Long-term imaging of living adult zebrafish.

The zebrafish has become a widely used animal model due, in large part, to its accessibility to and usefulness for high-resolution optical imaging. Although zebrafish research has historically focused mostly on early development, in recent years the fish has increasingly been used to study regeneration, cancer metastasis, behavior and other processes taking place in juvenile and adult animals. However, imaging of live adult zebrafish is extremely challenging, with survival of adult fish limited to a few tens of minutes using standard imaging methods developed for zebrafish embryos and larvae. Here, we describe a new method for imaging intubated adult zebrafish using a specially designed 3D printed chamber for long-term imaging of adult zebrafish on inverted microscope systems. We demonstrate the utility of this new system by nearly day-long observation of neutrophil recruitment to a wound area in living double-transgenic adult casper zebrafish with fluorescently labeled neutrophils and lymphatic vessels, as well as intubating and imaging the same fish repeatedly. We also show that Mexican cavefish can be intubated and imaged in the same way, demonstrating this method can be used for long-term imaging of adult animals from diverse aquatic species.

In vivo dissection of Rhoa function in vascular development using zebrafish.

The small monomeric GTPase RHOA acts as a master regulator of signal transduction cascades by activating effectors of cellular signaling, including the Rho-associated protein kinases ROCK1/2. Previous in vitro cell culture studies suggest that RHOA can regulate many critical aspects of vascular endothelial cell (EC) biology, including focal adhesion, stress fiber formation, and angiogenesis. However, the specific in vivo roles of RHOA during vascular development and homeostasis are still not well understood. In this study, we examine the in vivo functions of RHOA in regulating vascular development and integrity in zebrafish. We use zebrafish RHOA-ortholog (rhoaa) mutants, transgenic embryos expressing wild type, dominant negative, or constitutively active forms of rhoaa in ECs, pharmacological inhibitors of RHOA and ROCK1/2, and Rock1 and Rock2a/b dgRNP-injected zebrafish embryos to study the in vivo consequences of RHOA gain- and loss-of-function in the vascular endothelium. Our findings document roles for RHOA in vascular integrity, developmental angiogenesis, and vascular morphogenesis in vivo, showing that either too much or too little RHOA activity leads to vascular dysfunction.

Differential expression and hypoxia-mediated regulation of the N-myc downstream regulated gene family.

Many organisms rely on oxygen to generate cellular energy (adenosine triphosphate or ATP). During severe hypoxia, the production of ATP decreases, leading to cell damage or death. Conversely, excessive oxygen causes oxidative stress that is equally damaging to cells. To mitigate pathological outcomes, organisms have evolved mechanisms to adapt to fluctuations in oxygen levels. Zebrafish embryos are remarkably hypoxia-tolerant, surviving anoxia (zero oxygen) for hours in a hypometabolic, energy-conserving state. To begin to unravel underlying mechanisms, we analyze here the distribution of the N-myc Downstream Regulated Gene (ndrg) family, ndrg1-4, and their transcriptional response to hypoxia. These genes have been primarily studied in cancer cells and hence little is understood about their normal function and regulation. We show here using in situ hybridization that ndrgs are expressed in metabolically demanding organs of the zebrafish embryo, such as the brain, kidney, and heart. To investigate whether ndrgs are hypoxia-responsive, we exposed embryos to different durations and severity of hypoxia and analyzed transcript levels. We observed that ndrgs are differentially regulated by hypoxia and that ndrg1a has the most robust response, with a ninefold increase following prolonged anoxia. We further show that this treatment resulted in de novo expression of ndrg1a in tissues where the transcript is not observed under normoxic conditions and changes in Ndrg1a protein expression post-reoxygenation. These findings provide an entry point into understanding the role of this conserved gene family in the adaptation of normal cells to hypoxia and reoxygenation.

Highly Fluorescent Imidazole Probes for the Pico Molar Detection of CN(-) ion and Application in Living Cells.

