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To realize the detection of pesticide, this study developed a paper-based chip involving a 2D to 3D flow channel and automatic time delay for detecting pesticides. The time delay was controlled by different pullulan concentrations. The 3D channel was fabricated by hot embossing (optimized printing parameters were 28 kg at 80°C, and Advantec No. 1 filter paper was used) and transfer printing the hydrophobic material into the paper. Finally, the limit of the detected concentration of the pesticide was 4 ng/mL, which was achieved using 250 U/mL of acetylcholinesterase (AChE) and 2.5% pullulan solution.

Antifouling surfaces that are resistant to protein adsorption and cell adhesion are desirable for many biomedical devices, such as diagnostic devices, biosensors, and implants. In this study, we developed an antifouling hyperbranched polyglycerol (hPG) surface on hydroxyl poly-p-xylylene (PPX-OH). PPX-OH was deposited via chemical vapor deposition (CVD), and an hPG film was then developed via the ring-opening reaction of glycidol. The hPG film greatly reduced the adhesion of L929 cells and platelets as well as protein adsorption. The addition of alkenyl groups in the hPG layer allows the conjugation of biomolecules, such as peptides and biotin, and elicits specific biological interactions. Since the CVD deposition of PPX-OH could be applied to most types of materials, our approach makes it possible to decorate an antifouling hPG film on most types of materials. Our method could be applied to biosensors, diagnostics, and biomedical devices in the future.

We present the development of conductive nanoporous CNT/chitosan membrane for charge-selective transport of charged molecules, carboxylfluorescein (CF), substance P, and tumor necrosis factor-alpha (TNF-α). The membrane was made porous and conductive via gelatin nanoparticle leaching technique and addition of carbon nanotubes, respectively. These nanoporous membranes discriminate the diffusion of positive-charged molecules while inhibiting the passage of negative-charged molecules as positive potential was applied. The permeation selectivity of these membranes is reversed by converting the polarity of applied potential into negative. Based on this principle, charged molecules (carboxylfluorescein, substance P, and TNF-α) are successfully filtered through these membranes. This system shows 30 times more selective for CF than substance P as positive potential was applied, while 2.5 times more selective for substance P than CF as negative potential was applied.

Previously, we have demonstrated the induction of Src in lipopolysaccharide (LPS)-stimulated macrophages. In this study, we observed that pharmacological blockade or knockout of inducible nitric-oxide synthase (iNOS) reduced LPS-mediated Src induction and macrophage migration. Either SNAP (a NO donor) or 8-Br-cGMP (a cGMP analogue) could rescue these defects in iNOS-null macrophages, which indicated the participation of NO/cGMP in LPS-elicited Src expression and mobilization. In addition, Src family kinase (SFK)-specific inhibitor, PP2, inhibited SNAP- and 8-Br-cGMP-evoked motility implicating the involvement of SFKs downstream of NO/cGMP. Analysis of the expression of SFKs indicated LPS dramatically induced Src, which could be attributable to the increased level of the src transcript. Attenuation of Src by src-specific siRNA reduced LPS- and SNAP-evoked mobilization in Raw264.7 macrophages, and reintroduction of avian Src could rescue their motility. Furthermore, LPS-mediated Src induction led to increased FAK Pi-Tyr-397 and Pi-Tyr-861, which was also iNOS-dependent. With these findings, we concluded that iNOS was important for LPS-mediated macrophage locomotion and Src was a critical player in this process.

The purpose of this study was to develop kinds of multi-walled carbon nanotubes (MWCNT)-based materials, which could replace current autografts for neuron regeneration. The alternative MWCNT films was developed by encapsulated chitosan. To evaluate the characteristics of MWCNT films after modification, the measurements of electrical conductivity, contact angle and the degree of degradation were carried out. Two kinds of chitosan film preparation were designed, one was the dish film and the other was microscope slide film. The microscope slide film was exterior, however, the dish film was wrinkling. Comparing the thickness by micrometer caliper, the thickness of the dish film was 0.1950 to 0.0425 mm, and the microscope slide film was 0.0584 to 0.0109 mm. This result indicated the microscope slide film was more suitable for the following experiment because of less error value. The contact angle of the chitosan film was measured between 60~90 deg, it indicated the film was more hydrophobic. In order to achieve better biocompatibility, adding MWCNT significantly increased hydrophilicity of chitosan film.

