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International audience; Purpose: To describe bleeding and thrombotic events and their risk factors in patients receiving extracorporeal membrane oxygenation (ECMO) for severe coronavirus disease 2019 (COVID-19) and to evaluate their impact on in-hospital mortality.Methods: The ECMOSARS registry included COVID-19 patients supported by ECMO in France. We analyzed all patients included up to March 31, 2022 without missing data regarding bleeding and thrombotic events. The association of bleeding and thrombotic events with in-hospital mortality and pre-ECMO variables was assessed using multivariable logistic regression models.Results: Among 620 patients supported by ECMO, 29% had only bleeding events, 16% only thrombotic events and 20% both bleeding and thrombosis. Cannulation site (18% of patients), ear nose and throat (12%), pulmonary bleeding (9%) and intracranial hemorrhage (8%) were the most frequent bleeding types. Device-related thrombosis and pulmonary embolism/thrombosis accounted for most of thrombotic events. In-hospital mortality was 55.7%. Bleeding events were associated with in-hospital mortality (adjusted odds ratio (adjOR) = 2.91[1.94-4.4]) but not thrombotic events (adjOR = 1.02[0.68-1.53]). Intracranial hemorrhage was strongly associated with in-hospital mortality (adjOR = 13.5[4.4-41.5]). Ventilation duration before ECMO ≥ 7 days and length of ECMO support were associated with bleeding. Thrombosis-associated factors were fibrinogen ≥ 6 g/L and length of ECMO support.Conclusions: In a nationwide cohort of COVID-19 patients supported by ECMO, bleeding incidence was high and associated with mortality. Intracranial hemorrhage incidence was higher than reported for non-COVID patients and carried the highest risk of death. Thrombotic events were less frequent and not associated with mortality. Length of ECMO support was associated with a higher risk of both bleeding and thrombosis, supporting the development of strategies to minimize ECMO duration.

CD4+ T cells are central mediators of adaptive and innate immune responses and constitute a major reservoir for human immunodeficiency virus (HIV) in vivo. Detailed investigations of resting human CD4+ T cells have been precluded by the absence of efficient approaches for genetic manipulation limiting our understanding of HIV replication and restricting efforts to find a cure. Here we report a method for rapid, efficient, activation-neutral gene editing of resting, polyclonal human CD4+ T cells using optimized cell cultivation and nucleofection conditions of Cas9–guide RNA ribonucleoprotein complexes. Up to six genes, including HIV dependency and restriction factors, were knocked out individually or simultaneously and functionally characterized. Moreover, we demonstrate the knock in of double-stranded DNA donor templates into different endogenous loci, enabling the study of the physiological interplay of cellular and viral components at single-cell resolution. Together, this technique allows improved molecular and functional characterizations of HIV biology and general immune functions in resting CD4+ T cells., A nucleofection-based method to enable efficient gene editing of primary human resting CD4+ T cells.

This project explores whether e-cigarette (EC) flavours are associated with quitting success or longer term use of e-cigarettes when provided as stop smoking aids in trial contexts

Supplemental material, sj-pdf-1-ajs-10.1177_03635465211061150 for Predictors of Graft Failure in Young Active Patients Undergoing Hamstring Autograft Anterior Cruciate Ligament Reconstruction With or Without a Lateral Extra-articular Tenodesis: The Stability Experience by Andrew D. Firth, Dianne M. Bryant, Robert Litchfield, Robert G. McCormack, Mark Heard, Peter B. MacDonald, Tim Spalding, Peter C.M. Verdonk, Devin Peterson, Davide Bardana, Alex Rezansoff, STABILITY Study Group, Alan M.J. Getgood, Kevin Willits, Trevor Birmingham, Chris Hewison, Stacey Wanlin, Ryan Pinto, Ashley Martindale, Lindsey O���Neill, Morgan Jennings, Michal Daniluk, Dory Boyer, Mauri Zomar, Karyn Moon, Raely Moon, Brenda Fan, Bindu Mohan, Gregory M. Buchko, Laurie A. Hiemstra, Sarah Kerslake, Jeremy Tynedal, Greg Stranges, Sheila Mcrae, LeeAnne Gullett, Holly Brown, Alexandra Legary, Alison Longo, Mat Christian, Celeste Ferguson, Nick Mohtadi, Rhamona Barber, Denise Chan, Caitlin Campbell, Alexandra Garven, Karen Pulsifer, Michelle Mayer, Nicole Simunovic, Andrew Duong, David Robinson, David Levy, Matt Skelly, Ajaykumar Shanmugaraj, Fiona Howells, Murray Tough, Pete Thompson, Andrew Metcalfe, Laura Asplin, Alisen Dube, Louise Clarkson, Jaclyn Brown, Alison Bolsover, Carolyn Bradshaw, Larissa Belgrove, Francis Milan, Sylvia Turner, Sarah Verdugo, Janet Lowe, Debra Dunne, Kerri McGowan, Charlie-Marie Suddens, Geert Declerq, Kristien Vuylsteke and Mieke Van Haver in The American Journal of Sports Medicine

