Measuring, in solution, multiple-fluorophore labeling by combining Fluorescence Correlation Spectroscopy and photobleaching

Determining the number of fluorescent entities that are coupled to a given molecule (DNA, protein, etc.) is a key point of numerous biological studies, especially those based on a single molecule approach. Reliable methods are important, in this context, not only to characterize the labeling process, but also to quantify interactions, for instance within molecular complexes. We combined Fluorescence Correlation Spectroscopy (FCS) and photobleaching experiments to measure the effective number of molecules and the molecular brightness as a function of the total fluorescence count rate on solutions of cDNA (containing a few percent of C bases labeled with Alexa Fluor 647). Here, photobleaching is used as a control parameter to vary the experimental outputs (brightness and number of molecules). Assuming a Poissonian distribution of the number of fluorescent labels per cDNA, the FCS-photobleaching data could be easily fit to yield the mean number of fluorescent labels per cDNA strand (@ 2). This number could not be determined solely on the basis of the cDNA brightness, because of both the statistical distribution of the number of fluorescent labels and their unknown brightness when incorporated in cDNA. The statistical distribution of the number of fluorophores labeling cDNA was confirmed by analyzing the photon count distribution (with the cumulant method), which showed clearly that the brightness of cDNA strands varies from one molecule to the other.Comment: 38 pages (avec les figures

Orally administered 5-aminolevulinic acid for isolation and characterization of circulating tumor-derived extracellular vesicles in glioblastoma patients

Background: In glioblastoma (GB), tissue is required for accurate diagnosis and subtyping. Tissue can be obtained through resection or (stereotactic) biopsy, but these invasive procedures provide risks for patients. Extracellular vesicles (EVs) are small, cell-derived vesicles that contain miRNAs, proteins, and lipids, and possible candidates for liquid biopsies. GB-derived EVs can be found in the blood of patients, but it is difficult to distinguish them from circulating non-tumor EVs. 5-aminolevulinic acid (5-ALA) is orally administered to GB patients to facilitate tumor visualization and maximal resection, as it is metabolized to fluorescent protoporphyrin IX (PpIX) that accumulates in glioma cell

The Implicitome: A Resource for Rationalizing Gene-Disease Associations

High-throughput experimental methods such as medical sequencing and genome-wide association studies (GWAS) identify increasingly large numbers of potential relations between genetic variants and diseases. Both biological complexity (millions of potential gene-disease associations) and the accelerating rate of data production necessitate computational approaches to prioritize and rationalize potential gene-disease relations. Here, we use concept profile technology to expose from the biomedical literature both explicitly stated gene-disease relations (the explicitome) and a much larger set of implied gene-disease associations (the implicitome). Implicit relations are largely unknown to, or are even unintended by the original authors, but they vastly extend the reach of existing biomedical knowledge for identification and interpretation of gene-disease associations. The implicitome can be used in conjunction with experimental data resources to rationalize both known and novel associations. We demonstrate the usefulness of the implicitome by rationalizing known and novel gene-disease associations, including those from GWAS. To facilitate the re-use of implicit gene-disease associations, we publish our data in compliance with FAIR Data Publishing recommendations [https://www.force11.org/group/fairgroup] using nanopublications. An online tool (http://knowledge.bio) is available to explore established and potential gene-disease associations in the context of other biomedical relations.UB – Publicatie

Study protocol for the multicentre cohorts of Zika virus infection in pregnant women, infants, and acute clinical cases in Latin America and the Caribbean: The ZIKAlliance consortium

