In-cell PCR from mRNA: amplifying and linking the rearranged immunoglobulin heavy and light chain V-genes within single cells.

We describe a process for the identification of mRNAs within single cells, as demonstrated with the immunoglobulin (Ig) variable region (V) genes of two mouse hybridoma cell lines and the bcr-abl fusion gene of the human K562 myeloid leukaemia line. The cells were fixed and permeabilised, the mRNA reverse transcribed to cDNA and the cDNA amplified by the polymerase chain reaction (PCR). After using fluorescent PCR primers, the amplified DNA could be detected within the cells as demonstrated by confocal fluorescence microscopy and flow cytometry. Furthermore the amplified Ig VH and VL DNA could be assembled within the same cell using suitable PCR primers. We detected no cross-contamination of amplified DNA between cells: the DNA isolated from mixtures of two hybridoma cell lines (B1-8 and NQ10/12.5) treated to in-cell PCR and assembly, was shown by cloning to correspond to the combinations of VH and VL genes of the parent hybridomas. We forsee diverse applications of in-cell assembly by PCR, especially for the analysis of the combinations of chains of rearranged Ig or T cell receptor (TCR) V-genes in a population of cells, and the construction of human antibodies from the V-genes of immune B-lymphocytes.     

Coding characters from different life stages for phylogenetic reconstruction: a case study on dragonfly adults and larvae,including a description of the larval head anatomy of Epiophlebia superstes (Odonata: Epiophlebiidae)

The exclusive use of characters coding for specific life stages may bias tree reconstruction. If characters from several life stages are coded, the type of coding becomes important. Here, we simulate the influence on tree reconstruction of morphological characters of Odonata larvae incorporated into a data matrix based on the adult body under different coding schemes. For testing purposes, our analysis is focused on a well‐supported hypothesis: the relationships of the suborders Zygoptera, ‘Anisozygoptera’, and Anisoptera. We studied the cephalic morphology of Epiophlebia, a key taxon among Odonata, and compared it with representatives of Zygoptera and Anisoptera in order to complement the data matrix. Odonate larvae are characterized by a peculiar morphology, such as the specific head form, mouthpart configuration, ridge configuration, cephalic musculature, and leg and gill morphology. Four coding strategies were used to incorporate the larval data: artificial coding (AC), treating larvae as independent terminal taxa; non‐multistate coding (NMC), preferring the adult life stage; multistate coding (MC); and coding larval and adult characters separately (SC) within the same taxon. As expected, larvae are ‘monophyletic’ in the AC strategy, but with anisopteran and zygopteran larvae as sister groups. Excluding larvae in the NMC approach leads to strong support for both monophyletic Odonata and Epiprocta, whereas MC erodes phylogenetic signal completely. This is an obvious result of the larval morphology leading to many multistate characters. SC results in the strongest support for Odonata, and Epiprocta receives the same support as with NMC. Our results show the deleterious effects of larval morphology on tree reconstruction when multistate coding is applied. Coding larval characters separately is still the best approach in a phylogenetic framework. © 2015 The Linnean Society of London     

Importance of rare taxa for bacterial diversity in the rhizosphere of Bt- and conventional maize varieties

Ribosomal 16S rRNA gene pyrosequencing was used to explore whether the genetically modified (GM) Bt-maize hybrid MON 89034 × MON 88017, expressing three insecticidal recombinant Cry proteins of Bacillus thuringiensis, would alter the rhizosphere bacterial community. Fine roots of field cultivated Bt-maize and three conventional maize varieties were analyzed together with coarse roots of the Bt-maize. A total of 547 000 sequences were obtained. Library coverage was 100% at the phylum and 99.8% at the genus rank. Although cluster analyses based on relative abundances indicated no differences at higher taxonomic ranks, genera abundances pointed to variety specific differences. Genera-based clustering depended solely on the 49 most dominant genera while the remaining 461 rare genera followed a different selection. A total of 91 genera responded significantly to the different root environments. As a benefit of pyrosequencing, 79 responsive genera were identified that might have been overlooked with conventional cloning sequencing approaches owing to their rareness. There was no indication of bacterial alterations in the rhizosphere of the Bt-maize beyond differences found between conventional varieties. B. thuringiensis-like phylotypes were present at low abundance (0.1% of Bacteria) suggesting possible occurrence of natural Cry proteins in the rhizospheres. Although some genera indicated potential phytopathogenic bacteria in the rhizosphere, their abundances were not significantly different between conventional varieties and Bt-maize. With an unprecedented sensitivity this study indicates that the rhizosphere bacterial community of a GM maize did not respond abnormally to the presence of three insecticidal proteins in the root tissue.     

Temperature increase and fluctuation induce phytoplankton biodiversity loss – Evidence from a multi‐seasonal mesocosm experiment

Global climate change scenarios predict lake water temperatures to increase up to 4°C and extreme weather events, including heat waves and large temperature fluctuations, to occur more frequently. Such changes may result in a reorganization of the plankton community structure, causing shifts in diversity and structure toward a community dominated by fewer species that are more adapted to endure warmer and irregular temperature conditions. We designed a long‐term (8 months) mesocosm experiment to explore how ambient water temperature (C: control), induced increased temperature (T: +4°C), and temperature fluctuations (F: ±4°C relative to T) change phytoplankton phenology, taxonomical diversity, and community structure, and how such changes affected zooplankton abundance and composition. Synthesis. Our results show that T and F relative to C significantly decreased phytoplankton diversity. Moreover, there was a clear effect of the temperature treatments (T and F) on phytoplankton size structure that resulted in a significantly lower growth of large species (i.e., large Chlorophyta) compared to C. Decreased diversity and evenness in the T and F treatments pushed the community toward the dominance of only a few phytoplankton taxa (mainly Cyanobacteria and Chlorophyta) that are better adapted to endure warmer and more irregular temperature conditions. The observed shift toward Cyanobacteria dominance may affect trophic energy transfer along the aquatic food web.     

