Single-Cell Biochemical Multiplexing by Multidimensional Phasor Demixing and Spectral Fluorescence Lifetime Imaging Microscopy

Data Availability Statement: The original contributions presented in the study are included in the article/Supplementary Material; further inquiries can be directed to the corresponding author.Copyright © 2021 Haas, Fries, Venkitaraman and Esposito. Revealing mechanisms underpinning cell function requires understanding the relationship between different biochemical reactions in living cells. However, our capabilities to monitor more than two biochemical reactions in living cells are limited. Therefore, the development of methods for real-time biochemical multiplexing is of fundamental importance. Here, we show that data acquired with multicolor (mcFLIM) or spectrally resolved (sFLIM) fluorescence lifetime imaging can be conveniently described with multidimensional phasor transforms. We demonstrate a computational framework capable of demixing three Forster resonance energy transfer (FRET) probes and quantifying multiplexed biochemical activities in single living cells. We provide a comparison between mcFLIM and sFLIM suggesting that sFLIM might be advantageous for the future development of heavily multiplexed assays. However, mcFLIM—more readily available with commercial systems—can be applied for the concomitant monitoring of three enzymes in living cells without significant losses.Gates Foundation studentship; Medical Research Council core grants (MC_UU_12022/1 and MC_UU_12022/8); Wellcome Trust (090340/Z/09/Z); Cancer Research UK (C54674/A27487)

Alcohol affects neuronal substrates of response inhibition but not of perceptual processing of stimuli signalling a stop response

Alcohol impairs inhibitory control, including the ability to terminate an initiated action. While there is increasing knowledge about neural mechanisms involved in response inhibition, the level at which alcohol impairs such mechanisms remains poorly understood. Thirty-nine healthy social drinkers received either 0.4g/kg or 0.8g/kg of alcohol, or placebo, and performed two variants of a Visual Stop-signal task during acquisition of functional magnetic resonance imaging (fMRI) data. The two task variants differed only in their instructions: in the classic variant (VSST), participants inhibited their response to a “Go-stimulus” when it was followed by a “Stop-stimulus”. In the control variant (VSST_C), participants responded to the “Go-stimulus” even if it was followed by a “Stop-stimulus”. Comparison of successful Stop-trials (Sstop)>Go, and unsuccessful Stop-trials (Ustop)>Sstop between the three beverage groups enabled the identification of alcohol effects on functional neural circuits supporting inhibitory behaviour and error processing. Alcohol impaired inhibitory control as measured by the Stop-signal reaction time, but did not affect other aspects of VSST performance, nor performance on the VSST_C. The low alcohol dose evoked changes in neural activity within prefrontal, temporal, occipital and motor cortices. The high alcohol dose evoked changes in activity in areas affected by the low dose but importantly induced changes in activity within subcortical centres including the globus pallidus and thalamus. Alcohol did not affect neural correlates of perceptual processing of infrequent cues, as revealed by conjunction analyses of VSST and VSST_C tasks. Alcohol ingestion compromises the inhibitory control of action by modulating cortical regions supporting attentional, sensorimotor and action-planning processes. At higher doses the impact of alcohol also extends to affect subcortical nodes of fronto-basal ganglia- thalamo-cortical motor circuits. In contrast, alcohol appears to have little impact on the early visual processing of infrequent perceptual cues. These observations clarify clinically-important effects of alcohol on behaviour

TGF-beta(2)- and H2O2-Induced Biological Changes in Optic Nerve Head Astrocytes Are Reduced by the Antioxidant Alpha-Lipoic Acid

