High inter-laboratory variability in the assessment of HER2-low breast cancer: a national registry study on 50,714 Danish patients.

BACKGROUND: Considering the recent advancements in the treatment of breast cancer with low expression of human epidermal growth factor receptor 2 (HER2), we aimed to examine inter-laboratory variability in the assessment of HER2-low breast cancer across all Danish pathology departments. METHODS: From the Danish Breast Cancer Group, we obtained data on all women diagnosed with primary invasive breast cancer in 2007-2019 who were subsequently assigned for curatively intended treatment. RESULTS: Of 50,714 patients, HER2 score and status were recorded for 48,382, among whom 59.2% belonged to the HER2-low group (score 1+ or 2+ without gene amplification), 26.8% had a HER2 score of 0, and 14.0% were HER2 positive. The proportion of HER2-low cases ranged from 46.3 to 71.8% among pathology departments (P < 0.0001) and from 49.3 to 65.6% over the years (P < 0.0001). In comparison, HER2 positivity rates ranged from 11.8 to 17.2% among departments (P < 0.0001) and from 12.6 to 15.7% over the years (P = 0.005). In the eight departments with the highest number of patients, variability in HER2-low cases increased from 2011 to 2019, although the same immunohistochemical assay was used. By multivariable logistic regression, the examining department was significantly related to both HER2 score 0 and HER2 positivity (P < 0.0001) but showed greater dispersion in odds ratios in the former case (range 0.25-1.41 vs. 0.84-1.27). CONCLUSIONS: Our data showed high inter-laboratory variability in the assessment of HER2-low breast cancer. The findings cast doubt on whether the current test method for HER2 is robust and reliable enough to select HER2-low patients for HER2-targeted treatment in daily clinical practice.

Temporal regulation of temperature tolerances and gene expression in an arctic insect.

Terrestrial arthropods in the Arctic are exposed to highly variable temperatures that frequently reach cold and warm extremes. Yet, ecophysiological studies on arctic insects typically focus on the ability of species to tolerate low temperatures, whereas studies investigating physiological adaptations of species to periodically warm and variable temperatures are few. In this study, we investigated temporal changes in thermal tolerances and the transcriptome in the Greenlandic seed bug Nysius groenlandicus, collected in the field across different times and temperatures in Southern Greenland. We found that plastic changes in heat and cold tolerances occurred rapidly (within hours) and at a daily scale in the field, and that these changes are correlated with diurnal temperature variation. Using RNA sequencing, we provide molecular underpinnings of the rapid adjustments in thermal tolerance across ambient field temperatures and in the laboratory. We show that transcriptional responses are sensitive to daily temperature changes, and days characterized by high temperature variation induced markedly different expression patterns than thermally stable days. Further, genes associated with laboratory-induced heat responses, including expression of heat shock proteins and vitellogenins, were shared across laboratory and field experiments, but induced at time points associated with lower temperatures in the field. Cold stress responses were not manifested at the transcriptomic level.

Psychotherapy for patients with schizotypal personality disorder: A scoping review.

BACKGROUND: Treatment of schizotypal personality disorder is complex. Currently, there are no clear evidence-based recommendations for use of psychotherapy for individuals suffering from this mental illness, and studies are sparse. Our aim in this review is to map and describe the existing research and to answer the research question: What do we know about the use of psychotherapy for people with schizotypal personality disorder? METHODS: We conducted a scoping review using systematic searches in the Embase, MEDLINE and PsycINFO databases. Two reviewers screened possible studies and extracted data on subject samples, type of psychotherapy, outcomes and suggested mechanisms of change. The review is based on the PRISMA checklist for scoping reviews. RESULTS: Twenty-three papers were included, and we found a wide variety of study types, psychotherapeutic orientations and outcomes. Few studies emerged that focused solely on schizotypal personality disorder. CONCLUSION: Psychotherapy as a treatment for schizotypal personality disorder is understudied compared with diagnoses such as schizophrenia and borderline personality disorder. Our results included two randomized controlled studies, as well as mainly smaller studies with different approaches to diagnostic criteria, psychotherapeutic orientation and outcome measures. The findings are too sparse and too diverse to make any evidence-based recommendations. We found some indications that psychotherapy may support and assist individuals with schizotypal personality disorder.

