Hyori Kim

Ultrasensitive Near‐Infrared Raman Reporters for SERS‐Based In Vivo Cancer Detection

Surface-enhanced Raman spectroscopy (SERS) has recently emerged as an alternative to fluorescence-based spectroscopy in bioimaging, as it can minimize photobleaching, peak overlapping, and low signal-to-noise ratio in complex biological systems. SERS probes are based on the 10–10-fold scattering enhancement caused by the proximity of Ramanactive signature molecules to the surface of metal nanoparticles (NPs), which can be modulated with molecular recognition motifs to render diagnostic tools for optical imaging and therapeutic studies. However, the preparation of ultrasensitive SERS probes is hampered by the limited availability, sensitivity, and reproducibility of Raman-active compounds. This drawback is particularly important at the near-infrared (NIR) region, where the availability of reporters is restricted to a few Raman-active molecules. Herein, we report the first combinatorial approach to discover novel and highly sensitive NIR SERS reporters. The synthesis and screening of an 80-member tricarbocyanine library led to the identification of CyNAMLA-381 as a NIR SERS reporter with 12-fold higher sensitivity than the standard 3,3’-diethylthiatricarbocyanine (DTTC), and we validated its advantages for the construction of ultrasensitive in vivo SERS probes. A major bottleneck in SERS probe discovery is the development of highly sensitive Raman reporters. Most of the commonly used Raman signature molecules are active in the UV/Vis range (e.g., crystal violet, malachite green isothiocyanate, rhodamine-6G, Nile blue, 2-napthalenethiol, TRITC (tetramethylrhodamine-5-isothiocyanate), and XRITC (Xrhodamine-5-(and-6)-isothiocyanate), and thus have a restricted potential for in vivo imaging. The adequacy of the NIR region for in vivo studies has raised the interest in NIR surface-enhanced resonance Raman spectroscopy (SERRS)-active molecules. Although the cyanine derivative DTTC has been regarded as a standard in NIR SERRS studies, it shows only a moderate Raman intensity, which limits the preparation of highly sensitive probes for in vivo applications. Since little is known about the correlation between the cyanine scaffold and its Raman intensity, we designed a library of structurally diverse tricarbocyanines with the aim of discovering novel NIR SERRS-active compounds that surpass the sensitivity of DTTC. The tricarbocyanine core is an accessible NIR structure, the central chlorine atom of which can be replaced with different nucleophiles. We designed the synthesis of tricarbocyanine derivatives by substitution with different amines, and acetylated the resulting alkylor benzylamino groups to obtain compounds with NIR absorption properties and good chemical stability in aqueous media (CyNA). To prepare compounds that could be chemisorbed on gold nanoparticles (AuNPs), we prepared the scaffold 1 with an aminopropyl linker that could be later coupled to a disulfide-containing lipoic acid spacer (Scheme 1). The amine group of 1 was Boc-protected prior to the derivatization of the central chlorine atomwith 80 structurally different primary amines including heterocyclic, alkyl, and aromatic groups (for structures, see Chart S1 in the Supporting Information). After acetylation, the compounds were treated with an optimized TFA/dichloromethane (1:9) solution that overcame the lability of the tricarbocyanine core in acidic conditions. The final coupling to a lipoic acidactivated ester resin yielded 80 derivatives (CyNAMLA) with an average purity of 90% (for data of HPLC-determined purities, see Table S1 in the Supporting Information). CyNAMLA compounds proved to be remarkably NIRactive with absorbance maximumwavelengths around 800 nm (Table S1 in the Supporting Information). Their SERS [*] A. Samanta, X. Liao, Prof. Y. T. Chang Department of Chemistry & MedChem Program of Life Sciences Institute, National University of Singapore 117543 Singapore (Singapore) Fax: (+65)6779-1691 E-mail: chmcyt@nus.edu.sg Homepage: http://ytchang.science.nus.edu.sg

The pattern of bone marrow oedema on MRI in osteonecrosis of the femoral head

It has been suggested that transient osteoporosis or the bone marrow oedema syndrome (BMOS) may be the initial phase of osteonecrosis of the femoral head (ONFH) and that there may be a common pathophysiology. In this study, we have assessed the MR images of 200 consecutive patients with ONFH in respect of the BMO pattern in order to test this hypothesis. This pattern was not observed in the early stage of ONFH. The initial abnormal finding detected on the MR images was an abnormal band of intensity at the junction between the necrotic area and the normal bone. Structural damage of the head seems to result in the appearance of the BMO pattern and the development of pain in ONFH. There was no finding to support the existence of a continuum between BMOS and ONFH.

Solid-phase synthesis of BODIPY dyes and development of an immunoglobulin fluorescent sensor

The diversification of the BODIPY scaffold has been hindered by its controversial adaptability to solid-phase chemistry. Herein we report the first solid-phase synthesis of a BODIPY library in high purities. We screened the library against a set of proteins, identified an immunoglobulin fluorescent sensor (Ig Orange) and confirmed its binding by SPR experiments.

Application of bispecific antibody against antigen and hapten for immunodetection and immunopurification.

We present a bispecific antibody that recognizes an antigen and a hapten and can be applied to various biological assays, including immunoblotting and immunoprecipitation. In immunoblot analysis of serum, an anti-C5 × anti-cotinine bispecific tandem single-chain variable fragment (scFv)-Fc fusion protein and cotinine-conjugated horseradish peroxidase (HRP) generated a clean signal without the high background that was observed in a parallel experiment using HRP-conjugated goat anti-rabbit immunoglobulin G (Fc-specific) antibody. In immunoprecipitation analysis of serum, use of the bispecific tandem scFv-Fc fusion protein and cotinine-crosslinked magnetic beads significantly reduced the amount of protein contaminants compared with a parallel experiment done with protein A agarose beads. In subsequent immunoblot analysis, use of cotinine–HRP as the secondary probe instead of HRP-conjugated goat anti-rabbit IgG (Fc-specific) antibody successfully eliminated the band corresponding to the bispecific tandem scFv-Fc fusion protein.

