Quantitative single-molecule mapping of neuronal proteins at the nanoscale

The advent of super-resolution microscopy, also called nanoscopy, allowed a substantial improvement of spatial resolution, opening the door for the observation of biological structures beyond the diffraction limit impossible with conventional light microscopy. Among the super-resolution techniques, single-molecule localization microscopies have proven to be a powerful tool to address many biological issues, since they provide an imaging resolution of the order of tens of nanometers and the possibility to perform quantitative measurements. Neuroscience has been one of the fields in biology to benefit most from super-resolution microscopy. During the last years, single-molecule localization microscopies have been widely exploited to study diffraction-limited subcellular structures in neurons, allowing a deeper understanding of molecular mechanisms underlying neural network functioning and its impairments in pathologies. In this thesis, we developed a tool to investigate the distribution, spatial organization, clustering, and density of neural proteins at the nanoscale. In particular, we focused on the quantitative study of synaptic neurotransmitter receptors and focal adhesions. The knowledge of the distribution and stoichiometry of synaptic proteins is fundamental to understand the regulation of the synaptic transmission in neurons. However, a detailed characterization of the protein architecture within synapses can be achieved only by visualizing them at a nanometric level. Here we propose a quantitative approach based on stochastic optical reconstruction microscopy combined with cluster analysis to investigate the molecular rearrangement of GABAA receptors into subsynaptic domains during synaptic plasticity of the inhibitory neurotransmission. This approach was also applied to the study of the adhesion machinery of mammalian cells and neurons at the interface with single-layer graphene to investigate the interaction between cells and nanostructured materials. Due to their excellent properties and biocompatibility, graphene and its derivatives are the ideal candidates for many biomedical applications, such as neural tissue engineering. However, the adhesion processes at the graphene/neuron interface are still not clear nowadays. Our method offers an easy way to study how graphene substrates can affect adhesion and migration of different types of cells

Peri-operative red blood cell transfusion in neonates and infants: NEonate and Children audiT of Anaesthesia pRactice IN Europe: A prospective European multicentre observational study

BACKGROUND: Little is known about current clinical practice concerning peri-operative red blood cell transfusion in neonates and small infants. Guidelines suggest transfusions based on haemoglobin thresholds ranging from 8.5 to 12 g dl-1, distinguishing between children from birth to day 7 (week 1), from day 8 to day 14 (week 2) or from day 15 (≥week 3) onwards. OBJECTIVE: To observe peri-operative red blood cell transfusion practice according to guidelines in relation to patient outcome. DESIGN: A multicentre observational study. SETTING: The NEonate-Children sTudy of Anaesthesia pRactice IN Europe (NECTARINE) trial recruited patients up to 60 weeks' postmenstrual age undergoing anaesthesia for surgical or diagnostic procedures from 165 centres in 31 European countries between March 2016 and January 2017. PATIENTS: The data included 5609 patients undergoing 6542 procedures. Inclusion criteria was a peri-operative red blood cell transfusion. MAIN OUTCOME MEASURES: The primary endpoint was the haemoglobin level triggering a transfusion for neonates in week 1, week 2 and week 3. Secondary endpoints were transfusion volumes, 'delta haemoglobin' (preprocedure - transfusion-triggering) and 30-day and 90-day morbidity and mortality. RESULTS: Peri-operative red blood cell transfusions were recorded during 447 procedures (6.9%). The median haemoglobin levels triggering a transfusion were 9.6 [IQR 8.7 to 10.9] g dl-1 for neonates in week 1, 9.6 [7.7 to 10.4] g dl-1 in week 2 and 8.0 [7.3 to 9.0] g dl-1 in week 3. The median transfusion volume was 17.1 [11.1 to 26.4] ml kg-1 with a median delta haemoglobin of 1.8 [0.0 to 3.6] g dl-1. Thirty-day morbidity was 47.8% with an overall mortality of 11.3%. CONCLUSIONS: Results indicate lower transfusion-triggering haemoglobin thresholds in clinical practice than suggested by current guidelines. The high morbidity and mortality of this NECTARINE sub-cohort calls for investigative action and evidence-based guidelines addressing peri-operative red blood cell transfusions strategies. TRIAL REGISTRATION: ClinicalTrials.gov, identifier: NCT02350348

Quantitative Super-Resolution Microscopy to Assess Adhesion of Neuronal Cells on Single-Layer Graphene Substrates

Single Layer Graphene (SLG) has emerged as a critically important nanomaterial due to its unique optical and electrical properties and has become a potential candidate for biomedical applications, biosensors, and tissue engineering. Due to its intrinsic 2D nature, SLG is an ideal surface for the development of large-area biosensors and, due to its biocompatibility, can be easily exploited as a substrate for cell growth. The cellular response to SLG has been addressed in different studies with high cellular affinity for graphene often detected. Still, little is known about the molecular mechanism that drives/regulates the cellular adhesion and migration on SLG and SLG-coated interfaces with respect to other substrates. Within this scenario, we used quantitative super-resolution microscopy based on single-molecule localization to study the molecular distribution of adhesion proteins at the nanoscale level in cells growing on SLG and glass. In order to reveal the molecular mechanisms underlying the higher affinity of biological samples on SLG, we exploited stochastic optical reconstruction microscopy (STORM) imaging and cluster analysis, quantifying the super-resolution localization of the adhesion protein vinculin in neurons and clearly highlighting substrate-related correlations. Additionally, a comparison with an epithelial cell line (Chinese Hamster Ovary) revealed a cell dependent mechanism of interaction with SLG

