Antibiotic production in Streptomyces is organized by a division of labor through terminal genomic differentiation.

One of the hallmark behaviors of social groups is division of labor, where different group members become specialized to carry out complementary tasks. By dividing labor, cooperative groups increase efficiency, thereby raising group fitness even if these behaviors reduce individual fitness. We find that antibiotic production in colonies of Streptomyces coelicolor is coordinated by a division of labor. We show that S. coelicolor colonies are genetically heterogeneous because of amplifications and deletions to the chromosome. Cells with chromosomal changes produce diversified secondary metabolites and secrete more antibiotics; however, these changes reduced individual fitness, providing evidence for a trade-off between antibiotic production and fitness. Last, we show that colonies containing mixtures of mutants and their parents produce significantly more antibiotics, while colony-wide spore production remains unchanged. By generating specialized mutants that hyper-produce antibiotics, streptomycetes reduce the fitness costs of secreted secondary metabolites while maximizing the yield and diversity of these products.

Extracellular vesicles containing oncogenic mutant ß-catenin activate Wnt signalling pathway in the recipient cells.

Mutations in ß-catenin, especially at the residues critical for its degradation, render it constitutively active. Here, we show that mutant ß-catenin can be transported via extracellular vesicles (EVs) and activate Wnt signalling pathway in the recipient cells. An integrative proteogenomic analysis identified the presence of mutated ß-catenin in EVs secreted by colorectal cancer (CRC) cells. Follow-up experiments established that EVs released from LIM1215 CRC cells stimulated Wnt signalling pathway in the recipient cells with wild-type ß-catenin. SILAC-based quantitative proteomics analysis confirmed the transfer of mutant ß-catenin to the nucleus of the recipient cells. In vivo tracking of DiR-labelled EVs in mouse implanted with RKO CRC cells revealed its bio-distribution, confirmed the activation of Wnt signalling pathway in tumour cells and increased the tumour burden. Overall, for the first time, this study reveals that EVs can transfer mutant ß-catenin to the recipient cells and promote cancer progression.

Extracellular vesicles secreted by Saccharomyces cerevisiae are involved in cell wall remodelling.

Extracellular vesicles (EVs) are membranous vesicles that are released by cells. In this study, the role of the Endosomal Sorting Complex Required for Transport (ESCRT) machinery in the biogenesis of yeast EVs was examined. Knockout of components of the ESCRT machinery altered the morphology and size of EVs as well as decreased the abundance of EVs. In contrast, strains with deletions in cell wall biosynthesis genes, produced more EVs than wildtype. Proteomic analysis highlighted the depletion of ESCRT components and enrichment of cell wall remodelling enzymes, glucan synthase subunit Fks1 and chitin synthase Chs3, in yeast EVs. Interestingly, EVs containing Fks1 and Chs3 rescued the yeast cells from antifungal molecules. However, EVs from fks1∆ or chs3∆ or the vps23∆chs3∆ double knockout strain were unable to rescue the yeast cells as compared to vps23∆ EVs. Overall, we have identified a potential role for yeast EVs in cell wall remodelling.

Analysis of extracellular vesicles generated from monocytes under conditions of lytic cell death.

Extracellular vesicles (EVs) are an important class of membrane-bound structures that have been widely investigated for their roles in intercellular communication in the contexts of tumor progression, vascular function, immunity and regenerative medicine. Much of the current knowledge on the functions of EVs pertains to those derived from viable cells (e.g. exosomes and microvesicles) or apoptotic cells (e.g. apoptotic bodies) whilst the generation of EVs from dying cells under non-apoptotic conditions remains poorly characterized. Herein, the release of EVs from THP-1 monocytes under conditions of primary necrosis, secondary necrosis and pyroptosis, was investigated. A comprehensive analysis of THP-1-derived EVs revealed that cells undergoing lytic forms of cell death generated a high number of EVs compared with viable or apoptotic cells in vitro. Differential centrifugation via 16,000 g and 100,000 g revealed that dying THP-1 cells release both medium and small EVs, respectively, consistent with the known characteristics of microvesicles and/or exosomes. In addition, large EVs isolated via 2000 g centrifugation were also present in all samples. These findings suggest that lytic cell death under both sterile and non-sterile inflammatory conditions induces monocytes to generate EVs, which could potentially act as mediators of cell-to-cell communication.

