Manipulating and assembling metallic beads with Optoelectronic Tweezers

Optoelectronic tweezers (OET) or light-patterned dielectrophoresis (DEP) has been developed as a micromanipulation technology for controlling micro- and nano-particles with applications such as cell sorting and studying cell communications. Additionally, the capability of moving small objects accurately and assembling them into arbitrary 2D patterns also makes OET an attractive technology for microfabrication applications. In this work, we demonstrated the use of OET to manipulate conductive silver-coated Poly(methyl methacrylate) (PMMA) microspheres (50μm diameter) into tailored patterns. It was found that the microspheres could be moved at a max velocity of 3200μm/s, corresponding to 4.2 nano-newton (10−9N) DEP force, and also could be positioned with high accuracy via this DEP force. The underlying mechanism for this strong DEP force is shown by our simulations to be caused by a significant increase of the electric field close to the particles, due to the interaction between the field and the silver shells coating the microspheres. The associated increase in electrical gradient causes DEP forces that are much stronger than any previously reported for an OET device, which facilitates manipulation of the metallic microspheres efficiently without compromise in positioning accuracy and is important for applications on electronic component assembling and circuit construction

Channel integrated optoelectronic tweezer chip for microfluidic particle manipulation

Light patterned electrical fields have been widely used for the manipulation of microparticles, from cells to microscopic electronic components. In this work, we explore a novel electromechanical phenomenon for particle focusing and sorting where the electrical field patterns are shaped by a combination of the light patterned photoconductor and the channel geometry. This effect results from the combination of particle polarisation described by the Clausius–Mossotti relation and the engineering of large electric gradients produced by choosing the channels height to suit the size of the particles being manipulated. The matched geometry increases the distortion of the field created by a combination of the illuminated photoconductor and the particles themselves and hence the non-uniformity of the field they experience. We demonstrate a new channel integration strategy which allows the creation of precisely defined channel structures in the OET device. By defining channels in photoresist sandwiched between upper and lower ITO coated glass substrates we produce robust channels of well controlled height tailored to the particle. Uniquely, the top substrate is attached before photolithographically defining the channels. We demonstrate versatile control using this effect with dynamically reconfigurable light patterns allowing the retention against flow, focusing and sorting of micro particles within the channels. Contrary to traditional designs, this channel integrated device allows patterned micro channels to be used in conjunction with conductive top and bottom electrodes producing optimal conditions for the dielectrophoretic manipulation as demonstrated by the rapid flow (up to 5mm s−1 ) in which the particles can be focuse

Manufacturing with light - micro-assembly of opto-electronic microstructures

Optical micromanipulation allows the movement and patterning of discrete micro-particles within a liquid environment. However, for manufacturing applications it is desirable to remove the liquid, leaving the patterned particles in place. In this work, we have demonstrated the use of optoelectronic tweezers (OET) to manipulate and accurately assemble Sn62Pb36Ag2 solder microspheres into tailored patterns. A technique based on freeze-drying technology was then developed that allows the assembled patterns to be well preserved and fixed in place after the liquid medium in the OET device is removed. After removing the liquid from the OET device and subsequently heating the assembled pattern and melting the solder microspheres, electrical connections between the microspheres were formed, creating a permanent conductive bridge between two isolated metal electrodes. Although this method is demonstrated with 40 µm diameter solder beads arranged with OET, it could be applied to a great range of discrete components from nanowires to optoelectronic devices, thus overcoming one of the basic hurdles in using optical micromanipulation techniques in a manufacturing micro-assembly setting

A Novel, All-Optical Tool for Controllable and Non- Destructive Poration of Cells with Single-Micron Resolution

We demonstrate controllable poration within ≈1 µm regions of individual cells, mediated by a near-IR laser interacting with thin-layer amorphous silicon substrates. This technique will allow new experiments in single-cell biology, particularly in neuroscience. As our understanding of the fundamental mechanistic processes underpinning biology expands, so does the need for high-precision tools to allow the dissection of the heterogeneity and stochastic processes that dominate at the single- and sub-cellular level. Here, we demonstrate a highly controllable and reproducible optical technique for inducing poration within specific regions of a target cell’s plasma membrane, permitting localized delivery of payloads, depolarization and lysis experiments to be conducted in unprecedented detail. Experiments support a novel mechanism for the process, based upon a thermally-induced change triggered by the interactions of a near-IR laser with a biocompatible thin film substrate at powers substantially below that used in standard optoporation experiments

Genome-wide study of association and interaction with maternal cytomegalovirus infection suggests new schizophrenia loci.

