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The purpose of this study was to evaluate the differential effect of chemotherapy regimen in preoperative chemoradiotherapy (CRT) for locally advanced rectal cancer.The medical records of 279 patients who underwent preoperative CRT followed by surgery for cT3/4 rectal cancer from 2003 to 2010 were retrospectively reviewed. Thirty-four patients were treated with one cycle of i.v. bolus 5-fluorouracil (5-FU) during 1st week (group A), 214 patients with two cycles of i.v. bolus 5-FU during 1st and 5th week (group B), and 31 patients with oral capecitabine on the days with radiotherapy (group C). Propensity score matching was performed between three groups.Median follow-up was 60.1 months. Five-year locoregional recurrence-free survival (LRFS), distant metastasis-free survival (DMFS), disease-free survival (DFS) and overall survival (OS) rates were 91.2, 83.3, 75.0, and 84.5%, respectively. Thirty-one patients per group were allocated to three groups via propensity score matching. On univariate analysis, concurrent chemotherapy regimen was not a significant prognostic factor for survival outcomes in the matched group analysis (OS, p=0.175; DFS, p=0.481; DMFS, p=0.515; LRFS, p=0.456). In addition, there was no significant difference in the sphincter preserving surgery rate, circumferential resection margin status, and pathologic response between three groups (p=0.441, 1.000, 0.818, respectively). As regards to treatment-related toxicity, 9 patients showed grade 3 neutropenia in group B, while there was no grade 3 or higher toxicity in groups A and C.The concurrent chemotherapy regimen (5-FU #1 vs 5-FU #2 vs capecitabine) did not have a significant effect on treatment outcomes in locally advanced rectal cancer patients receiving neoadjuvant CRT.

We present measurements of the in situ, microscopic architecture of a self-assembled bilayer at the interface between a regularly nanopatterned surface and an aqueous sub-phase using neutron reflectometry. The substrate is patterned with a rectangular array of nanoscale holes. Because of the high quality of the pattern, using neutron reflectometry, we are able to map the surface-normal density distribution of the patterned silicon, the penetration of water into the pattern, and the distribution of a deposited film inside and outside of the etched holes. In this study, 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC) single bilayers were deposited on the hydrophilic patterned surface. For bilayers deposited either by vesicle fusion (VF) or by the Langmuir-Schaefer (L-S) technique, the most consistent model found to fit the data shows that the lipids form bilayer coatings on top of the substrate as well as the bottoms of the holes in an essentially conformal fashion. However, while there is a single bilayer on the unetched silicon surface, the lipids coating the bottoms of the holes form a complex bimodal structure consistent with a rough surface produced by the etching process. This study provides insight into film transfer both outside and inside regular nanopatterned features. Open image in new window

Two kinds of N,O-hydroxypropyl chitosans (HPCTOs) with degree of substitution (DS) and molar substitution (MS) ranging from 2.15 to 2.39 and 2.9 to 4.1, respectively, and five kinds of aliphatic acid esters of HPCTOs (HPCTOAms, m=0,2,4,7,9, the number of methylene units in aliphatic substituent) based on the HPCTOs were synthesized, and the thermotropic liquid crystalline properties of the derivatives were investigated. All the derivatives formed enantiotropic cholesteric phases whose optical pitches ('s) increased with increasing temperature. However, the glass and clearing temperatures, the magnitude of of the mesophase at the same temperature, and the temperature dependence of of the investigated derivatives highly depended on MS and m. The thermotropic mesophase properties of HPCTOAms were significantly different from those reported for the aliphatic acid esters of hydroxypropyl celluloses. The results indicate that the secondary amino group in the C-2 position plays an important role in the thermal stabilization and temperature dependence of of the cholesteric mesophase.

