A microfluidic device for high density hydrodynamic cell trapping, growth and super-resolution imaging

Super-Resolution imaging techniques such as Fluorescent Photo-Activation Localisation Microscopy (FPALM) have created a powerful new toolkit for investigating living cells, however a simple platform for growing, trapping, holding and controlling the cells is needed before the approach can become truly widespread. We present a microfluidic device formed in polydimethylsiloxane (PDMS) with a fluidic design which traps cells in a high-density array of wells and holds them very still throughout the life cycle, using hydrodynamic forces only. The device meets or exceeds all the necessary criteria for FPALM imaging of Schizosaccharomyces pombe and is designed to remain flexible, robust and easy to use. © 2011 IEEE.

The deposition of small particles from a turbulent air flow in a curved duct

The paper describes an experimental and theoretical study of the deposition of small spherical particles from a turbulent air flow in a curved duct. The objective was to investigate the interaction between the streamline curvature of the primary flow and the turbulent deposition mechanisms of diffusion and turbophoresis. The experiments were conducted with particles of uranine (used as a fluorescent tracer) produced by an aerosol generator. The particles were entrained in an air flow which passed vertically downwards through a long straight channel of rectangular cross-section leading to a 90° bend. The inside surfaces of the channel and bend were covered with tape to collect the deposited particles. Following a test run the tape was removed in sections, the uranine was dissolved in sodium hydroxide solution and the deposition rates established by measuring the uranine concentration with a luminescence spectrometer. The experimental results were compared with calculations of particle deposition in a curved duct using a computer program that solved the ensemble-averaged particle mass and momentum conservation equations. A particle density-weighted averaging procedure was used and the equations were expressed in terms of the particle convective, rather than total, velocity. This approach provided a simpler formulation of the particle turbulence correlations generated by the averaging process. The computer program was used to investigate the distance required to achieve a fully-developed particle flow in the straight entry channel as well as the variation of the deposition rate around the bend. The simulations showed good agreement with the experimental results. © 2012 Elsevier Ltd.

噬菌蛭弧菌的分离及初步鉴定

采用双层平板法,以滤膜过滤的方法来收集噬菌蛭弧菌,以嗜水气单胞菌(Aeromonas hudrophila)、荧光假单胞菌(Pseudomonas fluorescent)和绿脓杆菌(Pseudomonas aeruginosa)为宿主菌,进行噬菌蛭弧菌的分离研究;并在此基础上,通过接触酶检测和寄生性确认对噬菌蛭弧菌(Bdellovibrio bacteriovorus)进行了初步的鉴定。结果表明未使用滤膜过滤,采用自来水琼脂双层平板法分离噬菌蛭弧菌的效果较好;并经过接触酶和寄生性检测初步鉴定此BD-SP

Measuring EGFR Separations on Cells with ∼10 nm Resolution via Fluorophore Localization Imaging with Photobleaching

Detecting receptor dimerisation and other forms of clustering on the cell surface depends on methods capable of determining protein-protein separations with high resolution in the ∼10-50 nm range. However, this distance range poses a significant challenge because it is too large for fluorescence resonance energy transfer and contains distances too small for all other techniques capable of high-resolution in cells. Here we have adapted the technique of fluorophore localisation imaging with photobleaching to measure inter-receptor separations in the cellular environment. Using the epidermal growth factor receptor, a key cancer target molecule, we demonstrate ∼10 nm resolution while continuously covering the range of ∼10-80 nm. By labelling the receptor on cells expressing low receptor numbers with a fluorescent antagonist we have found inter-receptor separations all the way up from 8 nm to 59 nm. Our data are consistent with epidermal growth factor receptors being able to form homo-polymers of at least 10 receptors in the absence of activating ligands. © 2013 Needham et al.

Prospects of III-nitride optoelectronics grown on Si.

