Mrassf1a-Pap,a Novel Methylation-Based Assay for the Detection of Cell-Free Fetal DNA in Maternal Plasma

Objectives

RASSF1A has been described to be differentially methylated between fetal and maternal DNA and can therefore be used as a universal sex-independent marker to confirm the presence of fetal sequences in maternal plasma. However, this requires highly sensitive methods. We have previously shown that Pyrophosphorolysis-activated Polymerization (PAP) is a highly sensitive technique that can be used in noninvasive prenatal diagnosis. In this study, we have used PAP in combination with bisulfite conversion to develop a new universal methylation-based assay for the detection of fetal methylated RASSF1A sequences in maternal plasma.

Methods

Bisulfite sequencing was performed on maternal genomic (g)DNA and fetal gDNA from chorionic villi to determine differentially methylated regions in the RASSF1A gene using bisulfite specific PCR primers. Methylation specific primers for PAP were designed for the detection of fetal methylated RASSF1A sequences after bisulfite conversion and validated.

Results

Serial dilutions of fetal gDNA in a background of maternal gDNA show a relative percentage of ∼3% can be detected using this assay. Furthermore, fetal methylated RASSF1A sequences were detected both retrospectively as well as prospectively in all maternal plasma samples tested (n = 71). No methylated RASSF1A specific bands were observed in corresponding maternal gDNA. Specificity was further determined by testing anonymized plasma from non-pregnant females (n = 24) and males (n = 21). Also, no methylated RASSF1A sequences were detected here, showing this assay is very specific for methylated fetal DNA. Combining all samples and controls, we obtain an overall sensitivity and specificity of 100% (95% CI 98.4%–100%).

Conclusions

Our data demonstrate that using a combination of bisulfite conversion and PAP fetal methylated RASSF1A sequences can be detected with extreme sensitivity in a universal and sex-independent manner. Therefore, this assay could be of great value as an addition to current techniques used in noninvasive prenatal diagnostics.     

Resistance gene enrichment sequencing (RenSeq) enables reannotation of the NB‐LRR gene family from sequenced plant genomes and rapid mapping of resistance loci in segregating populations

RenSeq is a NB‐LRR (nucleotide binding‐site leucine‐rich repeat) gene‐targeted, Resistance gene enrichment and sequencing method that enables discovery and annotation of pathogen resistance gene family members in plant genome sequences. We successfully applied RenSeq to the sequenced potato Solanum tuberosum clone DM, and increased the number of identified NB‐LRRs from 438 to 755. The majority of these identified R gene loci reside in poorly or previously unannotated regions of the genome. Sequence and positional details on the 12 chromosomes have been established for 704 NB‐LRRs and can be accessed through a genome browser that we provide. We compared these NB‐LRR genes and the corresponding oligonucleotide baits with the highest sequence similarity and demonstrated that ~80% sequence identity is sufficient for enrichment. Analysis of the sequenced tomato S. lycopersicum ‘Heinz 1706’ extended the NB‐LRR complement to 394 loci. We further describe a methodology that applies RenSeq to rapidly identify molecular markers that co‐segregate with a pathogen resistance trait of interest. In two independent segregating populations involving the wild Solanum species S. berthaultii (Rpi‐ber2) and S. ruiz‐ceballosii (Rpi‐rzc1), we were able to apply RenSeq successfully to identify markers that co‐segregate with resistance towards the late blight pathogen Phytophthora infestans. These SNP identification workflows were designed as easy‐to‐adapt Galaxy pipelines.     

Regional Differences in Antifungal Susceptibility of the Prevalent Dermatophyte Trichophyton rubrum

Mycopathologia - In vitro susceptibility testing for Trichophyton rubrum has shown resistance to terbinafine, azoles and amorolfine, locally, but epidemiological cutoffs are not available. In order...     

Live Tracking of Inter-organ Communication by Endogenous Exosomes In Vivo

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Asexual sporulation facilitates adaptation: The emergence of azole resistance in Aspergillus fumigatus

Understanding the occurrence and spread of azole resistance in Aspergillus fumigatus is crucial for public health. It has been hypothesized that asexual sporulation, which is abundant in nature, is essential for phenotypic expression of azole resistance mutations in A. fumigatus facilitating subsequent spread through natural selection. Furthermore, the disease aspergilloma is associated with asexual sporulation within the lungs of patients and the emergence of azole resistance. This study assessed the evolutionary advantage of asexual sporulation by growing the fungus under pressure of one of five different azole fungicides over seven weeks and by comparing the rate of adaptation between scenarios of culturing with and without asexual sporulation. Results unequivocally show that asexual sporulation facilitates adaptation. This can be explained by the combination of more effective selection because of the transition from a multicellular to a unicellular stage, and by increased mutation supply due to the production of spores, which involves numerous mitotic divisions. Insights from this study are essential to unravel the resistance mechanisms of sporulating pathogens to chemical compounds and disease agents in general, and for designing strategies that prevent or overcome the emerging threat of azole resistance in particular.     

