Eunseok Seo

In vivo two-photon fluorescent imaging of fluoride with a desilylation-based reactive probe

A two-photon excitable molecular probe for fluoride, developed based on a fluoride-specific desilylation reaction, is demonstrated to be useful for fluorescent imaging of fluoride ions in live zebrafish by one-photon as well as two-photon microscopy for the first time.

Toward a selective, sensitive, fast-responsive, and biocompatible two-photon probe for hydrogen sulfide in live cells.

Hydrogen sulfide has emerged as an exciting endogenous gasotransmitter in addition to nitric oxide and carbon dioxide. Noninvasive detection methods for hydrogen sulfide thus become indispensable tools for studying its diverse roles in biological systems. Accordingly, fluorescent probes for hydrogen sulfide have received great attention in recent years. A practically useful fluorescent probe for bioimaging of hydrogen sulfide should be selective, sensitive, fast-responsive, biocompatible, observable in the biological optical window, and capable of deep-tissue imaging. These sensing properties, however, are extremely difficult to achieve at the same time. Disclosed here is the two-photon fluorescent probe that meets all of these criteria. The probe belongs to a Michael acceptor system, which raised a serious selectivity issue over the competing biothiols such as cysteine and glutathione. We have addressed the selectivity issue by optimizing the electronic and steric interactions between biothiols and the probe, in addition to achieving very high sensitivity, fast-response, and biocompatibility. Also, the sensing mechanism suggested in the literature was revised. The probe thus enables us to image the endogenously produced hydrogen sulfide with negligible interference from other biothiols in live cells. The excellent sensing properties of the probe combined with its capability of bioimaging thus make it a practically useful tool for further studying biological roles of hydrogen sulfide.

Gold nanoparticle-incorporated human red blood cells (RBCs) for X-ray dynamic imaging

Time-resolved dynamic imaging of bio-fluids can provide valuable information for clinical diagnosis and treatment of circulatory disorders. Quantitative information on non-transparent blood flows can be directly obtained by particle-tracing dynamic X-ray imaging, which needs better spatial resolution and enhanced image contrast compared to static imaging. For that use, tracer particles tagging along the flow streams are critically required. In this study, taking the advantage of high X-ray absorption, gold nanoparticles (AuNPs) are incorporated into human red blood cells (RBC) to produce contrast-enhanced tracers designed for dynamic X-ray imaging of blood flows. RBCs are advantageous tracers for blood flow measurements since they are natural and primary components of blood. The loading efficiency of AuNPs into RBCs is investigated in terms of the surface properties of the AuNPs. The AuNP-incorporated RBC provides a potential in the dynamic X-ray imaging of blood flows which can be used for clinical applications.

Controlled cellular uptake and drug efficacy of nanotherapeutics

Cellular uptake pathway of nanoparticle (NP) is different from that of free drugs. Therefore, NP-mediated nanotherapeutics can be designed to overcome the adverse effects of free drugs. However, synthetic NPs are typically trapped in the endosome and have difficulty to reach the cytosol because of the characteristic endocytosis, where the endosomal membranes wrap-up the introduced NPs. In this study, the Spacer molecules linking the apoptotic anticancer drug and the gold NP (AuNP) are designed and cellular uptake procedure and drug deployment in the cancer cells are controlled. X-ray nanoscopy and two-photon microscopy are employed to observe the AuNPs in a cell in-situ without additional dye molecule or imaging agent introduction on an AuNP. We confirm that the effective design of the Spacer molecules importantly control the cellular interaction of the AuNPs. This technology can be generalized to broad biomedical applications utilizing nanotherapeutics-mediated diagnosis and new-concepted disease treatment technologies.

Detection of Heparin in the Salivary Gland and Midgut of Aedes togoi

Mosquitoes secrete saliva that contains biological substances, including anticoagulants that counteract a hosts hemostatic response and prevent blood clotting during blood feeding. This study aimed to detect heparin, an anticoagulant in Aedes togoi using an immunohistochemical detection method, in the salivary canal, salivary gland, and midgut of male and female mosquitoes. Comparisons showed that female mosquitoes contained higher concentrations of heparin than male mosquitoes. On average, the level of heparin was higher in blood-fed female mosquitoes than in non-blood-fed female mosquitoes. Heparin concentrations were higher in the midgut than in the salivary gland. This indicates presence of heparin in tissues of A. togoi.

