Modulatory Effects on Laminar Neural Activity Induced by Near-Infrared Light Stimulation with a Continuous Waveform to the Mouse Inferior Colliculus In Vivo.

Infrared neural stimulation (INS) is a promising area of interest for the clinical application of a neuromodulation method. This is in part because of its low invasiveness, whereby INS modulates the activity of the neural tissue mainly through temperature changes. Additionally, INS may provide localized brain stimulation with less tissue damage. The inferior colliculus (IC) is a crucial auditory relay nucleus and a potential target for clinical application of INS to treat auditory diseases and develop artificial hearing devices. Here, using continuous INS with low to high-power density, we demonstrate the laminar modulation of neural activity in the mouse IC in the presence and absence of sound. We investigated stimulation parameters of INS to effectively modulate the neural activity in a facilitatory or inhibitory manner. A mathematical model of INS-driven brain tissue was first simulated, temperature distributions were numerically estimated, and stimulus parameters were selected from the simulation results. Subsequently, INS was administered to the IC of anesthetized mice, and the modulation effect on the neural activity was measured using an electrophysiological approach. We found that the modulatory effect of INS on the spontaneous neural activity was bidirectional between facilitatory and inhibitory effects. The modulatory effect on sound-evoked responses produced only an inhibitory effect to all examined stimulus intensities. Thus, this study provides important physiological evidence on the response properties of IC neurons to INS. Overall, INS can be used for the development of new therapies for neurological disorders and functional support devices for auditory central processing.

Transplantation of Human Embryonic Stem Cell-Derived Pericyte-Like Cells Transduced with Basic Fibroblast Growth Factor Promotes Angiogenic Recovery in Mice with Severe Chronic Hindlimb Ischemia.

Critical limb ischemia (CLI) is a state of severe peripheral artery disease, with no effective treatment. Cell therapy has been investigated as a therapeutic tool for CLI, and pericytes are promising therapeutic candidates based on their angiogenic properties. We firstly generated highly proliferative and immunosuppressive pericyte-like cells from embryonic stem (ES) cells. In order to enhance the angiogenic potential, we transduced the basic fibroblast growth factor (bFGF) gene into the pericyte-like cells and found a significant enhancement of angiogenesis in a Matrigel plug assay. Furthermore, we evaluated the bFGF-expressing pericyte-like cells in the previously established chronic hindlimb ischemia model in which bone marrow-derived MSCs were not effective. As a result, bFGF-expressing pericyte-like cells significantly improved blood flow in both laser Doppler perfusion imaging (LDPI) and dynamic contrast-enhanced MRI (DCE-MRI). These findings suggest that bFGF-expressing pericyte-like cells differentiated from ES cells may be a therapeutic candidate for CLI.

Turtle skull development unveils a molecular basis for amniote cranial diversity.

Amniote skulls display diverse architectural patterns including remarkable variations in the number of temporal arches surrounding the upper and lower temporal fenestrae. However, the cellular and molecular basis underlying this diversification remains elusive. Turtles are a useful model to understand skull diversity due to the presence of secondarily closed temporal fenestrae and different extents of temporal emarginations (marginal reduction of dermal bones). Here, we analyzed embryos of three turtle species with varying degrees of temporal emargination and identified shared widespread coexpression of upstream osteogenic genes Msx2 and Runx2 and species-specific expression of more downstream osteogenic genes Sp7 and Sparc in the head. Further analysis of representative amniote embryos revealed differential expression patterns of osteogenic genes in the temporal region, suggesting that the spatiotemporal regulation of Msx2, Runx2, and Sp7 distinguishes the temporal skull morphology among amniotes. Moreover, the presence of Msx2- and/or Runx2-positive temporal mesenchyme with osteogenic potential may have contributed to their extremely diverse cranial morphology in reptiles.

Low-Cost Electroencephalographic Recording System Combined with a Millimeter-Sized Coil to Transcranially Stimulate the Mouse Brain In Vivo.