Speed dating for enzymes! Finding the perfect phosphopantetheinyl transferase partner for your polyketide synthase.

The biosynthetic pathways for the fungal polyketides bikaverin and bostrycoidin, from Fusarium verticillioides and Fusarium solani respectively, were reconstructed and heterologously expressed in S. cerevisiae alongside seven different phosphopantetheinyl transferases (PPTases) from a variety of origins spanning bacterial, yeast and fungal origins. In order to gauge the efficiency of the interaction between the ACP-domains of the polyketide synthases (PKS) and PPTases, each were co-expressed individually and the resulting production of target polyketides were determined after 48 h of growth. In co-expression with both biosynthetic pathways, the PPTase from Fusarium verticillioides (FvPPT1) proved most efficient at producing both bikaverin and bostrycoidin, at 1.4 mg/L and 5.9 mg/L respectively. Furthermore, the remaining PPTases showed the ability to interact with both PKS's, except for a single PKS-PPTase combination. The results indicate that it is possible to boost the production of a target polyketide, simply by utilizing a more optimal PPTase partner, instead of the commonly used PPTases; NpgA, Gsp and Sfp, from Aspergillus nidulans, Brevibacillus brevis and Bacillus subtilis respectively.

Genomic variation predicts adaptive evolutionary responses better than population bottleneck history.

The relationship between population size, inbreeding, loss of genetic variation and evolutionary potential of fitness traits is still unresolved, and large-scale empirical studies testing theoretical expectations are surprisingly scarce. Here we present a highly replicated experimental evolution setup with 120 lines of Drosophila melanogaster having experienced inbreeding caused by low population size for a variable number of generations. Genetic variation in inbred lines and in outbred control lines was assessed by genotyping-by-sequencing (GBS) of pooled samples consisting of 15 males per line. All lines were reared on a novel stressful medium for 10 generations during which body mass, productivity, and extinctions were scored in each generation. In addition, we investigated egg-to-adult viability in the benign and the stressful environments before and after rearing at the stressful conditions for 10 generations. We found strong positive correlations between levels of genetic variation and evolutionary response in all investigated traits, and showed that genomic variation was more informative in predicting evolutionary responses than population history reflected by expected inbreeding levels. We also found that lines with lower genetic diversity were at greater risk of extinction. For viability, the results suggested a trade-off in the costs of adapting to the stressful environments when tested in a benign environment. This work presents convincing support for long-standing evolutionary theory, and it provides novel insights into the association between genetic variation and evolutionary capacity in a gradient of diversity rather than dichotomous inbred/outbred groups.

Non-GMO potato lines, synthesizing increased amylose and resistant starch, are mainly deficient in isoamylase debranching enzyme.

Solanum tuberosum potato lines with high amylose content were generated by crossing with the wild potato species Solanum sandemanii followed by repeated backcrossing to Solanum tuberosum lines. The trait, termed increased amylose (IAm), was recessive and present after three generations of backcrossing into S. tuberosum lines (6.25% S. sandemanii genes). The tubers of these lines were small, elongated and irregular with small and misshaped starch granules and high sugar content. Additional backcrossing resulted in less irregular tuber morphology, increased starch content (4.3%-9.5%) and increased amylose content (29%-37.9%) but indifferent sugar content. The amylose in the IAm starch granules was mainly located in peripheral spots, and large cavities were found in the granules. Starch pasting was suppressed, and the digestion-resistant starch (RS) content was increased. Comprehensive microarray polymer profiling (CoMPP) analysis revealed specific alterations of major pectic and glycoprotein cell wall components. This complex phenotype led us to search for candidate IAm genes exploiting its recessive trait. Hence, we sequenced genomic DNA of a pool of IAm lines, identified SNPs genome wide against the draft genome sequence of potato and searched for regions of decreased heterozygosity. Three regions, located on chromosomes 3, 7 and 10, respectively, displayed markedly less heterozygosity than average. The only credible starch metabolism-related gene found in these regions encoded the isoamylase-type debranching enzyme Stisa1. Decreased expression of mRNA (>500 fold) and reduced enzyme activity (virtually absent from IAm lines) supported Stisa1 as a candidate gene for IAm.

