Rapid Screening of Glucocorticoid Receptor (GR) Effectors Using Cortisol-Detecting Sensor Cells.

Cortisol, a stress hormone, plays key roles in mediating stress and anti-inflammatory responses. As abnormal cortisol levels can induce various adverse effects, screening cortisol and cortisol analogues is important for monitoring stress levels and for identifying drug candidates. A novel cell-based sensing system was adopted for rapid screening of cortisol and its functional analogues under complex cellular regulation. We used glucocorticoid receptor (GR) fused to a split intein which reconstituted with the counterpart to trigger conditional protein splicing (CPS) in the presence of targets. CPS generates functional signal peptides which promptly translocate the fluorescent cargo. The sensor cells exhibited exceptional performance in discriminating between the functional and structural analogues of cortisol with improved sensitivity. Essential oil extracts with stress relief activity were screened using the sensor cells to identify GR effectors. The sensor cells responded to peppermint oil, and L-limonene and L-menthol were identified as potential GR effectors from the major components of peppermint oil. Further analysis indicated L-limonene as a selective GR agonist (SEGRA) which is a potential anti-inflammatory agent as it attenuates proinflammatory responses without causing notable adverse effects of GR agonists.

Projections from the dorsomedial division of the bed nucleus of the stria terminalis to hypothalamic nuclei in the mouse.

As stressful environment is a potent modulator of feeding, we seek in the present work to decipher the neuroanatomical basis for an interplay between stress and feeding behaviors. For this, we combined anterograde and retrograde tracing with immunohistochemical approaches to investigate the patterns of projections between the dorsomedial division of the bed nucleus of the stria terminalis (BNST), well connected to the amygdala, and hypothalamic structures such as the paraventricular (PVH) and dorsomedial (DMH), the arcuate (ARH) nuclei and the lateral hypothalamic areas (LHA) known to control feeding and motivated behaviors. We particularly focused our study on afferences to proopiomelanocortin (POMC), agouti-related peptide (AgRP), melanin-concentrating-hormone (MCH) and orexin (ORX) neurons characteristics of the ARH and the LHA, respectively. We found light to intense innervation of all these hypothalamic nuclei. We particularly showed an innervation of POMC, AgRP, MCH and ORX neurons by the dorsomedial and dorsolateral divisions of the BNST. Therefore, these results lay the foundation for a better understanding of the neuroanatomical basis of the stress-related feeding behaviors.

Transient Gene Expression as a Tool to Monitor and Manipulate the Levels of Acidic Phospholipids in Plant Cells.

Anionic phospholipids represent only minor fraction of cell membranes lipids but they are critically important for many membrane-related processes, including membrane identity, charge, shape, the generation of second messengers, and the recruitment of peripheral proteins. The main anionic phospholipids of the plasma membrane are phosphoinositides phosphatidylinositol 4-phosphate (PI4P), phosphatidylinositol 4,5-bisphosphate (PI4,5P2), phosphatidylserine (PS), and phosphatidic acid (PA). Recent insights in the understanding of the nature of protein-phospholipid interactions enabled the design of genetically encoded fluorescent molecular probes that can interact with various phospholipids in a specific manner allowing their imaging in live cells. Here, we describe the use of transiently transformed plant cells to study phospholipid-dependent membrane recruitment.

Vacuole-Targeted Proteins: Ins and Outs of Subcellular Localization Studies.

Accurate and efficient demonstrations of protein localizations to the vacuole or tonoplast remain strict prerequisites to decipher the role of vacuoles in the whole plant cell biology and notably in defence processes. In this chapter, we describe a reliable procedure of protein subcellular localization study through transient transformations of Catharanthus roseus or onion cells and expression of fusions with fluorescent proteins allowing minimizing artefacts of targeting.

A Homogeneous Cell-Based Halide-Sensitive Yellow Fluorescence Protein Assay to Identify Modulators of the Cystic Fibrosis Transmembrane Conductance Regulator Ion Channel.

