Adiponectin secretion by perivascular adipose tissue supports impaired vasodilation in a mouse model of accelerated vascular smooth muscle cell and adipose tissue aging.

OBJECTIVE: Perivascular adipose tissue (PVAT) function during aging has not been investigated in detail so far and its effect on vasodilation remains to be fully elucidated. The aim of this study was to investigate endothelium-dependent vasodilation of thoracic aorta in a mouse model of accelerated, selective vascular smooth muscle and PVAT aging, induced by SM22α-Cre-driven genetic deletion of the endonuclease ERCC1 (SMC-KO mice) versus healthy littermates (LM). We hypothesized that PVAT enhances vasodilation in LM, possibly through adiponectin secretion, which might be compromised in SMC-KO animals. METHODS: Thoracic aorta was isolated from SMC-KO animals and LM and segments with and without PVAT were mounted in wire myography setups. The endothelium-dependent vasodilation was assessed via acetylcholine dose-response curves and pathway contribution was studied. Moreover, adiponectin secretion was measured after stimulating the aortic segments with PVAT with acetylcholine. RESULTS: Adiponectin, secreted by PVAT, led to increased NO-contribution to endothelium-dependent vasodilation in healthy LM, although this did not increase maximum relaxation due to loss of EDH. Endothelium-dependent vasodilation was decreased in SMC-KO animals due to reduced NO-contribution and complete EDH loss. Despite strong lipodystrophy the PVAT partially compensated for lost vasodilation in SMC-KO. LM PVAT contained acetylcholinesterase that attenuated acetylcholine responses. This was lost in SMC-KO. CONCLUSIONS: PVAT-derived adiponectin is able to partially compensate for age-related decline in NO-mediated vasodilation, even during strong lipodystrophy, in conditions of absence of compensating EDH. In aorta with healthy PVAT acetylcholinesterase modulates vascular tone, but this is lost during aging, further compensating for decreased acetylcholine responsiveness. Thus, preservation of adiponectin levels, through relatively increased production in lipodystrophic PVAT, and reduction of cholinesterase might be regulatory mechanisms of the PVAT to preserve cholinergic vasodilation during aging.

Presence of procoagulant peripheral blood mononuclear cells in severe COVID-19 patients relate to ventilation perfusion mismatch and precede pulmonary embolism.

PURPOSE: Pulmonary emboli (PE) contribute substantially to coronavirus disease 2019 (COVID-19) related mortality and morbidity. Immune cell-mediated hyperinflammation drives the procoagulant state in COVID-19 patients, resulting in immunothrombosis. To study the role of peripheral blood mononuclear cells (PBMC) in the procoagulant state of COVID-19 patients, we performed a functional bioassay and related outcomes to the occurrence of PE. Secondary aims were to relate this functional assay to plasma D-dimer levels, ventilation perfusion mismatch and TF expression on monocyte subsets. METHODS: PBMC from an ICU biobank were obtained from 20 patients with a computed tomography angiograph (CTA) proven PE and compared to 15 COVID-19 controls without a proven PE. Functional procoagulant properties of PBMC were measured using a modified fibrin generation time (MC-FGT) assay. Tissue factor (TF) expression on monocyte subsets were measured by flow cytometry. Additional clinical data were obtained from patient records including end-tidal to arterial carbon dioxide gradient. RESULTS: MC-FGT levels were highest in the samples taken closest to the PE detection, similar to the end-tidal to arterial carbon dioxide gradient (ETCO2 - PaCO2), a measurement to quantify ventilation-perfusion mismatch. In patients without proven PE, peak MC-FGT relates to an increase in end-tidal to arterial carbon dioxide gradient. We identified non-classical, CD16 positive monocytes as the subset with increased TF expression. CONCLUSION: We show that the procoagulant state of PBMC could aid in early detection of PE in COVID-19 ICU patients. Combined with end-tidal to ETCO2 - PaCO2 gradient, these tests could improve early detection of PE on the ICU.