A series of four imidazole based fluorescent receptors (R1-R4) containing two different signaling units were synthesized for the sensing of specific anions. R1 and R2 act as excellent colorimetric sensors via color change from yellow to orange and pink in presence of F (-) , AcO (-) and CN (-) ions. R1 and R2 show intramolecular charge transfer (ICT) band at 414 nm and 434 nm and were red shifted into 500 nm and 510 nm respectively, with the addition of above anions. Anion complexes of R1 and R2 were found to exhibit remarkable orange and red fluorescence under UV light which is consistent with an emission observed at 594 nm and 618 nm respectively. Likewise, quenching in the green fluorescence of R3 and R4 was found under UV light accompanied with quenching of emission at 530 nm and 541 nm. Selective colorimetric sensing of CN (-) ion is also achieved in 3 % aq. DMF medium. Intracellular uptake of CN (-) ion by R1 was achieved in living RAW 264.7 cells. Finally, practical application of R2 to detect CN (-) and F (-) ions present in aqueous solution of cassava flour and toothpaste also performed.

Colorimetric detection of in situ metal acetates and fluorides by a bipyridyl-linked Schiff base.

Here, we present a new bipyridyl moiety linked Schiff base (bipy-1) that is well characterized using spectroscopic techniques. Colorimetric and UV-vis titrations were used to study the photophysical properties of bipy-1 in the presence of various tetrabutyl ammonium salt of anions and metal salts containing different counter cations. bipy-1 showed selective recognition of dimethyl sulphoxide solution of tetrabutyl ammonium salt of F(-) ion accompanied with a UV-vis band at 529 nm and interesting binding of aqueous Co, Ni, and Cu acetates/fluorides, as confirmed by distinct color changes from fluorescent green to pink or orange and a strong band around 480-510 nm in the UV-vis spectrum. However, in the presence of Co, Ni, and Cu countercations, any form of metal acetate/fluorides was found to be able to respond to similar color changes from fluorescent green to pink or orange, showing a band around 480-510 nm. This type of output clearly indicates that the in situ formation of Co, Ni, and Cu acetates/fluorides also coordinates with bipyridyl nitrogen atoms.

Anti-counterfeiting fiber system with near-infrared wavelength selectivity based on photothermal and thermochromic dyes.

Anti-counterfeiting (ACF) technology plays a crucial role in distinguishing genuine products from counterfeits, as well as in identity verification. Moreover, it serves as a protective measure for safeguarding the rights of individuals, companies, and governments. In this study, a high-level ACF technology was developed using a color-conversion system based on the photothermal effect of near-infrared (NIR) wavelengths. Diimonium dye (DID), which is a photothermal dye, was selected because it is an NIR absorbing dye with over 98% transparency in the visible light (vis) region. Due to the photothermal properties of DID, the temperature increased to approximately 65 °C at 1064 nm and 39 °C at 808 nm, respectively. Additionally, we employed a donor-acceptor Stenhouse adduct dye, a thermochromic dye, which exhibits reversible color change due to heat (red color) and light (colorless). Our ACF technology was applied to the brand-protecting fiber utilizing the difference in photothermal temperature according to the NIR wavelength. We successfully implemented anti-counterfeit clothing using alphabet K labels that could distinguish between genuine and counterfeit products by irradiating with specific NIR wavelengths.

Influence of ITO electrode on the electrochromic performance outcomes of viologen-functionalized polyhedral oligomeric silsesquioxanes.

Electrochromic devices (ECDs) exhibit reversible optical changes under applied electrical stimuli. Transparent conducting electrodes (TCOs), generally constructed with indium tin oxide (ITO), are a vital component determining transparency and switching behaviors. ITO specifications for TCO materials have not drawn much attention despite the critical role of these materials. Herein we investigate the influence of ITO electrodes in achieving high-performance ECDs containing viologen-functionalized polyhedral oligomeric silsesquioxane (POSS-viologen). Indeed, ITO electrodes exert significant effects on the electrochromic characteristics. A high ITO thickness shows superior color-switching with high optical density and coloration efficiency levels. Enhanced electrical conductivity facilitates diffusion behaviors, an outcome beneficial for electrochromic switching. The surface-charge capacity ratio values are measured and found to be close to one, indicating that no residual current remains, and the prepared devices provide good reversibility during the coloring and bleaching process. Furthermore, with an increase in the ITO thickness, the current required for the coloring and bleaching processes decreases, and the power consumption needed for the operation of the device becomes low. The superiority of POSS-viologen should also be noted, especially when compared to normal viologens, in terms of the electrochromic properties and long-term operational stability. These results demonstrate the critical role of electrical conductivity in ITO electrodes, providing a valuable guideline for TCO specifications for ECD fabrication using viologen derivatives.

