Pneumatic Microballoons for Active Control of the Vibration-Induced Flow.

Vibration-induced flow (VIF), in which a mean flow is induced around a microstructure by applying periodic vibrations, is increasingly used as an active flow-control technique at the microscale. In this study, we have developed a microdevice that actively controls the VIF patterns using elastic membrane protrusions (microballoons) actuated by pneumatic pressure. This device enables on-demand spatial and temporal fluid manipulation using a single device that cannot be achieved using a conventional fixed-structure arrangement. We successfully demonstrated that the device achieved displacements of up to 38 µm using the device within a pressure range of 0 to 30 kPa, indicating the suitability of the device for microfluidic applications. Using this active microballoon array, we demonstrated that the device can actively manipulate the flow field and induce swirling flows. Furthermore, we achieved selective actuation of the microballoon using this system. By applying air pressure from a multi-input channel system through a connection tube, the microballoons corresponding to each air channel can be selectively actuated. This enabled precise control of the flow field and periodic switching of the flow patterns using a single chip. In summary, the proposed microdevice provides active control of VIF patterns and has potential applications in advanced microfluidics, such as fluid mixing and particle manipulation.

Ustekinumab during pregnancy and lactation: drug levels in maternal serum, cord blood, breast milk, and infant serum.

BACKGROUND: Patients with ulcerative colitis (UC) may be concerned about medication safety during preconception, pregnancy, and lactation, and they should be closely followed up to ensure that UC activity is controlled during the perinatal period. Reported information on the safety of ustekinumab during pregnancy and lactation is limited. In this case report, we examined the safety of ustekinumab in a fetus and breastfed infant with reference to drug concentrations in maternal serum, cord blood, breast milk, and infant serum. CASE PRESENTATION: A 36-year-old female who developed hematochezia and was diagnosed with ulcerative colitis at age 24 was pregnant with her first child. During pregnancy she was treated with subcutaneous bimonthly ustekinumab, at a dose of 90 mg, until 29 weeks of gestation. Her ulcerative colitis symptoms remained in remission. At 38 weeks of gestation she underwent cesarean section and delivered a healthy female infant weighing 3043 g and with no congenital malformations. The infant received routine vaccinations with no adverse events. Ustekinumab treatment was resumed at 7 weeks postpartum. The ustekinumab concentration in maternal serum at 12 days after injection (30.7 weeks of gestation) was 7968.5 ng/mL, and it decreased to 106.1 ng/mL at 114 days after the last dose. In cord blood, the ustekinumab concentration was 1131.2 ng/mL at 65 days after the last dose; this was 2.5 times higher than that in the maternal serum, which was consistent with a previous report. Ustekinumab was detected in infant serum collected at 71 days after the last maternal dose (299.0 ng/mL), with rapid elimination from the infant's body. In breast milk, the maximum ustekinumab concentrations were 13.6 ng/mL at 9 days after the last maternal dose, respectively. The ratio of the calculated areas under the time-concentration curves of ustekinumab in breast milk and maternal serum was 0.0008 (257.1/327632.7), which was comparable with a previous human study. CONCLUSION: The placental transfer and breast milk secretion of ustekinumab in our case were comparable with previous reports. Use of ustekinumab during pregnancy and lactation was feasible in this case. Further research is needed to clarify the safety of ustekinumab during pregnancy and lactation.

Microgripper Using Soft Microactuators for Manipulation of Living Cells.

We present a microgripper actuated by a soft microactuator for manipulating a single living cell. Soft actuators have attracted attention in recent years because their compliance which can adapt to soft targets. In this study, we propose a microgripper actuated by soft thermoresponsive hydrogels. The thermoresponsive gel swells in water when the temperature is low and shrinks when the temperature is high. Therefore, the microgripper can be driven by controlling the temperature of the thermoresponsive gel. The gels are actuated by irradiating with infrared (IR) laser to localize heating. The actuation characteristics of the gripper were theoretically analyzed and we designed a gripper that gripped a ≈10 µm size cell. Additionally, we succeeded in actuating the fabricated microgripper with laser irradiation and gripping a single living cell.