Chicken scFvs with an Artificial Cysteine for Site-Directed Conjugation

For the site-directed conjugation of chemicals and radioisotopes to the chicken-derived single-chain variable fragment (scFv), we investigated amino acid residues replaceable with cysteine. By replacing each amino acid of the 157 chicken variable region framework residues (FR, 82 residues on VH and 75 on VL) with cysteine, 157 artificial cysteine mutants were generated and characterized. At least 27 residues on VL and 37 on VH could be replaced with cysteine while retaining the binding activity of the original scFv. We prepared three VL (L5, L6 and L7) and two VH (H13 and H16) mutants as scFv-Ckappa fusion proteins and showed that PEG-conjugation to the sulfhydryl group of the artificial cysteine was achievable in all five mutants. Because the charge around the cysteine residue affects the in vivo stability of thiol-maleimide conjugation, we prepared 16 charge-variant artificial cysteine mutants by replacing the flanking residues of H13 with charged amino acids and determined that the binding activity was not affected in any of the mutants except one. We prepared four charge-variant H13 artificial cysteine mutants (RCK, DCE, ECD and ECE) as scFv-Ckappa fusion proteins and confirmed that the reactivity of the sulfhydryl group on cysteine is active and their binding activity is retained after the conjugation process.

Progress of antibody-based inhibitors of the HGF|[ndash]|cMET axis in cancer therapy

Dysregulated receptor tyrosine kinase signaling in human cancer cells leads to tumor progression, invasion and metastasis. The receptor tyrosine kinase cMET is frequently overexpressed in cancer tissue, and activation of cMET signaling is related to drug resistance and the processes of carcinogenesis, invasion and metastasis. For that reason, cMET and its ligand, hepatocyte growth factor (HGF), are considered prime targets for the development of anticancer drugs. At least eight anti-cMET and four anti-HGF antibodies have been tested or are being tested in clinical trials. However, to date none of these HGF/cMET inhibitors have shown significant efficacy in clinical trials. Furthermore, no receptor tyrosine kinase inhibitors primarily targeting cMET have been approved. Given that neutralization of HGF or cMET does not cause significant adverse effects, inhibition of the HGF/cMET signaling pathway appears to be safe. In this review, we summarized the completed and ongoing clinical trials testing antibody- or protein-based anticancer drugs targeting cMET and HGF.

Deep gray matter atrophy in neuromyelitis optica spectrum disorder and multiple sclerosis

We investigated changes in deep gray matter (DGM) volume and its relationship to cognition and clinical factors in a large cohort of patients with neuromyelitis optica spectrum disorder (NMOSD) and compared them with results from multiple sclerosis (MS).

In vitro and in vivo application of anti-cotinine antibody and cotinine-conjugated compounds

The combination of a high-affinity antibody to a hapten, and hapten-conjugated compounds, can provide an alternative to the direct chemical cross-linking of the antibody and compounds. An optimal hapten for in vitro use is one that is absent in biological systems. For in vivo applications, additional characteristics such as pharmacological safety and physiological inertness would be beneficial. Additionally, methods for cross-linking the hapten to various chemical compounds should be available. Cotinine, a major metabolite of nicotine, is considered advantageous in these aspects. A high-affinity anti-cotinine recombinant antibody has recently become available, and can be converted into various formats, including a bispecific antibody. The bispecific anti-cotinine antibody was successfully applied to immunoblot, enzyme immunoassay, immunoaffinity purification, and pre-targeted in vivo radioimmunoimaging. The anti-cotinine IgG molecule could be complexed with aptamers to form a novel affinity unit, and extended the in vivo half-life of aptamers, opening up the possibility of applying the same strategy to therapeutic peptides and chemical compounds. [BMB Reports 2014; 47(3): 130-134]

An antibody reactive to the Gly63-Lys68 epitope of NT-proBNP exhibits O-glycosylation-independent binding.

The N-terminal fragment of prohormone brain natriuretic peptide (NT-proBNP) is a commonly used biomarker for the diagnosis of congestive heart failure, although its biological function is not well known. NT-proBNP exhibits heavy O-linked glycosylation, and it is quite difficult to develop an antibody that exhibits glycosylation-independent binding. We developed an antibody that binds to the recombinant NT-proBNP protein and its deglycosylated form with similar affinities in an enzyme immunoassay. The epitope was defined as Gly63–Lys68 based on mimetic peptide screening, site-directed mutagenesis and a competition assay with a peptide mimotope. The nearest O-glycosylation residues are Thr58 and Thr71; therefore, four amino acid residues intervene between the epitope and those residues in both directions. In conclusion, we report that an antibody reactive to Gly63–Lys68 of NT-proBNP exhibits O-glycosylation-independent binding.

Highly task‐specific oromandibular dystonia in a telephone operator

S. Y. Kang, H. Kim, H.-I. Ma, Y. J. Kim, S.-B. Kwon, S. H. Hwang and Y. H. Sohn Department of Neurology, Hallym University College of Medicine, Seoul; Graduate Program in Speech and Language Pathology, Department of Rehabilitation Medicine, & Research Institute of Rehabilitation Medicine; and Department of Neurology and Brain Research Institute, Yonsei University College of Medicine, Seoul, Korea