The gelatin-tricalcium phosphate membranes were cross-linking with low concentration glutaraldehyde solution (GTG). This material has good mechanical property, biocompatibility, and is feasible for surgical manipulation. For axonal regeneration, nerve growth factors (NGF) were immobilized onto the composite (GTG) with carbodiimide. The purpose of this study was to evaluate the release characteristics and bioactivity of NGF after covalent immobilization onto the GTG membranes (GEN). NGF immobilized onto and released from the composite was quantified using ELISA method. PC 12 cells were cultured on the GTG and GEN composites. Cell survival, cytotoxicity, and cellular activity were evaluated by total protein content, LDH activity, and MTT assay respectively. Neurite outgrowth assay was used to evaluate the biological activity of NGF released from GEN composite. From ELISA measurement, the releasing curve for NGF showing two distinctive parts with different slopes indicated that NGF were released from the composite in diffusion-controlled mechanism and degradation-controlled mechanism respectively. While culturing with PC 12 cells, LDH leakage results implied that whether GTG composite cross-linked with NGF or not showed little cytotoxicity. The total protein content and cellular activity of PC 12 cells were lower on GTG and GEN membranes than control group. However, 56%±3.98 of PC 12 cells showed significant neurite outgrowth on GEN membranes which was statistically higher than GTG without NGF immobilization. In addition, sustained release of bioactive NGF for two months had been demonstrated by neurite outgrowth assay. From these experiments, it can be concluded that the technique used in the present study is capable of immobilizing NGF onto GTG membranes covalently and remaining the bioactivity of NGF. Therefore, GEN composite can be materials for sustained release of bioactive NGF and a candidate for future therapeutic application in nerve repair.

The thermal and degradable properties of carbodiimide (EDC) or glutaraldehyde (GTA) cross-linked gelatin membranes have been investigated in order to evaluate the effects of different concentrations of two kinds of cross-linking reagent on the stability of membranes. In the thermogram recorded from a gelatin membrane cross-linked with EDC solution, the endothermic peak of 0.8% EDC cross-linking gelatin was centered at about 61°C that was higher than other samples treated with EDC solutions. Denaturation temperature (Td) of gelatin samples increased on increasing EDC concentration (0.2% to 0.8%), in agreement with the simultaneous increased of the extent of cross-linking. But increasing GTA concentration from 0.05% to 0.6%, the Td values of gelatin samples were decreased from 66.2°C to 56.3°C . In addition, two endothermic peaks were observed in 0.4% and 0.6% GTA cross-linking groups because of the GTA concentration was too high to complete cross-linking reaction. Therefore, partial of gelatin membrane was cross-linked completely but others were not. In the thermogravimetric analysis, the proportion of cracking endothermic peak of 0.6% GTA cross-linking gelatin (g15G0.6) was higher than the peak of 0.6% EDC cross-linking gelatin (g15C0.6). Therefore, g15G0.6 cracked to smaller molecules has to absorb more calorific capacity than g15C0.6. The increase in the strength of covalent binding on increasing the proportion of endothermic peak was evident. The results of degradable rate were in agreement with the lower concentration of cross-linked reagent the faster degraded rate of gelatin membrane. The MTT assay showed that 15% gelatin cross-linked by 0.8% EDC has the least cytotoxicity, and cell activity of this group was similar to control group (blank dish). As the concentration of GTA in gelatin membranes was down to 0.05% or 0.1% the cell viability was returned to approach the value of control group.

The purpose of this study was to investigate if PPARγplays a role in the melanogenesis. B16/F10 cells were divided into five groups: control, melanin stimulating hormone (α-MSH),α-MSH+retinol,α-MSH+GW9662 (PPARγantagonist), and GW9662. Cells in the control group were cultured in the Dulbecco’s modified Eagle’s medium (DMEM) for 48 hrs. To initiate the melanogenesis, cells in allα-MSH groups were cultured in medium containingα-MSH (10 nM) for 48 hrs. Cells were treated simultaneously with retinol (5 μM) in theα-MSH+retinol group. Instead of retinol, GW9662 (10 μM) was cocultured in theα-MSH+GW9662 group. Cells in the final group were cultured in the DMEM with GW9662. All the analyses were carried out 48 hours after treatments. Theα-MSH was able to increase cell number, melanin production, and the activity of tyrosinase, the limiting enzyme in melanogenesis. Theseα-MSH-induced changes were prevented either by retinol or by GW9662. Further analyses of the activities of antioxidant enzymes including glutathione, catalase, and the superoxide dismutase (SOD) showed thatα-MSH treatment raised the activity of SOD which was dependent on PPARγlevel. According to our results, theα-MSH-induced melanogenesis was PPARγdependent, which also modulated the expression of SOD.

In this article, a nanoporous chitosan polymer membrane was successfully produced and applied as microdialysis membrane forin vitrosampling of biomolecules. With the use of nanoparticle leaching technique, porogenic gelatin nanoparticles formed nanopores in the chitosan-based membrane to create a secure implantable nanoporous membrane for biomolecule sampling. The gelatin nanoparticles size was in the range of 45 to 70 nm, and the pore size of the chitosan membrane was around 40 to 100 nm. The porosity of membrane was found to be dependent on the mixing ratio of chitosan solution and gelatin nanoparticles solution. The results of diffusion study showed that we can alter the mixing ratio of porogen to achieve size-selective molecular diffusion, which means that the porosity and cut-off size of porous membrane can be controlled. The recoveries of the probe fabricated from the chitosan-based membrane were examined for four different model compounds of different molecular weights: 2-NBDG, substance P, TNF-α, and FITC-BSA. The microdialysis probes showed linear responses and substantial recovery to various concentrations of biomolecules. These results indicated that the microdialysis probe constructed by chitosan nanoporous membrane could sample and monitor the biomoleculesin vitroand has the potential for the applicationin vivo.

