Optogenetic control of gut movements reveals peristaltic wave-mediated induction of cloacal contractions and reactivation of impaired gut motility.

Gut peristalsis, recognized as a wave-like progression along the anterior-posterior gut axis, plays a pivotal role in the transportation, digestion, and absorption of ingested materials. The embryonic gut, which has not experienced ingested materials, undergoes peristalsis offering a powerful model for studying the intrinsic mechanisms underlying the gut motility. It has previously been shown in chicken embryos that acute contractions of the cloaca (an anus-like structure) located at the posterior end of the hindgut are tightly coupled with the arrival of hindgut-derived waves. To further scrutinize the interactions between hindgut and cloaca, we here developed an optogenetic method that produced artificial waves in the hindgut. A variant form of channelrhodopsin-2 (ChR2(D156C)), permitting extremely large photocurrents, was expressed in the muscle component of the hindgut of chicken embryos using Tol2-mediated gene transfer and in ovo electroporation techniques. The D156C-expressing hindgut responded efficiently to local pulses of blue light: local contractions emerge at an ectopic site in the hindgut, which were followed by peristaltic waves that reached to the endpoint of the hindgut. Markedly, the arrival of the optogenetically induced waves caused concomitant contractions of the cloaca, revealing that the hindgut-cloaca coordination is mediated by signals triggered by peristaltic waves. Moreover, a cloaca undergoing pharmacologically provoked aberrant contractions could respond to pulsed blue light irradiation. Together, the optogenetic technology developed in this study for inducing gut peristalsis paves the way to study the gut movement and also to explore therapeutic methodology for peristaltic disorders.

Newly raised anti-c-Kit antibody visualizes morphology of interstitial cells of Cajal in the developing gut of chicken embryos.

The gut peristaltic movement, a wave-like propagation of a local contraction, is important for the transportation and digestion of ingested materials. Among three types of cells, the enteric nervous system (ENS), smooth muscle cells, and interstitial cells of Cajal (ICCs), the ICCs have been thought to act as a pacemaker, and therefore it is important to decipher the cellular functions of ICCs to further our understanding of gut peristalsis. c-Kit, a tyrosine kinase receptor, has widely been used as a marker for ICCs. Most studies with ICCs have been conducted in mammals using commercially available anti-c-Kit antibody. Recently, the chicken embryonic gut has emerged as a powerful model to study gut peristalsis. However, since the anti-c-Kit antibody for mammals does not work for chickens, cellular mechanisms by which ICCs are regulated have largely been unexplored. Here, we report a newly raised polyclonal antibody against the chicken c-Kit protein. The specificity of the antibody was validated by both western blotting analyses and immunocytochemistry. Co-immunostaining with the new antibody and anti-α smooth muscle actin (αSMA) antibody successfully visualized ICCs in the chicken developing hindgut in the circular muscle and longitudinal muscle layers. As previously shown in mice, common progenitors of ICCs and smooth muscle cells at early stages were double positive for αSMA and c-Kit, and at later stages, differentiated ICCs and smooth muscle cells exhibited only c-Kit and αSMA, respectively. A novel ICC population was also found that radially extended from the submucosal layer to the circular muscle layer. Furthermore, the new antibody delineated individual ICCs in a cleared hindgut. The antibody newly developed in this study will facilitate the study of peristaltic movement in chicken embryos.

Characterization of Extra-Cellular Vesicle Dielectrophoresis and Estimation of Its Electric Properties.

Dielectrophoresis (DEP) refers to a type of electrical motion of dielectric particles. Because DEP is caused by particle polarization, it has been utilized to characterize particles. This study investigated the DEP of three types of exosomes, namely bovine milk, human breast milk, and human breast cancer exosomes. Exosomes are kinds of extracellular vesicles. The crossover frequencies of the exosomes were determined by direct observation of their DEPs. Consequently, bovine and human milk exosomes showed similar DEP properties, whereas the cancer exosomes were significantly different from the others. The membrane capacitance and conductivity of the exosomes were estimated using determined values. A significant difference was observed between bovine and human milk exosomes on their membrane capacitance. It was revealed that the membrane capacitances of human breast milk and human breast cancer exosomes were almost identical to those of their host cells and the conductivity of the exosomes were much lower than that of the host cell. Based on these results, DEP separation of the human breast milk and cancer exosomes was demonstrated. These results imply that DEP can be utilized to separate and identify cancer exosomes rapidly. Additionally, our method can be utilized to estimate the electric property of other types of extracellular vesicles.

Distribution Map of Peristaltic Waves in the Chicken Embryonic Gut Reveals Importance of Enteric Nervous System and Inter-Region Cross Talks Along the Gut Axis.