Diagnosing and monitoring the characteristics of Acanthamoeba keratitis using slit scanning and laser scanning in vivo confocal microscopy.

INTRODUCTION: Acanthamoeba keratitis (AK) is a serious and potentially blinding ocular infection caused by the free-living amoeba, Acanthamoeba. In vivo confocal microscopy (IVCM) is a non-invasive device which has been proven of great use to diagnose Acanthamoeba infections immediately. The aim of this review was to establish different patterns and signs of AK that appear on the IVCM both before and after treatment. METHODS: A systematic review of the literature from 1974 until September 2021 was performed using Embase and PubMed, following The Preferred Reporting Items for Systematic reviews and Meta-Analyses (PRISMA) guidelines. RESULTS: Twenty different signs of AK were observed using IVCM. The included studies used vastly different criteria to diagnose infections, ranging from just 1 to 13 of the signs, demonstrating the current lack of a standardised diagnosis of this infection using the IVCM. The appearance of double wall cysts, trophozoites, signet rings, target signs and clusters were shown to be pathognomonic to AK infections. Bright spots located in the corneal epithelium were demonstrated as non-reliable predictors of AK. The presence of cysts in clusters and single file can predict the need for corneal transplantation. The morphological changes in cysts using the IVCM following treatment were described as breaking down to hollow forms and occasionally surrounded by black cavities. Using this information, a visual guideline for identifying AK signs in diagnosis and follow-up using IVCM was created. CONCLUSION: Increased awareness of the different signs and patterns of AK that appear on the IVCM is crucial in order to correctly identify an infection and increase the potential of this device. Our guidelines presented here can be used, but further studies are needed in order to determine the relationship and aetiology of these signs and cellular changes on the IVCM both before and after anti-amoeba treatment.

Aetiology, risk factors and preventive strategies for niche development: A review.

The increase in caesarean sections (CS) has resulted in an increase in women with a uterine niche. The exact aetiology of niche development has yet to be elucidated but is likely multifactorial. This study aimed to give a systematic overview of the available literature on histopathological features, risk factors and results of preventive strategies on niche development to gain more insight into the underlying mechanisms. Based on current published data histopathological findings associated with niche development were necrosis, fibrosis, inflammation, adenomyosis and insufficient approximation. Patient-related risk factors included multiple CS, BMI and smoking. Labour-related factors were CS before onset of labour, extended cervical dilatation, premature rupture of membranes and presenting part of the fetus at CS below the pelvic inlet. Preventive strategies should focus on the optimal level of incision, training of surgeons and full-thickness closure of the myometrium (single or double-layer) using non-locking sutures. Conflicting data exist concerning the effect of endometrial inclusion. Future studies without heterogeneity in population, using standardized performance of the CS after proper training and using standardized niche evaluation with a relevant core outcome set are required to allow meta-analyses and to develop evidence-based preventive strategies. These studies are needed to reduce the prevalence of niches and prevent complications in subsequent pregnancies such as caesarean scar pregnancies.

Scoping literature review and focus groups with healthcare professionals on psychosocial and lifestyle assessments for childhood obesity care.

BACKGROUND: Childhood obesity is a complex disease resulting from the interaction of multiple factors. The effective management of childhood obesity requires assessing the psychosocial and lifestyle factors that may play a role in the development and maintenance of obesity. This study centers on available scientific literature on psychosocial and lifestyle assessments for childhood obesity, and experiences and views of healthcare professionals with regard to assessing psychosocial and lifestyle factors within Dutch integrated care. METHODS: Two methods were used. First, a scoping review (in PubMed, Embase, PsycInfo, IBSS, Scopus and Web of Science) was performed by systematically searching for scientific literature on psychosocial and lifestyle assessments for childhood obesity. Data were analysed by extracting data in Microsoft Excel. Second, focus group discussions were held with healthcare professionals from a variety of disciplines and domains to explore their experiences and views about assessing psychosocial and lifestyle factors within Dutch integrated care. Data were analysed using template analysis, complemented with open coding in MAXQDA. RESULTS: The results provide an overview of relevant psychosocial and lifestyle factors that should be assessed and were classified as child, family, parental and lifestyle (e.g. nutrition, physical activity and sleep factors) and structured into psychological and social aspects. Insights into how to assess psychosocial and lifestyle factors were identified as well, including talking about psychosocial factors, lifestyle and weight; the professional-patient relationship; and attitudes of healthcare professionals. CONCLUSIONS: This study provides an overview of psychosocial and lifestyle factors that should be identified within the context of childhood obesity care, as they may contribute to the development and maintenance of obesity. The results highlight the importance of both what is assessed and how it is assessed. The results of this study can be used to develop practical tools for facilitating healthcare professionals in conducting a psychosocial and lifestyle assessment.

