Dihydroxanthene-based monoamine oxidase A-activated photosensitizers for photodynamic/photothermal therapy of tumors.

Small molecule photosensitizers for combined in vivo tailored cancer diagnostics and photodynamic/photothermal therapy are desperately needed. Monoamine oxidase A (MAO-A)-activated therapeutic and diagnostic compounds provide great selectivity because MAO-A can be employed as a biomarker for associated Tumors. In order to screen photosensitizers with photodynamic therapeutic potential, we have created a range of near-infrared fluorescent molecules in this work by combining dihydroxanthene parent with various heterocyclic fluorescent dyes. The NIR fluorescent diagnostic probe, DHMQ, was created by combining the screened fluorescent dye matrices with the propylamino group, which is the recognition moiety of MAO-A, based on the oxidative deamination mechanism of the enzyme. This probe has a low toxicity level and can identify MAO-A precisely. It has the ability to use fluorescence imaging on mice and cells to track MAO-A activity in real-time. It has strong phototoxicity and can produce singlet oxygen when exposed to laser light. The temperature used in photothermal imaging can get up to 50 °C, which can harm tumor cells permanently and have a positive phototherapeutic impact on tumors grown from SH-SY5Y xenograft mice. The concept of using MAO-A effectively in diseases is expanded by the MAO-A-activated diagnostic-integrated photosensitizers, which offer a new platform for in vivo cancer diagnostics and targeted anticancer treatment.

Monoamine oxidase B activatable red fluorescence probe for bioimaging in cells and zebrafish.

A real-time and specific for the detection of Monoamine Oxidase B (MAO-B) to investigate the MAO-B-relevant disease development and treatment process is urgently desirable. Here, we utilized MAO-B to catalyze the conversion of propylamino groups to aldehyde groups, which was then quickly followed by a ß-elimination process to produce fluorescent probes (FNJP) that may be used to detect MAO-B in vitro and in vivo. The FNJP probe possesses unique properties, including favorable reactivity (Km = 10.8 µM), high cell permeability, and NIR characteristics (λem = 610 nm). Moreover, the FNJP probe showed high selectivity for MAO-B and was able to detect endogenous MAO-B levels from a mixed population of NIH-3 T3 and HepG2 cells. MAO-B expression was found to be increased in cells under lipopolysaccharide-stimulated cellular oxidative stress in neuronal-like SH-SY5Y cells. In addition, the visualization of FNJP for MAO-B activity in zebrafish can be an effective tool for exploring the biofunctions of MAO-B. Considering these excellent properties, the FNJP probe may be a powerful tool for detecting MAO-B levels in living organisms and can be used for accurate clinical diagnoses of related diseases.

Functional analysis of a lily SHORT VEGETATIVE PHASE ortholog in flowering transition and floral development.

Lilium is a commercially important genus of bulbous flowers, investigating the flowering molecular mechanisms is important for flowering regulation of lily. MADS-box SHORT VEGETATIVE PHASE (SVP) orthologs are involved in the flowering transition and floral organ differentiation in many plants. In this study, we identified an SVP ortholog from L. × formolongi (LfSVP), which was closely related to Arabidopsis SVP according to phylogenetic analysis. Tissue-specific expression patterns indicated that LfSVP expression levels peaked in the leaves and showed low expression levels in flowering tepals. Stage-dependent expression patterns of LfSVP showed high transcription level in the flowering induction stage under different photoperiods and exhibited transcription peak in the floral budding development stage under long days. Overexpressed LfSVP led to delayed flowering and floral organ defects in Arabidopsis independent of photoperiod. Tobacco rattle virus -induced gene silencing of LfSVP caused a strongly earlier flowering time and floral organ defects of L. × formolongi. Moreover, LfSVP can interact with L. × formolongi APETALA1 (AP1) in both yeast and tobacco cells, and the two may interact to regulate floral organ differentiation. In conclusion, LfSVP is a flowering repressor and may be involved in the regulation of floral organ differentiation. This study will be helpful for the molecular breeding of short-life-period and rich floral patterns lily varieties.

Investigating Spatial Heterogeneity of Nanoparticles Movement in Live Cells with Pair-Correlation Microscopy and Phasor Analysis.