Genetic and environmental components as well as their interaction contribute to the risk of schizophrenia, making it highly relevant to include environmental factors in genetic studies of schizophrenia. This study comprises genome-wide association (GWA) and follow-up analyses of all individuals born in Denmark since 1981 and diagnosed with schizophrenia as well as controls from the same birth cohort. Furthermore, we present the first genome-wide interaction survey of single nucleotide polymorphisms (SNPs) and maternal cytomegalovirus (CMV) infection. The GWA analysis included 888 cases and 882 controls, and the follow-up investigation of the top GWA results was performed in independent Danish (1396 cases and 1803 controls) and German-Dutch (1169 cases, 3714 controls) samples. The SNPs most strongly associated in the single-marker analysis of the combined Danish samples were rs4757144 in ARNTL (P=3.78 × 10(-6)) and rs8057927 in CDH13 (P=1.39 × 10(-5)). Both genes have previously been linked to schizophrenia or other psychiatric disorders. The strongest associated SNP in the combined analysis, including Danish and German-Dutch samples, was rs12922317 in RUNDC2A (P=9.04 × 10(-7)). A region-based analysis summarizing independent signals in segments of 100 kb identified a new region-based genome-wide significant locus overlapping the gene ZEB1 (P=7.0 × 10(-7)). This signal was replicated in the follow-up analysis (P=2.3 × 10(-2)). Significant interaction with maternal CMV infection was found for rs7902091 (P(SNP × CMV)=7.3 × 10(-7)) in CTNNA3, a gene not previously implicated in schizophrenia, stressing the importance of including environmental factors in genetic studies

Structure of the hDmc1-ssDNA filament reveals the principles of its architecture

In eukaryotes, meiotic recombination is a major source of genetic diversity, but its defects in humans lead to abnormalities such as Down's, Klinefelter's and other syndromes. Human Dmc1 (hDmc1), a RecA/Rad51 homologue, is a recombinase that plays a crucial role in faithful chromosome segregation during meiosis. The initial step of homologous recombination occurs when hDmc1 forms a filament on single-stranded (ss) DNA. However the structure of this presynaptic complex filament for hDmc1 remains unknown. To compare hDmc1-ssDNA complexes to those known for the RecA/Rad51 family we have obtained electron microscopy (EM) structures of hDmc1-ssDNA nucleoprotein filaments using single particle approach. The EM maps were analysed by docking crystal structures of Dmc1, Rad51, RadA, RecA and DNA. To fully characterise hDmc1-DNA complexes we have analysed their organisation in the presence of Ca2+, Mg2+, ATP, AMP-PNP, ssDNA and dsDNA. The 3D EM structures of the hDmc1-ssDNA filaments allowed us to elucidate the principles of their internal architecture. Similar to the RecA/Rad51 family, hDmc1 forms helical filaments on ssDNA in two states: extended (active) and compressed (inactive). However, in contrast to the RecA/Rad51 family, and the recently reported structure of hDmc1-double stranded (ds) DNA nucleoprotein filaments, the extended (active) state of the hDmc1 filament formed on ssDNA has nine protomers per helical turn, instead of the conventional six, resulting in one protomer covering two nucleotides instead of three. The control reconstruction of the hDmc1-dsDNA filament revealed 6.4 protein subunits per helical turn indicating that the filament organisation varies depending on the DNA templates. Our structural analysis has also revealed that the N-terminal domain of hDmc1 accomplishes its important role in complex formation through domain swapping between adjacent protomers, thus providing a mechanistic basis for coordinated action of hDmc1 protomers during meiotic recombination

Smc5/6 coordinates formation and resolution of joint molecules with chromosome morphology to ensure meiotic divisions

During meiosis, Structural Maintenance of Chromosome (SMC) complexes underpin two fundamental features of meiosis: homologous recombination and chromosome segregation. While meiotic functions of the cohesin and condensin complexes have been delineated, the role of the third SMC complex, Smc5/6, remains enigmatic. Here we identify specific, essential meiotic functions for the Smc5/6 complex in homologous recombination and the regulation of cohesin. We show that Smc5/6 is enriched at centromeres and cohesin-association sites where it regulates sister-chromatid cohesion and the timely removal of cohesin from chromosomal arms, respectively. Smc5/6 also localizes to recombination hotspots, where it promotes normal formation and resolution of a subset of joint-molecule intermediates. In this regard, Smc5/6 functions independently of the major crossover pathway defined by the MutLγ complex. Furthermore, we show that Smc5/6 is required for stable chromosomal localization of the XPF-family endonuclease, Mus81-Mms4Eme1. Our data suggest that the Smc5/6 complex is required for specific recombination and chromosomal processes throughout meiosis and that in its absence, attempts at cell division with unresolved joint molecules and residual cohesin lead to severe recombination-induced meiotic catastroph

