A new technique for the culture of fern gametophytes

Fern gametophytes initiated from spores grew successfully when immobilised in reticulate polyurethane foam in liquid shake cultures. Colonisation of this matrix was complete seven days after medium inoculation and appeared to be a completely passive process. Yield (dry weight) was greater than that from liquid grown cultures.     

A high throughput barley stripe mosaic virus vector for virus induced gene silencing in monocots and dicots

Barley stripe mosaic virus (BSMV) is a single-stranded RNA virus with three genome components designated alpha, beta, and gamma. BSMV vectors have previously been shown to be efficient virus induced gene silencing (VIGS) vehicles in barley and wheat and have provided important information about host genes functioning during pathogenesis as well as various aspects of genes functioning in development. To permit more effective use of BSMV VIGS for functional genomics experiments, we have developed an Agrobacterium delivery system for BSMV and have coupled this with a ligation independent cloning (LIC) strategy to mediate efficient cloning of host genes. Infiltrated Nicotiana benthamiana leaves provided excellent sources of virus for secondary BSMV infections and VIGS in cereals. The Agro/LIC BSMV VIGS vectors were able to function in high efficiency down regulation of phytoene desaturase (PDS), magnesium chelatase subunit H (ChlH), and plastid transketolase (TK) gene silencing in N. benthamiana and in the monocots, wheat, barley, and the model grass, Brachypodium distachyon. Suppression of an Arabidopsis orthologue cloned from wheat (TaPMR5) also interfered with wheat powdery mildew (Blumeria graminis f. sp. tritici) infections in a manner similar to that of the A. thaliana PMR5 loss-of-function allele. These results imply that the PMR5 gene has maintained similar functions across monocot and dicot families. Our BSMV VIGS system provides substantial advantages in expense, cloning efficiency, ease of manipulation and ability to apply VIGS for high throughput genomics studies.     

A reproducible, high throughput method for fabricating fibrin gels

ABSTRACT: BACKGROUND: Fibrin gels are a promising biomaterial for tissue engineering. However, current fabrication methods are time intensive with inherent variation. There is a pressing need to develop new and consistent approaches for producing fibrin-based hydrogels for examination. RESULTS: We developed a high throughput method for creating fibrin gels using molds fabricated from polydimethylsiloxane (PDMS). Fibrin gels were produced by adding solutions of fibrinogen and thrombin to cylindrical defects in a PDMS sheet. Undisturbed gels were collected by removing the sheet, and fibrin gels were characterized. The characteristics of resulting gels were compared to published data by measuring compressive stiffness and osteogenic response of entrapped human mesenchymal stem cells (MSCs). Gels exhibited compressive moduli nearly identical to our previously reported fabrication method. Trends in alkaline phosphatase activity, an early marker of osteogenic differentiation in MSCs, were also consistent with previous data. CONCLUSIONS: These findings demonstrate a streamlined approach to fibrin gel production that drastically reduces the time required to make fibrin gels, while also reducing variability between gel batches. This fabrication technique provides a valuable tool for generating large numbers of gels in a cost-effective manner.     

A KV2.1 gating modifier binding assay suitable for high throughput screening

Gating modifier peptides alter gating of voltage-gated potassium (KV) channels by binding to the voltage sensor paddle and changing the energetics of channel opening. Since the voltage sensor paddle is a modular motif with low sequence similarity across families, targeting of this region should yield highly specific channel modifiers. To test this idea, we developed a binding assay with the KV2.1 gating modifier, GxTX-1E. Monoiodotyrosine-GxTX-1E (125I-GxTX-1E) binds with high affinity (IC50 = 4 nM) to CHO cells stably expressing hKV2.1 channels, but not to CHO cells expressing Maxi-K channels. Binding of 125I-GxTX-1E to K_V2.1 channels is inhibited by another K_V2.1 gating modifier, stromatoxin (IC50 = 30 nM), but is not affected by iberiotoxin or charybdotoxin, pore blocking peptides of other types of potassium channels, or by ProTx-II, a selective gating modifier peptide of the voltage-gated sodium channel Na_V1.7. Specific 125I-GxTX-1E binding is not detectable when CHO-K_V2.1 cells are placed in high external potassium, suggesting that depolarization favors dissociation of the peptide. The binding assay was adapted to a 384-well format, allowing high throughput screening of large compound libraries. Interestingly, we discovered that compounds related to PAC, a di-substituted cyclohexyl K_V channel blocker, displayed inhibitory binding activity. These data establish the feasibility of screening large libraries of compounds in an assay that monitors the displacement of a gating modifier from the channel's voltage sensor. Future screens using this approach will ultimately test whether the voltage sensor of K_V channels can be selectively targeted by small molecules to modify channel function.     