Heterologous Expression of the Core Genes in the Complex Fusarubin Gene Cluster of Fusarium Solani.

Through stepwise recreation of the biosynthetic gene cluster containing PKS3 from Fusarium solani, it was possible to produce the core scaffold compound of bostrycoidin, a red aza-anthraquinone pigment in Saccharomyces cerevisiae. This was achieved through sequential transformation associated recombination (TAR) cloning of FvPPT, fsr1, fsr2, and fsr3 into the pESC-vector system, utilizing the inducible bidirectional galactose promoter for heterologous expression in S. cerevisiae. The production of the core metabolite bostrycoidin was investigated through triplicate growth cultures for 1-4 days, where the maximum titer of bostrycoidin was achieved after 2 days of induction, yielding 2.2 mg/L.

Effect of wet storage conditions on potato tuber transcriptome, phytohormones and growth.

BACKGROUND: Stored potato (Solanum tuberosum L.) tubers are sensitive to wet conditions that can cause rotting in long-term storage. To study the effect of water on the tuber surface during storage, microarray analysis, RNA-Seq profiling, qRT-PCR and phytohormone measurements were performed to study gene expression and hormone content in wet tubers incubated at two temperatures: 4 °C and 15 °C. The growth of the plants was also observed in a greenhouse after the incubation of tubers in wet conditions. RESULTS: Wet conditions induced a low-oxygen response, suggesting reduced oxygen availability in wet tubers at both temperatures when compared to that in the corresponding dry samples. Wet conditions induced genes coding for heat shock proteins, as well as proteins involved in fermentative energy production and defense against reactive oxygen species (ROS), which are transcripts that have been previously associated with low-oxygen stress in hypoxic or anoxic conditions. Wet treatment also induced senescence-related gene expression and genes involved in cell wall loosening, but downregulated genes encoding protease inhibitors and proteins involved in chloroplast functions and in the biosynthesis of secondary metabolites. Many genes involved in the production of phytohormones and signaling were also affected by wet conditions, suggesting altered regulation of growth by wet conditions. Hormone measurements after incubation showed increased salicylic acid (SA), abscisic acid (ABA) and auxin (IAA) concentrations as well as reduced production of jasmonate 12-oxo-phytodienoic acid (OPDA) in wet tubers. After incubation in wet conditions, the tubers produced fewer stems and more roots compared to controls incubated in dry conditions. CONCLUSIONS: In wet conditions, tubers invest in ROS protection and defense against the abiotic stress caused by reduced oxygen due to excessive water. Changes in ABA, SA and IAA that are antagonistic to jasmonates affect growth and defenses, causing induction of root growth and rendering tubers susceptible to necrotrophic pathogens. Water on the tuber surface may function as a signal for growth, similar to germination of seeds.

Genomic prediction of starch content and chipping quality in tetraploid potato using genotyping-by-sequencing.

KEY MESSAGE: Genomic prediction models for starch content and chipping quality show promising results, suggesting that genomic selection is a feasible breeding strategy in tetraploid potato. Genomic selection uses genome-wide molecular markers to predict performance of individuals and allows selections in the absence of direct phenotyping. It is regarded as a useful tool to accelerate genetic gain in breeding programs, and is becoming increasingly viable for crops as genotyping costs continue to fall. In this study, we have generated genomic prediction models for starch content and chipping quality in tetraploid potato to facilitate varietal development. Chipping quality was evaluated as the colour of a potato chip after frying following cold induced sweetening. We used genotyping-by-sequencing to genotype 762 offspring, derived from a population generated from biparental crosses of 18 tetraploid parents. Additionally, 74 breeding clones were genotyped, representing a test panel for model validation. We generated genomic prediction models from 171,859 single-nucleotide polymorphisms to calculate genomic estimated breeding values. Cross-validated prediction correlations of 0.56 and 0.73 were obtained within the training population for starch content and chipping quality, respectively, while correlations were lower when predicting performance in the test panel, at 0.30-0.31 and 0.42-0.43, respectively. Predictions in the test panel were slightly improved when including representatives from the test panel in the training population but worsened when preceded by marker selection. Our results suggest that genomic prediction is feasible, however, the extremely high allelic diversity of tetraploid potato necessitates large training populations to efficiently capture the genetic diversity of elite potato germplasm and enable accurate prediction across the entire spectrum of elite potatoes. Nonetheless, our results demonstrate that GS is a promising breeding strategy for tetraploid potato.

