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Near-infrared (NIR) fluorophores have unique features that endow them with several advantages over conventional shorter wavelength emitting dyes. As a result, they have been widely utilized as fluorescence and photoacoustic imaging agents, as well as photodynamic and photothermal therapeutic agents. However, non-targeting NIR fluorescence-emitting organic molecules have the drawback of low selectivity toward tumors, which potentially results in severe side effects caused by damage to normal tissues. Thus, the development of NIR fluorophore-based substances that target tumors is a highly active area in medicinal chemistry research. Research efforts carried out thus far have led to the development of a number of NIR fluorophore-based, tumor imaging and therapeutic agents. The discussion in this review focuses on the results of research reported in the 2012-2021 period, giving particular emphasis to studies of NIR small organic dye-based imaging and therapeutic agents that are designed utilizing cancer-selective strategies.

Through their specific interactions with proteins, cellular glycans play key roles in a wide range of physiological and pathological processes. One of the main goals of research in the areas of glycobiology and glycomedicine is to understand glycan-protein interactions at the molecular level. Over the past two decades, glycan microarrays have become powerful tools for the rapid evaluation of interactions between glycans and proteins. In this review, we briefly describe methods used for the preparation of glycan probes and the construction of glycan microarrays. Next, we highlight applications of glycan microarrays to rapid profiling of glycan-binding patterns of plant, animal and pathogenic lectins, as well as other proteins. Finally, we discuss other important uses of glycan microarrays, including the rapid analysis of substrate specificities of carbohydrate-active enzymes, the quantitative determination of glycan-protein interactions, discovering high-affinity or selective ligands for lectins, and identifying functional glycans within cells. We anticipate that this review will encourage researchers to employ glycan microarrays in diverse glycan-related studies.

Recognition of glycans by proteins plays a crucial role in a variety of physiological processes in cells and living organisms. In addition, interactions of glycans with proteins are involved in the development of diverse diseases, such as pathogen infection, inflammation and tumor metastasis. It is well-known that multivalent glycans bind to proteins much more strongly than do their monomeric counterparts. Owing to this property, numerous multivalent glycans have been utilized to elucidate glycan-mediated biological processes and to discover glycan-based biomedical agents. In this review, we discuss recent advances (2014-2020) made in the development and biological and biomedical applications of synthetic multivalent glycans, including neoglycopeptides, neoglycoproteins, glycodendrimers, glycopolymers, glyconanoparticles and glycoliposomes. We hope this review assists researchers in the design and development of novel multivalent glycans with predictable activities.

Owing to the generation of heterogeneous glycoproteins in cells, it is highly difficult to study glycoprotein-mediated biological events and to develop biomedical agents. Thus, general and efficient methods to prepare homogeneous glycoproteins are in high demand. Herein, we report a general method for the efficient preparation of homogeneous glycoproteins that utilizes a combination of genetic code expansion and chemoselective ligation techniques. In the protocol to produce glycan-defined glycoproteins, an alkyne tag-containing protein, generated by genetic encoding of an alkynylated unnatural amino acid, was quantitatively coupled via click chemistry to versatile azide-appended glycans. The glycoproteins produced by the present strategy were found to recognize mammalian cell-surface lectins and enter the cells through lectin-mediated internalization. Also, cell studies exhibited that the glycoprotein containing multiple mannose-6-phosphate residues enters diseased cells lacking specific lysosomal glycosidases by binding to the cell-surface M6P receptor, and subsequently migrates to lysosomes for efficient degradation of stored glycosphingolipids.

Herein we report a strategy to eradicate pathogenic bacteria selectively, which utilizes bacterial lectin-targeting glycoconjugates that contain an epitope or a photosensitizer to promote antibody-dependent cellular cytotoxicity (ADCC) or photodynamic therapy (PDT), respectively. Our results show that death promoted by using the designed synthetic glycoconjugates coupled with ADCC or PDT takes place selectively in pathogenic bacteria expressing lectins on their surfaces.

Herein we describe a method for inducing cancer cell death, which relies on the use of a H2O2-responsive glycan metabolic precursor in conjunction with antibody-dependent cellular cytotoxicity (ADCC) or photodynamic therapy (PDT).

