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The regulation of hydrogen ion concentration (pH) is fundamental to cell viability, metabolism, and enzymatic function. Within the nervous system, the control of pH is also involved in diverse and dynamic processes including development, synaptic transmission, and the control of network excitability. As pH affects neuronal activity, and can also itself be altered by neuronal activity, the existence of tools to accurately measure hydrogen ion fluctuations is important for understanding the role pH plays under physiological and pathological conditions. Outside of their use as a marker of synaptic release, genetically encoded pH sensors have not been utilized to study hydrogen ion fluxes associated with network activity. By combining whole-cell patch clamp with simultaneous two-photon or confocal imaging, we quantified the amplitude and time course of neuronal, intracellular, acidic transients evoked by epileptiform activity in two separate in vitro models of temporal lobe epilepsy. In doing so, we demonstrate the suitability of three genetically encoded pH sensors: deGFP4, E(2)GFP, and Cl-sensor for investigating activity-dependent pH changes at the level of single neurons. Show less

AbstractWith the extensive use of two‐photon fluorescence microscopy (2PFM) in the biomedical field, the need for development of fluorescent probes with improved two‐photon fluorescence (2PF) properties has triggered extensive studies in the synthesis of new probes that undergo efficient two‐photon absorption (2PA). In order to provide a more comprehensive comparison of fluorophores for 2PF bioimaging, a figure of merit (FM) was developed by normalizing the 2PA action cross‐section, a commonly used parameter for characterizing bioimaging 2PF probes, by the photodecomposition quantum yield. Another important aspect of developing 2PA fluorophores is hydrophilicity. Although hydrophilic fluorophores are generally preferred in 2PFM bioimaging, hydrophobic fluorophores are typically easier to synthesize and purify, and have been used successfully in 2PFM bioimaging. The methodologies of dispersing hydrophobic fluorophores into aqueous media, such as in a DMSO/water mixture, micelles, silica nanoparticles, or forming polymer nanoparticles, are reviewed. The design and synthesis of hydrophilic 2PA fluorophores, achieved by introducing polyethylene glycol (PEG), anionic acid groups, cationic ammonium salt, and PAMAM dendrimers, is presented. Introduction of specificity to target certain biomarkers via labeling of antibodies, DNA, smallbioactive molecules, and peptides, and for the sensing of sepcific cations and pH, is also reviewed. Show less

The first bis(BF(2)) core complex containing a 1,8-naphthyridin derivative (1,2-bis(5,7-dimethyl-1,8-naphthyridin-2-yl)hydrazine) and with yellow-green emission as well as a high quantum yield was synthesized and structurally characterized, and the compound exhibits two-photon absorption and excited fluorescence properties. Show less

Fine-mapping of the cell-division cycle, notably the identification of mitotic kinase signaling pathways, provides novel opportunities for cancer-drug discovery. As a key regulator of multiple steps during mitotic progression across eukaryotic species, the serine/threonine-specific Polo-like kinase 1 (Plk1) is highly expressed in malignant cells and serves as a negative prognostic marker in specific human cancer types . Here, we report the discovery of a potent small-molecule inhibitor of mammalian Plk1, BI 2536, which inhibits Plk1 enzyme activity at low nanomolar concentrations. The compound potently causes a mitotic arrest and induces apoptosis in human cancer cell lines of diverse tissue origin and oncogenome signature. BI 2536 inhibits growth of human tumor xenografts in nude mice and induces regression of large tumors with well-tolerated intravenous dose regimens. In treated tumors, cells arrest in prometaphase, accumulate phosphohistone H3, and contain aberrant mitotic spindles. This mitotic arrest is followed by a surge in apoptosis, detectable by immunohistochemistry and noninvasive optical and magnetic resonance imaging. For addressing the therapeutic potential of Plk1 inhibition, BI 2536 has progressed into clinical studies in patients with locally advanced or metastatic cancers. Show less

Double-stranded RNA adenosine deaminase (ADAR1) is an ubiquitous enzyme in metazoa that edits pre-mRNA changing adenosine to inosine in regions of double-stranded RNA. Zalpha, an N-terminal domain of human ADAR1 encompassing 76 amino acid residues, shows apparent specificity for the left-handed Z-DNA conformation adopted by alternating (dGdC) polymers modified by bromination or methylation, as well as for (dGdC)13 inserts present in supercoiled plasmids. Here, a combination of circular dichroism, fluorescence, and gel-retardation studies is utilized to characterize recombinant Zalpha peptide and to examine its interaction with DNA. Results from laser-Raman spectroscopy experiments provide direct evidence for the existence of Z-DNA in peptide-DNA complexes. Show less