Parallel single cell analysis on an integrated microfluidic platform for cell trapping, lysis and analysis

We report here a novel and easily scalable microfluidic platform for the parallel analysis of hundreds of individual cells, with controlled single cell trapping, followed by their lysis and subsequent retrieval of the cellular content for on-chip analysis. The device consists of a main channel and an array of shallow side channels connected to the main channel via trapping structures. Cells are individually captured in dam structures by application of a negative pressure from an outlet reservoir, lyzed on site and the cellular content controllably extracted and transported in the individual side channels for on-chip analysis.\u

Cancer-ID:Toward Identification of Cancer by Tumor-Derived Extracellular Vesicles in Blood

Extracellular vesicles (EVs) have great potential as biomarkers since their composition and concentration in biofluids are disease state dependent and their cargo can contain disease-related information. Large tumor-derived EVs (tdEVs, >1μm) in blood from cancer patients are associated with poor outcome, and changes in their number can be used to monitor therapy effectiveness. Whereas, small tumor-derived EVs (<1μm) are likely to outnumber their larger counterparts, thereby offering better statistical significance, identification and quantification of small tdEVs are more challenging. In the blood of cancer patients, a subpopulation of EVs originate from tumor cells, but these EVs are outnumbered by non-EV particles and EVs from other origin. In the Dutch NWO Perspectief Cancer-ID program, we developed and evaluated detection and characterization techniques to distinguish EVs from non-EV particles and other EVs. Despite low signal amplitudes, we identified characteristics of these small tdEVs that may enable the enumeration of small tdEVs and extract relevant information. The insights obtained from Cancer-ID can help to explore the full potential of tdEVs in the clinic

Tests of the Equivalence Principle with Neutral Kaons

We test the Principle of Equivalence for particles and antiparticles, using CPLEAR data on tagged K0 and K0bar decays into pi^+ pi^-. For the first time, we search for possible annual, monthly and diurnal modulations of the observables |eta_{+-}| and phi_{+-}, that could be correlated with variations in astrophysical potentials. Within the accuracy of CPLEAR, the measured values of |eta_{+-}| and phi_{+-} are found not to be correlated with changes of the gravitational potential. We analyze data assuming effective scalar, vector and tensor interactions, and we conclude that the Principle of Equivalence between particles and antiparticles holds to a level of 6.5, 4.3 and 1.8 x 10^{-9}, respectively, for scalar, vector and tensor potentials originating from the Sun with a range much greater than the distance Earth-Sun. We also study energy-dependent effects that might arise from vector or tensor interactions. Finally, we compile upper limits on the gravitational coupling difference between K0 and K0bar as a function of the scalar, vector and tensor interaction range.Comment: 15 pages latex 2e, five figures, one style file (cernart.csl) incorporate

Test of CPT Symmetry and Quantum Mechanics with Experimental data from CPLEAR

We use fits to recent published CPLEAR data on neutral kaon decays to $\pi^+\pi^-$ and $\pi e\nu$ to constrain the CPT--violation parameters appearing in a formulation of the neutral kaon system as an open quantum-mechanical system. The obtained upper limits of the CPT--violation parameters are approaching the range suggested by certain ideas concerning quantum gravity.Comment: 9 pages of uuencoded postscript (includes 3 figures

Prevalence of H63D, S65C and C282Y hereditary hemochromatosis gene mutations in Slovenian population by an improved high-throughput genotyping assay

<p>Abstract</p> <p>Background</p> <p>Hereditary hemochromatosis (HH) is a common genetic disease characterized by excessive iron overload that leads to multi-organ failure. Although the most prevalent genotype in HH is homozygosity for C282Y mutation of the <it>HFE </it>gene, two additional mutations, H63D and S65C, appear to be associated with a milder form of HH. The aim of this study was to develop a high-throughput assay for <it>HFE </it>mutations screening based on TaqMan technology and to determine the frequencies of <it>HFE </it>mutations in the Slovenian population.</p> <p>Methods</p> <p>Altogether, 1282 randomly selected blood donors from different Slovenian regions and 21 HH patients were analyzed for the presence of <it>HFE </it>mutations by an in-house developed real-time PCR assay based on TaqMan technology using shorter non-interfering fluorescent single nucleotide polymorphism (SNP)-specific MGB probes. The assay was validated by RFLP analysis and DNA sequencing.</p> <p>Results</p> <p>The genotyping assay of the H63D, S65C and C282Y mutations in the <it>HFE </it>gene, based on TaqMan technology proved to be fast, reliable, with a high-throughput capability and 100% concordant with genotypes obtained by RFLP and DNA sequencing. The observed frequency of C282Y homozygotes in the group of HH patients was only 48%, others were of the heterogeneous <it>HFE </it>genotype. Among 1282 blood donors tested, the observed H63D, S65C and C282Y allele frequency were 12.8% (95% confidence interval (CI) 11.5 – 14.2%), 1.8% (95% CI 1.4 – 2.5%) and 3.6% (95% CI 3.0 – 4.5%), respectively. Approximately 33% of the tested subjects had at least one of the three HH mutations, and 1% of them were C282Y homozygotes or compound heterozygotes C282Y/H63D or C282Y/S65C, presenting an increased risk for iron overload disease. A significant variation in H63D allele frequency was observed for one of the Slovenian regions.</p> <p>Conclusion</p> <p>The improved real-time PCR assay for H63D, S65C and C282Y mutations detection is accurate, fast, cost-efficient and ready for routine screening and diagnostic procedures. The genotype frequencies in the Slovenian population agree with those reported for the Central European populations although some deviations where observed in comparison with other populations of Slavic origin. Regional distribution of the mutations should be considered when planning population screening.</p

Observation of an Excited Bc+ State

Using pp collision data corresponding to an integrated luminosity of 8.5 fb-1 recorded by the LHCb experiment at center-of-mass energies of s=7, 8, and 13 TeV, the observation of an excited Bc+ state in the Bc+π+π- invariant-mass spectrum is reported. The observed peak has a mass of 6841.2±0.6(stat)±0.1(syst)±0.8(Bc+) MeV/c2, where the last uncertainty is due to the limited knowledge of the Bc+ mass. It is consistent with expectations of the Bc∗(2S31)+ state reconstructed without the low-energy photon from the Bc∗(1S31)+→Bc+γ decay following Bc∗(2S31)+→Bc∗(1S31)+π+π-. A second state is seen with a global (local) statistical significance of 2.2σ (3.2σ) and a mass of 6872.1±1.3(stat)±0.1(syst)±0.8(Bc+) MeV/c2, and is consistent with the Bc(2S10)+ state. These mass measurements are the most precise to date

Monitoring one-electron photo-oxidation of guanine in DNA crystals using ultrafast infrared spectroscopy

To understand the molecular origins of diseases caused by ultraviolet and visible light, and also to develop photodynamic therapy, it is important to resolve the mechanism of photoinduced DNA damage. Damage to DNA bound to a photosensitizer molecule frequently proceeds by one-electron photo-oxidation of guanine, but the precise dynamics of this process are sensitive to the location and the orientation of the photosensitizer, which are very difficult to define in solution. To overcome this, ultrafast time-resolved infrared (TRIR) spectroscopy was performed on photoexcited ruthenium polypyridyl–DNA crystals, the atomic structure of which was determined by X-ray crystallography. By combining the X-ray and TRIR data we are able to define both the geometry of the reaction site and the rates of individual steps in a reversible photoinduced electron-transfer process. This allows us to propose an individual guanine as the reaction site and, intriguingly, reveals that the dynamics in the crystal state are quite similar to those observed in the solvent medium