Safety and tolerability of stereotactic radiotherapy combined with durvalumab with or without tremelimumab in advanced non-small cell lung cancer, the phase I SICI trial.

INTRODUCTION: This phase I study primarily addresses the safety and tolerability of Stereotactic radiotherapy on the primary tumor combined with double Immune Checkpoint Inhibition (SICI) in patients with non-small cell lung cancer (NSCLC). Increasing the release of neoantigens by radiotherapy might enhance response to immunotherapy. Especially, by targeting trunk mutations in the primary tumor. MATERIALS AND METHODS: In three sequential cohorts, immunotherapy regimes combined with stereotactic body radiotherapy (SBRT) on the primary tumor (1x20 Gy on 9 cc) were studied in stage IIIB/IV NSCLC patients progressing on chemotherapy. The first cohort (n = 3) received durvalumab. The second (n = 6) received a combination of tremelimumab and durvalumab followed by durvalumab monotherapy. The third cohort (n = 6) was similar except that the combination was reversed. Descriptive statistics were used to assess safety parameters and the exploratory outcomes of efficacy. Adverse events were reported using NCI CTCAE version 4.03. Exhaled breath was analyzed at baseline. RESULTS: Fifteen patients were included. Median irradiated volume was 9.13 cc, on a median primary tumor volume of 79 cc. There were seven patients with grade 1-2, and two patients with grade 3 treatment related adverse events. There was 1 dose limiting toxicity (colitis) with double immunotherapy. CONCLUSION: The combination of SBRT to the primary tumor and double immunotherapy in advanced NSCLC patients is safe and feasible.

Fusarium: more than a node or a foot-shaped basal cell.