Background: The European Commission (EC) Horizon 2020 (H2020)-funded ZIKAlliance Consortium designed a multicentre study including pregnant women (PW), children (CH) and natural history (NH) cohorts. Clinical sites were selected over a wide geographic range within Latin America and the Caribbean, taking into account the dynamic course of the ZIKV epidemic. Methods: Recruitment to the PW cohort will take place in antenatal care clinics. PW will be enrolled regardless of symptoms and followed over the course of pregnancy, approximately every 4 weeks. PW will be revisited at delivery (or after miscarriage/abortion) to assess birth outcomes, including microcephaly and other congenital abnormalities according to the evolving definition of congenital Zika syndrome (CZS). After birth, children will be followed for 2 years in the CH cohort. Follow-up visits are scheduled at ages 1-3, 4-6, 12, and 24 months to assess neurocognitive and developmental milestones. In addition, a NH cohort for the characterization of symptomatic rash/fever illness was designed, including follow-up to capture persisting health problems. Blood, urine, and other biological materials will be collected, and tested for ZIKV and other relevant arboviral diseases (dengue, chikungunya, yellow fever) using RT-PCR or serological methods. A virtual, decentralized biobank will be created. Reciprocal clinical monitoring has been established between partner sites. Substudies of ZIKV seroprevalence, transmissio

Strongly Reduced Penetration of Atomic Deuterium in Radiation-Damaged Tungsten

Radiation-damaged tungsten is exposed to high-flux, low-energy deuterium plasmas at self-bias conditions. We observe that the fraction of deuterium that penetrates is only 10-5–10-7 of the plasma flux and strongly dependent on the local surface temperature. We propose that deuterium does not directly penetrate bulk tungsten but that it thermalizes at the surface, where it forms a protective chemisorbed layer. We find an energy barrier of 1–2 eV between the surface and bulk, causing the influx of deuterium to be low as compared to the number of defects and leading to slow filling of the damaged layer

Reduced deuterium retention in self-damaged tungsten exposed to high-flux plasmas at high surface temperatures

We investigated the effect of surface temperature on deuterium retention in self-damaged tungsten exposed to high-flux deuterium plasmas. The retention saturates at a W 4+ fluence of about 3 × 10 17 m −2 and is strongly reduced for the present high surface temperatures of 800–1200 K as compared with previous experiments at 470–525 K. Combination of nuclear reaction analysis (NRA), thermal desorption spectroscopy and positron annihilation Doppler broadening (PADB) was used to investigate the reduction in deuterium retention. The NRA showed a strong reduction of retention at the surface at high surface temperatures. The PADB measurements suggest that during plasma exposure defects are mobile and cluster into larger clusters containing up to a few tens of vacancies. The Tritium Migration Analysis Program 7 simulations show that trapping and de-trapping rates are very high for defects with trapping energies below ∼1.5 eV. The strong reduction in retention seems to be caused by the reduced amount of mono-vacancies and small vacancy clusters in combination with their strong depopulation due to thermal trapping and de-trapping

Deuterium retention in tungsten and tungsten-tantalum alloys exposed to high-flux deuterium plasmas

A direct comparison of deuterium retention in samples of tungsten and two grades of tungsten-tantalum alloys-W-1% Ta and W-5% Ta, exposed to deuterium plasmas (ion flux similar to 10(24) m(-2) s(-1), ion energy at the biased target similar to 50 eV) at the plasma generator Pilot-PSI was performed using thermal desorption spectroscopy (TDS). No systematic difference in terms of total retention in tungsten and tungsten-tantalum was identified. The measured retention value for each grade did not deviate by more than 24% from the value averaged over the three grades exposed to the same conditions. No additional desorption peaks appeared in the TDS spectra of the W-Ta samples as compared with the W target, indicating that no additional kinds of traps are introduced by the alloying of W with Ta. In the course of the experiment the same samples were exposed to the same plasma conditions several times, and it is demonstrated that samples with the history of prior exposures yield an increase in deuterium retention of up to 130% under the investigated conditions compared with the samples that were not exposed before. We consider this as evidence that exposure of the considered materials to ions with energy below the displacement threshold generates additional traps for deuterium. The positions of the release peaks caused by these traps are similar for W and W-Ta, which indicates that the corresponding traps are of the same kind