Analysis of close associations of uropod-associated proteins in human T-cells using the proximity ligation assay

We have shown previously that the raft-associated proteins flotillin-1 and -2 are rapidly recruited to the uropods of chemoattractant-stimulated human neutrophils and T-cells and are involved in cell polarization. Other proteins such as the adhesion receptor PSGL-1, the actin-membrane linker proteins ezrin/radixin/moesin (ERM) and the signaling enzyme phosphatidylinositol-4-phosphate 5-kinase type Iγ90 (PIPKIγ90) also accumulate in the T-cell uropod. Using the in situ proximity ligation assay (PLA) we now have investigated putative close associations of these proteins in human freshly isolated T-cells before and after chemokine addition. The PLA allows in situ subcellular localization of close proximity of endogenous proteins at single-molecule resolution in fixed cells. It allows detection also of weaker and transient complexes that would not be revealed with co-immunoprecipitation approaches. We previously provided evidence for heterodimer formation of tagged flotillin-1 and -2 in T-cells before and after chemokine addition using fluorescence resonance energy transfer (FRET). We now confirm these findings using PLA for the endogenous flotillins in fixed human T-cells. Moreover, in agreement with the literature, our PLA findings confirm a close association of endogenous PSGL-1 and ERM proteins both in resting and chemokine-activated human T-cells. In addition, we provide novel evidence using the PLA for close associations of endogenous activated ERM proteins with PIPKIγ90 and of endogenous flotillins with PSGL-1 in human T-cells, before and after chemokine addition. Our findings suggest that preformed clusters of these proteins coalesce in the uropod upon cell stimulation

Simulation study of micelle formation by bile salts

We report coarse-grained, implicit-solvent simulations of aqueous solutions of bile salts. The parameters in our model were optimized to reproduce some of the experimentally known behavior of dihydroxy bile salts at “physiological” temperature and counterion concentration. We find that micelle formation in dihydroxy and trihydroxy bile salts is only weakly cooperative in the sense that there is barely a free energy barrier that stabilizes these micelles against disassembly. Bile molecules are found to pack in many different orientations in pure bile micelles. Both features may be physiologically relevant: the ability to pack in different orientations may be necessary to form mixed micelles with nutrients of a wide range of molecular lengths and shapes, and the reduced micelle stability may facilitate nutrient release once the mixed micelles reach the intestinal wall.FOM Institute-Scientific Research (NWO). Senter-Novem -Dutch Ministry of Economic; Affairs through the Food & Nutrition Delta 2 Program -grant DFN0642300; Royal Society of London (Wolfson Merit Award) and from the ERC (Advanced Grant agreement 227758)

Valores medios relacionados con puntos aleatorios dados en un cuerpo convexo y en particular en la esfera

Fil: Affentranger, Fernando. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina

Random Convex Hulls and Extreme Value Statistics

In this paper we study the statistical properties of convex hulls of $N$ random points in a plane chosen according to a given distribution. The points may be chosen independently or they may be correlated. After a non-exhaustive survey of the somewhat sporadic literature and diverse methods used in the random convex hull problem, we present a unifying approach, based on the notion of support function of a closed curve and the associated Cauchy's formulae, that allows us to compute exactly the mean perimeter and the mean area enclosed by the convex polygon both in case of independent as well as correlated points. Our method demonstrates a beautiful link between the random convex hull problem and the subject of extreme value statistics. As an example of correlated points, we study here in detail the case when the points represent the vertices of $n$ independent random walks. In the continuum time limit this reduces to $n$ independent planar Brownian trajectories for which we compute exactly, for all $n$, the mean perimeter and the mean area of their global convex hull. Our results have relevant applications in ecology in estimating the home range of a herd of animals. Some of these results were announced recently in a short communication [Phys. Rev. Lett. {\bf 103}, 140602 (2009)].Comment: 61 pages (pedagogical review); invited contribution to the special issue of J. Stat. Phys. celebrating the 50 years of Yeshiba/Rutgers meeting