Additional file 2 Comparison of health-related quality of life assessed by mean SF-36 scores in ECMO-rescued ARDS survivors according to the timing of delivery. ECMO, extracorporeal membrane oxygenation; ARDS, acute respiratory distress syndrome; ICU, intensive care unit; IES, impact of event scales; PTSD, post-traumatic stress disorder.

Additional file 1. Appendices 1–7

Supplemental material, sj-docx-1-mdm-10.1177_0272989X211053794 for The Use of Expert Elicitation among Computational Modeling Studies in Health Research: A Systematic Review by Christopher J. Cadham, Marie Knoll, Luz María Sánchez-Romero, K. Michael Cummings, Clifford E. Douglas, Alex Liber, David Mendez, Rafael Meza, Ritesh Mistry, Aylin Sertkaya, Nargiz Travis and David T. Levy in Medical Decision Making

Additional file 1: Supplement 1. Mathematical formulation of the SAVM model. Supplement 2. Estimation of the Switch Rate from Cigarette to NVP Use. Supplement 3. Further validation of the model.

Additional file 1 Supplementary Table 1. Surveys Description. Supplementary Table 2. Questions used to collect data about the number of days use in the past 30 days for each of the three national health surveys. Supplementary Table 3. List of Mutually Exclusive Categories of single, dual and poly tobacco product use. Supplementary Table 4. Population prevalence of exclusive, dual, and polytobacco users using three frequency of use thresholds for three national surveys*.

Supplemental material, sj-docx-1-mdm-10.1177_0272989X211053794 for The Use of Expert Elicitation among Computational Modeling Studies in Health Research: A Systematic Review by Christopher J. Cadham, Marie Knoll, Luz María Sánchez-Romero, K. Michael Cummings, Clifford E. Douglas, Alex Liber, David Mendez, Rafael Meza, Ritesh Mistry, Aylin Sertkaya, Nargiz Travis and David T. Levy in Medical Decision Making

Supplemental Material, sj-pdf-1-ahp-10.1177_0890117120964065 for Exclusive, Dual, and Polytobacco Use Among US Adults by Sociodemographic Factors: Results From 3 Nationally Representative Surveys by Jana L. Hirschtick, Delvon T. Mattingly, Beomyoung Cho, Luis Zavala Arciniega, David T. Levy, Luz Maria Sanchez-Romero, Jihyoun Jeon, Stephanie R. Land, Ritesh Mistry, Rafael Meza and Nancy L. Fleischer in American Journal of Health Promotion

Additional file 1: Table 1. Studies of flavored other tobacco product bans that excluded over 21 establishments.

Supplemental material, DS_10.1177_0363546519896333 for Lateral Extra-articular Tenodesis Reduces Failure of Hamstring Tendon Autograft Anterior Cruciate Ligament Reconstruction: 2-Year Outcomes From the STABILITY Study Randomized Clinical Trial by Alan M.J. Getgood, Dianne M. Bryant, Robert Litchfield, Mark Heard, Robert G. McCormack, Alex Rezansoff, Devin Peterson, Davide Bardana, Peter B. MacDonald, Peter C.M. Verdonk, Tim Spalding, Kevin Willits, Trevor Birmingham, Chris Hewison, Stacey Wanlin, Andrew Firth, Ryan Pinto, Ashley Martindale, Lindsey O’Neill, Morgan Jennings, Michal Daniluk, Dory Boyer, Mauri Zomar, Karyn Moon, Raely Pritchett, Krystan Payne, Brenda Fan, Bindu Mohan, Gregory M. Buchko, Laurie A. Hiemstra, Sarah Kerslake, Jeremy Tynedal, Greg Stranges, Sheila Mcrae, LeeAnne Gullett, Holly Brown, Alexandra Legary, Alison Longo, Mat Christian, Celeste Ferguson, Nick Mohtadi, Rhamona Barber, Denise Chan, Caitlin Campbell, Alexandra Garven, Karen Pulsifer, Michelle Mayer, Nicole Simunovic, Andrew Duong, David Robinson, David Levy, Matt Skelly, Ajaykumar Shanmugaraj, Fiona Howells, Murray Tough, Tim Spalding, Pete Thompson, Andrew Metcalfe, Laura Asplin, Alisen Dube, Louise Clarkson, Jaclyn Brown, Alison Bolsover, Carolyn Bradshaw, Larissa Belgrove, Francis Millan, Sylvia Turner, Sarah Verdugo, Janet Lowe, Debra Dunne, Kerri McGowan, Charlie-Marie Suddens, Geert Declercq, Kristien Vuylsteke and Mieke Van Haver in The American Journal of Sports Medicine