The use of III-nitride-based light-emitting diodes (LEDs) is now widespread in applications such as indicator lamps, display panels, backlighting for liquid-crystal display TVs and computer screens, traffic lights, etc. To meet the huge market demand and lower the manufacturing cost, the LED industry is moving fast from 2 inch to 4 inch and recently to 6 inch wafer sizes. Although Al2O3 (sapphire) and SiC remain the dominant substrate materials for the epitaxy of nitride LEDs, the use of large Si substrates attracts great interest because Si wafers are readily available in large diameters at low cost. In addition, such wafers are compatible with existing processing lines for 6 inch and larger wafers commonly used in the electronics industry. During the last decade, much exciting progress has been achieved in improving the performance of GaN-on-Si devices. In this contribution, the status and prospects of III-nitride optoelectronics grown on Si substrates are reviewed. The issues involved in the growth of GaN-based LED structures on Si and possible solutions are outlined, together with a brief introduction to some novel in situ and ex situ monitoring/characterization tools, which are especially useful for the growth of GaN-on-Si structures.

Identification of Prorocentrum minimum and Takayama pulchella by fluorescence in situ hybridization through epifluorescence microscopy and flow cytometry

Partial rDNA sequences of Prorocentrum minimum and Takayama pulchella were amplified, cloned and sequenced. and these sequence data were deposited in the GenBank. Eight oligonucleotide probes (DNA probes) were designed based on the sequence analysis. The probes were employed to detect and identify P. minimum and T. pulchella in unialgal and mixed algal samples with a fluorescence in situ hybridization method using flow cytometry. Epifluorescence micrographs showed that these specific probes labeled with fluorescein isothiocyanate entered the algal cells and bound to target sequences, and the fluorescence signal resulting from whole-cell hybridization varied from probe to probe. These DNA probes and the hybridization protocol we developed were specific and effective for P. minimum and T. pulchella, without any specific binding to other algal species. The hybridization efficiency of different probes specific to P. minimum was in the order: PM18S02 > PM28S02 > PM28S01 > PM18S01, and that of the probes specific to T. pulchella was TP18S02 > TP28S01 > TP28S02 > TP18S01. The different hybridization efficiency of the DNA probes could also be shown in the fluorescent signals between the labeled and unlabeled cells demonstrated using flow cytometry. The DNA probes PM18S02, PM28S02; TP18S02 and TP28S01, and the protocol, were also useful for the detection of algae in natural samples.

Cytochemical changes in the developmental process of Nostoc sphaeroides (cyanobacterium)

There are several apparent developmental stages in the life cycle of Nostoc sphaeroides Kutzing, an edible cyanobacterium found mainly in paddy fields in central China. The cytochemical changes in developmental stages such as hormogonia, aseriate stage, filamentous stage and colony in N. sphaeroides were examined using fluorescent staining and colorimetric methods. The staining of acidic and sulfated polysaccharides increased with development when hormogonia were used as the starting point. Acidic polysaccharides (AP) were most abundant at the aseriate stage and then decreased, while the sulfated polysaccharides (SP) were highest at the colony stage. Quantitatively, along the developmental process from hormogonia to colony, total carbohydrates first increased, then became stable, and then reached their highest level at the colony stage, while reducing sugars were highest at the hormogonia stage and then decreased sharply once development began. SP were not detectable in the hot water soluble polysaccharides (HWSP), and hormogonia had the lowest content of AP, while old colonies had the highest. The AP content of the aseriate stage, filamentous stage and young colony stage were very similar. The evolutionary relationships reflected in the developmental stages of N. sphaeroides are discussed.

Establishment of a Chinese sturgeon Acipenser sinensis tail-fin cell line and its susceptibility to frog iridovirus

Chinese sturgeon Acipenser sinensis, a cartilaginous ganoid, is a 'living fossil' on a deeply isolated evolutionary branch. A cell line was established from Chinese sturgeon tail-fin tissue (CSTF) . These epithelial CSTF cells grew well in Dulbecco's modified Eagle's medium at 25 degrees C. Karyotypic analysis revealed a normal diploid karyotype with 2n = 264 and large numbers of punctate chromosomes. A strain of frog iridoviruses [Rana grylio virus (RGV)] was used to test the susceptibility of this cell line to infection. Infection was confirmed by cytopathic effect, immunofluorescence and electron-microscope observations, which detected the viral antigens or particles in the cytoplasm of RGV-infected cells. Molecular analysis further suggested that c. 550 bp DNA fragment could be cloned from the RGV-infected CSTF cells' DNA with major capsid protein gene polymerase chain reaction primers. Furthermore, after transfection with pEGFP vector DNA, the CSTF cell line produced significant fluorescent signals indicating its utility in exogenous studies.

