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Sequestosome 1 (p62) is a multifunctional adapter protein involved in various physiological functions, such as selective autophagy and oxidative stress response. Hence, aberrant expression and defective regulation of p62 are thought to lead to the onset of various diseases, including cancer. The expression of p62 has been shown to be increased in breast cancer tissues, and is correlated with a poor prognosis. However, the role of p62 in the breast cancer pathophysiology is still unclear. Here, we aimed to analyze the effect of changes in p62 expression on breast cancer cell lines. DNA microarray analysis revealed that the expression of progesterone receptor (PR), which is one of the indices for the classification of breast cancer subtypes, was markedly suppressed by forced expression of p62. The protein expression of PR was also decreased by forced expression of p62, but increased by knockdown of p62. Moreover, we found that p62 knockdown induced the protein expression of argonaute 2 (AGO2). Luciferase reporter assay results showed that the gene expression of PR was promoted by AGO2. Furthermore, results revealed that overexpression of AGO2 partially rescued the decrease in PR expression induced by forced expression of p62. Collectively, our findings indicated that p62 accumulation suppressed the expression of AGO2, which in turn decreased the expression of PR, suggesting that p62 may serve as a marker of aggressive breast cancer and poor prognosis. Moreover, the p62-AGO2-PR axis was identified as a crucial signaling cascade in breast cancer progression.

We report the case of a bowel injury, which occurred after the percutaneous cryoablation (PCA) of large renal cell carcinoma (RCC). A 50-year-old man with RCC measuring 47 mm in diameter. First, we performed transarterial embolization for the tumor, followed by PCA with hydrodissection, which displaced the small intestine from the iceball. The procedure was completed without any complication on the procedural day; however, the patient complained of appetite loss and abdominal pain 2 days after PCA. Computed tomography revealed a bowel injury at the small intestine adjacent to the tumor. After 7 days, ileus tube insertion, and fasting, the patient recovered from the bowel injury and was discharged 10 days after PCA. He underwent a second PCA because of a small recurrent renal tumor 5 months after the first PCA without complications. This case indicated that a bowel injury after PCA for RCC could be treated conservatively. Keywords: Cryoablation, Renal cell carcinoma, Bowel injury, CT guided

A rapid and efficient lithium-ion recovery from seawater established by employing calix[4]arene derivatives as extraction reagents in a microreactor system. A tripropyl-monoacetic acid derivative of calix[4]arene (1Ac) exhibited a very high extraction ability for lithium-ion within 2.00 s by using a microreactor system. The complete stripping of the lithium ions achieved with 1.0 M HCl as a stripping reagent. In a competitive metal system containing lithium, sodium, potassium, rubidium, cesium, magnesium, calcium, strontium and barium ions; lithium-ion selectively loaded on 1Ac. Further, lithium recovery from seawater was carried out without any pretreatment or pH adjustment in the three-step process. In the first step of the process, 1Ac extracted 100% lithium and 2.56% sodium ions from the seawater. Both lithium and sodium ions stripped with HCl in the second step and then sodium in the stripped solution selectively re-extracted with monopropyl-triacetic acid calix[4]arene derivative (3Ac) thus yielding pure lithium chloride in the aqueous phase. Both 1Ac and 3Ac calix[4]arene derivatives regenerated after the stripping process.

Removal of Pb(II) ion over several other metal ions with 25,26,27,28-tetrakis(N,N-diethylaminocarbonylmethoxy)-5,11,17,23-tetrakis(1,1,3,3-tetramethylbutyl)calix[4]arene (EATOC) from nitric acid media was investigated in a solvent extraction system. Pb(II) ion was selectively extracted over Fe(III), Co(II), Zn(II), Cu(II) and Ni(II) in the competitive model system. Further, Pb(II) ion was easily stripped from the metal-laden EATOC complex with distilled water as stripping agent. However, Pb(II) extraction and stripping in the batch system took 90 min, 24 h, respectively, to reach an equilibrium in a batch system, but it was only 2.00 s with using droplet microfluidic reactor system, which is remarkable. A rapid and selective removal of Pb(II) from a simulated wastewater effluent was also achieved. Pb(II) complexation mechanism with EATOC was investigated by using ultraviolet, Fourier transform-infrared and proton nuclear magnetic resonance spectroscopies. It was found that the complexation between Pb(II) and EATOC takes place in 1:1 stoichiometric ratio through solvation mechanism. The carbonyl oxygen atoms and phenoxy oxygen atoms of EATOC played a pivotal role in Pb(II) complexation.