Recent publications have argued that there are potentially serious consequences for researchers in recognising distinct genera in the terminal fusarioid clade of the family Nectriaceae. Thus, an alternate hypothesis, namely a very broad concept of the genus Fusarium was proposed. In doing so, however, a significant body of data that supports distinct genera in Nectriaceae based on morphology, biology, and phylogeny is disregarded. A DNA phylogeny based on 19 orthologous protein-coding genes was presented to support a very broad concept of Fusarium at the F1 node in Nectriaceae. Here, we demonstrate that re-analyses of this dataset show that all 19 genes support the F3 node that represents Fusarium sensu stricto as defined by F. sambucinum (sexual morph synonym Gibberella pulicaris). The backbone of the phylogeny is resolved by the concatenated alignment, but only six of the 19 genes fully support the F1 node, representing the broad circumscription of Fusarium. Furthermore, a re-analysis of the concatenated dataset revealed alternate topologies in different phylogenetic algorithms, highlighting the deep divergence and unresolved placement of various Nectriaceae lineages proposed as members of Fusarium. Species of Fusarium s. str. are characterised by Gibberella sexual morphs, asexual morphs with thin- or thick-walled macroconidia that have variously shaped apical and basal cells, and trichothecene mycotoxin production, which separates them from other fusarioid genera. Here we show that the Wollenweber concept of Fusarium presently accounts for 20 segregate genera with clear-cut synapomorphic traits, and that fusarioid macroconidia represent a character that has been gained or lost multiple times throughout Nectriaceae. Thus, the very broad circumscription of Fusarium is blurry and without apparent synapomorphies, and does not include all genera with fusarium-like macroconidia, which are spread throughout Nectriaceae (e.g., Cosmosporella, Macroconia, Microcera). In this study four new genera are introduced, along with 18 new species and 16 new combinations. These names convey information about relationships, morphology, and ecological preference that would otherwise be lost in a broader definition of Fusarium. To assist users to correctly identify fusarioid genera and species, we introduce a new online identification database, Fusarioid-ID, accessible at www.fusarium.org. The database comprises partial sequences from multiple genes commonly used to identify fusarioid taxa (act1, CaM, his3, rpb1, rpb2, tef1, tub2, ITS, and LSU). In this paper, we also present a nomenclator of names that have been introduced in Fusarium up to January 2021 as well as their current status, types, and diagnostic DNA barcode data. In this study, researchers from 46 countries, representing taxonomists, plant pathologists, medical mycologists, quarantine officials, regulatory agencies, and students, strongly support the application and use of a more precisely delimited Fusarium (= Gibberella) concept to accommodate taxa from the robust monophyletic node F3 on the basis of a well-defined and unique combination of morphological and biochemical features. This F3 node includes, among others, species of the F. fujikuroi, F. incarnatum-equiseti, F. oxysporum, and F. sambucinum species complexes, but not species of Bisifusarium [F. dimerum species complex (SC)], Cyanonectria (F. buxicola SC), Geejayessia (F. staphyleae SC), Neocosmospora (F. solani SC) or Rectifusarium (F. ventricosum SC). The present study represents the first step to generating a new online monograph of Fusarium and allied fusarioid genera (www.fusarium.org).

Human Respiratory Syncytial Virus Subgroup A and B Infections in Nasal, Bronchial, Small-Airway, and Organoid-Derived Respiratory Cultures.

Human respiratory syncytial virus (HRSV) is the leading cause of bronchiolitis in infants. Two subgroups of HRSV (A and B) routinely cocirculate. Most research has been performed with HRSV-A strains because these are easier to culture than HRSV-B strains. In this study, we aimed to compare the replicative fitness and HRSV-induced innate cytokine responses of HRSV-A and HRSV-B strains in disease-relevant cell culture models. We used two recombinant (r) clinical isolate-based HRSV strains (A11 and B05) and one recombinant laboratory-adapted HRSV strain (A2) to infect commercially available nasal, bronchial, and small-airway cultures. Epithelial cells from all anatomical locations were susceptible to HRSV infection despite the induction of a dominant type III interferon response. Subgroup A viruses disseminated and replicated faster than the subgroup B virus. Additionally, we studied HRSV infection and innate responses in airway organoids (AOs) cultured at air-liquid interface (ALI). Results were similar to the commercially obtained bronchial cells. In summary, we show that HRSV replicates well in cells from both the upper and the lower airways, with a slight replicative advantage for subgroup A viruses. Lastly, we showed that AOs cultured at ALI are a valuable model for studying HRSV ex vivo and that they can be used in the future to study factors that influence HRSV disease severity.IMPORTANCE Human respiratory syncytial virus (HRSV) is the major cause of bronchiolitis and pneumonia in young infants and causes almost 200,000 deaths per year. Currently, there is no vaccine or treatment available, only a prophylactic monoclonal antibody (palivizumab). An important question in HRSV pathogenesis research is why only a fraction (1 to 3%) of infants develop severe disease. Model systems comprising disease-relevant HRSV isolates and accurate and reproducible cell culture models are indispensable to study infection, replication, and innate immune responses. Here, we used differentiated AOs cultured at ALI to model the human airways. Subgroup A viruses replicated better than subgroup B viruses, which we speculate fits with epidemiological findings that subgroup A viruses cause more severe disease in infants. By using AOs cultured at ALI, we present a highly relevant, robust, and reproducible model that allows for future studies into what drives severe HRSV disease.

