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We report an improved approach for the integration of flow synthesis and continuous chromatography, for applications in the end-to-end preparation of pharmaceutically-relevant small molecules. It involves the combination of sequential microreactors and twin-column counter-current chromatography based on the often-used C18 columns. The column loading method ensures that the product breaking through a fully loaded first column is loaded onto the second one, avoiding waste of precious material and technological complexity associated with the use of four-to-six columns typical of simulated moving bed chromatography. The system was applied to synthesize biphenyl via Suzuki–Miyaura reaction, and was also demonstrated for other structurally-different compounds. Compared to the discontinuous and other traditional approaches, our method leads to higher isolated yields (ca. +60%), higher productivity (ca. +30%), and reduced solvent consumption (ca. −80%). A circularity and life-cycle analysis was also conducted to demonstrate the environmental benefits of the flow process. Funding from the European Commission (GV, grant agreement 101031710), Bracco Imaging (AS, VR, and GV), Fondazione Bracco (GV), and YMC Japan (TKK and MS) is gratefully acknowledged.

Optical diffraction tomography (ODT), an emerging imaging technique that does not require fluorescent staining, can measure the three-dimensional distribution of the refractive index (RI) of organelles. In this study, we used ODT to characterize the pathological characteristics of human eosinophils derived from asthma patients presenting with eosinophilia. In addition to morphological information about organelles appearing in eosinophils, including the cytoplasm, nucleus, and vacuole, we succeeded in imaging specific granules and quantifying the RI values ​​of the granules. Interestingly, ODT analysis showed that the RI (i.e., molecular density) of granules was significantly different between eosinophils from asthma patients and healthy individuals without eosinophilia, and that vacuoles were frequently found in the cells of asthma patients. Our results suggest that the physicochemical properties of eosinophils derived from patients with asthma can be quantitatively distinguished from those of healthy individuals. The method will provide insight into efficient evaluation of the characteristics of eosinophils at the organelle level for various diseases with eosinophilia.

Conjugation of biopharmaceuticals to polyethylene glycol chains, known as PEGylation, is nowadays an efficient and widely exploited strategy to improve critical properties of the active molecule, including stability, biodistribution profile, and reduced clearance. A crucial step in the manufacturing of PEGylated drugs is the purification. The reference process in industrial settings is single-column chromatography, which can meet the stringent purity requisites only at the expenses of poor product recoveries. A valuable solution to this trade-off is the Multicolumn Countercurrent Solvent Gradient Purification (MCSGP), which allows the internal and automated recycling of product-containing side fractions that are typically discarded in the batch processes. In this study, an ad hoc design procedure was applied to the single-column batch purification of an industrially relevant PEGylated protein, with the aim of defining optimal collection window, elution duration and elution buffer ionic strength to be then transferred to the MCSGP. This significantly alleviates the design of the continuous operation, subjected to manifold process parameters. The MCSGP designed by directly transferring the optimal parameters allowed to improve the yield and productivity by 8.2% and 17.8%, respectively, when compared to the corresponding optimized batch process, ensuring a purity specification of 98.0%. Once the efficacy of MCSGP was demonstrated, a detailed analysis of its cost of goods was performed and compared to the case of single-column purification. To the best of our knowledge, this is the first example of a detailed economic investigation of the MCSGP across different manufacturing scenarios and process cadences of industrial relevance, which demonstrated not only the viability of this continuous technology but also its flexibility.

Background: Tumor-derived exosomal microRNAs are key elements of the cell-cell communications response to lots of stimuli. However, various functions of the exosome in tumor suppression by radiotherapy (RT) are not clearly understood. Our study showed a previously unknown interaction of p53 and histone deacetylase 5 (HDAC5) by radiation exposure in hepatocellular carcinoma (HCC). Methods: Using serial ultracentrifugation methods, radiation and non-radiation exosomes were purified to investigate the radioresistance of miRNA151a-3p. Radiation doses were treated in 2gy and 4gy using radiation equipment X-RAD 320 to observe the expression of HDAC5 and p53 in hepatic cancer cells. Exosomal miRNA bioinformatics analysis was conducted to find a variation in the miRNA configuration inside Exosome after radiation exposure.Results: HDAC5 and p53 interacted by exposure to radiation, which increased exosome release and altered microRNAs' composition within exosomes. Also, we have described the intercommunication occurring between irradiated and untreated cells via exosomal microRNAs that affect tumor proliferation. In particular, the expression of exosomal microR151a-3p was markedly reduced by radiation treatment. We confirmed that inhibition of exosomal microR151a-3p promotes suppression of non-irradiated cancer cells, thereby increasing RT sensitivity. Conclusion: our present findings demonstrated HDAC5 is a key component of the p53-mediated release of exosomes resulting in tumor suppression through exosomal microRNA-151a-3p in response to radiation. Finally, we highlight the important role of exosomal microRNA 151a-3p as a biomarker in enhancing RT sensitivity.