Purpose To employ bioluminescence imaging (BLI) as a quantitative imaging biomarker to assess preclinical evaluation of cryoablation in a murine model. Materials and Methods In vitro, Colon26-Luc (C26-Luc) cells were seeded at 6 different concentrations in 35-mm dishes. These were divided into 6 groups: group 0 (G0), a control group without treatment; and groups 1–5 (G1–G5) according to the number of freeze–thaw cycles, with each cycle consisting of freezing at −80°C for 10 min followed by thawing at room temperature for 5 minutes. BLI and flow-cytometric analysis were performed after cryotherapy. In vivo, 20 tumor-bearing mice with C26-Luc cells were divided into 4 groups: group 0 (G0), a control group; and groups 1–3 (G1–G3) according to the number of freeze–thaw cycles. Each cryoablation procedure was performed for 30 seconds with liquid nitrogen (−170°C) applied with cotton-tipped applicators. BLI was acquired at 6 hours and 1, 3, and 7 days after treatments. Results In vitro, BLI signal showed a negative correlation with the number of freeze–thaw cycles (r = –0.86, P = .02). In vivo, there was no difference in tumor volume at 1 day after cryoablation among all groups, but the BLI signals were significantly different between G0 and G2/G3 (P = .03 and P = .02, respectively) and between G1 and G3 (P = .04). BLI signals reflected tumor growth speed and survival ratio. Conclusions This study demonstrates the direct validation of BLI as a quantitative tool for the early assessment of therapeutic effects of cryoablation.

Chondromas are benign tumors that can be found at several sites in the body, while those arising from the dura mater are extremely rare. Among them, although chondromas arising from the cranial dura mater have been reported, those arising from the spinal dura mater have not been reported in the literature to date. A 66-year-old woman presented with right-sided continuous backache which she had developed recently. After detailed examinations, an epidural tumor at the thoracic level was detected. The patient underwent surgery and a total en-bloc resection was accomplished. From the clinical and pathological findings, the tumor was revealed as chondroma arising from the spinal dura mater. A recent comprehensive study has identified the isocitrate dehydrogenase 1 (IDH1) and IDH2 mutations in conventional central and periosteal cartilaginous tumors, and it has subsequently been analyzed in intracranial chondrosarcoma and chondroma, including chondroma of the convexity dura mater. Herein, we describe a novel case of chondroma arising from the spinal dura mater, with mutation analysis of IDH1 and IDH2 both of which revealed wild-type.

We have discovered and reported potent p53-MDM2 interaction inhibitors possessing dihydroimidazothiazole scaffold. Our lead showed strong activity in vitro, but did not exhibit antitumor efficacy in vivo for the low metabolic stability. In order to obtain orally active compounds, we executed further optimization of our lead by the improvement of physicochemical properties. Thus we furnished optimal compounds by introducing an alkyl group onto the pyrrolidine at the C-2 substituent to prevent the metabolism; and modifying the terminal substituent of the proline motif improved solubility. These optimal compounds exhibited good PK profiles and significant antitumor efficacy with oral administration on a xenograft model using MV4-11 cells having wild type p53.