Background/Aims: The goal of the present study was to determine whether transforming growth factor-beta(2) (TGF-beta(2))- and oxidative stress-induced cellular changes in cultured human optic nerve head (ONH) astrocytes could be reduced by pretreatment with the antioxidant alpha-lipoic acid (LA). Methods: Cultured ONH astrocytes were treated with 1.0 ng/ml TGF-beta(2) for 24 h or 200 mu M hydrogen peroxide (H2O2) for 1 h. Lipid peroxidation was measured by a decrease in cis-pari-naric acid fluorescence. Additionally, cells were pretreated with different concentrations of LA before TGF-beta 2 or H2O2 exposure. Expressions of the heat shock protein (Hsp) alpha B-crystallin and Hsp27, the extracellular matrix (ECM) component fibronectin and the ECM-modulating protein connective tissue growth factor (CTGF) were examined with immunohistochemistry and real-time PCR analysis. Results: Both TGF-beta(2) and H2O2 increased lipid peroxidation. Treatment of astrocytes with TGF-beta(2) and H2O2 upregulated the expression of alpha B-crystallin, Hsp27, fibronectin and CTGF. Pretreatment with different concentrations of LA reduced the TGF-beta(2)- and H2O2-stimulated gene expressions. Conclusion: We showed that TGF-beta(2)- and H2O2-stimulated gene expressions could be prevented by pretreatment with the antioxidant LA in cultured human ONH astrocytes. Therefore, it is tempting to speculate that the use of antioxidants could have protective effects in glaucomatous optic neuropathy. Copyright (C) 2012 S. Karger AG, Base

Task-Related Effects on the Temporal and Spatial Dynamics of Resting-State Functional Connectivity in the Default Network

Recent evidence points to two potentially fundamental aspects of the default network (DN), which have been relatively understudied. One is the temporal nature of the functional interactions among nodes of the network in the resting-state, usually assumed to be static. The second is possible influences of previous brain states on the spatial patterns (i.e., the brain regions involved) of functional connectivity (FC) in the DN at rest. The goal of the current study was to investigate modulations in both the spatial and temporal domains. We compared the resting-state FC of the DN in two runs that were separated by a 45 minute interval containing cognitive task execution. We used partial least squares (PLS), which allowed us to identify FC spatiotemporal patterns in the two runs and to determine differences between them. Our results revealed two primary modes of FC, assessed using a posterior cingulate seed – a robust correlation among DN regions that is stable both spatially and temporally, and a second pattern that is reduced in spatial extent and more variable temporally after cognitive tasks, showing switching between connectivity with certain DN regions and connectivity with other areas, including some task-related regions. Therefore, the DN seems to exhibit two simultaneous FC dynamics at rest. The first is spatially invariant and insensitive to previous brain states, suggesting that the DN maintains some temporally stable functional connections. The second dynamic is more variable and is seen more strongly when the resting-state follows a period of task execution, suggesting an after-effect of the cognitive activity engaged during task that carries over into resting-state periods

Vitamin D supplementation to prevent acute respiratory tract infections: systematic review and meta-analysis of individual participant data

This study was supported by a grant from the National Institute for Health Research (NIHR) under its Health Technology Assessment programme (reference No 13/03/25, to ARM

The NAMPT inhibitor FK866 reverts the damage in spinal cord injury

<p>Abstract</p> <p>Background</p> <p>Emerging data implicate nicotinamide phosphoribosyl transferase (NAMPT) in the pathogenesis of cancer and inflammation. NAMPT inhibitors have proven beneficial in inflammatory animal models of arthritis and endotoxic shock as well as in autoimmune encephalitis. Given the role of inflammatory responses in spinal cord injury (SCI), the effect of NAMPT inhibitors was examined in this setting.</p> <p>Methods</p> <p>We investigated the effects of the NAMPT inhibitor FK866 in an experimental compression model of SCI.</p> <p>Results</p> <p>Twenty-four hr following induction of SCI, a significant functional deficit accompanied widespread edema, demyelination, neuron loss and a substantial increase in TNF-α, IL-1β, PAR, NAMPT, Bax, MPO activity, NF-κB activation, astrogliosis and microglial activation was observed. Meanwhile, the expression of neurotrophins BDNF, GDNF, NT3 and anti-apoptotic Bcl-2 decreased significantly. Treatment with FK866 (10 mg/kg), the best known and characterized NAMPT inhibitor, at 1 h and 6 h after SCI rescued motor function, preserved perilesional gray and white matter, restored anti-apoptotic and neurotrophic factors, prevented the activation of neutrophils, microglia and astrocytes and inhibited the elevation of NAMPT, PAR, TNF-α, IL-1β, Bax expression and NF-κB activity.</p> <p>We show for the first time that FK866, a specific inhibitor of NAMPT, administered after SCI, is capable of reducing the secondary inflammatory injury and partly reduce permanent damage. We also show that NAMPT protein levels are increased upon SCI in the perilesional area which can be corrected by administration of FK866.</p> <p>Conclusions</p> <p>Our findings suggest that the inflammatory component associated to SCI is the primary target of these inhibitors.</p