Chrysogine Biosynthesis Is Mediated by a Two-Module Nonribosomal Peptide Synthetase.

Production of chrysogine has been reported from several fungal genera including Penicillium, Aspergillus, and Fusarium. Anthranilic acid and pyruvic acid, which are expected precursors of chrysogine, enhance production of this compound. A possible route for the biosynthesis using these substrates is via a nonribosomal peptide synthetase (NRPS). Through comparative analysis of the NRPSs from genome-sequenced producers of chrysogine we identified a candidate NRPS cluster comprising five additional genes named chry2-6. Deletion of the two-module NRPS (NRPS14 = chry1) abolished chrysogine production in Fusarium graminearum, indicating that the gene cluster is responsible for chrysogine biosynthesis. Overexpression of NRPS14 enhanced chrysogine production, suggesting that the NRPS is the bottleneck in the biosynthetic pathway.

In vivo gene expression in a Staphylococcus aureus prosthetic joint infection characterized by RNA sequencing and metabolomics: a pilot study.

BACKGROUND: Staphylococcus aureus gene expression has been sparsely studied in deep-sited infections in humans. Here, we characterized the staphylococcal transcriptome in vivo and the joint fluid metabolome in a prosthetic joint infection with an acute presentation using deep RNA sequencing and nuclear magnetic resonance spectroscopy, respectively. We compared our findings with the genome, transcriptome and metabolome of the S. aureus joint fluid isolate grown in vitro. RESULT: From the transcriptome analysis we found increased expression of siderophore synthesis genes and multiple known virulence genes. The regulatory pattern of catabolic pathway genes indicated that the bacterial infection was sustained on amino acids, glycans and nucleosides. Upregulation of fermentation genes and the presence of ethanol in joint fluid indicated severe oxygen limitation in vivo. CONCLUSION: This single case study highlights the capacity of combined transcriptome and metabolome analyses for elucidating the pathogenesis of prosthetic infections of major clinical importance.

Catalase and NO CATALASE ACTIVITY1 promote autophagy-dependent cell death in Arabidopsis.

Programmed cell death often depends on generation of reactive oxygen species, which can be detoxified by antioxidative enzymes, including catalases. We previously isolated catalase-deficient mutants (cat2) in a screen for resistance to hydroxyurea-induced cell death. Here, we identify an Arabidopsis thaliana hydroxyurea-resistant autophagy mutant, atg2, which also shows reduced sensitivity to cell death triggered by the bacterial effector avrRpm1. To test if catalase deficiency likewise affected both hydroxyurea and avrRpm1 sensitivity, we selected mutants with extremely low catalase activities and showed that they carried mutations in a gene that we named NO CATALASE ACTIVITY1 (NCA1). nca1 mutants showed severely reduced activities of all three catalase isoforms in Arabidopsis, and loss of NCA1 function led to strong suppression of RPM1-triggered cell death. Basal and starvation-induced autophagy appeared normal in the nca1 and cat2 mutants. By contrast, autophagic degradation induced by avrRpm1 challenge was compromised, indicating that catalase acted upstream of immunity-triggered autophagy. The direct interaction of catalase with reactive oxygen species could allow catalase to act as a molecular link between reactive oxygen species and the promotion of autophagy-dependent cell death.

Cytosolic glutamine synthetase is important for photosynthetic efficiency and water use efficiency in potato as revealed by high-throughput sequencing QTL analysis.