A novel, inexpensive and efficient method for the preparation of laccase-immobilized microreactors has been proposed, based on the formation of an enzyme-polymeric membrane on the inner wall of microtubes (500 μm inner diameter) as a result of the cross-linking polymerization reaction between the laccase and bifunctional cross-linkers agents (paraformaldehyde and glutaraldehyde). Under the optimum conditions, an immobilization yield of 72% and an activity of 45 μM/min, determined using 2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonate) (ABTS) as substrate in a continuous-flow assay, were detected. The biochemical characterization of the laccase-immobilized microreactors demonstrated their enhanced characteristics: they exhibited a broad range of optimum pH and temperature and excellent stability under different conditions of pH, temperature, chemical inactivating agent, storage and long-term operation. The laccase-immobilized microreactors were applied for the biotransformation of model compounds to demonstrate their efficiency and performance. Important reaction yields were obtained, even at lower residence times compared with conventional bioreactors. Furthermore, it was designed a two-stage bioreactor for the application of laccase-mediated reactions, preventing the biocatalyst from inactivation. The great performance of the microreactor system, possibly due to the more rapid mass transfer and the larger area to volume ratio, makes the presented technologies excellent platforms for increasing the laccase uses and improving its catalytic action in several fields such as biotransformations or bioanalyses.

With the aim of discovering potent inhibitors of the p53-MDM2 interaction and thus obtaining a potent anticancer drug, we have pursued synthesis and optimization of dihydroimidazothiazole derivatives, which have been discovered via scaffold hopping by mimicing the mode of interaction between MDM2 and Nutlins. Upon the discovery we encountered a problem involving the chemical instability of the scaffold, that is, susceptibility to oxidation which led to imidazothiazole. In order to solve this problem and to obtain further potent compounds, we executed medicinal research and thus furnished the optimal compounds by incorporating the methyl group onto the C-6 position to avoid the oxidation, and by modifying the C-2 moiety of the additional proline motif, which furnished high potency. The incorporation of the pyrrolidine moiety at the C-2 position raised another hydrophobic interaction site with MDM2 protein, which was generated by the induced-fitting observed by co-crystal structure analysis. These optimal molecules showed significant improvement in potency when compared with the early lead (+)-1 or Nutlin-3a.

Starting with Nutlins as an initial lead, we designed and generated bicyclic scaffolds aiming to place cis-bischlorophenyl moiety at the equivalent location where the hydrophobic interaction with MDM2 could be expected. As a result, we discovered novel MDM2 inhibitors possessing a dihydroimidazothiazole scaffold. Further exploration of the side chains on the dihydroimidazothiazole scaffold aided by molecular modeling resulted in compounds exhibiting almost comparable in vitro potency to Nutlin-3a.

Proteolysis by sequence-specific proteases is the key step for positive sequencing in proteomic studies integrated with mass spectrometry (MS). The conventional method of in-solution digestion of protein is a time-consuming procedure and has limited sensitivity. In this study, we report a simple and rapid system for the analysis of protein sequence and protein posttranslational modification by multienzymatic reaction in a continuous flow using the enzyme (trypsin, chymotrypsin, or alkaline phosphatase)-immobilized microreactor. The feasibility and performance of the single microreactor and tandem microreactors that were connected by the different microreactors were determined by the digestion of nonphosphoprotein (cytochrome c) and phosphoproteins (β-casein and pepsin A). The single microreactor showed rapid digestion compared with that of in-solution digestions. Multiple digestion by the tandem microreactors showed higher sequence coverage compared with that by in-solution or the single microreactor. Moreover, the tandem microreactor that was made by using the combination of protease-immobilized microreactor and phosphatase-immobilized microreactor showed the capability for phosphorylation site analysis in phosphoproteins without the use of any enrichment strategies or radioisotope labeling techniques. This approach provides a strategy that can be applied to various types of linking microreactor-based multienzymatic reaction systems for proteomic analysis.

