Our understanding of the cellular implementation of systems-level neural processes like action, thought and emotion has been limited by the availability of tools to interrogate specific classes of neural cells within intact, living brain tissue. Here we identify and develop an archaeal light-driven chloride pump (NpHR) from Natronomonas pharaonis for temporally precise optical inhibition of neural activity. NpHR allows either knockout of single action potentials, or sustained blockade of spiking. NpHR is compatible with ChR2, the previous optical excitation technology we have described, in that the two opposing probes operate at similar light powers but with well-separated action spectra. NpHR, like ChR2, functions in mammals without exogenous cofactors, and the two probes can be integrated with calcium imaging in mammalian brain tissue for bidirectional optical modulation and readout of neural activity. Likewise, NpHR and ChR2 can be targeted together to Caenorhabditis elegans muscle and cholinergic motor neurons to control locomotion bidirectionally. NpHR and ChR2 form a complete system for multimodal, high-speed, genetically targeted, all-optical interrogation of living neural circuits.

Multimodal fast optical interrogation of neural circuitry

These investigations and several others that have beenpublishedwithin recentyears compel us us to hold the view that the ratio of the vital capacity to the body length, trunk length, chest circumference,surfacearea or weight or any combination of thesemeasurements, is too variable to admit of any workable standardor normal value. On the other hand the vital capacity of each individual, after he had becomeaccustomedto the use of the spirometer,will be found to be subjectto but small variations as long as good health is maintained. Thereseems to beevidenceto show that a reductionin the vital capacityis ofen the first sign of a progressivedamageto the respiratorytissue.

The cancer cell

Photobiomodulation also known as low-level laser (or light) therapy (LLLT), has been known for almost 50 years but still has not gained widespread acceptance, largely due to uncertainty about the molecular, cellular, and tissular mechanisms of action. However, in recent years, much knowledge has been gained in this area, which will be summarized in this review. One of the most important chromophores is cytochrome c oxidase (unit IV in the mitochondrial respiratory chain), which contains both heme and copper centers and absorbs light into the near-infrared region. The leading hypothesis is that the photons dissociate inhibitory nitric oxide from the enzyme, leading to an increase in electron transport, mitochondrial membrane potential, and adenosine triphosphate production. Another hypothesis concerns light-sensitive ion channels that can be activated allowing calcium (Ca2+) to enter the cell. After the initial photon absorption events, numerous signaling pathways are activated via reactive oxygen species, cyclic AMP, NO, and Ca2+, leading to activation of transcription factors. These transcription factors can lead to increased expression of genes related to protein synthesis, cell migration and proliferation, anti-inflammatory signaling, anti-apoptotic proteins, and antioxidant enzymes. Stem cells and progenitor cells appear to be particularly susceptible to LLLT.

Proposed Mechanisms of Photobiomodulation or Low-Level Light Therapy

Photoacoustic imaging is a promising method for visualizing biological tissues with optical absorbers. This article provides an overview of the rapidly developing field of photoacoustic imaging. Photoacoustics, the physical basis of photoacoustic imaging, is analyzed briefly. The merits of photoacoustic technology, compared with optical imaging and ultrasonic imaging, are described. Various imaging techniques are also discussed, including scanning tomography, computed tomography and original detection of photoacoustic imaging. Finally, some biomedical applications of photoacoustic imaging are summarized.

Photoacoustic imaging in biomedicine

Stochastic gene expression has been implicated in a variety of cellular processes, including cell differentiation and disease. In this issue of Cell, Weinberger et al. (2005) take an integrated computational-experimental approach to study the Tat transactivation feedback loop in HIV-1 and show that fluctuations in a key regulator, Tat, can result in a phenotypic bifurcation. This phenomenon is observed in an isogenic population where individual cells display two distinct expression states corresponding to latent and productive infection by HIV-1. These findings demonstrate the importance of stochastic gene expression in molecular "decision-making."

Stochastic Gene Expression in a Lentiviral Positive Feedback Loop: HIV-1 Tat Fluctuations Drive Phenotypic Diversity

A facile pretreatment process for SEM: The use of room temperature ionic liquids (RTILs) provides an interesting method for SEM of biological specimens. We used a novel and concise method of pretreatment, excluding fixation or Au sputtering steps. Fine and smooth-textured SEM images of a wide variety of biological specimens treated in this way were observed without artefacts.

SEM observation of wet biological specimens pretreated with room-temperature ionic liquid.

