Direct Capture and Amplification of Small Fragmented DNAs Using Nitrogen-Mustard-Coated Microbeads.

Circulating cell-free DNA (cfDNA) has been implicated as an important biomarker and has been intensively studied for "liquid biopsy" applications in cancer diagnostics. Owing to its small fragment size and its low concentration in circulation, cfDNA extraction and purification from serum samples are complicated, and the extraction yield affects the precision of subsequent molecular diagnostic tests. Here, we report a novel approach using nitrogen-mustard-coated DNA capture beads (NMD beads) that covalently capture DNA and allow direct subsequent polymerase chain reaction (PCR) amplification from the NMD bead without elusion. The complex DNA extraction and purification processes are not required. To illustrate the diagnostic use of the NMD beads, we detected short DNA fragments (142 bp) that were spiked into fetal bovine serum (as a model serum sample). The spiked DNAs were captured directly from serum samples and detected using real-time PCR at concentrations as low as 10 fg/mL. We anticipate that this DNA capture bead technique has the potential to simplify the preanalytical processes required for cfDNA detection, which could significantly expand the diagnostic applications of liquid biopsy.

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.

A novel method for successful induction of interdigitating process formation in conditionally immortalized podocytes from mice, rats, and humans.

Formation of processes in podocytes is regarded as the hallmark of maturity and normal physical condition for the cell. There are many accumulated findings about molecular mechanisms that cause retraction of podocyte processes; however, there is little knowledge of the positive mechanisms that promote process formation in vitro, and most previous reports about this topic have been limited to low-density cultures. Here, we found that process formation can be induced in 100% confluent cultures of conditionally immortalized podocytes in mouse, rat, and human species by combining serum depletion and Y-27632 ROCK inhibitor supplementation on the scaffold of laminin-521(L521). We noted the cytoskeletal reorganization of the radial extension pattern of vimentin filaments and downregulation of actin stress fiber formation under that condition. We also found that additional standard amount of serum, depletion of ROCK inhibitor, or slight mismatch of the scaffold as laminin-511(L511) hinder process formation. These findings suggest that the combination of reduced serum, podocyte-specific scaffold, and intracellular signaling to reduce the overexpression of ROCK are required factors for process formation.

Attoampere Nanoelectrochemistry.

Electrochemical microscopy techniques have extended the understanding of surface chemistry to the micrometer and even sub-micrometer level. However, fundamental questions related to charge transport at the solid-electrolyte interface, such as catalytic reactions or operation of individual ion channels, require improved spatial resolutions down to the nanoscale. A prerequisite for single-molecule electrochemical sensitivity is the reliable detection of a few electrons per second, that is, currents in the atto-Ampere (10-18 A) range, 1000 times below today's electrochemical microscopes. This work reports local cyclic voltammetry (CV) measurements at the solid-liquid interface on ferrocene self-assembled monolayer (SAM) with sub-atto-Ampere sensitivity and simultaneous spatial resolution < 80 nm. Such sensitivity is obtained through measurements of the charging of the local faradaic interface capacitance at GHz frequencies. Nanometer-scale details of different molecular organizations with a 19% packing density difference are resolved, with an extremely small dispersion of the molecular electrical properties. This is predicted previously based on weak electrostatic interactions between neighboring redox molecules in a SAM configuration. These results open new perspectives for nano-electrochemistry like the study of quantum mechanical resonance in complex molecules and a wide range of applications from electrochemical catalysis to biophysics.

Real-Time Detection of Glyphosate by a Water-Gated Organic Field-Effect Transistor with a Microfluidic Chamber.

This paper reports the development of a real-time monitoring system utilizing the combination of a water-gated organic field-effect transistor (WG-OFET) and a microfluidic chamber for the detection of the herbicide glyphosate (GlyP). For the realization of the real-time sensing with the WG-OFET, the surface of a polymer semiconductor was utilized as a sensing unit. The aqueous solution including the target analyte, which is employed as a gate dielectric of the WG-OFET, flows into a designed microfluidic chamber on the semiconductor layer and the gate electrode. As the sensing mechanism, the WG-OFET-based sensor utilizes the competitive complexation among carboxylate-functionalized polythiophene, a copper(II) (Cu2+) ion, and GlyP. The reversible accumulation and desorption of the positively charged Cu2+ ion on the semiconductor surface induced a change in the electrical double-layer capacitance (EDLC). The optimization of the microfluidic chamber enables a uniform water flow and contributes to real-time quantitative sensing of GlyP at a micromolar level. Thus, this study would lead to practical real-time sensing in water for various fields including environmental assessment.