Tumor-derived exosomes (TEXs) contain enriched miRNAs, and exosomal miRNAs can affect tumor growth, including cell proliferation, metastasis, and drug resistance through cell-to-cell communication. We investigated the role of exosomal miR-1260b derived from non-small cell lung cancer (NSCLC) in tumor progression. Exosomal miR-1260b induced angiogenesis by targeting homeodomain-interacting protein kinase-2 (HIPK2) in human umbilical vein endothelial cells (HUVECs). Furthermore, exosomal miR-1260b or suppression of HIPK2 led to enhanced cellular mobility and cisplatin resistance in NSCLC cells. In patients with NSCLC, the level of HIPK2 was significantly lower in tumor tissues than in normal lung tissues, while that of miR-1260b was higher in tumor tissues. HIPK2 and miR-1260b expression showed an inverse correlation, and this correlation was strong in distant metastasis. Finally, the expression level of exosomal miR-1260b in plasma was higher in patients with NSCLC than in healthy individuals, and higher levels of exosomal miR-1260b were associated with high-grade disease, metastasis, and poor survival. In conclusion, exosomal miR-1260b can promote angiogenesis in HUVECs and metastasis of NSCLC by regulating HIPK2 and may serve as a prognostic marker for lung cancers.

Since their development in the 1960s, immuno-gold techniques have been steadily used in biomedical science, because these techniques are applicable to all kinds of antigens, from viruses to animal tissues. Immuno-gold staining exploits antigen-antibody reactions and is used to investigate locations and interactions of components in the ultrastructure of tissues, cells, and particles. These methods are increasingly used with advanced technologies, such as correlative light and electron microscopy and cryo-techniques. In this protocol, we introduce the principles and technical details of recent advances in this area and discuss their advantages and limitations.

Various silica-based fluorescent nanoparticles ((Si-FNP)) with magnetic or metal cores represent a standard class of nanoparticles offering new opportunities for high-resolution cellular imaging and biomedicine applications, such as drug delivery. Their high solubility, homogeneity, biocompatibility, and chemical inertness Si-FNPs make them attractive probes for correlative light and electron microscopy (CLEM) studies, offering novel insights into nanoparticle-cell interactions in detail. In the present chapter, we present a procedure for imaging silica-based fluorescent magnetic core-shell nanoparticles (Si-FMNP) at the single-particle scale in cells. Our method facilitates the acquisition of information on the extracellular and intercellular distribution of nanoparticles and their various interactions with various cellular organelles when cells are cultured and electroporated by NPs. In addition, such information could facilitate the evaluation of the efficacy of nanocarriers designed for drug delivery.

A segmented-core (SC) structure has been widely used for high-power-density (HP) motors. However, the SC motor is associated with a number of problems due to the complexity of both the structure and the manufacturing process. To address these issues, a novel structure of a HP motor is proposed, referred to as the ring-coupled segmented-stator (RSS) model here. The proposed RSS can increase the reliability, the stability, and the manufacturability of motor. Furthermore, useful thermal analysis and design flow which take into account the RSS and asymmetric overhang structure of the motor are proposed in this research. The proposed lumped parameter thermal network (LPTN) for the thermal analysis shows a good agreement with experimental data within 9.8% difference. The proposed analysis and design method can be used for the diverse kinds of motor requiring the HP. The usefulness of the proposed RSS motor, the analysis method, and the design method are verified through the experiment in this research.

Since their development in the 1960s, immuno-gold techniques have been steadily used in biomedical science, because these techniques are applicable to all kinds of antigens, from viruses to animal tissues. Immuno-gold staining exploits antigen–antibody reactions and is used to investigate locations and interactions of components in the ultrastructure of tissues, cells, and particles. These methods are increasingly used with advanced technologies, such as correlative light and electron microscopy and cryo-techniques. In this protocol, we introduce the principles and technical details of recent advances in this area and discuss their advantages and limitations.

Various silica-based fluorescent nanoparticles ((Si-FNP)) with magnetic or metal cores represent a standard class of nanoparticles offering new opportunities for high-resolution cellular imaging and biomedicine applications, such as drug delivery. Their high solubility, homogeneity, biocompatibility, and chemical inertness Si-FNPs make them attractive probes for correlative light and electron microscopy (CLEM) studies, offering novel insights into nanoparticle-cell interactions in detail. In the present chapter, we present a procedure for imaging silica-based fluorescent magnetic core-shell nanoparticles (Si-FMNP) at the single-particle scale in cells. Our method facilitates the acquisition of information on the extracellular and intercellular distribution of nanoparticles and their various interactions with various cellular organelles when cells are cultured and electroporated by NPs. In addition, such information could facilitate the evaluation of the efficacy of nanocarriers designed for drug delivery.