How nanoparticles distribute in living cells and overcome cellular barriers are important criteria in the design of drug carriers. Pair-correlation microscopy is a correlation analysis of fluctuation in the fluorescence intensity obtained by a confocal line scan that can quantify the dynamic properties of nanoparticle diffusion including the number of mobile nanoparticles, diffusion coefficient, and transit time across a spatial distance. Due to the potential heterogeneities in nanoparticle properties and the complexity within the cellular environment, quantification of averaged auto- and pair-correlation profiles may obscure important insights into the ability of nanoparticles to deliver drugs. To overcome this issue, we used phasor analysis to develop a data standardizing method, which can segment the scanned line into several subregions according to diffusion and address the spatial heterogeneity of nanoparticles moving inside cells. The phasor analysis is a fit-free method that represents autocorrelation profiles for each pixel relative to free diffusion on the so-called phasor plots. Phasor plots can then be used to select subpopulations for which the auto- and pair-correlation analysis can be performed separately. We demonstrate the phasor analysis for pair-correlation microscopy for investigating 16 nm, Cy5-labeled silica nanoparticles diffusing across the plasma membrane and green fluorescent proteins (GFP) diffusing across nuclear envelope in MCF-7 cells.

Mitochondrial-Targeted and Near-Infrared Fluorescence Probe for Bioimaging and Evaluating Monoamine Oxidase A Activity in Hepatic Fibrosis.

Monoamine oxidase A (MAO-A) is a promising diagnostic marker for cancer, depression, Parkinson's disease, and liver disease. The fluorescence detection of MAO-A in living animals is of extreme importance for the early diagnosis of related diseases. However, the development of specific and mitochondrial-targeted and near-infrared (NIR) fluorescence MAO-A probes is still inadequate. Here, we designed and synthesized four NIR fluorescence probes containing a dihydroxanthene (DH) skeleton to detect MAO-A in complex biological systems. The specificity of our representative probe DHMP2 displays a 31-fold fluorescence turn-on in vitro, and it can effectively accumulate in the mitochondria and specifically detect the endogenous MAO-A concentrations in PC-3 and SH-SY5Y cell lines. Furthermore, the probe DHMP2 can be used to visualize the endogenous MAO-A activity in zebrafish and tumor-bearing mice. More importantly, it is the first time that the MAO-A activity of hepatic fibrosis tissues is detected through the probe DHMP2. The present study shows that the synthesized DHMP2 might serve as a potential tool for monitoring MAO-A activity in vivo and diagnosing related diseases.

Design and Synthesis of a Ratiometric Photoacoustic Probe for In Situ Imaging of Zinc Ions in Deep Tissue In Vivo.

As a noninvasive deep-tissue imaging technique, photoacoustic (PA) imaging has great application potential in biomedicine and molecular diagnosis. The zinc ion (Zn2+), which is a necessary metal ion in the human body, plays a very important role in the regulation of gene transcription and metalloenzyme function. The imbalance of Zn2+ homeostasis is also associated with a variety of neurological diseases. Therefore, it is critically important to accurately image the steady-state changes of Zn2+ in vivo. However, no PA imaging method is currently available for Zn2+. To this end, we designed and synthesized the first PA probe of Zn2+, namely, CR-1 for in situ ratiometric imaging of Zn2+ in deep tissue in vivo. The CR-1molecule, combined with Zn2+, weakened the conjugation system of the π-electron in the CR-1 molecule, which resulted in the blue shift of its absorption peak from 710 nm to 532 nm. The PA signal intensity decreased at 710 nm and increased at 532 nm, and the ratiometric PA signal at these two wavelengths (PA532/PA710) showed a good linear relationship with the concentration of Zn2+ in the range of 0-50 µM, with a detection limit as low as 170 nM. Furthermore, this probe exhibits extremely fast responsiveness, is highly selective, and has excellent biocompatibility. We have used the developed PA probe for the ratiometric PA imaging of Zn2+ in the thigh tissue of mice, and we still can accurately image Zn2+ after covering chicken breast tissue on the surface of mice thigh. In light of these outstanding features, the developed PA probe has high potential for imaging Zn2+ in deep tissues; thus, it will open up new avenues for the study of the complex biochemical processes involving Zn2+ in vivo.

Conformational States Control Lck Switching between Free and Confined Diffusion Modes in T Cells.

T cell receptor phosphorylation by Lck is an essential step in T cell activation. It is known that the conformational states of Lck control enzymatic activity; however, the underlying principles of how Lck finds its substrate over the plasma membrane remain elusive. Here, single-particle tracking is paired with photoactivatable localization microscopy to observe the diffusive modes of Lck in the plasma membrane. Individual Lck molecules switched between free and confined diffusion in both resting and stimulated T cells. Lck mutants locked in the open conformation were more confined than Lck mutants in the closed conformation. Further confinement of kinase-dead versions of Lck suggests that Lck confinement was not caused by phosphorylated substrates. Our data support a model in which confined diffusion of open Lck results in high local phosphorylation rates, and inactive, closed Lck diffuses freely to enable long-range distribution over the plasma membrane.

A near infrared dye-coated silver nanoparticle/carbon dot nanocomposite for targeted tumor imaging and enhanced photodynamic therapy.

