A paper describing a potential new pipeline for structural genomics based on small angle X-ray scattering was far and away the most popular paper of the August issue. It will be very interesting to see what kind of impact it has on the field. While it may not provide high-resolution structures like x-ray crystalography, it is certainly faster and has a higher success rate, both of which are critical parameters for high-throughput pipelines. A paper from Helicos describing new terminator nucleotides for single-molecule next-generation sequencing (or should this be 2nd or 3rd generation?) made it to the #5 spot.

Top 7 research papers published in the August issue
1. Robust, high-throughput solution structural analyses by small angle X-ray scattering (SAXS)
2. Digital RNA allelotyping reveals tissue-specific and allele-specific gene expression in human
3. Mass spectrometry of membrane transporters reveals subunit stoichiometry and interactions
4. SHOREmap: simultaneous mapping and mutation identification by deep sequencing
5. Virtual terminator nucleotides for next-generation DNA sequencing
6. Global discovery of adaptive mutations
7. Metabolic network analysis integrated with transcript verification for sequenced genomes

The top five spots in the ten most popular papers published prior to our August issue and downloaded during August are unchanged since last month. We have a surprise appearance of an old and slightly controversial paper at position #6. This appearance appears to be the result of their publication of a follow-up paper in PNAS at the beginning of August. Squeaking in at #9 and #10 are two papers from the July issue. The top downloaded paper in July was also close behind but didn't quite make it.

Top 10 research papers published prior to the August issue
1. Mapping and quantifying mammalian transcriptomes by RNA-Seq
2. mRNA-Seq whole-transcriptome analysis of a single cell
3. Universal sample preparation method for proteome analysis
4. Stem cell transcriptome profiling via massive-scale mRNA sequencing
5. Isolation of human iPS cells using EOS lentiviral vectors to select for pluripotency
6. The development of a bioengineered organ germ method
7. Genome-wide profiles of STAT1 DNA association using chromatin immunoprecipitation and massively parallel sequencing
8. Stable knockdown of microRNA : in vivo: by lentiviral vectors
9. In vivo fluorescence imaging with high-resolution microlenses
10. Mapping the structure and conformational movements of proteins with transition metal ion FRET

Posted by Daniel Evanko at 10:42 AM | Permalink | Comments (0)

Deep sequencing of metagenomic datasets by next-generation technology, has revealed a richness and diversity of microbial species that far exceeded previous expectations.

In 2006 Mitchell Sogin presented the concept of a rare biosphere, low-abundance microbial populations that have persisted over large evolutionary time scales. Sogin described it as a ‘potentially inexhaustible reservoir of genomic innovation’ which lets microbial communities recover from environmental assault and allows them to adapt to changed circumstances.

How do scientists determine microbial diversity? Sequence reads covering the hypervariable regions of 16S rRNA genes are often used for classification. These ‘pyrotags’ (named thus since they are obtained on Roche’s 454 sequencer) can be classified by matching them against a rRNA sequence database.

The challenge is that for most microbial species the 16S rRNA gene is not known. An alternative is shotgun sequencing and grouping of reads into operational taxonomic units; i.e. a cluster of sequences with a defined difference to a neighboring cluster — commonly a sequence difference of 3% is required at the species level, 5% at the genus level.

But with predictions of an ever larger rare biosphere voices of caution are starting to be heard saying that its size is overestimated. Is everything that is classified as a new and rare species indeed real, or simply a sequencing error?

Most recently we featured an error correction program in our own pages that addressed this issue; and a note of caution by Phil Hugenholtz and colleagues about ‘Wrinkles in the rare biosphere’ just appeared in "Early View" in Environmental Microbiology.

How well founded are the concerns of an artificially inflated rare biosphere? Are current estimates of the rare biosphere really 10-fold too high? If so, what are the consequences — does it mean that the rare biosphere is far less important than assumed? Is it not such an inexhaustible reservoir after all but just background noise?

We understand that these are very controversial issues and we would love to hear from you.

Posted by Daniel Evanko at 01:08 PM | Permalink | Comments (0) | TrackBacks (0)

Two Correspondences made the list of top downloads for July coming in at #3 and #4, demonstrating that while this format may not report new methods it does have information of high interest to readers. The two top downloads seem to highlight a high level of interest in assaying single cells and using FRET to examine protein dynamics.