Handwriting fluency and visuospatial generativity at primary school

Abstract Handwriting is a complex activity that involves continuous interaction between lowerlevel perceptual-motor and higher-level cognitive processes. All handwriting models describe involvement of executive functions (EF) in handwriting development. Particular EF domains associated with handwriting include maintenance of information in working memory, inhibition of prepotent responses, and shifting and sustaining of attention. Generativity, an EF domain that has not been extensively studied, may play a role in the acquisition and automation of handwriting. We hypothesized that neuropsychological performances and handwriting fluency would be mutually associated, and that visuospatial generativity could have a specific role in handwriting speed. We enrolled 96 children who completed a comprehensive neuropsychological battery. The study found that handwriting speed was associated with visuospatial fluency, which involves behavioural execution and initiation. In learning and performing handwriting, there is an inherent non-exclusive role of fine motor skills

Quantitative Super-Resolution Microscopy of Proteins at the Synaptic Level

Single-molecule localization (SML) techniques provide a powerful tool to answer biological questions requiring the observation of subcellular structures at the nanoscale. Quantitative single-molecule analysis allows quantifying the number and distribution of molecules in several biological systems beyond the diffraction limit [1]. In the last few years, many computational methods employing clustering analysis algorithms have been developed to extract quantitative information from SML data sets. In neuroscience, quantitative SML has been applied to reveal density and spatial organization of synaptic protein

Unveiling the Inhibitory Synapse Organization Using Superresolution Microscopy

The advent of super-resolution microscopy provided both a substantial improvement of the spatial resolution and the possibility to perform quantitative measurements at a nanometric level. In particular, single-molecule localization (SML) techniques provide a powerful tool to answer biological questions that require the observation of subcellular structures. Quantitative single-molecule analysis allows quantifying the number and observing the distribution of molecules in several biological systems beyond the diffraction limit [1]. In the last few years, many computational methods employing clustering analysis algorithms [2] have been developed to extract quantitative information from SML data sets

Effects of Natural, Urban and Historical-Urban Environments on Children affected by ADHD in Italy

Attention is essential to development and is important for executive functioning. Unfortunately, a growing number of school children suffer of chronic attention deficit such as the Attention Deficit Hyperactivity Disorder (ADHD). It is important to consider that many of these very young patients are pharmacologically treated and need to deal with side effects that may occur. Most literature on attention functioning in developing age is focused on the role that social environment plays in child development whereas scarce attention is paid on the physical environment, both built and natural. Based on Kaplan’s Attention Restoration Theory (Kaplan & Kaplan, 1989; ART), researchers have widely shown a great impact of Nature contact on cognitive functioning, among other benefits, virtually at zero costs and without side effects. We present two consecutive studies with the aim of evaluating how a walk in three different environments may benefit children with ADHD (Study 1) and how a possible correlation could exist between symptoms’ severity (assessed by children themselves, teachers and parents), connectedness with Nature (assessed by children themselves and both parents) as well as the frequency of contact with Nature during the weekend and afterschool activities in the last 12 months (Study 2). In the first study, ART is tested on children aged 7-14 affected by ADHD in Italy (N=21). Attention, impulsivity, emotions were tested before and after the treatment (baseline and post-test); in addition, after the baseline and before the treatment, “mental fatigue” (Kaplan, 1995) was inducted through 15 minutes puzzle and other tiring tasks (maths exercise and scattegories game) and attention performance was remeasured afterwards to check the effectivity of the manipulation. The perception of the restorative value of the place and the environmental preference were also measured among conditions after treatment, by using the Italian version for children (Berto et al., 2007) of the Perceived Restorativeness Scale (Bagot et al., 2004, 2007; Hartig et al., 1997). Treatment consisted into a 20 minutes individually guided walk in a within-subject design and in three different sequential order (between-subject) among the three following environmental conditions: a countryside openfield for the Nature condition, a Medieval well-kept village for the historical-urban condition, a Town for standard-urban condition. The effects of the three environments on attention and impulsivity (core ADHD components), as well as on emotions and on perceived restorativeness are discussed. Some preliminary results show better environmental perception in the Nature condition, than in the Standard-urban condition and the Historical-urban condition. In addition, in Study 2, we argue whether in the same children (N=21) a relation exists between connectedness with Nature, frequency of contact with Nature, symptoms severity and performance tests of Study 1. Study 2 also includes a more numerous sample of children with ADHD to test the above mentioned variables. Results on both studies are integrated and discussed at the conference