The cell nucleus is a three-dimensional, dynamic organelle organized into subnuclear compartments such as chromatin and nucleoli. The structure and function of these compartments are maintained by diffusion and interactions between related factors as well as by dynamic and structural changes. Recent studies using fluorescent microscopic techniques suggest that protein factors can access and are freely mobile in heterochromatin and in mitotic chromosomes, despite their densely packed structure. However, the physicochemical properties of the chromosome during cell division are not fully understood. In the present study, characteristic properties such as the refractive index (RI), volume of the mitotic chromosomes, and diffusion coefficient (D) of fluorescent probes inside the chromosome were quantified using an approach combining label-free optical diffraction tomography with complementary confocal laser-scanning microscopy and fluorescence correlation spectroscopy. Variations in these parameters correlated with osmotic conditions, suggesting that changes in RI are consistent with those of the diffusion coefficient for mitotic chromosomes and cytosol. Serial RI tomography images of chromosomes in live cells during mitosis were compared with three-dimensional confocal micrographs to demonstrate that compaction and decompaction of chromosomes induced by osmotic change were characterized by linked changes in chromosome RI, volume, and the mobilities of fluorescent proteins.

Label-free optical diffraction tomography (ODT), an imaging technology that does not require fluorescent labeling or other pre-processing, can overcome the limitations of conventional cell imaging technologies, such as fluorescence and electron microscopy. In this study, we used ODT to characterize the cellular organelles of three different stem cells-namely, human liver derived stem cell, human umbilical cord matrix derived mesenchymal stem cell, and human induced pluripotent stem cell-based on their refractive index and volume of organelles. The physical property of each stem cell was compared with that of fibroblast. Based on our findings, the characteristic physical properties of specific stem cells can be quantitatively distinguished based on their refractive index and volume of cellular organelles. Altogether, the method employed herein could aid in the distinction of living stem cells from normal cells without the use of fluorescence or specific biomarkers.

// Je-won Ryu 1, * , Sung Sik Choe 2, * , Seung-Hee Ryu 1 , Eun-Young Park 1 , Byoung Wook Lee 3 , Tae Keun Kim 4 , Chang Hoon Ha 3 , Sang-wook Lee 1 1 Department of Radiation Oncology, Asan Medical Center and University of Ulsan College of Medicine, Seoul, Republic of Korea 2 Department of Biological Sciences, Institute of Molecular Biology and Genetics, National Creative Research Institutive Center for Adipose Tissue Remodeling, Seoul National University, Seoul, Republic of Korea 3 Asan Institute for Life Science, Asan Medical Center and University of Ulsan College of Medicine, Seoul, Republic of Korea 4 Department of Life Science, College of Natural Science, Hallym University, Kyeongki Province, Republic of Korea * These authors contributed equally to this work Correspondence to: Chang Hoon Ha, email: chhoonha@gmail.com Sang-Wook Lee, email: lsw@amc.seoul.kr Keywords: Epidermal Growth Factor (EGF), Purinergic Receptor 2(P2Y), Redox Factor-1(Ref-1), Zinc Finger-containing Transcriptional Regulator 1(EGR1), Phosphatase and Tensin Homolog (PTEN) Received: September 30, 2016 Accepted: November 24, 2016 Published: December 07, 2016 ABSTRACT Epidermal growth factor (EGF) signaling promotes cell proliferation and survival in several types of cancer. Here, however, we showed that EGF inhibits proliferation and promotes programmed cell death in non-small cell lung cancer (NSCLC) cells. In A549 cells, EGF increased redox factor-1 (Ref-1) expression and the association of Ref-1 with zinc finger-containing transcriptional regulator (EGR1) via activation of p22 phox , RAC1, and an NADPH oxidase subunit. EGF increased p22 phox and RAC1 expression through activation of purinergic receptors (P2Y). Elevated Ref-1/EGR1 levels increased phosphatase and tensin homolog (PTEN) levels, leading to inhibition of the Akt pathway. EGF-induced PTEN upregulation increased apoptosis and autophagy-induced damage in A549 cells, whereas Ref-1 knockdown blocked EGF-induced PTEN upregulation in an NADPH oxidase p22 phox subunit-independent manner. In addition, p22 phox knockdown restored EGF-induced effects, implying that changes in P2Y activity caused by EGF, which activates NADPH oxidase via RAC1, influenced Ref-1-mediated redox regulation. Finally, EGF similarly attenuated cell proliferation and promoted autophagy and apoptosis in vivo in a xenograft model using A549 cells. These findings reveal that EGF-induced redox signaling is linked to Ref-1-induced death in NSCLC cells.