Contribution of AMPA and NMDA receptors in the spontaneous firing patterns of single neurons in autaptic culture.

Single neurons in an autaptic culture exhibit various types of firing pattern with different firing durations and rhythms. However, a neuron with autapses has often been modeled as an oscillator providing a monotonic firing pattern with a constant periodicity because of the lack of a mathematical model. In the work described in this study, we use computational simulation and whole-cell patch-clamp recording to elucidate and model the mechanism by which such neurons generate various firing pattens. In the computational simulation, three types of spontaneous firing pattern, i.e., short, long-lasting, and periodic burst firing patterns are realized by changing the combination ratio of N-methyl-d-aspartate (NMDA) to α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate (AMPA) conductance. These three types of firing patterns are also observed in the experiments where neurons are cultured in isolation on micropatterned substrates. Using the AMPA and NMDA current models, we discuss that, in principle, autapses can regulate rhythmicity and information selection in neuronal networks.

Raman image-activated cell sorting.

The advent of image-activated cell sorting and imaging-based cell picking has advanced our knowledge and exploitation of biological systems in the last decade. Unfortunately, they generally rely on fluorescent labeling for cellular phenotyping, an indirect measure of the molecular landscape in the cell, which has critical limitations. Here we demonstrate Raman image-activated cell sorting by directly probing chemically specific intracellular molecular vibrations via ultrafast multicolor stimulated Raman scattering (SRS) microscopy for cellular phenotyping. Specifically, the technology enables real-time SRS-image-based sorting of single live cells with a throughput of up to ~100 events per second without the need for fluorescent labeling. To show the broad utility of the technology, we show its applicability to diverse cell types and sizes. The technology is highly versatile and holds promise for numerous applications that are previously difficult or undesirable with fluorescence-based technologies.

Author Correction: A practical guide to intelligent image-activated cell sorting.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

High-speed microparticle isolation unlimited by Poisson statistics.

High-speed isolation of microparticles (e.g., microplastics, heavy metal particles, microbes, cells) from heterogeneous populations is the key element of high-throughput sorting instruments for chemical, biological, industrial and medical applications. Unfortunately, the performance of continuous microparticle isolation or so-called sorting is fundamentally limited by the trade-off between throughput, purity, and yield. For example, at a given throughput, high-purity sorting needs to sacrifice yield, or vice versa. This is due to Poisson statistics of events (i.e., microparticles, microparticle clusters, microparticle debris) in which the interval between successive events is stochastic and can be very short. Here we demonstrate an on-chip microparticle sorter with an ultrashort switching window in both time (10 µs) and space (10 µm) at a high flow speed of 1 m s-1, thereby overcoming the Poisson trade-off. This is made possible by using femtosecond laser pulses that can produce highly localized transient cavitation bubbles in a microchannel to kick target microparticles from an acoustically focused, densely aligned, bumper-to-bumper stream of microparticles. Our method is important for rare-microparticle sorting applications where both high purity and high yield are required to avoid missing rare microparticles.

Label-free chemical imaging flow cytometry by high-speed multicolor stimulated Raman scattering.

Combining the strength of flow cytometry with fluorescence imaging and digital image analysis, imaging flow cytometry is a powerful tool in diverse fields including cancer biology, immunology, drug discovery, microbiology, and metabolic engineering. It enables measurements and statistical analyses of chemical, structural, and morphological phenotypes of numerous living cells to provide systematic insights into biological processes. However, its utility is constrained by its requirement of fluorescent labeling for phenotyping. Here we present label-free chemical imaging flow cytometry to overcome the issue. It builds on a pulse pair-resolved wavelength-switchable Stokes laser for the fastest-to-date multicolor stimulated Raman scattering (SRS) microscopy of fast-flowing cells on a 3D acoustic focusing microfluidic chip, enabling an unprecedented throughput of up to ∼140 cells/s. To show its broad utility, we use the SRS imaging flow cytometry with the aid of deep learning to study the metabolic heterogeneity of microalgal cells and perform marker-free cancer detection in blood.

A practical guide to intelligent image-activated cell sorting.