Octa[8-{4-(4'-cyanophenylazo)phenoxy}]octyl and octa[8-{4-(4'-cyanophenylazo)phenoxycarbonyl}]heptanoated disaccharide derivatives were synthesized by reacting cellobiose, maltose, and lactose with 1-{4-(4'-cyanophenylazo) phenoxy}octylbromide or 1-{4-(4'-cyanophenylazo)phenoxycarbonyl}]heptanoyl chloride, and their thermotropic liquid crystalline and photochemical phase transition behavior were investigated. All the {(cyanophenylazo)phenoxy} octyl disaccharide ethers (CADETs) formed monotropic nematic (N) phases, whereas all the {(cyanophenylazo) phenoxycarbonyl}heptanoated disaccharide esters (CADESs) exhibited enantiotropic N phases. Compared with CADETs, CADESs showed higher isotropic (I)-to-N phase transition temperatures. Photoirradiation of the disaccharide derivatives in a glass cell or in a cell with a rubbed polyimide (PI) alignment layer at a N phase resulted in disappearance of the N phase due to trans-cis photoisomerization of azobenzene, and the initial N phase was recovered when the irradiated sample was kept in the dark because of cis-trans thermal isomerization and reorientation of trans-azobenzenes. The rates of the photochemical N-I and the thermal I-N phase transition of disaccharide derivatives in a cell with a rubbed PI alignment layer were faster than those in a glass cell, and were significantly different from those observed for the monomesogenic compounds containing cyanoazobenzene and the 4-{4'-(cyanophenylazo)phenoxy}octyl glucose and cellulose ethers. The results were discussed in terms of difference in cooperative motion of azobenzene groups due to the flexibility of the main chain, the number of mesogenic units per repeating units, and the distance between the azobenzene groups.

Various types of high-temperature superconducting (HTS) devices have been developed, some of which have completed field testing and are ready for use in commercial applications. In order to facilitate the commercialization of HTS electric equipment for extra high voltage levels, cryogenic dielectric technologies should be established. In this work, in order to develop partial discharge (PD) measurement techniques for HTS transformers, artificial defects were installed both in liquid nitrogen and insulating oil. Experimental investigations were carried out for the diagnosis of the HTS transformer, and various PD patterns, which occurred due to the artificial defects of immersion in both insulating oil and liquid nitrogen, were measured. Throughout this work, the PD patterns that occurred in liquid nitrogen were detected, analyzed, and compared to those in insulating oil through the use of a high frequency current transformer (HFCT) diagnostic method. The partial discharge measuring techniques we developed could be fundamental in establishing HTS transformer diagnosis technologies for power applications.

The recent increase in the number of failures during the installation and insulation of gas-insulated switchgear (GIS) has led to a need for a reliable risk assessment technique. Accordingly, in this study, a fundamental database of UHF partial discharge (PD) patterns corresponding to different types of defects is presented for risk assessment and for the observation and diagnosis of the state of insulation of GIS at field sites; the patterns have been obtained by modelling a number of defects that have been reported to be the most critical in GIS. For the realisation of a system that can simultaneously detect and analyse UHF PD (it is internationally accepted that the use of such a system is the most effective method to diagnose GIS insulation), a wideband UHF sensor and amplifier are designed and fabricated. The system operation is then investigated. In addition, a chaotic analysis of partial discharge (CAPD) is proposed. This analysis can identify the type of defect by means of PD pattern classification without employing the phase information of the applied voltage signal. The proposed CAPD can replace the conventional phase-resolved partial discharge (PRPD) analysis and can be employed at field sites when the phase information is unavailable. Especially, the PD patterns of free-moving conducting particles, known to be very important in GIS, can be distinguished from those of other defects when using the CAPD method, which is not the case when the PRPD analysis is being performed.

Because these coupling reactions require tailored purified pro-teins, purification of the target protein was required. Difficultiesassociated with obtaining a diversity of purified proteins, atime-consuming and labor-intensive process, have no doubtlimited the number of studies using functional protein arrays.

A novel technique has been proposed and related experimental works have been performed in order to detect the partial discharges and the location of the possible defects introduced into the Gas Insulated Switchgear. For this purpose, a prototype HY Pockels sensor has been developed and then employed in order to investigate its field applicability for finding the location of the defects using a 170kV GIS mock-up. Our proposed sensor enables us to measure the electric field variation due to the PD occurrence. In addition, the different PD patterns are observed, which might be dependant on the location and the distance of the sensor with respect to the PD source. Throughout this work, its linear response has been proved to be admissible as a function of the applied voltage. And also the position of the PD source might be distinguished by comparing the PD patterns.

