Cyan Fluorescence Research Articles

A selective turn-off fluorescence detection of nitroexplosive 2,4,6-trinitrophenol by pyridinium-based chemosensor in pure aqueous medium

A chemosensor 1-(2-(6-oxobenzo [4,5]imidazo [1,2-c]quinazolin-5(6H)-yl)ethyl)pyridin-1-ium bromide (BZQZPy) bearing benzimidazo-quinazolinone and pyridinium moieties has been developed for the nanomolar detection of 2,4,6-trinitrophenol (TNP) in aqueous medium. BZQZPy exhibited excellent cyan blue fluorescence that is quenched on the addition of TNP via turn-off fluorescence response. The UV–visible, fluorescence and 1H NMR spectroscopy, lifetime measurements and theoretical calculations suggested that ground state charge transfer complex formation and energy transfer mechanisms are possible causes for this fluorescence quenching. BZQZPy is highly sensitive and selective towards TNP with high quenching constant (Ksv) of 1.53 × 104 M−1 and low detection limit of 20 nM. The proposed chemosensor is also applicable for the on-site determination of TNP on filter paper and in real water samples.

Investigation of crystal structure, nonlinear optical and magnetic properties of a new tetrachlorocobaltate(II) anion salt with benzyl pyridinium cation based on DFT calculation

The crystal of a new tetrachlorocobalt(II) anion salt, [BzPy]2[CoCl4] (1) ([BzPy]+ = benzyl pyridinium), was grown by the slow evaporation solution growth method at room temperature. The salt 1 crystallizes in the monoclinic space group P2(1)/c and consists of two [BzPy]+ cations and one [CoCl4]2− anion in which the Co(II) ion is coordinated by four Cl atoms in a close to tetrahedral geometry. The crystal packing is stabilized by electrostatic interactions and C − H···Cl hydrogen bonds between [BzPy]+ cations and [CoCl4]2− anions. Powder XRD diffraction study confirms the crystalline nature and purity of the title compound. The Fourier transform infrared (FT-IR) spectrum was recorded and analyzed with the aid of DFT calculations in order to make a suitable assignment of the observed bands. The thermal stability of the hybrid crystal was analyzed by TG-DTA techniques revealing that the title crystal was stable up to 195 °C. The title salt exhibits violet and cyan fluorescence emissions upon excitation at 345 nm and weak antiferromagnetic interactions in the temperature range 2-300 K. The nonlinear optical (NLO) effect of this compound was predicted using DFT, suggesting that the title crystal is a good material for NLO applications.

Biomass-derived nitrogen self-doped carbon dots via a simple one-pot method: Physicochemical, structural, and luminescence properties

The efficient disposal and high-value utilization of biomass are crucial for easing the worsening environmental, resource, and energy situations. “Green” carbon dots (CDs) have got a lot of attention recently and biomass is a good “green” carbon source. In this work, cellulose and protein were used as typical components, and peanut shell, cotton stalk, and soymeal were utilized as typical biomass to synthesize nitrogen self-doped CDs with outstanding optical properties via a one-step and green microwave-assisted hydrothermal method without any additive. The reaction mechanism was briefly analyzed, and cellulose and protein differentiated the functional characteristics of the biomass-based CDs. A close connection was observed between optical properties and physicochemical structure, such as sp2 C, N-doped surface states, and the synergy among them. Pyridinic N, amine N, and sp2 C can reduce the bandgap. Quaternary N is beneficial for anti-interference in the CDs due to the large Stokes shift. The absolute photoluminescence quantum yields were measured as 2.83%–6.81%. The solid state of the CDs from protein, cotton stalk, and soymeal can emit clear cyan and claybank fluorescence under ultraviolet light. CDs prepared from “cellulosic” biomass and “protein-rich” biomass had significant differences in physicochemical, structural and luminescence characteristics.

Room-Temperature Dual Fluorescence of a Locked Green Fluorescent Protein Chromophore Analogue.