A KV2.1 gating modifier binding assay suitable for high throughput screening

Gating modifier peptides alter gating of voltage-gated potassium (KV) channels by binding to the voltage sensor paddle and changing the energetics of channel opening. Since the voltage sensor paddle is a modular motif with low sequence similarity across families, targeting of this region should yield highly specific channel modifiers. To test this idea, we developed a binding assay with the KV2.1 gating modifier, GxTX-1E. Monoiodotyrosine-GxTX-1E (125I-GxTX-1E) binds with high affinity (IC50 = 4 nM) to CHO cells stably expressing hKV2.1 channels, but not to CHO cells expressing Maxi-K channels. Binding of 125I-GxTX-1E to KV2.1 channels is inhibited by another KV2.1 gating modifier, stromatoxin (IC50 = 30 nM), but is not affected by iberiotoxin or charybdotoxin, pore blocking peptides of other types of potassium channels, or by ProTx-II, a selective gating modifier peptide of the voltage-gated sodium channel NaV1.7. Specific 125I-GxTX-1E binding is not detectable when CHO-KV2.1 cells are placed in high external potassium, suggesting that depolarization favors dissociation of the peptide. The binding assay was adapted to a 384-well format, allowing high throughput screening of large compound libraries. Interestingly, we discovered that compounds related to PAC, a di-substituted cyclohexyl KV channel blocker, displayed inhibitory binding activity. These data establish the feasibility of screening large libraries of compounds in an assay that monitors the displacement of a gating modifier from the channel's voltage sensor. Future screens using this approach will ultimately test whether the voltage sensor of KV channels can be selectively targeted by small molecules to modify channel function.     

A high throughput profiling method for cutinolytic esterases

A procedure for identifying and profiling cutinolytic esterases was developed by combining traditional plate screen assays with an automated robotic system. In the first phase, the micro-organisms were screened on agar plates with cutin or the model substrate polycaprolactone as the sole carbon sources. In the second phase, p-nitrophenyl esters of fatty acids were used as the substrates in an automated activity assay of liquid culture media. The variables used were pH and the carbon chain length of the fatty acid moiety of the p-nitrophenyl substrate. Finally, ³H-labelled cutin was used as a specific substrate to verify the positive hits and to validate the screening procedure. With pH as the variable in the automatic screen, esterase production of cutinase positive strains typically proceeded in two stages: first an esterase with neutral activity optimum was produced, after which a strong esterolytic response in the alkaline range was detected. With carbon chain length of the fatty acid as the variable best correlation with cutinase production was obtained with strains showing a high ratio of activities towards p-nitrophenyl-butyrate and p-nitrophenyl-palmitate.     

Developmental morphology of strap-shaped gametophytes of Colysis decurrens: a new look at meristem development and function in fern gametophytes

Background and Aims

The gametophytes of most homosporous ferns are cordate–thalloid in shape. Some are strap- or ribbon-shaped and have been assumed to have evolved from terrestrial cordate shapes as an adaptation to epiphytic habitats. The aim of the present study was to clarify the morphological evolution of the strap-shaped gametophyte of microsoroids (Polypodiaceae) by precise analysis of their development.

Methods

Spores of Colysis decurrens collected in Kagoshima, Japan, were cultured and observed microscopically. Epi-illuminated micrographs of growing gametophytes were captured every 24 h, allowing analysis of the cell lineage of meristems. Light microscopy of resin-sections and scanning electron microscopy were also used.