The aim of this paper is to study the possibility of sentimental relationships between human and digital beings. We are interested in what kind of “other” a digital being can be for a human subject because it is the first step in understanding how our intimate lives will be shaped by the introduction of new digital technologies. Today computer technologies are growing fast, and they are becoming pervasive. They are intertwining their digital content with every aspect of our everyday lives and they are placing themselves as our “companions”. This co-existence is so tight that it is possible to think of sentimental relationships growing between users and these devices. We will analyse these relationships from a phenomenological perspective by introducing the Japanese term riajuu [ ] which tackles the problem of having a sentimental and intimate relationship with a digital being. Moreover, thanks to Husserl’s phenomenology, we will show how it is important to discern the digital content of the “other” from how this entity relates to the subject. We will show if the subject can build with a digital other an intimate relationship even when users know it is not a human person they are dealing with.

As Grids are loosely-coupled congregations of geographically distributed heterogeneous resources, the efficient utilization of the resources requires the support of a sound Performance Prediction System (PPS). The performance prediction of grid resources is helpful for both Resource Management Systems and grid users to make optimized resource usage decisions. There have been many PPS projects that span over several grid resources in several dimensions. In this paper the taxonomy for describing the PPS architecture is discussed. The taxonomy is used to categorize and identify approaches which are followed in the implementation of the existing PPSs for Grids. The taxonomy and the survey results are used to identify approaches and issues that have not been fully explored in research., Comment: 35 pages,4 figures,2 tables

This is a proposal for a focused panel discussion session cum workshop on the topic of how institutions have used CDIO Standards to meet Accreditation Expectations. It will comprise elevator pitches followed by the panel discussion with audience participation to address the issued highlighted in the elevator pitches. We will require formal paper submissions from the panel members. They will speak briefly to their papers in their elevator pitches

[EN] The present invention provides a material that comprises hygroscopic or deliquescent particles encapsulated in a transparent matrix which may or may not be on a support or substrate, the process for obtaining said material and the use thereof for the manufacturing of devices with controlled variable optical transmission., Consejo Superior de Investigaciones Científicas, A1 Solicitud de patente con informe sobre el estado de la técnica

Simulation models (SMs) combine information from a variety of sources to provide a useful tool for examining how the effects of obesity unfold over time and impact population health. SMs can aid in the understanding of the complex interaction of the drivers of diet and activity and their relation to health outcomes. As emphasized in a recently released report of the Institute or Medicine, SMs can be especially useful for considering the potential impact of an array of policies that will be required to tackle the obesity problem. The purpose of this paper is to present an overview of existing SMs for obesity. First, a background section introduces the different types of models, explains how models are constructed, shows the utility of SMs, and discusses their strengths and weaknesses. Using these typologies, we then briefly review extant obesity SMs. We categorize these models according to their focus: health and economic outcomes, trends in obesity as a function of past trends, physiologically-based behavioral models, environmental contributors to obesity, and policy interventions. Finally, we suggest directions for future research.

La presente invención proporciona un material que comprende partículas higroscópicas o delicuescentes encapsuladas en una matriz transparente que pueden o no estar sobre un soporte o sustrato, al procedimiento de obtención de dicho material y al uso para la fabricación de dispositivos con transmisión óptica variable y controlada., Consejo Superior de Investigaciones Científicas (España), A1 Solicitud de patente con informe sobre el estado de la técnica

Fecha de solicitud: 07-08-2008.- Titular: Consejo Superior de Investigaciones Científicas (CSIC), The device comprises a series of layers of photochromic material (1-2-3), superimposed and obtained by the "sol-gel" method with maximum absorption peaks in different ranges of the visible spectrum and arranged on transparent glass substrate (4), in such a way that the assembly is applicable to any conventional camera. The method for obtaining images consists in arrangement of the device on the b/n sensor for image detection, after which the three colours (blue, red and yellow) are activated by means of a simultaneous ultraviolet flash, thereby obtaining the contribution of each colour to the image from images taken according to the thermal whitening times for each colour by subtracting images with coefficients for each colour calibrated with a model colour sample in such a way that the coloured image of the scene is recovered by processing the images obtained using colour algebra techniques., El dispositivo comprende una serie de capas del material de tipo fotocrómico (1-2-3), el superpuesto y obtenido por el método del “sol-gel” con los picos máximos de la absorción en los diferentes intervalos del espectro visible y dispuestos en el sustrato del vidrio transparente (4), de una manera tal que el conjunto sea aplicable a cualquier cámara convencional. El método para obtener imágenes consiste en la disposición del dispositivo en el sensor de b/n para la detección de la imagen, después de lo cual el son de tres colores (azules, rojo y amarillo) activado por medio de una resolución rápida simultánea de la ultravioleta, de tal modo obteniendo la contribución de cada color a la imagen de las imágenes tenidas según el tiempo que blanquea térmico para cada color restando imágenes con los coeficientes para cada color calibrado con una muestra del color del modelo de una manera tal que la imagen coloreada de la escena sea recuperada por el tratamiento las imágenes obtenidas usando las técnicas de la álgebra del color.