Aspergillus niger uses the peroxisomal CoA-dependent ß-oxidative genes to degrade the hydroxycinnamic acids caffeic acid, ferulic acid, and p-coumaric acid.

Aromatic compounds are important molecules which are widely applied in many industries and are mainly produced from nonrenewable sources. Renewable sources such as plant biomass are interesting alternatives for the production of aromatic compounds. Ferulic acid and p-coumaric acid, a precursor for vanillin and p-vinyl phenol, respectively, can be released from plant biomass by the fungus Aspergillus niger. The degradation of hydroxycinnamic acids such as caffeic acid, ferulic acid, and p-coumaric acid has been observed in many fungi. In A. niger, multiple metabolic pathways were suggested for the degradation of hydroxycinnamic acids. However, no genes were identified for these hydroxycinnamic acid metabolic pathways. In this study, several pathway genes were identified using whole-genome transcriptomic data of A. niger grown on different hydroxycinnamic acids. The genes are involved in the CoA-dependent ß-oxidative pathway in fungi. This pathway is well known for the degradation of fatty acids, but not for hydroxycinnamic acids. However, in plants, it has been shown that hydroxycinnamic acids are degraded through this pathway. We identified genes encoding hydroxycinnamate-CoA synthase (hcsA), multifunctional ß-oxidation hydratase/dehydrogenase (foxA), 3-ketoacyl CoA thiolase (katA), and four thioesterases (theA-D) of A. niger, which were highly induced by all three tested hydroxycinnamic acids. Deletion mutants revealed that these genes were indeed involved in the degradation of several hydroxycinnamic acids. In addition, foxA and theB are also involved in the degradation of fatty acids. HcsA, FoxA, and KatA contained a peroxisomal targeting signal and are therefore predicted to be localized in peroxisomes. KEY POINTS: • Metabolism of hydroxycinnamic acid was investigated in Aspergillus niger • Using transcriptome data, multiple CoA-dependent ß-oxidative genes were identified. • Both foxA and theB are involved in hydroxycinnamate but also fatty acid metabolism.

Distal muscle weakness and optic atrophy without central nervous system involvement in a patient with a homozygous missense mutation in the C19ORF12-gene.

Variants of the C19ORF12-gene have been described in patients with spastic paraplegia type 43 and in patients with mitochondrial membrane protein-associated neurodegeneration (MPAN), a subtype of neurodegeneration associated with brain iron accumulation (NBIA). In both subtypes optic atrophy and neuropathy have been frequently described. This case report describes a patient with bilateral optic atrophy and severe distal muscle weakness based on motor neuropathy without involvement of the central nervous system. Exome sequencing revealed a homozygous pathogenic missense variant (c.187G>C;p.Ala63Pro) of the C19ORF12-gene while iron deposits were absent on repeat MR-imaging of the brain, thus showing that peripheral neuropathy and optic neuropathy can be the sole manifestations of the C19ORF12-related disease spectrum whereby iron accumulation in the brain may be absent.

Case Report: Adequate T and B Cell Responses in a SARS-CoV-2 Infected Patient After Immune Checkpoint Inhibition.

After the COVID-19 outbreak, non-evidence based guidelines were published to advise clinicians on the adjustment of oncological treatment during this pandemic. As immune checkpoint inhibitors directly affect the immune system, concerns have arisen about the safety of immunotherapy during this pandemic. However, data on the immune response in oncology patients treated with immunotherapy are still lacking. Here, we present the adaptive immune response in a SARS-CoV-2 infected patient who was treated with immune checkpoint inhibitors for advanced renal cell cancer. To evaluate the immune response in this patient, the number of T cells and their major subsets were measured according to expression of markers for co-signalling, maturation, and chemotaxis at baseline, during therapy, and during the SARS-CoV-2 infection. In addition, plasma samples were analyzed for IgM and IgG antibodies and the ability of these antibodies to neutralise SARS-CoV-2. Despite several risk factors for an impaired immune response to SARS-CoV-2, both T- and B-cell responses were observed. Moreover, after treatment with immune checkpoint inhibitors, a sufficient cellular and humoral immune response was achieved in this SARS-CoV-2 infected patient. These findings warrant renewed discussion on withholding of immune checkpoint inhibitors during an ongoing COVID-19 pandemic.

