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This paper highlights the behavior of microdroplets used for portable biosensors. Microdroplets have been applied for various Lab-on-a-chip applications, in which portability/wearable is becoming a trend word. However, the resonance (sloshing phenomena) of the microdroplets could be an issue. For example, bilayer lipid membrane, fragile characteristics due to its thickness of 5 nm, easily ruptures by jiggling. We therefore prepared various dimensions of droplets by changing the well shape, and examined the resulting waves depending on the vibration frequency between 20 and 100 Hz, including daily life frequencies. Moreover, we investigated an influence of surface coating of the wells by the same tests. The results of the first examination showed that the sloshing was effectively suppressed by reducing the well length along the vibration axis. We also found that the sloshing was suppressed by reducing surface energy of the microwells. We succeeded in clarifying the characteristics of microdroplets at the vibration in daily life frequency, which will be useful information for development of portable biosensors.

Recombinant transmembrane adenylate cyclase (AC) was incorporated into membranes of giant liposomes using membrane fusion between liposomes and baculovirus-budded virus (BV). AC genes were constructed into transfer vectors in a form fused with fluorescent protein or polyhistidine at the C-terminus. The recombinant BVs were collected by ultracentrifugation and AC expression was verified using western blotting. The BVs and giant liposomes generated using gentle hydration were fused under acidic conditions; the incorporation of AC into giant liposomes was demonstrated by confocal laser scanning microscopy through the emission of fluorescence from their membranes. The AC-expressing BVs were also fused with liposomes containing the substrate (ATP) with/without a specific inhibitor (SQ 22536). An enzyme immunoassay on extracts of the sample demonstrated that cAMP was produced inside the liposomes. This procedure facilitates direct introduction of large transmembrane proteins into artificial membranes without solubilization.

In this paper, we present an efficient methodology to manipulate a single micro-object using round-tip positive dielectrophoresis-based tweezers. The tweezers consist of a glass needle with a round-tip and a pair of thin gold-film electrodes. The round-tip, which has a radius of 3µm, is formed by melting a finely pulled glass needle and concentrates the electric field at the tip of the tweezers, which allows the individual manipulation of single micro-objects. The tweezers successfully captured, conveyed, and positioned single cell-sized liposomes with diameters of 5-23µm, which are difficult to manipulate with conventional manipulation methodologies, such as optical tweezers or glass micropipettes, due to the similarities between their optical properties and those of the media, as well as the ease with which they are deformed or broken. We used Stokes' drag theory to experimentally evaluate the positive dielectrophoresis (pDEP) force generated by the tweezers as a function of the liposome size, the content of the surrounding media, and the applied AC voltage and frequency. The results agreed with the theoretically deduced pDEP force. Finally, we demonstrated the separation of labeled single cells from non-labeled cells with the tweezers. This device can be used as an efficient tool for precisely and individually manipulating biological micro-objects that are typically transparent and flexible., (Copyright © 2013 Elsevier B.V. All rights reserved.)

CD40L is excessively produced in both human and murine lupus and plays a role in lupus pathogenesis. To address how excess CD40L induces autoantibody production, we crossed CD40L-transgenic mice with the anti-DNA H-chain transgenic mouse lines 3H9 and 56R, well-characterized models for studying B-cell tolerance to nuclear antigens. Excess CD40L did not induce autoantibody production in 3H9 mice in which anergy maintains self-tolerance, nor did it perturb central tolerance, including deletion and receptor editing, of anti-DNA B cells in 56R mice. In contrast, CD40L/56R mice restored a large number of marginal zone (MZ) B cells reactive to Sm/ribonucleoprotein (RNP) and produced autoantibody, whereas these B cells were deleted by apoptosis in MZ of 56R mice. Thus, excess CD40L efficiently blocked tolerance of Sm/RNP-reactive MZ B cells, leading to production of anti-Sm/RNP antibody implicated in the pathogenesis of lupus. These results suggest that self-reactive B cells such as anti-Sm/RNP B cells, which somehow escape tolerance in the bone marrow and migrate to MZ, are tolerized by apoptotic deletion in MZ and that a break in this tolerance may play a role in the pathogenesis of lupus.

N-cadherin (CDH2) proteins were reconstituted with liposomes using a baculovirus expression-liposome fusion method. CDH2 budded viruses were fused with giant liposomes containing dioleoylphophogycerol/dioleoylphosphatidylcholine (DOPG/DOPC) at pH 4.5 and the localization of CDH2 on the liposome membrane was observed by confocal laser scanning microscopy. CDH2 liposomes showed Ca(2+)-dependent association. CDH2-mediated association/dissociation in CDH2 liposomes was specific to Ca(2+) and reversible. CDH2-expressing LN-229 cells (human glioblastoma cell) adhered to CDH2 liposomes and small CDH2 liposomes (diameter approximately 150 nm), in particular, were internalized by endocytosis and partly escaped endosomes. Cadherin-containing liposomes show high potential as a new cell-specific proteoliposome. The baculovirus expression-liposome fusion method is useful as a new enabling technology for biomedical applications of functional proteoliposomes., (Crown Copyright © 2011. Published by Elsevier Ltd. All rights reserved.)