A low-cost electroencephalographic (EEG) recording system is proposed here to drive transcranial magnetic stimulation (TMS) of the mouse brain in vivo, utilizing a millimeter-sized coil. Using conventional screw electrodes combined with a custom-made, flexible, multielectrode array substrate, multi-site recording can be carried out from the mouse brain. In addition, we explain how a millimeter-sized coil is produced using low-cost equipment usually found in laboratories. Practical procedures for fabricating the flexible multielectrode array substrate and the surgical implantation technique for screw electrodes are also presented, which are necessary to produce low-noise EEG signals. Although the methodology is useful for recording from the brain of any small animal, the present report focuses on electrode implementation in an anesthetized mouse skull. Furthermore, this method can be easily extended to an awake small animal that is connected with tethered cables via a common adapter and fixed with a TMS device to the head during recording.The present version of the EEG-TMS system, which can include a maximum of 32 EEG channels (a device with 16 channels is presented as an example with fewer channels) and one TMS channel device, is described. Additionally, typical results obtained by the application of the EEG-TMS system to anesthetized mice are briefly reported.

Creating morphological diversity in reptilian temporal skull region: A review of potential developmental mechanisms.

Reptilian skull morphology is highly diverse and broadly categorized into three categories based on the number and position of the temporal fenestrations: anapsid, synapsid, and diapsid. According to recent phylogenetic analysis, temporal fenestrations evolved twice independently in amniotes, once in Synapsida and once in Diapsida. Although functional aspects underlying the evolution of tetrapod temporal fenestrations have been well investigated, few studies have investigated the developmental mechanisms responsible for differences in the pattern of temporal skull region. To determine what these mechanisms might be, we first examined how the five temporal bones develop by comparing embryonic cranial osteogenesis between representative extant reptilian species. The pattern of temporal skull region may depend on differences in temporal bone growth rate and growth direction during ontogeny. Next, we compared the histogenesis patterns and the expression of two key osteogenic genes, Runx2 and Msx2, in the temporal region of the representative reptilian embryos. Our comparative analyses suggest that the embryonic histological condition of the domain where temporal fenestrations would form predicts temporal skull morphology in adults and regulatory modifications of Runx2 and Msx2 expression in osteogenic mesenchymal precursor cells are likely involved in generating morphological diversity in the temporal skull region of reptiles.

Efficient four-wave mixing wavelength conversion in a hybrid silicon slot and polymer microring resonator.

We present a silicon slot microring resonator for efficient frequency conversion via four-wave mixing (FWM). The slot consists of a narrow silicon waveguide pair with a gap of 80 nm, which is filled with a nonlinear optical polymer. The group velocity dispersion for the microring is controlled by engineering the geometry of the slot structure. Because of the large buildup factor of the slot microring, an FWM conversion efficiency of -27.4 dB is achieved with an optical pump power of less than 1.0 mW. From the measured power dependence of FWM generation, a nonlinear refractive index coefficient of 1.31 × 10-17 m2 W-1 is obtained at a wavelength of 1562 nm. This work presents a hybrid silicon slot and polymer microring as a potential nonlinear device for applications in integrated photonic devices.

Efficient silicon and side-cladding waveguide modulator with electro-optic polymer.

Efficient electro-optic (EO) modulation can be generated in the hybrid silicon modulator with EO polymer in the form of an in-plane coplanar waveguide and electrode structure. Strong confinement of the optical field in the hybrid structure is critical to performing efficient electric poling and modulation of the EO polymer. The waveguide consists of silica-based side claddings and an EO core for increasing the integral of the optical field and the overlap interaction between the optical field and the modulated electric field within the EO polymer. We discuss in detail the volume resistivity dependence of the efficiency of electric poling and modulation for various side-cladding materials. In a Mach-Zehnder interferometer modulator, the measured half-wave-voltage length product (VπL) is 1.9 V·cm at an optical communication wavelength of 1,550 nm under the TE optical mode operation. The high-speed signaling of the device is demonstrated by generating on-off-keying transmission at signal rates up to 52 Gbit/s with a Q factor of 6.1 at a drive voltage of 2.0 Vpp.

A novel model of chronic limb ischemia to therapeutically evaluate the angiogenic effects of drug candidates.

