Genome organization and DNA accessibility control antigenic variation in trypanosomes.

Many evolutionarily distant pathogenic organisms have evolved similar survival strategies to evade the immune responses of their hosts. These include antigenic variation, through which an infecting organism prevents clearance by periodically altering the identity of proteins that are visible to the immune system of the host1. Antigenic variation requires large reservoirs of immunologically diverse antigen genes, which are often generated through homologous recombination, as well as mechanisms to ensure the expression of one or very few antigens at any given time. Both homologous recombination and gene expression are affected by three-dimensional genome architecture and local DNA accessibility2,3. Factors that link three-dimensional genome architecture, local chromatin conformation and antigenic variation have, to our knowledge, not yet been identified in any organism. One of the major obstacles to studying the role of genome architecture in antigenic variation has been the highly repetitive nature and heterozygosity of antigen-gene arrays, which has precluded complete genome assembly in many pathogens. Here we report the de novo haplotype-specific assembly and scaffolding of the long antigen-gene arrays of the model protozoan parasite Trypanosoma brucei, using long-read sequencing technology and conserved features of chromosome folding4. Genome-wide chromosome conformation capture (Hi-C) reveals a distinct partitioning of the genome, with antigen-encoding subtelomeric regions that are folded into distinct, highly compact compartments. In addition, we performed a range of analyses-Hi-C, fluorescence in situ hybridization, assays for transposase-accessible chromatin using sequencing and single-cell RNA sequencing-that showed that deletion of the histone variants H3.V and H4.V increases antigen-gene clustering, DNA accessibility across sites of antigen expression and switching of the expressed antigen isoform, via homologous recombination. Our analyses identify histone variants as a molecular link between global genome architecture, local chromatin conformation and antigenic variation.

GT-rich promoters can drive RNA pol II transcription and deposition of H2A.Z in African trypanosomes.

Genome-wide transcription studies are revealing an increasing number of "dispersed promoters" that, unlike "focused promoters", lack well-conserved sequence motifs and tight regulation. Dispersed promoters are nevertheless marked by well-defined chromatin structures, suggesting that specific sequence elements must exist in these unregulated promoters. Here, we have analyzed regions of transcription initiation in the eukaryotic parasite Trypanosoma brucei, in which RNA polymerase II transcription initiation occurs over broad regions without distinct promoter motifs and lacks regulation. Using a combination of site-specific and genome-wide assays, we identified GT-rich promoters that can drive transcription and promote the targeted deposition of the histone variant H2A.Z in a genomic context-dependent manner. In addition, upon mapping nucleosome occupancy at high resolution, we find nucleosome positioning to correlate with RNA pol II enrichment and gene expression, pointing to a role in RNA maturation. Nucleosome positioning may thus represent a previously unrecognized layer of gene regulation in trypanosomes. Our findings show that even highly dispersed, unregulated promoters contain specific DNA elements that are able to induce transcription and changes in chromatin structure.

Exploiting CRISPR-Cas9 technology to investigate individual histone modifications.

Despite their importance for most DNA-templated processes, the function of individual histone modifications has remained largely unknown because in vivo mutational analyses are lacking. The reason for this is that histone genes are encoded by multigene families and that tools to simultaneously edit multiple genomic loci with high efficiency are only now becoming available. To overcome these challenges, we have taken advantage of the power of CRISPR-Cas9 for precise genome editing and of the fact that most DNA repair in the protozoan parasite Trypanosoma brucei occurs via homologous recombination. By establishing an episome-based CRISPR-Cas9 system for T. brucei, we have edited wild type cells without inserting selectable markers, inserted a GFP tag between an ORF and its 3'UTR, deleted both alleles of a gene in a single transfection, and performed precise editing of genes that exist in multicopy arrays, replacing histone H4K4 with H4R4 in the absence of detectable off-target effects. The newly established genome editing toolbox allows for the generation of precise mutants without needing to change other regions of the genome, opening up opportunities to study the role of individual histone modifications, catalytic sites of enzymes or the regulatory potential of UTRs in their endogenous environments.

Resistance of thermophilic spore formers isolated from milk and whey products towards cleaning-in-place conditions: Influence of pH, temperature and milk residues.