Hybrid cytomegalovirus-U6 promoter-based plasmid vectors improve efficiency of RNA interference in zebrafish

Short hairpin RNA (shRNA) directed by RNA polymerase III (Pol III) or Pol II promoter was shown to be capable of silencing gene expression, which should permit analyses of gene functions or as a potential therapeutic tool. However, the inhibitory effect of shRNA remains problematic in fish. We demonstrated that silencing efficiency by shRNA produced from the hybrid construct composed of the CMV enhancer or entire CMV promoter placed immediately upstream of a U6 promoter. When tested the exogenous gene, silencing of an enhanced green fluorescent protein (EGFP) target gene was 89.18 +/- 5.06% for CMVE-U6 promoter group and 88.26 +/- 6.46% for CMV-U6 promoter group. To test the hybrid promoters driving shRNA efficiency against an endogenous gene, we used shRNA against no tail (NTL) gene. When vectorized in the zebrafish, the hybrid constructs strongly repressed NTL gene expression. The NTL phenotype occupied 52.09 +/- 3.06% and 51.56 +/- 3.68% for CMVE-U6 promoter and CMV-U6 promoter groups, respectively. The NTL gene expression reduced 82.17 +/- 2.96% for CMVE-U6 promoter group and 83.06 +/- 2.38% for CMV-U6 promoter group. We concluded that the CMV enhancer or entire CMV promoter locating upstream of the U6-promoter could significantly improve inhibitory effect induced by the shRNA for both exogenous and endogenous genes compared with the CMV promoter or U6 promoter alone. In contrast, the two hybrid promoter constructs had similar effects on driving shRNA.

The cytomegalovirus promoter-driven short hairpin RNA constructs mediate effective RNA interference in zebrafish in vivo

The ability to utilize the RNA interference (RNAi) machinery for silencing target-gene expression has created a lot of excitement in the research community. In the present study, we used a cytomegalovirus (CMV) promoter-driven DNA template approach to induce short hairpin RNA (shRNA) triggered RNAi to block exogenous Enhanced Green Fluorescent Protein (EGFP) and endogenous No Tail (NTL) gene expressions. We constructed three plasmids, pCMV-EGFP-CMV-shGFP-SV40, pCMV-EGFP-CMV-shNTL-SV40, and pCMV-EGFP-CMV-shScrambled-SV40, each containing a CMV promoter driving an EGFP reporter cDNA and DNA coding for one shRNA under the control of another CMV promoter. The three shRNA-generating plasmids and pCMV-EGFP control plasmid were introduced into zebrafish embryos by microinjection. Samples were collected at 48 h after injection. Results were evaluated by phenotype observation and real-time fluorescent quantitative reverse-transcription polymerase chain reaction (Q-PCR). The shGFP-generating plasmid significantly inhibited the EGFP expression viewed under fluorescent microscope and reduced by 70.05 +/- 1.26% of exogenous EGFP gene mRNA levels compared with controls by Q-PCR. The shRNA targeting endogenous NTL gene resulted in obvious NTL phenotype of 30 +/- 4% and decreased the level of their corresponding mRNAs up to 54.52 +/- 2.05% compared with nontargeting control shRNA. These data proved the feasibility of the CMV promoter-driven shRNA expression technique to be used to inhibit exogenous and endogenous gene expressions in zebrafish in vivo.