Arthroscopic surgery is a minimally invasive surgery which provides a number of benefits, such as faster recovery and minimal incision. However, due to the limited field of view caused by the use of an arthroscope and the difficulties associated with maneuvering surgical instruments through a narrow portal, a highly skilled surgeon is often required. To cope with this, various surgical training simulators have been developed. This study proposes novel designs for the surgical instruments to be used for a shoulder joint arthroscopic surgery simulator. In-depth analysis on the surgical instruments used in the shoulder joint arthroscopic surgery training has been conducted, and novel surgical instruments for a training simulator is developed. The developed surgical instrument is equipped with sensors and actuators to deliver haptic sensation to the users.

This paper deals with the development of a relief robot system that can provide first aid to the injured person by remote control in disaster environment and battlefield. To perform safe first aid, relief robot has hardware and controller that can feed back the contact force while following the command of remote controller. In addition, a controller capable of maintaining system stability under the influence of time delay and packet loss due to communication state during remote control is mounted. The developed relief robot system is evaluated through experiments simulating actual first aid procedures.

The instability of recombinant basic fibroblast growth factor (bFGF) is a major disadvantage for its therapeutic use and means frequent applications to cells or tissues are required for sustained effects. Originating from silkworm hemolymph, 30Kc19α is a cell-penetrating protein that also has protein stabilization properties. Herein, it was investigated whether fusing 30Kc19α to bFGF could enhance the stability and skin penetration properties of bFGF, which may consequently increase its therapeutic efficacy. The fusion of 30Kc19α to bFGF protein increased protein stability, as confirmed by ELISA. 30Kc19α-bFGF also retained the biological activity of bFGF as it facilitated the migration and proliferation of fibroblasts and angiogenesis of endothelial cells. It was discovered that 30Kc19α could improve the transdermal delivery of a small molecular fluorophore through the skin of hairless mice. Importantly, it increased the accumulation of bFGF and further facilitated its translocation into the skin through follicular routes. Finally, when applied to a skin wound model in vivo, 30Kc19α-bFGF penetrated the dermis layer effectively, which promoted cell proliferation, tissue granulation, angiogenesis, and tissue remodeling. Consequently, our findings suggest that 30Kc19α improves the therapeutic functionalities of bFGF, and would be useful as a protein stabilizer and/or a delivery vehicle in therapeutic applications.

The plasmonic coupling, the enhanced electromagnetic field occurring through a uniform and small separation between metallic particles, is required for better application to localized surface plasmon resonance. Graphene has been studied as a good spacer candidate because of its precise controllability at subnanoscale. Here, the enhancement of plasmonic coupling among metallic nanoparticles (NPs) uniformly spread out on both sides of a graphene spacer is experimentally and simulatively investigated. Additionally, the post-evaporated flat structure is rippled along one direction to reduce the separation between nanoparticles. As the amount of rippling increases, the enhancement factor (EF) of the plasmonic coupling increases almost linearly or quadratically depending on the size of nanoparticles. Such a highly rippled nanostructure is believed to not only increase the plasmonic coupling in either side of the spacer but lead to a higher density of “hot spots” through the spacer gap also. The observed EFs of a structure with the MLG spacer are consistent with the simulation results obtained from the classical electrodynamics. On the other hand, the SLG case appears to be inconsistent with such a classical approach, indicating that the plasmon tunneling through the thin barrier is prevalent in the case of the SLG spacer.

This study was performed to establish a production system for in situ and ex situ conservation of Elsholtzia minima Nakai, an endemic plant grown in Jeju Island. Moreover, this study aimed to identify root-growth characteristics according to the use of pre-treatment agents and seedling growth effect according to fertilizer application. The mean temperature was similar in greenhouse and vinyl-moist chamber, but air humidity was higher in vinyl-moist chamber than in greenhouse. After stem planting of Elsholtzia minima Nakai, initial root growth was observed after 10 days in greenhouse and after 7 days in vinyl-moist chamber. Root growth rate was more rapid in vinyl-moist chamber. Moreover, survival rate, root growth rate and root number was slightly higher in vinyl-moist chamber than in greenhouse, indicating that vinyl-moist chamber is more effective in plant growth. When pre-treatment agents were used to remove root growth-inhibiting substances, a higher root growth rate of more than 95% was found in pre-treatment groups, excluding the group treated with AgNO3 at 77.5%. Thus, Elsholtzia minima Nakai is thought to have less root growth inhibitors. In the analysis of nitrogen application rate and Osmocote application by seedling container, a difference was found in survival rate and growth according to application rate and container conditions. When Osmocote, a slow release fertilizer, was applied to the soil surface around each culture container, survival rate and the growth of aerial and root parts were most favorable. Thus, Osmocote fertilizer is thought to be desirable for seedling propagation of Elsholtzia minima Nakai.