A structurally locked green fluorescent protein (GFP) chromophore with a phenyl group at C(2) of the imidazolone has been synthesized. Rotation around the exocyclic double bond is hindered, resulting in room-temperature fluorescence. The quantum yield in water is 500 times greater than that of unlocked analogues. Unlike the methyl-substituted analogue, the phenyl analogue exhibits a dual emission (cyan and red) that can be used for ultrasensitive ratiometric measurements and fluorescence microscopy. To explain this dual emission, DFT calculations were carried out along with fluorescence upconversion experiments. The Z-isomer was found to be emissive, while the origin of the dual emission was dependent on the phenyl group in the Z-isomer, which stabilizes the Franck-Condon state, resulting in a cyan fluorescence, while the zwitterionic tautomer fluoresces red. These results bring important new insights into the photophysics of the GFP chromophore and provide a new scaffold capable of dual emission with utility in biotechnology.

MolecuLight i:X: A new tool for wound infection diagnosis

Background. The MolecuLight i:X device is intended to assist healthcare practitioners managing patients with wounds by enabling real-time visualisation of potentially harmful bacteria. Healthcare practitioners are provided with a visual indication of bacterial presence, load and location within and around wounds. Objectives. We decided to use the device in a cohort of burns patients in an attempt to test its effectiveness and applicability in an adult burn centre. Methods. We were given permission by our hospital to use the MolecuLight i:X device for a test period of 1 month, and elected to do a pilot observational study to test its application on burn wounds. A total of 10 patients were assessed for presence of infection (red or green flare). Results . Seven patients showed either cyan or red fluorescence. Pseudomonas aeruginosa was confirmed on wound swabs in the 4 patients with cyan fluorescence. The red fluorescence in 3 patients showed on wound swabs as either MRSA, Streptococcus pyogenes or Proteus mirabilis . The remaining 3 patients did not show any fluorescence, and there was no growth on wound swabs. Conclusion . The MolecuLight i:X is a new device for identifying Gram-positive and Gram-negative bacteria on an open wound. It is of great practical benefit to the surgeon in making decisions about the adequacy of debridement before subsequent skin grafts, and it also identifies target areas for antiseptics.

Chemically-modulated turn-on fluorescence for rapid and visual discrimination of norepinephrine and epinephrine and its application for dopamine-β-hydroxylase detection

Monitoring the concentration of norepinephrine (NE), epinephrine (Ep) and dopamine-β-hydroxylase (DβH) is of great importance in studying and diagnosing related diseases. In this study, a simple chemically-modulated turn-on fluorescence strategy is proposed for rapid, selective and visual discrimination of trace NE and Ep. Herein, Ep is differentiated from other catecholamines via its fast fluorescence response in 1.0 M NaOH medium. Furthermore, NE is alkalized with 0.01 M NaOH and is then immediately triggered by polyethyleneimine to emit bright cyan fluorescence, whereas Ep and DA show no any fluorescence response. The fluorescence intensity of resultant solution is linearly proportional to the concentration of Ep and NE in the range of 50.0–10000.0 nM and 10.0–10000.0 nM, respectively. Moreover, NE and Ep as low as 500.0 nM can be fluorescently identified with naked-eye, which is convenient for rapidly preliminary diagnosis and self-test of hypercatecholaminism. Inspired by the catalytic property of DβH transforming DA into NE, the proposed strategy enables a simple and high-efficient fluorescence method to monitor DβH by using trace DA as substrate and directly quantifying NE. The method is simple, smart, highly efficient and eco-friendly, and has been applied to the rapid assay of NE, Ep and DβH in real samples.

