Development of PDMS-based Microfluidic Device for Cell-based Assays

In a single step photolithography, muhi-level microfluidic device is fabricated by printing novel architectures on a film photomasks. The whole fabrication process is executed by classical PCB technology without the need to access clean room facilities. Different levels of protruding features on PCB master are produced by exposing a photomask with specifically arranged "windows and rims" architectures, followed by chemical wet etching. Poly(dimethylsiloxane)(PDMS) is then molded against the positive relief master to generate microfluidic device featured with multi-level sandbag structure and peripheral microchannels. This sandbag structure is an analog to traditional dam or weir for particle entrapment. The microstructure does not collapse when subjected to applied pressure, which is suitable for operation on elastic PDMS substrate.Typical immunocytochemcial staining assays were performed in the microdevice to demonstrate the applicability of the sandbag structure for cellular analysis. This simplified microfabrication process employs low-cost materials and minimal specialized equipment and can reproducibly produce mask lines with about 20 μm in width, which is sufficient for most microfluidic applications.     

Site-Specific <Emphasis Type="Italic">N</Emphasis>-Glycosylation of Recombinant Pentameric and Hexameric Human IgM

Glycosylation is known to play an important role in IgG antibody structure and function. Polymeric IgM, the largest known antibody in humans, displays five potential N-glycosylation sites on each heavy chain monomer. IgM can exist as a pentamer with a connecting singly N-glycosylated J-chain (with a total of 51 glycosylation sites) or as a hexamer (60 glycosylation sites). In this study, the N-glycosylation of recombinant pentameric and hexameric IgM produced by the same human cell type and culture conditions was site-specifically profiled by RP-LC-CID/ETD-MS/MS using HILIC-enriched tryptic and GluC glycopeptides. The occupancy of all putative N-glycosylation sites on the pentameric and hexameric IgM were able to be determined. Distinct glycosylation differences were observed between each of the five N-linked sites on the IgM heavy chains. While Asn171, Asn332, and Asn395 all had predominantly complex type glycans, differences in glycan branching and sialylation were observed between the sites. Asn563, a high mannose-rich glycosylation site that locates in the center of the IgM polymer, was only approximately 60% occupied in both the pentameric and hexameric IgM forms, with a difference in relative abundance of the glycan structures between the pentamer and hexamer. This study highlights the information obtained by characterization of the site-heterogeneity of a highly glycosylated protein of high molecular mass with quaternary structure, revealing differences that would not be seen by global glycan or deglycosylated peptide profiling.

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Graphical Abstract ?

     

Bullvalene trisepoxide and its stereospecific rearrangement to 2,8,12-trioxahexacyclo[8.3.0.0(3,9)0(4,6)0(5,13)0(7,11)]tridecane: two new C3-symmetrical oligocycles with propeller chirality

Epoxidation of bullvalene (1) with a neutralized solution of Oxone gave racemic trisepoxide rac-6 in 93 % isolated yield. Its structure was examined by X-ray crystallography. The two enantiomers of 6 were separated by preparative HPLC and exhibited specific rotations of [alpha](25)(D)= +160, [alpha](25)(365)= +567 (c=0.946, CHCl(3)) for the firstly eluted and [alpha](25)(D)= -157, [alpha](25)(365)= -554 (c=0.986, CHCl3) for the secondly eluted enantiomer of 6. The geometry of (+)-6 and the absolute configuration of (-)-6 were determined by X-ray crystal structure analysis and anomalous diffraction, respectively. According to this, (-)-6 possesses (3R,5S,7S,9R,11R,13S)- and (+)-6 has (3S,5R,7R,9S,11S,13R)-configuration. Upon treatment with BF(3)Et(2)O at -78 degrees C, trisepoxide rac-6 rearranges with retention of the skeletal three-membered carbocycle to give the cage trisether rac-8, as proved by X-ray crystal structure analysis, in virtually quantitative yield. Enantiomers of rac-8 were separated by preparative HPLC and exhibited specific rotations of [alpha](25)(D)= +49, [alpha](25)(365)= +170 (c=1.01, CHCl3) (firstly eluting) and [alpha](25)(D)= -46, [alpha](25)(365)= -160 (c=1.02, CHCl(3)) (secondly eluting enantiomer). The absolute configuration of (-)-8 was determined by anomalous diffraction to be (1R,3R,7R,9R,11R,13R). DFT computations at the TD-B3 LYP/6-31+G(d,p)//B3 LYP/6-31+G(d) level of theory for (3R,5S,7S,9R,11R,13S)-6 and (1R,3R,7R,9R,11R,13R)-8 predicted specific rotations of -206.7 and -83.4, respectively. Acid-catalyzed isomerization of the enantiomerically pure (+)-6 proceeded without racemization to give exclusively (-)-8, and (-)-6 provided only (+)-8. Thus, this isomerization occurs with ring opening of the three C--O bonds in the epoxide moieties in the alpha-position relative to the three-membered carbocycle rather than in the beta-position.     