Cystic fibrosis (CF), an inherited genetic disease, is caused by mutation of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene, which encodes an ion channel involved in hydration maintenance by anion homeostasis. Ninety percent of CF patients possess one or more copies of the F508del CFTR mutation. This mutation disrupts trafficking of the protein to the plasma membrane and diminishes function of mature CFTR. Identifying small molecule modulators of mutant CFTR activity or biosynthesis may yield new tools for discovering novel CF treatments. One strategy utilizes a 384-well, cell-based fluorescence-quenching assay, which requires extensive wash steps, but reports sensitive changes in fluorescence-quenching kinetic rates. In this study, we describe the methods of adapting the protocol to a homogeneous, miniaturized 1,536-well format and further optimization of this functional F508del CFTR assay. The assay utilizes a cystic fibrosis bronchial epithelial (CFBE41o-) cell line, which was engineered to report CFTR-mediated intracellular flux of iodide by a halide-sensitive yellow fluorescence protein (YFP) reporter. We also describe the limitations of quench rate analysis and the subsequent incorporation of a novel, kinetic data analysis modality to quickly and efficiently find active CFTR modulators. This format yields a Z' value interval of 0.61 ± 0.05. As further evidence of high-throughput screen suitability, we subsequently completed a screening campaign of >645,000 compounds, identifying 2,811 initial hits. After completing secondary and tertiary follow-up assays, we identified 187 potential CFTR modulators, which EC50's < 5 µM. Thus, the assay has integrated the advantages of a phenotypic screen with high-throughput scalability to discover new small-molecule CFTR modulators.

Analysis of Pelagia noctiluca proteome Reveals a Red Fluorescent Protein, a Zinc Metalloproteinase and a Peroxiredoxin.

Pelagia noctiluca is the most venomous jellyfish in the Mediterranean Sea where it forms dense blooms. Although there is several published research on this species, until now none of the works has been focused on a complete protein profile of the all body constituents of this organism. Here, we have performed a detailed proteomics characterization of the major protein components expressed by P. noctiluca. With that aim, we have considered the study of jellyfish proteins involved in defense, body constituents and metabolism, and furthered explore the significance and potential application of such bioactive molecules. P. noctiluca body proteins were separated by1D SDS-PAGE and 2DE followed by characterization by nanoLC-MS/MS and MALDI-TOF/TOF techniques. Altogether, both methods revealed 68 different proteins, including a Zinc Metalloproteinase, a Red Fluorescent Protein (RFP) and a Peroxiredoxin. These three proteins were identified for the first time in P. noctiluca. Zinc Metalloproteinase was previously reported in the venom of other jellyfish species. Besides the proteins described above, the other 65 proteins found in P. noctiluca body content were identified and associated with its clinical significance. Among all the proteins identified in this work we highlight: Zinc metalloproteinase, which has a ShK toxin domain and therefore should be implicated in the sting toxicity of P. noctiluca.; the RFP which are a very important family of proteins due to its possible application as molecular markers; and last but not least the discovery of a Peroxiredoxin in this organism makes it a new natural resource of antioxidant and anti-UV radiation agents.

Improved fluorescence assays to measure the defects associated with F508del-CFTR allow identification of new active compounds.