Particle sorting using acoustofluidics has enormous potential but widespread adoption has been limited by complex device designs and low throughput. Here, we report high-throughput separation of particles and T lymphocytes (600 μL min(-1)) by altering the net sonic velocity to reposition acoustic pressure nodes in a simple two-channel device. The approach is generalizable to other microfluidic platforms for rapid, high-throughput analysis.

Neutrophils are the predominant inflammatory cells found in the vaginal discharges of patients infected with Trichomonas vaginalis . Although chemoattractants, such as leukotriene B 4 and interleukin-8 (IL-8), are found in the vaginal discharges of symptomatic trichomoniasis patients, little is known about the mechanism of how neutrophils accumulate or mediate initial inflammatory response after acute T. vaginalis infection. We examined IL-8 production in neutrophils activated by T. vaginalis and evaluated the factors involved in T. vaginalis adherence that might affect IL-8 production. When human neutrophils were stimulated with live trophozoites, T. vaginalis lysate, or T. vaginalis excretory-secretory products, the live trichomonads induced higher levels of IL-8 production than the lysate or products did. When live trichomonads were pretreated with various inhibitors of proteinase, microtubule, microfilament, or adhesin (which are all known to participate in the adherence of T. vaginalis to vaginal epithelial cells), IL-8 production significantly decreased compared with the untreated controls. Furthermore, an NF-κB inhibitor (pyrrolidine dithiocarbamate), a mitogen-activated protein (MAP) kinase (MEK) inhibitor (PD98059), and a p38 MAP kinase inhibitor (SB203580) significantly suppressed IL-8 synthesis in neutrophils. These results suggest that live T. vaginalis , particularly adherent trophozoites, can induce IL-8 production in neutrophils and that this action may be mediated through the NF-κB and MAP kinase signaling pathways. In other words, T. vaginalis -induced neutrophil recruitment may be mediated via the IL-8 expressed by neutrophils in response to activation by live T. vaginalis .

The molecular level details of the displacement of surface adsorbed fibrinogen from silica substrates were studied by atomic force microscopy, immunochemical assays, fluorescence microscopy, and vibrational sum frequency spectroscopy. The results showed that human plasma fibrinogen (HPF) can be readily displaced from the interface by other plasma proteins near neutral pH because the positively charged alpha C domains on HPF sit between the rest of the macromolecule and the underlying surface. The alpha C domains make weak electrostatic contact with the substrate, which is manifest by a high degree of alignment of Lys and Arg residues. Upon cycling through acidic pH, however, the alpha C domains are irreversibly removed from this position and the rest of the macromolecule is free to engage in stronger hydrogen bonding, van der Waals, and hydrophobic interactions with the surface. This results in a 170-fold decrease in the rate at which HPF can be displaced from the interface by other proteins in human plasma.

The photodegradation of pentachlorophenol in water was examined with a high-pressure mercury lamp. A number of photodegradation products of pentachlorophenol, such as less-chlorinated phenols through reductive dechlorination, and catechol and trihydroxylated products via the addition of hydroxyl radicals, were observed as major products. Moreover, polychlorinated biphenylethers, hydroxylated polychlorobiphenylethers and polychlorinated dibenzo-p-dioxins as minor products were also formed by dimerization process during the photolysis of pentachlorophenol. These compounds were identified by gas chromatography-mass spectrometry. The degradation rate of PCP in water was measured against the UV-irradiation time. After the irradiation for 15 min, 75% of PCP was converted into a number of products through reductive dechlorination and oxidation. On the basis of the identified products, the photodegradation pathways of PCP under UV irradiation were proposed.

