Lipids organize the activity of the cell's proteome through a complex network of interactions. The assembly of comprehensive atlases embracing all protein-lipid interactions is an important challenge ...that requires innovative methods. We recently developed a liposome-microarray-based assay (LiMA) that integrates liposomes, microfluidics and fluorescence microscopy and which is capable of measuring protein recruitment to membranes in a quantitative and high-throughput manner. Compared with previous assays that are labor-intensive and difficult to scale up, LiMA improves the protein-lipid interaction assay throughput by at least three orders of magnitude. Here we provide a step-by-step LiMA protocol that includes the following: (i) the serial and generic production of the liposome microarray; (ii) its integration into a microfluidic format; (iii) the measurement of fluorescently labeled protein (either purified proteins or from cell lysate) recruitment to liposomal membranes using high-throughput microscopy; (iv) automated image analysis pipelines to quantify protein-lipid interactions; and (v) data quality analysis. In addition, we discuss the experimental design, including the relevant quality controls. Overall, the protocol-including device preparation, assay and data analysis-takes 6-8 d. This protocol paves the way for protein-lipid interaction screens to be performed on the proteome and lipidome scales.
Lipids have a role in virtually all biological processes, acting as structural elements, scaffolds and signaling molecules, but they are still largely under-represented in known biological networks. ...Here we describe a liposome microarray-based assay (LiMA), a method that measures protein recruitment to membranes in a quantitative, automated, multiplexed and high-throughput manner.
Many cellular processes involve the recruitment of proteins to specific membranes, which are decorated with distinctive lipids that act as docking sites. The phosphoinositides form signaling hubs, ...and we examine mechanisms underlying recruitment. We applied a physiological, quantitative, liposome microarray-based assay to measure the membrane-binding properties of 91 pleckstrin homology (PH) domains, the most common phosphoinositide-binding target. 10,514 experiments quantified the role of phosphoinositides in membrane recruitment. For most domains examined, the observed binding specificity implied cooperativity with additional signaling lipids. Analyses of PH domains with similar lipid-binding profiles identified a conserved motif, mutations in which—including some found in human cancers—induced discrete changes in binding affinities in vitro and protein mislocalization in vivo. The data set reveals cooperativity as a key mechanism for membrane recruitment and, by enabling the interpretation of disease-associated mutations, suggests avenues for the design of small molecules targeting PH domains.
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•The lipid-binding properties of 91 pleckstrin homology domains have been quantified•Over 10,000 individual protein-lipid interaction experiments have been performed•The binding specificity and affinity imply cooperativity between signaling lipids•Mutations found in cancer biopsies fine-tune lipid-binding specificities
Vonkova et al. systematically quantify the lipid-binding properties of 91 pleckstrin homology (PH) domains using a physiological, quantitative, liposome microarray-based assay. The data set reveals that cooperativity between lipids is a key mechanism for membrane recruitment of PH domains.
The present paper reports the development of a novel silver nanoparticles-derivatized polystyrene substrate useful for for protein microarray application. The silvered coating was prepared by a novel ...photochemical method. Various coatings were tested and characterized by means of chemical-physical methods. It was demonstrated that these methods are useful for the preparation of protein microarray for the analysis of human antibody by sandwich methods of fluorescence immunoassay (Alexa labeled). Comparative studies were carried out with different printing buffers and various surface derivatization to find the optimal conditions for antigen immobilization. Therein described assay, the human myeloperoxidase antigen (MPO) was quantitative detected directly from human serum. The cross-reactivity was here evaluated by reaction with recombinant human tissue transglutaminase antigen (htTG), a linearity response from 20 to 2000 ng/μl was investigated by human IgG probes. The data indicate an increase of both signal-to-noise ratio value and reproducibility for the silver nanoparticle coating respect the value obtained for the polystyrene commercial slides.