The occurrence of thermophilic spore formers in dairy powders is a major concern for producers worldwide. This study aims to investigate the resistance of thermophilic endospores towards cleaning solutions typically used for cleaning-in-place in dairy manufacturing plants. From eleven tested strains, all were able to survive an alkaline treatment (NaOH) at 65 °C for 10 min (0.5%), whereas at concentrations of 2% eight strains withstood the treatment. Acid solutions were more sporicidal. At 0.5% of HNO3, only three strains survived the treatment. Milk impurities reduced the inactivation effect of the NaOH solutions towards thermophilic spore formers. For two selected strains, a detailed kinetic inactivation in NaOH and HNO3 solutions at different temperatures was performed and non-log-linear inactivation curves were observed. This study highlights the risk of reusing cleaning solutions in dairies.

Genome-wide analysis of chromatin structures in Trypanosoma brucei using high-resolution MNase-ChIP-seq.

Specific DNA-protein interactions are the basis for many important cellular mechanisms like the regulation of gene expression or replication. Knowledge about the precise genomic locations of DNA-protein interactions is important because it provides insight into the regulation of these processes. Recently, we have adapted an approach that combines micrococcal nuclease (MNase) digestion of chromatin with chromatin immunoprecipitation in Trypanosoma brucei. Here, we describe in detail how this method can be used to map the genome-wide distribution of nucleosomes or other DNA-binding proteins at high resolution in T. brucei.

[Quality assurance in tissue biobanking-an overview]. / Qualitätssicherung im Gewebebiobanking ­ Ein Überblick.

Tissue biobanks are important resource and technology platforms for biomedical research, which deals with molecular pathogenesis and the prevention, diagnosis and treatment of diseases.Due to this central role in the standardised collection, storage and distribution of human tissue and its derivatives, a practised quality management is one of the most important measures to achieve and maintain a comprehensive quality assurance of all biobanking processes. At the same time, this promotes acceptance and credibility. External quality assurance of biobanks can be achieved through accreditation. Within the German biobanking community, increasing harmonisation of biobanking processes has also been achieved through the provision of various quality assurance measures by the German Biobank Node (GBN).In the following, challenges and opportunities in the implementation of a comprehensive quality assurance in biobanking will be discussed and solutions for tissue biobanking will be presented using the example of the tissue bank of the National Centre for Tumour Diseases (NCT).

High counts of thermophilic spore formers in dairy powders originate from persisting strains in processing lines.

During the last decades, thermophilic spore counts became a very important quality parameter for manufacturers with regard to powdered dairy products. Low-spore count powders are highly demanded but challenging to produce when high production volume and long process times are intended. In this study a detailed monitoring of microbial levels in three skim milk powder plants was conducted. Anoxybacillus flavithermus was found to be primarily responsible for increased spore levels with increasing spore numbers being detected after 6-8 h already during initial processing steps. Simultaneously, the species composition shifted from a diverse bulk tank milk microbiota where different Bacillus species represented around 90% of the thermophilic bacteria to a dominance of A. flavithermus in the end product. The analysis of A. flavithermus isolates from different powder batches with RAPD PCR revealed recurring patterns in each of the eight German manufacturers sampled over several months. The high relatedness of isolates exhibiting identical RAPD patterns was exemplified by cgMLST based on whole genome sequences. We assume that A. flavithermus strains persisted in production plants and were not eliminated by cleaning. It is concluded that such persisting strains recurrently recontaminated subsequent powder productions. The data highlight that a targeted optimization of cleaning and disinfection procedures is the most promising measure to effectively reduce thermophilic spore counts in German dairy powders.

Thermal treatment of skim milk concentrates in a novel shear-heating device: Reduction of thermophilic spores and physical properties.

Endospores of thermophilic bacilli are a major concern for producers of dairy powders. In this study, we heat treated 10 different spore suspensions at 110 °C in skim milk and skim milk concentrate (36% dry matter) of the species Geobacillus stearothermophilus (10 min) and Anoxybacillus flavithermus (5 min) in a new shear-heating device. The highest log reduction in skim milk concentrate was 3.5. The death behavior of the spores was strain dependent. Particle formation and Maillard reaction were observed. By increasing the shear-rate up to 1500 s-1 the particle size was reduced for both heating times (D90 reduction: 57.4 and 77.0%, respectively). The particle size was lessened by a reduction of dry matter of 27%, compared to 36%. This work emphasizes, that heat treatment of concentrated dairy products represents a technological option to reduce thermophilic spores in skim milk concentrate and powders produced thereof.