Influence of FTO rs9939609 and Mediterranean diet on body composition and weight loss: a randomized clinical trial

Background The Mediterranean diet (MeD) plays a key role in the prevention of obesity. Among the genes involved in obesity, the Fat mass and obesity-associated gene (FTO) is one of the most known, but its interaction with MeD remained uncertain so far. Methods We carried out a study on a sample of 188 Italian subjects, analyzing their FTO rs9939609 alleles, and the difference in body composition between the baseline and a 4-weeks nutritional intervention. The sample was divided into two groups: the control group of 49 subjects, and the MeD group of 139 subjects. Results We found significant relations between MeD and both variation of total body fat (ΔTBFat) (p = 0.00) and gynoid body fat (p = 0.04). ∆TBFat (kg) demonstrated to have a significant relation with the interaction diet-gene (p = 0.04), whereas FTO was associated with the variation of total body water (p = 0.02). Conclusions MeD demonstrated to be a good nutritional treatment to reduce the body fat mass, whereas data about FTO remain uncertain. Confirming or rejecting the hypothesis of FTO and its influence on body tissues during nutritional treatments is fundamental to decide whether its effect has to be taken into consideration during both development of dietetic plans and patients monitoring. Trial Registration ClinicalTrials.gov Id: NCT01890070. Registered 01 July 2013, https://clinicaltrials.gov/ct2/show/NCT0189007

The integrated action framework of Rete Natura 2000 Basilicata

Basilicata Natura 2000 network consists of 50 Sites of Community Importance (SCI) and 17 Special Protection Zones (SPZs), covering alltogether more than 17% of the regional area. This network, partially overlapping other forms of land protection, represents a valuable environmental, agricultural and cultural heritage, in which the safeguard of natural resources and landscapes has to be coupled with the needs of the local population; especially in relation to development and social welfare. The Natura 2000 project involved a panel of experts belonging to 15 different institutions, to form a steering committee with the following professional and scientific skills: vegetation, landscape, fauna, geology, agriculture, forestry, sea, architecture and planning, territorial analysis and representation. Along the 4 years project, the steering committee designed and coordinated the activities of over 150 professionals, mostly from Basilicata, who carried out field surveys and data analysis aimed at assessing the environmental conditions in the SCI and SPZs, proposing measures and plans, implementing thematic databases. SCI/SPZ management plans, by themselves, may not be sufficient to fulfill the requirements of an effective environmental policy, which has to go along with the awareness of people, citizens and local administrators about the instances of a sustainable policy. For this reason, the activities of surveying and management have been coupled with a communication project that involves all the experts and a relevant part of professionals who participated to the Natura 2000 project. The communication activity implies the use and the creation of several tools (publishing, video, websites, meetings, photo contests, social networks ...) targeted to different groups: policy makers (local, regional, national, European ), organizations, citizens, schools, farmers, small and medium enterprises (www.natura2000basilicata.it). A further goal is to feature the environmental highlights of Basilicata which are linked to a specific and often surprising integration of an ancient human presence with the natural elements, and the role performed by the traditional farming activities in the maintenance of ecosystem dynamics and services (in particular with agriculture). In fact, a good number of Natura 2000 sites can be considered High Nature Value Farmlands (HNVF, sensu E. Andersen, 2003), in which a virtuous relationship was established a long time ago between traditional practices and the environment itself. In this context, it combines the convergence between the activities carried on the Natura 2000 network and the project Agrival (http://utagri.enea.it/projects/agrival), a research project led by ENEA in Val d'Agri, in order to experimentally contribute to the methodology for the identification of the High Nature Value Farmlands and make them cohabit with the other economic activities in the local context. The process started with the project Basilicata Natura 2000 Network is therefore an interesting methodological model that, in coherence with the financial planning of the European Community for the period 2014-2020 (Brussels, 12.12.2011, COMM. 874) puts together projects on environmental issues to boost the meeting of agricultural, environmental, cultural and productive policies, fostered by the EC, and enhancing the implementation of the "Prioritized Actions Frameworks" (PAF), pointed out by the European Commission as the optimal tools for the management of Natura 2000 networks