Search

Nucleosome repositioning in cancer highlights changes of many aspects of genome organisation and regulation. Understanding it is important both from the fundamental point of view and for medical diagnostics based on cell-free DNA (cfDNA), which originates from genomic DNA regions protected from digestion by nucleosomes. Here we generated ultra-high resolution nucleosome maps in paired tumour and normal tissues from the same breast cancer patients using MNase-assisted histone H3 ChIP-seq and compared them with the corresponding cfDNA from blood plasma. We detected cancer-specific single-nucleosome repositioning at key regulatory regions in a patient-specific manner, as well as common patterns across patients. The nucleosomes gained in tumour versus normal tissue were particularly informative of cancer pathways, with 20-fold enrichment at CpG islands, a large fraction of which marked promoters of genes encoding DNA-binding proteins. Tumour tissues were characterised by 5-10 bp decrease in average distances between nucleosomes (nucleosome repeat length, NRL). These effects were modulated by GC content and DNA sequence repeats and correlated with differential DNA methylation and binding of linker histone variants H1.4 and H1X. Our findings provide missing mechanistic understanding of nucleosome positioning in tumour tissues and can be valuable for nucleosomics analyses in liquid biopsies.

The Antarctic krill (Euphausia superba Dana) is a keystone species in the Southern Ocean that uses an arsenal of hydrolases for biomacromolecule decomposition to effectively digest its omnivorous diet. The present study builds on a hybrid-assembled transcriptome (13,671 ORFs) combined with comprehensive proteome profiling. The analysis of individual krill compartments allowed detection of significantly more different proteins compared to that of the entire animal (1464 vs. 294 proteins). The nearby krill sampling stations in the Bransfield Strait (Antarctic Peninsula) yielded rather uniform proteome datasets. Proteins related to energy production and lipid degradation were particularly abundant in the abdomen, agreeing with the high energy demand of muscle tissue. A total of 378 different biomacromolecule hydrolysing enzymes were detected, including 250 proteases, 99 CAZymes, 14 nucleases and 15 lipases. The large repertoire in proteases is in accord with the protein-rich diet affiliatedwith E. superba’s omnivorous lifestyle and complex biology. The richness in chitin-degrading enzymes allows not only digestion of zooplankton diet, but also the utilisation of the discharged exoskeleton after moulting.

The increasing incidence of bloodstream infections including sepsis is a major challenge in intensive care units worldwide. However, current diagnostics for pathogen identification mainly depend on culture- and molecular-based approaches, which are not satisfactory regarding specificity, sensitivity, and time to diagnosis. Herein, we established a complete diagnostic workflow for real-time high-throughput sequencing of cell-free DNA from plasma based on nanopore sequencing for the detection of the causative agents, which was applied to the analyses of eight samples from four septic patients and three healthy controls, and subsequently validated against standard next-generation sequencing results. By optimization of library preparation protocols for short fragments with low input amounts, a 3.5-fold increase in sequencing throughput could be achieved. With tailored bioinformatics workflows, all eight septic patient samples were found to be positive for relevant pathogens. When considering time to diagnosis, pathogens were identified within minutes after start of sequencing. Moreover, an extrapolation of real-time sequencing performance on a cohort of 239 septic patient samples revealed that more than 90% of pathogen hits would have also been detected using the optimized MinION workflow. Reliable identification of pathogens based on circulating cell-free DNA sequencing using optimized workflows and real-time nanopore-based sequencing can be accomplished within 5 to 6 hours following blood draw. Therefore, this approach might provide therapy-relevant results in a clinically critical timeframe.

Simple Summary Gastrointestinal (GI) cancers are a common cancer, affecting both men and women, normally diagnosed through tissue biopsies in combination with imaging techniques and standardized biomarkers leading to patient selection for local or systemic therapies. Liquid biopsies (LBs)—due to their non-invasive nature as well as low risk—are the current focus of cancer research and could be a promising tool for early cancer detection and treatment surveillance, thus leading to better patient outcomes. In this review, we provide an overview of different types of LBs enabling early detection and monitoring of GI cancers and their clinical application. Abstract Worldwide, gastrointestinal (GI) cancers account for a significant amount of cancer-related mortality. Tests that allow an early diagnosis could lead to an improvement in patient survival. Liquid biopsies (LBs) due to their non-invasive nature as well as low risk are the current focus of cancer research and could be a promising tool for early cancer detection. LB involves the sampling of any biological fluid (e.g., blood, urine, saliva) to enrich and analyze the tumor’s biological material. LBs can detect tumor-associated components such as circulating tumor DNA (ctDNA), extracellular vesicles (EVs), and circulating tumor cells (CTCs). These components can reflect the status of the disease and can facilitate clinical decisions. LBs offer a unique and new way to assess cancers at all stages of treatment, from cancer screenings to prognosis to management of multidisciplinary therapies. In this review, we will provide insights into the current status of the various types of LBs enabling early detection and monitoring of GI cancers and their use in in vitro diagnostics.