Author Correction: Rapid de novo assembly of the European eel genome from nanopore sequencing reads.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has not been fixed in the paper.

Exosomes from N-Myc amplified neuroblastoma cells induce migration and confer chemoresistance to non-N-Myc amplified cells: implications of intra-tumour heterogeneity.

Neuroblastoma accounts for 15% of childhood cancer mortality. Amplification of the oncogene N-Myc is a well-established poor prognostic marker for neuroblastoma. Whilst N-Myc amplification status strongly correlates with higher tumour aggression and resistance to treatment, the role of N-Myc in the aggressiveness of the disease is poorly understood. Exosomes are released by many cell types including cancer cells and are implicated as key mediators in cell-cell communication via the transfer of molecular cargo. Hence, characterising the exosomal protein components from N-Myc amplified and non-amplified neuroblastoma cells will improve our understanding on their role in the progression of neuroblastoma. In this study, a comparative proteomic analysis of exosomes isolated from cells with varying N-Myc amplification status was performed. Label-free quantitative proteomic profiling revealed 968 proteins that are differentially abundant in exosomes released by the neuroblastoma cells. Gene ontology-based analysis highlighted the enrichment of proteins involved in cell communication and signal transduction in N-Myc amplified exosomes. Treatment of SH-SY5Y cells with N-Myc amplified SK-N-BE2 cell-derived exosomes increased the migratory potential, colony forming abilities and conferred resistance to doxorubicin induced apoptosis. Incubation of exosomes from N-Myc knocked down SK-N-BE2 cells abolished the transfer of resistance to doxorubicin induced apoptosis. These findings suggest that exosomes could play a pivotal role in N-Myc-driven aggressive neuroblastoma and transfer of chemoresistance between cells. Abbreviations: RNA = ribonucleic acid; DNA = deoxyribonucleic acid; FCS = foetal calf serum; NTA = nanoparticle tracking analysis; LC-MS = liquid chromatography-mass spectrometry; KD = knockdown; LTQ = linear trap quadropole; TEM = transmission electron microscopy.

Serum Neurofilament light correlates with CADASIL disease severity and survival.

Objective: To validate whether serum Neurofilament Light-chain (NfL) levels correlate with disease severity in CADASIL, and to determine whether serum NfL predicts disease progression and survival. Methods: Fourty-one (pre-) manifest individuals with CADASIL causing NOTCH3 mutations and 22 healthy controls were recruited from CADASIL families. At baseline, MRI-lesion load and clinical severity was determined and serum was stored. Disease progression was measured in 30/41 patients at 7-year follow-up, and survival of all individuals was determined at 17-year follow-up. Serum NfL levels were quantified using an ultra-sensitive molecule array. Generalized estimated equation regression (GEE) was used to analyze association between serum NfL, MRI-lesion load, disease severity, and disease progression. With GEE-based Cox regression, survival was analyzed. Results: At baseline, serum NfL levels correlated with MRI-lesion load [lacune count (s = 0.64, P = 0.002), brain atrophy (r = -0.50, P = 0.001), and microbleed count (s = 0.48, P = 0.044)], cognition [CAMCOG (s = -0.45, P = 0.010), MMSE (r = -0.61, P = 0.003), GIT (r = -0.61, P < 0.001), TMT-A (r = 0.70, P < 0.001)) and disability (mRS (r = 0.70, P = 0.002)]. Baseline serum NfL predicted 7-year changes in disability (B = 0.34, P < 0.001) and cognition (CAMCOG B = -4.94, P = 0.032), as well as 17-year survival. Higher NfL levels were associated with increased mortality (HR=1.8 per twofold increase in NfL levels, P = 0.006). Interpretation: Serum NfL levels correlate with disease severity, disease progression and 17-year survival in CADASIL patients. Serum NfL is a promising biomarker to monitor and predict disease course in CADASIL, as well as potentially assessing therapeutic response in future clinical trials.