Enzyme-linked immunosorbent assay of changes in serum levels of growth hormone (cGH) in common carps (Cyprinus carpio)

The aim of the present study was to purify the common native carp growth hormone (ncGH), produce monoclonal antibodies (mAbs) to common native carp growth hormone (ncGH), and further enhance the sensitivity of enzyme-linked immunosorbent assays (ELISA) for ncGH. Additionally, we investigated changes in serum ncGH levels in carps raised in different environmental conditions. The recombinant grass carp (Ctenopharyngodon idella) growth hormone was purified and used as antigen to immunize the rabbit. The natural ncGH was isolated from the pituitaries of common carp. SDS-PAGE and Western blot utilizing the polyclonal anti-rgcGH antibody confirmed the purification of ncGH from pituitaries. Purified ncGH was then used as an immunogen in the B lymphocyte hybridoma technique. A total of 14 hybridoma cell lines (FMU-cGH 1-14) were established that were able to stably secrete mAbs against ncGH. Among them, eight clones (FMU-cGH1-6, 12 and 13) were successfully used for Western blot while nine clones (FMU-cGH 1-7, 9 and 10) were used in fluorescent staining and immunohistochemistry. Epitope mapping by competitive ELISA demonstrated that these mAbs recognized five different epitopes. A sensitive sandwich ELISA for detection of ncGH was developed using FMU-cGH12 as the coating mAb and FMU-cGH6 as the enzyme labeled mAb. This detection system was found to be highly stable and sensitive, with detection levels of 70 pg/mL. Additionally, we found that serum ncGH levels in restricted food group and in the net cage group increased 6.9-and 5.8-fold, respectively, when compared to controls, demonstrating differences in the GH stress response in common carp under different living conditions.

Knock down of gfp and no tail expression in zebrafish embryo by in vivo-transcribed short hairpin RNA with T7 plasmid system

A short-hairpin RNA (shRNA) expression system, based on T7 RNA polymerase (T7RP) directed transcription machinery, has been developed and used to generate a knock down effect in zebrafish embryos by targeting green fluorescent protein (gfp) and no tail (ntl) mRNA. The vector pCMVT7R harboring T7RP driven by CMV promoter was introduced into zebrafish embryos and the germline transmitted transgenic individuals were screened out for subsequent RNAi application. The shRNA transcription vectors pT7shRNA were constructed and validated by in vivo transcription assay. When pT7shGFP vector was injected into the transgenic embryos stably expressing T7RP, gfp relative expression level showed a decrease of 68% by analysis of fluorescence real time RT-PCR. As a control, injection of chemical synthesized siRNA resulted in expression level of 40% lower than the control when the injection dose was as high as 2 mu g/mu l. More importantly, injection of pT7shNTL vector in zebrafish embryos expressing T7RP led to partial absence of endogenous ntl transcripts in 30% of the injected embryos when detected by whole mount in situ hybridization. Herein, the T7 transcription system could be used to drive the expression of shRNA in zebrafish embryos and result in gene knock down effect, suggesting a potential role for its application in RNAi studies in zebrafish embryos.

Applicability of the MTT assay for measuring viability of cyanobacteria and algae, specifically for Microcystis aeruginosa (Chroococcales, Cyanobacteria)

Several assay methods were screened for viability assessment in cyanobacteria using Microcystis aeruginosa FACHB 905. Compared with fluorescent diacetate (FDA), Evan's Blue and autofluorescence, the 3-[4,5-dimethylthiazol-2-yl]2,5-diphenyl tetrazolium bromide (MTT) assay, which was based on the ability of viable cells to reduce MTT to formazan, was found to be reliable and was selected for further study. MTT concentration, incubation time and temperature were optimized for M. aeruginosa. Improvements to the sensitivity and reproducibility of the MTT assay included performing it in the dark to reduce the effects of formazan light sensitivity when extracted in DMSO. Another improvement involved collecting viability data by cell by counting rather than colourimetrically, which was concluded from the fact that oxidoreductase activity, responsible for MTT reduction, would elevate or decrease under stress conditions. Half-life of oxidoreductase in dead cell was calculated to be 3 h. The MTT assay was also found to be applicable to other cyanobacteria and diatoms, including field samples, but not for algae belonging to Chlorophyta, Euglenophyta, Pyrrophyta or Chrysophyta. Based on the above results, we proposed an optimized procedure for the MTT method on Microcystis strains. The use of this assay may be of importance to better understand the dynamics of bloom and the fate of Microcystis under natural or disturbed conditions.