Mortality in infective endocarditis (IE) is still high, and the long term prognosis remains uncertain. This study aimed to identify predictors of long-term mortality for any cause, adverse event ...rate, relapse rate, valvular and ventricular dysfunction at follow-up, in a real-world surgical centre.
We retrospectively analyzed 363 consecutive episodes of IE (123 women, 34%) admitted to our department with a definite diagnosis of non-device-related IE. Median follow-up duration was 2.9 years. Primary endpoints were predictors of mortality, recurrent endocarditis, and major non-fatal adverse events (hospitalization for any cardiovascular cause, pace-maker implantation, new onset of atrial fibrillation, sternal dehiscence), and ventricular and valvular dysfunction at follow-up.
Multivariate analysis independent predictors of mortality showed age (HR per unit 1.031, p < 0.003), drug abuse (HR 3.5, p < 0.002), EUROSCORE II (HR per unit 1.017, p < 0.0006) and double valve infection (HR 2.3, p < 0.001) to be independent predictors of mortality, while streptococcal infection remained associated with a better prognosis (HR 0.5, p < 0.04). Major non-fatal adverse events were associated with age (HR 1.4, p < 0.022). New episodes of infection were correlated with S aureus infection (HR 4.8, p < 0.001), right-sided endocarditis (HR 7.4, p < 0.001), spondylodiscitis (HR 6.8, p < 0.004) and intravenous drug abuse (HR 10.3, p < 0.001). After multivariate analysis, only drug abuse was an independent predictor of new episodes of endocarditis (HR 8.5, p < 0.001). Echocardiographic follow-up, available in 95 cases, showed a worsening of left ventricular systolic function (p < 0.007); severe valvular dysfunction at follow-up was reported only in 4 patients, all of them had mitral IE (p < 0.03).
The present study highlights some clinical, readily available factors that can be useful to stratify the prognosis of patients with IE.
Abstract
Background
Surgery is performed in 50% to 60% of infective endocarditis. In patients with definite surgical indication for hearth failure and with large vegetation, early surgery prevents ...embolic events. The optimal timing of surgery for other indications is still debated. Moreover, patients with large vegetation as unique indication to surgery, have a weak class of recommendation to it. Accurate risk stratification for embolic events is desirable to optimize selection of surgical candidates.
Materials and methods
We retrospectively analyzed 195 consecutive patients (72 women and 123 men) admitted to our department between 2013 and 2017 with definite IE according to modified Duke University criteria. Transesophageal echocardiography and blood cultures were performed in all patients for confirmation of diagnosis. Systemic embolism was soughton admission clinically and with imaging techniques (Brain and chest CT plus abdominal CT or US scan). Seventy-seven percent of patients underwent surgery (valve repair or replacement). Outcome following discharge was systematically assessed by structured telephone interviews.
Results
Of the 195 patients with left sided IE, 151 underwent surgery, 29 were low risk and treated medically, 5 refused surgery and 10 were not operated due to high surgical risk. Overall survival was 78% at 4 years. Patients excluded from surgery had the worst prognosis, while operated patients with high-risk IE showed comparable survival to non-complicated infections treated medically. Early surgery (<2 weeks from diagnosis) was associated with similar survival compared to later intervention. Euroscore II was the main predictor of mortality when above a threshold of 7 before 2015 and 16 after 2015, reflecting surgical management of higher risk patients over time.In left sided IE, mean vegetation length was 11.1 mm; embolic events before diagnosis occurred in 35% of cases and Staphylococcus aureus etiologywas the main risk factor associated with embolism (OR 4, p<0.05). Vegetation size >10 mm was also independently associated with embolic risk (p=0.033) whereas renal failure, age, sex, endocarditis location (mitral or aortic), type of valve (native or prosthetic), perivalvular extension and degree of valveregurgitation were not.
Conclusions
Compared to low-risk IE patients treated medically, those at high-risk showed comparable survival when managed surgically, whereas a conservative approach was associated with adverse prognosis. In patients with left sided IE and intermediate vegetation length, S. aureus infection was the best independent predictor of systemic embolic events. Our data support extensive surgical referral in high risk IE and suggest that its etiology represents an important factor in decision-making.