Proteomic Profiling of Exosomes Secreted by Breast Cancer Cells with Varying Metastatic Potential.

Cancer cells actively release extracellular vesicles, including exosomes, into the surrounding microenvironment. Exosomes play pleiotropic roles in cancer progression and metastasis, including invasion, angiogenesis, and immune modulation. However, the proteome profile of exosomes isolated from cells with different metastatic potential and the role of these exosomes in driving metastasis remains unclear. Here, we conduct a comparative proteomic analysis of exosomes isolated from several genetically related mouse breast tumor lines with different metastatic propensity. The amount of exosomes produced and the extent of cancer-associated protein cargo vary significantly between nonmetastatic and metastatic cell-derived exosomes. Metastatic cell-derived exosomes contain proteins that promote migration, proliferation, invasion, and angiogenesis while the nonmetastatic cell-derived exosomes contain proteins involved in cell-cell/cell-matrix adhesion and polarity maintenance. The metastatic exosomes contain a distinct set of membrane proteins including Ceruloplasmin and Metadherin which could presumably aid in targeting the primary cancer cells to specific metastatic sites. Hence, it can be concluded that the exosomes contain different protein cargo based on the host cells metastatic properties and can facilitate in the dissemination of the primary tumors to distant sites.

Rapid de novo assembly of the European eel genome from nanopore sequencing reads.

We have sequenced the genome of the endangered European eel using the MinION by Oxford Nanopore, and assembled these data using a novel algorithm specifically designed for large eukaryotic genomes. For this 860 Mbp genome, the entire computational process takes two days on a single CPU. The resulting genome assembly significantly improves on a previous draft based on short reads only, both in terms of contiguity (N50 1.2 Mbp) and structural quality. This combination of affordable nanopore sequencing and light weight assembly promises to make high-quality genomic resources accessible for many non-model plants and animals.

Insulin Mediated Activation of PI3K/Akt Signalling Pathway Modifies the Proteomic Cargo of Extracellular Vesicles.

Epidemiological studies suggest that diabetes and obesity increases the risk of colorectal cancer (CRC) and lowers the patient survival rate. An important attribute in diabetes and obesity is the presence of high levels of growth factors including insulin in blood which can activate the PI3K/Akt signalling pathway. Dysregulation of PI3K/Akt signalling pathway leads to sustained proliferative signals thereby allowing the cells susceptible to cancer. Extracellular vesicles (EVs), secreted nanovesicles of endocytic origin, are implicated in mediating the transfer of oncogenic cargo in the tumour microenvironment. In this study, CRC cells were treated with insulin to activate PI3K/Akt signaling pathway. Insulin treatment significantly increased the number of EVs secreted by CRC cells. Furthermore, pAkt was exclusively packaged in EVs secreted by PI3K/Akt activated cells. Quantitative proteomics analysis confirmed that the protein cargo of EVs are modified upon activation of PI3K/Akt signaling pathway. Bioinformatics analysis highlighted the enrichment of proteins implicated in cell proliferation in EVs secreted by PI3K/Akt activated cells. Furthermore, incubation of EVs secreted by PI3K/Akt activated cells induced proliferation in recipient CRC cells. These findings suggest that EVs can amplify the signal provided by the growth factors in the tumor microenvironment and hence aid in cancer progression.

Bovine milk-derived exosomes from colostrum are enriched with proteins implicated in immune response and growth.