KEY MESSAGE: WUE phenotyping and subsequent QTL analysis revealed cytosolic GS genes importance for limiting N loss due to photorespiration under well-watered and well-fertilized conditions. Potato (Solanum tuberosum L.) closes its stomata at relatively low soil water deficits frequently encountered in normal field conditions resulting in unnecessary annual yield losses and extensive use of artificial irrigation. Therefore, unraveling the genetics underpinning variation in water use efficiency (WUE) of potato is important, but has been limited by technical difficulties in assessing the trait on individual plants and thus is poorly understood. In this study, a mapping population of potatoes has been robustly phenotyped, and considerable variation in WUE under well-watered conditions was observed. Two extreme WUE bulks of clones were identified and pools of genomic DNA from them as well as the parents were sequenced and mapped to reference potato genome. Following a novel data analysis approach, two highly resolved QTLs were found on chromosome 1 and 9. Interestingly, three genes encoding isoforms of cytosolic glutamine synthase were located in the QTL at chromosome 1 suggesting a major contribution of this enzyme to photosynthetic efficiency and thus WUE in potato. Indeed, Glutamine synthetase enzyme activity of leaf extracts was measured and found to be correlated with contrasting WUE phenotypes.

The morphological growth patterns of colorectal liver metastases are prognostic for overall survival.

Colorectal metastases in the liver grow according to three histological patterns: a pushing pattern, a replacement pattern, and a desmoplastic pattern. The objective of the current study was to explore the prognostic significance of these three growth patterns for survival. The study included 217 consecutive patients, liver resected between 2007 and 2011 due to hepatic metastases from colorectal adenocarcinoma. The growth patterns were assessed on archival hematoxylin and eosin-stained tissue sections. In 150 metastases, the density of the immune cell infiltrate at the tumor periphery was judged by a semi-quantitative method. The prevalence of the pushing-type, the desmoplastic-type, and the replacement-type was 33%, 32%, and 11%, respectively; 24% of the metastases displayed a mixed pattern. Kaplan-Meier analysis and Cox regression demonstrated a prognostic significance of the growth patterns (P=0.0006, log-rank test), as the replacement pattern appeared as an independent predictor of poor overall survival. For patients with replacement growth, the hazard of death was 2-2.5 times higher than for patients with pushing growth (P=0.004, cox regression) or mixed growth (P=0.01), and nearly four times higher than for patients with desmoplastic growth (P<0.0001). The negative prognostic effect of the replacement growth pattern was even more pronounced after adjusting for tumor size. Desmoplastic growth corresponded with small tumor size, dense lymphocytic infiltration and a more favorable prognosis. Eventually, the growth patterns may contribute to a histology-based prognostic biomarker for patients with colorectal liver metastases.

The effect of marker types and density on genomic prediction and GWAS of key performance traits in tetraploid potato.

Genomic prediction and genome-wide association studies are becoming widely employed in potato key performance trait QTL identifications and to support potato breeding using genomic selection. Elite cultivars are tetraploid and highly heterozygous but also share many common ancestors and generation-spanning inbreeding events, resulting from the clonal propagation of potatoes through seed potatoes. Consequentially, many SNP markers are not in a 1:1 relationship with a single allele variant but shared over several alleles that might exert varying effects on a given trait. The impact of such redundant "diluted" predictors on the statistical models underpinning genome-wide association studies (GWAS) and genomic prediction has scarcely been evaluated despite the potential impact on model accuracy and performance. We evaluated the impact of marker location, marker type, and marker density on the genomic prediction and GWAS of five key performance traits in tetraploid potato (chipping quality, dry matter content, length/width ratio, senescence, and yield). A 762-offspring panel of a diallel cross of 18 elite cultivars was genotyped by sequencing, and markers were annotated according to a reference genome. Genomic prediction models (GBLUP) were trained on four marker subsets [non-synonymous (29,553 SNPs), synonymous (31,229), non-coding (32,388), and a combination], and robustness to marker reduction was investigated. Single-marker regression GWAS was performed for each trait and marker subset. The best cross-validated prediction correlation coefficients of 0.54, 0.75, 0.49, 0.35, and 0.28 were obtained for chipping quality, dry matter content, length/width ratio, senescence, and yield, respectively. The trait prediction abilities were similar across all marker types, with only non-synonymous variants improving yield predictive ability by 16%. Marker reduction response did not depend on marker type but rather on trait. Traits with high predictive abilities, e.g., dry matter content, reached a plateau using fewer markers than traits with intermediate-low correlations, such as yield. The predictions were unbiased across all traits, marker types, and all marker densities >100 SNPs. Our results suggest that using non-synonymous variants does not enhance the performance of genomic prediction of most traits. The major known QTLs were identified by GWAS and were reproducible across exonic and whole-genome variant sets for dry matter content, length/width ratio, and senescence. In contrast, minor QTL detection was marker type dependent.

