Visible light promoted deaminative difluoroalkylation of aliphatic amines with difluoroenoxysilanes.

A visible light promoted deaminative strategy for the difluoroalkylation reaction utilizing pyridinium-activated aliphatic primary amines and difluoroenoxysilane as substrates has been developed. This protocol is characteriazed by its mild reaction conditions and broad substrate scope, which converted a diverse array of amine-containing molecules to the alkyl-CF2COPh products. Moreover, the resulting products can be easily transformed into a vast array of structurally novel and interesting difluoro-containing moieties, therefore providing a facile route for applications in medicinal chemistry and the life sciences.

A PDM-based 128-Gb/s PAM4 fibre-FSO convergent system with OBPFs for polarisation de-multiplexing.

A polarisation-division-multiplexing (PDM)-based four-level pulse amplitude modulation (PAM4) fibre-free-space optical (FSO) convergent system with optical band-pass filters (OBPFs) for polarisation de-multiplexing is feasibly demonstrated for the first time. In a PDM scenario with PAM4 modulation, the transmission capacity of fibre-FSO convergent systems is enhanced four times with an aggregate channel capacity of 128 Gb/s (64 Gb/s PAM4/polarisation × 2 polarisations). With an OBPF, polarisation-tracking free de-multiplexing is attained by eliminating other optical carrier with orthogonal polarisation. An OBPF is a simple polarisation de-multiplexing scheme in which the polarisation-orthogonal carrier can be effectively de-multiplexed and the cross-polarisation interference can be nearly eliminated. Compared with traditional PDM-based fibre-FSO convergent systems with sophisticated polarisation-tracking mechanism and elaborate digital signal processing (DSP) approach, it reveals a noteworthy one with the advantage of simplicity. Through 25 km single-mode fibre transport and 500 m FSO link, sufficiently low bit error rate and qualified PAM4 eye diagrams are attained. This proposed polarisation-tracking free PDM-based fibre-FSO convergent system is notable because it not only incorporates the fibre backbone and optical wireless feeder, but it also simplifies the framework since complicated polarisation-tracking mechanism and DSP approach are not involved.

A PDM-based bi-directional fibre-FSO integration with two RSOAs scheme.

A polarization-division-multiplexing (PDM)-based bi-directional fibre-free-space optical (FSO) integration with two reflective semiconductor optical amplifiers (RSOAs) scheme to efficiently wipe off the modulated data for upstream modulation is proposed and successfully demonstrated. For downstream modulation, a high-speed 128 Gb/s vestigial sideband (VSB)-four-level pulse amplitude modulation (PAM4) fibre-FSO integration is feasibly established. The transmission capacity is increased up to four times through PDM operation and VSB-PAM4 modulation. For uplink transmission, a 10 Gb/s non-return-to-zero fibre-FSO integration with two RSOAs scheme to effectually erase the downstream modulated data is practically constructed. The upstream performance exhibits noticeable enhancement by using of two RSOAs scheme to wipe off the modulated data clearly. Such illustrated PDM-based bi-directional 128 Gb/s (downstream)/10 Gb/s (upstream) fibre-FSO integration is shown to be prominent not only due to its enhancement in the convergence of fibre backhaul and optical wireless reach extender but also because of its benefit in bi-directional transmission for affording high transmission capacity with long-reach optical wireless link and improved upstream performance.

A Novel Hyperspectral Microscopic Imaging System for Evaluating Fresh Degree of Pork.