An excellent photosensitizer for imaging-guided high efficiency photodynamic therapy (PDT) requires certain features, such as near-infrared (NIR) light emission, high singlet-to-triplet intersystem crossing (ISC) efficiency, and tumor targeting. However, synthetizing photosensitizers that meet the aforementioned characteristics still remains a challenge. In this study, we synthetized a NIR dye (CyOH)-coated silver nanoparticle/carbon dot nanocomposite (CyOH-AgNP/CD) as a novel nanophotosensitizer for targeted tumor imaging and high-efficiency PDT. The CyOH-AgNP/CD nanophotosensitizer was constructed using a NIR dye (CyOH) and an AgNP/CD nanohybrid via Ag-O interaction. Relative to the AgNP/CD nanohybrid, CyOH-AgNP/CD exhibited a high singlet oxygen yield, mitochondrial accumulation, superior tissue penetration of 660 nm laser irradiation, and enhanced tumor targeting. The developed nanophotosensitizer exerted a higher antitumor effect than the CyOH dye or AgNP/CD nanohybrid. This result provides a new idea for the design of excellent photosensitizers that can benefit high-efficiency PDT.

Inhibitor structure-guided design and synthesis of near-infrared fluorescent probes for monoamine oxidase A (MAO-A) and its application in living cells and in vivo.

A series of near-infrared fluorescent probes based on inhibitor (clorgyline) structure-guided design were synthesized for the specific detection of MAO-A in cells and in vivo. Moreover, we have successfully used the high specificity of one of the probes to visualize MAO-A activity in zebrafish and tumor tissue for the first time.

Discovery of anti-inflammatory terpenoids from Mallotus conspurcatus croizat.

ETHNOPHARMACOLOGICAL RELEVANCE: Mallotus conspurcatus croizat (Euphorbiaceae), a plant native to Jinxiu in Guangxi, is popularly used in folk medicine to treat pelvic inflammatory disease. The anti-inflammatory activities of the compounds obtained from M. conspurcatus root were evaluated in this study. AIM OF THE STUDY: This study explored the major anti-inflammatory components of this plant. MATERIALS AND METHODS: The ethyl acetate fraction of the ethanol extract from M. conspurcatus was separated using chromatographic techniques. The structures of the isolates were elucidated from NMR, MS and X-ray data as well as from ECD. The anti-inflammatory activities of the isolates from M. conspurcatus were evaluated using LPS-stimulated RAW 264.7 cell models. The production of NO, TNF-α and PGE-2 was determined by ELISA and Griess tests. The expression levels of COX-2, NF-κB/p65 and iNOS were measured by western blotting. RESULTS: Two new diterpenoids, malloconspur A (1) and malloconspur B (2), and sixteen known terpenoids (3-18) were identified by comprehensive spectroscopic analyses and comparison with literature data. Malloconspur B (2) and 17-hydroxycleistantha-12,15-dien-3-one (3) substantially inhibited the release of NO with IC50 values of 10.47 µM and 9.32 µM, respectively. Compounds 1, 2 and 3 markedly decreased the secretion of PGE2 and TNF-α (P < 0.01) by LPS-induced RAW264.7 cells. Compounds 2 and 3 markedly decreased iNOS, NF-κB/p65 and COX-2 protein expression. CONCLUSIONS: Our identification of these diterpenoids provides strong evidence for the use of M. conspurcatus among the Yao people as a medicinal plant for the treatment of inflammation. The dramatic differences in the chemical structures of the active diterpenoids of this plant from those on the market suggest these compounds have potential as anti-inflammatory lead compounds for follow-up research.

A mobile endocytic network connects clathrin-independent receptor endocytosis to recycling and promotes T cell activation.

Endocytosis of surface receptors and their polarized recycling back to the plasma membrane are central to many cellular processes, such as cell migration, cytokinesis, basolateral polarity of epithelial cells and T cell activation. Little is known about the mechanisms that control the organization of recycling endosomes and how they connect to receptor endocytosis. Here, we follow the endocytic journey of the T cell receptor (TCR), from internalization at the plasma membrane to recycling back to the immunological synapse. We show that TCR triggering leads to its rapid uptake through a clathrin-independent pathway. Immediately after internalization, TCR is incorporated into a mobile and long-lived endocytic network demarked by the membrane-organizing proteins flotillins. Although flotillins are not required for TCR internalization, they are necessary for its recycling to the immunological synapse. We further show that flotillins are essential for T cell activation, supporting TCR nanoscale organization and signaling.

Design, synthesis and biological evaluation of novel 1-hydroxyl-3-aminoalkoxy xanthone derivatives as potent anticancer agents.