Serum levels of sIL-2R can be used to monitor in vivo immune activation and its elevation have been shown to be correlated with T cell mediated immune disease such as atopic dermatitis, psoriasis, lymphoma and systemic sclerosis. Vitiligo is the disease of depigmentation caused by destruction of melanocytes, and there have been extensive studies on the immune pathogenesis. If the pathogenesis of vitiligo is correlated with the activation of T lymphocytes, the change of IL-2R will be detected compared with that of normal control. Therefore we sought the change in sIL-2R to determine whether T lymphocytes from patients with vitiligo show abnormal biological behavior. The quantitation of sIL-2R was done by sandwich enzyme-linked immunosorbent assay (ELISA) from the sera of 79 vitiligo patients and 40 normal controls. The results were summarized as following. The sIL-2R level in vitiligo patients (671.91 +/- 368.59 U/ml) was significantly increased compared with that of controls (370.8 +/- 71.8 U/ml; P < 0.005). According to clinical types, sIL-2R level in focal type of vitiligo patients was significantly higher than those in other types (segmental or generalized; P < 0.05). The sIL-2R level in patients less than 1 year duration was significantly higher than in patients more than 1 year duration (P < 0.05). The sIL-2R levels were not significantly different between active and inactive group. There was no significant differences among sIL-2R levels according to sex or age of onset. Our study showed that sIL-2R level was higher in vitiligo patients compared with that of normal controls, so the activation of T lymphocytes would be an important component in the pathogenesis of vitiligo. The higher sIL-2R levels in recent onset group would suggest that sIL-2R level might be an acute immunologic marker in vitiligo patients.

The photodegradation of DDT in water was examined with a high-pressure mercury lamp. A total of 17 photodegradation products of DDT including DDD, DDE and DDMU were identified by gas chromatography-mass spectrometry (GC-MS). Mass spectrometric analyses were done with electron impact (EI), methane positive chemical ionization (PCI) and electron capture negative ion chemical ionization (ENCI). The degradation rate of DDT in the presence of acetone was measured against the irradiation time. After the irradiation for 10min, 80% of DDT was converted into a number of products, including DDD, DDE, DDMU and dichlorobenzophenone, through reductive dechlorination, oxidation, isomerization and chlorination. On the basis of the identified products, the photodegradation pathways of DDT are proposed.

Cell-free systems offer a simplified and flexible context that enables important biological reactions while removing complicating factors such as fitness, division, and mutation that are associated with living cells. However, cell-free expression in unconfined spaces is missing important elements of expression in living cells. In particular, the small volume of living cells can give rise to significant stochastic effects, which are negligible in bulk cell-free reactions. Here, we confine cell-free gene expression reactions to cell-relevant 20 fL volumes (between the volumes of Escherichia coli and Saccharomyces cerevisiae ), in polydimethylsiloxane (PDMS) containers. We demonstrate that expression efficiency varies widely among different containers, likely due to non-Poisson distribution of expression machinery at the observed scale. Previously, this phenomenon has been observed only in liposomes. In addition, we analyze gene expression noise. This analysis is facilitated by our use of cell-free systems, which allow the mapping of the measured noise properties to intrinsic noise models. In contrast, previous live cell noise analysis efforts have been complicated by multiple noise sources. Noise analysis reveals signatures of translational bursting, while noise dynamics suggest that overall cell-free expression is limited by a diminishing translation rate. In addition to offering a unique approach to understanding noise in gene circuits, our work contributes to a deeper understanding of the biophysical properties of cell-free expression systems, thus aiding efforts to harness cell-free systems for synthetic biology applications.

Introduction: Situs inversus totalis (SIT), occurring at a frequency of 1:5000–1:10,000 live births,1 is characterized by a complete right-to-left transposition of the thoracic and abdominal organs. Only five cases of laparoscopic colon surgery in an SIT patient have been reported. The present case describes a laparoscopic hemicolectomy necessitated by ascending colon cancer in a patient with SIT. Patient and Methods: A 54-year-old woman with ascending colon cancer was referred to our hospital. Preoperative chest radiography showed dextrocardia, and computed tomography showed complete transposition of the abdominal viscera, confirming a diagnosis of SIT. The patient, free of distant metastasis, underwent laparoscopic right hemicolectomy and radical lymphadenectomy, performed by a surgeon experienced in laparoscopic colectomies. After inducing general anesthesia, the patient was placed in a modified lithotomy position, with the operator and scopist situated on the patient's right and the first ass...