This study proposed a rapid microscopic examination method for pork freshness evaluation by using the self-assembled hyperspectral microscopic imaging (HMI) system with the help of feature extraction algorithm and pattern recognition methods. Pork samples were stored for different days ranging from 0 to 5 days and the freshness of samples was divided into three levels which were determined by total volatile basic nitrogen (TVB-N) content. Meanwhile, hyperspectral microscopic images of samples were acquired by HMI system and processed by the following steps for the further analysis. Firstly, characteristic hyperspectral microscopic images were extracted by using principal component analysis (PCA) and then texture features were selected based on the gray level co-occurrence matrix (GLCM). Next, features data were reduced dimensionality by fisher discriminant analysis (FDA) for further building classification model. Finally, compared with linear discriminant analysis (LDA) model and support vector machine (SVM) model, good back propagation artificial neural network (BP-ANN) model obtained the best freshness classification with a 100 % accuracy rating based on the extracted data. The results confirm that the fabricated HMI system combined with multivariate algorithms has ability to evaluate the fresh degree of pork accurately in the microscopic level, which plays an important role in animal food quality control.

Effect of substrate stiffness on hepatocyte migration and cellular Young's modulus.

Hepatic fibrosis progress accompanied by an unbalanced extracellular matrix (ECM) degradation and deposition leads to an increased tissue stiffness. Hepatocytes interplay with all intrahepatic cell populations inside the liver. However, how hepatocytes migration and cellular Young's modulus influenced by the substrate stiffness are not well understood. Here, we established a stiffness-controllable in vitro cell culture model by using a polyvinyl alcohol (PVA) hydrogel that mimicked the same physical stiffness as a fibrotic liver. Three levels of stiffness were used in our experiment that corresponded to the stiffness levels found in normal liver tissue (4.5 kPa), the early (19 kPa) and late stages (37 kPa) of fibrotic liver tissues. Cytoskeleton of hepatocyte was influenced by substrate stiffness. Soft substrate promoted the cellular migration and directionality. The cellular Young's modulus firstly increased and then decreased with increasing substrate stiffness. Integrin-ß1 and ß-catenin expression on cytomembrane were up-regulated and down-regulated with the increase of substrate stiffness, respectively. Our data not only suggested that hepatocytes were sensitive to substrate stiffness, but also suggested that there may be a potential relationship among substrate stiffness, cellular Young's modulus and the dynamic balance of integrin-ß1 and ß-catenin pathways. These results may provide us a new insight in mechanism investigation of mechano-dependent diseases, especially like fibrosis related diseases.

Mechanosensing of cells in 3D gel matrices based on natural and synthetic materials.

Cells in vivo typically are found in 3D matrices, the mechanical stiffness of which is important to the cell and tissue-scale biological processes. Although it is well characterized that as to how cells sense matrix stiffness in 2D substrates, the scenario in 3D matrices needs to be explored. Thus, materials that can mimic native 3D environments and possess wide, physiologically relevant elasticity are highly desirable. Natural polymer-based materials and synthetic hydrogels could provide an better 3D platforms to investigate the mechano-response of cells with stiffness comparable to their native environments. However, the limited stiffness range together with interdependence of matrix stiffness and adhesive ligand density are inherent in many kinds of materials, and hinder efforts to demonstrate the true effects contributed by matrix stiffness. These problems have been addressed by the recently emerging exquisitely designed materials based on native matrix components, designer matrices, and synthetic polymers. In this review, a variety of materials with a wide stiffness range that mimic the mechanical environment of native 3D matrices and the independent affection of stiffness for cellular behavior and tissue-level processes are discussed.

Responsiveness of voltage-gated calcium channels in SH-SY5Y human neuroblastoma cells on quasi-three-dimensional micropatterns formed with poly (l-lactic acid).