BACKGROUND AND PURPOSE: Cystic fibrosis (CF) is a debilitating disease caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which codes for a Cl-/HCO3 - channel. F508del, the most common CF-associated mutation, causes both gating and biogenesis defects in the CFTR protein. This paper describes the optimization of two fluorescence assays, capable of measuring CFTR function and cellular localization, and their use in a pilot drug screen. EXPERIMENTAL APPROACH: HEK293 cells expressing YFP-F508del-CFTR, in which halide sensitive YFP is tagged to the N-terminal of CFTR, were used to screen a small library of compounds based on the VX-770 scaffold. Cells expressing F508del-CFTR-pHTomato, in which a pH sensor is tagged to the fourth extracellular loop of CFTR, were used to measure CFTR plasma membrane exposure following chronic treatment with the novel potentiators. KEY RESULTS: Active compounds with efficacy ~50% of VX-770, micromolar potency, and structurally distinct from VX-770 were identified in the screen. The F508del-CFTR-pHTomato assay suggests that the hit compound MS131A, unlike VX-770, does not decrease membrane exposure of F508del-CFTR. CONCLUSIONS AND IMPLICATIONS: Most known potentiators have a negative influence on F508del-CFTR biogenesis/stability, which means membrane exposure needs to be monitored early during the development of drugs targeting CFTR. The combined use of the two fluorescence assays described here provides a useful tool for the identification of improved potentiators and correctors. The assays could also prove useful for basic scientific investigations on F508del-CFTR, and other CF-causing mutations.

The need for calcium imaging in nonhuman primates: New motor neuroscience and brain-machine interfaces.

A central goal of neuroscience is to understand how populations of neurons coordinate and cooperate in order to give rise to perception, cognition, and action. Nonhuman primates (NHPs) are an attractive model with which to understand these mechanisms in humans, primarily due to the strong homology of their brains and the cognitively sophisticated behaviors they can be trained to perform. Using electrode recordings, the activity of one to a few hundred individual neurons may be measured electrically, which has enabled many scientific findings and the development of brain-machine interfaces. Despite these successes, electrophysiology samples sparsely from neural populations and provides little information about the genetic identity and spatial micro-organization of recorded neurons. These limitations have spurred the development of all-optical methods for neural circuit interrogation. Fluorescent calcium signals serve as a reporter of neuronal responses, and when combined with post-mortem optical clearing techniques such as CLARITY, provide dense recordings of neuronal populations, spatially organized and annotated with genetic and anatomical information. Here, we advocate that this methodology, which has been of tremendous utility in smaller animal models, can and should be developed for use with NHPs. We review here several of the key opportunities and challenges for calcium-based optical imaging in NHPs. We focus on motor neuroscience and brain-machine interface design as representative domains of opportunity within the larger field of NHP neuroscience.

Immune Response and Protective Efficacy of a Heterologous DNA-Protein Immunization with Leishmania Superoxide Dismutase B1.

Growing evidence shows that antioxidant proteins of Leishmania could be used as vaccine candidates. In this study, we report the efficacy of Leishmania donovani iron superoxide dismutase B1 (LdFeSODB1) as a vaccine antigen in BALB/c mice in a DNA-protein prime-boost immunization regimen in the presence or absence of murine granulocyte macrophage colony stimulating factor (mGMCSF) DNA adjuvant. The expression study confirmed that LdFeSODB1 is expressed in mammalian cells and mGMCSF fusion mediates the secretion of the recombinant protein. Heterologous immunization with LdFeSODB1 induced a strong antibody- and cell-mediated immune response in mice. Immunization triggered a mixed Th1/Th2 response as evidenced by the ratio of IgG2a to IgG1. Antigen-stimulated spleen cells from the immunized mice produced high level IFN-γ. Multiparametric flow cytometry data showed that immunization with LdFeSODB1 induced significantly higher expression of TNF-α or IL-2 by antigen-stimulated T cells. Eight weeks after L. major infection, immunization with the antigen shifted the immune response to a more Th1 type than the controls as demonstrated by IgG2a/IgG1 ratio. Moreover, IFN-γ production by antigen-stimulated spleen cells from immunized mice remained high. The footpad swelling experiment showed that immunization with LdFeSODB1 resulted in partial protection of mice from a high dose L. major infection.

Development of Leishmania donovani stably expressing DsRed for flow cytometry-based drug screening using chalcone thiazolyl-hydrazone as a new antileishmanial target.