EFFECTS OF NATURAL, URBAN AND HISTORICAL-URBAN ENVIRONMENTS ON CHILDREN AFFECTED BY ADHD IN ITALY

Attention is essential to development and is important for executive functioning. Unfortunately, a growing number of school children suffer of chronic attention deficit such as the Attention Deficit Hyperactivity Disorder (ADHD). It is important to consider that many of these very young patients are pharmacologically treated and need to deal with side effects that may occur. Most literature on attention functioning in developing age is focused on the role that social environment plays in child development whereas scarce attention is paid on the physical environment, both built and natural. Based on Kaplan’s Attention Restoration Theory (Kaplan & Kaplan, 1989; ART), researchers have widely shown a great impact of Nature contact on cognitive functioning, among other benefits, virtually at zero costs and without side effects. We present two consecutive studies with the aim of evaluating how a walk in three different environments may benefit children with ADHD (Study 1) and how a possible correlation could exist between symptoms’ severity (assessed by children themselves, teachers and parents), connectedness with Nature (assessed by children themselves and both parents) as well as the frequency of contact with Nature during the weekend and afterschool activities in the last 12 months (Study 2). In the first study, ART is tested on children aged 7-14 affected by ADHD in Italy (N=21). Attention, impulsivity, emotions were tested before and after the treatment (baseline and post-test); in addition, after the baseline and before the treatment, “mental fatigue” (Kaplan, 1995) was inducted through 15 minutes puzzle and other tiring tasks (maths exercise and scattegories game) and attention performance was remeasured afterwards to check the effectivity of the manipulation. The perception of the restorative value of the place and the environmental preference were also measured among conditions after treatment, by using the Italian version for children (Berto et al., 2007) of the Perceived Restorativeness Scale (Bagot et al., 2004, 2007; Hartig et al., 1997). Treatment consisted into a 20 minutes individually guided walk in a within-subject design and in three different sequential order (between-subject) among the three following environmental conditions: a countryside openfield for the Nature condition, a Medieval well-kept village for the historical-urban condition, a Town for standard-urban condition. The effects of the three environments on attention and impulsivity (core ADHD components), as well as on emotions and on perceived restorativeness are discussed. Some preliminary results show better environmental perception in the Nature condition, than in the Standard-urban condition and the Historical-urban condition. In addition, in Study 2, we argue whether in the same children (N=21) a relation exists between connectedness with Nature, frequency of contact with Nature, symptoms severity and performance tests of Study 1. Study 2 also includes a more numerous sample of children with ADHD to test the above mentioned variables. Results on both studies are integrated and discussed at the conference

The Breast Cancer Protooncogenes HER2, BRCA1 and BRCA2 and Their Regulation by the iNOS/NOS2 Axis

The expression of inducible nitric oxide synthase (iNOS; NOS2) and derived NO in various cancers was reported to exert pro- and anti-tumorigenic effects depending on the levels of expression and the tumor types. In humans, the breast cancer level of iNOS was reported to be overexpressed, to exhibit pro-tumorigenic activities, and to be of prognostic significance. Likewise, the expression of the oncogenes HER2, BRCA1, and BRCA2 has been associated with malignancy. The interrelationship between the expression of these protooncogenes and oncogenes and the expression of iNOS is not clear. We have hypothesized that there exist cross-talk signaling pathways between the breast cancer protooncogenes, the iNOS axis, and iNOS-mediated NO mutations of these protooncogenes into oncogenes. We review the molecular regulation of the expression of the protooncogenes in breast cancer and their interrelationships with iNOS expression and activities. In addition, we discuss the roles of iNOS, HER2, BRCA1/2, and NO metabolism in the pathophysiology of cancer stem cells. Bioinformatic analyses have been performed and have found suggested molecular alterations responsible for breast cancer aggressiveness. These include the association of BRCA1/2 mutations and HER2 amplifications with the dysregulation of the NOS pathway. We propose that future studies should be undertaken to investigate the regulatory mechanisms underlying the expression of iNOS and various breast cancer oncogenes, with the aim of identifying new therapeutic targets for the treatment of breast cancers that are refractory to current treatments

ADHD children: what role does the outdoor environment play into alleviating or worsening their symptoms? A comparison of three settings.

Attention is essential to development and healthy life. Unfortunately, a growing number of school children suffer of chronic attention deficit such as the Attention Deficit Hyperactivity Disorder (ADHD). Most literature is focused on the role that social environment plays in child development whereas scarce attention is paid on the physical environment, both built and natural. METHOD We present two studies with the aim of evaluating how a walk in three different outdoor environments (natural, built-historical, built-modern) may affect children with ADHD (Study 1) and the possible correlation between symptoms’ severity and their general frequency of contact with Nature (Study 2). RESULTS In Study 1, some preliminary results show better environmental perception in the Nature condition, than in the Standard-urban condition and the Historical-urban condition and better attention only in the natural context. In Study 2, we expect a positive correlation between Frequency of Contact with Nature, perceived restorativeness and symptoms severity. DISCUSSION In accordance to previous research, our findings show a great impact of Nature contact on cognitive functioning, among other benefits. The absence of costs, virtually, and side effects should motivate into applying research outcomes with the aim of improving the quality of life of atypical children