Intelligent image-activated cell sorting (iIACS) is a machine-intelligence technology that performs real-time intelligent image-based sorting of single cells with high throughput. iIACS extends beyond the capabilities of fluorescence-activated cell sorting (FACS) from fluorescence intensity profiles of cells to multidimensional images, thereby enabling high-content sorting of cells or cell clusters with unique spatial chemical and morphological traits. Therefore, iIACS serves as an integral part of holistic single-cell analysis by enabling direct links between population-level analysis (flow cytometry), cell-level analysis (microscopy), and gene-level analysis (sequencing). Specifically, iIACS is based on a seamless integration of high-throughput cell microscopy (e.g., multicolor fluorescence imaging, bright-field imaging), cell focusing, cell sorting, and deep learning on a hybrid software-hardware data management infrastructure, enabling real-time automated operation for data acquisition, data processing, intelligent decision making, and actuation. Here, we provide a practical guide to iIACS that describes how to design, build, characterize, and use an iIACS machine. The guide includes the consideration of several important design parameters, such as throughput, sensitivity, dynamic range, image quality, sort purity, and sort yield; the development and integration of optical, microfluidic, electrical, computational, and mechanical components; and the characterization and practical usage of the integrated system. Assuming that all components are readily available, a team of several researchers experienced in optics, electronics, digital signal processing, microfluidics, mechatronics, and flow cytometry can complete this protocol in ~3 months.

High-throughput label-free molecular fingerprinting flow cytometry.

Flow cytometry is an indispensable tool in biology for counting and analyzing single cells in large heterogeneous populations. However, it predominantly relies on fluorescent labeling to differentiate cells and, hence, comes with several fundamental drawbacks. Here, we present a high-throughput Raman flow cytometer on a microfluidic chip that chemically probes single live cells in a label-free manner. It is based on a rapid-scan Fourier-transform coherent anti-Stokes Raman scattering spectrometer as an optical interrogator, enabling us to obtain the broadband molecular vibrational spectrum of every single cell in the fingerprint region (400 to 1600 cm-1) with a record-high throughput of ~2000 events/s. As a practical application of the method not feasible with conventional flow cytometry, we demonstrate high-throughput label-free single-cell analysis of the astaxanthin productivity and photosynthetic dynamics of Haematococcus lacustris.

Numerical and Experimental Analyses of Three- Dimensional Unsteady Flow around a Micro-Pillar Subjected to Rotational Vibration.

The steady streaming (SS) phenomenon is gaining increased attention in the microfluidics community, because it can generate net mass flow from zero-mean vibration. We developed numerical simulation and experimental measurement tools to analyze this vibration-induced flow, which has been challenging due to its unsteady nature. The validity of these analysis methods is confirmed by comparing the three-dimensional (3D) flow field and the resulting particle trajectories induced around a cylindrical micro-pillar under circular vibration. In the numerical modeling, we directly solved the flow in the Lagrangian frame so that the substrate with a micro-pillar becomes stationary, and the results were converted to a stationary Eulerian frame to compare with the experimental results. The present approach enables us to avoid the introduction of a moving boundary or infinitesimal perturbation approximation. The flow field obtained by the micron-resolution particle image velocimetry (micro-PIV) measurement supported the three-dimensionality observed in the numerical results, which could be important for controlling the mass transport and manipulating particulate objects in microfluidic systems.

Impact of modular organization on dynamical richness in cortical networks.

As in many naturally formed networks, the brain exhibits an inherent modular architecture that is the basis of its rich operability, robustness, and integration-segregation capacity. However, the mechanisms that allow spatially segregated neuronal assemblies to swiftly change from localized to global activity remain unclear. Here, we integrate microfabrication technology with in vitro cortical networks to investigate the dynamical repertoire and functional traits of four interconnected neuronal modules. We show that the coupling among modules is central. The highest dynamical richness of the network emerges at a critical connectivity at the verge of physical disconnection. Stronger coupling leads to a persistently coherent activity among the modules, while weaker coupling precipitates the activity to be localized solely within the modules. An in silico modeling of the experiments reveals that the advent of coherence is mediated by a trade-off between connectivity and subquorum firing, a mechanism flexible enough to allow for the coexistence of both segregated and integrated activities. Our results unveil a new functional advantage of modular organization in complex networks of nonlinear units.

