The evolutionary trajectory of the mating-type (mat) genes in Neurospora relates to reproductive behavior of taxa

<p>Abstract</p> <p>Background</p> <p>Comparative sequencing studies among a wide range of taxonomic groups, including fungi, have led to the discovery that reproductive genes evolve more rapidly than other genes. However, for fungal reproductive genes the question has remained whether the rapid evolution is a result of stochastic or deterministic processes. The mating-type (<it>mat</it>) genes constitute the master regulators of sexual reproduction in filamentous ascomycetes and here we present a study of the molecular evolution of the four <it>mat</it>-genes (<it>mat a-1</it>, <it>mat A-1</it>, <it>mat A-2 </it>and <it>mat A-3</it>) of 20 <it>Neurospora </it>taxa.</p> <p>Results</p> <p>We estimated nonsynonymous and synonymous substitution rates of genes to infer their evolutionary rate, and confirmed that the <it>mat</it>-genes evolve rapidly. Furthermore, the evolutionary trajectories are related to the reproductive modes of the taxa; likelihood methods revealed that positive selection acting on specific codons drives the diversity in heterothallic taxa, while among homothallic taxa the rapid evolution is due to a lack of selective constraint. The latter finding is supported by presence of stop codons and frame shift mutations disrupting the open reading frames of <it>mat a-1</it>, <it>mat A-2 </it>and <it>mat A-3 </it>in homothallic taxa. Lower selective constraints of <it>mat</it>-genes was found among homothallic than heterothallic taxa, and comparisons with non-reproductive genes argue that this disparity is not a nonspecific, genome-wide phenomenon.</p> <p>Conclusion</p> <p>Our data show that the <it>mat</it>-genes evolve rapidly in <it>Neurospora</it>. The rapid divergence is due to either adaptive evolution or lack of selective constraints, depending on the reproductive mode of the taxa. This is the first instance of positive selection acting on reproductive genes in the fungal kingdom, and illustrates how the evolutionary trajectory of reproductive genes can change after a switch in reproductive behaviour of an organism.</p

Snail regulates BMP and TGFβ pathways to control the differentiation status of glioma-initiating cells

Glioblastoma multiforme is a brain malignancy characterized by high heterogeneity, invasiveness, and resistance to current therapies, attributes related to the occurrence of glioma stem cells (GSCs). Transforming growth factor β (TGFβ) promotes self-renewal and bone morphogenetic protein (BMP) induces differentiation of GSCs. BMP7 induces the transcription factor Snail to promote astrocytic differentiation in GSCs and suppress tumor growth in vivo. We demonstrate that Snail represses stemness in GSCs. Snail interacts with SMAD signaling mediators, generates a positive feedback loop of BMP signaling and transcriptionally represses the TGFB1 gene, decreasing TGFβ1 signaling activity. Exogenous TGFβ1 counteracts Snail function in vitro, and in vivo promotes proliferation and re-expression of Nestin, confirming the importance of TGFB1 gene repression by Snail. In conclusion, novel insight highlights mechanisms whereby Snail differentially regulates the activity of the opposing BMP and TGFβ pathways, thus promoting an astrocytic fate switch and repressing stemness in GSCs

Profiling surface proteins on individual exosomes using a proximity barcoding assay

Exosomes have been implicated in numerous biological processes, and they may serve as important disease markers. Surface proteins on exosomes carry information about their tissues of origin. Because of the heterogeneity of exosomes it is desirable to investigate them individually, but this has so far remained impractical. Here, we demonstrate a proximity-dependent barcoding assay to profile surface proteins of individual exosomes using antibody-DNA conjugates and next-generation sequencing. We first validate the method using artificial streptavidin-oligonucleotide complexes, followed by analysis of the variable composition of surface proteins on individual exosomes, derived from human body fluids or cell culture media. Exosomes from different sources are characterized by the presence of specific combinations of surface proteins and their abundance, allowing exosomes to be separately quantified in mixed samples to serve as markers for tissue-specific engagement in disease

Characterization of the prion protein in relation to normal cellular function and in disease