Gut peristaltic movements recognized as the wave-like propagation of a local contraction are crucial for effective transportation and digestion/absorption of ingested materials. Although the physiology of gut peristalsis has been well studied in adults, it remains largely unexplored how the cellular functions underlying these coordinated tissue movements are established along the rostral-caudal gut axis during development. The chicken embryonic gut serves as an excellent experimental model for elucidating the endogenous potential and regulation of these cells since peristalsis occurs even though no ingested material is present in the moving gut. By combining video-recordings and kymography, we provide a spatial map of peristaltic movements along the entire gut posterior to the duodenum: midgut (jejunum and ileum), hindgut, caecum, and cloaca. Since the majority of waves propagate bidirectionally at least until embryonic day 12 (E12), the sites of origin of peristaltic waves (OPWs) can unambiguously be detected in the kymograph. The spatial distribution map of OPWs has revealed that OPWs become progressively confined to specific regions/zones along the gut axis during development by E12. Ablating the enteric nervous system (ENS) or blocking its activity by tetrodotoxin perturb the distribution patterns of OPWs along the gut tract. These manipulations have also resulted in a failure of transportation of inter-luminally injected ink. Finally, we have discovered a functional coupling of the endpoint of hindgut with the cloaca. When surgically separated, the cloaca ceases its acute contractions that would normally occur concomitantly with the peristaltic rhythm of the hindgut. Our findings shed light on the intrinsic regulations of gut peristalsis, including unprecedented ENS contribution and inter-region cross talk along the gut axis.

Cis-acting mutation affecting GJA5 transcription is underlying the Melanotic within-feather pigmentation pattern in chickens.

Melanotic (Ml) is a mutation in chickens that extends black (eumelanin) pigmentation in normally brown or red (pheomelanin) areas, thus affecting multiple within-feather patterns [J. W. Moore, J. R. Smyth Jr, J. Hered. 62, 215-219 (1971)]. In the present study, linkage mapping using a back-cross between Dark Cornish (Ml/Ml) and Partridge Plymouth Rock (ml+/ml+ ) chickens assigned Ml to an 820-kb region on chromosome 1. Identity-by-descent mapping, via whole-genome sequencing and diagnostic tests using a diverse set of chickens, refined the localization to the genomic region harboring GJA5 encoding gap-junction protein 5 (alias connexin 40) previously associated with pigmentation patterns in zebrafish. An insertion/deletion polymorphism located in the vicinity of the GJA5 promoter region was identified as the candidate causal mutation. Four different GJA5 transcripts were found to be expressed in feather follicles and at least two showed differential expression between genotypes. The results showed that Melanotic constitutes a cis-acting regulatory mutation affecting GJA5 expression. A recent study established the melanocortin-1 receptor (MC1R) locus and the interaction between the MC1R receptor and its antagonist agouti-signaling protein as the primary mechanism underlying variation in within-feather pigmentation patterns in chickens. The present study advances understanding the mechanisms underlying variation in plumage color in birds because it demonstrates that the activity of connexin 40/GJA5 can modulate the periodic pigmentation patterns within individual feathers.

Rapid and low-cost amplicon visualization for nucleic acid amplification tests using magnetic microbeads.

This study presents a rapid and low-cost amplicon detection method in which amplicons are attached to magnetic microbeads, suspended in deionized water, and subjected to a magnetic field on a hydrophilic surface resulting in the circular agglomeration of amplicon-conjugated microbeads, visible to the naked eye.

Avian Pigment Pattern Formation: Developmental Control of Macro- (Across the Body) and Micro- (Within a Feather) Level of Pigment Patterns.

Animal color patterns are of interest to many fields, such as developmental biology, evolutionary biology, ethology, mathematical biology, bio-mimetics, etc. The skin provides easy access to experimentation and analysis enabling the developmental pigment patterning process to be analyzed at the cellular and molecular level. Studies in animals with distinct pigment patterns (such as zebrafish, horse, feline, etc.) have revealed some genetic information underlying color pattern formation. Yet, how the complex pigment patterns in diverse avian species are established remains an open question. Here we summarize recent progress. Avian plumage shows color patterns occurring at different spatial levels. The two main levels are macro- (across the body) and micro- (within a feather) pigment patterns. At the cellular level, colors are mainly produced by melanocytes generating eumelanin (black) and pheomelanin (yellow, orange). These melanin-based patterns are regulated by melanocyte migration, differentiation, cell death, and/or interaction with neighboring skin cells. In addition, non-melanin chemical pigments and structural colors add more colors to the available palette in different cell types or skin regions. We discuss classic and recent tissue transplantation experiments that explore the avian pigment patterning process and some potential molecular mechanisms. We find color patterns can be controlled autonomously by melanocytes but also non-autonomously by dermal cells. Complex plumage color patterns are generated by the combination of these multi-scale patterning mechanisms. These interactions can be further modulated by environmental factors such as sex hormones, which generate striking sexual dimorphic colors in avian integuments and can also be influenced by seasons and aging.

