Retrograde axonal transport of Poliovirus and EV71 in motor neurons

Poliovirus (PV) can spread through neural pathway to the central nervous system and replicates in motor neurons, which leads to poliomyelitis. Enterovirus 71 (EV71), which is closely related to PV, is one of the causative agents of hand-foot-and-mouth disease and can cause severe neurological diseases similar to poliomyelitis. Since PV is similar to EV71 in its motor neurotoxicity, we tried to understand if the results obtained with PV are of general applicability to EV71 and other viruses with similar characteristics. Using microfluidic devices, we demonstrated that both PV capsid and the PV genome undergo axonal retrograde transport with human PV receptor (hPVR), and the transported virus replicated in the soma of hPVR-expressing motor neurons. Similar to PV in hPVR-transgenic (Tg) mice, neural pathway ensuring spreading of EV71 has been shown in adult human scavenger receptor class B, member 2 (hSCARB2)-Tg mice. We have validated this finding in microfluidic devices by showing that EV71 is retrogradely transported together with hSCARB2 to the cell body where it replicates in an hSCARB2-dependent manner

Regeneration of Bone- and Tendon/Ligament-Like Tissues Induced by Gene Transfer of Bone Morphogenetic Protein-12 in a Rat Bone Defect

Members of the bone morphogenetic protein (BMP) family have diverse physiological roles. For instance, BMP-2 stimulates osteogenesis, while BMP-12 induces the formation of tendon/ligament-like tissues. Here, we designed a study to determine whether BMP-12 has bone and/or cartilage regeneration abilities similar to those of BMP-2. We implanted plasmid vectors encoding either BMP-2 or BMP-12 in rats with femur defects, and monitored the bone healing process for 8-weeks. The BMP-12 transgene induced prominent fibrogenesis by 2 weeks, with bone substitution occurring by 8 weeks. BMP-2, however, was associated predominantly with osteogenesis throughout the 8 week period. Thus, we conclude that BMP-12 does not function similarly to BMP-2 during bone healing. Further work is needed to better understand the mechanisms by which it stimulates bony growths to replace the connective tissues formed during the first stages of bone healing

Discovery of widespread transcription initiation at microsatellites predictable by sequence-based deep neural network

Using the Cap Analysis of Gene Expression (CAGE) technology, the FANTOM5 consortium provided one of the most comprehensive maps of transcription start sites (TSSs) in several species. Strikingly, ~72% of them could not be assigned to a specific gene and initiate at unconventional regions, outside promoters or enhancers. Here, we probe these unassigned TSSs and show that, in all species studied, a significant fraction of CAGE peaks initiate at microsatellites, also called short tandem repeats (STRs). To confirm this transcription, we develop Cap Trap RNA-seq, a technology which combines cap trapping and long read MinION sequencing. We train sequence-based deep learning models able to predict CAGE signal at STRs with high accuracy. These models unveil the importance of STR surrounding sequences not only to distinguish STR classes, but also to predict the level of transcription initiation. Importantly, genetic variants linked to human diseases are preferentially found at STRs with high transcription initiation level, supporting the biological and clinical relevance of transcription initiation at STRs. Together, our results extend the repertoire of non-coding transcription associated with DNA tandem repeats and complexify STR polymorphism

Hybridization in a Microfluidic Device for DNA Tile Self-Assembly

Abstract- In order to self-assemble desired DNA nanostructure, it is necessary to suppress assembly errors caused by fluctuations of temperature and concentration. We propose a microfluidic device to directly control such environmental parameters of DNA self-assembly. In this paper, some experimental results concerned with key processes of self-assembly in the device, such as immobilizing DNA strands in a reaction chamber and relation rate of hybridization under flow condition are reported. 1

A Rapid Method for Optimizing Running Temperature of Electrophoresis through Repetitive On-Chip CE Operations

In this paper, a rapid and simple method to determine the optimal temperature conditions for denaturant electrophoresis using a temperature-controlled on-chip capillary electrophoresis (CE) device is presented. Since on-chip CE operations including sample loading, injection and separation are carried out just by switching the electric field, we can repeat consecutive run-to-run CE operations on a single on-chip CE device by programming the voltage sequences. By utilizing the high-speed separation and the repeatability of the on-chip CE, a series of electrophoretic operations with different running temperatures can be implemented. Using separations of reaction products of single-stranded DNA (ssDNA) with a peptide nucleic acid (PNA) oligomer, the effectiveness of the presented method to determine the optimal temperature conditions required to discriminate a single-base substitution (SBS) between two different ssDNAs is demonstrated. It is shown that a single run for one temperature condition can be executed within 4 min, and the optimal temperature to discriminate the SBS could be successfully found using the present method

DNA Separation using a PDMS (Polydimethylsiloxane)-based Electrophoresis Device

特集 マイクロ・ナノメカトロニク

Characterization of the receptor binding residues of kisspeptins by positional scanning using peptide photoaffinity probes.

Kisspeptins, endogenous peptide ligands for GPR54, play an important role in GnRH secretion. Since in vivo administration of kisspeptins induces increased plasma LH levels, GPR54 agonists hold promise as therapeutic agents for the treatment of hormonal secretion diseases. To facilitate the design of novel potent GPR54 ligands, residues in kisspeptins that involve in the interaction with GPR54 were investigated by kisspeptin-based photoaffinity probes. Herein, we report the design and synthesis of novel kisspeptin-based photoaffinity probes, and the application to crosslinking experiments for GPR54-expressing cells

Highly efficient single cell arraying by integrating acoustophoretic cell pre-concentration and deialctrophoretic cell trapping

To array rare cells at the single-cell level, the volumetric throughput may become a bottleneck in the cell trapping and the subsequent single-cell analysis, since the target cells per definition commonly exist in a large sample volume after purification from the original sample. Here, we present a novel approach for high throughput single cell arraying by integrating two original microfluidic devices: an acoustofluidic chip and an electroactive microwell array. The velocity of the cells is geared down in the acoustofluidic chip while maintaining a high volume flow rate at the inlet of the microsystem, and the cells are subsequently trapped one by one into the microwell array using dielectrophoresis. The integrated system exhibited a 10 times improved sample throughput compared to trapping with the electroactive microwell array chip alone, while maintaining a highly efficient cell recovery above 90%. The results indicate that the serial integration of the acoustophoretic pre-concentration with the dielectrophoretic cell trapping drastically improves the performance of the electroactive microwell array for highly efficient single cell analysis. This simple and effective system for high throughput single cell arraying with further possible integration of additional functions, including cell sorting and downstream analysis after cell trapping, has potential for development to a highly integrated and automated platform for single-cell analysis of rare cells

An integrated acousto- and dielectrophoresis device for tumor cell separation, concentration, and single-cell trapping

Many microfluidic devices have been developed to separate rare cells, e.g. circulating tumor cells. The collection and analysis of the separated rare cells pose a challenge as it may lead to loss of target cells since extremely small number of cells are usually suspended in a large volume after separation. Here, we present an integrated device that allows acoustophoretic target cell separation and concentration, followed by dielectrophoretic single-cell trapping. We show that the human prostate cancer cell line DU145 were efficiently separated from peripheral blood mononuclear cells (PBMCs), and 81.7% of DU145 were trapped, with a contamination of 1.7% PBMC