Nowadays, it is widely used the term Nanosatellite when we refer to those satellites whose mass are less than about 10 kg. But it is not only a question of size and power consumption when we talk about this new kind of satellites; it is also a new spacecraft design philosophy. In this paper, we present a small LEO (Low Earth Orbit) satellite, named ‘NANOSAT’, designed and developed by the Spanish Space Agency INTA (Instituto Nacional de Técnica Aeroespacial), currently in the manufacturing and testing phase. We will show here the general design philosophy, which has conducted the project to this stage, giving a description of each subsystem inside the satellite as well as the interfaces between them.

On January 24, 2000, the Council on Library and Information Resources (CLIR) convened a group of experts from different domains of the information resources community to address the question, “What is an authentic digital object?” Five writers–an archivist, a digital library expert, a documentary editor and special collections librarian, an expert on documentary theory, and a computer scientist–were asked to write position papers that identify the attributes that define authentic digital data over time. These papers, together with a brief reflection on the major outcomes of the workshop, are presented in this document. The papers are: “Authentication of Digital Objects: Lessons from a Historian’s Research” (Charles T. Cullen); “Archival Authenticity in a Digital Age” (Peter B. Hirtle); “Where’s Waldo? Reflections on Copies and Authenticity in a Digital Environment” (David M. Levy); “Authenticity and Integrity in the Digital Environment: An Exploratory Analysis of the Central Role of Trust” (Clifford Lynch); “Preserving Authentic Digital Information” (Jeff Rothenberg); and “Authenticity in Perspective” (Abby Smith).

Fecha de solicitud: 16.03.2000.- Consejo Superior de Investigaciones Científicas (CSIC).-Instituto Nacional de Tecnica Aeroespacial "Esteban Terradas"., [EN]Holographic recording material based on silica gel impregnated with methyl methacrylate. A silica gel impregnated with methyl methacrylate is proposed as a holographic recording material. Several characteristics of this hybrid material, such as its good homogeneity, high purity levels, dimensional stability, easy and precise control on the composition, inherent porosity with the controllable pore size, reactivity of the internal surface, possibility both of depositing films as substrates of large dimensions as well as of impregnating the silica matrix with a large variety of organic and inorganic materials are of interest whenever this material is used as a holographic recording medium. The material is appropriate for registering holographic grids of volume, of high and low spatial frequency, and of high diffraction efficiency (88%). [EN] Material holográfico de grabación a base de gel de sílice impregnado con metilmetacrilato. Un gel de sílice impregnado con metacrilato de metilo se propone como un material de registro holográfico. Varias características de este material híbrido, como su buena homogeneidad, altos niveles de pureza, estabilidad dimensional, control fácil y precisa sobre la composición, porosidad inherente con el tamaño de poro controlable, la reactividad de la superficie interna posibilidad, tanto de depositar películas como sustratos de grandes dimensiones, así como la impregnación de la matriz de sílice con una gran variedad de materiales orgánicos e inorgánicos de interés cada vez que este material se utiliza como un medio de grabación holográfica. El material es adecuado para el registro de redes holográficas de volumen, la frecuencia espacial alta y baja, y de la eficiencia de difracción de alta (88%).

Parte de este trabajo ha sido financiado por la CICYT (proyecto PTR94-0115).

Se ha estudiado un material de registro holográfico para almacenamiento de la información óptica compuesto por Azul de Metileno, Acrilamida, N,N'-metilenbisacrilamida, Trietanolamina y Alcohol Polivinílico como soporte. Se ha optimizado la composición siendo el rendimiento en difracción del 80% y sensibilidad entorno a 300 mJ/cm^2 para redes de difracción con frecuencias espaciales de 1000 líneas/mm. The holographic recording material for optical storage composed by methylene blue, acrylamide, N,N'-methylenbisacrylame, triethanolamine and polyvinylalcohol as binder is studied. Optimum composition is obtained with a diffraction efficiency of ~80% achieved with an energetic sensitivity betwen 300 mJ/cm^2, and the spatial resolution is up to 1000 lines/mm. Este trabajo ha sido financiado por el proyecto de investigación PTR94-0115 de la Comisión Interministerial de Ciencia y Tecnología (CICYT).