This article reports the enhancement of thermal stability involving normal duplex and mutation-carrying DNA duplexes in microchannel laminar flow. The application of an in-house temperature-controllable microchannel-type flow cell is demonstrated for improved discrimination of mismatch base pairs such as A-G and T-G that are difficult to distinguish due to the rather small thermal destabilizations. Enhancement in thermal stability is reflected by an increased thermal melting temperature achieved in microchannel laminar flow as compared with batch reactions. To examine the kinetics and thermodynamics of duplex-coil equilibrium of DNA oligomers, denaturation-renaturation hysteresis curves were measured. The influence of microchannel laminar flow on DNA base mismatch analysis was described from the kinetic and thermodynamic perspectives. An increasing trend was observed for association rate constant as flow rate increased. In contrast, an apparent decrease in dissociation rate constant was observed with increasing flow rate. The magnitudes of the activation energies of dissociation were nearly constant for both the batch and microchannel laminar flow systems at all flow rates. In contrast, the magnitudes of activation energies of association decreased as flow rate increased. These results clearly show how microchannel laminar flow induces change in reaction rate by effecting change in activation energy. We anticipate, therefore, that this approach based on microchannel laminar flow system holds great promise for improved mismatch discrimination in DNA analyses, particularly on single-base-pair mismatch, by pronouncedly enhancing thermal stability.

This paper reports the direct microchannel circular dichroism (CD) measurement method for analyzing conformational changes and orientations of macromolecules in a microchannel flow. CD is a sensitive probe for secondary structure. For that reason, we apply direct CD measurement for microchannels. Herein, we conducted the direct microchannel CD measurement method by devising an optical system in the sample chamber. Furthermore, using this CD microchannel measurement method, we studied conformational changes and orientations of long-strand DNA in an elongational flow.

Conformational control of macromolecules is useful for efficient chemical and biochemical reactions. This article reports a direct observation method for macromolecules, such as long-strand DNA, in microchannel flow as well as a simple method for stretching DNA strands by microfluidics. Stretching and orientation of DNA molecules by control of flow within a microchannel was observed by optical microscopy. This DNA stretching is explained by coil–stretch transition of polymer molecules. This technique is useful for creating chemical reactions with macromolecules. It offers high selectivity and efficiency that are impossible to achieve in bulk solution. We also demonstrate that our microfluidic stretching method can accomplish efficient hybridization of long-strand DNA. This method will be useful for direct hybridization assay of long-strand DNA.

A simple method using self-assembly of colloidal particles was applied to modify a microcapillary inner surface. It was possible to arrange the particles on to the capillary inner wall and to control particle layer thickness and layer patterning of by choosing adequate combinations of solvents and drying temperature. Furthermore, anatase type TiO2 coated SiO2 composite particles were shown to be applicable for the particle arrangement process in the microreactor to carry anatase type TiO2 catalyst. This process was applied as a simple catalyst carrying method onto the micro-reactor inner wall. It was applied for a few catalytic reaction systems including enzyme reaction and photocatalytic reaction. Enzyme reaction was enhanced because of the increase in the reactor surface area. Furthermore, reasonably good enhancement of the photocatalytic reaction was observed for a reaction in a micro-reactor.

A microfluidic system offers superior controllability of fluids. This study developed a novel separation method for complexes formed in a microchannel. It presents an application for DNA sensor. This method is based on the molecular sieving effect, which is achieved by superior controllability of fluidics. Use of confocal fluorescence microscopy observation and computer simulation confirmed that the laminar secondary flow at the curing part of a microchannel enables this microfluidic separation. Also in this study, we applied this molecular sieving effect for sequence-selective DNA sensor. This measurement showed that the response of a complementary sequence target DNA was higher than that of a non-complementary sequence. Moreover, the response of a longer target was higher than a different-length complementary sequence target. This method does not require immobilization of probe or target DNA: solutions are merely injected into the microchannel and all reactions occur in the liquid phase. Such features might lower the experimental error and difference in data by operators.

Without using additional surfactant as structure-directing agent, well dispersed ZnO nanorods were directly synthesized in the dimethyl sulfoxide (DMSO) and alcohol mixture solution by a one-step wet chemical method. The experiment results demonstrated that alcohol molecular with longer chain is helpful for the growth of ZnO nanorods. The absorption and emission spectra of as-synthesized ZnO nanorods were also presented in this paper.