Apiospora arundinis, a panoply of carbohydrate-active enzymes and secondary metabolites.

The Apiospora genus comprises filamentous fungi with promising potential, though its full capabilities remain undiscovered. In this study, we present the first genome assembly of an Apiospora arundinis isolate, demonstrating a highly complete and contiguous assembly estimated to 48.8 Mb, with an N99 of 3.0 Mb. Our analysis predicted a total of 15,725 genes, with functional annotations for 13,619 of them, revealing a fungus capable of producing very high amounts of carbohydrate-active enzymes (CAZymes) and secondary metabolites. Through transcriptomic analysis, we observed differential gene expression in response to varying growth media, with several genes related to carbohydrate metabolism showing significant upregulation when the fungus was cultivated on a hay-based medium. Finally, our metabolomic analysis unveiled a fungus capable of producing a diverse array of metabolites.

The fungal symbiont of Acromyrmex leaf-cutting ants expresses the full spectrum of genes to degrade cellulose and other plant cell wall polysaccharides.

BACKGROUND: The fungus gardens of leaf-cutting ants are natural biomass conversion systems that turn fresh plant forage into fungal biomass to feed the farming ants. However, the decomposition potential of the symbiont Leucocoprinus gongylophorus for processing polysaccharides has remained controversial. We therefore used quantifiable DeepSAGE technology to obtain mRNA expression patterns of genes coding for secreted enzymes from top, middle, and bottom sections of a laboratory fungus-garden of Acromyrmex echinatior leaf-cutting ants. RESULTS: A broad spectrum of biomass-conversion-relevant enzyme genes was found to be expressed in situ: cellulases (GH3, GH5, GH6, GH7, AA9 [formerly GH61]), hemicellulases (GH5, GH10, CE1, GH12, GH74), pectinolytic enzymes (CE8, GH28, GH43, PL1, PL3, PL4), glucoamylase (GH15), α-galactosidase (GH27), and various cutinases, esterases, and lipases. In general, expression of these genes reached maximal values in the bottom section of the garden, particularly for an AA9 lytic polysaccharide monooxygenase and for a GH5 (endocellulase), a GH7 (reducing end-acting cellobiohydrolase), and a GH10 (xylanase), all containing a carbohydrate binding module that specifically binds cellulose (CBM1). Although we did not directly quantify enzyme abundance, the profile of expressed cellulase genes indicates that both hydrolytic and oxidative degradation is taking place. CONCLUSIONS: The fungal symbiont of Acromyrmex leaf-cutting ants can degrade a large range of plant polymers, but the conversion of cellulose, hemicellulose, and part of the pectin occurs primarily towards the end of the decomposition process, i.e. in the bottom section of the fungus garden. These conversions are likely to provide nutrients for the fungus itself rather than for the ants, whose colony growth and reproductive success are limited by proteins obtained from ingesting fungal gongylidia. These specialized hyphal tips are hardly produced in the bottom section of fungus gardens, consistent with the ants discarding old fungal biomass from this part of the garden. The transcripts that we found suggest that actively growing mycelium in the bottom of gardens helps to maintain an optimal water balance to avoid hyphal disintegration, so the ants can ultimately discard healthy rather than decaying and diseased garden material, and to buffer negative effects of varying availability and quality of substrate across the seasons.

Metagenomes obtained by 'deep sequencing' - what do they tell about the enhanced biological phosphorus removal communities?