The influence of prenatal and intrapartum antibiotics on intestinal microbiota colonisation in infants: A systematic review.

OBJECTIVES: The intestinal microbiota develops in early infancy and is essential for health status early and later in life. In this review we focus on the effect of prenatal and intrapartum maternally administered antibiotics on the infant intestinal microbiota. METHODS: A systematic literature search was conducted in PubMed and EMBASE. All studies reporting effect on diversity or microbiota profiles were included. RESULTS: A total of 4.030 records were encountered. A total of 24 articles were included in the final analysis. Infants from mothers exposed to antibiotics during delivery showed a decreased microbial diversity compared to non-exposed infants. The microbiota of infants exposed to antibiotics was characterised by a decreased abundance of Bacteriodetes and Bifidobacteria, with a concurrent increase of Proteobacteria. These effects were most pronounced in term vaginally born infants. CONCLUSION: Maternal administration of antibiotics seems to have profound effects on the infant gut microbiota colonisation. Interpretation of microbiota aberrations in specific populations, such as preterm and caesarean born infants, is complicated by multiple confounding factors and by lack of high quality studies and high heterogeneity in study design. Further research is needed to investigate the potential short- and long-term clinical consequences of these microbial alterations.

Evolutionary adaptation of Aspergillus niger for increased ferulic acid tolerance.

AIMS: To create an Aspergillus niger mutant with increased tolerance against ferulic acid using evolutionary adaptation. METHODS AND RESULTS: Evolutionary adaptation of A. niger N402 was performed by consecutive growth on increasing concentrations of ferulic acid in the presence of 25 mmol l-1 d-fructose, starting from 0·5 mmol l-1 and ending with 5 mmol l-1 ferulic acid. The A. niger mutant obtained after six months, named Fa6, showed increased ferulic acid tolerance compared to the parent. In addition, Fa6 has increased ferulic acid consumption and a higher conversion rate, suggesting that the mutation affects aromatic metabolism of this species. Transcriptome analysis of the evolutionary mutant on ferulic acid revealed a distinct gene expression profile compared to the wild type. Further analysis of this mutant and the parent strain provided the first experimental confirmation that A. niger converts coniferyl alcohol to ferulic acid. CONCLUSIONS: The evolutionary adaptive A. niger mutant Fa6 has beneficial mutations that increase the tolerance, conversion rate and uptake of ferulic acid. SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrates that evolutionary adaptation is a powerful tool to modify micro-organisms towards increased tolerance to harsh conditions, which is beneficial for various industrial applications.

Prediction of response to anti-PD-1 therapy in patients with non-small-cell lung cancer by electronic nose analysis of exhaled breath.

BACKGROUND: Immune checkpoint inhibitors have improved survival outcome of advanced non-small-cell lung cancer (NSCLC). However, most patients do not benefit. Therefore, biomarkers are needed that accurately predict response. We hypothesized that molecular profiling of exhaled air may capture the inflammatory milieu related to the individual responsiveness to anti-programmed death ligand 1 (PD-1) therapy. This study aimed to determine the accuracy of exhaled breath analysis at baseline for assessing nonresponders versus responders to anti-PD-1 therapy in NSCLC patients. METHODS: This was a prospective observational study in patients receiving checkpoint inhibitor therapy using both a training and validation set of NSCLC patients. At baseline, breath profiles were collected in duplicate by a metal oxide semiconductor electronic nose (eNose) positioned at the rear end of a pneumotachograph. Patients received nivolumab or pembrolizumab of which the efficacy was assessed by Response Evaluation Criteria in Solid Tumors (RECIST) version 1.1 at 3-month follow-up. Data analysis involved advanced signal-processing and statistics based on independent t-tests followed by linear discriminant and receiver operating characteristic (ROC) analysis. RESULTS: Exhaled breath data of 143 NSCLC patients (training: 92, validation: 51) were available at baseline. ENose sensors contributed significantly (P < 0.05) at baseline in differentiating between patients with different responses at 3 months of anti-PD-1 treatment. The eNose sensors were combined into a single biomarker with an ROC-area under the curve (AUC) of 0.89 [confidence interval (CI) 0.82-0.96]. This AUC was confirmed in the validation set: 0.85 (CI 0.75-0.96). CONCLUSION: ENose assessment was effective in the noninvasive prediction of individual patient responses to immunotherapy. The predictive accuracy and efficacy of the eNose for discrimination of immunotherapy responder types were replicated in an independent validation set op patients. This finding can potentially avoid application of ineffective treatment in identified probable nonresponders.