Despite antifungal prophylaxis following liver transplantation (LTX), patients are at risk for the development of subsequent opportunistic infections, such as an invasive fungal disease (IFD). However, culture-based diagnostic procedures are associated with relevant weaknesses. Culture and next-generation sequencing (NGS)-based fungal findings as well as corresponding plasma levels of s-D-glucan (BDG), galactomannan (GM), interferon gamma (IFN-γ), tumor necrosis factor alpha (TNF-α), interleukin (IL)-2, -4, -6, -10, -17A and mid-regional proadrenomedullin (MR-proADM) were evaluated in 93 patients at 6 consecutive time points within 28 days following LTX. A NGS-based diagnostic approach was shown to be suitable for the early identification of fungal pathogens in patients following LTX. Moreover, MR-proADM and IL-17A in plasma proved suitable for the identification of patients with an IFD. Plasma measurements of MR-proADM and IL-17A as well as a NGS-based diagnostic approach were shown to be attractive methodologies to attenuate the weaknesses of routinely used culture-based diagnostic procedures for the determination of an IFD in patients following LTX. However, an additional confirmation within a larger multicenter trial needs to be recommended. German Clinical Trials Register: DRKS00005480 .

The Antarctic krill (Euphausia superba Dana) is a keystone species in the Southern Ocean that uses an arsenal of hydrolases for biomacromolecule decomposition to effectively digest its omnivorous diet. The present study builds on a hybrid-assembled transcriptome (13,671 ORFs) combined with comprehensive proteome profiling. The analysis of individual krill compartments allowed detection of significantly more different proteins compared to that of the entire animal (1,464 vs. 294 proteins). The nearby krill sampling stations in the Bransfield Strait (Antarctic Peninsula) yielded rather uniform proteome datasets. Proteins related to energy production and lipid degradation were particularly abundant in the abdomen, agreeing with the high energy demand of muscle tissue. A total of 378 different biomacromolecule hydrolysing enzymes were detected, including 250 proteases, 99 CAZymes, 14 nucleases and 15 lipases. The large repertoire in proteases is in accord with the protein-rich diet affiliated with E. superba’s omnivorous lifestyle and complex biology. The richness in chitin-degrading enzymes allows not only digestion of zooplankton diet, but also the utilization of the discharged exoskeleton after moulting.

The importance of viral infections as a leading cause of morbidity and mortality is well documented in severely immunosuppressed patients undergoing allogeneic stem cell transplantation. By contrast, viral infections generally receive less attention in patients with malignant disorders undergoing chemotherapy, where the onset of neutropenic fever is mostly associated with bacterial or fungal infections, and screening for viral infections is not routinely performed. To address the occurrence of invasive viral infections in a clinical setting commonly associated with less pronounced immunosuppression, we have prospectively screened 237 febrile neutropenic episodes in pediatric (n = 77) and adult (n = 69) patients undergoing intensive chemotherapy, primarily for treatment of acute leukemia. Serial peripheral blood specimens were tested by RQ-PCR assays for the presence and quantity of the clinically relevant viruses CMV, EBV, HHV6 and HAdV, commonly reactivated in highly immunocompromised patients. Viremia was documented in 36 (15%) episodes investigated, including the detection of HHV6 (n = 14), EBV (n = 15), CMV (n = 6), or HAdV (n = 1). While low or intermediate levels of viremia (104 copies/mL) was documented in patients without evidence for other infections, raising the possibility that at least in some instances the onset of fever may have been attributable to the virus detected. The observations suggest that viral infections, potentially resulting from reactivation, might also play a clinically relevant role in patients receiving chemotherapy for treatment of malignant neoplasms, and routine screening for viremia in this clinical setting might be warranted.

INTRODUCTION: Sepsis and septic shock are the most severe forms of infection affecting predominantly elderly people, preterm and term neonates, and young infants. Even in high-income countries sepsis causes about 8% of admissions to pediatric intensive care units (PICUs). Early diagnosis, rapid anti-infective treatment, and prompt hemodynamic stabilization are crucial for patient survival. In this context, it is essential to identify the causative pathogen as soon as possible to optimize antimicrobial treatment. To date, culture-based diagnostic procedures (e.g., blood cultures) represent the standard of care. However, they have 2 major problems: on the one hand, in the case of very small sample volumes (and thus usually in children), they are not sufficiently sensitive. On the other hand, with a time-to-result of 2 to 5 days, blood cultures need a relatively long time for the anti-infective therapy to be calculated. To overcome these problems, culture-independent molecular diagnostic procedures such as unbiased sequence analysis of circulating cell-free DNA (cfDNA) from plasma samples of septic patients by next-generation sequencing (NGS) have been tested successfully in adult septic patients. However, these results still need to be transferred to the pediatric setting. METHODS: The Next GeneSiPS-Trial is a prospective, observational, non-interventional, multicenter study used to assess the diagnostic performance of an NGS-based approach for the identification of causative pathogens in (preterm and term) neonates (d1-d28, n = 50), infants (d29 to