Ratiometric photothermal detection of silver ions using diimmonium salts.

Selective detection and determination of silver ions are tricky due to the poor reactivity and the interference of various metal ions and halides. Only a few cases have been reported concerning sulfur-based functionality. This work proposes silver-selective probes exhibiting peculiar colorimetric and photothermal responses without the need for sulfur-functional groups. Tetrakis(4-dialklyaminophenyl)1,4-phenylenediamine containing hexyl and methoxytriethyleneglycol substituents, PTS1 and PTS2, respectively, are selectively oxidized by silver ions to form dicationic diimmonium salts. The resulting quinone diimminium salts show strong absorption in the near-infrared (NIR) region with intense color changes. The striking color change is observed from pale brown to intense olive green with immediate responses. PTS1 shows a higher binding constant and nanomolar detection limit, whereas PTS2 shows a sub-micromolar detection limit. Job's plot studies reveal the stoichiometric ratio as 1:2 (PTS1: Ag+). Due to the strong NIR absorption of the oxidized PTS probes, the temperature rise is monitored, after adding silver ions, by a thermal camera and in-contact thermal sensors. The concentration of the actual samples can be determined based on a calibration plot with a temperature change as a function of silver ion concentration. The current results offer highly selective and ultrasensitive probes for silver ions under multiple detection mediums, including naked eye and photothermal detection.

Near-Infrared Absorption Properties of Neutral Bis(1,2-dithiolene) Platinum(II) Complexes Using Density Functional Theory.

Small metal complexes are highly interesting for bioimaging because of their excellent near-infrared (NIR) absorption properties. In this study, neutral complexes of platinum(II) connected to two monoreduced 1,3-diisopropylimidazoline-2,4,5-trithione ligands-namely, [Pt(iPr2timdt)2]-were investigated. Theoretical studies using the density functional theory (DFT) and GW-BSE approximation verified the effects of the geometry of the isopropyl moieties on the NIR absorption spectra. The calculated absorption spectra showed excellent correspondence with the experimental results. The geometry of the isopropyl groups considerably influenced the electronic structures of the metal complexes, which altered the absorption profiles of the respective geometries, as demonstrated in this research.

N-myc downstream regulated gene 1 (ndrg1) functions as a molecular switch for cellular adaptation to hypoxia.

Lack of oxygen (hypoxia and anoxia) is detrimental to cell function and survival and underlies many disease conditions. Hence, metazoans have evolved mechanisms to adapt to low oxygen. One such mechanism, metabolic suppression, decreases the cellular demand for oxygen by downregulating ATP-demanding processes. However, the molecular mechanisms underlying this adaptation are poorly understood. Here, we report on the role of ndrg1a in hypoxia adaptation of the anoxia-tolerant zebrafish embryo. ndrg1a is expressed in the kidney and ionocytes, cell types that use large amounts of ATP to maintain ion homeostasis. ndrg1a mutants are viable and develop normally when raised under normal oxygen. However, their survival and kidney function is reduced relative to WT embryos following exposure to prolonged anoxia. We further demonstrate that Ndrg1a binds to the energy-demanding sodium-potassium ATPase (NKA) pump under anoxia and is required for its degradation, which may preserve ATP in the kidney and ionocytes and contribute to energy homeostasis. Lastly, we show that sodium azide treatment, which increases lactate levels under normoxia, is sufficient to trigger NKA degradation in an Ndrg1a-dependent manner. These findings support a model whereby Ndrg1a is essential for hypoxia adaptation and functions downstream of lactate signaling to induce NKA degradation, a process known to conserve cellular energy.

Optical and Electronic Properties of Organic NIR-II Fluorophores by Time-Dependent Density Functional Theory and Many-Body Perturbation Theory: GW-BSE Approaches.