In vitro spermatogenesis in two-dimensionally spread mouse testis tissues.

PURPOSE: Mouse in vitro spermatogenesis is possible with classical organ culture methods, by placing the testis tissue at the interphase between culture medium and air. In this condition, however, a tissue piece tends to round up to be compact, whose central region suffers from shortage of nutrients and oxygen. In this study, the authors improved the culture condition by spreading each tissue thin and flat, by which they were able to get better access to the oxygen and nutrients. METHODS: Immature mouse testis tissues placed on agarose gel block were forced to spread flat by covering with a polydimethylsiloxane (PDMS) ceiling chip (PC chip). They were then cultured for weeks and evaluated by the transgene expression of Acr-Gfp, which reflects the progression of spermatogenesis. RESULTS: Testis tissues covered with PC chip initiated and maintained spermatogenesis in its wider region than those without PC chip covering. Flow cytometric analysis demonstrated that the PC method yielded more numerous meiotic germ cells than those without PC. Immunohistochemical examination confirmed the authentic histological figure of spermatogenesis from spermatogonia up to round or elongating spermatids. CONCLUSIONS: The PC chip method is simple and effective to improve the efficiency of in vitro spermatogenesis in the organ culture system.

A monolayer microfluidic device supporting mouse spermatogenesis with improved visibility.

In our previous study, we produced a microfluidic device (MFD) which successfully maintained spermatogenesis for over 6 months in mouse testis tissues loaded in the device. In the present study, we developed a new MFD, a monolayer device (ML-D) with a barrier structure consisting of pillars and slits, which is simpler in design and easier to make. This ML-D was also effective for inducing mouse spermatogenesis and maintained it for a longer period than the conventional culture method. In addition, we devised a way of introducing sample tissue into the device during its production, just before bonding the upper layer of polydimethylsiloxane (PDMS) and bottom glass slide. The tissue can obtain nutrients horizontally from the medium running beside it and oxygen vertically from above through PDMS. In addition, the glass slide set at the bottom improved the visibility of the sample tissue with an inverted microscope. When we took photos of cultured tissue of the Acr-Gfp transgenic mouse testis in ML-D sequentially every day, morphological changes of the acrosome during spermiogenesis were successfully recorded. The ML-D is simple in design and useful for culturing testis tissue for inducing and maintaining spermatogenesis with clearer visibility. Due to the new method of sample loading, tissues other than testis should also be applicable.

Neonatal testis growth recreated in vitro by two-dimensional organ spreading.

Organ culture experiments can be hampered by central degeneration or necrosis due to the inadequate permeation of oxygen and nutrients, which deteriorates the function and growth of cultured tissues. In the current study, we aimed to overcome this limitation of organ culture through spreading the tissue two dimensionally on an agarose gel stand and molding into a disc shape by placing a ceiling of polydimethylsiloxane (PDMS) chip, which is highly oxygen permeable. By this, every part of the tissue can receive a sufficient supply of oxygen through PDMS as well as nutrients through the agarose gel below. This method not only prevented central necrosis of tissues, but also supported the tissue growth over time. In addition, such growth, as volume enlargement, could be easily measured. Under these conditions, we examined the effect of several factors on the growth of neonatal mouse testis, and found that follicle stimulating hormone (FSH) and insulin significantly promoted the growth. These results are in good agreement with previous in vivo reports. Notably, the growth achieved over 7 days in our in vitro system is almost comparable to, about 80% of, that observed in vivo. Thus, we successfully monitored the promotion of tissue growth beyond the limits of the conventional organ culture method. This extremely simple method could offer a unique platform to evaluate the growth as well as functional properties of organs, not only the testis but also others as well.

Comparison of the transcriptomic profile of hepatic human induced pluripotent stem like cells cultured in plates and in a 3D microscale dynamic environment.

We have compared the transcriptomic profiles of human induced pluripotent stem cells after their differentiation in hepatocytes like cells in plates and microfluidic biochips. The biochips provided a 3D and dynamic support during the cell differentiation when compared to the 2D static cultures in plates. The microarray have demonstrated the up regulation of important pathway related to liver development and maturation during the culture in biochips. Furthermore, the results of the transcriptomic profile, coupled with immunostaining, and RTqPCR analysis have shown typical biomarkers illustrating the presence of responders of biliary like cells, hepatocytes like cells, and endothelial like cells. However, the overall tissue still presented characteristic of immature and foetal patterns. Nevertheless, the biochip culture provided a specific micro-environment in which a complex multicellular differentiation toward liver could be oriented.