Parte de este trabajo ha sido financiado por la CICYT (proyecto PTR94-0115).

This study examines the relationship between diversification and predation, focusing on the firm's investments in sunk cost assets. Unlike the single product firm, the diversified firm may employ assets that are not sunk to a product. The ability to transfer these assets among uses or locations may lower the costs of predation. The "long-purse" and "multimarket reputation" may be viewed as special cases of this phenomenon. The likelihood of predation may be reduced when the diversified firm is the target, since it may be able to transfer assets back into an industry when the predator raises the price. Copyright 1989 by Blackwell Publishing Ltd.

Additional file 1. Fig. S1. Reporter viruses employed in this study. Diagram of the main characteristics of the reporter HIV-1 viruses employed in this study (see methods). Figure S2. Stability of GFP and HSA reporter proteins following apoptosis. T cells infected or not with GFP (NLENG1-ES-IRES) or HSA (NLENHSA-ES-IRES) reporter viruses were treated or not with staurosporine (1 μM) at 5 dpi. Twenty-four hours later GFP fluorescence and HSA expression (following anti-HSA staining) were measured by flow cytometry. Figure S3. Toxicity of NNRTI treatment on uninfected cells. Additional data from experiment in Fig. 1c, d here showing effects of NNRTIs on mock infected T cells. Figure S4. EFV killing of Infected resting T cells. Additional data from experiment in Fig. 2a showing killing with EFV. Figure S5. Addition of fresh IL-7 does not prevent NNRTI killing. IL-7 treated resting CD4 T cells infected with HSA reporter were treated at 5 dpi with EFV in the presence of freshly supplemented IL-7 or not. Cell death was analyzed by flow cytometry measuring FSC reduction of HSA+ cells after 24 h. Data are representative of 2 independent experiments. Figure S6. Inhibition of NNRTI killing by PIs IDV, SQV and TPV. T cells infected with NL4-3 virus were treated at 5 dpi with RPV for 4 h in the presence of various concentrations of the PIs IDV, SQV and TPV. Data represent the percentage of inhibition of RPV killing. Productively infected cells were identified by intracellular p24Gag staining. Data are representative of 2 experiments.