Critical limb ischemia (CLI) is a severe state of peripheral artery disease with high unmet clinical needs. Further, there are no effective treatment options for patients with CLI. Based on preclinical study results, predicting the clinical efficacy of CLI treatments is typically difficult because conventional hindlimb ischemia (HLI) rodent models display spontaneous recovery from ischemia, which is not observed in patients with CLI. Therefore, we aimed to develop a novel chronic and severe HLI model to properly evaluate the therapeutic effects of drug candidates for CLI. Severe HLI mice (Type-N) were generated by increasing the excised area of blood vessels in a hindlimb of NOG mice. Immunohistochemistry and gene expression analysis at 9 wk after the Type-N operation revealed that the ischemic limb was in a steady state with impaired angiogenesis, like that observed in patients with CLI. We did selection of chronic Type-N mice based on the number of necrotic nails and blood flow rate at 2 wk after surgery because some Type-N mice showed mild symptoms. Therapeutic treatment with cilostazol, which is used for intermittent claudication, did not restore blood flow in chronic Type-N mice. In contrast, therapeutic transplantation of pericytes and vascular endothelial cells, which can form new blood vessels in vivo, significantly improved blood flow in a subset of Type-N mice. These findings suggest that this novel chronic and severe HLI model may be a valuable standard animal model for therapeutic evaluation of the angiogenic effects of CLI drug candidates.NEW & NOTEWORTHY We developed a chronic and severe hindlimb ischemia (HLI) mouse model for preclinical research on critical limb ischemia (CLI). This model partially reflects human CLI pathology in that it does not show spontaneous restoration of blood flow or expression of angiogenic genes in the ischemic limb. This novel model may be valuable for therapeutic evaluation of the angiogenic effects of CLI drug candidates.

New tools and functions in data-out activities at Protein Data Bank Japan (PDBj).

The Protein Data Bank Japan (PDBj), a member of the worldwide Protein Data Bank (wwPDB), accepts and processes the deposited data of experimentally determined biological macromolecular structures. In addition to archiving the PDB data in collaboration with the other wwPDB partners, PDBj also provides a wide range of original and unique services and tools, which are continuously improved and updated. Here, we report the new RDB PDBj Mine 2, the WebGL molecular viewer Molmil, the ProMode-Elastic server for normal mode analysis, a virtual reality system for the eF-site protein electrostatic molecular surfaces, the extensions of the Omokage search for molecular shape similarity, and the integration of PDBj and BMRB searches.

Low driving voltage Mach-Zehnder interference modulator constructed from an electro-optic polymer on ultra-thin silicon with a broadband operation.

An electro-optic (EO) polymer waveguide using an ultra-thin silicon hybrid has been designed and fabricated. The silicon core has the thickness of 50 nm and a width of 5 µm. The waveguide was completed after covering the cladding with the high temperature stable EO polymer. We have demonstrated a low half-wavelength voltage of 0.9 V at the wavelength of 1.55 µm by using a Mach-Zehnder interference modulator with TM mode operation. The measured modulation corresponded to an effective in-device EO coefficient of 165 pm/V. By utilizing the traveling-wave electrode on the modulator the high-frequency response was tested up to 40 GHz. The 3 dB modulation bandwidth was measured to be 23 GHz. In addition, the high frequency sideband spectral measurement revealed that a linear response of the modulation index against the RF power was confirmed up to 40 GHz signal.

Contributions of humic substances to the dissolved organic carbon pool in wetlands from different climates.

Wetlands are an important source of DOM. However, the quantity and quality of wetlands' DOM from various climatic regions have not been studied comprehensively. The relationship between the concentrations of DOM (DOC), humic substances (HS) and non-humic substances (NHS) in wetland associated sloughs, streams and rivers, in cool temperate (Hokkaido, Japan), sub-tropical (Florida, USA), and tropical (Sarawak, Malaysia) regions was investigated. The DOC ranged from 1.0 to 15.6 mg CL(-1) in Hokkaido, 6.0-24.4 mg CL(-1) in Florida, and 18.9-75.3 mg CL(-1) in Sarawak, respectively. The relationship between DOC and HS concentrations for the whole sample set was regressed to a primary function with y-intercept of zero (P<0.005) and a slope value of 0.841. A similar correlation was observed between DOC and NHS concentrations, with a smaller slope value of 0.159. However, the correlation coefficient of the latter was much larger when the data was regressed to a logarithmic curve. These observations suggest the presence of a general tendency that the increased DOC in the river waters was mainly due to the increased supply of HS from wetland soils, whereas the rate of the increase in the NHS supply has an upper limit which may be controlled by primary productivity.

The (pro)renin receptor/ATP6AP2 is essential for vacuolar H+-ATPase assembly in murine cardiomyocytes.