Top 8 research papers published in the July issue
1. Quantitative analysis of gene expression in a single cell by qPCR
2. Mapping the structure and conformational movements of proteins with transition metal ion FRET
3. Limitations and possibilities of small RNA digital gene expression profiling
4. Enabling IMAC purification of low abundance recombinant proteins from E. coli lysates
5. In vivo fluorescence imaging with high-resolution microlenses
6. Protein interaction platforms: visualization of interacting proteins in yeast
7. Agouti C57BL/6N embryonic stem cells for mouse genetic resources
8. Reaching the protein folding speed limit with large, sub-microsecond pressure jumps

There has been very little movement in the ten most popular papers published prior to our July issue and downloaded during July. The exception is the appearance of the HUPO test sample paper from June appearing at #7. The papers below this have changed but mostly because a large number of papers have very similar download numbers and they shuffle around from month to month.

Top 10 research papers published prior to the July issue
1. Mapping and quantifying mammalian transcriptomes by RNA-Seq
2. mRNA-Seq whole-transcriptome analysis of a single cell
3. Universal sample preparation method for proteome analysis
4. Stem cell transcriptome profiling via massive-scale mRNA sequencing
5. Isolation of human iPS cells using EOS lentiviral vectors to select for pluripotency
6. Genome-wide profiles of STAT1 DNA association using chromatin immunoprecipitation and massively parallel sequencing
7. A HUPO test sample study reveals common problems in mass spectrometry–based proteomics
8. Generation of transgene-free induced pluripotent mouse stem cells by the piggyBac transposon
9. Amplification-free Illumina sequencing-library preparation facilitates improved mapping and assembly of (G+C)-biased genomes
10. A versatile tool for conditional gene expression and knockdown

Posted by Daniel Evanko at 05:07 PM | Permalink | Comments (0)

Here are the top downloads for June. Due to lack of time I have to forego any comments this month but I've posted the rankings of the five most popular papers published in our June issue based on PDF downloads during June.

Top 5 research papers published in the June issue
1. Predicting microRNA targets and functions: traps for the unwary
2. Automated unrestricted multigene recombineering for multiprotein complex production
3. A HUPO test sample study reveals common problems in mass spectrometry–based proteomics
4. Versatile P[acman] BAC libraries for transgenesis studies in Drosophila melanogaster
5. High-throughput ethomics in large groups of Drosophila

There has been quite a reshuffling of the ten most popular papers published prior to our June issue and downloaded during June. Part of this movement may be due to the fact that beginning this month I have decided to use the number of successfully downloaded PDFs rather than combined PDF and HTML views but I think most of the explanation is the high quality of the papers published in our May issue which have displaced some of the older papers.

Top 10 research papers published prior to the June issue
1. Mapping and quantifying mammalian transcriptomes by RNA-Seq
2. mRNA-Seq whole-transcriptome analysis of a single cell
3. Universal sample preparation method for proteome analysis
4. Isolation of human iPS cells using EOS lentiviral vectors to select for pluripotency
5. Super-resolution video microscopy of live cells by structured illumination
6. Generation of transgene-free induced pluripotent mouse stem cells by the piggyBac transposon
7. Stem cell transcriptome profiling via massive-scale mRNA sequencing
8. Genetically encoded fluorescent indicator for intracellular hydrogen peroxide
9. Genome-wide profiles of STAT1 DNA association using chromatin immunoprecipitation and massively parallel sequencing
10. Enzymatic assembly of DNA molecules up to several hundred kilobases

Posted by Daniel Evanko at 07:02 PM | Permalink | Comments (0)

Below I've posted the rankings of the six most popular papers published in our May issue based on downloads and page-views during May. The most popular paper by a rather large margin was a paper describing mRNA-Seq analysis of single cells. Regular readers of this blog won't be surprised by this given the high level of interest that next-generation sequencing papers generally receive.

A surprising omission from the list is the paper by Allan Bradley and colleagues describing the use of transposons carrying reprogramming factors to generate iPS cells followed by removal of the transposons from the genome. After analyzing the download stats it looks like the explanation for this is the fact that we express-published the paper online ahead of the other papers from the May issue so it would appear soon after a similar paper in Nature. So although initial downloads were very high, they were trailing off during May. In contrast, downloads of newer papers from the May issue were still at their peak during May.

