Sorting and pooling strategy: a novel tool to map a virus proteome for CD8 T-cell epitopes

Human cytomegalovirus (CMV) is a major cause of morbidity in immunocompromised individuals. However, no efficient vaccine has been developed to date. Identification of T-cell target proteins and epitopes is crucial not only for developing a successful immunization strategy, but also for new approaches using adoptive transfer of antigen-specific T-cells. The CMV genome has more than 200 open reading frames potentially coding for as many proteins. Here, we describe a robust, fast, and simple SPOT synthesis strategy, which allowed us to micro-synthesize every possible CD8 T-cell epitope in the entire potential CMV proteome. So far, 9069 of these peptides have been tested in an ex vivo T-cell stimulation assay. As well as confirming a number of previously known epitopes, we identified several new ones.     

Cardiac tissue engineering: regeneration of the wounded heart

New solutions are needed to regenerate hearts damaged by myocardial infarction, to overcome bad prognosis of patients with heart failure, and to address the shortage of heart donors. In the past few years, cardiac tissue engineering has emerged as a new and ambitious approach that combines knowledge from material chemistry with cell biology and medicine. In this short review, we present an overview on the most promising materials and cell-therapy strategies used in the past few years for the regeneration of the wounded heart.     

A dual effect on protein synthesis and degradation modulates the tubulin level in rice cells treated with oryzalin

The effect of the anti-microtubular drug oryzalin on growth and morphology of cultured rice (Oryza sativa L., cv. Roncarolo) cells was evaluated with specific reference to mechanisms that control intracellular tubulin levels. The addition of oryzalin caused a great reduction in the level of both alpha- and beta-tubulin polypeptides, as detected by Western blot analysis. However, no appreciable decrease was observed in the population of total or isotype-specific alpha- and beta-tubulin mRNAs. Only within the first 24 h of the oryzalin treatment, when the level of both alpha- and beta-tubulin polypeptides was still undiminished, was a consistent reduction in the amount of total beta-tubulin mRNA observed. Pulse-chase experiments performed on rice cells grown in the presence of 1 microM oryzalin revealed the presence of two distinct mechanisms that negatively control alpha- and beta-tubulin polypeptide levels. (i) There was an immediate effect on protein synthesis, which resulted in a reduction in the level of newly synthesized tubulin. (ii) There was a delayed response characterized by a substantial degradation of both alpha- and beta-tubulin monomers; this degradation occurred after 24 h of herbicide treatment. The possible involvement of Ca2+ in the degradation of the unincorporated tubulin monomers is also documented and discussed.     

High-throughput generation of sequence indexes from T-DNA mutagenized Arabidopsis thaliana lines

A pipeline has been created for the characterization of Arabidopsis thaliana mutants by generating flanking sequence tags (FSTs) and optimized for economic, high-throughput production. The GABI-Kat collection of T-DNA mutagenized A. thaliana plants was used as a source of independent transgenic lines. The pipeline included robotized extraction of genomic DNA in a 96-well format, an adapter-ligation PCR method for amplification of plant sequences adjacent to T-DNA borders, automated purification and sequencing of PCR products, and computational trimming of the resulting sequence files. Data quality was significantly improved by (i) restriction digestion of the adaptor-ligation products to reduce trivial sequences caused by co-amplification of fragments derived from the free plasmid, and (ii) the design of the adaptor primers for the second amplification step to enhance selective generation of single PCR fragments, even from lines with multiple T-DNA insertions. Gel-purification was avoided by including these steps, the number of amplification reactions per line was reduced from four to three, and the percentage of lines that yielded at least one FST was increased from 66% to 86%. More than 58,000 FSTs have been submitted to GenBank and are available at http://www.mpiz-koeln.mpg.de/GABI-Kat/.     

Membrane trafficking and polar growth in root hairs and pollen tubes

Root hairs and pollen tubes extend by rapid elongation that occurs exclusively at the tip. Fundamental for such local, tip-focused growth (so-called 'tip growth') is the polarization of the cytoplasm that directs secretory events to the tip, and the presence of internal gradients and transmembrane flux of ions, notably Ca2+, H+, K+, and Cl-. Electrophysiological and imaging studies using fluorescent markers have sought to link ion gradients with growth and membrane trafficking. Current models recognize membrane trafficking as fundamental to tip growth, notably its role in supplying lipid and protein to the new plasma membrane and cell wall that extend the apex of the cell, and a complementary role for endocytosis in retrieving excess membrane and in recycling various protein fractions. The current state of knowledge is reviewed here in order to highlight the major gaps in the present understanding of trafficking as it contributes to polar growth in these cells and recent results, that suggest a role for membrane trafficking in the active regulation of ion channel turnover and activity during polar tip growth, are discussed.     