A Microfluidic Diffusion Cell for Fast and Easy Percutaneous Absorption Assays.

PURPOSE: Percutaneous absorption assays of molecules for pharmaceutical and cosmetology purposes are important to determine the bioavailability of new compounds, once topically applied. The current method of choice is to measure the rate of diffusion through excised human skin using a diffusion cell. This method however entails significant drawbacks such as scarce availability and poor reproducibility of the sample, low sampling rate, and tedious assay setup. METHODS: The objective of the present work is to propose an alternative method that overcomes these issues by integrating an experimental model of the skin (artificial stratum corneum) and online optical sensors into a microfluidic device. RESULTS: The measurement of the diffusion profile followed by the calculation of the permeability coefficients and time lag were performed on seven different molecules and obtained data positively fit with those available from literature on human skin penetration. The coating of the lipid mixture to generate the artificial stratum corneum also proved robust and reproducible. The results show that the proposed device is able to give fast, real-time, accurate, and reproducible data in a user-friendly manner, and can be produced at a large scale. CONCLUSION: These assets should help both the cosmetics and pharmaceutics fields where the skin is the target or a pathway of a formulated compound, by allowing more candidate molecules or formulations to be assessed during the various stages of their development.

Dynamic DNA-toolbox reaction circuits: a walkthrough.

In living organisms, the integration of signals from the environment and the molecular computing leading to a cellular response are orchestrated by Gene Regulatory Networks (GRN). However, the molecular complexity of in vivo genetic regulation makes it next to impossible to describe in a quantitative manner. Reproducing, in vitro, reaction networks that could mimic the architecture and behavior of in vivo networks, yet lend themselves to mathematical modeling, represents a useful strategy to understand, and even predict, the function of GRN. In this paper, we define a set of in vitro, DNA-based molecular transformations that can be linked to each other in such a way that the product of one transformation can activate or inhibit the production of one or several other DNA compounds. Therefore, these reactions can be wired in arbitrary networks. This approach provides an experimental way to reproduce the dynamic features of genetic regulation in a test tube. We introduce the rules to design the necessary DNA species, a guide to implement the chemical reactions and ways to optimize the experimental conditions. We finally show how this framework, or "DNA toolbox", can be used to generate an inversion module, though many other behaviors, including oscillators and bistable switches, can be implemented.

Population transcriptomics with single-cell resolution: a new field made possible by microfluidics: a technology for high throughput transcript counting and data-driven definition of cell types.

Tissues contain complex populations of cells. Like countries, which are comprised of mixed populations of people, tissues are not homogeneous. Gene expression studies that analyze entire populations of cells from tissues as a mixture are blind to this diversity. Thus, critical information is lost when studying samples rich in specialized but diverse cells such as tumors, iPS colonies, or brain tissue. High throughput methods are needed to address, model and understand the constitutive and stochastic differences between individual cells. Here, we describe microfluidics technologies that utilize a combination of molecular biology and miniaturized labs on chips to study gene expression at the single cell level. We discuss how the characterization of the transcriptome of each cell in a sample will open a new field in gene expression analysis, population transcriptomics, that will change the academic and biomedical analysis of complex samples by defining them as quantified populations of single cells.

Bottom-up construction of in vitro switchable memories.

Reaction networks displaying bistability provide a chemical mechanism for long-term memory storage in cells, as exemplified by many epigenetic switches. These biological systems are not only bistable but switchable, in the sense that they can be flipped from one state to the other by application of specific molecular stimuli. We have reproduced such functions through the rational assembly of dynamic reaction networks based on basic DNA biochemistry. Rather than rewiring genetic systems as synthetic biology does in vivo, our strategy consists of building simplified dynamic analogs in vitro, in an artificial, well-controlled milieu. We report successively a bistable system, a two-input switchable memory element, and a single-input push-push memory circuit. These results suggest that it is possible to build complex time-responsive molecular circuits by following a modular approach to the design of dynamic in vitro behaviors. Our approach thus provides an unmatched opportunity to study topology/function relationships within dynamic reaction networks.

Involvement of the rabies virus phosphoprotein gene in neuroinvasiveness.