Additional file 1: Figure S1 (supplement to Fig. 1). Integrase D116N mutant expression in resting CD4 T cells. An experiment performed independently from those in Figs. 1 and 2 was performed using a different donor. (A) Kinetics of virus expression with and without stimulation. (B) Loss of GFPhi population similar to raltegravir treatment of WT virus. Figure S2 (supplement to Fig. 1). Protein, RNA and DNA in GFP+ and GFP- cells at day 14 post infection. (A) HIV-GFP expression in cells at day 14 for analysis in E. Data are representative of >5 independent experiments using cells from different donors. MFI = mean fluorescence intensity of GFP. (B) Intracellular HIV-1 RNA in sorted cells from integration permissive (No RAL) and integration inhibited (+RAL) cultures. Early Rev-independent Fully spliced RNA, and late Rev-dependent unspliced RNA were quantified by RT-qPCR (see methods). Data are normalized to Fully spliced RNA in the GFP+ No RAL cells set as 100%. Data from one of two similar experiments is shown. Standard deviations are from PCR triplicates. Non-proliferated cells were sorted for this analysis (Fig S4A-B). (C) qPCR analysis of Total and Integrated HIV-1 DNA in cells from Fig. 1E-F. Infection in the presence of raltegravir resulted in less than 1 in 500 GFP+ cells containing an integrated provirus. Figure S3 (supplement to Fig. 1). Lack of influence of MOI on the timing of HIV-1 gene expression and on No RAL vs. + RAL latency in resting CD4 T cells. (A) Cells were infected as in Figure 1 except that different doses of HIV-1 were applied. Figure 1 employs 300ng p24gag per million cells. Note that the Y axis scales are adjusted to allow comparison of relative kinetics at different MOI. (B) Relative amounts of latency for No RAL and +RAL infections remain constant with varying MOI. GFP-negative cells from each infection were sorted at Day 14 p.i. by FACS and stimulated with Pro/TSA in triplicates or DMSO. Expression of GFP one day after stimulation is shown. Data representative of 3 independent experiments with 3 different donors. Figure S4 (supplement to Fig 1). Homeostatic proliferation of IL-4 treated cells and resting status of the non-productively infected cells. (A) eFluor670 and GFP expression on day 14 p.i. Sorting gates are indicated by red boxes. Data are representative of >5 independent experiments using cells from different donors. (B) Analysis of cell proliferation on the day of sorting. The FlowJo Proliferation Platform was used to calculate the percentage of cells that had divided at least once. (C) Expression of T cell activation markers CD69, HLA-DR and CD25 on the eFluorhiGFP-negative cells one day after sorting. Data are representative of three independent experiments. (D) 7AAD-Pyronin Y staining of eFluorhiGFP-negative cells one day after sort. As a positive control for cell cycle progression, uninfected CD4 T cells were activated with αCD3/CD28 beads four days prior to analysis. The arrow with * indicates cells with increased DNA content but low RNA levels that are apparently preparing for homeostatic proliferation. Data are representative of two independent experiments. Figure S5 (supplement to Fig 2). Expanded panel of latency reversing agents in a donor different from Fig. 2. Grey and black vertical lines represent expression levels from the IL-4-only negative controls for No RAL and +RAL conditions in order to better visualize induction from latency. (A) Percent GFP+ cells as in Fig. 2 for the expanded panel. (B) Mean fluorescence intensity (MFI) of the GFP+ cells. Table S1 (supplement to Fig 4). Calculations of ratios of uDNA to iDNA in sorted No RAL cells. Calculations of the ratios of integrated to total DNA in sorted cell populations for Fig. 4A and C. Table S2 (Supplement to Fig 4). Percentage of cells with only uDNA from 4 additional experiments. Results from 4 additional independent experiments utilizing different donor cells for comparison with Fig. 4. Figure S6 (supplement to Fig 5). Activation of productively infected cells accelerates death only when integration is allowed. HSA+ cells were isolated 8 days after infection and stimulated or not with αCD3/CD28 activation beads. Viability was assessed at the indicated times by flow cytometry as in Fig. 5E. Similar results were obtained using the GFP virus and sorted GFP+ cells (not shown). Figure S7 (supplement to Fig 6). Two rounds of stimulation by Pro/TSA did not reverse all HIV-1 latency. (A) Experimental design. Infected cells were treated with DMSO (B) or Pro/TSA (C) on day 8 p.i., then washed and re-treated with the same compounds on day 11 post infection. GFP-neg. cells were sorted on day 14 p.i. and treated with IL-4, Pro/TSA or αCD3/CD28 activation beads + IL-2 for 2 days before analysis. Indinavir was added at day 0 and 7 p.i. as an additional precaution against second round infection.

