Lifetime estimation on moving sub-cellular objects in frequency domain FLIM imaging

International audienceFluorescence lifetime is usually defined as the average nanosecond-scale delay between excitation and emission of fluorescence. It has been established that lifetime measurement yields numerous indications on cellular processes such as inter-protein and intra-protein mechanisms through fluorescent tagging and Förster resonance energy transfer (FRET). In this area, frequency domain fluorescence lifetime imaging microscopy (FD FLIM) is particularly well appropriate to probe a sample non-invasively and quantify these interactions in living cells. The aim is then to measure fluorescence lifetime in the sample at each location in space from fluorescence variations observed in a temporal sequence of images obtained by phase modulation of the detection signal. This leads to a sensitivity of lifetime determination to other sources of fluorescence variations such as intracellular motion. In this paper, we propose a robust statistical method for lifetime estimation on both background and small moving structures with a focus on intracellular vesicle trafficking

Genotypic variability enhances the reproducibility of an ecological study

Many scientific disciplines are currently experiencing a “reproducibility crisis” because numerous scientific findings cannot be repeated consistently. A novel but controversial hypothesis postulates that stringent levels of environmental and biotic standardization in experimental studies reduces reproducibility by amplifying impacts of lab-specific environmental factors not accounted for in study designs. A corollary to this hypothesis is that a deliberate introduction of controlled systematic variability (CSV) in experimental designs may lead to increased reproducibility. We tested this hypothesis using a multi-laboratory microcosm study in which the same ecological experiment was repeated in 14 laboratories across Europe. Each laboratory introduced environmental and genotypic CSV within and among replicated microcosms established in either growth chambers (with stringent control of environmental conditions) or glasshouses (with more variable environmental conditions). The introduction of genotypic CSV led to lower among-laboratory variability in growth chambers, indicating increased reproducibility, but had no significant effect in glasshouses where reproducibility was generally lower. Environmental CSV had little effect on reproducibility. Although there are multiple causes for the “reproducibility crisis”, deliberately including genetic variation may be a simple solution for increasing the reproducibility of ecological studies performed in controlled environments

Stem Cell Res

Primary hyperoxaluria type 1 (PH1) is a rare autosomal recessive disorder of the liver metabolism due to functional deficiency of the peroxisomal enzyme alanine:glyoxylate aminotransferase (AGT). AGT deficiency results in overproduction of oxalate which complexes with calcium to form insoluble calcium-oxalate salts in urinary tracts, ultimately leading to end-stage renal disease. Currently, the only curative treatment for PH1 is combined liver-kidney transplantation, which is limited by donor organ shortage and lifelong requirement for immunosuppression. Transplantation of genetically modified autologous hepatocytes is an attractive therapeutic option for PH1. However, the use of fresh primary hepatocytes suffers from limitations such as organ availability, insufficient cell proliferation, loss of function, and the risk of immune rejection. We developed patient-specific induced pluripotent stem cells (PH1-iPSCs) free of reprogramming factors as a source of renewable and genetically defined autologous PH1-hepatocytes. We then investigated additive gene therapy using a lentiviral vector encoding wild-type AGT under the control of the liver-specific transthyretin promoter. Genetically modified PH1-iPSCs successfully provided hepatocyte-like cells (HLCs) that exhibited significant AGT expression at both RNA and protein levels after liver-specific differentiation process. These results pave the way for cell-based therapy of PH1 by transplantation of genetically modified autologous HLCs derived from patient-specific iPSCs

An Orthotopic Model of Glioblastoma Is Resistant to Radiodynamic Therapy with 5-AminoLevulinic Acid

Radiosensitization of glioblastoma is a major ambition to increase the survival of this incurable cancer. The 5-aminolevulinic acid (5-ALA) is metabolized by the heme biosynthesis pathway. 5-ALA overload leads to the accumulation of the intermediate fluorescent metabolite protoporphyrin IX (PpIX) with a radiosensitization potential, never tested in a relevant model of glioblastoma. We used a patient-derived tumor cell line grafted orthotopically to create a brain tumor model. We evaluated tumor growth and tumor burden after different regimens of encephalic multifractionated radiation therapy with or without 5-ALA. A fractionation scheme of 5 × 2 Gy three times a week resulted in intermediate survival [48-62 days] compared to 0 Gy (15-24 days), 3 × 2 Gy (41-47 days) and, 5 × 3 Gy (73-83 days). Survival was correlated to tumor growth. Tumor growth and survival were similar after 5 × 2 Gy irradiations, regardless of 5-ALA treatment (RT group (53-67 days), RT+5-ALA group (40-74 days), HR = 1.57, p = 0.24). Spheroid growth and survival were diminished by radiotherapy in vitro, unchanged by 5-ALA pre-treatment, confirming the in vivo results. The analysis of two additional stem-like patient-derived cell lines confirmed the absence of radiosensitization by 5-ALA. Our study shows for the first time that in a preclinical tumor model relevant to human glioblastoma, treated as in clinical routine, 5-ALA administration, although leading to important accumulation of PpIX, does not potentiate radiotherapy

Characterization of a Recombinant Adeno-Associated Virus Type 2 Reference Standard Material