INTRODUCTION: In this study, quasi-three-dimensional (3D) microwell patterns were fabricated with poly (l-lactic acid) for the development of cell-based assays, targeting voltage-gated calcium channels (VGCCs). METHODS AND MATERIALS: SH-SY5Y human neuroblastoma cells were interfaced with the microwell patterns and found to grow as two dimensional (2D), 3D, and near two dimensional (N2D), categorized on the basis of the cells' location in the pattern. The capability of the microwell patterns to support 3D cell growth was evaluated in terms of the percentage of the cells in each growth category. Cell spreading was analyzed in terms of projection areas under light microscopy. SH-SY5Y cells' VGCC responsiveness was evaluated with confocal microscopy and a calcium fluorescent indicator, Calcium Green™-1. The expression of L-type calcium channels was evaluated using immunofluorescence staining with DM-BODIPY. RESULTS: It was found that cells within the microwells, either N2D or 3D, showed more rounded shapes and less projection areas than 2D cells on flat poly (l-lactic acid) substrates. Also, cells in microwells showed a significantly lower VGCC responsiveness than cells on flat substrates, in terms of both response magnitudes and percentages of responsive cells, upon depolarization with 50 mM K(+). This lower VGCC responsiveness could not be explained by the difference in L-type calcium channel expression. For the two patterns addressed in this study, N2D cells consistently exhibited an intermediate value of either projection areas or VGCC responsiveness between those for 2D and 3D cells, suggesting a correlative relation between cell morphology and VGCC responsiveness. CONCLUSION: These results suggest that the pattern structure and therefore the cell growth characteristics were critical factors in determining cell VGCC responsiveness and thus provide an approach for engineering cell functionality in cell-based assay systems and tissue engineering scaffolds.

[Secretion and expression of vascular endothelial growth factor and interleukin-8 by SH-SY5Y human neuroblastoma cells].

To establish vascular endothelial growth factor (VEGF) and interleukin-8 (IL-8) as secretary biomarkers for cell growth on topographic substrates, we have evaluated the secretion and expression of these 2 factors by SH-SY5Y human neuroblastoma cells on poly-L-lactide (PLLA) micropillar arrayed topographic substrates. We fabricated topographic substrates with UV lithography, silicon etching and polydimethylsiloxane-based replica molding, and interfaced SH-SY5Y human neuroblastoma cells with both the topographic substrates and PLLA flat substrates. Cell morphology and spreading were examined with scanning electron microscopy. The secretion and mRNA expression of VEGF and IL-8 were evaluated with enzyme linked immunosorbent assay (ELISA) and real time qPCR, respectively, 24 hours after cell plating. We successfully achieved 4 topographic substrates with a nominal pillar diameter of 2 microm and 4 microm, and a nominal pillar spacing of 2 microm and 7 microm. We found that the secretion and mRNA expression of VEGF and/or IL-8 by SH-SY5Y cells on 2-2 microm (pillar diameter-spacing), 4-2 microm and 4-7 microm topographic substrates were upregulated in comparison to those by cells on PLLA flat substrate, 24 hours after cell plating. Furthermore, both cytokines were even more substantially upregulated on the 2-7 microm substrate than on the other 3 topographic substrates. Compared to those on PLLA flat substrate, cells on topographic substrates showed significant changes in morphology (spreading area, perimeter and roundness), and the increase in the secretion and mRNA expression of VEGF and IL-8 was accompanied with a decrease in cell spreading areas. These results provided evidence that pillar arrayed topography was an important microenvironmental factor in affecting VEGF and IL-8 expression or secretion, and VEGF and IL-8 might serve as important secretary biomarkers for growth on topographic substrates by SH-SY5Y cells.

[Fabrication of three-dimensional microwell patterns and their integration with C17. 2 neural stem cells].

UV photolithography and hydrofluoric acid wet etching were used to produce silicon master molds and polydimethylsiloxane (PMDS)-based soft lithography was adopted to fabricate three-dimensional poly(lactic-co-glycolic acid) (PLGA) and PDMS microwell patterns with high aspect ratio and channel connection. Nine microwell patterns were thus obtained with different structural dimensions. Patterns were treated with oxygen plasma etching and polylysine coating to enhance hydrophilicity and cell compatibility for subsequent culture of C17. 2 neural stem cells. With proliferation during the culture, C17. 2 cells gradually distributed within the microwells, showing an obviously three-dimensional (3-D) growth behavior. The presence of channel structures greatly favored the 3-D growth of C17. 2 neural stem cells on the microwell patterns. Multi-layered scanning with confocal microscopy and 3-D rendering after carboxyfluorescein diacetate succinimidyl ester (CFDA-SE) staining showed that most C17. 2 cells grew within a range of 30 to 90 microm from the microwell bottom. Immunofluorescence staining indicated that C17. 2 cells within 3-D microwell patterns were uniformly nestin-positive on day 2 after cell plating. It could well be concluded that the microwell patterns thus fabricated were suitable for the 3-D culture and subsequent differentiation of C17. 2 neural stem cells. And the cells can be maintained with uniform stemness properties while cultured in these microwell patterns.