Additional file 1: Figure S1 (supplement to Fig. 1). Integrase D116N mutant expression in resting CD4 T cells. An experiment performed independently from those in Figs. 1 and 2 was performed using a different donor. (A) Kinetics of virus expression with and without stimulation. (B) Loss of GFPhi population similar to raltegravir treatment of WT virus. Figure S2 (supplement to Fig. 1). Protein, RNA and DNA in GFP+ and GFP- cells at day 14 post infection. (A) HIV-GFP expression in cells at day 14 for analysis in E. Data are representative of >5 independent experiments using cells from different donors. MFI = mean fluorescence intensity of GFP. (B) Intracellular HIV-1 RNA in sorted cells from integration permissive (No RAL) and integration inhibited (+RAL) cultures. Early Rev-independent Fully spliced RNA, and late Rev-dependent unspliced RNA were quantified by RT-qPCR (see methods). Data are normalized to Fully spliced RNA in the GFP+ No RAL cells set as 100%. Data from one of two similar experiments is shown. Standard deviations are from PCR triplicates. Non-proliferated cells were sorted for this analysis (Fig S4A-B). (C) qPCR analysis of Total and Integrated HIV-1 DNA in cells from Fig. 1E-F. Infection in the presence of raltegravir resulted in less than 1 in 500 GFP+ cells containing an integrated provirus. Figure S3 (supplement to Fig. 1). Lack of influence of MOI on the timing of HIV-1 gene expression and on No RAL vs. + RAL latency in resting CD4 T cells. (A) Cells were infected as in Figure 1 except that different doses of HIV-1 were applied. Figure 1 employs 300ng p24gag per million cells. Note that the Y axis scales are adjusted to allow comparison of relative kinetics at different MOI. (B) Relative amounts of latency for No RAL and +RAL infections remain constant with varying MOI. GFP-negative cells from each infection were sorted at Day 14 p.i. by FACS and stimulated with Pro/TSA in triplicates or DMSO. Expression of GFP one day after stimulation is shown. Data representative of 3 independent experiments with 3 different donors. Figure S4 (supplement to Fig 1). Homeostatic proliferation of IL-4 treated cells and resting status of the non-productively infected cells. (A) eFluor670 and GFP expression on day 14 p.i. Sorting gates are indicated by red boxes. Data are representative of >5 independent experiments using cells from different donors. (B) Analysis of cell proliferation on the day of sorting. The FlowJo Proliferation Platform was used to calculate the percentage of cells that had divided at least once. (C) Expression of T cell activation markers CD69, HLA-DR and CD25 on the eFluorhiGFP-negative cells one day after sorting. Data are representative of three independent experiments. (D) 7AAD-Pyronin Y staining of eFluorhiGFP-negative cells one day after sort. As a positive control for cell cycle progression, uninfected CD4 T cells were activated with αCD3/CD28 beads four days prior to analysis. The arrow with * indicates cells with increased DNA content but low RNA levels that are apparently preparing for homeostatic proliferation. Data are representative of two independent experiments. Figure S5 (supplement to Fig 2). Expanded panel of latency reversing agents in a donor different from Fig. 2. Grey and black vertical lines represent expression levels from the IL-4-only negative controls for No RAL and +RAL conditions in order to better visualize induction from latency. (A) Percent GFP+ cells as in Fig. 2 for the expanded panel. (B) Mean fluorescence intensity (MFI) of the GFP+ cells. Table S1 (supplement to Fig 4). Calculations of ratios of uDNA to iDNA in sorted No RAL cells. Calculations of the ratios of integrated to total DNA in sorted cell populations for Fig. 4A and C. Table S2 (Supplement to Fig 4). Percentage of cells with only uDNA from 4 additional experiments. Results from 4 additional independent experiments utilizing different donor cells for comparison with Fig. 4. Figure S6 (supplement to Fig 5). Activation of productively infected cells accelerates death only when integration is allowed. HSA+ cells were isolated 8 days after infection and stimulated or not with αCD3/CD28 activation beads. Viability was assessed at the indicated times by flow cytometry as in Fig. 5E. Similar results were obtained using the GFP virus and sorted GFP+ cells (not shown). Figure S7 (supplement to Fig 6). Two rounds of stimulation by Pro/TSA did not reverse all HIV-1 latency. (A) Experimental design. Infected cells were treated with DMSO (B) or Pro/TSA (C) on day 8 p.i., then washed and re-treated with the same compounds on day 11 post infection. GFP-neg. cells were sorted on day 14 p.i. and treated with IL-4, Pro/TSA or αCD3/CD28 activation beads + IL-2 for 2 days before analysis. Indinavir was added at day 0 and 7 p.i. as an additional precaution against second round infection.

Aim: The aim of this study was to examine homeopathic practice as a lived phenomenon. This thesis explores the interior of clinical practice. Background: Homeopathy is a globally practiced discipline in the arena of complementary and alternative medicine. Despite sustained practice it is epistemically contentious, yet little is known about the reasoning and decision making practices of its practitioners, what actually happens in practice. Methods: Using interpretative phenomenological analysis, participants were extensively observed, recorded and interviewed. Data were coded, categorised and analysed. Results: Themes representing the sources and forms of reasoning were generated. Practice is underpinned by theory, core texts and clinical authority. Beyond these sources of authority, practice is guided by clinical experience, by praxis and professional wisdom. Clinical reasoning does not exclusively depend on knowledge; it is performed between practitioners and their patients, built upon a therapeutic relationship. Discussion: This thesis enhances knowledge and understanding of clinical reasoning practice. It raises pedagogical and philosophical questions about how clinical practice can be interpreted and understood. It also argues that questions about forms of evidence for disciplines including homeopathy need to consider the diverse goals and preferences of patients and health professionals alike. Conclusions: Research into the lived experience confirms that homeopathic clinical reasoning is highly complex, driven by multiple sources of evidence.

Additional file 1. Fig. S1. Reporter viruses employed in this study. Diagram of the main characteristics of the reporter HIV-1 viruses employed in this study (see methods). Figure S2. Stability of GFP and HSA reporter proteins following apoptosis. T cells infected or not with GFP (NLENG1-ES-IRES) or HSA (NLENHSA-ES-IRES) reporter viruses were treated or not with staurosporine (1 μM) at 5 dpi. Twenty-four hours later GFP fluorescence and HSA expression (following anti-HSA staining) were measured by flow cytometry. Figure S3. Toxicity of NNRTI treatment on uninfected cells. Additional data from experiment in Fig. 1c, d here showing effects of NNRTIs on mock infected T cells. Figure S4. EFV killing of Infected resting T cells. Additional data from experiment in Fig. 2a showing killing with EFV. Figure S5. Addition of fresh IL-7 does not prevent NNRTI killing. IL-7 treated resting CD4 T cells infected with HSA reporter were treated at 5 dpi with EFV in the presence of freshly supplemented IL-7 or not. Cell death was analyzed by flow cytometry measuring FSC reduction of HSA+ cells after 24 h. Data are representative of 2 independent experiments. Figure S6. Inhibition of NNRTI killing by PIs IDV, SQV and TPV. T cells infected with NL4-3 virus were treated at 5 dpi with RPV for 4 h in the presence of various concentrations of the PIs IDV, SQV and TPV. Data represent the percentage of inhibition of RPV killing. Productively infected cells were identified by intracellular p24Gag staining. Data are representative of 2 experiments.