Discerning readers will note that the last paper in the list is actually a Correspondence. Because Correspondences sometimes contain original data we will now be including these in the rankings with original research papers when appropriate.

Top 6 research papers published in the May issue
1. mRNA-Seq whole-transcriptome analysis of a single cell
2. Universal sample preparation method for proteome analysis
3. Super-resolution video microscopy of live cells by structured illumination
4. Isolation of human iPS cells using EOS lentiviral vectors to select for pluripotency
5. Single molecule–sensitive probes for imaging RNA in live cells
6. Massively parallel exon capture and library-free resequencing across 16 genomes

As in past months, there has been very little movement in the list of most popular papers published in months prior to the issue month being analyzed. The main changes this month are the appearances of two papers from the April issue and an old paper from 2007 describing new far-red fluorescent proteins Katushka and mKate. The only explanation I come come up with for this is the appearance of an article in Science from Roger Tsien that compared the performance of a new fluorescent protein they developed to mKate instead of the more recent improved variant mKate2 published in this paper in The Biochemical Journal.

Top 10 research papers published prior to the May issue
1. Mapping and quantifying mammalian transcriptomes by RNA-Seq
2. Genome-wide analysis of transcription factor binding sites based on ChIP-Seq data
3. Stem cell transcriptome profiling via massive-scale mRNA sequencing
4. Amplification-free Illumina sequencing-library preparation facilitates improved mapping and assembly of (G+C)-biased genomes
5. Photoactivatable mCherry for high-resolution two-color fluorescence microscopy
6. Stable knockdown of microRNA in vivo by lentiviral vectors
7. Bright far-red fluorescent protein for whole-body imaging
8. Global mapping of protein-DNA interactions by digital genomic footprinting
9. Genome-wide profiles of STAT1 DNA association using chromatin immunoprecipitation and massively parallel sequencing
10. Lifeact: a versatile marker to visualize F-actin

Posted by Daniel Evanko at 02:39 PM | Permalink | Comments (0)

Our June issue, which went live online yesterday, includes an Analysis paper describing the results of a large-scale study to try to get to the root causes of irreproducibility in mass spectrometry-based proteomics. Despite the novel and valuable biological applications made possible by proteomics and the continuing impressive technological advances in mass spectrometry, the technology has been unable to completely shed its reputation of being poorly reproducible.

To attempt to pinpoint the sources of irreproducibility, John Bergeron and colleagues, as part of a Human Proteome Organization (HUPO) effort, sent a test sample consisting of 20 purified proteins at equal concentrations to 27 different proteomics labs. The study designers asked these labs to identify the 20 proteins by whatever mass spectrometry instrumentation and workflows they were used to using. Initially, only 7 labs correctly reported all 20 proteins! However, when the study designers re-analyzed the data from the labs that failed in the task, they found that almost all actually did have mass spectra for all 20 proteins in hand. Most of the problems therefore stemmed from the database searching approaches used to go from the raw spectra to a protein identification. Many of the labs also reported ‘false positives’ – proteins that were not actually in the test samples. However, it turned out that many of these false positives were real; they were contaminants introduced during the sample handling process.

This study reaches several interesting conclusions. First of all, and reassuringly, the authors found that the mass spectrometry technology itself is reproducible. However, because of the number of complicated steps required to go from an unknown sample to a protein identification, the success of each of the groups varied widely, demonstrating the need for careful sample handling and proper training. The authors also state that improvements in database search engines and the proteomic databases themselves are direly needed.

This work also shows the value of examining the reproducibility of new technologies and methods on a large scale, especially between labs, using carefully prepared test samples. These studies can be expensive and time-consuming, but they are highly beneficial. Broad guidelines for Analysis papers are provided in our April 2008 editorial, and authors interested in submitting such studies are encouraged to contact the editors beforehand.

This Analysis will be freely available for one month. Be sure to also check out the News and Views article by Ruedi Aebersold which accompanies the paper.

Posted by Allison Doerr at 04:42 PM | Permalink | Comments (0)

The June issue of Nature Methods goes live today, with several papers, coincidentally, reporting on methods for the study of fruit flies.