16S rRNA gene-based oligonucleotide microarray for environmental monitoring of the betaproteobacterial order "Rhodocyclales"

For simultaneous identification of members of the betaproteobacterial order "Rhodocyclales" in environmental samples, a 16S rRNA gene-targeted oligonucleotide microarray (RHC-PhyloChip) consisting of 79 probes was developed. Probe design was based on phylogenetic analysis of available 16S rRNA sequences from all cultured and as yet uncultured members of the "Rhodocyclales." The multiple nested probe set was evaluated for microarray hybridization with 16S rRNA gene PCR amplicons from 29 reference organisms. Subsequently, the RHC-PhyloChip was successfully used for cultivation-independent "Rhodocyclales" diversity analysis in activated sludge from an industrial wastewater treatment plant. The implementation of a newly designed "Rhodocyclales"-selective PCR amplification system prior to microarray hybridization greatly enhanced the sensitivity of the RHC-PhyloChip and thus enabled the detection of "Rhodocyclales" populations with relative abundances of less than 1% of all bacteria (as determined by fluorescence in situ hybridization) in the activated sludge. The presence of as yet uncultured Zoogloea-, Ferribacterium/Dechloromonas-, and Sterolibacterium-related bacteria in the industrial activated sludge, as indicated by the RHC-PhyloChip analysis, was confirmed by retrieval of their 16S rRNA gene sequences and subsequent phylogenetic analysis, demonstrating the suitability of the RHC-PhyloChip as a novel monitoring tool for environmental microbiology.     

Recognition of 5-hydroxymethylcytosine by the Uhrf1 SRA domain

Recent discovery of 5-hydroxymethylcytosine (5hmC) in genomic DNA raises the question how this sixth base is recognized by cellular proteins. In contrast to the methyl-CpG binding domain (MBD) of MeCP2, we found that the SRA domain of Uhrf1, an essential factor in DNA maintenance methylation, binds 5hmC and 5-methylcytosine containing substrates with similar affinity. Based on the co-crystal structure, we performed molecular dynamics simulations of the SRA:DNA complex with the flipped cytosine base carrying either of these epigenetic modifications. Our data indicate that the SRA binding pocket can accommodate 5hmC and stabilizes the flipped base by hydrogen bond formation with the hydroxyl group.     

Landscape simplification increases vineyard pest outbreaks and insecticide use

Diversifying agricultural landscapes may mitigate biodiversity declines and improve pest management. Yet landscapes are rarely managed to suppress pests, in part because researchers seldom measure key variables related to pest outbreaks and insecticides that drive management decisions. We used a 13‐year government database to analyse landscape effects on European grapevine moth (Lobesia botrana) outbreaks and insecticides across c. 400 Spanish vineyards. At harvest, we found pest outbreaks increased four‐fold in simplified, vineyard‐dominated landscapes compared to complex landscapes in which vineyards are surrounded by semi‐natural habitats. Similarly, insecticide applications doubled in vineyard‐dominated landscapes but declined in vineyards surrounded by shrubland. Importantly, pest population stochasticity would have masked these large effects if numbers of study sites and years were reduced to typical levels in landscape pest‐control studies. Our results suggest increasing landscape complexity may mitigate pest populations and insecticide applications. Habitat conservation represents an economically and environmentally sound approach for achieving sustainable grape production.     

Tapasin and other chaperones: models of the MHC class I loading complex

MHC (major histocompatibility complex) class I molecules bind intracellular virus-derived peptides in the endoplasmic reticulum (ER) and present them at the cell surface to cytotoxic T lymphocytes. Peptide-free class I molecules at the cell surface, however, could lead to aberrant T cell killing. Therefore, cells ensure that class I molecules bind high-affinity ligand peptides in the ER, and restrict the export of empty class I molecules to the Golgi apparatus. For both of these safeguard mechanisms, the MHC class I loading complex (which consists of the peptide transporter TAP, the chaperones tapasin and calreticulin, and the protein disulfide isomerase ERp57) plays a central role. This article reviews the actions of accessory proteins in the biogenesis of class I molecules, specifically the functions of the loading complex in high-affinity peptide binding and localization of class I molecules, and the known connections between these two regulatory mechanisms. It introduces new models for the mode of action of tapasin, the role of the class I loading complex in peptide editing, and the intracellular localization of class I molecules.     

Rapid parasite adaptation drives selection for high recombination rates

The Red Queen hypothesis proposes that sex is maintained through selection pressure imposed by coevolving parasites: susceptible hosts are able to escape parasite pressure by recombining their genome to create resistant offspring. However, previous theoretical studies have shown that the Red Queen typically selects against sex unless selection is strong, arguing that high rates of recombination cannot evolve when parasites are of low virulence. Here we show that under the biologically plausible assumption of a severe fitness cost for parasites that fail to infect, the Red Queen can cause selection for high recombination rates, and that the strength of virulence is largely irrelevant to the direction of selection for increased recombination rates. Strong selection on parasites and short generation times make parasites usually better adapted to their hosts than vice versa and can thus favor higher recombination rates in hosts. By demonstrating the importance of host-imposed selection on parasites, our findings resolve previously reported conflicting results.