Transmissible spongiform encephalopathies (TSEs), also known as prion diseases, are a group of rare and fatal neurodegenerative disorders that can affect both human and animals. Evidence indicates that the key event in prion disease pathogenesis is the conformational conversion of the normal cellular prion protein (PrPC) into an aggregated isoform called the scrapie prion protein (PrPSc). The normal function of PrPC is still not known but the protein is essential for transmission of prion diseases. Defining the physiological activities of PrPC is crucial for understanding the normal function and pathogenesis of prion diseases. In this thesis, the proteolytic cleavages and shedding of PrPC were studied. PrPC was shown to be released from the cell by three different mechanisms. The first mechanism released a N-terminal fragment of PrPC by α-cleavage, the second released the full length PrPC and a C-terminal fragment without GPI-anchor via an extreme C-terminal cleavage and a third mechanism released PrPC in association with exosomes. It was also shown that a deletion in the α-cleavage site inhibited the α-cleavage of PrPC and that the α-cleavage likely took place at the cell surface. Metalloproteases have been suggested to be involved in the different cleavages. Here, it was shown that metalloproteases were involved in the cleavage of the extreme C-terminal end, but not in the α-cleavage of PrPC. Nor98/atypical scrapie was identified in Norway for the first time in 1998. Characterization of the molecular and genetic properties in two Swedish cases of Nor98 showed that unique proteinase K (PK)-resistant fragments are present in Nor98-affected sheep. The existence of two PK-resistant fragments that share overlapping regions suggests that at least two distinct PrP conformations are present in brain extracts from affected sheep. Chronic wasting disease (CWD) affects cervids in North America. Here, it was shown that the PrP sequence of cervids in Scandinavia is similar to genotypes connected with CWD susceptibility in North American cervid species. Also, the European moose was shown to have a unique variation in codon 109 with 109K/Q and 109Q/Q

Detecting individual extracellular vesicles using a multicolor in situ proximity ligation assay with flow cytometric readout

Flow cytometry is a powerful method for quantitative and qualitative analysis of individual cells. However, flow cytometric analysis of extracellular vesicles (EVs), and the proteins present on their surfaces has been hampered by the small size of the EVs - in particular for the smallest EVs, which can be as little as 40 nm in diameter, the limited number of antigens present, and their low refractive index. We addressed these limitations for detection and characterization of EV by flow cytometry through the use of multiplex and multicolor in situ proximity ligation assays (in situ PLA), allowing each detected EV to be easily recorded over background noise using a conventional flow cytometer. By targeting sets of proteins on the surface that are specific for distinct classes of EVs, the method allows for selective recognition of populations of EVs in samples containing more than one type of EVs. The method presented herein opens up for analyses of EVs using flow cytometry for their characterization and quantification

Stability of Proteins in Dried Blood Spot Biobanks.

An important motivation for the construction of biobanks is to discover biomarkers that identify diseases at early, potentially curable stages. This will require biobanks from large numbers of individuals, preferably sampled repeatedly, where the samples are collected and stored under conditions that preserve potential biomarkers. Dried blood samples are attractive for biobanking because of the ease and low cost of collection and storage. Here we have investigated their suitability for protein measurements. 92 proteins with relevance for oncology were analyzed using multiplex proximity extension assays (PEA) in dried blood spots collected on paper and stored for up to 30 years at either +4&amp;deg;C or -24&amp;deg;C.&lt;/p&gt; &lt;p&gt;Our main findings were that 1) the act of drying only slightly influenced detection of blood proteins (average correlation of 0.970), and in a reproducible manner (correlation of 0.999), 2) detection of some proteins was not significantly affected by storage over the full range of three decades (34% and 76% of the analyzed proteins at +4&amp;deg;C and -24&amp;deg;C, respectively), while levels of others decreased slowly during storage with half-lives in the range of 10 to 50 years, and 3) detectability of proteins was less affected in dried samples stored at -24&amp;deg;C compared to at +4&amp;deg;C, as the median protein abundance had decreased to 80% and 93% of starting levels after 10 years of storage at +4&amp;deg;C or -24&amp;deg;C, respectively. The results of our study are encouraging as they suggest an inexpensive means to collect large numbers of blood samples, even by the donors themselves, and to transport, and store biobanked samples as spots of whole blood dried on paper. Combined with emerging means to measure hundreds or thousands of protein, such biobanks could prove of great medical value by greatly enhancing discovery as well as routine analysis of blood biomarkers

A fine-needle aspiration-based protein signature discriminates benign from malignant breast lesions

There are increasing demands for informative cancer biomarkers, accessible via minimally invasive procedures, both for initial diagnostics and to follow-up personalized cancer therapy. Fine-needle aspiration (FNA) biopsy provides ready access to relevant tissues; however, the minute sample amounts require sensitive multiplex molecular analysis to achieve clinical utility. We have applied proximity extension assays (PEA) and NanoString (NS) technology for analyses of proteins and of RNA, respectively, in FNA samples. Using samples from patients with breast cancer (BC, n=25) or benign lesions (n=33), we demonstrate that these FNA-based molecular analyses (a) can offer high sensitivity and reproducibility, (b) may provide correct diagnosis in shorter time and at a lower cost than current practice, (c) correlate with results from routine analysis (i.e., benchmarking against immunohistochemistry tests for ER, PR, HER2, and Ki67), and (d) may also help identify new markers related to immunotherapy. A specific 11-protein signature, including FGF binding protein 1, decorin, and furin, distinguished all cancer patient samples from all benign lesions in our main cohort and in smaller replication cohort. Due to the minimally traumatic sampling and rich molecular information, this combined proteomics and transcriptomic methodology is promising for diagnostics and evaluation of treatment efficacy in BC