Biodistribution and Efficacy of L-Annamycin in a Novel Imageable AML Mouse Model

Annamycin (Ann) is an anti-tumoral anthracycline whose anti-leukemia activity is relatively unaffected by P-glycoprotein-related multidrug resistance. Unlike for the related doxorubicin (DOX), Ann accumulates in multidrug resistant cell lines, which is accompanied by DNA damage and apoptosis. In preclinical toxicology studies, in contrast to DOX, free Ann displayed a greatly reduced cardiotoxicity, while L-Ann appeared to be non-cardiotoxic. A liposomal formulation of Ann, termed L-Annamycin (L-Ann), is currently evaluated in patients with acute myeloid leukemia (AML). Anti-leukemia activity of Ann was demonstrated in several leukemia models as judged by circulating blast cytoreduction and extension of overall survival. However, the efficacy of L-Ann in the microenvironment of the bone marrow and other organ tissues remains unclear. In the current study, we assessed the anti-AML efficacy of Ann in a novel AML model that allows visualizing the dynamics of individual AML cells in vivo by two-photon microscopy. In this model, mouse AML cells bearing the MLL/ENL-FLT3/ITD[p53-/-] mutations co-express high levels of the cyan fluorescent protein mTurquoise2. Upon intravenous infusion of several tens of thousands cells into syngeneic immunocompetent C57BL6 mice, lethal AML disease reliably develops within 2 weeks. Using host mice expressing appropriate fluorescence reporter genes, the bright cyan fluorescence enables sensitive intravital imaging of individual AML cells in the context of organ architecture. Using this model in Thy1-RFP reporter mice expressing red fluorescence in all organ tissues with the blood flow marked by BSA-AF647 fluorescence, we evaluated AML cellularity reduction in the bone marrow and other organs after a single dose of L-Ann as well as in response to chronic treatment. In addition, we assessed the localization of the surviving AML cells at a high spatial resolution. We evaluated the in vivo organ biodistribution of intravenously infused L-Ann in C57BL6 mice by flow cytometry and two-photon microscopy based on the intrinsic fluorescence of the drug. In addition, we visualized the intracellular compartmentalization of L-Ann using confocal microscopy. Consistent with in vitro findings, we observed a rapid and deep reduction of AML blasts in the peripheral blood after a single dose of L-Ann in a dose-dependent manner (1-4 mg/kg). This reduction was strongly correlated with prolongation of animal survival from 14 days (vehicle) to 37 days (L-Ann 4 mg/kg once weekly started on day 10). In vitro and intravital microscopy revealed a distinct pattern of L-Ann distribution in organ tissues, which correlated in part with the local index of AML cellularity reduction and residual disease localization. Interestingly, in addition to the expected uptake of Ann in the cell's nucleus, Ann was also accumulated in the cytosol of the cells. This bi-compartmental intracellular distribution pattern contrasted with the nuclear-only localization of DOX. Administration of L-Ann early in the course of AML resulted in occasional complete responses some of which associated with resistance to AML re-challenge, suggesting capacity for anti-AML immune memory induction. This study confirms the efficacy of the drug in the model setting of syngeneic, immune-competent AML. Besides reinforcing the rationale for further development of Annamycin in AML, this study demonstrates a highly advantageous AML mouse model that is highly informative in studies of AML pharmacology, minimum residual disease (MRD), microenvironment and immunology. Disclosures Fokt: Moleculin Biotech, Inc.: Equity Ownership, Research Funding. Andreeff:Oncoceutics: Equity Ownership; Senti Bio: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Daiichi Sankyo, Inc.: Consultancy, Patents & Royalties: Patents licensed, royalty bearing, Research Funding; Jazz Pharmaceuticals: Consultancy; Celgene: Consultancy; Amgen: Consultancy; AstaZeneca: Consultancy; 6 Dimensions Capital: Consultancy; Reata: Equity Ownership; Aptose: Equity Ownership; Eutropics: Equity Ownership; Leukemia Lymphoma Society: Membership on an entity's Board of Directors or advisory committees; NCI-RDCRN (Rare Disease Cliln Network): Membership on an entity's Board of Directors or advisory committees; CLL Foundation: Membership on an entity's Board of Directors or advisory committees; BiolineRx: Membership on an entity's Board of Directors or advisory committees; German Research Council: Membership on an entity's Board of Directors or advisory committees; NCI-CTEP: Membership on an entity's Board of Directors or advisory committees; Cancer UK: Membership on an entity's Board of Directors or advisory committees; Oncolyze: Equity Ownership; Breast Cancer Research Foundation: Research Funding; CPRIT: Research Funding; NIH/NCI: Research Funding; Center for Drug Research & Development: Membership on an entity's Board of Directors or advisory committees. Priebe:Moleculin Biotech, Inc.: Consultancy, Equity Ownership, Research Funding. Zal:VueBio.com: Equity Ownership; BioLineRx: Research Funding; Daiichi-Sankyo: Research Funding; Moleculin Biotech, Inc.: Research Funding; NIH-CTEP: Research Funding; CPRIT: Research Funding; NIH/NCI: Research Funding.