Latently infected CD4 T cells form a stable reservoir of HIV that leads to life-long viral persistence; the mechanisms involved in establishment of this latency are not well understood. Three scenarios have been proposed: 1) an activated, proliferating cell becomes infected and reverts back to a resting state; 2) an activated cell becomes infected during its return to resting; or 3) infection is established directly in a resting cell. The aim of this study was, therefore, to investigate the relationship between T cell activation and proliferation and the establishment of HIV latency. Isolated primary CD4 cells were infected at different time points before or after TCR-induced stimulation. Cell proliferation within acutely infected cultures was tracked using CFSE viable dye over 14 days; and cell subsets that underwent varying degrees of proliferation were isolated at end of culture by flow cytometric sorting. Recovered cell subpopulations were analyzed for the amount of integrated HIV DNA, and the ability to produce virus, upon a second round of cell stimulation. We show that cell cultures exposed to virus, prior to stimulus addition, contained the highest levels of integrated and replication-competent provirus after returning to quiescence; whereas, cells infected during the height of cell proliferation retained the least. Cells that did not divide or exhibited limited division, following virus exposure and stimulation contained greater amounts of integrated and inducible HIV than did cells that had divided many times. Based on these results, co-culture experiments were conducted to demonstrate that latent infection could be established directly in non-dividing cells via cell-to-cell transmission from autologous productively infected cells. Together, the findings from our studies implicate the likely importance of direct infection of sub-optimally activated T cells in establishment of latently infected reservoirs in vivo, especially in CD4 lymphocytes that surround productive viral foci within immune tissue microenvironments.

In this chapter, we introduce the vision of the H2020 project ``Precision Farming of Hazelnut Orchards'' (PANTHEON), which is to develop the agricultural equivalent of an industrial Supervisory Control and Data Acquisition (SCADA) system to be used for precision farming of orchards. PANTHEON's objective is to design an integrated system where a relatively limited number of heterogeneous unmanned robotic components (including terrestrial and aerial robots) move within the orchard to collect data and perform typical farming operations. In addition, an Internet-of-Things (IoT) agrometeorological solar-powered network is deployed to continuously monitor the environmental conditions of the orchard. The information so collected is then stored in a central operative unit that integrates the data to perform automatic feedback actions (e.g., to regulate the irrigation system) and to support the decisions of the agronomists and farmers in charge of the orchard. The proposed SCADA system will acquire information at the resolution of the individual plant, to drastically increase, compared to current best practices, the detection of possible limiting factors at the level of the individual plant and to react accordingly. Differently from the current state of the art in precision farming for large-scale orchards, the capability of monitoring the state and the evolution of each single tree will be the enabling technology to allow more focused interventions. This will lead to a better average health of the orchard and to an increased effectiveness of integrated pest management (IPM) activities. In conclusion, the ongoing implemented architecture has the potential to increase production while, at the same time, being more cost-effective and environmentally friendly. To summarize, we believe that the proposed SCADA paradigm for Precision Agriculture may represent an attractive opportunity for the design of a novel real-time software architecture. In other words, by allowing the processing of massive amounts of datasets derived from the SCADA architecture, it will be possible to step up the current effectiveness of Precision Agriculture (PA) methodologies by providing real-time answers to the questions posed by farm managers, when in need of timely decisions.

The zebrafish has emerged over the last decade as a powerful model system for understanding molecular and cellular aspects of vertebrate development and disease processes. Because of the implications for, and applications to human health, the vast majority of work on JAK and STAT function has been done in mammalian systems, primarily in the mouse and in human cell lines. However, apart from natural curiosity, there are two important reasons to consider JAK and STAT biology in non-human systems. Firstly, an evolutionary perspective will shed light on which features of mammalian JAK/STAT function are conserved, and which are peculiarities of mammalian development and physiology. Second, the technical advantages that many organisms possess over mammals, particularly in genetic screening and embryological manipulation means that certain questions are relatively simple to answer in these systems, producing insight that would be impossible or financially prohibitive to achieve in mammals. Thus, the exploration of JAK/STAT function in non-mammalian model systems is a critical complementary line of investigation for a thorough understanding of these fascinating signaling molecules.