Matched Ascertainment of Informative Families for Complex Genetic Modelling

Family data are used extensively in quantitative genetic studies to disentangle the genetic and environmental contributions to various diseases. Many family studies based their analysis on population-based registers containing a large number of individuals composed of small family units. For binary trait analyses, exact marginal likelihood is a common approach, but, due to the computational demand of the enormous data sets, it allows only a limited number of effects in the model. This makes it particularly difficult to perform joint estimation of variance components for a binary trait and the potential confounders. We have developed a data-reduction method of ascertaining informative families from population-based family registers. We propose a scheme where the ascertained families match the full cohort with respect to some relevant statistics, such as the risk to relatives of an affected individual. The ascertainment-adjusted analysis, which we implement using a pseudo-likelihood approach, is shown to be efficient relative to the analysis of the whole cohort and robust to mis-specification of the random effect distribution

Anti-Angiogenic Activity of a Small Molecule STAT3 Inhibitor LLL12

Background: Recent data indicate the Signal Transducer and Activator of Transcription 3 (STAT3) pathway is required for VEGF production and angiogenesis in various types of cancers. STAT3 inhibitors have been shown to reduce tumor microvessel density in tumors but a direct anti-angiogenic activity has not been described. Methodology/Principal Findings: We investigated the direct action of a small molecule inhibitor of STAT3 (LLL12) in human umbilical cord vascular endothelial cells (HUVECs) in vitro, in a Matrigel model for angiogenesis in vivo, and its antitumor activity in a xenograft model of osteosarcoma. LLL12 (100 nM) significantly inhibited VEGF-stimulated STAT3 phosphorylation in HUVECs, reduced their proliferation/migration and inhibited VEGF-induced tube formation. Morphologic analysis of LLL12 treated HUVECs demonstrated marked changes in actin/tubulin distribution and bundling. In scid mice, LLL12 reduced microvessel invasion into VEGF-infused Matrigel plugs by,90 % at a dose of 5 mg/kg daily. Following a period of tumor progression (2 weeks), LLL12 completely suppressed further growth of established OS-1 osteosarcoma xenografts. Pharmacodynamic studies showed robust phosphorylated STAT3 in control tumors, whereas phospho-STAT3 was not detected in LLL12-treated OS-1 tumors. Treated tumors demonstrated decreased proliferation (Ki67 staining), and decreased microvessel density (CD34 staining), but no significant increase in apoptosis (TUNEL staining), relative to controls. Assay of angiogenic factors, using an antibody array, showed VEGF, MMP-9, Angiopoietin1/2, Tissue Factor and FGF-

Efficacy and pharmacokinetic/pharmacodynamic evaluation of the Aurora kinase A inhibitor MLN8237 against preclinical models of pediatric cancer

To gain a greater understanding of the potential of the Aurora kinase A inhibitor MLN8237 in the treatment of pediatric malignancies. The activity of MLN8237 was evaluated against 28 neuroblastoma and Ewing sarcoma cell lines, and its in vivo efficacy was studied over a range of doses against 12 pediatric tumor xenograft models. Pharmacokinetic, pharmacodynamic, and genomic studies were undertaken. In vitro neuroblastoma cell lines were generally more sensitive to MLN8237 than Ewing sarcoma lines. MLN8237 demonstrated significant activity in vivo against solid tumor models at the maximum tolerated dose (MTD); however, only 2 of 6 neuroblastoma models had objective responses at 0.25MTD. In contrast, MLN8237 induced objective responses at its MTD and at 0.5MTD in three ALL models and in two out of three at 0.25MTD. Pharmacokinetic studies at 0.5MTD demonstrated a T (max) of 0.5 h, C (max) of 24.8 mu M, AUC((0-24)) of 60.3 mu M h, and 12 h trough level of 1.2 mu M. Mitotic indices increased 6-12 h after MLN8237 administration. AURKA copy number variation was frequent in xenografts, and expression was highly correlated with copy number. Objective responses were more frequent in tumors with decreased AURKA copy number (5/8) compared to those with increased gene copy number (2/14). This report confirms the significant activity against both solid tumor and ALL xenografts at the MTD, with a steep dose response. These data support clinical development of MLN8237 in childhood cancer. Because of the steep dose-response relationship, such studies should target achieving trough levels of 1 mu M or higher for sustained periods of treatment