Intravital Imaging of Bright Cyan-Fluorescent AML Model Reveals Impact of CXCR4 Inhibitor on AML and Immune Cell Dynamics

INTRODUCTION: CXCR4 chemokine receptor inhibitors such as BL-8040 (BioLineRx) have been investigated by us and others as possible anti-leukemic drugs due to their ability to "mobilize" leukemia cells out of the BM and into the circulation, where they are more sensitive to chemotherapy. However, the exact mode of cell relocation remains unclear. CXCR4/CXCL12 signaling pathway also participates in BM homing of immune cells, including both central memory T cells and immunosuppressive CD4+FoxP3+ T-regulatory cells (T-reg). Therefore, CXCR4 inhibition has the potential to either counteract or enhance the process of AML immune surveillance. Therefore, we sought to develop a syngeneic AML model for intravital 2-photon microscopy (TPM) compatible with existing immune reporter mouse strains, which typically occupy the green, yellow and red fluorescence channels. HYPOTHESIS: CXCR4 inhibition decreases AML and T cell BM cellularity by increasing the rate of intravascular cell entry and/or decreasing the rate of circulating cell homing back to BM. MODEL: The cyan-colored fluorescent protein mTurquoise2 was lentivirally introduced into C57BL6-origin AML cells containing the MLL, ENL-FLT3, ITD, and p53-/- mutations, termed AML1-mTurq2. Syngeneic FoxP3-GFP/CD11c-YFP/hCD2-DsRed reporter mice were generated by inter-breeding of the corresponding strains, respectively highlighting T-reg, myeloid antigen presenting cells, and all T cells. After intravenous infusion of 1E5 AML1-mTurq2 cells, 1-2% blasts appeared in peripheral blood on day 9, increasing to 70% on day 15-20 when animals had to be euthanized. TREATMENT: Mice with >1% blasts were given BL-8040 I.P. in two daily 400 µg doses followed by imaging 24 h later, or intravenously during imaging 10 µg and 50 µg one hour later. ANALYSIS: Disease progression was characterized by blood flow cytometry, symptom scoring and thick-mount organ tissue fluorescence microscopy. Intravital TPM of the calvarial bone marrow (BM) was performed through intact bone under general anesthesia. By interline multiplexing dual femtosecond lasers with four-sensor detection for 8 distinct channels, mTurquoise2 and SHG were recorded by the same sensor at, respectively, 860 and 990 nm excitation, along with GFP, YFP, DsRed and dextran-TRITC (blood tracer). AML and T cell subsets were 3-D tracked using Imaris software. RESULTS: AML1-mTurq2 cells stably and uniformly expressed bright cyan fluorescence, suitable for intravital TPM with low incident laser powers and fast imaging rates in deep tissue locations. In C57BL6 mice, sparse AML cell clusters were found in BM perivascular spaces on day 1 after cell infusion. AML cells were slowly motile (~4 um/min) and highly proliferative, gradually filling BM spaces and emerging in other organs. T cells and CD11c dendritic cells were present in leukemic BM, and the vasculature appeared largely intact and well perfused. T cells interacted with AML cells and the stroma, migrating with high average velocities (~10 µm/min) and slowing down to ~3 µm/min in late-stage disease. After 2 days of BL-8040 treatment, disease symptom scores improved from 3 to 1 while the untreated controls progressed from 3 to 4 (range 0-6). TPM revealed a 4-fold reduction of AML cellularity in BM. Cellular velocities of both AML and T cells were unchanged by BL-8040 treatment. After acute drug administration, a fraction of stromal AML cells begun entering capillary vessel lumens by amoeboid movement. The intravasated AML cells adhered to vessel wall for 1-2 minutes before rapid detachment. Some cells remained tethered while already loose in the blood stream. CONCLUSIONS: A novel, brightly cyan-fluorescent syngeneic AML1-mTurq2 AML model is advantageous for 6-color intravital microscopy of cell trafficking and immune surveillance in optimal compatibility with green, yellow and red reporters of cell lineages and tissue architecture. Using this model, we show that CXCR4 inhibitor BL-8040 decreases AML BM cellularity by increasing the frequency of intravasation without increasing AML migratory velocity. Disclosures Zal: Daiichi-Sankyo: Research Funding; NIH-CTEP: Research Funding; BioLineRx: Research Funding; VueBio.com: Equity Ownership; NIH/NCI: Research Funding; CPRIT: Research Funding; Moleculin Biotech, Inc.: Research Funding. Andreeff:BiolineRx: Membership on an entity's Board of Directors or advisory committees; Aptose: Equity Ownership; Eutropics: Equity Ownership; Senti Bio: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Oncoceutics: Equity Ownership; Oncolyze: Equity Ownership; Breast Cancer Research Foundation: Research Funding; CPRIT: Research Funding; CLL Foundation: Membership on an entity's Board of Directors or advisory committees; NCI-RDCRN (Rare Disease Cliln Network): Membership on an entity's Board of Directors or advisory committees; Leukemia Lymphoma Society: Membership on an entity's Board of Directors or advisory committees; German Research Council: Membership on an entity's Board of Directors or advisory committees; NCI-CTEP: Membership on an entity's Board of Directors or advisory committees; Cancer UK: Membership on an entity's Board of Directors or advisory committees; Center for Drug Research & Development: Membership on an entity's Board of Directors or advisory committees; NIH/NCI: Research Funding; Reata: Equity Ownership; 6 Dimensions Capital: Consultancy; AstaZeneca: Consultancy; Amgen: Consultancy; Celgene: Consultancy; Daiichi Sankyo, Inc.: Consultancy, Patents & Royalties: Patents licensed, royalty bearing, Research Funding; Jazz Pharmaceuticals: Consultancy.