Proposal for a new therapy for drug-resistant malaria using Plasmodium synthetic lethality inference

Graphical abstract

Bubble-free and pulse-free fluid delivery into microfluidic devices

The bubble-free and pulse-free fluid delivery is critical to reliable operation of microfluidic devices. In this study, we propose a new method for stable bubble-free and pulse-free fluid delivery in a microfluidic device. Gas bubbles are separated from liquid by using the density difference between liquid and gas in a closed cavity. The pulsatile flow caused by a peristaltic pump is stabilized via gas compressibility. To demonstrate the proposed method, a fluidic chamber which is composed of two needles for inlet and outlet, one needle for a pinch valve and a closed cavity is carefully designed. By manipulating the opening or closing of the pinch valve, fluids fill up the fluidic chamber or are delivered into a microfluidic device through the fluidic chamber in a bubble-free and pulse-free manner. The performance of the proposed method in bubble-free and pulse-free fluid delivery is quantitatively evaluated. The proposed method is then applied to monitor the temporal variations of fluidic flows of rat blood circulating within a complex fluidic network including a rat, a pinch valve, a reservoir, a peristaltic pump, and the microfluidic device. In addition, the deformability of red blood cells and platelet aggregation are quantitatively evaluated from the information on the temporal variations of blood flows in the microfluidic device. These experimental demonstrations confirm that the proposed method is a promising tool for stable, bubble-free, and pulse-free supply of fluids, including whole blood, into a microfluidic device. Furthermore, the proposed method will be used to quantify the biophysical properties of blood circulating within an extracorporeal bypass loop of animal models.

Recombinant mussel proximal thread matrix protein promotes osteoblast cell adhesion and proliferation

Backgroundvon Willebrand factor (VWF) is a key load bearing domain for mamalian cell adhesion by binding various macromolecular ligands in extracellular matrix such as, collagens, elastin, and glycosaminoglycans. Interestingly, vWF like domains are also commonly found in load bearing systems of marine organisms such as in underwater adhesive of mussel and sea star, and nacre of marine abalone, and play a critical load bearing function. Recently, Proximal Thread Matrix Protein1 (PTMP1) in mussel composed of two vWF type A like domains has characterized and it is known to bind both mussel collagens and mammalian collagens.ResultsHere, we cloned and mass produced a recombinant PTMP1 from E. coli system after switching all the minor codons to the major codons of E. coli. Recombinant PTMP1 has an ability to enhance mouse osteoblast cell adhesion, spreading, and cell proliferation. In addition, PTMP1 showed vWF-like properties as promoting collagen expression as well as binding to collagen type I, subsequently enhanced cell viability. Consequently, we found that recombinant PTMP1 acts as a vWF domain by mediating cell adhesion, spreading, proliferation, and formation of actin cytoskeleton.ConclusionsThis study suggests that both mammalian cell adhesion and marine underwater adhesion exploits a strong vWF-collagen interaction for successful wet adhesion. In addition, vWF like domains containing proteins including PTMP1 have a great potential for tissue engineering and the development of biomedical adhesives as a component for extra-cellular matrix.

Biophysiochemical properties of endothelial cells cultured on bio-inspired collagen films

BackgroundIn this study, we investigated the effect of the extracellular matrix on endothelial dysfunction by careful observation of human umbilical vein endothelial cells (HUVECs) cultured on denatured collagen film.ResultsHUVECs on denatured collagen film showed relatively high surface roughness compared with normal HUVECs. The expression levels of MMP-1, MMP-2 and CD146 increased in the ECs on denatured collagen film. In addition, we examined the accumulation of fluorescent beads on HUVEC layers subjected to circulatory flow. The number of accumulated fluorescent beads increased on the disorganized HUVEC layers.ConclusionsThe proposed in vitro study using bio-inspired collagen films could potentially be used in the size- and ligand-based design of drugs to treat endothelial dysfunction caused by circulatory vascular diseases.

Detection of circulating tumor cells via an X-ray imaging technique

Detailed information on the location and the size of tumor cells circulating through lymphatic and blood vessels is useful to cancer diagnosis. Metastasis of cancers to other non-adjacent organs is reported to cause 90% of deaths not from the primary tumors. Therefore, effective detection of circulating tumors cells (CTCs) related to metastasis is emphasized in cancer treatments. With the use of synchrotron X-ray micro-imaging techniques, high-resolution images of individual flowing tumor cells were obtained. Positively charged gold nanoparticles (AuNPs) which were inappropriate for incorporation into human red blood cells were selectively incorporated into tumor cells to enhance the image contrast. This approach enables images of individual cancer cells and temporal movements of CTCs to be captured by the high X-ray absorption efficiency of selectively incorporated AuNPs. This new technology for in vivo imaging of CTCs would contribute to improve cancer diagnosis and cancer therapy prognosis.