Key Results

Contrary to previous assumptions that strap-shaped Colysis gametophytes have no organized meristem, three different types of meristems are formed during development: (1) apical-cell based – responsible for early growth; (2) marginal – further growth, including gametophyte branching; and (3) multicellular – formation of cushions with archegonia. The cushion is two or three layers thick and intermittent. The apical-cell and multicellular meristems are similar to those of cordate gametophytes of other ferns, but the marginal meristem is unique to the strap-shaped gametophyte of this fern.

Conclusions

The strap-shaped gametophytes of C. decurrens may have evolved from ancestors with a cordate shape by insertion of the marginal meristem phase between the first apical-cell-based meristem and subsequent multicellular meristem phases. Repeated retrieval of the marginal meristem at the multicellular meristem phase would result in indefinite prolongation of gametophyte growth, an ecological adaptation to epiphytic habitats.     

A geminivirus-induced gene silencing system for gene function validation in cassava

We have constructed an African cassava mosaic virus (ACMV) based gene-silencing vector as a reverse genetics tool for gene function analysis in cassava. The vector carrying a fragment from the Nicotiana tabacumsulfur gene (su), encoding one unit of the chloroplast enzyme magnesium chelatase, was used to induce the silencing of the cassava orthologous gene resulting in yellow–white spots characteristic of the inhibition of su expression. This result suggests that well developed sequence databases from model plants like Arabidopsis thaliana, Nicotiana benthamiana, N. tabacum, Lycopersicon esculentum and others could be used as a major source of information and sequences for functional genomics in cassava. Furthermore, a fragment of the cassava CYP79D2endogenous gene, sharing 89% homology with CYP79D1endogenous gene was inserted into the ACMV vector. The resultant vector was inducing the down regulation of the expression of these two genes which catalyze the first-dedicated step in the synthesis of linamarin, the major cyanogenic glycoside in cassava. At 21 days post-inoculation (dpi), a 76% reduction of linamarin content was observed in silenced leaves. Using transgenic plants expressing antisense RNA of CYP79D1and CYP79D2, Siritunga and Sayre (2003) obtained several lines with a reduction level varying from 60% to 94%. This result provides the first example of direct comparison of the efficiency of a virus-induced gene silencing (VIGS) system and the transgenic approach for suppression of a biosynthetic pathway. The ACMV VIGS system will certainly be a complement and in some cases an alternative to the transgenic approach, for gene discovery and gene function analysis in cassava.     

A system for performing high throughput assays of synaptic function

Unbiased, high-throughput screening has proven invaluable for dissecting complex biological processes. Application of this general approach to synaptic function would have a major impact on neuroscience research and drug discovery. However, existing techniques for studying synaptic physiology are labor intensive and low-throughput. Here, we describe a new high-throughput technology for performing assays of synaptic function in primary neurons cultured in microtiter plates. We show that this system can perform 96 synaptic vesicle cycling assays in parallel with high sensitivity, precision, uniformity, and reproducibility and can detect modulators of presynaptic function. By screening libraries of pharmacologically defined compounds on rat forebrain cultures, we have used this system to identify novel effects of compounds on specific aspects of presynaptic function. As a system for unbiased compound as well as genomic screening, this technology has significant applications for basic neuroscience research and for the discovery of novel, mechanism-based treatments for central nervous system disorders.     

A high throughput method for genome-wide analysis of retroviral integration

Retroviral and lentiviral vectors integrate their DNA into the host cell genome leading to stable transgene expression. Integration preferentially occurs in the proximity of active genes, and may in some case disturb their activity, with adverse toxic consequences. To efficiently analyze high numbers of lentiviral insertion sites in the DNA of transduced cells, we developed an improved high-throughput method called vector integration tag analysis (VITA). VITA is based on the identification of Genomic Tags associated to the insertion sites, which are used as signatures of the integration events. We use the capacity of MmeI to cleave DNA at a defined distance of its recognition site, in order to generate 21 bp long tags from libraries of junction fragments between vector and cellular DNA. The length of the tags is sufficient in most cases, to identify without ambiguity an unique position in the human genome. Concatenation, cloning and sequencing of the tags allow to obtain information about 20–25 insertion sites in a single sequencing reaction. As a validation of this method, we have characterized 1349 different lentiviral vector insertion sites in transduced HeLa cells, from only 487 sequencing reactions, with a background of <2% false positive tags.     