Workshop on the validation and regulatory acceptance of innovative 3R approaches in regulatory toxicology - Evolution versus revolution.

At a joint workshop organized by RIVM and BfR, international experts from governmental institutes, regulatory agencies, industry, academia and animal welfare organizations discussed and provided recommendations for the development, validation and implementation of innovative 3R approaches in regulatory toxicology. In particular, an evolutionary improvement of our current approach of test method validation in the context of defined approaches or integrated testing strategies was discussed together with a revolutionary approach based on a comprehensive description of the physiological responses of the human body to chemical exposure and the subsequent definition of relevant and predictive in vitro, in chemico or in silico methods. A more comprehensive evaluation of biological relevance, scientific validity and regulatory purpose of new test methods and assessment strategies together with case studies that provide practical experience with new approaches were discussed as essential steps to build up the necessary confidence to facilitate regulatory acceptance.

Genomic and exoproteomic diversity in plant biomass degradation approaches among Aspergilli.

We classified the genes encoding carbohydrate-active enzymes (CAZymes) in 17 sequenced genomes representing 16 evolutionarily diverse Aspergillus species. We performed a phylogenetic analysis of the encoding enzymes, along with experimentally characterized CAZymes, to assign molecular function to the Aspergilli CAZyme families and subfamilies. Genome content analysis revealed that the numbers of CAZy genes per CAZy family related to plant biomass degradation follow closely the taxonomic distance between the species. On the other hand, growth analysis showed almost no correlation between the number of CAZyme genes and the efficiency in polysaccharide utilization. The exception is A. clavatus where a reduced number of pectinolytic enzymes can be correlated with poor growth on pectin. To gain detailed information on the enzymes used by Aspergilli to breakdown complex biomass, we conducted exoproteome analysis by mass spectrometry. These results showed that Aspergilli produce many different enzymes mixtures in the presence of sugar beet pulp and wheat bran. Despite the diverse enzyme mixtures produced, species of section Nigri, A. aculeatus, A. nidulans and A. terreus, produce mixtures of enzymes with activities that are capable of digesting all the major polysaccharides in the available substrates, suggesting that they are capable of degrading all the polysaccharides present simultaneously. For the other Aspergilli, typically the enzymes produced are targeted to a subset of polysaccharides present, suggesting that they can digest only a subset of polysaccharides at a given time.

Duplications and losses of genes encoding known elements of the stress defence system of the Aspergilli contribute to the evolution of these filamentous fungi but do not directly influence their environmental stress tolerance.