(a) An introductory sentence indicating the purposes of the study Distant metastasis is the major cause of cancer-related death in colorectal cancer (CRC) patients. Although the Calponin family has emerged as a distinguishing feature in epithelial-to-mesenchymal transition (EMT) in human cancer, the role of CNN3 member in the metastatic CRC has not been investigated. (b) A brief description of pertinent experimental procedures. Three pairs of primary CRC and corresponding matched metastasis cell lines were analyzed for gene expression by microarray and western blotting. CNN3 was transduced to CRC cell line with lower CNN3 expression level (SW-480), and CNN3 with higher CNN3 expression CRC cell lines (SW-620, HCT-116) was suppressed by lentiviral-induced shRNA. Functional analysis of CNN3 overexpression and suppression was investigated in these CRC cell lines for proliferation and invasion. Expression of several potential CNN3 target genes (ERK1/2) and EMT markers (E-cadherin and vimentin) in primary CRC and matching metastasis CRC cell lines was validated. (c) A summary of the new, unpublished data. Global CNN3 expression levels were significantly higher in metastasis cell lines compared with the matched primary cell lines in both mRNA (FC = 2.3, p = 0.017) and protein levels. CRC cell lines with suppressed CNN3 showed decreased cell proliferation and reduced invasive behavior in CRC cell lines. Suppression of CNN3 in CRC cell lines with higher CNN3 expression resulted in increased E-cadherin and decreased vimentin. pERK1/2 were decreased in accordance with suppression of CNN3 in both SW-620 and HCT-116. Overexpression of CNN3 in CRC cell lines caused reduced E-cadherin in SW-480 and HT-29. (d) A statement of the conclusions CNN3 is involved in mediating EMT and metastatic behavior in the colon. Its expression is accordantly related to EMT markers, and CNN3 may serve as a possible diagnostic marker and therapeutic target for patients with CRC. Citation Format: Soon-Chan Kim, Chang-Won Hong, Sang-Geun Jang, Yeh-Ah Kim, Seung-Yong Jung, Jea-Gab Park, Ja-Lok Ku. Calponin-3, acidic (CNN3) mediates EMT-related genes in human colorectal cancer metastasis. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1642.

This paper describes stepwise on-demand generation and fusion of femtolitre aqueous droplets based on interfacial tension. Sub-millisecond reaction times from droplet fusion were demonstrated, as well as a reversible chemical toggle switch based on alternating fusion of droplets containing acidic or basic solution, monitored with the pH-dependent emission of fluorescein.

This paper describes stepwise on-demand generation and fusion of femtolitre aqueous droplets based on interfacial tension. Sub-millisecond reaction times from droplet fusion were demonstrated, as well as a reversible chemical toggle switch based on alternating fusion of droplets containing acidic or basic solution, monitored with the pH-dependent emission of fluorescein.

This paper presents an effective technique for measuring the propogation velocity of the partial discharge (PD) electromagnetic (EM) in a gas-insulated switchgear. The propagation velocity varies with the type of gas insulated switchgear. Because of this, it is necessary to evaluate the propagation of a PD EM wave front between different points in the gas insulated switchgear. The signal time delay is determined by locating the initial PD in the cumulative energy plot, as obtained by the correlation of the signals from two UHF sensors. The efficiency of this method is illustrated with experimental results.

We developed a microfluidic platform for splitting well-mixed, femtoliter-volume droplets from larger water-in-oil plugs, where the sizes of the daughter droplets were not limited by channel width. These droplets were separated from mother plugs at a microfabricated T-junction, which enabled the study of how increased confinement affected enzyme kinetics in droplets 4-10 microm in diameter. Initial rates for enzyme catalysis in the mother plugs and the largest daughter drops were close to the average bulk rate, while the rates in smaller droplets decreased linearly with increasing surface to volume ratio. Rates in the smallest droplets decreased by a factor of 4 compared to the bulk rate. Traditional methods for detecting nonspecific adsorption at the water-oil interface were unable to detect evidence of enzyme adsorption, including pendant drop tensiometry, laser scanning confocal microscopy of drops containing labeled proteins in microemulsions, and epifluorescence microscopy of plugs and drops generated on-chip. We propose the slowing of enzyme reaction kinetics in the smaller droplets was the result of increased adsorption and inactivation of enzymes at the water-oil interface arising from transient interfacial shear stresses imparted on the daughter droplets as they split from the mother plugs and passed through the constricted opening of the T-junction. Such stresses are known to modulate the interfacial area and density of surfactant molecules that can passivate the interface. Bright field images of the splitting processes at the junction indicate that these stresses scaled with increasing surface to volume ratios of the droplets but were relatively insensitive to the average flow rate of plugs upstream of the junction.