Rabies virus (RABV), which is transmitted via a bite wound caused by a rabid animal, infects peripheral nerves and then spreads to the central nervous system (CNS) before causing severe neurological symptoms and death in the infected individual. Despite the importance of this ability of the virus to spread from a peripheral site to the CNS (neuroinvasiveness) in the pathogenesis of rabies, little is known about the mechanism underlying the neuroinvasiveness of RABV. In this study, to obtain insights into the mechanism, we conducted comparative analysis of two fixed RABV strains, Nishigahara and the derivative strain Ni-CE, which cause lethal and asymptomatic infections, respectively, in mice after intramuscular inoculation. Examination of a series of chimeric viruses harboring the respective genes from Nishigahara in the genetic background of Ni-CE revealed that the Nishigahara phosphoprotein (P) gene plays a major role in the neuroinvasiveness by mediating infection of peripheral nerves. The results obtained from both in vivo and in vitro experiments strongly suggested that the Nishigahara P gene, but not the Ni-CE P gene, is important for stable viral replication in muscle cells. Further investigation based on the previous finding that RABV phosphoprotein counteracts the host interferon (IFN) system demonstrated that the Nishigahara P gene, but not the Ni-CE P gene, functions to suppress expression of the beta interferon (IFN-ß) gene (Ifn-ß) and IFN-stimulated genes in muscle cells. In conclusion, we provide the first data strongly suggesting that RABV phosphoprotein assists viral replication in muscle cells by counteracting the host IFN system and, consequently, enhances infection of peripheral nerves.

Quencher-free multiplexed monitoring of DNA reaction circuits.

We present a simple yet efficient technique to monitor the dynamics of DNA-based reaction circuits. This technique relies on the labeling of DNA oligonucleotides with a single fluorescent modification. In this quencher-free setup, the signal is modulated by the interaction of the 3'-terminus fluorophore with the nucleobases themselves. Depending on the nature of the fluorophore's nearest base pair, fluorescence intensity is decreased or increased upon hybridization. By tuning the 3'-terminal nucleotides, it is possible to obtain opposite changes in fluorescence intensity for oligonucleotides whose hybridization site is shifted by a single base. Quenching by nucleobases provides a highly sequence-specific monitoring technique, which presents a high sensitivity even for small oligonucleotides. Compared with other sequence-specific detection methods, it is relatively non-invasive and compatible with the complex dynamics of DNA reaction circuits. As an application, we show the implementation of nucleobase quenching to monitor a DNA-based chemical oscillator, allowing us to follow in real time and quantitatively the dephased oscillations of the components of the network. This cost-effective monitoring technique should be widely implementable to other DNA-based reaction systems.

Spatial waves in synthetic biochemical networks.

We report the experimental observation of traveling concentration waves and spirals in a chemical reaction network built from the bottom up. The mechanism of the network is an oscillator of the predator-prey type, and this is the first time that predator-prey waves have been observed in the laboratory. The molecular encoding of the nonequilibrium behavior relies on small DNA oligonucleotides that enforce the network connectivity and three purified enzymes that control the reactivity. Wave velocities in the range 80-400 µm min(-1) were measured. A reaction-diffusion model in quantitative agreement with the experiments is proposed. Three fundamental parameters are easy to tune in nucleic acid reaction networks: the topology of the network, the rate constants of the individual reactions, and the diffusion coefficients of the individual species. For this reason, we expect such networks to bring unprecedented opportunities for assaying the principles of spatiotemporal order formation in chemistry.

Single-step CE for miniaturized and easy-to-use system.

We developed a novel single-step capillary electrophoresis (SSCE) scheme for miniaturized and easy to use system by using a microchannel chip, which was made from the hydrophilic material polymethyl methacrylate (PMMA), equipped with a capillary stop valve. Taking the surface tension property of liquids into consideration, the capillary effect was used to introduce liquids and control capillary stop valves in a partial barrier structure in the wall of the microchannel. Through the combined action of stop valves and air vents, both sample plug formation for electrophoresis and sample injection into a separation channel were successfully performed in a single step. To optimize SSCE, different stop valve structures were evaluated using actual microchannel chips and the finite element method with the level set method. A partial barrier structure at the bottom of the channel functioned efficiently as a stop valve. The stability of stop valve was confirmed by a shock test, which was performed by dropping the microchannel chip to a floor. Sample plug deformation could be reduced by minimizing the size of the side partial barrier. By dissolving hydroxyl ethyl cellulose and using it as the sample solution, the EOF and adsorption of the sample into the PMMA microchannel were successfully reduced. Using this method, a 100-bp DNA ladder was concentrated; good separation was observed within 1 min. At a separation length of 5 mm, the signal was approximately 20-fold higher than a signal of original sample solution by field-amplified sample stacking effect. All operations, including liquid introduction and sample separation, can be completed within 2 min by using the SSCE scheme.