Herein, we describe a novel approach for the practical synthesis of thiadiazine 1-oxides 10. The first example of an intramolecular cyclization with 2-N-cyano-sulfonimidoyl amides 9 to form the desired thiadiazine 1-oxides 10 was developed. One-pot acid-induced hydrolysis of the cyano group and the intramolecular cyclocondensation protocol readily provided various heterocyclic frameworks in good to moderate yields. Notably, the crystal structures of N-urea sulfoximine 11 and thiadiazine 1-oxide 10i have been determined using X-ray crystallography.

Carotenoids are phytochemicals that are precursors of vitamin A and effective antioxidants, required for human health. The mechanisms and underlying genetic network responsible for regulating carotenoid production in plants, however, is poorly understood, despite the carotenoid biosynthesis pathway being known. We found that a single amino acid insertion in lycopene β-cyclase2 (LCYB2) caused catalytic failure, possibly due to a flux down of lycopene to the carotenoids which may be the molecular basis for the color of red carrot roots.

Recently, we showed that synthetic anion transporters DSC4P-1 and SA-3 had activity related to cancer cell death. They were found to increase intracellular chloride and sodium ion concentrations. They were also found to induce apoptosis (DSC4P-1) and both induce apoptosis and inhibit autophagy (SA-3). However, determinants underlying these phenomenological findings were not elucidated. The absence of mechanistic understanding has limited the development of yet-improved systems. Here, we show that three synthetic anion transporters, DSC4P-1, SA-3, and 8FC4P, induce osmotic stress in cells by increasing intracellular ion concentrations. This triggers the generation of reactive oxygen species via a sequential process and promotes caspase-dependent apoptosis. In addition, two of the transporters, SA-3 and 8FC4P, induce autophagy by increasing the cytosolic calcium ion concentration promoted by osmotic stress. However, they eventually inhibit the autophagy process as a result of their ability to disrupt lysosome function through a transporter-mediated decrease in a lysosomal chloride ion concentration and an increase in the lysosomal pH.

Lysosomal pHs are maintained at low values by the cooperative action of a proton pump and a chloride channel to maintain electroneutrality. Owing to the biological significance of lysosomal chloride ions, measurements of their levels are of great importance to understand lysosome-associated biological events. However, appropriate probes to selectively detect Cl- ions within acidic lysosomes have not been developed to date. In this study, we prepared MQAE-MP, a lysosomal Cl--selective fluorescent probe, and applied it to gain information about biological processes associated with lysosomes. The fluorescence of MQAE-MP is pH-insensitive over physiological pH ranges and is quenched by Cl- with a Stern-Volmer constant of 204 M-1. Because MQAE-MP detects lysosomal Cl- selectively, it was employed to assess the effects of eleven substances on lysosomal Cl- concentrations. The results show that lysosomal Cl- concentrations decrease in cells treated with substances that inhibit proteins responsible for lysosomal membrane stabilization, induce lysosomal membrane permeabilization, and transport lysosomal Cl- to the cytosol. In addition, we investigated the effect of lysosomal chloride ions on the fusion of autophagosomes with lysosomes to generate autolysosomes during autophagy inhibition promoted by substances. It was found that changes in lysosomal Cl- concentrations did not affect the fusion of autophagosomes with lysosomes but an increase in the cytosolic Ca2+ concentration blocked the fusion process. We demonstrate from the current study that MQAE-MP has great potential as a lysosomal Cl--selective fluorescent probe for studies of biological events associated with lysosomes.

Carbohydrate microarrays, containing glycosylated fluorescent probes, have been constructed using N-hydroxysuccinimide (NHS) ester-conjugated BSA modified surfaces. When the carbohydrate moieties were cleaved from the fluorescent probes in the conjugates by glycosidases, the fluorescence signals of the probes were enhanced. In this study, we have applied these microarrays to profile glycosidase activities and have employed them to determine IC50 values of glycosidase inhibitors.