Green fluorescent protein produces significant fluorescence and is extremely stable, however its excitation maximum is close to the ultraviolet range and thus can damage living cells. Hence, Leishmania donovani stably expressing DsRed were developed and their suitability for flow cytometry-based antileishmanial screening was assessed by evaluating the efficacies of standard drugs as well as newly synthesised chalcone thiazolyl-hydrazone compounds. The DsRed gene was successfully integrated at the 18S rRNA locus of L. donovani and transfectants (LdDsRed) were selected using hygromycin B. Enhanced expression of DsRed and a high level of infectivity to J774A.1 macrophages were achieved, which was confirmed by fluorescence microscopy and flow cytometry. Furthermore, these LdDsRed transfectants were utilised for development of an in vitro screening assay using the standard antileishmanial drugs miltefosine, amphotericin B, pentamidine and paromomycin. The response of transfectants to standard drugs correlated well with previous reports. Subsequently, the suitability of this system was further assessed by screening a series of 18 newly synthesised chalcone thiazolyl-hydrazone compounds in vitro for their antileishmanial activity, wherein 8 compounds showed moderate antileishmanial activity. The most active compound 5g, with ca. 73% splenic parasite reduction, exerted its activity via generating nitric oxide and reactive oxygen species and inducing apoptosis in LdDsRed-infected macrophages. Thus, these observations established the applicability of LdDsRed transfectants for flow cytometry-based antileishmanial screening. Further efforts aimed at establishing a high-throughput screening assay and determining the in vivo screening of potential antileishmanial leads are required.

A human liver microphysiology platform for investigating physiology, drug safety, and disease models.

This paper describes the development and characterization of a microphysiology platform for drug safety and efficacy in liver models of disease that includes a human, 3D, microfluidic, four-cell, sequentially layered, self-assembly liver model (SQL-SAL); fluorescent protein biosensors for mechanistic readouts; as well as a microphysiology system database (MPS-Db) to manage, analyze, and model data. The goal of our approach is to create the simplest design in terms of cells, matrix materials, and microfluidic device parameters that will support a physiologically relevant liver model that is robust and reproducible for at least 28 days for stand-alone liver studies and microfluidic integration with other organs-on-chips. The current SQL-SAL uses primary human hepatocytes along with human endothelial (EA.hy926), immune (U937) and stellate (LX-2) cells in physiological ratios and is viable for at least 28 days under continuous flow. Approximately, 20% of primary hepatocytes and/or stellate cells contain fluorescent protein biosensors (called sentinel cells) to measure apoptosis, reactive oxygen species (ROS) and/or cell location by high content analysis (HCA). In addition, drugs, drug metabolites, albumin, urea and lactate dehydrogenase (LDH) are monitored in the efflux media. Exposure to 180 µM troglitazone or 210 µM nimesulide produced acute toxicity within 2-4 days, whereas 28 µM troglitazone produced a gradual and much delayed toxic response over 21 days, concordant with known mechanisms of toxicity, while 600 µM caffeine had no effect. Immune-mediated toxicity was demonstrated with trovafloxacin with lipopolysaccharide (LPS), but not levofloxacin with LPS. The SQL-SAL exhibited early fibrotic activation in response to 30 nM methotrexate, indicated by increased stellate cell migration, expression of alpha-smooth muscle actin and collagen, type 1, alpha 2. Data collected from the in vitro model can be integrated into a database with access to related chemical, bioactivity, preclinical and clinical information uploaded from external databases for constructing predictive models.

JC virus-iLOV fluorescent strains enable the detection of early and late viral protein expression.