Simple microfluidic device for detecting the negative dielectrophoresis of DNA labeled microbeads.

We propose a new microfluidic device that can be used to determine the change in the negative dielectrophoresis (n-DEP) of dielectric microbeads when a small amount of DNA is attached to them. We previously proposed a DNA detection method based on changes in the DEP of microbeads induced by the attachment of DNA. When target DNA is attached to the microbeads having n-DEP property, the DEP changes from negative to positive. This occurs because electric charges of the DNA increase the surface conductance of the microbeads. Thus, only the DNA-labeled microbeads are attracted to a microelectrode by positive DEP. The trapped DNA-labeled microbeads can be counted by dielectrophoretic impedance measurements. A large amount of DNA (approximately 105 DNA molecules) is required to change the DEP from negative to positive. Even though this method can be combined with DNA amplification, reducing the amount of DNA required can help us to shorten the reaction time. In this study, we aimed to detect DNA less than 105 DNA molecules by determining the change in the n-DEP change. To achieve this, we proposed a simple microfluidic device consisting of a single microchannel and a single pair of microelectrodes. Numerical simulations revealed that the device can identify the slight change in the n-DEP of the microbeads corresponding to the attachment of a small amount of DNA. In practical experiments, the fabricated device distinguished 10-1000 DNA molecules per microbead. This method represents a fast and easy method of DNA detection when combined with DNA amplification techniques.

Correlation between the Carbon Nanotube Growth Rate and Byproducts in Antenna-Type Remote Plasma Chemical Vapor Deposition Observed by Vacuum Ultraviolet Absorption Spectroscopy.

For sp2 or sp3 carbon material growth, it is important to investigate the precursors or intermediates just before growth. In this study, the density of ethylene (C2 H4 ) outside the plasma discharge space and just before reaching the carbon nanotube (CNT) growth region is investigated by vacuum ultraviolet absorption spectroscopy for plasma discharge in an antenna-type remote plasma chemical vapor deposition with a CH4 /H2 system, with which the growth of very long (≈0.5 cm) CNT forests is achieved. Single-wall CNT forests have the potential for application as electrodes in battery cells, vertical wiring for high current applications, and thermal interface materials. It is observed that the plasma discharge decomposes the CH4 source gas and forms C2 Hx species, which reversibly reform to C2 H4 in the plasma-off state. In addition, the density of the formed C2 H4 has a strong correlation to the CNT growth rate. Therefore, the C2 H4 density is a good indicator of the density of C2 Hx species for CNT growth in the CH4 /H2 plasma system.

Instructive role of melanocytes during pigment pattern formation of the avian skin.

Animal skin pigment patterns are excellent models to study the mechanism of biological self-organization. Theoretical approaches developed mathematical models of pigment patterning and molecular genetics have brought progress; however, the responsible cellular mechanism is not fully understood. One long unsolved controversy is whether the patterning information is autonomously determined by melanocytes or nonautonomously determined from the environment. Here, we transplanted purified melanocytes and demonstrated that melanocytes could form periodic pigment patterns cell autonomously. Results of heterospecific transplantation among quail strains are consistent with this finding. Further, we observe that developing melanocytes directly connect with each other via filopodia to form a network in culture and in vivo. This melanocyte network is reminiscent of zebrafish pigment cell networks, where connexin is implicated in stripe formation via genetic studies. Indeed, we found connexin40 (cx40) present on developing melanocytes in birds. Stripe patterns can form in quail skin explant cultures. Several calcium channel modulators can enhance or suppress pigmentation globally, but a gap junction inhibitor can change stripe patterning. Most interestingly, in ovo, misexpression of dominant negative cx40 expands the black region, while overexpression of cx40 expands the yellow region. Subsequently, melanocytes instruct adjacent dermal cells to express agouti signaling protein (ASIP), the regulatory factor for pigment switching, which promotes pheomelanin production. Thus, we demonstrate Japanese quail melanocytes have an autonomous periodic patterning role during body pigment stripe formation. We also show dermal agouti stripes and how the coupling of melanocytes with dermal cells may confer stable and distinct pigment stripe patterns.

Turing patterning with and without a global wave.