Andreprevotia sp. strain IGB-42 is a chitin-degrading bacterium isolated from the soil of an anthill. The genome contains 4.7 Mb, a G+C content of 61.31%, 4,257 predicted open reading frames, and a set of industrially interesting chitinase genes.

Cytochrome P450 mono-oxygenases (P450) are versatile enzymes which play essential roles in C-source assimilation, secondary metabolism, and in degradations of endo- and exogenous xenobiotics. In humans, several P450 isoforms constitute the largest part of phase I metabolizing enzymes and catalyze oxidation reactions which convert lipophilic xenobiotics, including drugs, to more water soluble species. Recombinant human P450s and microorganisms are applied in the pharmaceutical industry for the synthesis of drug metabolites for pharmacokinetics and toxicity studies. Compared to the membrane-bound eukaryotic P450s, prokaryotic ones exhibit some advantageous features, such as high stability and generally easier heterologous expression. Here, we describe a novel P450 from Streptomyces platensis DSM 40041 classified as CYP107L that efficiently converts several commercial drugs of various size and properties. This P450 was identified by screening of actinobacterial strains for amodiaquine and ritonavir metabolizing activities, followed by genome sequencing and expression of the annotated S. platensis P450s in Escherichia coli. Performance of CYP107L in biotransformations of amodiaquine, ritonavir, amitriptyline, and thioridazine resembles activities of the main human metabolizing P450s, namely CYPs 3A4, 2C8, 2C19, and 2D6. For application in the pharmaceutical industry, an E. coli whole-cell biocatalyst expressing CYP107L was developed and evaluated for preparative amodiaquine metabolite production.

This study aimed to uncover microbial dynamics and transcriptional adaptations during mesophilic AD of maize silage and slurry. While one digester performed under optimal conditions, the investigations also evaluated the microbiome during a temperature drop mediated process failure accompanied by acidification and how it contributed to a process recovery. Composition and pathway activities were analyzed by whole genome shotgun (WGS) and metatranscriptome sequencing, respectively. A biodiversity of 112 species was observed with noticeable shifts over process time. Although four distinct groups of microbes could be identified with a correlating versatility according to substrate and to process disturbance, also tremendous effects on gene expression were monitored especially of the archaeal methane metabolism. Particularly, the expression of acetogenotrophic methanogenesis related genes was identified to be relevant for process regeneration.

Members of the Cysteine-rich secretory protein, Antigen 5 and Pathogenesis-related 1 (CAP) protein superfamily are important virulence factors in fungi but remain poorly characterized on molecular level. Here, we investigate the cellular localization and molecular function of Rbe1p and Rbt4p, two CAP family members from the human pathogen Candida albicans. We unexpectedly found that Rbe1p localizes to budding sites of yeast cells in a disulfide bond-dependent manner. Furthermore, we show that Rbe1p and Rbt4p bind free cholesterol in vitro and export cholesteryl acetate in vivo. These findings suggest a previously undescribed role for Rbe1p in cell wall- associated processes and a possible connection between the virulence attributes of fungal CAP proteins and sterol binding.

Although various strategies exist for chronic constipation therapy, the pathogenesis of chronic constipation is still not completely understood. The aim of this exploratory experimental study is to elucidate alterations of the autonomous enteric nervous system at the molecular level in patients with obstructed defecation, who represent one of the most predominant groups of constipated patients. Full-thickness rectal wall samples of patients with obstructed defecation were analyzed and compared with controls. Differential gene expression analyses by RNA-Seq transcriptome profiling were performed and gene expression profiles were assigned to gene ontology pathways by application of different biological libraries. Analysis of the transcriptome showed that genes associated with the enteric nervous system functions were significantly downregulated in patients with obstructed defecation. These affected functions included developmental processes and synaptic transmission. Our results therefore indicate that obstructed defecation may represent an enteric neuropathy, comparable to Hirschsprung disease and slow-transit constipation.