Exosomes are extracellular vesicles secreted by multiple cell types into the extracellular space. They contain cell-state specific cargos which often reflects the (patho)physiological condition of the cells/organism. Milk contains high amounts of exosomes and it is unclear whether their cargo is altered based on the lactation stage of the organism. Here, we isolated exosomes from bovine milk that were obtained at various stages of lactation and examined the content by quantitative proteomics. Exosomes were isolated by OptiPrep density gradient centrifugation from milk obtained from cow after 24, 48 and 72 h post calving. As control, exosomes were also isolated from cows during mid-lactation period which has been referred to as mature milk (MM). Biochemical and biophysical characterization of exosomes revealed the high abundance of exosomes in colostrum and MM samples. Quantitative proteomics analysis highlighted the change in the proteomic cargo of exosomes based on the lactation state of the cow. Functional enrichment analysis revealed that exosomes from colostrum are significantly enriched with proteins that can potentially regulate the immune response and growth. This study highlights the importance of exosomes in colostrum and hence opens up new avenues to exploit these vesicles in the regulation of the immune response and growth.

Sengers Syndrome-Associated Mitochondrial Acylglycerol Kinase Is a Subunit of the Human TIM22 Protein Import Complex.

Acylglycerol kinase (AGK) is a mitochondrial lipid kinase that catalyzes the phosphorylation of monoacylglycerol and diacylglycerol to lysophosphatidic acid and phosphatidic acid, respectively. Mutations in AGK cause Sengers syndrome, which is characterized by congenital cataracts, hypertrophic cardiomyopathy, skeletal myopathy, exercise intolerance, and lactic acidosis. Here we identified AGK as a subunit of the mitochondrial TIM22 protein import complex. We show that AGK functions in a kinase-independent manner to maintain the integrity of the TIM22 complex, where it facilitates the import and assembly of mitochondrial carrier proteins. Mitochondria isolated from Sengers syndrome patient cells and tissues show a destabilized TIM22 complex and defects in the biogenesis of carrier substrates. Consistent with this phenotype, we observe perturbations in the tricarboxylic acid (TCA) cycle in cells lacking AGK. Our identification of AGK as a bona fide subunit of TIM22 provides an exciting and unexpected link between mitochondrial protein import and Sengers syndrome.

Detecting PKD1 variants in polycystic kidney disease patients by single-molecule long-read sequencing.

A genetic diagnosis of autosomal-dominant polycystic kidney disease (ADPKD) is challenging due to allelic heterogeneity, high GC content, and homology of the PKD1 gene with six pseudogenes. Short-read next-generation sequencing approaches, such as whole-genome sequencing and whole-exome sequencing, often fail at reliably characterizing complex regions such as PKD1. However, long-read single-molecule sequencing has been shown to be an alternative strategy that could overcome PKD1 complexities and discriminate between homologous regions of PKD1 and its pseudogenes. In this study, we present the increased power of resolution for complex regions using long-read sequencing to characterize a cohort of 19 patients with ADPKD. Our approach provided high sensitivity in identifying PKD1 pathogenic variants, diagnosing 94.7% of the patients. We show that reliable screening of ADPKD patients in a single test without interference of PKD1 homologous sequences, commonly introduced by residual amplification of PKD1 pseudogenes, by direct long-read sequencing is now possible. This strategy can be implemented in diagnostics and is highly suitable to sequence and resolve complex genomic regions that are of clinical relevance.

De novo whole-genome assembly of a wild type yeast isolate using nanopore sequencing.

Background: The introduction of the MinION TM sequencing device by Oxford Nanopore Technologies may greatly accelerate whole genome sequencing. It has been shown that the nanopore sequence data, in combination with other sequencing technologies, is highly useful for accurate annotation of all genes in the genome. However, it also offers great potential for de novo assembly of complex genomes without using other technologies. In this manuscript we used nanopore sequencing as a tool to classify yeast strains. Methods: We compared various technical and software developments for the nanopore sequencing protocol, showing that the R9 chemistry is, as predicted, higher in quality than R7.3 chemistry. The R9 chemistry is an essential improvement for assembly of the extremely AT-rich mitochondrial genome. Results: In this study, we used this new technology to sequence and de novo assemble the genome of a recently isolated ethanologenic yeast strain, and compared the results with those obtained by classical Illumina short read sequencing. This strain was originally named Candida vartiovaarae ( Torulopsis vartiovaarae) based on ribosomal RNA sequencing. We show that the assembly using nanopore data is much more contiguous than the assembly using short read data. Conclusions: The mitochondrial and chromosomal genome sequences showed that our strain is clearly distinct from other yeast taxons and most closely related to published Cyberlindnera species. In conclusion, MinION-mediated long read sequencing can be used for high quality de novo assembly of new eukaryotic microbial genomes.