A colorimetric and fluorometric probe for hydrazine through subsequent ring-opening and closing reactions: Its environmental applications

Hydrazine (N2H4), as one of the essential industrial chemicals, is main water contaminant and highly toxic for human health. Therefore, the rapid, selective and sensitive detection strategies for hydrazine are needed. The present study describes the first example of a receptor based on pyrazolone and hydrazide formation by subsequent ring-opening and closing reactions displayed distinct naked eye colorimetric and visual fluorescence sensing towards hydrazine with rapid reactivity and very good selectivity over various anions, cations and amines. The probe BC was obtained in good yield by three stages. The sensing mechanism was confirmed by 1H NMR and 13C NMR spectra. Upon interaction with hydrazine, coumarin ring was easily opened and pink color followed by purple was formed. The more hydrazine addition triggered pyrazolone ring formation with yellow colored solution in company with the remarkable cyan blue turn-on fluorescence output. An easy-to-produce TLC test strip enables to determine hydrazine in the solution. The results were successfully shown to be convenient and efficient for hydrazine detection. Therefore, the present strategy is supposed to be widely applied to fabricate fluorescent sensors for detection of hydrazine.

Investigation of autofluorescence in zooplankton for use in classification of larval salmon lice.

We present autofluorescence of six zooplankton species, including salmon lice (Lepeophtheirus salmonis), for the purpose of classification in marine environments. Using a 410nm excitation wavelength, we find that all measured zooplankton species exhibit broad cyan fluorescence centered around 510-520nm. Furthermore, salmon lice show an absence of red fluorescence from undigested chlorophyll, which is measured from the gut of the herbivorous zooplankton species. We show the capability to distinguish noneating species, including salmon lice, from algae-eating species using a dual-band analysis of the fluorescence spectra. This shows the potential of autofluorescence as an important signature in real-time monitoring and classification of salmon lice.

Fluorescence imaging guided dressing change frequency during negative pressure wound therapy: a case series.

Knowledge of wound bioburden can guide selection of therapies, for example, the use of negative pressure wound therapy (NPWT) devices with instillation in a heavily contaminated wound. Wound and periwound bacteria can be visualised in real-time using a novel, non-contact, handheld fluorescence imaging device that emits a safe violet light. This device was used to monitor bacterial burden in patients undergoing NPWT. Diverse wounds undergoing NPWT were imaged for bacterial (red or cyan) fluorescence as part of routine wound assessments. We assessed 11 wounds undergoing NPWT. Bacterial fluorescence was detected under sealed, optically-transparent (routine) adhesive before dressing changes, on foam dressings, within the wound bed, and on periwound tissues. Bacterial visualisation in real-time helped to guide: (1) bioburden-based, personalised treatment regimens, (2) clinician selection of NPWT, with or without instillation of wound cleansers, and (3) the extent and location of wound cleaning during dressing changes. The ability to visualise bacteria before removal of dressings led to expedited dressing changes when heavy bioburden was detected and postponement of dressing changes for 24 hours when red fluorescence was not observed, avoiding unnecessary disturbance of the wound bed. Fluorescence imaging of bacteria prompted and helped guide the timing of dressing changes, the extent of wound cleaning, and selection of the appropriate and most cost-effective NPWT (standard versus instillation). These results highlight the capability of bacterial fluorescence imaging to provide invaluable real-time information on a wound's bioburden, contributing to clinician treatment decisions in cases where bacterial contamination could impede wound healing.