The dependence of yield stress on strain rate as determined from ball-indentation tests

A ball-indentation experiment was conducted to explore the possibility of using this test for the investigation of strain-rate dependence of yield properties of materials. The initial velocities of indentation used were between 0.0002 and 3 ips. Force and depth of penetration were measured continuously during a test. Using these and the results given in a previous report, the dependence of yield pressure of the indentation test on penetration velocity is shown for a range of velocities from 0.0002 to 300 ips. Results are presented for annealed C1018 steel, annealed 1100 F and annealed 6061-T6 aluminums. For all materials tested, the dependence of yield stress on strain rate seems to be derivable from the ball-indentation results by using Tabor's empirical formulas and an equation relating strain rate and velocity of penetration. The increase of yield pressure over the range of indentation velocities is 100 percent for steel, 30 percent for 1100 aluminum and 20 percent for 6061 aluminum.     

Efficient green coumarin dopants for organic light-emitting devices

We have synthesized a new green fluorescent dopant C-545P having incorporated five strategically placed "methyl" steric spacers on the julolidyl ring system. C-545P has good thermal properties and photostability, and when fabricated as a dopant in an Alq(3)-hosted OLED device, it shows notable improvement in luminance efficiency and is more resistant to concentration quenching than C-545T, particularly in the doping concentration range between 1 and 2% v/v, while achieving comparable device stability. [structure: see text]     

Bioinformatics approaches for disulfide connectivity prediction

Protein structure prediction with computational methods has gained much attention in the research fields of protein engineering and protein folding studies. Due to the vastness of conformational space, one of the major tasks is to restrain the flexibility of protein structure and reduce the search space. Many studies have revealed that, with the information of disulfide connectivity available, the search in conformational space can be dramatically reduced and lead to significant improvements in the prediction accuracy. As a result, predicting disulfide connectivity using bioinformatics approaches is of great interest nowadays. In this mini-review, the prediction of disulfide connectivity in proteins will be discussed in four aspects: (1) how the problem formulated and the computational techniques used in the literatures; (2) the effects of the features adopted to encode the information and the biological meanings implied; (3) the problems encountered and limitations of disulfide connectivity prediction; and (4) the practical usages of predicted disulfide bond information in molecular simulation and the prospects in the future.     

Remarkable enhancement of magnetization in the superconducting state of In/Ni nanoparticle composites by inhomogeneous spin anti-screening

The enzymatic hydrolysis of quinizarin diester in silica nanoparticle (NP) of 200 nm diameter is investigated by confocal fluorescence microscopy. The quinizarin diester substrate and the intermediate quinizarin monoester are non-fluorescent species and only the end product—quinizarin formed by enzymatic hydrolysis produces intense fluorescence of the silica NP. The enzyme activity of lipase adsorbed into silica NP was similar to that observed for lipase chemically bound to silica surface. In both situations, partial aggregation of the silica NP dispersed in thin film of polyvinylpyrrolidone was observed from fluorescence and scanning electron microscopy images. The fluorescence decay of the end product—quinizarin in silica NP was biexponential with decay times of 0.49 and 2.17 ns. These two decay times found are ascribed to quinizarin adsorbed in silica NP and dispersed in the surrounding medium, respectively.