As discussed in our editorial, Michael Dickinson, Pietro Perona and colleagues use machine vision to track individual flies as they interact within a group (Branson et al); this may prove useful to study social behaviour and how it is influenced by specific genes or neural circuits. In a News and Views Michael Reiser proposes that, with approaches such as these, phenotyping methods may be catching up with the well-established molecular genetics toolkit available for the fly. But of course, improvements in genetic resources are still needed. Also in the June issue, two groups (those of Hugo Bellen and Pavel Tomancak) present libraries that cover the entire genomes of Drosophila melanogaster and Drosophila pseudoobscura: Venken et al present high-coverage BAC libraries in the P[acman] system for D. melanogaster and Ejsmont et al present fosmid libaries for D. melanogaster and D. pseudoobscura. These resources allow modification of genes by recombineering (for instance, to make mutants or to add tags for visualization) as well as integration into precise sites in the genome, and should facilitate a wide variety of studies – including behavioural analyses – that require transgenesis in the fly.

Posted by Natalie De Souza at 07:50 AM | Permalink | Comments (0)

We've now been posting information on the most-downloaded papers in Nature Methods for three months now so I think we can dispense with the introduction. Please see the earlier posts for basic information on how these rankings were determined.

As I have commented previously, the next-generation sequencing papers continue to generate the most interest and occupy the top three spots for most-downloaded papers published in the April issue. A variation on an established, but relatively unknown, scanning probe method for imaging the surface topography of cells squeeked in at number four.

Top 4 research papers published in the April issue
1. Global mapping of protein-DNA interactions by digital genomic footprinting
2. Amplification-free Illumina sequencing-library preparation facilitates improved mapping and assembly of (G+C)-biased genomes
3. Quantification of rare allelic variants from pooled genomic DNA
4. Nanoscale live-cell imaging using hopping probe ion conductance microscopy

[Update on 5/13/09: A question came up regarding the popularity of Correspondences. If these are included in the above list, the Correspondence describing the UCSC Cancer Genomics Browser would be ranked #1.]

There has been very little movement in the list of most popular papers published in months prior to the issue month being analyzed. The top seven positions just moved around a bit. Only the last three positions see the appearance of new arrivals. The Lifeact paper makes an appearance due to a Correspondence published in the May issue of Nature Methods and the proteomics paper has retained its popularity after being published in the March issue. A surprise appearance is made by a paper we published in August 2006. This is the STORM super-resolution imaging paper that scooped the competing PALM paper in Science by one day. Looking at data from past months shows that this paper has been hanging out just under the number ten spot but has been seeing a slow but steady increase in the number of downloads. It is tempting to speculate about the cause of this but I'll avoid doing so.

Top 10 research papers published prior to the April issue
1. Mapping and quantifying mammalian transcriptomes by RNA-Seq
2. Stem cell transcriptome profiling via massive-scale mRNA sequencing
3. Genome-wide analysis of transcription factor binding sites based on ChIP-Seq data
4. Stable knockdown of microRNA in vivo by lentiviral vectors
5. Photoactivatable mCherry for high-resolution two-color fluorescence microscopy
6. Genome-wide profiles of STAT1 DNA association using chromatin immunoprecipitation and massively parallel sequencing
7. miRNA in situ hybridization in formaldehyde and EDC-fixed tissues
8. Lifeact: a versatile marker to visualize F-actin
9. Quantitative interaction proteomics using mass spectrometry
10. Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM)

Posted by Daniel Evanko at 08:51 AM | Permalink | Comments (0)

It's a new month and time to see how papers published in Nature Methods have been received by our readers. I alway find it fascinating to see how the numbers of downloads compare to our opinion of each paper. I've listed the original research papers we published that received the most unique views (HTML) and downloads (PDF) in March in order of popularity and broken them down between papers published in the March issue and papers published in prior months.

Of course downloads for the March issue are based on nothing more than the title of the paper and maybe the editor's extraordinarily brief summary. It isn't until later that the number of downloads is more indicative of the quality and impact of a paper. For this reason the downloads of papers published prior to the March issue can be more interesting since they are more likely to be influenced by community discussion of which papers should be read and cited.