Generating Cyan Fluorescence with De Novo Tripeptides: An In Vitro Mutation Study on the Role of Single Amino Acid Residues and Their Sequence.

Amino acids are natural choices as building blocks when developing biofunctional entities owing to their superior diversity and versatile physicochemical properties compared to nucleotide bases. A simple permutation of the amino acids creates a broad palette of proteins and these have been successfully engineered into useful biofunctional agents. For example, the intrinsic ultraviolet fluorescence of phenylalanine and tryptophan has been engineered to emit in the visible spectrum, which has broad applications for imaging/sensing probes, photothermal therapy agents, optogenetic switches, etc. Nature produces more colorful coats/furs, feathers/hairs, and eyes through various biochemical modifications of tyrosine-based pigmentation. However, it is challenging to modulate the fluorescence wavelength from the UV to the visible region through oligopeptides. Herein, we report an innovative approach to obtain cyan fluorescence by using de novo tripeptides containing glycine, tyrosine, and lysine, which form robust dimer structures under moderate oxidizing conditions. Through an in vitro mutation approach, we deduce that both the amino acids and their sequence play significant roles in modulating the fluorescence. We believe this work holds great promise for developing novel cell imaging and resonance energy-transfer-based fluorescent probes.

Europium(III) modified silicone nanoparticles for ultrasensitive visual determination of tetracyclines by employing a fluorescence color switch.

Silicon nanoparticles (SiNPs) modified with Eu(III) were synthesized and are shown to be a viable ratiometric fluorescent probe for tetracycline antibiotics. SiNPs/Eu under 405nm excitation display two emissions, viz. a strong cyan colored fluorescence peaking at 497nm and a weak pink fluorescence peaking at 622nm. On addition of tetracyclines (chlortetracycline, tetracycline, doxycycline), the fluorescence at 497nm is reduced, while the one at 622nm is increased. Thus, the visible color of fluorescence changes from cyan to pink. This was exploited to design ratiometric fluorometric method for detecting tetracyclines. The method has a limit of detection that is lower by a factor of about 1000 when compared to the use of SiNPs only. A test paper was prepared with the SiNPs/Eu and then applied for the visual semi-quantitative detection of tetracyclines. With the addition of tetracyclines, the test paper exhibited a dosage-sensitive color conversion from cyan to pink with a visually discernible scale as low as 0.4μM. Graphical abstract Tetracyclines decrease the fluorescence at 497nm of europium (III) modified silicon nanoparticles (SiNPs/Eu) due to the inner filter effect and increase the one at 622nm due to an antenna effect. Thus the fluorescence color of SiNPs/Eu changes from cyan to pink. Based on this color switch, a ultrasensitive and visual determination strategy for tetracyclines is proposed.

Role of c-MET Inhibitors in Overcoming Drug Resistance in Spheroid Models of Primary Human Pancreatic Cancer and Stellate Cells.

Pancreatic stellate cells (PSCs) are a key component of tumor microenvironment in pancreatic ductal adenocarcinoma (PDAC) and contribute to drug resistance. c-MET receptor tyrosine kinase activation plays an important role in tumorigenesis in different cancers including PDAC. In this study, effects of PSC conditioned medium (PCM) on c-MET phosphorylation (by immunocytochemistry enzyme-linked immunosorbent assay (ELISA)) and drug response (by sulforhodamine B assay) were investigated in five primary PDAC cells. In novel 3D-spheroid co-cultures of cyan fluorescence protein (CFP)-firefly luciferase (Fluc)-expressing primary human PDAC cells and green fluorescence protein (GFP)-expressing immortalized PSCs, PDAC cell growth and chemosensitivity were examined by luciferase assay, while spheroids’ architecture was evaluated by confocal microscopy. The highest phospho-c-MET expression was detected in PDAC5 and its subclone sorted for “stage specific embryonic antigen-4” (PDAC5 (SSEA4)). PCM of cells pre-incubated with PDAC conditioned medium, containing increased hepatocyte growth factor (HGF) levels, made PDAC cells significantly more resistant to gemcitabine, but not to c-MET inhibitors. Hetero-spheroids containing both PSCs and PDAC5 (SSEA4) cells were more resistant to gemcitabine compared to PDAC5 (SSEA4) homo-spheroids. However, c-MET inhibitors (tivantinib, PHA-665752 and crizotinib) were equally effective in both spheroid models. Experiments with primary human PSCs confirmed the main findings. In conclusion, we developed spheroid models to evaluate PSC–PDAC reciprocal interaction, unraveling c-MET inhibition as an important therapeutic option against drug resistant PDAC.