A device with femtoliter-scale chambers and controlled reaction initiation was developed for single-molecule enzymology. Initially separated substrate and enzyme streams were rapidly mixed in a microfluidic device and encapsulated in an array of individual microreactors, allowing for enzyme kinetics to be monitored with millisecond dead times and single-molecule sensitivity. Because the arrays of chambers were fabricated by micromolding in PDMS, the chambers were monodisperse in size, and the chamber volume could be systematically controlled. Microreactors could be purged and replenished with fresh reactants for consecutive rounds of observation. Repeated experiments with statistically identical initial conditions could be performed rapidly, with zero cross-talk between chambers in the array.

Herein, we describe the development of a novel lithographic technique to pattern artificial lipid bilayer microdomains by exploiting the limited mobility of gel-phase phospholipids in two-component mixtures. Reconstituted supported phospholipid bilayers are formed by the self-assembly of lipids into two opposing leaflets on a hydrophilic surface. They are useful model systems for studying the physics and chemistry of biological membranes because of similarities with natural analogues, as well as accessibility to fluorescence microscopy and atomic force microscopy. Spatially addressable supported lipid bilayers hold great promise for the development of integrated biological solid-state devices, such as rapid screening assays for proteins or other biomolecules that associate with membranes, enabling new high-throughput studies for many important cellular functions. They are also highly ordered structures that effectively resist nonspecific binding of many molecules. Patterning of supported lipid bilayer microarrays has been demonstrated with various lateral diffusion barriers, such as metals or metal oxides, proteins, photoresist, mechanical scratches, and voids following either resist lift-off or photo-oxidative degradation of lipids. Patterning has also been demonstrated by direct deposition or removal of lipid material with polymeric stamps. Recently, metastable membrane microdomains with lipid mixtures that resemble the composition of putative natural lipid rafts were patterned photolithographically into supported bilayers. Our approach is to use kinetically “trapped” separation of components in a binary mixture to form stable lipid patterns. Previous investigators have shown that microdomains can spontaneously form in supported bilayer membranes, which contain two or more lipid components. Lipid phases segregate due to different head groups, chain lengths, and/or varying degrees of saturation of their hydrocarbon chains. 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), which has a longer chain length and a higher transition temperature (Tm 41 8C), was used to form ordered gel-like regions, while 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC) (Tm 1 8C) was used to form liquid crystalline regions. Differential scanning calorimetry of multi-bilayers, as well as confocal microscopy and fluorescence correlation spectroscopy of giant unilamellar vesicles, have shown that DLPC and DPPC lipids have limited miscibility and can mix over time to form a two-phase region of coexisting ordered (rich in DPPC) and fluid (rich in DLPC) phases at equilibrium. However, the lateral mobility of lipid molecules in the ordered phase is 250 times below that of the fluid phase. Lipid mixing in patterned areas in this case will be an activated process with a large energy barrier, which suggests that, at room temperature, segregated regions of pure DLPC and pure DPPC will remain kinetically stable. Poly(dimethylsiloxane) (PDMS) stamps were used to create both positive and negative pattern transfers of bilayer microdomains, as depicted in Figure 1. Fluid microdomains could be patterned into a solid gel-like matrix (Figure 1A). Alternatively, gel-like lipids could be patterned in a sea of fluid lipids (Figure 1B). For the first scenario shown in Figure 1A, a PDMS stamp with square features in bas relief was held in contact with a clean planar glass coverslip in aqueous solution. This served as a mold around which the gel-phase DPPC bilayer was cast, using the vesicle fusion method. A solution containing small unilamellar vesicles (SUVs) of DPPC at 45 8C, which is above the transition temperature for this lipid, formed a fluid bilayer around the PDMS posts on the coverslip. Upon cooling to room temperature, the DPPC bilayer solidified into a gel. After rinsing away any residual debris and removal of the PDMS, a solution containing DLPC vesicles was introduced, which resulted in the formation of fluid bilayers in the areas previously protected by the PDMS posts. Generally, the fluid-to-gel phase transition in supported phospholipid bilayers results in the formation of submicron [a] Dr. S.-Y. Jung, Prof. C. P. Collier Division of Chemistry and Chemical Engineering California Institute of Technology, M/C 127–72 Pasadena, CA 91125–7200 (USA) Fax: (+1)626-568-8824 E-mail : collier@caltech.edu [b] Dr. M. A. Holden University of Oxford, Chemistry Research Laboratory Mansfield Road, Oxford, OX1 3TA (UK) [c] Prof. P. S. Cremer Department of Chemistry, Texas A&M University, P.O. Box 30012 College Station, Texas 77843–3012 (USA) Supporting information for this article is available on the WWW under http://www.chemphyschem.org or from the author.