Organic-molecule fluorophores with emission wavelengths in the second near-infrared window (NIR-II, 1000-1700 nm) have attracted substantial attention in the life sciences and in biomedical applications because of their excellent resolution and sensitivity. However, adequate theoretical levels to provide efficient and accurate estimations of the optical and electronic properties of organic NIR-II fluorophores are lacking. The standard approach for these calculations has been time-dependent density functional theory (TDDFT). However, the size and large excitonic energies of these compounds pose challenges with respect to computational cost and time. In this study, we used the GW approximation combined with the Bethe-Salpeter equation (GW-BSE) implemented in many-body perturbation theory approaches based on density functional theory. This method was used to perform calculations of the excited states of two NIR molecular fluorophores (BTC980 and BTC1070), going beyond TDDFT. In this study, the optical absorption spectra and frontier molecular orbitals of these compounds were compared using TDDFT and GW-BSE calculations. The GW-BSE estimates showed excellent agreement with previously reported experimental results.

Fluorescence turn-on chemodosimetric sensing of cyanide by cyanovinylterpyridine modified phthalonitrile and subphthalocyanine.

Terpyridine-attached phthalonitrile (Pn-TP) linked by cyanovinyl bond has been synthesized and employed for the preparation of subphthalocyanine (SubPc-TP) bearing conjugated terpyridine moieties. Both Pn-TP and SubPc-TP exhibited highly selective fluorescence turn-on in the presence of cyanide anions (CN-) based on chemodosimetric sensing mechanism. The conjugation of the Pn-TP molecule was interrupted by the addition of CN- at the cyanovinyl bond, showing the ratiometric fluorescence turn-on behavior. This sensing mechanism was further supported by density functional theory calculation and nuclear magnetic resonance titration studies. Optical and photophysical responses of SubPc-TP towards CN- were also investigated, in which similar fluorescence enhancement was observed due to the addition of CN- at the reactive boron trimer. The detection limit was estimated to be 94 nM, much below the World Health Organization-allowed level (1.9 µM) of CN- in water.

Sub-phthalocyanine-incorporated Fe(ii) metallo-supramolecular polymer exhibiting blue-to-transmissive electrochromic transition with high transmittance and coloration efficiency.

The preparation of a new Fe(ii) metallo-supramolecular polymer (poly-subPc-Fe) constructed from a terpyridine-functionalized sub-phthalocyanine with axially substituted polyisobutylene is presented. The as-prepared poly-subPc-Fe exhibited an excellent blue-to-transmissive electrochromic transition with high transmittance of 91.8%, superior coloration efficiency of 325.3 cm2 C-1, and stable redox cycling even after 1000 cycles.

Ultrastructure and phylogenetic placement within Heterolobosea of the previously unclassified, extremely halophilic heterotrophic flagellate Pleurostomum flabellatum (Ruinen 1938).

Although Pleurostomum was described almost a century ago, flagellates assigned to this taxon have been recorded only in very occasional faunistic studies of highly saline habitats, and their phylogenetic position has remained uncertain. We report the cultivation, ultrastructure, and phylogenetic relationships of Pleurostomum flabellatum isolated from a Korean saltern pond of 313 per thousand salinity. This isolate is biflagellated with a cytostomal groove, and is not distinguishable from previous accounts of P. flabellatum from saturated brines in India and Australia. Pleurostomum flabellatum shows ultrastructural features characteristic of many Heterolobosea: (1) a striated rhizoplast, (2) an absence of stacked Golgi bodies, (3) parallel basal bodies and flagella, and (4) a large number of peripheral microtubules supporting a rostrum. 18S rRNA gene phylogenies strongly confirm the affinities of P. flabellatum within Heterolobosea. Furthermore, the 18S rRNA gene of P. flabellatum has the heterolobosean-specific helix 17_1, and a group I intron in the same position as in Acrasis rosea. Within Heterolobosea, the 'amoeboflagellate' genera Naegleria and Willaertia were its closest relatives with high bootstrap support and posterior probability. P. flabellatum was observed only as a flagellate, and never as an amoeba. Since light microscopy and electron microscopy observations indicate that P. flabellatum flagellates are capable both of feeding and division, there might be no amoeba stage. Being morphologically distinct from its closest relatives and phylogenetically distant from other flagellate-only Heterolobosea, P. flabellatum cannot be moved into any previously described heterolobosean genus. Instead, we move Pleurostomum into Heterolobosea, and assign as the type species Pleurostomum salinum Namyslowski 1913, a species that closely resembles P. flabellatum. The optimal temperature for growth of P. flabellatum is 40 degrees C. Interestingly, P. flabellatum grows optimally at 300 per thousand salinity and fails to grow below 200 per thousand salinity, indicating that it is an 'extreme halophile'. The optimal salinity for growth is the highest for any eukaryote examined to date.