Fresh common (Agaricus bisporus) and high-temperature mushrooms (A. bitorquis) were irradiated with ultraviolet-C (UV-C) for 0, 0.5, 1, and 2 h at 12 °C. Fresh common, shiitake (Lentinula edodes), and straw mushrooms (Volvariella volvacea) were irradiated with UV-B for 0, 0.5, 1, and 2 h at 12 °C. After UV-C irradiation for 2 h, vitamin D2 contents in common and high-temperature mushrooms increased from 2.20 and 4.01 μg/g of dry weight to 7.30 and 5.32 μg/g, respectively. After UV-B irradiation for 2 h, the vitamin D2 content in common mushrooms reached 12.48 μg/g. UV-B irradiation resulted in higher vitamin D2 conversion for common mushrooms. After UV-B irradiation for 2 h, vitamin D2 contents in shiitake and straw mushrooms increased from 2.16 and 3.86 μg/g to 6.58 and 7.58 μg/g, respectively. The increase rates in shiitake and straw mushrooms were not as high as in common mushrooms. Keywords: Mushrooms; Agaricus bisporus; Agaricus bitorquis; Lentinula edodes; Volvariella volvacea; ultraviolet-B; ultrav...

According to the multiple alignment of various dihydrolipoamide dehydrogenases (E3s) sequences, three human mutant E3s of the conserved residues in the center domain, N286D, N286Q, and D320N were created, over-expressed and purified. We characterized these mutants to investigate the reaction mechanism of human dihydrolipoamide dehydrogenases. The specific activities of N286D, N286Q, and D320N are 30.84%, 24.57% and 48.60% to that of the wild-type E3 respectively. The FAD content analysis indicated that these mutant E3s about 96.0%, 99.4% and 82.7% of FAD content compared to that of wild-type E3 respectively. The molecular weight analysis showed that these three mutant proteins form the dimer. Kinetic's data demonstrated that the K(cat) of both forward and reverse reactions of these mutant proteins were decreased. These results suggest that N286 and D320 play a role in the catalytic function of the E3.

In order to modulate the mechanical properties of gelatin, we previously developed a biodegradable composite composed by tricalcium phosphate and glutaraldehyde crosslinking gelatin (GTG) feasible for surgical manipulation. In this study, we evaluated the in vivo applications of GTG conduit for peripheral nerve repair. The effect of sciatic nerve reconstruction was compared between resorbable permeable GTG conduits and durable impermeable silicone tubes. Traditional methods of assessing nerve recovery following peripheral nerve repair including histomorphometric and electrophysiologic features were conducted in our study. In addition, autotomy score and sciatic function index (SFI) in walking tract analysis were used as additional parameters for assessing the return of nerve function. Twenty-four weeks after sciatic nerve repair, the GTG conduits were harvested. Microscopically, regeneration of nerves was observed in the cross-section at the mid portion of all implanted GTG conduits. The cross-sectional area of regenerated nerve of the GTG group was significant larger than that of the silicone group. In the compound muscle action potentials (CMAP), the mean recovery index of CMAP amplitude was 0.24 ± 0.02 for the silicone group, 0.41 ± 0.07 for the GTG group. The mean SFI increased with time in the GTG group during the evaluation period until 24 weeks. Walking tract analysis showed a higher SFI score in the GTG group at both 12 and 24 weeks. The difference reached a significant level at 24 weeks. Thus, the histomorphometric, electrophysiologic, and functional assessments demonstrate that GTG can be a candidate for peripheral nerve repair. © 2005 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 2006

We previously developed a biodegradable composite with potentially good biocompatibility composed by tricalcium phosphate and gluataraldehyde cross-linking gelatin (GTG) with good mechanical property feasible for surgical manipulation. The purpose of this study was to evaluate the feasibility of immobilizing nerve growth factor (NGF) onto the composite (GTG) with carbodiimide (GEN composite). Cultured Schwann cells were seeded onto the GTG and GEN composites. For comparison, GTG membrane soaked in NGF solution without carbodiimide (GN composite) as cross-linking agent was also used to culture Schwann cells. Cell morphology was observed by a scanning electron microscope. Cell survival, cytotoxicity and cellular metabolism on the NGF-grafted GTG membrane were assessed quantitatively in terms of cell protein content, leakage of cytosolic lactate dehydrogenase (LDH) activity and by the well-established MTT assay, respectively. The result of LDH study did not show significant difference among GTG, NGF-modified GTG and control group. This indicated that GTG composite, whether cross-linking with NGF or not, has little cytotoxic effect. Comparing the protein content and MTT assay among GEN, GN composite and control group, the data confirmed more attachment of Schwann cells on GEN composite. Although GTG cross-linking with NGF did not promote Schwann cell proliferation, the techniques we used in this study provided a method to fabricate a novel biomaterial incorporation of Schwann cells and covalently immobilized NGF.