Acquisition of genome information from single-celled unculturable organisms (radiolaria) by exploiting genome profiling (GP)

BACKGROUND: There is no effective method to obtain genome information from single-celled unculturable organisms such as radiolarians. Even worse, such organisms are often very difficult to collect. Sequence analysis of 18S rDNA has been carried out, but obtaining the data has been difficult and it has provided a rather limited amount of genome information. In this paper, we have developed a method which provides a sufficient amount of data from an unculturable organism. The effectiveness of this method was demonstrated by applying it to the provisional classification of a set of unculturable organisms (radiolarians). RESULTS: Dendrogram was drawn regarding the single-celled unculturable species based on the similarity score termed PaSS, offering a consistent result with the conventional taxonomy of them built up based on phenotypes. This fact has shown that genome profiling-based technology developed here can obtain genome information being sufficient for identifying and classifying species from a single-celled organism. CONCLUSION: Since this method is so simple, general, and yet powerful, it can be applied to various organisms and cells, especially single-celled, uncluturable ones, for their genome analysis

A high-throughput direct FRET-based assay for analysing apoptotic proteases using flow cytometry and fluorescence-lifetime measurements

International audienceCytometry is a versatile and powerful method applicable to different fields, particularly pharmacology and biomedical studies. Based on the data obtained, cytometric studies are classified into high-throughput (HTP) or high-content screening (HCS) groups. However, assays combining the advantages of both are required to facilitate research. In this study, we developed a high-throughput system to profile cellular populations in terms of time- or dose-dependent responses to apoptotic stimulations, since apoptotic inducers are potent anti-cancer drugs. We previously established assay systems involving protease to monitor live cells for apoptosis using tuneable FRET-based bioprobes. These assays can be used for microscopic analyses or fluorescence-activated cell sorting. In this study, we developed FRET-based bioprobes to detect the activity of the apoptotic markers caspase-3 and caspase-9 via changes in bioprobe fluorescence lifetimes using a flow cytometer for direct estimation of FRET efficiencies. Different patterns of changes in the fluorescence lifetimes of these markers during apoptosis were observed, indicating a relationship between discrete steps in the apoptosis process. The findings demonstrate the feasibility of evaluating collective cellular dynamics during apoptosis

Novel concept microarray enabling PCR and multistep reactions through pipette-free aperture-to-aperture parallel transfer

<p>Abstract</p> <p>Background</p> <p>The microarray has contributed to developing the omic analysis. However, as it depends basically on the surface reaction, it is hard to perform bulk reactions and sequential multistep reactions. On the other hand, the popular microplate technology, which has a great merit of being able to perform parallel multistep reactions, has come to its limit in increasing the number of wells (currently, up to 9600) and reducing the volume to deal with due to the difficulty in operations.</p> <p>Results</p> <p>Here, we report a novel microarray technology which enables us to explore advanced applications, termed <it>microarray-with-manageable volumes </it>(MMV). The technical essence is in the pipette-free direct parallel transfer from well to well performed by centrifugation, evading the evaporation and adsorption-losses during handling. By developing the MMV plate, accompanying devices and techniques, generation of multiple conditions (256 kinds) and performance of parallel multistep reactions, including PCR and <it>in vitro tr</it>anslation reactions, have been made possible. These were demonstrated by applying the MMV technology to searching lysozyme-crystallizing conditions and selecting peptides aimed for Aβ-binding or cathepsin E-inhibition.</p> <p>Conclusions</p> <p>With the introduction of a novel concept microarray (MMV) technology, parallel and multistep reactions in sub-μL scale have become possible.</p

A Solution for Universal Classification of Species Based on Genomic DNA

Traditionally, organisms have been classified on the basis of their phenotype. Recently, genotype-based classification has become possible through the development of sequencing technology. However, it is still difficult to apply sequencing approaches to the analysis of a large number of species due to the cost and labor. In most biological fields, the analysis of complex systems comprising various species has become an important theme, demanding an effective method for handling a vast number of species. In this paper, we have demonstrated, using plants, fish, and insects, that genome profiling, a compact technology for genome analysis, can classify organisms universally. Surprisingly, in all three of the domains of organisms tested, the phylogenetic trees generated from the phenotype topologically matched completely those generated from the genotype. Furthermore, a single probe was sufficient for the genome profiling, thereby demonstrating that this methodology is universal and compact

Peptide aptamer-modified single-walled carbon nanotube-based transistors for high-performance biosensors

Biosensors employing single-walled carbon nanotube field-effect transistors (SWCNT FETs) offer ultimate sensitivity. However, besides the sensitivity, a high selectivity is critically important to distinguish the true signal from interference signals in a non-controlled environment. This work presents the first demonstration of the successful integration of a novel peptide aptamer with a liquid-gated SWCNT FET to achieve highly sensitive and specific detection of Cathepsin E (CatE), a useful prognostic biomarker for cancer diagnosis. Novel peptide aptamers that specifically recognize CatE are engineered by systemic in vitro evolution. The SWCNTs were firstly grown using the thermal chemical vapor deposition (CVD) method and then were employed as a channel to fabricate a SWCNT FET device. Next, the SWCNTs were functionalized by noncovalent immobilization of the peptide aptamer using 1-pyrenebutanoic acid succinimidyl ester (PBASE) linker. The resulting FET sensors exhibited a high selectivity (no response to bovine serum albumin and cathepsin K) and label-free detection of CatE at unprecedentedly low concentrations in both phosphate-buffered saline (2.3 pM) and human serum (0.23 nM). Our results highlight the use of peptide aptamer-modified SWCNT FET sensors as a promising platform for near-patient testing and point-of-care testing applications

Genome Profiling (GP) Method Based Classification of Insects: Congruence with That of Classical Phenotype-Based One

Ribosomal RNAs have been widely used for identification and classification of species, and have produced data giving new insights into phylogenetic relationships. Recently, multilocus genotyping and even whole genome sequencing-based technologies have been adopted in ambitious comparative biology studies. However, such technologies are still far from routine-use in species classification studies due to their high costs in terms of labor, equipment and consumables.Here, we describe a simple and powerful approach for species classification called genome profiling (GP). The GP method composed of random PCR, temperature gradient gel electrophoresis (TGGE) and computer-aided gel image processing is highly informative and less laborious. For demonstration, we classified 26 species of insects using GP and 18S rDNA-sequencing approaches. The GP method was found to give a better correspondence to the classical phenotype-based approach than did 18S rDNA sequencing employing a congruence value. To our surprise, use of a single probe in GP was sufficient to identify the relationships between the insect species, making this approach more straightforward.The data gathered here, together with those of previous studies show that GP is a simple and powerful method that can be applied for actually universally identifying and classifying species. The current success supported our previous proposal that GP-based web database can be constructible and effective for the global identification/classification of species

An Efficient Ligation Method in the Making of an in vitro Virus for in vitro Protein Evolution

The “in vitro virus” is a molecular construct to perform evolutionary protein engineering. The “virion (=viral particle)” (mRNA-peptide fusion), is made by bonding a nascent protein with its coding mRNA via puromycin in a test tube for in vitro translation. In this work, the puromycin-linker was attached to mRNA using the Y-ligation, which was a method of two single-strands ligation at the end of a double-stranded stem to make a stem-loop structure. This reaction gave a yield of about 95%. We compared the Y-ligation with two other ligation reactions and showed that the Y-ligation gave the best productivity. An efficient amplification of the in vitro virus with this “viral genome” was demonstrated