Inhibition of adhesion of hepatocellular carcinoma cells to basement membrane components by receptor competition with RGD- or YIGSR-containing synthetic peptides.

A micropipette technique was used to investigate the effects of four synthetic peptides, YIGSR, CDPGYIGSR, RGDS and GRGDTP, on the adhesion of hepatocellular carcinoma (HCC) cells onto type IV collagen/laminin/fibronectin coated surfaces. Adhesion of HCC cells to laminin was found to be YIGSR- or CDPGYIGSR-dependent while that to fibronectin and type IV collagen was RGDS- or GRGDTP-dependent. The reduction in adhesion strengths of HCC cells was slight to moderate (up to 55%), and was dependent on the peptide concentration. The decrease in adhesion strengths was reversed by an increase in ligand coating concentration and was compromised by prolonged interaction of the cells with the surfaces. These results suggested that the inhibition was due to competitive retardation rather than to a blockade of adhesion strengthening. A simple asymptotic function was adopted to fit the correlation between the mean of cell adhesion strengths and peptide concentration within defined concentration ranges. Regression analysis showed that cell adhesion strengths appeared to approach a plateau with increasing concentration of the inhibitory peptides, which was not always uniform over the entire concentration range tested. Further reduction in adhesion strengths was observed at higher peptide concentrations. It is suggested that the constants obtained by fitting over a low peptide concentration range might be kinetically representative of the inhibition during early events of adhesion or attachment.

[Effects of mechanical strain on the proliferation of pulmonary epithelial cells and reorganization of integrins].

Using a cyclic strain unit, the proliferation and reorganization of alpha(5), beta(1 ) integrins in human pulmonary epithelial A549 cells that underwent mechanical strain were studied. Flow cytometry revealed that cellular proliferative index reduced significantly after cells were subjected to 15% elongation at frequency 20 cycles/min or 40 cycles/min for 48 h. Confocal microscopy revealed that alpha(5), beta(1 ) integrins in human pulmonary epithelial A549 cells transferred from the apic al layer to the basal layer and created adhesion plaques after 24 h exposure to 15% elongation at frequency 40 cycles/min. It is concluded that alpha(5), beta(1 ) integrins in pulmonary epithelial cells play an important role in transducing mechanical stress into intracellular signals.

[Investigation on culture of rat pulmonary microvascular endothelial cells and their viscoelasticity].

It is the infent of this study to establish a simple method for cultivation of rat pulmonary microvascular endothelial cells(PMVECs) and investigate the viscoelasticity of PMVECs. First, we obtained rat's peripheral pulmonary tissue, which then was cut into small pieces and cultured with 3 ml DMEM containing 20% bovine calf serum, 90 U/ml heparin, 4 mmol L-glutamine, 100 U/ml penicillin and 100 micrograms/ml streptomycin. Next, moved away the pulmonary tissue pieces 60 h later, and started passage 2-4 days after continued culture. Last, digested and separated PMVECs and studied viscoelastic coefficients of PMVECs by using micropipette aspiration technique. The results revealed that the cultured PMVECs showed regular cobblestone morphology and conformed with endothelial cells morphological characterization by phase contrast microscopy. PMVECs elastic modulus K1 was 49.3 +/- 9.2 Pa, K2 was 73.2 +/- 24.8 Pa, and it's viscosity factor mu was 19.2 +/- 7.2 Pa. s. These data demonstrate that it is feasible to cultivate PMVECs with tissue pieces method, and PMVECs is of greater rigidity.