Intelligent Image-Activated Cell Sorting.

A fundamental challenge of biology is to understand the vast heterogeneity of cells, particularly how cellular composition, structure, and morphology are linked to cellular physiology. Unfortunately, conventional technologies are limited in uncovering these relations. We present a machine-intelligence technology based on a radically different architecture that realizes real-time image-based intelligent cell sorting at an unprecedented rate. This technology, which we refer to as intelligent image-activated cell sorting, integrates high-throughput cell microscopy, focusing, and sorting on a hybrid software-hardware data-management infrastructure, enabling real-time automated operation for data acquisition, data processing, decision-making, and actuation. We use it to demonstrate real-time sorting of microalgal and blood cells based on intracellular protein localization and cell-cell interaction from large heterogeneous populations for studying photosynthesis and atherothrombosis, respectively. The technology is highly versatile and expected to enable machine-based scientific discovery in biological, pharmaceutical, and medical sciences.

Microfluidic single-particle chemical analyzer with dual-comb coherent Raman spectroscopy.

Label-free particle analysis is a powerful tool in chemical, pharmaceutical, and cosmetic industries as well as in basic sciences, but its throughput is significantly lower than that of fluorescence-based counterparts. Here we present a label-free single-particle analyzer based on broadband dual-comb coherent Raman scattering spectroscopy operating at a spectroscopic scan rate of 10 kHz. As a proof-of-concept demonstration, we perform broadband coherent anti-Stokes Raman scattering measurements of polystyrene microparticles flowing on an acoustofluidic chip at a high throughput of >1000 particles per second. This high-throughput label-free particle analyzer has the potential for high-precision statistical analysis of a large number of microparticles including biological cells.

Dependence and Homeostasis of Membrane Impedance on Cell Morphology in Cultured Hippocampal Neurons.

The electrical impedance of cell membranes is important for excitable cells, such as neurons, because it strongly influences the amount of membrane potential change upon a flow of ionic current across the membrane. Here, we report on an investigation of how neuronal morphology affects membrane impedance of cultured hippocampal neurons. Microfabricated substrates with patterned scaffolding molecules were used to restrict the neurite growth of hippocampal neurons, and the impedance was measured via whole-cell patch-clamp recording under the inhibition of voltage-dependent ion channels. Membrane impedance was found to depend inversely on the dendrite length and soma area, as would be expected from the fact that its electrical property is equivalent to a parallel RC circuit. Moreover, we found that in biological neurons, the membrane impedance is homeostatically regulated to impede changes in the membrane area. The findings provide direct evidence on cell-autonomous regulation of neuronal impedance and pave the way towards elucidating the mechanism responsible for the resilience of biological neuronal networks.

A surgical simulator for peeling the inner limiting membrane during wet conditions.

The present study was performed to establish a novel ocular surgery simulator for training in peeling of the inner limited membrane (ILM). This simulator included a next-generation artificial ILM with mechanical properties similar to the natural ILM that could be peeled underwater in the same manner as in actual surgery. An artificial eye consisting of a fundus and eyeball parts was fabricated. The artificial eye was installed in the eye surgery simulator. The fundus part was mounted in the eyeball, which consisted of an artificial sclera, retina, and ILM. To measure the thickness of the fabricated ILM on the artificial retina, we calculated the distance of the step height as the thickness of the artificial ILM. Two experienced ophthalmologists then assessed the fabricated ILM by sensory evaluation. The minimum thickness of the artificial ILM was 1.9 ± 0.3 µm (n = 3). We were able to perform the peeling task with the ILM in water. Based on the sensory evaluation, an ILM with a minimum thickness and 1000 degrees of polymerization was suitable for training. We installed the eye model on an ocular surgery simulator, which allowed for the performance of a sequence of operations similar to ILM peeling. In conclusion, we developed a novel ocular surgery simulator for ILM peeling. The artificial ILM was peeled underwater in the same manner as in an actual operation.