Periodic patterning represents a fundamental process in tissue morphogenesis. In chicken dorsal skin, feather formation starts from the midline; then the morphogenetic wave propagates bilaterally, leaving a regular hexagonal array of feather germs. Yet, in vitro reconstitution showed feather germs appear simultaneously, leading to the hypothesis that the feather-forming wave results from the coupling of local Turing patterning processes with an unidentified global event. In this issue, Ho and colleagues showed such a global event in chicken feathers involves a spreading Ectodysplasin A (EDA) wave and Fibroblast Growth Factor 20 (FGF20)-cell aggregate-based mechanochemical coupling. In flightless birds, feather germs form periodically but without precise hexagonal patterning due to the lack of global wave.

Vertical-type two-dimensional hole gas diamond metal oxide semiconductor field-effect transistors.

Power semiconductor devices require low on-resistivity and high breakdown voltages simultaneously. Vertical-type metal-oxide-semiconductor field-effect transistors (MOSFETs) meet these requirements, but have been incompleteness in diamond. Here we show vertical-type p-channel diamond MOSFETs with trench structures and drain current densities equivalent to those of n-channel wide bandgap devices for complementary inverters. We use two-dimensional hole gases induced by atomic layer deposited Al2O3 for the channel and drift layers, irrespective of their crystal orientations. The source and gate are on the planar surface, the drift layer is mainly on the sidewall and the drain is the p+ substrate. The maximum drain current density exceeds 200 mA mm-1 at a 12 µm source-drain distance. On/off ratios of over eight orders of magnitude are demonstrated and the drain current reaches the lower measurement limit in the off-state at room temperature using a nitrogen-doped n-type blocking layer formed using ion implantation and epitaxial growth.

Role of Carboxyl and Amine Termination on a Boron-Doped Diamond Solution Gate Field Effect Transistor (SGFET) for pH Sensing.

In this paper, we report on the effect of carboxyl- and amine terminations on a boron-doped diamond surface (BDD) in relation to pH sensitivity. Carboxyl termination was achieved by anodization oxidation in Carmody buffer solution (pH 7). The carboxyl-terminated diamond surface was exposed to nitrogen radicals to generate an amine-terminated surface. The pH sensitivity of the carboxyl- and amine-terminated surfaces was measured from pH 2 to pH 12. The pH sensitivities of the carboxyl-terminated surface at low and high pH are 45 and 3 mV/pH, respectively. The pH sensitivity after amine termination is significantly higher—the pH sensitivities at low and high pH are 65 and 24 mV/pH, respectively. We find that the negatively-charged surface properties of the carboxyl-terminated surface due to ionization of ⁻COOH causes very low pH detection in the high pH region (pH 7⁻12). In the case of the amine-terminated surface, the surface properties are interchangeable in both acidic and basic solutions; therefore, we observed pH detection at both low and high pH regions. The results presented here may provide molecular-level understanding of surface properties with charged ions in pH solutions. The understanding of these surface terminations on BDD substrate may be useful to design diamond-based biosensors.

Durability-enhanced two-dimensional hole gas of C-H diamond surface for complementary power inverter applications.

Complementary power field effect transistors (FETs) based on wide bandgap materials not only provide high-voltage switching capability with the reduction of on-resistance and switching losses, but also enable a smart inverter system by the dramatic simplification of external circuits. However, p-channel power FETs with equivalent performance to those of n-channel FETs are not obtained in any wide bandgap material other than diamond. Here we show that a breakdown voltage of more than 1600 V has been obtained in a diamond metal-oxide-semiconductor (MOS) FET with a p-channel based on a two-dimensional hole gas (2DHG). Atomic layer deposited (ALD) Al2O3 induces the 2DHG ubiquitously on a hydrogen-terminated (C-H) diamond surface and also acts as both gate insulator and passivation layer. The high voltage performance is equivalent to that of state-of-the-art SiC planar n-channel FETs and AlGaN/GaN FETs. The drain current density in the on-state is also comparable to that of these two FETs with similar device size and VB.

Pigment pattern formation by contact-dependent depolarization.

Although recent experimental studies have suggested that the interactions among the pigment cells play a key role in the skin pattern formation, details of the mechanism remain largely unknown. By using an in vitro cell culture system, we have detected interactions between the two pigment cell types, melanophores and xanthophores, in the zebrafish skin. During primary culture, the melanophore membrane transiently depolarizes when contacted with the dendrites of a xanthophore. This depolarization triggers melanophore migration to avoid further contact with the xanthophores. Cell depolarization and repulsive movement were not observed in pigment cells with the jaguar mutant, which shows defective segregation of melanophores and xanthophores. The depolarization-repulsion of wild-type pigment cells may explain the pigment cell behaviors generating the stripe pattern of zebrafish.