Background Biogas production is an attractive technology for a sustainable generation of renewable energy. Although the microbial community is fundamental for such production, the process control is still limited to technological and chemical parameters. Currently, most of the efforts on microbial management system (MiMaS) are focused on process-specific marker species and community dynamics, but a practical implementation is in its infancy. The high number of unknown and uncharacterized microorganisms in general is one of the reasons hindering further advancements. Results A Biogas Metagenomics Hybrid Assembly (BioMETHA) database, derived from microbiomes of biogas plants, was generated using a dedicated assembly strategy for different metagenomic datasets. Long reads from nanopore sequencing (MinION) were combined with short, more accurate second-generation sequencing reads (Illumina). The hybrid assembly resulted in 231 genomic bins each representing a taxonomic unit with an average completeness of 47%. Functional annotation identified 13,190 non-redundant genes covering roughly 207 k coding sequences. Mapping rates of metagenomics DNA derived from diverse biogas plants and laboratory reactors increased up to 73%. In addition, an EC (enzyme commission) reference sequence collection (ERSC) was generated whose genes are crucial for biogas-related processes, consisting of 235 unique EC numbers organized in 52 metabolic modules. Mapping rates of metatranscriptomic data to this ERSC revealed coverages of up to 93%. Process parameters and imbalances of laboratory reactors could be reconstructed by evaluating abundance of biogas-specific metabolic modules using metatranscriptomic data derived from various fermenter systems. Conclusion This newly established metagenomic hybrid assembly in combination with an EC reference sequence collection might help to shed light on the microbial dark matter of biogas plants by contributing to the development of a reference for biogas plant microbiome-specific gene sequences. Considering a biogas microbiome as a complex meta-organism expressing a meta-transcriptome, the approach established here could lay the foundation for a function-based microbial management system.

Jatropha curcas, a multipurpose plant attracting a great deal of attention due to its high oil content and quality for biofuel, is recognized as a drought-tolerant species. However, this drought tolerance is still poorly characterized. This study aims to contribute to uncover the molecular background of this tolerance, using a combined approach of transcriptional profiling and morphophysiological characterization during a period of water-withholding (49 d) followed by rewatering (7 d). Morphophysiological measurements showed that J. curcas plants present different adaptation strategies to withstand moderate and severe drought. Therefore, RNA sequencing was performed for samples collected under moderate and severe stress followed by rewatering, for both roots and leaves. Jatropha curcas transcriptomic analysis revealed shoot- and root-specific adaptations across all investigated conditions, except under severe stress, when the dramatic transcriptomic reorganization at the root and shoot level surpassed organ specificity. These changes in gene expression were clearly shown by the down-regulation of genes involved in growth and water uptake, and up-regulation of genes related to osmotic adjustments and cellular homeostasis. However, organ-specific gene variations were also detected, such as strong up-regulation of abscisic acid synthesis in roots under moderate stress and of chlorophyll metabolism in leaves under severe stress. Functional validation further corroborated the differential expression of genes coding for enzymes involved in chlorophyll metabolism, which correlates with the metabolite content of this pathway.

The basidiomycetous fungus Pseudozyma aphidis is able to convert vegetable oils to abundant amounts of the biosurfactant mannosylerythritol lipid (MEL) with a unique product pattern of MEL-A, MEL-B, MEL-C, and MEL-D. To investigate the metabolism of MEL production, we analyzed the transcriptome of P. aphidis DSM 70725 under MEL-inducing and non-inducing conditions using deep sequencing. Following manual curation of the previously described in silico gene models based on RNA-Seq data, we were able to generate an experimentally verified gene annotation containing 6347 genes. Using this database, our expression analysis revealed that only four of the five cluster genes required for MEL synthesis were clearly induced by the presence of soybean oil. The acetyltransferase encoding gene PaGMAT1 was expressed on a much lower level, which may explain the secretion of MEL with different degrees of acetylation in P. aphidis. In parallel to MEL synthesis, microscopic observations showed morphological changes accompanied by expression of genes responsible for cell development, indicative of a coregulation between MEL synthesis and cell morphology. In addition a set of transcription factors was identified which may be responsible for regulation of MEL synthesis and cell development. The upregulation of genes required for nitrogen metabolism and other assimilation processes indicate additional metabolic pathways required under the MEL-inducing conditions used. We also searched for a conserved gene cluster for cellobiose lipids (CL) but only found seven genes with limited homology distributed over the genome. However, we detected characteristic TLC spots in fermentations using P. aphidis DSM 70725, indicative of CL secretion.

