Integrated microfluidic biosensing platform for simultaneous confocal microscopy and electrophysiological measurements on bilayer lipid membranes and ion channels

Combining high-resolution imaging and electrophysiological recordings is key for various types of experimentation on lipid bilayers and ion channels. Here, we propose an integrated biosensing platform consisting of a microfluidic cartridge and a dedicated chip-holder to conduct such dual measurements on suspended lipid bilayers, in a user-friendly manner. To illustrate the potential of the integrated platform, we characterize lipid bilayers in terms of thickness and fluidity while simultaneously monitoring single ion channel currents. For that purpose, POPC lipid bilayers are supplemented with a fluorescently-tagged phospholipid (NBD-PE, 1% mol) for Fluorescence Recovery After Photobleaching (FRAP) measurements and a model ion channel (gramicidin, 1 nM). These combined measurements reveal that NBD-PE has no effect on the lipid bilayer thickness while gramicidin induces thinning of the membrane. Furthermore, the presence of gramicidin does not alter the lipid bilayer fluidity. Surprisingly, in lipid bilayers supplemented with both probes, a reduction in gramicidin open probability and lifetime is observed compared to lipid bilayers with gramicidin only, suggesting an influence of NBD-PE on the gramicidin ion function. Altogether, our proposed microfluidic biosensing platform in combination with the herein presented multi-parametric measurement scheme paves the way to explore the interdependent relationship between lipid bilayer properties and ion channel function

Sonoprinting liposomes on tumor spheroids by microbubbles and ultrasound

Ultrasound-triggered drug-loaded microbubbles have great potential for drug delivery due to their ability to locally release drugs and simultaneously enhance their delivery into the target tissue. We have recently shown that upon applying ultrasound, nanoparticle-loaded microbubbles can deposit nanoparticles onto cells grown in 2D monolayers, through a process that we termed "sonoprinting". However, the rigid surfaces on which cell monolayers are typically growing might be a source of acoustic reflections and aspherical microbubble oscillations, which can influence microbubble-cell interactions. In the present study, we aim to reveal whether sonoprinting can also occur in more complex and physiologically relevant tissues, by using free-floating 3D tumor spheroids as a tissue model. We show that both monospheroids (consisting of tumor cells alone) and cospheroids (consisting of tumor cells and fibroblasts, which produce an extracellular matrix) can be sonoprinted. Using doxorubicin-liposome-loaded microbubbles, we show that sonoprinting allows to deposit large amounts of doxorubicin-containing liposomes to the outer cell layers of the spheroids, followed by doxorubicin release into the deeper layers of the spheroids, resulting in a significant reduction in cell viability. Sonoprinting may become an attractive approach to deposit drug patches at the surface of tissues, thereby promoting the delivery of drugs into target tissues

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

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

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

Allele frequencies of hemojuvelin gene (HJV) I222N and G320V missense mutations in white and African American subjects from the general Alabama population

BACKGROUND: Homozygosity or compound heterozygosity for coding region mutations of the hemojuvelin gene (HJV) in whites is a cause of early age-of-onset iron overload (juvenile hemochromatosis), and of hemochromatosis phenotypes in some young or middle-aged adults. HJV coding region mutations have also been identified recently in African American primary iron overload and control subjects. Primary iron overload unexplained by typical hemochromatosis-associated HFE genotypes is common in white and black adults in Alabama, and HJV I222N and G320V were detected in a white Alabama juvenile hemochromatosis index patient. Thus, we estimated the frequency of the HJV missense mutations I222N and G320V in adult whites and African Americans from Alabama general population convenience samples. METHODS: We evaluated the genomic DNA of 241 Alabama white and 124 African American adults who reported no history of hemochromatosis or iron overload to detect HJV missense mutations I222N and G320V using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technique. Analysis for HJV I222N was performed in 240 whites and 124 African Americans. Analysis for HJV G320V was performed in 241 whites and 118 African Americans. RESULTS: One of 240 white control subjects was heterozygous for HJV I222N; she was also heterozygous for HFE C282Y, but had normal serum iron measures and bone marrow iron stores. HJV I222N was not detected in 124 African American subjects. HJV G320V was not detected in 241 white or 118 African American subjects. CONCLUSIONS: HJV I222N and G320V are probably uncommon causes or modifiers of primary iron overload in adult whites and African Americans in Alabama. Double heterozygosity for HJV I222N and HFE C282Y may not promote increased iron absorption

Search for CP violation in D+→K−K+π+D^{+} \to K^{-}K^{+}\pi^{+} decays

A model-independent search for direct CP violation in the Cabibbo suppressed decay $D^+ \to K^- K^+\pi^+$ in a sample of approximately 370,000 decays is carried out. The data were collected by the LHCb experiment in 2010 and correspond to an integrated luminosity of 35 pb$^{-1}$. The normalized Dalitz plot distributions for $D^+$ and $D^-$ are compared using four different binning schemes that are sensitive to different manifestations of CP violation. No evidence for CP asymmetry is found.Comment: 13 pages, 8 figures, submitted to Phys. Rev.