Top 4 research papers published in the March issue
1. High-efficiency labeling of sialylated glycoproteins on living cells
2. Quantitative interaction proteomics using mass spectrometry
3. Automated light-based mapping of motor cortex by photoactivation of channelrhodopsin-2 transgenic mice
4. Analysis of receptor oligomerization by FRAP microscopy

It is interesting to note that the two photoactivatable fluorescent protein papers from February made it into the March "Top 10" list below with the 3D STORM paper from last year. Super-resolution imaging related papers continue to be of high interest. It is clear though that next-generation sequencing papers continue to be the hottest papers in the journal and have barely budged from their standings last month.

Top 10 research papers published prior to the March issue
1. Mapping and quantifying mammalian transcriptomes by RNA-Seq
2. Photoactivatable mCherry for high-resolution two-color fluorescence microscopy
3. Genome-wide analysis of transcription factor binding sites based on ChIP-Seq data
4. Stem cell transcriptome profiling via massive-scale mRNA sequencing
5. Stable knockdown of microRNA in vivo by lentiviral vectors
6. miRNA in situ hybridization in formaldehyde and EDC-fixed tissues
7. Genome-wide profiles of STAT1 DNA association using chromatin immunoprecipitation and massively parallel sequencing
8. Whole-cell 3D STORM reveals interactions between cellular structures with nanometer-scale resolution
9. A bright and photostable photoconvertible fluorescent protein
10. Microfluidic control of cell pairing and fusion

Posted by Daniel Evanko at 07:28 AM | Permalink | Comments (2) | TrackBacks (0)

Intuitively most of us would agree that standardized conditions in scientific experiments ensure reproducibility. If I know how something was done and exactly replicate the setup and protocol, I should get the same results. The authors of a Perspective in the April issue of Nature Methods argue otherwise when it comes to behavioral experiments done with live animals.

Hanno Wϋrbel and his co-workers present their view that standardizing environmental conditions in animal experiments actually leads to poor reproducibility, since it makes the results more vulnerable to the most subtle local differences that can result in conflicting results between different labs. Instead, the authors advocate systematic environmental heterogenization. In their opinion this will increase reproducibility and thus decrease the ethical cost of animal experiments.

To prove their point the authors performed a data-mining experiment in which they group together the results from several behavioral tests performed in a multi-laboratory study with mice. They re-analyzed the published data by either maximizing or minimizing the environmental variation between the groups. The results from the heterogeneous group—those within the maximized environmental variation group—were less variable than those from the standardized group.

With this analysis Wϋrbel and co-workers challenge the current paradigm that standardization is essential in animal experiments. Their view is likely to spark discussion and we invite you to contribute to this discussion here.

Posted by Nicole Rusk at 01:00 PM | Permalink | Comments (0) | TrackBacks (0)

It is always interesting to see how papers we publish are received by the community and one useful metric is the number of times a paper is viewed or downloaded. Authors also like to see this information when their paper was one of the most heavily viewed papers.

Each month we will report the original research papers we published that received the most unique views (HTML) and downloads (PDF) the previous month. This will be broken down between papers published in that month's issue and papers published in prior months. Now for the most popular downloads for the month of February.

Top 4 research papers published in the February issue
1. Microfluidic control of cell pairing and fusion
2. Photoactivatable mCherry for high-resolution two-color fluorescence microscopy
3. A bright and photostable photoconvertible fluorescent protein
4. miRNA in situ hybridization in formaldehyde and EDC–fixed tissues

Top 10 research papers published prior to the February issue
1. Mapping and quantifying mammalian transcriptomes by RNA-Seq
2. Stable knockdown of microRNA in vivo by lentiviral vectors
3. Genome-wide analysis of transcription factor binding sites based on ChIP-Seq data
4. Stem cell transcriptome profiling via massive-scale mRNA sequencing
5. mirWIP: microRNA target prediction based on microRNA-containing ribonucleoprotein-enriched transcripts
6. Genome-wide profiles of STAT1 DNA association using chromatin immunoprecipitation and massively parallel sequencing
7. Whole-cell 3D STORM reveals interactions between cellular structures with nanometer-scale resolution
8. Bright far-red fluorescent protein for whole-body imaging
9. High-resolution mapping of copy-number alterations with massively parallel sequencing
10. A versatile tool for conditional gene expression and knockdown

Posted by Daniel Evanko at 04:10 PM | Permalink | Comments (0) | TrackBacks (0)

It has been appreciated for a while that fruit flies interact with each other, but studying these interactions is quite difficult. In fact, this is a general problem in understanding animal behavior – the measurements are incredibly tedious to make and to quantify. In a paper published yesterday in Nature Methods (Dankert et al, 2009), Heiko Dankert, Pietro Perona, David Anderson and colleagues now automate the process, using the power of machine vision to analyse social interactions in pairs of fruit flies.