We have functionalized gels with a putative cell-binding (-Arg-Gly-Asp-) (RGD) domain in an effort to regulate mammalian cell behavior in cells entrapped with gel. Adhesion molecules composed of Gly-Arg-Gly-Asp-Ser (GRGDS) peptides and cell recognition ligands were inculcated into thermo-reversible hydrogel composed of N-isopropylacrylamide, with a small amount of succinyl poly(ethylene glycol) (PEG) acrylate (MW 2000) used as a biomimetic extracellular matrix (ECM). The GRGDS-containing p(NiPAAm-co-PEG) copolymer gel was studied in vitro for its ability to promote cell spreading and to increase the viability of cells by introducing PEG spacers. Hydrogel lacking the adhesion molecules proved to be a poor ECM for adhesion, permitting only a 20% spread of the seeded cells after 10 d. When PEG spacer arms, immobilized by a peptide linkage, had been integrated into the hydrogel, conjugation of RGD promoted cell spread by 300% in a 28-d trial. In addition, in a serum-free medium, only GRGDS peptides conjugated with the spacer arm were able to promote cell spread.

beta-catenin mutations have been identified in a variety of human malignancies; most of these are missense mutations restricted at hot-spot areas in exon 3. beta-catenin mutations are known to be highly associated with colorectal cancers with microsatellite instability (MSI). More than 70 beta-catenin mutations have been reported in colorectal cancers, and approximately 90% of beta-catenin mutations have been found in 11 codons (codons 29, 31, 32, 33, 34, 35, 37, 38, 41, 45, and 48) as missense mutations or in-frame deletions. We have developed an oligonucleotide microarray for detecting beta-catenin mutations at these 11 codons. The developed oligonucleotide microarray can detect a total of 110 types of beta-catenin mutations, including the 60 mutations reported previously. Nine beta-catenin mutations were identified in this study by five different methods, i.e., PCR- single-strand conformational polymorphism, denaturing high performance liquid chromatography, direct sequencing, cloning-sequencing, and with an oligonucleotide microarray. All nine of the mutations were identified by denaturing high performance liquid chromatography, cloning-sequencing, and by the oligonucleotide microarray. However, PCR-single-strand conformational polymorphism missed 1 beta-catenin mutation and direct sequencing missed 2. Five beta-catenin mutations from 74 colorectal carcinomas (34 proximal colon cancers and 40 distal colorectal cancers) and 4 beta-catenin mutations from 31 colorectal cancer cell lines (7 from the proximal colon, 6 from the distal colorectum, and 18 unknown) were identified. In colorectal carcinomas, all 5 of the beta-catenin mutations were found in proximal colon tumors. In colorectal cancer cell lines, 2 of 4 cell lines with beta-catenin mutations originated from the proximal colon, and the remaining 2 cell lines were simply described as having originated from the colon. Considering the relationships among beta-catenin mutations, MSI, and tumor location, the frequency of beta-catenin mutations was found to be meaningfully higher in colorectal carcinomas with MSI than in those with microsatellite stability (P0.001); moreover, MSI was found to be more frequent in proximal colon tumors (P0.01). In addition, beta-catenin mutations were also found to be associated with proximal colon cancer (P = 0.017).