Metagenomics enables studies of the genomic potential of complex microbial communities by sequencing bulk genomic DNA directly from the environment. Knowledge of the genetic potential of a community can be used to formulate and test ecological hypotheses about stability and performance. In this study deep metagenomics and fluorescence in situ hybridization (FISH) were used to study a full-scale wastewater treatment plant with enhanced biological phosphorus removal (EBPR), and the results were compared to an existing EBPR metagenome. EBPR is a widely used process that relies on a complex community of microorganisms to function properly. Insight into community and species level stability and dynamics is valuable for knowledge-driven optimization of the EBPR process. The metagenomes of the EBPR communities were distinct compared to metagenomes of communities from a wide range of other environments, which could be attributed to selection pressures of the EBPR process. The metabolic potential of one of the key microorganisms in the EPBR process, Accumulibacter, was investigated in more detail in the two plants, revealing a potential importance of phage predation on the dynamics of Accumulibacter populations. The results demonstrate that metagenomics can be used as a powerful tool for system wide characterization of the EBPR community as well as for a deeper understanding of the function of specific community members. Furthermore, we discuss and illustrate some of the general pitfalls in metagenomics and stress the need of additional DNA extraction independent information in metagenome studies.

Comparative genomic study of the Penicillium genus elucidates a diverse pangenome and 15 lateral gene transfer events.

The Penicillia are known to produce a wide range natural products-some with devastating outcome for the agricultural industry and others with unexploited potential in different applications. However, a large-scale overview of the biosynthetic potential of different species has been lacking. In this study, we sequenced 93 Penicillium isolates and, together with eleven published genomes that hold similar assembly characteristics, we established a species phylogeny as well as defining a Penicillium pangenome. A total of 5612 genes were shared between ≥ 98 isolates corresponding to approximately half of the average number of genes a Penicillium genome holds. We further identified 15 lateral gene transfer events that have occurred in this collection of Penicillium isolates, which might have played an important role, such as niche adaption, in the evolution of these fungi. The comprehensive characterization of the genomic diversity in the Penicillium genus supersedes single-reference genomes, which do not necessarily capture the entire genetic variation.

A model system for assessing and comparing the ability of exon microarray and tag sequencing to detect genes specific for malignant B-cells.

BACKGROUND: Malignant cells in tumours of B-cell origin account for 0.1% to 98% of the total cell content, depending on disease entity. Recently, gene expression profiles (GEPs) of B-cell lymphomas based on microarray technologies have contributed significantly to improved sub-classification and diagnostics. However, the varying degrees of malignant B-cell frequencies in analysed samples influence the interpretation of the GEPs. Based on emerging next-generation sequencing technologies (NGS) like tag sequencing (tag-seq) for GEP, it is expected that the detection of mRNA transcripts from malignant B-cells can be supplemented. This study provides a quantitative assessment and comparison of the ability of microarrays and tag-seq to detect mRNA transcripts from malignant B-cells. A model system was established by eight serial dilutions of the malignant B-cell lymphoma cell line, OCI-Ly8, into the embryonic kidney cell line, HEK293, prior to parallel analysis by exon microarrays and tag-seq. RESULTS: We identified 123 and 117 differentially expressed genes between pure OCI-Ly8 and HEK293 cells by exon microarray and tag-seq, respectively. There were thirty genes in common, and of those, most were B-cell specific. Hierarchical clustering from all dilutions based on the differentially expressed genes showed that neither technology could distinguish between samples with less than 1% malignant B-cells from non-B-cells. A novel statistical concept was developed to assess the ability to detect single genes for both technologies, and used to demonstrate an inverse proportional relationship with the sample purity. Of the 30 common genes, the detection capability of a representative set of three B-cell specific genes--CD74, HLA-DRA, and BCL6 - was analysed. It was noticed that at least 5%, 13% and 22% sample purity respectively was required for detection of the three genes by exon microarray whereas at least 2%, 4% and 51% percent sample purity of malignant B-cells were required for tag-seq detection. CONCLUSION: A sample purity-dependent loss of the ability to detect genes for both technologies was demonstrated. Taq-seq, in comparison to exon microarray, required slightly less malignant B-cells in the samples analysed in order to detect the two most abundantly expressed of the selected genes. The results show that malignant cell frequency is an important variable, with fundamental impact when interpreting GEPs from both technologies.