BackgroundNeutrophils are traditionally considered transcriptionally inactive. Compared to other immune cells, little is known about their transcriptional profile during interaction with pathogens.MethodsWe analyzed the meta-transcriptome of the neutrophil-Candida albicans interplay and the transcriptome of C. albicans challenged with neutrophil extracellular traps (NETs) by RNA-Seq, considering yeast and hypha individually in each approach.ResultsThe neutrophil response to C. albicans yeast and hyphae was dominated by a morphotype-independent core response. However, 11 % of all differentially expressed genes were regulated in a specific manner when neutrophils encountered the hyphal form of C. albicans. While involving genes for transcriptional regulators, receptors, and cytokines, the neutrophil core response lacked typical antimicrobial effectors genes. Genes of the NOD-like receptor pathway, including NLRP3, were enriched.Neutrophil- and NET-provoked responses in C. albicans differed. At the same time, the Candida transcriptome upon neutrophil encounter and upon NET challenge included genes from various metabolic processes and indicate a mutual role of the regulators Tup1p, Efg1p, Hap43p, and Cap1p. Upon challenge with neutrophils and NETs, the overall Candida response was partially morphotype-specific. Yet again, actual oppositional regulation in yeasts and hyphae was only detected for the arginine metabolism in neutrophil-infecting C. albicans.

Background: Neutrophils are traditionally considered transcriptionally inactive. Compared to other immune cells, little is known about their transcriptional profile during interaction with pathogens. Methods: We analyzed the meta-transcriptome of the neutrophil-Candida albicans interplay and the transcriptome of C. albicans challenged with neutrophil extracellular traps (NETs) by RNA-Seq, considering yeast and hypha individually in each approach. Results: The neutrophil response to C. albicans yeast and hyphae was dominated by a morphotype-independent core response. However, 11 % of all differentially expressed genes were regulated in a specific manner when neutrophils encountered the hyphal form of C. albicans. While involving genes for transcriptional regulators, receptors, and cytokines, the neutrophil core response lacked typical antimicrobial effectors genes. Genes of the NOD-like receptor pathway, including NLRP3, were enriched. Neutrophil-and NET-provoked responses in C. albicans differed. At the same time, the Candida transcriptome upon neutrophil encounter and upon NET challenge included genes from various metabolic processes and indicate a mutual role of the regulators Tup1p, Efg1p, Hap43p, and Cap1p. Upon challenge with neutrophils and NETs, the overall Candida response was partially morphotype-specific. Yet again, actual oppositional regulation in yeasts and hyphae was only detected for the arginine metabolism in neutrophil-infecting C. albicans. Conclusions: Taken together, our study provides a comprehensive and quantitative transcript profile of the neutrophil-C. albicans interaction. By considering the two major appearances of both, neutrophils and C. albicans, our study reveals yet undescribed insights into this medically relevant encounter. Hence, our findings will facilitate future research and potentially inspire novel therapy developments. Originally published in manuscript form with title [RNA-Seq transcription profile of the neutrophil: Candida albicans in vitro interaction]Errata BMC Genomics (2017) 18:696 DOI: 10.1186/s12864-017-4097-4

Fungi are of increasing importance in sepsis. However, culture-based diagnostic procedures are associated with relevant weaknesses. Therefore, culture- and next-generation sequencing (NGS)-based fungal findings as well as corresponding plasma levels of β-d-glucan, interferon gamma (INF-γ), tumor necrosis factor alpha (TNF-α), interleukin (IL)-2, -4, -6, -10, -17A, and mid-regional proadrenomedullin (MR-proADM) were evaluated in 50 septic patients at six consecutive time points within 28 days after sepsis onset. Furthermore, immune-response patterns during infections with Candida spp. were studied in a reconstituted human epithelium model. In total, 22% (n = 11) of patients suffered from a fungal infection. An NGS-based diagnostic approach appeared to be suitable for the identification of fungal pathogens in patients suffering from fungemia as well as in patients with negative blood cultures. Moreover, MR-proADM and IL-17A in plasma proved suitable for the identification of patients with a fungal infection. Using RNA-seq., adrenomedullin (ADM) was shown to be a target gene which is upregulated early after an epithelial infection with Candida spp. In summary, an NGS-based diagnostic approach was able to close the diagnostic gap of routinely used culture-based diagnostic procedures, which can be further facilitated by plasmatic measurements of MR-proADM and IL-17A. In addition, ADM was identified as an early target gene in response to epithelial infections with Candida spp.

The three Actinobacteria strains Streptomyces platensis DSM 40041, Pseudonocardia autotrophica DSM 535, and Streptomyces fradiae DSM 40063 were described to selectively oxyfunctionalize several drugs. Here, we present their draft genomes to unravel their gene sets encoding promising cytochrome P450 monooxygenases associated with the generation of drug metabolites.

The three Actinobacteria strains Streptomyces platensis DSM 40041, Pseudonocardia autotrophica DSM 535, and Streptomyces fradiae DSM 40063 were described to selectively oxyfunctionalize several drugs. Here, we present their draft genomes to unravel their gene sets encoding promising cytochrome P450 monooxygenases associated with the generation of drug metabolites.