Pathogens infect hosts often through initial binding of their cell surface lectins to glycans expressed on the exterior of host cells. Thus, methods to evaluate the glycan-binding properties of pathogens are of great importance. Because of the multivalent nature of interactions of pathogens with glycans, the ability to assess the glycan density-dependent binding of pathogens is particularly important. In this study, we developed a facile technique to construct multivalent carbohydrate microarrays through immobilization of unmodified glycans on multivalent hydrazide-derivatized glass surfaces. This immobilization strategy does not require the use of multivalent glycoconjugates, which are typically prepared by using multistep sequences. The results of analysis of microarray images, obtained after incubation of multivalent glycan microarrays with cholera toxin B and pathogens such as uropathogenic E. coli and H. pylori, show that the binding affinities of toxins and pathogens for glycans are highly glycan density-dependent. Specifically, toxins and pathogens bind to glycans more strongly as the valency of the glycans on the microarrays is increased from 1 to 4. It is anticipated that the newly developed immobilization method will be applicable to the preparation of multivalent carbohydrate microarrays that are employed to evaluate multivalent glycan binding properties of a variety of pathogens and toxins.

Fifty-five mono- and disaccharide analogues were prepared and used for the construction of microarrays to uncover lectin-selective ligands. The microarray study showed that two disaccharide analogues, 28' and 44', selectively bind to Solanum tuberosum lectin (STL) and wheat germ agglutinin (WGA), respectively. Cell studies indicated that 28' and 44' selectively block the binding of STL and WGA to mammalian cells, unlike the natural ligand LacNAc, which suppresses binding of both STL and WGA to cells.

For both fluorescence imaging and isolation of glycosidases in cells, we prepared novel activity-based, trifunctional fluorogenic probes that consist of (1) a sugar moiety as a glycosidase substrate, (2) a fluoromethylated coumarin for fluorescent labeling, and (3) an alkyne tag for click reaction to enable isolation of the labeled enzyme. One probe, β-GlcNAc-CM-F, was employed to fluorescently detect endogenous O-GlcNAcase in cells and to isolate the labeled enzyme by affinity chromatography.

MicroRNA-155, one of the most potent miRNAs that suppress apoptosis in human cancer, is overexpressed in numerous cancers, and it displays oncogenic activity. Peptide microarrays, constructed by immobilizing 185 peptides containing the C-terminal hydrazide onto epoxide-derivatized glass slides, were employed to evaluate peptide binding properties of pre-miRNA-155 and to identify its binding peptides. Two peptides, which were identified based on the results of peptide microarray and in vitro Dicer inhibition studies, were found to inhibit generation of mature miRNA-155 catalyzed by Dicer and to enhance expression of miRNA-155 target genes in cells. In addition, the results of cell experiments indicate that peptide inhibitors promote apoptotic cell death via a caspase-dependent pathway. Finally, observations made in NMR and molecular modeling studies suggest that a peptide inhibitor preferentially binds to the upper bulge and apical stem-loop region of pre-miRNA-155, thereby suppressing Dicer-mediated miRNA-155 processing.

Not only are glycan-mediated binding processes in cells and organisms essential for a wide range of physiological processes, but they are also implicated in various pathological processes. As a result, elucidation of glycan-associated biomolecular interactions and their consequences is of great importance in basic biological research and biomedical applications. In 2002, we and others were the first to utilize glycan microarrays in efforts aimed at the rapid analysis of glycan-associated recognition events. Because they contain a number of glycans immobilized in a dense and orderly manner on a solid surface, glycan microarrays enable multiple parallel analyses of glycan-protein binding events while utilizing only small amounts of glycan samples. Therefore, this microarray technology has become a leading edge tool in studies aimed at elucidating roles played by glycans and glycan binding proteins in biological systems. In this Account, we summarize our efforts on the construction of glycan microarrays and their applications in studies of glycan-associated interactions. Immobilization strategies of functionalized and unmodified glycans on derivatized glass surfaces are described. Although others have developed immobilization techniques, our efforts have focused on improving the efficiencies and operational simplicity of microarray construction. The microarray-based technology has been most extensively used for rapid analysis of the glycan binding properties of proteins. In addition, glycan microarrays have been employed to determine glycan-protein interactions quantitatively, detect pathogens, and rapidly assess substrate specificities of carbohydrate-processing enzymes. More recently, the microarrays have been employed to identify functional glycans that elicit cell surface lectin-mediated cellular responses. Owing to these efforts, it is now possible to use glycan microarrays to expand the understanding of roles played by glycans and glycan binding proteins in biological systems.