RATIONALE: The (pro)renin receptor [(P)RR], encoded in ATP6AP2, plays a key role in the activation of local renin-angiotensin system (RAS). A truncated form of (P)RR, termed M8.9, was also found to be associated with the vacuolar H(+)-ATPase (V-ATPase), implicating a non-RAS-related function of ATP6AP2. OBJECTIVE: We investigated the role of (P)RR/ATP6AP2 in murine cardiomyocytes. METHODS AND RESULTS: Cardiomyocyte-specific ablation of Atp6ap2 resulted in lethal heart failure; the cardiomyocytes contained RAB7- and lysosomal-associated membrane protein 2 (LAMP2)-positive multivesicular vacuoles, especially in the perinuclear regions. The myofibrils and mitochondria remained at the cell periphery. Cardiomyocyte death was accompanied by numerous autophagic vacuoles that contained undigested cellular constituents, as a result of impaired autophagic degradation. Notably, ablation of Atp6ap2 selectively suppressed expression of the V(O) subunits of V-ATPase, resulting in deacidification of the intracellular vesicles. Furthermore, the inhibition of intracellular acidification by treatment with bafilomycin A1 or chloroquine reproduced the phenotype observed for the (P)RR/ATP6AP2-deficient cardiomyocytes. CONCLUSIONS: Genetic ablation of Atp6ap2 created a loss-of-function model for V-ATPase. The gene product of ATP6AP2 is considered to act as in 2 ways: (1) as (P)RR, exerting a RAS-related function; and (2) as the V-ATPase-associated protein, exerting a non-RAS-related function that is essential for cell survival.

Role of MGAT2 and DGAT1 in the release of gut peptides after triglyceride ingestion.

Triglyceride ingestion releases gut peptides from enteroendocrine cells located in the intestinal epithelia and provides feedback regulations of gastrointestinal function. The precise mechanisms sensing lipids in the intestinal wall, however, are not well characterized. In the current study, we investigated the release of gut peptides following oral triglyceride loading in mice deficient for monoacylglycerol acyltransferase 2 (MGAT2KO) and diacylglycerol acyltransferase 1 (DGAT1KO), enzymes that sequentially re-synthesize triglyceride to secrete as chylomicron at the small intestine. In wild-type (Wt) mice, oral triglyceride loading resulted in hypertriglycemia. In addition, plasma glucose-dependent insulinotropic polypeptide (GIP), glucagon-like peptide-1 (GLP-1) and peptide YY (PYY) were significantly increased 30 min after triglyceride loading, before decaying in 2h. In MGAT2KO and DGAT1KO mice, oral triglyceride loading did not result in hypertriglycemia and the increase in GIP was significantly suppressed in both KO mouse strains. In contrast, the increases in plasma GLP-1 and PYY in both KO mouse strains were comparable to Wt mice 30 min after triglyceride loading, however, they remained elevated in DGAT1KO mice even 2h after triglyceride loading. In parallel to the changes in GLP-1 and PYY, gastric emptying was delayed after oral triglyceride loading in MGAT2KO mice comparably to Wt type mice and was further delayed in DGAT1KO mice. STC-1 and GLUTag, GLP-1-producing intestinal endocrine L-cell lines, displayed a significant level of DGAT1 activity but not MGAT activity. These findings suggest that synthesis and/or secretion of triglyceride-rich lipoproteins play an important role in the release of GIP. Moreover, DGAT1 may directly regulate the release of GLP-1 and PYY in L-cells.

Stable generation of serum- and feeder-free embryonic stem cell-derived mice with full germline-competency by using a GSK3 specific inhibitor.

C57BL/6 (B6)-derived embryonic stem (ES) cells are not widely used to generate knockout mice despite the advantage of a well-defined genetic background because of poor developmental potential. We newly established serum- and feeder-free B6 ES cells with full developmental potential by using leukemia inhibitory factor (LIF) and 6-bromoindirubin-3'-oxime (BIO), a glycogen synthase kinase-3 (GSK3) inhibitor. BIO treatment significantly increased the expression levels of 364 genes including pluripotency markers such as Nanog and Klf family. Unexpectedly, by aggregating or microinjecting those ES cells to each eight-cell-stage diploid embryo, we stably generated germline-competent ES-derived mice. Furthermore, founder mice completely derived from female XO, heterozygous, or homozygous mutant B6 ES cells were directly available for intercross breeding and phenotypic analysis. We hereby propose that serum- and feeder-free B6 ES cells stimulated with LIF plus GSK3 inhibitor are valuable for generating mouse models on B6 background.