JC virus (JCV) is highly prevalent in humans, and may cause progressive multifocal leukoencephalopathy (PML), JCV granule cell neuronopathy (JCV GCN), JCV encephalopathy (JCVE) and JCV meningitis (JCVM) in immunocompromised individuals. There is no treatment for JCV, and a growing number of multiple sclerosis patients treated with immunomodulatory medications have developed PML. Antiviral agents against JCV are therefore highly desirable but remain elusive, due to the difficulty of determining their effect in vitro. A JCV strain carrying a fluorescent protein gene would greatly simplify and accelerate the drug screening process. To achieve this goal, we selected the 366bp improved Light, Oxygen or Voltage-sensing domain (iLOV) of plant phototropin gene and created two full-length JCV-iLOV constructs on the prototype JCV Mad1 backbone. The iLOV gene was inserted either before the early regulatory T gene (iLOV-T), or after the late Agno gene (iLOV-Agno). Both JCV iLOV strains were replication-competent in vitro and emitted a fluorescent signal detectable by confocal microscope, but JCV iLOV-T exhibited higher cellular and supernatant viral loads compared to JCV iLOV-Agno. JCV iLOV-T could also produce infectious pseudovirions. These data suggest that JCV iLOV constructs may become valuable tools for anti-JCV drug screening.

High-throughput analysis of meiotic crossover frequency and interference via flow cytometry of fluorescent pollen in Arabidopsis thaliana.

During meiosis, reciprocal exchange between homologous chromosomes occurs as a result of crossovers (COs). CO frequency varies within genomes and is subject to genetic, epigenetic and environmental control. As robust measurement of COs is limited by their low numbers, typically 1-2 per chromosome, we adapted flow cytometry for use with Arabidopsis transgenic fluorescent protein-tagged lines (FTLs) that express eCFP, dsRed or eYFP fluorescent proteins in pollen. Segregation of genetically linked transgenes encoding fluorescent proteins of distinct colors can be used to detect COs. The fluorescence of up to 80,000 pollen grains per individual plant can be measured in 10-15 min using this protocol. A key element of CO control is interference, which inhibits closely spaced COs. We describe a three-color assay for the measurement of CO frequency in adjacent intervals and calculation of CO interference. We show that this protocol can be used to detect changes in CO frequency and interference in the fancm zip4 double mutant. By enabling high-throughput measurement of CO frequency and interference, these methods will facilitate genetic dissection of meiotic recombination control.

Subcellular calcium measurements in mammalian cells using jellyfish photoprotein aequorin-based probes.

The jellyfish Aequorea victoria produces a 22-kDa protein named aequorin that has had an important role in the study of calcium (Ca(2+)) signaling. Aequorin reacts with Ca(2+) via oxidation of the prosthetic group, coelenterazine, which results in emission of light. This signal can be detected by using a special luminescence reader (called aequorinometer) or luminescence plate readers. Here we describe the main characteristics of aequorin as a Ca(2+) probe and how to measure Ca(2+) in different intracellular compartments of animal cells (cytosol, different mitochondrial districts, nucleus, endoplasmic reticulum (ER), Golgi apparatus, peroxisomes and subplasma-membrane cytosol), ranging from single-well analyses to high-throughput screening by transfecting animal cells using DNA vectors carrying recombinant aequorin chimeras. The use of aequorin mutants and modified versions of coelenterazione increases the range of calcium concentrations that can be recorded. Cell culture and transfection takes ∼3 d. An experiment including signal calibration and the subsequent analyses will take ∼1 d.

Tagging of genomic STAT3 and STAT1 with fluorescent proteins and insertion of a luciferase reporter in the cyclin D1 gene provides a modified A549 cell line to screen for selective STAT3 inhibitors.