The contribution of stress protein duplication and deletion events to the evolution of the Aspergilli was studied. We performed a large-scale homology analysis of stress proteins and generated and analysed three stress defence system models based on Saccharomyces cerevisiae, Schizosaccharomyces pombe and Aspergillus nidulans. Although both yeast-based and A. nidulans-based models were suitable to trace evolutionary changes, the A. nidulans-based model performed better in mapping stress protein radiations. The strong Mantel correlation found between the positions of species in the phylogenetic tree on the one hand and either in the A. nidulans-based or S. cerevisiae-based models on the other hand demonstrated that stress protein expansions and reductions contributed significantly to the evolution of the Aspergilli. Interestingly, stress tolerance attributes correlated well with the number of orthologs only for a few stress proteins. Notable examples are Ftr1 iron permease and Fet3 ferro-O2-oxidoreductase, elements of the reductive iron assimilation pathway, in the S. cerevisiae-based model, as well as MpkC, a HogA-like mitogen activated protein kinase in the A. nidulans-based model. In the case of the iron assimilation proteins, the number of orthologs showed a positive correlation with H2O2-induced stress tolerance while the number of MpkC orthologs correlated positively with Congo Red induced cell wall stress, sorbitol induced osmotic stress and H2O2 induced oxidative stress tolerances. For most stress proteins, changes in the number of orthologs did not correlate well with any stress tolerance attributes. As a consequence, stress tolerance patterns of the studied Aspergilli did not correlate with either the sets of stress response proteins in general or with the phylogeny of the species studied. These observations suggest that stress protein duplication and deletion events significantly contributed to the evolution of stress tolerance attributes of Aspergilli. In contrast, there are other processes, which may counterbalance the effects of stress gene duplications or deletions including (i) alterations in the structures of stress proteins leading to changes in their biological activities, (ii) varying biosynthesis of stress proteins, (iii) rewiring stress response regulatory networks or even (iv) acquiring new stress response genes by horizontal gene transfer. All these multilevel changes are indispensable for the successful adaptation of filamentous fungi to altering environmental conditions, especially when these organisms are entering new ecological niches.

The gold-standard genome of Aspergillus niger NRRL 3 enables a detailed view of the diversity of sugar catabolism in fungi.

The fungal kingdom is too large to be discovered exclusively by classical genetics. The access to omics data opens a new opportunity to study the diversity within the fungal kingdom and how adaptation to new environments shapes fungal metabolism. Genomes are the foundation of modern science but their quality is crucial when analysing omics data. In this study, we demonstrate how one gold-standard genome can improve functional prediction across closely related species to be able to identify key enzymes, reactions and pathways with the focus on primary carbon metabolism. Based on this approach we identified alternative genes encoding various steps of the different sugar catabolic pathways, and as such provided leads for functional studies into this topic. We also revealed significant diversity with respect to genome content, although this did not always correlate to the ability of the species to use the corresponding sugar as a carbon source.

Templated co-assembly into nanorods of polyanions and artificial virus capsid proteins.

Recombinant triblock polypeptides C-Sn-B, where C is a 400 amino acid long hydrophilic random coil block, Sn is a multimer of the silk-like octapeptide S = (GAGAGAGQ), and B = K12 is an oligolysine, have previously been shown to encapsulate double stranded DNA into rod-shaped, virus-like particles. In order to gain insight of the co-assembly process, and in order to be able to use these proteins for templating other types of nanorods, we here explore their co-assembly with a range of polyanionic templates: poly(acrylic acids) (PAA) of a wide range of lengths, poly(styrene sulphonate) (PSS) and the stiff anionic polysaccharide xanthan. The formation of the complexes was characterized using Dynamic Light Scattering (DLS), cryogenic Transmission Electronic Microscopy (Cryo-TEM) and Atomic Force Microscopy (AFM). Except at very high molar masses, we find that flexible anionic PAA and PSS lead to co-assembly of proteins with single polyanion chains into nanorods, with a packing factor as expected on the basis of charge stochiometry. Only for very long PAA templates (8 × 105 Da) we find evidence for heterogeneous complexes with thin and thick sections. For the very stiff xanthan chains, we find that its stiffness precludes co-assembly with the artificial viral capsid proteins into condensed and regular nanorods. Given the simple and robust formation of rod-like structures with a range of polyanionic templates, we anticipate that the artificial virus proteins will be useful for preparing high-aspect ratio nanoparticles and scaffolds of precise size and find applications in nanotechnology and materials science for which currently natural rod-like viruses are being explored.