented with 0.1% (w/v) l-arabinose. The average of three independent experiments is reported with standard deviations; statistical significance with respect to <i>P</i>. <i>aeruginosa</i> PAO1 (pHERD30T) is indicated with one asterisk (<i>p</i> < 0.01).</p

PA3699 purification and P<i>lasR</i>-binding assay.

<p>(A) SDS-PAGE analysis of samples withdrawn at different steps of PA3699 purification. Lane L, PageRuler Unstained Protein Ladder (Fermentas); lane 1, non-induced protein crude extract; lane 2, induced protein crude extract; lane 3, soluble fraction of the induced protein crude extract; lane 4, purified protein; line 5, purified protein after thrombin cleavage. (B) Western blot analysis performed with mouse anti-6xHis primary antibody and anti-mouse peroxidase-conjugated secondary antibody on a gel identical to the one shown in (A). (C) Autoradiography of an EMSA showing direct interaction between a DNA probe encompassing the <i>lasR</i> promoter region and purified PA3699. PA3699 concentration (μM) is indicated above each lane. An unspecific probe was added in the reaction mixture as control. The PA3699-P<i>lasR</i> complex and the free DNA probes are indicated.</p

A New Transcriptional Repressor of the <i>Pseudomonas aeruginosa</i> Quorum Sensing Receptor Gene <i>lasR</i>

<div><p><i>Pseudomonas aeruginosa</i> pathogenic potential is controlled <i>via</i> multiple regulatory pathways, including three quorum sensing (QS) systems. LasR is a key QS signal receptor since it acts as a global transcriptional regulator required for optimal expression of main virulence factors. <i>P. aeruginosa</i> modulates the QS response by integrating this cell density-dependent circuit to environmental and metabolic cues. Hence, QS also controls the adaptation to challenging environmental niches, such as infection sites. However, little is known about the molecular mechanisms connecting QS and other signalling pathways. In this work, DNA-affinity chromatography was used to identify new <i>lasR</i> transcriptional regulators. This approach led to the identification and functional characterization of the TetR-like transcriptional repressor PA3699. This protein was purified and shown to directly bind to the <i>lasR</i> promoter region <i>in vitro</i>. The induction of PA3699 expression in <i>P. aeruginosa</i> PAO1 cultures repressed <i>lasR</i> promoter activity and the production of LasR-dependent virulence factors, such as elastase, pyocyanin, and proteases. These findings suggest a role for PA3699 in <i>P. aeruginosa</i> pathogenicity. <i>P. aeruginosa</i> genome encodes at least 38 TetR-family proteins, and PA3699 is the eighth member of this group functionally characterized so far and the first one shown to bind the <i>lasR</i> promoter <i>in vitro.</i></p> </div

Data_Sheet_1_Evolving trends in epidemiology and natural history of cardiac amyloidosis: 30-year experience from a tertiary referral center for cardiomyopathies.docx

ObjectiveNatural history of cardiac amyloidosis (CA) is poorly understood. We aimed to examine the changing mortality of different types of CA over a 30-year period.Patients and methodsConsecutive patients included in the “Trieste CA Registry” from January 1, 1990 through December 31, 2021 were divided into a historical cohort (diagnosed before 2016) and a contemporary cohort (diagnosed after 2016). Light chain (AL), transthyretin (ATTR) and other forms of CA were defined according to international recommendations. The primary and secondary outcome measures were all-cause mortality and cardiac death, respectively.ResultsWe enrolled 182 patients: 47.3% AL-CA, 44.5% ATTR-CA, 8.2% other etiologies. The number of patients diagnosed with AL and ATTR-CA progressively increased over time, mostly ATTR-CA patients (from 21% before 2016 to 67% after 2016) diagnosed non-invasively. The more consistent increase in event-rate was observed in the long-term (after 50 months) in ATTR-CA compared to the early increase in mortality in AL-CA. In the contemporary cohort, during a median follow up of 16 [4–30] months, ATTR-CA was associated with improved overall and cardiac survival compared to AL-CA. At multivariable analysis, ATTR-CA (HR 0.42, p = 0.03), eGFR (HR 0.98, p = 0.033) and ACE-inhibitor therapy (HR 0.24, p ConclusionIncidence and prevalence rates of ATTR-CA and, to a less extent, of AL-CA have been increasing over time, with significant improvements in 2-year survival of ATTR-CA patients from the contemporary cohort. Reaching an early diagnosis and starting disease-modifying treatments will improve long-term survival in CA.</p