Low reactive level laser therapy (LLLT) is mainly focused on the activation of intracellular or extracellular chromophore and the initiation of cellular signaling by using low power lasers. Over the past forty years, it was realized that the laser therapy had the potential to improve wound healing and reduce pain and inflammation. In recent years, the term LLLT has become widely recognized in the field of regenerative medicine. In this review, we will describe the mechanisms of action of LLLT at a cellular level and introduce the application to mesenchymal stem cells and mesenchymal stromal cells (MSCs) therapies. Finally, our recent research results that LLLT enhanced the MSCs differentiation to osteoblast will also be described.

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Low Reactive Level Laser Therapy for Mesenchymal Stromal Cells Therapies

Objective: The purpose of this study was to measure intracellular reactive oxygen species (ROS) production after laser irradiation in various types of cells. Background data: ROS are considered to be the key secondary messengers produced by low-level laser therapy (LLLT). Although various mechanisms for the effects of LLLT have been proposed, and intracellular ROS were indicated as the one of the key factors, direct measurement of intracellular ROS of several types of cells after different wavelength lasers irradiation has not been reported. Materials and methods: Various types of cells were used in this study: mouse preadipocytes (3T3-L1), prechondrocytes (ATDC5), myoblasts (C2C12), mesenchymal stromal cells (KUSA-A1), lung cancer cells (LLC), insulinoma cells (MIN6), fibroblasts (NIH-3T3), human cervix adenocarcinoma cells (HeLa), macrophages differentiated from lymphocytes (THP-1) after treatment with phorbol ester, and rat basophilic leukemia cells (RBL-2H3). Cells were irradiated with a blue...

Blue Laser Irradiation Generates Intracellular Reactive Oxygen Species in Various Types of Cells

Low-level laser therapy (LLLT) has been used as a method for biostimulation. Cartilage develops through the differentiation of mesenchymal cells into chondrocytes, and differentiated chondrocytes in articular cartilage maintain cartilage homeostasis by synthesizing cartilage-specific extracellular matrix. The aim of this study is to evaluate the enhancement of chondrocyte differentiation and the expression levels of chondrogenic mRNA in prechondrogenic ATDC5 cells after laser irradiation. For chondrogenic induction, ATDC5 cells were irradiated with a blue laser (405 nm, continuous wave) at 100 mW/cm(2) for 180 s following incubation in chondrogenic differentiation medium. Differentiation after laser irradiation was quantitatively evaluated by the measurement of total collagen contents and chondrogenesis-related mRNAs. The total amount of collagen and mRNA levels of aggrecan, collagen type II, SOX-9, and DEC-1 were increased relative to those of a non-laser irradiated group after 14 days of laser irradiation. On the other hand, Ap-2alpha mRNA, a negative transcription factor of chondrogenesis, was dramatically decreased after laser irradiation. In addition, intracellular reactive oxygen species (ROS) were generated after laser irradiation. These results, for the first time, provide functional evidence that mRNA expression relating to chondrogenesis is increased, and Ap-2alpha is decreased immediately after laser irradiation. As this technique could readily be applied in situ to control the differentiation of cells at an implanted site within the body, this approach may have therapeutic potential for the restoration of damaged or diseased tissue.

Chondrogenic mRNA expression in prechondrogenic cells after blue laser irradiation

Background Data and Objective: Mesenchymal stem cells (MSCs) are multipotent cells present in adult bone marrow that replicate as undifferentiated cells and can differentiate to lineages of mesenchymal tissues. Homeostatic control of bone remodeling maintains bone mass by ensuring that bone resorption and bone formation occur sequentially and in a balanced manner. As most homeostatic functions occur in a circadian manner, a circadian clock could control bone mass. Here we show that laser irradiation can direct the extracellular calcification of mouse MSCs by altering the intracellular localization of the circadian rhythm protein cryptochrome 1 (CRY1). Materials and Methods: MSCs were irradiated with a blue laser (wavelength 405 nm) for 180 sec via a fiber attached to the bottom of the culture dish. After laser irradiation, the MSCs were incubated in osteogenic differentiation medium for 5 d. After laser irradiation, circadian rhythm protein CRY1 was immunostained and histochemical staining for ex...

Toshihiro Kushibiki

Papers

SEM observation of wet biological specimens pretreated with room-temperature ionic liquid.

Low Reactive Level Laser Therapy for Mesenchymal Stromal Cells Therapies

Blue Laser Irradiation Generates Intracellular Reactive Oxygen Species in Various Types of Cells

Chondrogenic mRNA expression in prechondrogenic cells after blue laser irradiation

Blue Laser Irradiation Enhances Extracellular Calcification of Primary Mesenchymal Stem Cells