Establishment, characterization, and virus susceptibility of a new marine cell line from red spotted grouper (Epinephelus akaara)

A marine fish cell line from the snout of red spotted grouper Epinephelus akaara, a protogynous hermaphrodite, was established, characterized, and subcultured with more than 60 passages. The grouper snout cell line (GSC) cells multiplied well in Dulbecco's modified Eagle's medium (DMEM) medium supplemented with 10% fetal bovine serum. The optimal growth temperature was 25 degrees C, and morphologically the cells were fibroblastic. Chromosome analysis revealed that the GSC cell line has a normal diploid karyotype with 2n = 8st + 40t. A virus titration study indicated that the cells were susceptible to turbot Scophthalmus Maximus rhabdovirus (SMRV) (10(8.5) TCID50 ml(-1)), while the viral titer of frog Rana grylio virus 9807 (RGV(9807)) reached 10(3.5) TCID50 ml-1. The infection was confirmed by cytopathic effect (CPE), immunofluorescence, and electron microscopy experiments, which detected the viral particles in the cytoplasm of virus-infected cells, respectively. Further, significant fluorescent signals were observed when the GSC cells were transfected with pEGFP vector DNA, indicating their potential utility for transgenic and genetic manipulation studies.

Solar UV radiation drives CO2 fixation in marine phytoplankton: A double-edged sword

Photosynthesis by phytoplankton cells in aquatic environments contributes to more than 40% of the global primary production (Behrenfeld et al., 2006). Within the euphotic zone (down to 1% of surface photosynthetically active radiation [PAR]), cells are exposed not only to PAR (400-700 nm) but also to UV radiation (UVR; 280-400 nm) that can penetrate to considerable depths (Hargreaves, 2003). In contrast to PAR, which is energizing to photosynthesis, UVR is usually regarded as a stressor (Hader, 2003) and suggested to affect CO2-concentrating mechanisms in phytoplankton (Beardall et al., 2002). Solar UVR is known to reduce photosynthetic rates (Steemann Nielsen, 1964; Helbling et al., 2003), and damage cellular components such as D1 proteins (Sass et al., 1997) and DNA molecules (Buma et al., 2003). It can also decrease the growth (Villafane et al., 2003) and alter the rate of nutrient uptake (Fauchot et al., 2000) and the fatty acid composition (Goes et al., 1994) of phytoplankton. Recently, it has been found that natural levels of UVR can alter the morphology of the cyanobacterium Arthrospira (Spirulina) platensis (Wu et al., 2005b). On the other hand, positive effects of UVR, especially of UV- A (315-400 nm), have also been reported. UV- A enhances carbon fixation of phytoplankton under reduced (Nilawati et al., 1997; Barbieri et al., 2002) or fast-fluctuating (Helbling et al., 2003) solar irradiance and allows photorepair of UV- B-induced DNA damage (Buma et al., 2003). Furthermore, the presence of UV-A resulted in higher biomass production of A. platensis as compared to that under PAR alone (Wu et al., 2005a). Energy of UVR absorbed by the diatom Pseudo-nitzschia multiseries was found to cause fluorescence (Orellana et al., 2004). In addition, fluorescent pigments in corals and their algal symbiont are known to absorb UVR and play positive roles for the symbiotic photosynthesis and photoprotection (Schlichter et al., 1986; Salih et al., 2000). However, despite the positive effects that solar UVR may have on aquatic photosynthetic organisms, there is no direct evidence to what extent and howUVR per se is utilized by phytoplankton. In addition, estimations of aquatic biological production have been carried out in incubations considering only PAR (i. e. using UV-opaque vials made of glass or polycarbonate; Donk et al., 2001) without UVR being considered (Hein and Sand-Jensen, 1997; Schippers and Lurling, 2004). Here, we have found that UVR can act as an additional source of energy for photosynthesis in tropical marine phytoplankton, though it occasionally causes photoinhibition at high PAR levels. While UVR is usually thought of as damaging, our results indicate that UVR can enhance primary production of phytoplankton. Therefore, oceanic carbon fixation estimates may be underestimated by a large percentage if UVR is not taken into account.