Put two wild-type male fruit flies together, and they will lunge, tussle and threaten each other, actions classified as aggressive (however, not all interactions between male flies are aggressive). Put a male and a female fruit fly together though and, predictably, their interactions will be largely of the courtship variety; the male circles the female, extends his wings in a courtship ‘song’, and sometimes, things work out!

Dankert et al show in their paper that they can take simple videos of fly-pairs, and use new software to accurately identify these stereotypical aggression and courtship actions. This allows them to observe many such fly-pairs and to describe these behaviors quantitatively. What’s more, the software detects expected differences in fly behavior. Male flies in which octopaminergic neurons have been silenced or where the fruitless gene is spliced into a female-specific form are much less aggressive. Male flies in which cholinergic neurons have been feminized now court other males. The software sees these differences.

Importantly, the measurements can be made quickly and without researcher bias or fatigue. For instance, Dankert et al estimate that experiments that would take them about a hundred hours to perform manually can now be completed in a few minutes. This opens up the study of these behaviors to genetic and other screens, and it will be interesting indeed to see how such studies play out.

Posted by Natalie De Souza at 06:51 PM | Permalink | Comments (0)

Many receptors form functional dimers or higher order oligomers on the cell surface. A number of reports have also shown with various levels of certainty that G protein-coupled receptors (GPCRs) di- or oligomerize. In December 2006 we published an Article by Simon Davis and colleagues that challenged how bioluminescence resonance energy transfer (BRET) was being used to investigate GPCR dimerization. In particular, conclusions about β-adrenergic receptor (β-AR) dimerization were challenged. The paper was accompanied by a News and Views article by Martin Lohse.

This controversial paper was quickly followed by Correspondences by Michel Bouvier and colleagues and Salahpour and colleagues arguing against the findings. The controversy also seemed to attract a number of papers describing alternative methodologies for examining receptor oligomerization, including time-resolved FRET and snap-tag technologies (Maurel et al., 2008) and sequential BRET-FRET (Carriba et al., 2008).

Fast forward to this week and we see the return of Martin Lohse in a paper where he, Moritz Bűnemann and colleagues describe a dual-color fluorescence recovery after photobleaching (FRAP) microscopy approach relying on antibody-mediated receptor immobilization to analyze receptor oligomerization. They focus on β-AR oligomerization (the subject of controversy in the original papers) and show that β1-AR shows transient interactions while β2-AR can form stable oligomers.

What does this mean for the conclusions of the original study? That is difficult to say. FRAP can't distinguish between direct and indirect interactions so it is even less suitable than BRET for determining the type of interaction that is occuring, but it can distinguish between association of two vs many receptors.

It does appear that the Davis paper highlighted the need for the community to be more conscientious in providing experimental controls using constitutive interacting and non-interacting receptors. This is certainly a positive result and the tools now seem to be in place that will help researchers settle these questions.

Posted by Daniel Evanko at 09:46 AM | Permalink | Comments (0) | TrackBacks (0)

In June, Nature Methods focuses on the latest trends in single-molecule analysis.

Leaders in the field review the methodologies allowing to examine the mechanism of action of fundamental biological processes molecule by molecule. The Review and Perspective articles also provide practical guidance for some of the techniques that are most integral to in vitro single-molecule experiments.

They cover single-molecule fluorescence methods, microfluidic flow cells to manipulate experimental conditions during experiments, and force spectroscopy techniques such as atomic force and optical traps to manipulate individual molecules.

In addition, two timely original research articles in the same issue describe complementary advances in nanoscale visualization.

All these articles are accessible free of charge for the entire month of June. Check them out!

Posted by Veronique Kiermer at 01:00 PM | Permalink | Comments (0) | TrackBacks (0)

Last December we published an Article by Simon Davis and colleagues challenging the conventional methodology being used in BRET studies that detect dimerization of G protein coupled receptors. The conclusions were contested in a Correspondence by Michel Bouvier and colleagues. In a new Correspondence, a former member of the Bouvier lab further argues against the results and conclusions of the Davis study with experimental results using the 'Type 2' BRET assay of Davis and colleagues.