Pax-5 is essential for kappa sterile transcription during Ig kappa chain gene rearrangement.

Pax-5 is the key regulator in B cell development. Pax-5-deficient mice show defects in B cell commitment and recombination of IgH chain gene rearrangement from DJ to VDJ. Previously, we found that Pax-5 bound to KI and KII sites, which play a crucial role in kappa-chain gene rearrangement. However, the function of Pax-5 in Ig kappa chain gene rearrangement has not been investigated. To address this issue, we newly established pre-BI cell lines expressing the pre-B cell receptor from Pax-5-deficient mice and used them in an in vitro culture system, in which kappa-chain gene rearrangement is induced by removing IL-7. By examining the Pax-5-deficient pre-BI (knockout (KO)) cells, we show in this study that, despite recombination-activating gene 1 and 2 expression, these KO cells did not rearrange the kappa-chain gene following the absence of kappa sterile transcription. Consistent with these data, fluorescent in situ hybridization analyses revealed that the J(kappa) locus in KO cells was located at the nuclear periphery as a repressive compartment. Transfection of KO cells with Pax-5 constructs indicated that the transactivation domain of Pax-5 was required for kappa sterile transcription and kappa-chain gene rearrangement. Moreover, the hormone-inducible system in KO cells demonstrated that Pax-5 directly functioned in kappa sterile transcription. These results indicate that Pax-5 is necessary for kappa sterile transcription during Ig kappa chain gene rearrangement.

EBF-regulating Pax5 transcription is enhanced by STAT5 in the early stage of B cells.

Pax5 is an essential transcription factor for B cell development, and it is reported that Pax5 expression was reduced in the IL-7 receptor (IL-7R) knockout mouse. To investigate whether signals from the IL-7R regulate Pax5 transcription, we searched the consensus sequence of signal transducers and activators of transcription (STAT) in the Pax5 promoter region, since STAT is one of the components of cytokine signal transduction. A STAT-binding motif, termed SBM, was identified at 1,118 bp upstream of the transcriptional start site, and SBM completely overlapped with the binding site for early B cell factor (EBF). STAT5 was phosphorylated in the presence of IL-7 in the IL-7-dependent preB cell line, PreBR1, and phosphorylated-STAT5 as well as EBF was found to bind to the SBM. Moreover, we also revealed STAT5 binding to SBM in PreBR1 cells by chromatin immunoprecipitation assay. Transient co-transfection of reporter genes together with expression vectors of a constitutive active form of STAT5 and EBF into NIH3T3 cells demonstrated that STAT5 enhanced EBF-regulating transcription. Our results suggest that STAT5 phosphorylated by IL-7 can directly up-regulate Pax5 transcription in early B cells.

TRANCE together with IL-7 induces pre-B cells to proliferate.

TRANCE (TNF-related activation-induced cytokine)-deficient mice completely lack osteoclasts, and develop severe osteopetrosis. These mice also show a defect in their pre-B cell differentiation. In the present study, the expression of TRANCE was examined in pre-B cell lines using flow cytometry and reverse transcription-PCR. Three pre-B cell lines, 18-81, B3P816-1, and 38B9, expressed TRANCE on their surface, and two pre-B cell lines, 7OZ/3 and NFS5, at the late pre-B cell stage, expressed it at low levels, although their mRNA expression was normal. Another pre-B cell line, 38-C-13, at the intermediate stage between pre-B and immature B cells, did not express TRANCE. The IL-7-dependent pre-B cell line PreBR, which expresses the pre-B cell receptor on the cell surface, also expressed TRANCE. When differentiation of PreBR cells was induced in vitro by removing IL-7 from cultures, TRANCE expression dropped; it was restored by the addition of IL-7, suggesting that TRANCE functions in cooperation with IL-7. To examine the function of TRANCE, we introduced the TRANCE gene into PreBR cells and established two transfectants that constitutively expressed TRANCE, even in the absence of IL-7. In these transfectants, after removal of IL-7, the number of cells that succeeded in kappa chain rearrangement was decreased to one third; and CD40 expression decreased to less than one tenth. Moreover, the percentage of cells in the S/G2/M phase was increased by 50% over the mock transfectant. These findings indicate that, before kappa chain rearrangement occurs, TRANCE together with IL-7 induces pre-B cells to proliferate and makes this rearrangement more efficient.