Candida albicans is an inhabitant of mucosal surfaces in healthy individuals but also the most common cause of fungal nosocomial blood stream infections, associated with high morbidity and mortality. As such life-threatening infections often disseminate from superficial mucosal infections we aimed to study the use of probiotic Lactobacillus rhamnosus GG (LGG) in prevention of mucosal C. albicans infections. Here, we demonstrate that LGG protects oral epithelial tissue from damage caused by C. albicans in our in vitro model of oral candidiasis. Furthermore, we provide insights into the mechanisms behind this protection and dissect direct and indirect effects of LGG on C. albicans pathogenicity. C. albicans viability was not affected by LGG. Instead, transcriptional profiling using RNA-Seq indicated dramatic metabolic reprogramming of C. albicans. Additionally, LGG had a significant impact on major virulence attributes, including adhesion, invasion, and hyphal extension, whose reduction, consequently, prevented epithelial damage. This was accompanied by glucose depletion and repression of ergosterol synthesis, caused by LGG, but also due to blocked adhesion sites. Therefore, LGG protects oral epithelia against C. albicans infection by preventing fungal adhesion, invasion and damage, driven, at least in parts, by metabolic reprogramming due to nutrient limitation caused by LGG. © 2017 Mailänder-Sánchez et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Pseudonocardia autotrophica strain DSM 43083 is a filamentous actinobacterium and was described to degrade or modify lignin. Here, we present its draft genome sequence, with a size of 5.8 Mb, to unravel the gene set coding for promising monooxygenases, dioxygenases, and DyP-type peroxidases associated with aromatic metabolism and lignin modification.

The fungal pathogen Candida albicans colonizes basically all human epithelial surfaces, including the skin. Under certain conditions, such as immunosuppression, invasion of the epithelia occurs. Not much is known about defense mechanisms against C. albicans in subepithelial layers such as the dermis. Using immune cell-supplemented 3D skin models we defined a new role for fibroblasts in the dermis and identified a minimal set of cell types for skin protection against C. albicans invasion. Dual RNA sequencing of individual host cell populations and C. albicans revealed that dermal invasion is directly impeded by dermal fibroblasts. They are able to integrate signals from the pathogen and CD4+ T cells and shift toward an antimicrobial phenotype with broad specificity that is dependent on Toll-like receptor 2 and interleukin 1β. These results highlight a central function of dermal fibroblasts for skin protection, opening new possibilities for treatment of infectious diseases.

Human skin is a niche for various fungal species which either colonize the surface of this tissue as commensals or, primarily under conditions of immunosuppression, invade the skin and cause infection. Here we present a method for generation of a human in vitro skin model supplemented with immune cells of choice. This model represents a complex yet amenable tool to study molecular mechanisms of host-fungi interactions at human skin.

Sequencing the whole genome of an organism is invaluable for its comprehensive molecular characterization and has been drastically facilitated by the advent of high-throughput sequencing techniques. Especially in clinical microbiology the impact of sequenced strains increases as resistance and virulence markers can easily be detected. Here, we describe a combined approach for sequencing a fungal genome and transcriptome from initial nucleic acid isolation through the generation of ready-to-load DNA libraries for the Illumina platform and the final step of genome assembly with subsequent gene annotation.

Sequencing the whole genome of an organism is invaluable for its comprehensive molecular characterization and has been drastically facilitated by the advent of high-throughput sequencing techniques. Especially in clinical microbiology the impact of sequenced strains increases as resistance and virulence markers can easily be detected. Here, we describe a combined approach for sequencing a fungal genome and transcriptome from initial nucleic acid isolation through the generation of ready-to-load DNA libraries for the Illumina platform and the final step of genome assembly with subsequent gene annotation.

Human skin is a niche for various fungal species which either colonize the surface of this tissue as commensals or, primarily under conditions of immunosuppression, invade the skin and cause infection. Here we present a method for generation of a human in vitro skin model supplemented with immune cells of choice. This model represents a complex yet amenable tool to study molecular mechanisms of host-fungi interactions at human skin.