Herein we describe a new class of microfluidic immunoassays based upon solid supported lipid bilayers. Two-dimensionally fluid bilayer material, which can accommodate multivalent binding between surface-bound ligands and aqueous receptors, was coated on the surface of poly(dimethylsiloxane) microchannels. The bilayers contained dinitrophenyl (DNP)-conjugated lipids for binding with bivalent anti-DNP antibodies. Twelve independent data points of surface coverage versus bulk protein concentration could be made simultaneously by forming a linear array of channels and flowing fluorescently labeled antibodies into them. This enabled an entire binding curve to be obtained in a single experiment. The measured apparent binding constant for the DNP/anti-DNP system was 1.8 microM. The methodology for performing heterogeneous assays developed here not only produces rapid results but also requires much less protein than traditional procedures and eliminates some standard sources of experimental error.

Solid supported lipid bilayers are rapidly delaminated when drawn through the air/water interface. We have discovered that a close packed monolayer of specifically bound protein prevents this process. The protection mechanism worked in two ways. First, when protein-protected bilayers were drawn through the air/water interface, a thin bulk water layer was visible over the entire bilayer region, thereby preventing air from contacting the surface. Second, a stream of nitrogen was used to remove all bulk water from a protected bilayer, which remained fully intact as determined by fluorescence microscopy. The condition of this dried bilayer was further probed by fluorescence recovery after photobleaching. It was found that lipids were not two-dimensionally mobile in dry air. However, when the bilayer was placed in a humid environment, 91% of the bleached fluorescence signal was recovered, indicating long-range two-dimensional mobility. The diffusion coefficient of lipids under humid conditions was an order of magnitude slower than the same bilayer under water. Protected bilayers could be rehydrated after drying, and their characteristic diffusion coefficient was reestablished. Insights into the mechanism of bilayer preservation were suggested.

The keratinocyte culture model has previously been used as an in vitro method for testing skin irritating potential of common skin irritants. However, solubility limits its use for finished products. Shampoo is very soluble in water which should make it an ideal product category for the cell culture model. To determine the skin irritant potential of several commercial shampoos, we employed cultured human keratinocytes as an in vitro model. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide test (MTT) and lactic dehydrogenase release (LDH) test were used to document cell toxicity. 7 volunteers were patch tested and their reactions evaluated using laser Doppler flowmetry and compared with the in vitro data. MTT and LDH have a good negative correlation with each other. Patch test reaction, especially at high concentrations, correlates relatively well with the in vitro test, especially with shampoos of strong and weak irritancy. However, the rank order of the shampoos of moderate toxicity was not the same as in the in vitro data. This suggests that the cell culture technique cannot directly replace in vivo methods, and that data obtained by the cell culture method should be interpreted carefully.

Water in oil droplets has been an important research topic because of the possibility of small-volume biochemical reaction vessels in which the reaction time and chemical concentration can be precisely controlled. Here, a new method for producing femtoliter-scale (10−17 L) aqueous droplets on-demand has been developed based on pressure driven formation of droplets at the intersection of microchannels. Different aqueous solutions in two apposed microchannels with 1 micrometer in width were forced into an oil-filled microchannel to form two aqueous droplets, which then collided with each other to start biochemical reactions by diffusional/convectional mixing. These monodisperse micro-reactors can enable the characterization of catalytic or biochemical reaction dynamics in confined environments, as well as the development of reaction vessels for highly localized sampling and dosing.

We describe a method for creating discrete femtolitre-scale water-in-oil droplets on demand, based solely on a geometrically induced reduction in oil/water interfacial area at microfabricated junction orifices. This on-demand generation method is driven by self-shear of droplets due to interfacial tension induced forces resulting from a localized transition in microchannel height. The magnitudes of shear stresses involved appear to be significantly less than the shearing instabilities used to split off daughter droplets from aqueous mother plugs at microfabricated junctions in continuous water-in-oil segmented flows, which implies that this method may be better suited for studying biochemical reactions and reaction kinetics in droplets of decreased volume without loss of chemical reactivity due to redistribution of surfactant density used to passivate the oil/water interface. Predictable droplet generation rates under constant pressure conditions or the gated formation of one, two or more droplets at a time with fixed pressure pulses have been demonstrated in a similar manner to active on-demand droplet generation strategies, but with a simpler system not needing actuation and sensing equipment beyond a pressure regulator.