Rapid hit to lead evaluation of pyrazolo[3,4-d]pyrimidin-4-one as selective and orally bioavailable mGluR1 antagonists

Our HTS effort yielded a preferential mGluR1 pyrimidinone antagonist 1 with lead-like characteristics. Rapid hit to lead (HTL) study identified compounds with improved functional activity and selectivity such as 1b with little improvements in ADME properties. Addition of an aminosulfonyl group on the N-1 aromatic ring led to 2f, a compound with similar in vitro biochemical profiles as those of 1b but drastically improved in vitro ADME properties. These improvements were paralleled by rat PK study characterized by low clearance and quantitative bioavailability. Compound 2f represented a true lead-like molecule that is amenable for further lead optimization (LO) evaluation.     

Exploiting single-molecule transcript sequencing for eukaryotic gene prediction

We develop a method to predict and validate gene models using PacBio single-molecule, real-time (SMRT) cDNA reads. Ninety-eight percent of full-insert SMRT reads span complete open reading frames. Gene model validation using SMRT reads is developed as automated process. Optimized training and prediction settings and mRNA-seq noise reduction of assisting Illumina reads results in increased gene prediction sensitivity and precision. Additionally, we present an improved gene set for sugar beet (Beta vulgaris) and the first genome-wide gene set for spinach (Spinacia oleracea). The workflow and guidelines are a valuable resource to obtain comprehensive gene sets for newly sequenced genomes of non-model eukaryotes.

Electronic supplementary material

The online version of this article (doi:10.1186/s13059-015-0729-7) contains supplementary material, which is available to authorized users.     

Chroman and tetrahydroquinoline ureas as potent TRPV1 antagonists

Novel chroman and tetrahydroquinoline ureas were synthesized and evaluated for their activity as TRPV1 antagonists. It was found that aryl substituents on the 7- or 8-position of both bicyclic scaffolds imparted the best in vitro potency at TRPV1. The most potent chroman ureas were assessed in chronic and acute pain models, and compounds with the ability to cross the blood-brain barrier were shown to be highly efficacious. The tetrahydroquinoline ureas were found to be potent CYP3A4 inhibitors, but replacement of bulky substituents at the nitrogen atom of the tetrahydroisoquinoline moiety with small groups such as methyl can minimize the inhibition.     

Plasmodium falciparum: differential selection of drug resistance alleles in contiguous urban and peri-urban areas of Brazzaville, Republic of Congo

The African continent is currently experiencing rapid population growth, with rising urbanization increasing the percentage of the population living in large towns and cities. We studied the impact of the degree of urbanization on the population genetics of Plasmodium falciparum in urban and peri-urban areas in and around the city of Brazzaville, Republic of Congo. This field setting, which incorporates local health centers situated in areas of varying urbanization, is of interest as it allows the characterization of malaria parasites from areas where the human, parasite, and mosquito populations are shared, but where differences in the degree of urbanization (leading to dramatic differences in transmission intensity) cause the pattern of malaria transmission to differ greatly. We have investigated how these differences in transmission intensity affect parasite genetic diversity, including the amount of genetic polymorphism in each area, the degree of linkage disequilibrium within the populations, and the prevalence and frequency of drug resistance markers. To determine parasite population structure, heterozygosity and linkage disequilibrium, we typed eight microsatellite markers and performed haplotype analysis of the msp1 gene by PCR. Mutations known to be associated with resistance to the antimalarial drugs chloroquine and pyrimethamine were determined by sequencing the relevant portions of the crt and dhfr genes, respectively. We found that parasite genetic diversity was comparable between the two sites, with high levels of polymorphism being maintained in both areas despite dramatic differences in transmission intensity. Crucially, we found that the frequencies of genetic markers of drug resistance against pyrimethamine and chloroquine differed significantly between the sites, indicative of differing selection pressures in the two areas.