A recombinant adeno-associated virus serotype 2 Reference Standard Material (rAAV2 RSM) has been produced and characterized with the purpose of providing a reference standard for particle titer, vector genome titer, and infectious titer for AAV2 gene transfer vectors. Production and purification of the reference material were carried out by helper virus–free transient transfection and chromatographic purification. The purified bulk material was vialed, confirmed negative for microbial contamination, and then distributed for characterization along with standard assay protocols and assay reagents to 16 laboratories worldwide. Using statistical transformation and modeling of the raw data, mean titers and confidence intervals were determined for capsid particles ({X}, 9.18 × 1011 particles/ml; 95% confidence interval [CI], 7.89 × 1011 to 1.05 × 1012 particles/ml), vector genomes ({X}, 3.28 × 1010 vector genomes/ml; 95% CI, 2.70 × 1010 to 4.75 × 1010 vector genomes/ml), transducing units ({X}, 5.09 × 108 transducing units/ml; 95% CI, 2.00 × 108 to 9.60 × 108 transducing units/ml), and infectious units ({X}, 4.37 × 109 TCID50 IU/ml; 95% CI, 2.06 × 109 to 9.26 × 109 TCID50 IU/ml). Further analysis confirmed the identity of the reference material as AAV2 and the purity relative to nonvector proteins as greater than 94%. One obvious trend in the quantitative data was the degree of variation between institutions for each assay despite the relatively tight correlation of assay results within an institution. This relatively poor degree of interlaboratory precision and accuracy was apparent even though attempts were made to standardize the assays by providing detailed protocols and common reagents. This is the first time that such variation between laboratories has been thoroughly documented and the findings emphasize the need in the field for universal reference standards. The rAAV2 RSM has been deposited with the American Type Culture Collection and is available to the scientific community to calibrate laboratory-specific internal titer standards. Anticipated uses of the rAAV2 RSM are discussed

18th ITC Specialist Seminar on Quality of Experience [Elektronisk resurs]

The notion and topic of Quality of Experience (QoE) keeps attracting the attention of manufacturers, operators and researchers. It links user perception and expectations on one side and technical Quality of Service (QoS) parameters, management, pricing schemes etc. on the other side. Such links are needed in order to balance user satisfaction and economic aspects of service provisioning. However, the notion of QoE as such is not without controversy. Technicians, used to a world of objective and clearly definable parameters, tend to fear the subjective, somehow fuzzy parts associated with end user perception. Vice versa, customer relationship and marketing departments might find themselves uncomfortable with technical parameters which might not reflect the user perception in some tense situations. Nevertheless, appearance and utility of a networked service depend on the underlying technical solutions and their performance. Thus, we face the challenge of bringing it all together, which essentially describes the spirit of the 18th ITC Specialists Seminar on Quality of Experience (ITC-SS 18). ITC Specialist Seminars have a very good reputation in gathering experts and their high-quality contributions around a performance-oriented topic of mutual interest. ITC-SS 18 is intended as a meeting place between experts, researchers, practitioners, vendors and customers. It is devoted to presentations and discussions of QoE concepts, analysis, management approaches etc., both from industry and academia. While many conferences are dominated by academia, one third of the submissions to ITC-SS 18 originated from industry. The contributions have been peer-reviewed by at least three independent reviewers and finally, we selected 18 papers to be presented. Additionally, two keynote speeches reflect one industrial and one academic approach to QoE analysis and implementation. For the ITC, the leading conference for performance modeling and analysis of communication networks &amp; systems, ITC-SS 18 opens a window towards the end user. ITC-SS 18 takes place in Karlskrona on May 29—30, 2008. It is organized by the Dept. of Telecommunication Systems (ATS) within the School of Engineering (TEK) at Blekinge Institute of Technology (BTH) in cooperation with the International Advisory Council (IAC) of ITC.</p

18th ITC Specialist Seminar on Quality of Experience

The notion and topic of Quality of Experience (QoE) keeps attracting the attention of manufacturers, operators and researchers. It links user perception and expectations on one side and technical Quality of Service (QoS) parameters, management, pricing schemes etc. on the other side. Such links are needed in order to balance user satisfaction and economic aspects of service provisioning. However, the notion of QoE as such is not without controversy. Technicians, used to a world of objective and clearly definable parameters, tend to fear the subjective, somehow fuzzy parts associated with end user perception. Vice versa, customer relationship and marketing departments might find themselves uncomfortable with technical parameters which might not reflect the user perception in some tense situations. Nevertheless, appearance and utility of a networked service depend on the underlying technical solutions and their performance. Thus, we face the challenge of bringing it all together, which essentially describes the spirit of the 18th ITC Specialists Seminar on Quality of Experience (ITC-SS 18). ITC Specialist Seminars have a very good reputation in gathering experts and their high-quality contributions around a performance-oriented topic of mutual interest. ITC-SS 18 is intended as a meeting place between experts, researchers, practitioners, vendors and customers. It is devoted to presentations and discussions of QoE concepts, analysis, management approaches etc., both from industry and academia. While many conferences are dominated by academia, one third of the submissions to ITC-SS 18 originated from industry. The contributions have been peer-reviewed by at least three independent reviewers and finally, we selected 18 papers to be presented. Additionally, two keynote speeches reflect one industrial and one academic approach to QoE analysis and implementation. For the ITC, the leading conference for performance modeling and analysis of communication networks & systems, ITC-SS 18 opens a window towards the end user. ITC-SS 18 takes place in Karlskrona on May 29—30, 2008. It is organized by the Dept. of Telecommunication Systems (ATS) within the School of Engineering (TEK) at Blekinge Institute of Technology (BTH) in cooperation with the International Advisory Council (IAC) of ITC