A series of novel 1-hydroxyl-3-aminoalkoxy xanthone derivatives were designed, synthesized and evaluated for in vitro anticancer activity against four selected human cancer cell lines (nasopharyngeal neoplasm CNE, liver cancer BEL-7402, gastric cancer MGC-803, lung adenocarcinoma A549). Most of the synthesized compounds exhibit effective cytotoxic activity against the four tested cancer cell lines with the IC50 values at micromolar concentration level. Some preliminary structure-activity relationships were also discussed. In this series of derivatives, compound 3g shows excellent broad spectrum anticancer activity with IC50 values ranging from 3.57 to 20.07 µM. The in vitro anticancer activity effect and action mechanism of compound 3g on human gastric carcinoma MGC-803 cell were further investigated. The results showed that compound 3g exhibits dose- and time-dependent anticancer effects on MGC-803 cells through apoptosis, which might be associated with its decreasing intracellular calcium and the mitochondrial membrane potential.

Conformational states of the kinase Lck regulate clustering in early T cell signaling.

Phosphorylation of the T cell antigen receptor (TCR) by the tyrosine kinase Lck is an essential step in the activation of T cells. Because Lck is constitutively active, spatial organization may regulate TCR signaling. Here we found that Lck distributions on the molecular level were controlled by the conformational states of Lck, with the open, active conformation inducing clustering and the closed, inactive conformation preventing clustering. In contrast, association with lipid domains and protein networks were not sufficient or necessary for Lck clustering. Conformation-driven Lck clustering was highly dynamic, so that TCR triggering resulted in Lck clusters that contained phosphorylated TCRs but excluded the phosphatase CD45. Our data suggest that Lck conformational states represent an intrinsic mechanism for the intermolecular organization of early T cell signaling.

The Hsp40 family chaperone protein DnaJB6 enhances Schlafen1 nuclear localization which is critical for promotion of cell-cycle arrest in T-cells.

Tight control of cell-cycle progression is critical for T-lymphocytes to function properly. Slfn1 (Schlafen1) has been reported to play an important role in the establishment and maintenance of quiescence in T-lymphocytes. However, how Slfn1 accomplishes this critical function remains poorly understood. In the present study, we show that nuclear localization is a prerequisite for Slfn1 to induce cell-cycle arrest, with DnaJB6, identified as a new Slfn1-binding protein, playing a pivotal role in this process. DnaJB6, a chaperone protein of the DnaJ/Hsp (heat-shock protein) 40 family, stabilizes Slfn1 together with its partner Hsp70, and, more importantly, it enhances the nuclear import of Slfn1. Overexpression of DnaJB6 was found to increase Slfn1 nuclear accumulation and resulted in cell-cycle arrest, whereas, in DnaJB6 knock-down cells, Slfn1 was mainly sequestered in the cytoplasm and no cell-cycle arrest was observed. Furthermore, transgenic expression of DnaJB6 in T-lineage cells inhibited Slfn1's degradation, promoted its nuclear import and ultimately led to suppression of T-cell proliferation upon TCR (T-cell receptor) activation. In addition, DnaJB6 increased Slfn1's effect on its downstream target cyclin D1 in co-transfected cells. Altogether, our results demonstrate that DnaJB6 is necessary for translocation of Slfn1 into the nucleus, where Slfn1 down-regulates cyclin D1, induces cell-cycle arrest and programmes a quiescent state of T-cells.

Screening of an antigen target for immunocontraceptives from cross-reactive antigens between human sperm and Ureaplasma urealyticum.

Epidemiologic studies indicated that some infertile men who were infected with Ureaplasma urealyticum displayed positive antisperm antibodies in their serum and/or semen. The purpose of this study was to investigate the possible mechanism of antisperm antibodies production after infection with U. urealyticum and to analyze the relationship between U. urealyticum and infertility. The existence of cross-reactive antigens (61, 50, and 25 kDa) between U. urealyticum and human sperm membrane proteins was confirmed. Among the cross-reactive antigens, the urease complex component UreG of U. urealyticum was determined. By searching the Swiss-Prot protein database, a pentapeptide identity (IERLT) between UreG and human nuclear autoantigenic sperm protein (NASP) was found. Furthermore, using Western blot analysis and enzyme-linked immunosorbent assay, the cross-reaction between the NASP and UreG was verified. Both anti-rUreG antibody and the antiserum against the synthetic peptide NASP393-408 containing the pentapeptide inhibited mouse sperm egg binding and fusion. After immunization by rUreG or the synthetic peptide, 81.2 and 75% female mice became sterile, respectively. The effect on fertility in mice immunized with the synthetic peptide was reversible. These findings proved for the first time that it was feasible to screen antigens for immunocontraceptives from cross-reactive antigens between sperm and microorganisms which induce infertility.