Ocular Effects of Sildenafil in Naïve Mice and a Mouse Model of Optic Nerve Crush.

To investigate the potential neuroprotective effect of sildenafil on the ocular circulation in mice with/without optic nerve crush (ONC). Male adult mice (n = 63) were treated with intravitreal (IVT) sildenafil 24 μg/3 μL, intraperitoneal (IP) sildenafil 24 μg/300 μL, or IP saline immediately before right ONC induction (ONC group). A second group (n = 123) received the same treatments without ONC induction (naïve group). Evaluations included fluorescein angiography (naïve group; day 0), molecular studies (days 1 and 3), and retinal and optic nerve histology (day 21). Maximal retinal vessel dilatation and increased choroidal effusion were detected within 30 minutes of sildenafil injection. In the ONC group, moderate retinal ganglion cell (RGC) loss was noted at 21 days. However, molecular studies showed increased stress induced gene expression (IP superoxide dismutase [SOD]-1: 3.1-fold; heme oxygenase [HO]-1: 5.8-fold; IVT SOD-1: 1.47-fold), proapoptotic gene expression (IP BAX/B-cell lymphoma [BCL]-2 10.8-/2.3-fold), and glial gene expression (IP glial fibrillary acidic protein [GFAP]: 2.8- and myelin basic protein [MBP]: 2.5-fold). In the naïve group, IVT sildenafil was not associated with RGC loss or optic nerve stroke on histology, although in two samples, molecular parameters were compatible with stroke, showing increased gene expression of HO-1 (3.8-fold) and BCL-2 (2.5-fold). In the IP sildenafil subgroup, optic neuropathy was observed in 6/120 optic nerves, including 3 cyan fluorescence protein (CFP)-Thy-1 mice. Levels of antiapoptosis and anti-ischemia genes were decreased (<0.5-fold) except for three outliers. Sildenafil affects retinal and choroidal perfusion in mice. When injected immediately before ONC, molecular parameters showed a preconditioning neuroprotective effect while histologic studies did not. In the absence of ONC, it is associated with neuropathy, possibly dose-dependent.

Synthesis, physical properties and electroluminescence of functionalized pyrene derivative

Developing novel polycyclic aromatic hydrocarbons with high stability is of great importance, as well for opto-electronic applications as to provide novel materials for organic soft matter devices. Here the synthesis of a cyclized arene 13 and the corresponding acene-functionalized pyrene derivative 8 are described. Both molecules have been characterized by FT-IR, NMR spectroscopy, and mass spectroscopy. Their opto-electronic properties were determined from their absorption and emission spectra and electrochemistry and by theoretical calculations. Molecules 13 and 8 exhibit respectively a strong orange and cyan fluorescence in dichloromethane with high fluorescence quantum yields of 0.45 and 0.84 respectively, and present enhanced photo- and thermal stability compared to other large acenes. The organic light-emitting diodes using 13 and 8 as emitters were fabricated by via spin-coating way. After optimization they exhibited a maximum brightness of 2647 cd m−2 at 16.4 V with CIE coordinate of (0.61, 0.39) and 5620 cd m−2 at 7.2 V with CIE coordinate of (0.28, 0.64), respectively.

PH-Lemon, a Fluorescent Protein-Based pH Reporter for Acidic Compartments.