Dynamic reorganization of flotillins in chemokine-stimulated human T-lymphocytes

<p>Abstract</p> <p>Background</p> <p>Different types of membrane microdomains (rafts) have been postulated to be present in the rear and front of polarized migrating T-lymphocytes. Disruption of rafts by cholesterol sequestration prevents T-cell polarization and migration. Reggie/flotillin-1 and -2 are two highly homologous proteins that are thought to shape membrane microdomains. We have previously demonstrated the enrichment of flotillins in the uropod of human neutrophils. We have now investigated mechanisms involved in chemokine-induced flotillin reorganization in human T-lymphocytes, and possible roles of flotillins in lymphocyte polarization.</p> <p>Results</p> <p>We studied flotillin reorganization and lateral mobility at the plasma membrane using immunofluorescence staining and FRAP (fluorescence recovery after photobleaching). We show that flotillins redistribute early upon chemokine stimulation, and form very stable caps in the uropods of human peripheral blood T-lymphocytes, colocalizing with the adhesion molecule PSGL-1 and activated ezrin/radixin/moesin (ERM) proteins. Chemokine-induced formation of stable flotillin caps requires integrity and dynamics of the actin cytoskeleton, but is not abolished by inhibitors suppressing Rho-kinase or myosin II activity. Tagged flotillin-2 and flotillin-1 coexpressed in T-lymphocytes, but not singly expressed proteins, colocalize in stable caps at the tips of uropods. Lateral mobility of coexpressed flotillins at the plasma membrane is already partially restricted in the absence of chemokine. Incubation with chemokine results in almost complete immobilization of flotillins. Capping is abolished when wild-type flotillin-1 is coexpressed with a mutant of flotillin-2 (G2A) that is unable to interact with the plasma membrane, or with a deletion mutant of flotillin-2 that lacks a putative actin-binding domain. Wild-type flotillin-2 in contrast forms caps when coexpressed with a mutant of flotillin-1 unable to interact with membranes. Transfection of T-lymphocytes with flotillin-2-G2A reduces cell polarization and uropod recruitment of endogenous flotillin-1 and PSGL-1.</p> <p>Conclusions</p> <p>Our data suggest that stable flotillin cap formation in the rear of polarized T-lymphocytes requires flotillin heterooligomer formation, as well as direct F-actin interactions of flotillin-2 and raft/membrane association of flotillin-2, but not -1. Our data also implicate flotillin-rich actin-dependent membrane microdomains in T-lymphocyte uropod formation.</p

Caregiver alignment with triage acuity levels and drivers for discrepancy between caregiver assessment and triage acuity levels: a cross-sectional questionnaire based study.

Caregiver concern is the main driver to paediatric emergency departments visits. Understanding caregiver worries is crucial to guide patients to the most appropriate healthcare setting. Previous research shows mixed findings on the accordance between caregiver assessment and professional triage. We assessed data from two questionnaire-based studies conducted over 27 months in two tertiary paediatric emergency departments in Switzerland to compare caregiver perception of their child's medical acuity and standard nurse triage. Furthermore, we examined socioeconomic factors associated with caregiver perception. Our study of 2,126 children seen in the two paediatric emergency departments showed that caregiver assessment aligned well with the acuity assigned by professional triage in 89% (1,901/2,126) of cases. In 142 cases (7%, 142/2,126), caregivers rating their child's severity higher than nurse's triage while in 83 cases (4%, 83/2,126), they rated it lower. In an univariable analysis, we found that family's difficulties paying bills (20% vs. 12%) and low maternal education (19% vs. 10%) were associated with a higher percentage of caregiver rating their child's severity higher than nurse's triage. Fever as the main complaint was associated with caregiver rating their child's severity lower than nurse's triage. This questionnaire-based study shows that caregiver's assessment of the severity of the child and nurse triage are concordant in most situations. Our study sheds light on the association between caregiver assessment and professional triage in two paediatric emergency departments in Switzerland, revealing some of the factors leading to discordance. These factors most probably reflect health illiteracy. It is important that healthcare professionals recognize and address factors influencing caregiver assessments to facilitate accurate decision-making and enhanced paediatric emergency care outcomes

Prognostic value of pretreatment inflammatory markers in localised prostate cancer before radical prostatectomy