药物筛选新方法--高通量筛选

介绍了高通量筛选的概念、原理及其各个组成部分,着重阐明了高通量筛选的筛选模式,检测方法和应用实例,并简单介绍了国内在这方面的研究进展。     

A simplified miRNA-based gene silencing method for Drosophila melanogaster

MicroRNA-based RNA interference is commonly used to produce loss-of-function phenotypes in mammalian systems, but is used only sparingly in invertebrates such as Caenorhabditis elegans and Drosophila melanogaster. Here, we evaluate this method in transgenic strains of D. melanogaster and cultured S2 cells. High throughput-ready expression vectors were developed that permit rapid cloning of synthetic hairpin RNAs. As proof of concept, this method was used for the efficient silencing of dpp gene activity in the adult wing, and the analysis of the general RNA Polymerase II (Pol II) elongation factor, Nelf-E.     

A model-based high throughput method for fecundity estimation in fruit fly studies

ABSTRACT

The ability to quantify fecundity is critically important to a wide range of experimental applications, particularly in widely-used model organisms such as Drosophila melanogaster. However, the standard method of manually counting eggs is time consuming and limits the feasibility of large-scale experiments. We develop a predictive model to automate the counting of eggs from images of eggs removed from the media surface and washed onto dark filter paper. Our method uses the simple relationship between the white area in an image and the number of eggs present to create a predictive model that performs well even at high egg densities where clumping can complicate the individual identification of eggs. A cross-validation approach demonstrates our method performs well, with a correlation between predicted and manually counted values of 0.88. We show how this method can be applied to a large data set where egg densities vary widely.     

A TMA de-arraying method for high throughput biomarker discovery in tissue research

Background

Tissue MicroArrays (TMAs) represent a potential high-throughput platform for the analysis and discovery of tissue biomarkers. As TMA slides are produced manually and subject to processing and sectioning artefacts, the layout of TMA cores on the final slide and subsequent digital scan (TMA digital slide) is often disturbed making it difficult to associate cores with their original position in the planned TMA map. Additionally, the individual cores can be greatly altered and contain numerous irregularities such as missing cores, grid rotation and stretching. These factors demand the development of a robust method for de-arraying TMAs which identifies each TMA core, and assigns them to their appropriate coordinates on the constructed TMA slide.

Methodology

This study presents a robust TMA de-arraying method consisting of three functional phases: TMA core segmentation, gridding and mapping. The segmentation of TMA cores uses a set of morphological operations to identify each TMA core. Gridding then utilises a Delaunay Triangulation based method to find the row and column indices of each TMA core. Finally, mapping correlates each TMA core from a high resolution TMA whole slide image with its name within a TMAMap.

Conclusion

This study describes a genuine robust TMA de-arraying algorithm for the rapid identification of TMA cores from digital slides. The result of this de-arraying algorithm allows the easy partition of each TMA core for further processing. Based on a test group of 19 TMA slides (3129 cores), 99.84% of cores were segmented successfully, 99.81% of cores were gridded correctly and 99.96% of cores were mapped with their correct names via TMAMaps. The gridding of TMA cores were also extensively tested using a set of 113 pseudo slide (13,536 cores) with a variety of irregular grid layouts including missing cores, rotation and stretching. 100% of the cores were gridded correctly.     

A colorimetric microplate assay method for high throughput analysis of lipase activity

The present work describes a colorimetric microplate assay for lipase activity based on the reaction between 5,5'-dithiobis(2-nitro benzoic acid) (DTNB) and the hydrolysis product of 2,3-dimercapto-1-propanol tributyrate (DMPTB). Reaction mixtures containing DTNB, DMPTB, and lipase were prepared in microplate wells, and the absorbance at 405nm was recorded after incubation at 37 degrees C for 30 min. A linear relationship was obtained in the range of 0.1-1 U of lipase activity by this method. The reaction conditions were also optimized for the range of 0.01-0.1 U or 1-10 U. When assaying crude tissue extracts, the reaction of DTNB with non-specific reducing agents created a major source of error. However, this error was corrected by the use of blank samples that did not contain DMPTB.