Signal transducer and activator of transcription 3 (STAT3) is an oncogenic protein that is constitutively activated in numerous cancer cell lines and human cancers. Another STAT family member, STAT1, possesses cancer-inhibitory properties and can promote apoptosis in tumor cells upon activation. To better characterize these important cancer related genes, we tagged STAT3 and STAT1 loci with fluorescent protein (FP) sequences (RFP and GFP respectively) by targeted integration via zinc finger nuclease (ZFN)--mediated homologous recombination in A549 cells that express aberrantly activated STAT3. We inserted the FP transgenes at the N-terminus of the STAT3 locus and at the C-terminus of the STAT1 locus. The integration resulted in endogenous expression of fluorescent STAT3 and STAT1 chimeric fusion proteins. When stimulated with IL-6 or IFN-γ, the cells showed robust nuclear translocation of RFP-STAT3 or STAT1-GFP, respectively. Pre-incubation of cells with a known specific STAT3 inhibitor showed that IFN-γ-induced translocation of STAT1-GFP was not impaired. STAT3 activates multiple downstream targets such as genes involved in cell cycle progression - e.g. cyclin D1. To detect changes in expression of endogenous cyclin D1, we used ZFN technology to insert a secreted luciferase reporter behind the cyclin D1 promoter and separated the luciferase and cyclin D1 coding regions by a 2A sequence to induce a translational skip. The luciferase insertion was made in the RFP-STAT3/STAT1-GFP cell line to have all three reporters in a single cell line. Addition of a STAT3 inhibitor led to suppression of cyclin D1 promoter activity and cell growth arrest. The triple-modified cell line provides a simple and convenient method for high-content screening and pre-clinical testing of potential STAT3 inhibitors in live cells while ensuring that the STAT1 pathway is not affected. This approach of reporting endogenous gene activities using ZFN technology could be applied to other cancer targets.

Development of a multiplex phenotypic cell-based high throughput screening assay to identify novel hepatitis C virus antivirals.

Hepatitis C virus (HCV) infection is a global health concern with chronic liver damage threatening 3% of the world's population. To date, the standard of care is a combination of pegylated interferon-alpha with ribavirin, and recently two direct acting antivirals have entered the clinics. However, because of side effects, drug resistance and viral genotype-specific differences in efficacy current and potentially also future therapies have their limitations. Here, we describe the development of a phenotypic high-throughput assay to identify new cross-genotype inhibitors with novel mechanism of action, by combining a genotype (gt) 1 replicon with the infectious HCV gt2 cell culture system. To develop this phenotypic multiplex assay, HCV reporter cells expressing RFP-NLS-IPS and gt1b replicon cells expressing NS5A-GFP were co-plated and treated with compounds followed by inoculation with gt2a HCV. At 72h post treatment, RFP translocation as a marker for HCV infection and GFP fluorescence intensity as a marker for gt1 RNA replication were measured. Additionally, the total cell number, which serves as an indicator of cytotoxicity, was determined. This phenotypic strategy supports multi-parameter data acquisition from a single well to access cross-genotypic activity, provides an indication of the stage of the viral life cycle targeted, and also assesses compound cytotoxicity. Taken together, this multiplex phenotypic platform facilitates the identification of novel compounds for drug development and chemical probes for continuing efforts to understand the HCV life cycle.

Real-time imaging of apoptosis induction of human breast cancer cells by the traditional Chinese medicinal herb tubeimu.

Traditional Chinese Medicine (TCM) has been used for thousands of years, including treatment for cancer. Use of modern technology and the scientific method to evaluate the efficacy of TCM for cancer should enable its more widespread use. In the present study, the efficacy of the TCM tubeimu, extracted from the tuber of the plant Bolbostemma paniculatum, on the MDA-MB-231 human breast cancer cell line was evaluated. The MDA-MB-231 cell line was engineered to express red fluorescent protein (RFP) in the cytoplasm and green fluorescent protein (GFP) linked to histone H2B in the nucleus, which allows real-time imaging of nuclear-cytoplasmic dynamics. Apoptosis was readily visualized in these cells by nuclear shape changes and fragmentation. The MDA-MB-231 RFP-GFP cells were cultured either in two-dimensions on plastic or in three-dimensions on Gelfoam®. Cells were treated with a dichloromethane extract of fresh tubeimu. Apoptosis was further monitored by DNA fragmentation determined by gel electrophoresis. Tubeimu induced apoptosis of MDA-MB-231 cells, as observed by fluorescence microscopy, as early as 24 hours of treatment in vitro in two-dimensional culture. By 48 hours' treatment, DNA fragmentation could be observed. The frequency of apoptosis increased through at least 72 hours' treatment, with most of the cells being killed. Tubeimu also induced apoptosis of MDA-MB-231 cells in three-dimensional culture on Gelfoam®, but to a lesser extent than in 2D culture. The results of the present study indicate the potential of tubeimu in breast cancer therapy.