Sin3 is a transcriptional corepressor for REST silencing machinery that represses multiple neuronal genes in non-neuronal cells. However, functions of Sin3 (Sin3A and Sin3B) in suppression of neuronal phenotypes are not well characterized. Herein we show that Sin3A knockdown impedes the repressive activity of REST and enhances differentiation of pluripotent P19 cells into electrophysiologically active neurons without inducing astrogenesis. It is also found that silencing Sin3B induces neurogenesis of P19 cells with a lower efficiency than Sin3A knockdown. The results suggest that Sin3A has a more profound effect on REST repressive machinery for silencing neuronal genes in P19 cells than Sin3B. Furthermore, we show that a peptide inhibitor of Sin3A-REST interactions promotes differentiation of P19 cells into functional neurons. Observations made in studies using genetic deletion and a synthetic inhibitor suggests that Sin3A plays an important role in the repression of neuronal genes by the REST regulatory mechanism.

Summary In this study we developed an efficient method to prepare glycoengineered β-N-acetylhexosaminidase containing multiple mannose-6-phosphates (M6Ps) by combining genetic code expansion with bioorthogonal ligation techniques. We found that multiple M6P-conjugated enzymes were produced with a high efficiency by using combined techniques. Importantly, glycoengineered enzymes entered lysosomes of patient-derived primary cells, which lack endogenous lysosomal β-N-acetylhexosaminidase, more readily than commercialized human β-hexosaminidase. Moreover, glycoengineered enzymes successfully removed GM2-ganglioside stored in lysosomes of diseased cells, indicating that its activity is restored in diseased cells. We also synthesized and applied a lysosome-targeting fluorogenic substrate to monitor endogenous and supplemental glycoengineered β-N-acetylhexosaminidase activities in lysosomes. The results of this study indicate that the present strategy, which relies on genetic code expansion and bioorthogonal ligation techniques, is highly attractive to generate multi-M6P-containing lysosomal enzymes that can be used to study lysosomal storage disorders associated with lysosomal enzyme deficiencies.

Reactive oxygen (ROS) and nitrogen (RNS) species cause oxidative and nitrosative stresses, respectively. These stresses are implicated not only in diverse physiological processes but also in various pathological processes, including cancer and neurodegenerative disorders. In addition, some ROS and RNS in the environment are pollutants that threaten human health. As a consequence of these effects, sensitive methods, which can be employed to selectively monitor ROS and RNS in live cells, tissues and organisms as well as in environmental samples, are needed so that their biological roles can be understood and their concentrations in environmental samples can be determined. In this review, fluorescent, luminescent and colorimetric ROS and RNS probes, which have been developed since 2011, are comprehensively discussed.

Tomato hypocotyl can generally be one of two colors, purple or green. Genetically, this trait is controlled by a single dominant gene. Hypocotyl tissue specific color expression is one of many visible genetic marker sources used to select tomato progeny. However, the visible marker does not show a clear distinction between homozygous genotype and heterozygous genotype from the breeding lines. Therefore, to identify a hypocotyl pigmentation related marker, we screened DNA polymorphisms in thirteen tomato lines showing purple or green hypocotyls. The markers used for screening consisted of primer set information obtained from anthocyanin related genes, conserved ortholog set Ⅱ (COS Ⅱ) marker sets localized near anthocyanin related genes, and restriction fragment length polymorphism (RFLP) markers localized near COS Ⅱ markers, which produce polymorphisms between purple and green tomatoes. One primer from a RFLP fragment resulted in a polymorphism on agarose gel electrophoresis. From the RFLP fragment, a cleaved amplified polymorphic sequence (CAPS) marker was developed to distinguish between purple and green hypocotyls. The genotypes of 135 F2 individuals were analyzed using the CAPS marker, and among them, 132 individuals corresponded to the phenotypes of hypocotyl pigmentation.

Carbohydrate microarrays were used for the simultaneous screening of various glycans whose binding to the cell-surface lectin elicits cellular response., Carbohydrate microarrays have become robust and powerful tools for the rapid analysis of glycan-associated binding events. However, this microarray technology has rarely been applied in studies of glycan-mediated cellular responses. Herein we describe a carbohydrate microarray-based approach for the rapid screening of biologically active glycans that stimulate the production of reactive oxygen species (ROS) through binding to the cell-surface lectin. We employed a microarray assay and a fluorescent ROS probe to identify the functional glycans which enhance ROS production. Cells binding to glycans on the microarrays produced ROS, whose levels were decreased in the presence of a ROS scavenger or a NADPH oxidase inhibitor. The present study leads us to suggest that glycan microarrays are applicable to the simultaneous screening of various glycans whose binding to the cell-surface lectin elicits cellular response.