Tim29 is a novel subunit of the human TIM22 translocase and is involved in complex assembly and stability.

The TIM22 complex mediates the import of hydrophobic carrier proteins into the mitochondrial inner membrane. While the TIM22 machinery has been well characterised in yeast, the human complex remains poorly characterised. Here, we identify Tim29 (C19orf52) as a novel, metazoan-specific subunit of the human TIM22 complex. The protein is integrated into the mitochondrial inner membrane with it's C-terminus exposed to the intermembrane space. Tim29 is required for the stability of the TIM22 complex and functions in the assembly of hTim22. Furthermore, Tim29 contacts the Translocase of the Outer Mitochondrial Membrane, TOM complex, enabling a mechanism for transport of hydrophobic carrier substrates across the aqueous intermembrane space. Identification of Tim29 highlights the significance of analysing mitochondrial import systems across phylogenetic boundaries, which can reveal novel components and mechanisms in higher organisms.

A novel mechanism of generating extracellular vesicles during apoptosis via a beads-on-a-string membrane structure.

Disassembly of apoptotic cells into smaller fragments (a form of extracellular vesicle called apoptotic bodies) can facilitate removal of apoptotic debris and intercellular communication. However, the mechanism underpinning this process is unclear. While observing monocytes undergoing apoptosis by time-lapse microscopy, we discovered a new type of membrane protrusion that resembles a 'beads-on-a-string' structure. Strikingly, the 'beads' are frequently sheared off the 'string' to form apoptotic bodies. Generation of apoptotic bodies via this mechanism can facilitate a sorting process and results in the exclusion of nuclear contents from apoptotic bodies. Mechanistically, generation of 'beads-on-a-string' protrusion is controlled by the level of actomyosin contraction and apoptopodia formation. Furthermore, in an unbiased drug screen, we identified the ability of sertraline (an antidepressant) to block the formation of 'beads-on-a-string' protrusions and apoptotic bodies. These data uncover a new mechanism of apoptotic body formation in monocytes and also compounds that can modulate this process.

Proteogenomic analysis reveals exosomes are more oncogenic than ectosomes.

Extracellular vesicles (EVs) include the exosomes (30-100 nm) that are produced through the endocytic pathway via the multivesicular bodies and the ectosomes (100-1000 nm) that are released through the budding of the plasma membrane. Despite the differences in the mode of biogenesis and size, reliable markers that can distinguish between exosomes and ectosomes are non-existent. Moreover, the precise functional differences between exosomes and ectosomes remains poorly characterised. Here, using label-free quantitative proteomics, we highlight proteins that could be exploited as markers to discriminate between exosomes and ectosomes. For the first time, a global proteogenomics analysis unveiled the secretion of mutant proteins that are implicated in cancer progression through tumor-derived EVs. Follow up integrated bioinformatics analysis highlighted the enrichment of oncogenic cargo in exosomes and ectosomes. Interestingly, exosomes induced significant cell proliferation and migration in recipient cells compared to ectosomes confirming the oncogenic nature of exosomes. These findings ascertain that cancer cells facilitate oncogenesis by the secretion of mutant and oncoproteins into the tumor microenvironment via exosomes and ectosomes. The integrative proteogenomics approach utilized in this study has the potential to identify disease biomarker candidates which can be later assayed in liquid biopsies obtained from cancer patients.