A novel innovative approach towards a marketable lab-on-chip system for point-of-care in vitro diagnostics is reported. In a consortium of seven Fraunhofer Institutes a lab-on-chip system called "Fraunhofer ivD-platform" has been established which opens up the possibility for an on-site analysis at low costs. The system features a high degree of modularity and integration. Modularity allows the adaption of common and established assay types of various formats. Integration lets the system move from the laboratory to the point-of-need. By making use of the microarray format the lab-on-chip system also addresses new trends in biomedicine. Research topics such as personalized medicine or companion diagnostics show that multiparameter analyses are an added value for diagnostics, therapy as well as therapy control. These goals are addressed with a low-cost and self-contained cartridge, since reagents, microfluidic actuators and various sensors are integrated within the cartridge. In combination with a fully automated instrumentation (read-out and processing unit) a diagnostic assay can be performed in about 15 min. Via a user-friendly interface the read-out unit itself performs the assay protocol, data acquisition and data analysis. So far, example assays for nucleic acids (detection of different pathogens) and protein markers (such as CRP and PSA) have been established using an electrochemical read-out based on redoxcycling or an optical read-out based on total internal reflectance fluorescence (TIRF). It could be shown that the assay performance within the cartridge is similar to that found for the same assay in a microtiter plate. Furthermore, recent developments are the integration of sample preparation and polymerase chain reaction (PCR) on-chip. Hence, the instrument is capable of providing heating-and-cooling cycles necessary for DNA-amplification. In addition to scientific aspects also the production of such a lab-on-chip system was part of the development since this heavily affects the success of a later market launch. In summary, the Fraunhofer ivD-platform covers the whole value chain ranging from microfluidics, material and polymer sciences, assay and sensor development to the production and assembly design. In this consortium the gap between diagnostic needs and available technologies can be closed.

Infectious diseases have long been regarded as losing their threat to mankind. However, in the recent decades infectious diseases have been regaining grounds and are back in the focus of research. This is also due to the fact that medical progress has enabled us to treat and cure a much higher fraction of severe diseases or trauma, resulting in a significant proportion of temporarily or constantly immune-suppressed patients. Infectious diseases result from the interplay between pathogenic microorganisms and the hosts they infect, especially their defense systems. Consequently, immune-suppressed patients are at high risk to succumb from opportunistic infections, like Candida infections. To study the balance between host and C. albicans with regard to the establishment of disease or asymptomatic, commensal colonisation, we developed host-pathogen interaction systems to study both the adaptation of C. albicans to different epithelia as well as to investigate the sensors of the innate immune system, the pattern recognition receptors. These host-pathogen interaction systems, as well as some of the results gained are described in this review.

Differential gene expression profiling has become of central importance for the analysis of cellular systems at the transcriptional level. By now, many platform technologies including DNA-microarrays, serial analysis of gene expression or RNA-seq have been established in order to facilitate transcriptional profiling. However, these technologies are all subjected to specific limitations, as they require a priori knowledge of annotated genome sequences or are based on substantial bioinformatic infrastructure, for example. As an unbiased alternative we describe here a multidimensional electrophoretic system of separation for the analysis of gene expression for the global transcriptional profiling in any eukaryotic organism. This approach is compatible with standard laboratory equipment comprising high-resolution separation of complex cDNA-probes using two-dimensional DNA-gel electrophoresis. In this context cDNA fragments are separated using non-denaturing PAGE in the first dimension with subsequent denaturing gradient gel electrophoresis in the second dimension. Two-dimensional spot patterns are quantified by well-established bioinformatic algorithms and selected spots are identified using DNA sequencing. Neither does this method necessarily depend on annotated genome sequences, nor does it require sophisticated instrumentation. Strikingly, quantitative data on differential gene expression derived from multidimensional electrophoretic system of separation for the analysis of gene expression highly correlate with corresponding data from quantitative RT-PCR even for transcriptional profiles of limited amounts of total RNA.

For a more eco-friendly production of energy and chemicals (e.g. lactic acid), green biorefineries are implementing an environmentally conscious technique of using green biomass. To increase the amount of lactic acid in grass and rye silage, different ensiling treatments were conducted. Additionally, after separating the organic juice, the specific methane yield of the remaining solid residue of the ensiled material was determined. The amount of lactic acid was increased by 168.8 % (149.7 ± 20.9 g kg−1 dry matter (DM)) through applying homofermentative lactic acid bacteria together with carbonated lime to the raw material grass. For rye, while having a stable silage, the highest increase in lactic acid was achieved by chopping the raw material to a theoretical length of cut of 1 mm. As a result, an increase of 46.3 % (57.5 ± 0.6 g kg−1 DM) was attained. Taxonomic profiling by 16S amplicon sequencing revealed that the homofermentative species Lactobacillus plantarum was the most dominant species on both substrates with highest lactic acid production rate, though its growth on rye led to unstable silage conditions with butyric acid producing Clostridia. The specific methane yields of the corresponding solid residues were determined to be 335.7 ± 7.2 lN kg−1 organic dry matter (ODM) for grass and at 235.0 ± 2.6 lN kg−1 ODM for rye.