Distinct subcellular pH levels, especially in lysosomes and endosomes, are essential for the degradation, modification, sorting, accumulation, and secretion of macromolecules. Here, we engineered a novel genetically encoded pH probe by fusing the pH-stable cyan fluorescent protein (FP) variant, mTurquoise2, to the highly pH-sensitive enhanced yellow fluorescent protein, EYFP. This approach yielded a ratiometric biosensor—referred to as pH-Lemon—optimized for live imaging of distinct pH conditions within acidic cellular compartments. Protonation of pH-Lemon under acidic conditions significantly decreases the yellow fluorescence while the cyan fluorescence increases due to reduced Förster resonance energy transfer (FRET) efficiency. Because of its freely reversible and ratiometric responses, pH-Lemon represents a fluorescent biosensor for pH dynamics. pH-Lemon also shows a sizable pH-dependent fluorescence lifetime change that can be used in fluorescence lifetime imaging microscopy as an alternative observation method for the study of pH in acidic cellular compartments. Fusion of pH-Lemon to the protein microtubule-associated protein 1A/1B-light chain 3B (LC3B), a specific marker of autophagic membranes, resulted in its targeting within autolysosomes of HeLa cells. Moreover, fusion of pH-Lemon to a glycophosphatidylinositol (GPI) anchor allowed us to monitor the entire luminal space of the secretory pathway and the exoplasmic leaflet of the plasma membrane. Utilizing this new pH probe, we revealed neutral and acidic vesicles and substructures inside cells, highlighting compartments of distinct pH throughout the endomembrane system. These data demonstrate, that this novel pH sensor, pH-Lemon, is very suitable for the study of local pH dynamics of subcellular microstructures in living cells.

Metastasis‐inducing protein S100A4 interacts with p53 in the nuclei of living cells

AbstractObjectiveS100A4 can bind to p53 in vitro. However, it is unclear whether the proteins interact in the nuclei of living cells.MethodsInitially, using an optical biosensor, the ability of CFP‐S100A4 to bind with p53‐YFP was examined in vitro. The fluorescence resonance energy transfer (FRET) technique using laser confocal microscopy was applied to detect the interaction of S100A4 with p53 in living cells.ResultsImaging FRET in HeLa cells expressing S100A4 and p53 showed an increase in cyan fluorescent protein fluorescence intensity, after photobleaching yellow fluorescent protein in cell nuclei. In comparison, the imaging of cell nuclei expressing mutant S100A4‐C and p53 or S100A4 and yellow fluorescent protein as the negative control showed the occurrence of FRET, and demonstrated that the interaction between S100A4 and p53 occurred in the nuclei of living HeLa cells. However, FRET did not occur in the cytoplasm of cells, showing that S100A4 did not interact with p53 in the cytoplasm.ConclusionS100A4 can interact directly with p53 in the nuclei of living cells. This might be a molecular basis for metastasis‐inducing S100A4 in vivo. The results also suggest that the inhibition of the calcium‐binding site of S100A4 might be a possible anticancer therapy.

Pyromellitic acid-derived highly fluorescent N-doped carbon dots for the sensitive and selective determination of 4-nitrophenol

Highly fluorescent N-doped carbon dots (NCDs) were fabricated using pyromellitic acid (PA) and ethylene diamine (EDA) as novel precursors. The NCDs exhibited excellent fluorescent properties with a quantum yield as high as 75% due to the enhancement effect of the N dopant on the surface of the NCDs. Moreover, owing to its strong cyan fluorescence, the newly synthesized NCDs were applied effectively as a fluorescence platform for the sensitive and selective determination of 4-nitrophenol (4-NP) without further chemical modification. The NCDs fabricated in this study exhibited a wide linear range, low detection limit, and excellent selectivity towards other organic compounds.

Self-Assembly Fluorescent Cationic Cellulose Nanocomplex via Electrostatic Interaction for the Detection of Fe3+ Ions.

In this work, an aggregation-induced emission (AIE) sensor for the detection of Fe3+ ions was fabricated through the electrostatic interaction between 1,1,2-triphenyl-2-[4-(3-sulfonatopropoxyl)-phenyl]-ethene sodium salt (SPOTPE) and quaternized cellulose (QC). The structure and properties of the SPOTPE/QC nanocomplex were studied by using 1H NMR, spectrofluorophotometer, transmission electron microscopy (TEM), and dynamic laser light scattering (DLS). An aqueous solution of SPOTPE and QC resulted in a remarkably enhanced cyan fluorescence in comparison to that of the SPOTPE solution. Strong through-space electrostatic interaction between SPOTPE and QC is the main cause for the fluorescence emerging. The fluorescence of the SPOTPE/QC solutions show good stability over a wide pH range of 5.0–10.0. When introducing Fe3+ ions into the SPOTPE/QC solution, the fluorescence quenched within 5 s. SPOTPE/QC solutions exhibited high selectivity and sensitivity for the detection of Fe3+ ions with ignored interferences from other ions, and the detection limit was determined to be 2.92 × 10−6 M. The quenching mechanism was confirmed to be the consequence of the binding interactions between Fe3+ ions and SPOTPE/QC complex.