Extracellular vesicles including exosomes are mediators of signal transduction: are they protective or pathogenic?

Extracellular vesicles (EVs) are signaling organelles that are released by many cell types and is highly conserved in both prokaryotes and eukaryotes. Based on the mechanism of biogenesis, these membranous vesicles can be classified as exosomes, shedding microvesicles, and apoptotic blebs. It is becoming clearer that these EVs mediate signal transduction in both autocrine and paracrine fashion by the transfer of proteins and RNA. While the role of EVs including exosomes in pathogenesis is well established, very little is known about their function in normal physiological conditions. Recent evidences allude that EVs can mediate both protective and pathogenic effects depending on the precise state. In this review, we discuss the involvement of EVs as mediators of signal transduction in neurodegenerative diseases and cancer. In addition, the role of EVs in mediating Wnt and PI3K signaling pathways is also discussed. Additional findings on the involvement of EVs in homeostasis and disease progression will promote a better biological understanding, advance future therapeutic, and diagnostic applications.

Proteogenomic analysis of the Venturia pirina (Pear Scab Fungus) secretome reveals potential effectors.

A proteogenomic analysis is presented for Venturia pirina, a fungus that causes scab disease on European pear (Pyrus communis). V. pirina is host-specific, and the infection is thought to be mediated by secreted effector proteins. Currently, only 36 V. pirina proteins are catalogued in GenBank, and the genome sequence is not publicly available. To identify putative effectors, V. pirina was grown in vitro on and in cellophane sheets mimicking its growth in infected leaves. Secreted extracts were analyzed by tandem mass spectrometry, and the data (ProteomeXchange identifier PXD000710) was queried against a protein database generated by combining in silico predicted transcripts with six frame translations of a whole genome sequence of V. pirina (GenBank Accession JEMP00000000 ). We identified 1088 distinct V. pirina protein groups (FDR 1%) including 1085 detected for the first time. Thirty novel (not in silico predicted) proteins were found, of which 14 were identified as potential effectors based on characteristic features of fungal effector protein sequences. We also used evidence from semitryptic peptides at the protein N-terminus to corroborate in silico signal peptide predictions for 22 proteins, including several potential effectors. The analysis highlights the utility of proteogenomics in the study of secreted effectors.

Genome-wide genotyping demonstrates a polygenic risk score associated with white matter hyperintensity volume in CADASIL.

BACKGROUND AND PURPOSE: White matter hyperintensities (WMH) on MRI are a quantitative marker for sporadic cerebral small vessel disease and are highly heritable. To date, large-scale genetic studies have identified only a single locus influencing WMH burden. This might in part relate to biological heterogeneity of sporadic WMH. The current study searched for genetic modifiers of WMH volume in cerebral autosomal-dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL), a monogenic small vessel disease. METHODS: We performed a genome-wide association study to identify quantitative trait loci for WMH volume by combining data from 517 CADASIL patients collected through 7 centers across Europe. WMH volumes were centrally analyzed and quantified on fluid attenuated inversion recovery images. Genotyping was performed using the Affymetrix 6.0 platform. Individuals were assigned to 2 distinct genetic clusters (cluster 1 and cluster 2) based on their genetic background. RESULTS: Four hundred sixty-six patients entered the final genome-wide association study analysis. The phenotypic variance of WMH burden in CADASIL explained by all single nucleotide polymorphisms in cluster 1 was 0.85 (SE=0.21), suggesting a substantial genetic contribution. Using cluster 1 as derivation and cluster 2 as a validation sample, a polygenic score was significantly associated with WMH burden (P=0.001) after correction for age, sex, and vascular risk factors. No single nucleotide polymorphism reached genome-wide significance. CONCLUSIONS: We found a polygenic score to be associated with WMH volume in CADASIL subjects. Our findings suggest that multiple variants with small effects influence WMH burden in CADASIL. The identification of these variants and the biological pathways involved will provide insights into the pathophysiology of white matter disease in CADASIL and possibly small vessel disease in general.