InBrassica, self-incompatibility (SI) can be overcome by CO2 application, an effective method for obtaining numerous inbred lines for F, commercial seed. We previously reported two different S-alleles ofBrassica campestris, S733 and S734, with extremely different degrees of susceptibility to this gas. In the current study, we raised a cross-population between those two genetic lines, and analyzed their reaction level of self-incompatibility to CO2 (RLSICO2). Here, all 40 of our progeny from the F1 cross-population were susceptible, maintaining high values of RLSICO2. This suggests that the susceptible line, S734, is dominant to the insusceptible line, S733. We also generated an F2 selfing-population of each crossed progeny, S733♀ S734♂ and S733♂ S734♀, to assess the RLSICO2 of each individual. PCR-RFLP analysis was performed to determine the S-genotype of the F2 population. The S734 allele segregated in a theoretical ratio of the dominant trait, and the RLSICO2 was consistent with the dominance relationship. Therefore, we have now demonstrated that high RLSICO2 in β.campestris is controlled by a dominant gene.

Hsc70 is known to be involved in clathrin-mediated endocytosis (CME) by which cells take up various extracellular materials. More specifically, this protein promotes the disassembly of clathrin-coated vesicles (CCVs) by directly binding to clathrin during CME. As the ATPase activity of Hsc70 is required for its association with clathrin, we have investigated the effect of an inhibitor (apoptozole, Az) of an ATPase domain of Hsc70 on CME. The results of biochemical studies show that Az binds to Hsc70 and Hsp70 without binding to other types of heat shock proteins. Structure-activity relationship studies provide information on the structural features responsible for the inhibition of the ATPase activity of Hsc70. The results obtained from cell experiments reveal that Az disrupts the interaction of Hsc70 with clathrin in cells, thereby leading to the accumulation of transferrin in CCVs and suppression of release of free Fe(3+) from CCVs during transferrin receptor-mediated endocytosis.

Pepper is a recalcitrant plant for Agrobacterium-mediated genetic transformation. Several obstacles to genetic transformation remain such as extremely low transformation rates; the choice of correct genotype is critical; and there is a high frequency of false positives due to direct shoot formation. Here, we report a useful protocol with a suitable selection method. The most important aspect of the pepper transformation protocol is selecting shoots growing from the callus, which is referred to as callus-mediated shoot formation. This protocol is a reproducible and reliable system for pepper transformation.

Pepper is a recalcitrant plant for Agrobacterium-mediated genetic transformation. Several obstacles to genetic transformation remain such as extremely low transformation rates; the choice of correct genotype is critical; and there is a high frequency of false positives due to direct shoot formation. Here, we report a useful protocol with a suitable selection method. The most important aspect of the pepper transformation protocol is selecting shoots growing from the callus, which is referred to as callus-mediated shoot formation. This protocol is a reproducible and reliable system for pepper transformation.

Date Presented 4/7/2016 This survey study explored perceived role identity, value, and quality of life and their relationships among parent caregivers of children with and without disabilities in Korea. From this in-depth examination, the study attempted to capture aspects of disability cultures within a frame of family. Primary Author and Speaker: Ji-Young Hyun Additional Author and Speaker: Sun Lee

SummaryThe heat shock protein HSP70 plays antiapoptotic and oncogenic roles, and thus its inhibition has been recognized as a potential avenue for anticancer therapy. Here we describe the small molecule, apoptozole (Az), which inhibits the ATPase activity of HSP70 by binding to its ATPase domain and, as a result, induces an array of apoptotic phenotypes in cancer cells. Affinity chromatography provides evidence that Az binds HSP70 but not other types of heat shock proteins including HSP40, HSP60, and HSP90. We also demonstrate that Az induces cancer cell death via caspase-dependent apoptosis by disrupting the interaction of HSP70 with APAF-1. Animal studies indicate that Az treatment retards tumor growth in a xenograft mouse model without affecting mouse viability. These studies suggest that Az will aid the development of new cancer therapies and serve as a chemical probe to gain a better understanding of the diverse functions of HSP70.