PURPOSE There is growing evidence of an association between inflammatory processes and cancer development and progression. In different solid tumor entities, a pronounced inflammatory response is associated with worse oncological outcome. In this study, we aim to evaluate the prognostic role of clinically established pretreatment inflammatory markers in patients with localised prostate cancer (PCa) before radical prostatectomy (RP). METHODS A total of 641 men met our inclusion criteria and were followed prospectively for a median of 2.85 years. Univariable logistic and Cox regression analysis were performed to analyse associations between preoperative inflammatory markers and tumor characteristics, and biochemical recurrence free survival (BRFS). RESULTS Median age at RP was 64 years. Gleason Score (GS) 7a (263, 41%) was the most prevalent histology, whereas high-risk PCa (≥ GS 8) was present in 156 (24%) patients. Lympho-nodal metastasis and positive surgical margin (PSM) were detected in 69 (11%) and 180 (28%) patients, respectively. No statistically relevant association could be shown between pretreatment inflammatory markers with worse pathological features like higher tumor stage or grade, nodal positive disease or PSM (for all p > 0.05). Additionally, pretreatment inflammatory markers were not associated with a shorter BRFS (p > 0.05). Known risk factors (tumor grade, tumor stage, nodal positivity and positive surgical margins) were all associated with a shorter BRFS (for all p < 0.0001). CONCLUSION In this large prospective cohort, preoperative inflammatory markers were not associated with worse outcome

Preoperative smoking cessation program in patients undergoing intermediate to high-risk surgery: a randomized, single-blinded, controlled, superiority trial

BACKGROUND At present, effectively implementing smoking cessation programs in the health care system constitutes a major challenge. A unique opportunity to initiate smoking cessation focuses on smokers scheduled for surgery. These patients are not only highly motivated to quit smoking but also likely to benefit from a reduction in postoperative complications which may translate into a decrease of costs. Nevertheless, surgical patients are not routinely informed about the benefits of preoperative smoking cessation. Potential reasons for this missed opportunity may be the lack of time and training of surgeons and anaesthesiologists. We therefore aim to analyse the impact of a preoperative high-intensity smoking cessation intervention on surgical complications up to a 90-day postoperative period in patients of various surgical disciplines. The hypothesis is that a preoperative smoking cessation program improves outcomes in smokers undergoing intermediate to high-risk surgery. METHODS The present study is a single-centre, randomized trial with two parallel groups of smokers scheduled for surgery comparing surgery alone and surgery with preoperative smoking cessation. We plan to randomize 251 patients. The primary objective is to compare complications between patients with an institutional multifaceted smoking cessation intervention starting 4 weeks before surgery compared to patients in the advice-only group (control group) within a 90-day postoperative period. The primary endpoint is the Comprehensive Complication Index (CCI®) within 90 days of surgery. Secondary outcomes include the length of hospital stay, cost of care, quality of life, smoking abstinence, and reduction in nicotine consumption. DISCUSSION The hypothesis is that a preoperative smoking cessation program improves outcomes in smokers undergoing surgery. TRIAL REGISTRATION BASEC #2021-02004; ClinicalTrials.gov: NCT05192837 . Registered on January 14, 2022

Microbiome landscapes of the bladder, intestine, and vagina in bladder cancer: a systematic review

BACKGROUND Microbiomes have been linked to oncogenesis, e.g. the intestinal microbiome and colon cancer or HPV-associated cervical cancer. A connection between microbiomes of different body cavities and tumor oncogenesis was shown. The gut microbiome's influence on bladder cancer was established, raising the question whether nearby microbiomes (rectum, vagina) also influence bladder cancer due to their proximity. OBJECTIVE Considering the influence of various body cavities and the broader microbial components, this systematic review aims to investigate differences in the bladder, vaginal, and intestinal microbiota-including bacterial, viral, fungal and archaea-between patients with bladder cancer and healthy controls. METHODS Databases (PubMed, Scopus, Embase) were searched until April 2022. Three types of studies were included: "(1) studies using bladder cancer and control groups (case-controlled studies) (2) studies that provided information on the presence or abundance of microbial taxa (3) studies that provided information on increased or decreased taxa in bladder cancer and/or control groups.". Risk of bias was assessed using the Newcastle Ottawa Scale. RESULTS Fourteen studies (695 samples: 403 bladder cancer, 292 controls) were analyzed. Bacterial taxa that have been detected in at least two studies, the genera Geobacillus and Rubrobacter were more frequently in bladder cancer patients; while Streptococcus and Roseomonas were more prevalent in controls. No consistent taxa were identified across stool or bladder tissue samples. CONCLUSION The microbiota in bladder cancer patients show significant variation across studies. Standardized methods and expanded investigations into viral and fungal components are needed to clarify the role of microbiota in bladder cancer