Bioluminescence methodology for the detection of protein-protein interactions within the voltage-gated sodium channel macromolecular complex.

Protein-protein interactions are critical molecular determinants of ion channel function and emerging targets for pharmacological interventions. Yet, current methodologies for the rapid detection of ion channel macromolecular complexes are still lacking. In this study we have adapted a split-luciferase complementation assay (LCA) for detecting the assembly of the voltage-gated Na+ (Nav) channel C-tail and the intracellular fibroblast growth factor 14 (FGF14), a functionally relevant component of the Nav channelosome that controls gating and targeting of Nav channels through direct interaction with the channel C-tail. In the LCA, two complementary N-terminus and C-terminus fragments of the firefly luciferase were fused, respectively, to a chimera of the CD4 transmembrane segment and the C-tail of Nav1.6 channel (CD4-Nav1.6-NLuc) or FGF14 (CLuc-FGF14). Co-expression of CLuc-FGF14 and CD4-Nav1.6-NLuc in live cells led to a robust assembly of the FGF14:Nav1.6 C-tail complex, which was attenuated by introducing single-point mutations at the predicted FGF14:Nav channel interface. To evaluate the dynamic regulation of the FGF14:Nav1.6 C-tail complex by signaling pathways, we investigated the effect of kinase inhibitors on the complex formation. Through a platform of counter screenings, we show that the p38/MAPK inhibitor, PD169316, and the IκB kinase inhibitor, BAY 11-7082, reduce the FGF14:Nav1.6 C-tail complementation, highlighting a potential role of the p38MAPK and the IκB/NFκB pathways in controlling neuronal excitability through protein-protein interactions. We envision the methodology presented here as a new valuable tool to allow functional evaluations of protein-channel complexes toward probe development and drug discovery targeting ion channels implicated in human disorders.

Chemical and biological approaches to improve the efficiency of homologous recombination in human cells mediated by artificial restriction DNA cutter.

A chemistry-based artificial restriction DNA cutter (ARCUT) was recently prepared from Ce(IV)/EDTA complex and a pair of pseudo-complementary peptide nucleic acids. This cutter has freely tunable scission-site and site specificity. In this article, homologous recombination (HR) in human cells was promoted by cutting a substrate DNA with ARCUT, and the efficiency of this bioprocess was optimized by various chemical and biological approaches. Of two kinds of terminal structure formed by ARCUT, 3'-overhang termini provided by 1.7-fold higher efficiency than 5'-overhang termini. A longer homology length (e.g. 698 bp) was about 2-fold more favorable than shorter one (e.g. 100 bp). When the cell cycle was synchronized to G2/M phase with nocodazole, the HR was promoted by about 2-fold. Repression of the NHEJ-relevant proteins Ku70 and Ku80 by siRNA increased the efficiency by 2- to 3-fold. It was indicated that appropriate combination of all these chemical and biological approaches should be very effective to promote ARCUT-mediated HR in human cells.

Emergence of intrinsic representations of images by feedforward and feedback processes and bioluminescent photons in early retinotopic areas.

Recently, we put forward a redox molecular hypothesis involving the natural biophysical substrate of visual perception and imagery. Here, we explicitly propose that the feedback and feedforward iterative operation processes can be interpreted in terms of a homunculus looking at the biophysical picture in our brain during visual imagery. We further propose that the brain can use both picture-like and language-like representation processes. In our interpretation, visualization (imagery) is a special kind of representation i.e., visual imagery requires a peculiar inherent biophysical (picture-like) mechanism. We also conjecture that the evolution of higher levels of complexity made the biophysical picture representation of the external visual world possible by controlled redox and bioluminescent nonlinear (iterative) biochemical reactions in the V1 and V2 areas during visual imagery. Our proposal deals only with the primary level of visual representation (i.e. perceived "scene").