Ultra-high density protein spots achieved by on chip digitalized protein synthesis.

Current methodologies for arraying proteins using cell-free protein synthesis on a chip have spatial limitations that prevent reaching ultra-high density necessary for high throughput analysis. To circumvent this, we developed an on-chip method based on microcompartmentalization of protein synthesis. Proteins are synthesized in arrayed micrometer scale chambers from confined DNA template molecules. On-chip protein expression is highly efficient and the method can be used with a minimal amount of template i.e. single DNA molecules to perform digitalized cell-free protein synthesis (d-CFPS). A functionalized surface at the floor of the tightly sealed microchambers enables direct capture of expressed proteins. A density of 104 spots per mm² was achieved, which represents a gain by more than 3 orders of magnitude over conventional methods. This technique of forming such densely arrayed small protein spots is the first step towards the development of a general method that would allow fabrication of ultra-high density protein arrays for high-throughput analysis.

In vitro spermatogenesis in isolated seminiferous tubules of immature mice.

Mouse spermatogenesis, from spermatogonial stem cell proliferation to sperm formation, can be reproduced in vitro by culturing testis tissue masses of neonatal mice. However, it remains to be determined whether this method is also applicable when testis tissues are further divided into tiny fragments, such as segments of the seminiferous tubule (ST), a minimal anatomical unit for spermatogenesis. In this study, we investigated this issue using the testis of an Acrosin-GFP/Histone H3.3-mCherry (Acr/H3) double-transgenic mouse and monitored the expression of GFP and mCherry as indicators of spermatogenic progression. Initially, we noticed that the cut and isolated stretches of ST shrunk rapidly and conglomerated. We therefore maintained the isolation of STs in two ways: segmental isolation without truncation or embedding in soft agarose. In both cases, GFP expression was observed by fluorescence microscopy. By whole-mount immunochemical staining, meiotic spermatocytes and round and elongating spermatids were identified as Sycp3-, crescent-form GFP-, and mCherry-positive cells, respectively. Although the efficiency was significantly lower than that with tissue mass culture, we clearly showed that spermatogenesis can be induced up to the elongating spermatid stage even when the STs were cut into short segments and cultured in isolation. In addition, we demonstrated that lowered oxygen tension was favorable for spermatogenesis both for meiotic progression and for producing elongating spermatids in isolated STs. Culturing isolated STs rather than tissue masses is advantageous for explicitly assessing the various environmental parameters that influence the progression of spermatogenesis.

Enhanced podocyte differentiation and changing drug toxicity sensitivity through pressure-controlled mechanical filtration stress on a glomerulus-on-a-chip.

Podocytes, localized in the glomerulus, are a prognostic factor of proteinuria in kidney disease and are exposed to distinct physiological stimuli from basal to apical filtration flow. Research studies on drug discovery and disease modeling for glomerulopathy have developed a glomerulus-on-a-chip and studied podocyte mechanobiology to realize alternative methods to animal experiments. However, the effect of filtration stimulus on podocytes has remained unclear. Herein, we report the successful development of a user-friendly filtration culture device and system that can precisely control the filtration flow using air pressure control by incorporating a commercially available culture insert. It allows mouse podocytes to be cultured under filtration conditions for three days with a guarantee of maintaining the integrity of the podocyte layer. Using our system, this study demonstrated that podocyte damage caused by hyperfiltration resulting from glomerular hypertension, a common pathophysiology of many glomerulopathies, was successfully recapitulated and that filtration stimulus promotes the maturation of podocytes in terms of their morphology and gene expression. Furthermore, we demonstrated that filtration stimulus induced different drug responsiveness in podocytes than those seen under static conditions, and that the difference in drug responsiveness was dependent on the pharmacological mechanism. Overall, this study has revealed differentiating and pharmacodynamic properties of filtration stimulus and brings new insights into the research field of podocyte mechanobiology towards the realization of glomerulus-on-a-chip.