Continue reading "Receptor dimerization revisited" »

Posted by Daniel Evanko at 04:30 PM | Permalink | Comments (0)

An Article in the June issue of Nature Methods uses simulated data sets to evaluate programs used for metagenomics data analysis. The author of a News & Views argues that although the results indicate existing programs do work, new algorithms are needed as well as model metagenomics systems for use as test beds.

Posted by Daniel Evanko at 02:00 PM | Permalink | Comments (1) | TrackBacks (0)

A Correspondence in the May issue of Nature Methods points to the fact that the widely used human cytomegalovirus promoter shows dramatic differences of expression in various cell types.

The Correspondence focuses on potential problems associated with CMV in the brain in relation to a paper by Wickersham and Callaway who respond to the criticism.

Share your experience with CMV in neurons or other cell types by adding your voice to the discussion.

Posted by Veronique Kiermer at 11:29 AM | Permalink | Comments (3) | TrackBacks (0)

A paper by Takashi Tsuji and colleagues describing a general method to reconstitute ectodermal organs such as teeth and whiskers has met with criticism by members of the community who claim there is nothing substantively new reported. The development and transplantation of engineered teeth was previously reported but the changes described in the new report, though small, do seem to represent an important advance as attested to by others in the field.

Read the paper by Nakao et al. and some of the press coverage listed below and give us your thoughts.

The Scientist - Teeth, whiskers bioengineered
Chemistry World - Researchers sink their teeth in

Posted by Daniel Evanko at 05:15 PM | Permalink | Comments (2) | TrackBacks (0)

Stefan Hell and colleagues propose that the use of pulsed excitation with a delay of at least one microsecond between pulses can dramatically reduce photobleaching and increase the effective fluorescence signal in both single and multi-photon fluorescence microscopy. Is this something you would consider trying in your lab? Do you think the described benefits are worth the necessary equipment changes?

Read the paper here and then add your opinion.

Posted by Daniel Evanko at 04:38 PM | Permalink | Comments (3) | TrackBacks (0)

Using a new quantitative framework for evaluation of receptor dimerization using bioluminescence energy transfer (BRET), Simon Davis and colleagues at the University of Oxford present evidence that the signals arising from BRET measurements, and interpreted as evidence of dimerization, are the result of random interactions and not evidence of stable complexes.

Continue reading "Assessing receptor dimerization by BRET" »

Posted by Daniel Evanko at 03:09 PM | Permalink | Comments (1) | TrackBacks (0)

A group of investigators from various institutions have written a Commentary in which they articulate their experience about caveats of large-scale RNAi screens. These authors propose that the use of standard controls should be adopted across the community of RNAi users to minimize the risk of reporting false positives among screen results.

Far from trying to dictate rules, this Commentary seeks to open a community-wide discussion leading to a consensus of best practices that can realistically be implemented across all experimental settings. Therefore, we welcome your reactions and hope for a constructive dialog for the benefit of all. Please post your comments below.

The urgent need for such debate has been highlighted in two recent papers by Ma et al. in Nature and Kulkarni et al. in Nature Methods. These two studies demonstrate that RNAi screens in the Drosophila melanogaster system—in which long dsRNAs are used as RNAi triggers—are affected by off-target effects to a much wider extent than previously thought.

As far as the use of RNAi in mammalian systems is concerned, off-target effects have been a constant preoccupation. Nature Methods’ recent focus, RNA interference - A user’s guide, contains practical discussion of issues revolving around the use of RNAi as a research tool in mammalian systems, and includes notably a Perspective by Yi Pei and Tom Tuschl on effective siRNA design and a Perspective by Bryan Cullen on experimental design to enhance and confirm specificity. The latter is, however, focused on experimental situations in which one aims at knocking down the expression of a single, specific gene of interest in mammalian cells.

For those involved in genome-wide screens in various model organisms, like the Commentary's authors, the recommended controls may be technically difficult to implement—hence, the need for a discussion of appropriate measures to strike the right balance between practical feasibility and accuracy of published screen results.

Posted by Veronique Kiermer at 12:20 PM | Permalink | Comments (4) | TrackBacks (0)