Background Bloodstream infections remain one of the major challenges in intensive care units, leading to sepsis or even septic shock in many cases. Due to the lack of timely diagnostic approaches with sufficient sensitivity, mortality rates of sepsis are still unacceptably high. However a prompt diagnosis of the causative microorganism is critical to significantly improve outcome of bloodstream infections. Although various targeted molecular tests for blood samples are available, time-consuming blood culture-based approaches still represent the standard of care for the identification of bacteria. Methods Here we describe the establishment of a complete diagnostic workflow for the identification of infectious microorganisms from seven septic patients based on unbiased sequence analyses of free circulating DNA from plasma by next-generation sequencing. Results We found significant levels of DNA fragments derived from pathogenic bacteria in samples from septic patients. Quantitative evaluation of normalized read counts and introduction of a sepsis indicating quantifier (SIQ) score allowed for an unambiguous identification of Gram-positive as well as Gram-negative bacteria that exactly matched with blood cultures from corresponding patient samples. In addition, we also identified species from samples where blood cultures were negative. Reads of non-human origin also comprised fragments derived from antimicrobial resistance genes, showing that, in principle, prediction of specific types of resistance might be possible. Conclusions The complete workflow from sample preparation to species identification report could be accomplished in roughly 30 h, thus making this approach a promising diagnostic platform for critically ill patients suffering from bloodstream infections. Electronic supplementary material The online version of this article (doi:10.1186/s13073-016-0326-8) contains supplementary material, which is available to authorized users.

Nucleo-cytoplasmic transport of proteins is mostly mediated by specific interaction between transport receptors of the importin beta family and signal sequences present in their cargo. While several signal sequences, in particular the classical nuclear localization signal (NLS) recognized by the heterodimeric importin alpha/beta complex are well known, the signals recognized by other importin beta-like transport receptors remain to be characterized in detail. Here we present the systematic analysis of the nuclear import of Saccharomyces cerevisiae Asr1p, a nonessential alcohol-responsive Ring/PHD finger protein that shuttles between nucleus and cytoplasm but accumulates in the nucleus upon alcohol stress. Nuclear import of Asr1p is constitutive and mediated by its C-terminal domain. A short sequence comprising residues 243-280 is sufficient and necessary for active targeting to the nucleus. Moreover, the nuclear import signal is conserved from yeast to mammals. In vitro, the nuclear localization signal of Asr1p directly interacts with the importins Kap114p, Kap95p, Pse1p, Kap123p, or Kap104p, interactions that are sensitive to the presence of RanGTP. In vivo, these importins cooperate in nuclear import. Interestingly, the same importins mediate nuclear transport of histone H2A. Based on mutational analysis and sequence comparison with a region mediating nuclear import of histone H2A, we identified a ;novel type of NLS with the consensus sequence R/KxxL(x)(n)V/YxxV/IxK/RxxxK/R that is recognized by five yeast importins and connects them into a highly efficient network for nuclear import of proteins.

Amantichitinum ursilacus IGB-41 is a new species of chitin-degrading bacterium isolated from soil, which secretes potential industrial enzymes. The genome of A. ursilacus was sequenced, and the gene set encoding chitinases was identified. Here, we present the draft genome of 4.9 Mb, comprising 38 contigs, and the corresponding annotation.

Adhesion to mammalian epithelia is one of the prerequisites that are essential to accomplish pathogenesis of Candida albicans in the mammalian host. In this context C. albicans is able to adhere to a plethora of different cell types providing different microenvironments for colonization. To study the response of C. albicans adhering to different surfaces on the transcriptional level we have established an in vitro adhesion assay exploiting confluent monolayers of the human colorectal carcinoma cell line Caco-2 or epidermoid vulvo-vaginal A-431 cells. Candida albicans very efficiently adheres to these epithelia growing as hyphae. Using whole-genome DNA microarrays comprising probes for almost 7000 predicted ORFs we found that transcriptional profiles of C. albicans adhering to Caco-2 or to A-431 cells, although very similar, still significantly differ from those of Candida cells adhering to plastic surfaces. Differences became even more obvious when comparing C. albicans cells either growing in an adherent manner or in suspension culture. Correspondingly, we found for several cell surface genes, including PRA1, PGA23, PGA7 and HWP1, an adhesion-dependent induction of transcription. Obviously, C. albicans is able to respond specifically to very subtle differences in the environment during adhesion to various growth substrates.

Summary: Several actual developments in medical therapy are focussing on the potential of a surface-dependent selection and proliferation of special cell types. For an improvement of biocompatibility it is interesting to understand the interactions between cells and material surfaces in order to create devices with the respective characteristics. In this paper the possibility to isolate three different tumor cells lines are studied: HEK293, RINm5f and KYM-1. A procedure by means of plasma polymerization is demonstrated to create hydrophilic microstructures (precursor: AAc) on a hydrophobic (precursor: CHF3) substrate and shown to effectively select RINm5f cells. Moreover, by studying the interaction between culture media and deposited polymers, it is shown that Ca2+ ions and protein adsorption play a fundamental role in the cell-substrate adhesion and proliferation.