Comprehensive review of intracranial chordoma.

Intracranial chordoma is a locally aggressive and relatively rare tumor of the skull base that is thought to originate from embryonic remnants of the primitive notochord. Both computed tomography (CT) and magnetic resonance (MR) imaging are usually required for evaluation of intracranial chordomas due to bone involvement and the proximity of these tumors to many critical soft-tissue structures. At CT, intracranial chordoma typically appears as a centrally located, well-circumscribed, expansile soft-tissue mass that arises from the clivus with associated extensive lytic bone destruction. However, MR imaging is the single best imaging modality for both pre- and posttreatment evaluation of intracranial chordoma. On T1-weighted MR images, intracranial chordomas demonstrate intermediate to low signal intensity and are easily recognized within the high-signal-intensity fat of the clivus. On T2-weighted MR images, they characteristically demonstrate very high signal intensity, a finding that likely reflects the high fluid content of vacuolated cellular components. Moderate to marked enhancement is common and often heterogeneous on contrast material-enhanced images. Combination treatment with radical surgical resection and proton beam radiation therapy achieves the best results.

Highly selective response of bipyridyl-incorporated acetyelene dye for zinc acetate.

In this paper, we prepared bpy-modified acetylene dye 2 that contains 2,2'-bipyridine moiety in the center with symmetrically substituted flexible derivatives at both ends. Upon exposure toward various metal salts, 2 revealed a highly selective optical response to Zn(2+) with AcO(-) as a counteranion, which is evidenced by large red shift and significant enhancement in emission intensity. Such a peculiar response to single zinc compound, as far as we are aware, has not been reported elsewhere. Compound 2 will find important usefulness in chemo-sensor application due to its highly selective binding to zinc acetate.

Lithium-induced supramolecular hydrogel.

We describe a novel anisotropic supramolecular gel made of cyclodextrin-dye, in which physical gelation is completed by lithium salt. Rheological experiment reveals the elastic behaviors of the hydrogel, and high ionic conductivity represents a good mobility of ions inside the gel matrix.

Halocafeteria seosinensis gen. et sp. nov. (Bicosoecida), a halophilic bacterivorous nanoflagellate isolated from a solar saltern.

Recently, heterotrophic nanoflagellates (HNF) have been reported to actively ingest prokaryotes in high salinity waters. We report the isolation and culture of an HNF from a Korean saltern pond of 300 per thousand salinity. The organism is biflagellated with an acronematic anterior flagellum and never glides on surfaces. The mitochondria have tubular cristae. Neither transitional helix nor spiral fiber were observed in the transition zones of the flagella. The cell has a cytostome supported by an arc of eight microtubules, suggesting that our isolate is a bicosoecid. Our isolate had neither mastigonemes, lorica, body scales, nor cytopharynx and thus could not be placed in any of the presently described bicosoecid genera. Phylogenetic analysis of 18S rRNA gene sequences from stramenopiles confirmed the bicosoecid affinities of our isolate, but did not place it within any established genus or family. Its closest relatives include Caecitellus and Cafeteria. The optimal range of growth temperature was 30-35 degrees C. The isolated HNF grew optimally at 150 per thousand salinity and tolerated up to 363 per thousand salinity, but it failed to grow below 75 per thousand salinity, indicating that it could be a borderline extreme halophile. On the basis of its morphological features and position in 18S rRNA trees we propose a novel genus for our isolate; Halocafeteria, n. gen. The species name Halocafeteria seosinensis sp. nov. is proposed.

Reduced fluorescence quenching of cyclodextrin-acetylene dye rotaxanes.

An acetylene dye rotaxane with alpha-CD has been synthesized using the Heck-Cassar-Sonogashira-Hagihara-type reaction in aqueous solution. Free dye with tetracarboxylic acids is found to be highly sensitive to various metal ions, showing high Stern-Volmer constants, KSV. As CD encapsulation protects and stabilizes the threaded chromophore against an outside quencher, metal-insensitive biological tags are an obvious application for this class of molecules.