Three-Dimensional Blood Vessel Model with Temperature-Indicating Function for Evaluation of Thermal Damage during Surgery.

Surgical simulators have recently attracted attention because they enable the evaluation of the surgical skills of medical doctors and the performance of medical devices. However, thermal damage to the human body during surgery is difficult to evaluate using conventional surgical simulators. In this study, we propose a functional surgical model with a temperature-indicating function for the evaluation of thermal damage during surgery. The simulator is made of a composite material of polydimethylsiloxane and a thermochromic dye, which produces an irreversible color change as the temperature increases. Using this material, we fabricated a three-dimensional blood vessel model using the lost-wax process. We succeeded in fabricating a renal vessel model for simulation of catheter ablation. Increases in the temperature of the materials can be measured by image analysis of their color change. The maximum measurement error of the temperature was approximately -1.6 °C/+2.4 °C within the range of 60 °C to 100 °C.

On-chip cell sorting by high-speed local-flow control using dual membrane pumps.

Although researchers have proposed various methods of on-chip cell sorting, high-throughput sorting of large cells remains hampered by the difficulty of controlling high-speed flow over a wide sorting area. To overcome this problem, we proposed high-speed local-flow control using dual membrane pumps driven by piezoelectric actuators placed on the outside of a microfluidic chip in this paper. We evaluated the controllability of shifting the flow profile by the local-flow. The results indicated that we could sort large cells up to approximately 150 µm in size with an equivalent throughput of 31 kHz. Because our method can control the flow profiles, it is applicable not only to large cells but also to small cells. The cell-sorting efficacy of the proposed method was experimentally evaluated on Euglena gracilis NIES-48 (E. gracilis) cells as large target cells and GCIY-EGFP (GCIY) cells derived from a gastric cancer cell line as small target cells. In E. gracilis cells sorting, the throughput is 23 kHz with a 92.8% success rate, 95.8% purity, and 90.8% cell viability. In GCIY sorting, the throughput is 11 kHz with a 97.8% success rate, 98.9% purity, and 90.7% cell viability. These results confirm that the proposed method sorts differently sized cells with high throughput and hence, overcomes the throughput-size trade-off that exists in conventional on-chip cell sorters.

The first case of feline sinonasal aspergillosis due to Aspergillus fischeri in Japan.

Feline upper respiratory tract infection due to Aspergillus spp. is considered an emerging disease, with the number of reported cases continuing to rise. In this study, we report the first case of feline sinonasal aspergillosis caused by Aspergillus fischeri in Japan. The patient presented after 2 months of progressive facial deformity around the nose and nasal discharge. The isolate from this case was susceptible to itraconazole (ITZ), voriconazole and micafungin, but was resistant to amphotericine B. However, the infected cat died approximately 1 month after referral, despite treatment for 12 days ITZ administered orally at 10 mg/kg.

[Present status and problems of laparoscopic surgery for colorectal cancer, especially indications and limitations].

Ten years have passed since laparoscopic surgery for colorectal cancer was performed for the first time in Japan. Health insurance has covered laparoscopic surgery for every stage of colorectal cancer since April 2002, indicating that this method will become an established operative procedure in the 21st century. As lymph node dissection is performed not only in D1 or D1 + alpha but also in D2 or D3, this method is being used in advanced as well as early cancers. When extensive colorectal resection with appropriate lymph node dissection is performed in laparoscopic surgery, the laparoscopic mobilization of the colon and rectum and lymph node dissection are essential points, which require understanding of the anatomic characteristics of the colon and rectum. It is generally recognized that there is no difference in D3 lymph node dissection except for no. 223 and in lateral lymph node dissection between this method and the conventional method. However, this method involves various problems such as intraoperative accidents, difficulties in lymph node dissection and rectal exfoliation and excision, cost-effect issues, technical problems, port site recurrences, and long-term prognosis. The most decisive factor in the future development of this method is the concern about long-term prognosis. The results of a randomized controlled trial conducted in the USA/Europe will have considerable effect in determining the indications for this method. Care should be taken not to expand the indications for laparoscopic surgery in the absence of skilled techniques.