Most cases of infective endocarditis (IE) involve a single valve, and little is known concerning IE that simultaneously affects two valves. The involvement of more than one valve may imply more ...severe and extensive cardiac lesions. In these patients, surgery may be challenging. We aimed to determine the clinical characteristics, the therapeutic strategy, and the prognostic impact of double-valve IE (DVIE). We retrospectively included in the analysis that 440 consecutive patients with definite active IE in a single surgical centre. DVIE occurred in 75 of the total enrolled 440 patients (17%) and involved mostly the combination of mitral and aortic valves (
N
= 63, 84%). Most patients had double-native IE (
N
= 45, 60%). Staphylococci were less frequent in patients with double-valve than single-valve IE (SVIE). The proportion of patients undergoing valve repair among those treated surgically was higher for patients with DVIE than for SVIE (
p
< 0.03). Valve repair of at least one valve was associated with non-significant better survival than double replacement. DVIE was associated with higher all-cause mortality than SVIE (
p
< 0.013) and a higher relapse rate (
p
= 0.023). DVIE was not associated with a higher risk of composite non-fatal adverse events. DVIE represents a considerable proportion of overall cases of IE, mainly involving aortic and mitral valves, with a jet lesion on the mitral valve; Staphylococcus is significantly less frequent than in SVIE; DVIE is independently associated with higher mortality and relapse rate; finally, mitral valve repair is feasible in a considerable proportion of surgical cases.
The prevalence of obesity in children has increased dramatically during the past decades in Europe and understanding physical fitness and its components in children is critical to design and ...implement effective interventions. The objective of the present study was to analyse the association between physical fitness (aerobic, speed, agility, power, flexibility and balance) and body mass index (BMI) in pre-pubertal children. A total of 2411 healthy schoolchildren (7-11 years) participated in this study. Anthropometric characteristics and body composition were assessed by skinfold thickness. Physical fitness was measured by nine physical fitness tests: endurance running, 20 m running speed, agility, handgrip strength, standing long jump and squat jump, sit and reach, medicine ball forward throw and static balance. No relevant differences were observed between boys and girls regarding anthropometric characteristics, body composition and physical fitness. However, overweight and obese children showed significantly lower physical fitness levels in endurance running, speed and agility (mean: +18.8, +5.5 and +14.5% of time to complete tasks, respectively), lower limb power normalised to body mass (-23.3%) and balance tests (number of falls: +165.5%) than their normal weight counterparts. On the other hand, obesity did not affect handgrip, throwing and flexibility. In conclusion, increased BMI was associated with lower performance capabilities limiting proper motor skill development, which directly affects the ability of children to take on sports skills. Actions undertaken to promote children's wellness and fitness should be prioritised and introduced early in life with the aim of enhancing physical fitness as well as preventing overweight and obesity.
Intravenous drug abuse (IDA) is a known risk factor for infective endocarditis (IE) and is associated with frequent relapses, but its prognostic impact is still debated. The potential futility of ...surgery in this population is a further issue under discussion. We aimed to describe the clinical characteristics, the therapeutic strategy, and the prognosis associated with IDA in IE. We retrospectively analysed 440 patients admitted to a single surgical centre for definite active IE from January 2012 to December 2020. IE secondary to IDA affects younger patients than those with IE not associated with IDA. Probably due to this difference, IE secondary to IDA is not associated with significantly higher mortality, whereas the negative, long-term prognostic impact of IDA emerges in multivariate analysis. Considering the good prognosis of patients with uncomplicated IE treated medically, surgery should be reserved to patients with a strict- guidelines-based indication. However, since there are no clear predictors of an unfavourable risk-benefit ratio of surgery in patients with surgical indication, all patients with a complicated IE should be operated, irrespective of a history of IDA.
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