The impact of genetic diversity in protozoa on molecular diagnostics

Detection of intestinal parasitic protists, commonly referred to as 'intestinal protozoa,' by PCR is increasingly used not only for identification or confirmation but also as a first-line diagnostic tool. Apart from the ability to sample correctly and extract parasite DNA directly from faeces, primer and probe specificity and sensitivity affect predictive values and hence the utility of diagnostic assays. Molecular characterization of intestinal protists is necessary to design primers and probes because this is the basic material for current and future improved diagnostic PCRs for either detecting all genetic variants or specifically differentiating among such variants. As an example, this paper highlights the existence of interspecific and intraspecific genetic diversity among intestinal, unicellular parasites and its implications for nucleic acid-based diagnostic assays.     

Cell cycle phosphorylation of mitotic exit network (MEN) proteins

Phosphorylation of proteins is an important mechanism used to regulate most cellular processes. Recently, we completed an extensive phosphoproteomic analysis of the core proteins that constitute the Saccharomyces cerevisiae centrosome. Here, we present a study of phosphorylation sites found on the mitotic exit network (MEN) proteins, most of which are associated with the cytoplasmic face of the centrosome. We identified 55 sites on Bfa1, Cdc5, Cdc14 and Cdc15. Eight sites lie in cyclin-dependent kinase motifs (Cdk, S/T-P), and 22 sites are completely conserved within fungi. More than half of the sites were found in centrosomes from mitotic cells, possibly in preparation for their roles in mitotic exit. Finally, we report phosphorylation site information for other important cell cycle and regulatory proteins.Key words: in vivo phosphorylation, yeast centrosome, mitotic exit network (MEN), cell cycle, protein kinase, Cdk (cyclin-dependent kinase)/Cdc28, Plk1 (polo-like kinase)/Cdc5Reversible protein phosphorylation leads to changes in targeting, structure and stability of proteins and is used widely to modulate biochemical reactions in the cell. We are interested in phosphoregulation of centrosome duplication and function in the yeast Saccharomyces cerevisiae. Centrosomes nucleate microtubules and, upon duplication during the cell cycle, form the two poles of the bipolar mitotic spindle used to segregate replicated chromosomes into the two daughter cells. Timing and spatial cues are highly regulated to ensure that elongation of the mitotic spindle and separation of sister chromatids occur prior to progression into late telophase and initiation of mitotic exit. The mitotic exit network (MEN) regulates this timing through a complex signaling cascade activated at the centrosome that triggers the end of mitosis, ultimately through mitotic cyclin-dependent kinase (Cdk) inactivation (reviewed in ref. ¹).The major components of the MEN pathway (Fig. 1) are a Ras-like GTPase (Tem1), an activator (Lte1) with homology to nucleotide exchange factors, a GTPase-activating protein (GAP) complex (Bfa1/Bub2), several protein kinases [Cdc5 (Plk1 in humans), Cdc15 and Dbf2/Mob1] and Cdc14 phosphatase (reviewed in ref. ^2–5). Tem1 initiates the signal for the MEN pathway when switched to a GTP-active state. Prior to activation at anaphase, it is held at the centrosome in an inactive GDP-bound state by an inhibiting GAP complex, Bfa1/Bub2.⁶ The Bfa1/Bub2 complex and the inactive Tem1 are localized at the mother centrosome destined to move into the budded cell upon chromosome segregation, whereas the activator Lte1 is localized at the tip of the budded cell. These separate localizations ensure that Lte1 and Tem1 only interact in late anaphase, when the mitotic spindle elongates.^7,8 Lte1 has been thought to activate Tem1 as a nucleotide exchange factor, although more recent evidence suggests that it may instead affect Bfa1 localization.⁹ In addition, full activation of Tem1 is achieved through Cdc5 phosphorylation of the negative regulator Bfa1 ¹⁰ and potentially through phosphorylation of Lte1. GTP-bound Tem1 is then able to recruit Cdc15 to the centrosome, allowing for Dbf2 activation.³ The final step in the MEN pathway is release of Cdc14 from the nucleolus, which is at least partially due to phosphorylation by Dbf2¹¹ an leads to mitotic cyclin degradation and inactivation of the mitotic kinase.²Open in a separate windowFigure 1Schematic representation of the MEN proteins and pathway. MEN protein localization is shown within a metaphase cell when mitotic exit is inhibited and in a late anaphase cell when mitotic exit is initiated. Primary inhibition and activation events are described below the cells.Recently, we performed a large-scale analysis of phosphorylation sites on the 18 core yeast centrosomal proteins present in enriched centrosomal preparations.¹² In total, we mapped 297 sites on 17 of the 18 proteins and described their cell cycle regulation, levels of conservation and demonstrated defects in centrosome assembly and function resulting from mutating selected sites. MEN proteins were also identified in the centrosome preparations. This was expected, because Nud1, one of the 18 core centrosome components, is known to recruit several MEN proteins to the centrosome¹³ as part of its function in mitotic exit.^14,15 As phosphorylation is essential to several steps in the MEN pathway, beginning with recruitment of Bfa1/Bub2 by phosphorylated Nud1,¹⁵ we were interested in mapping in vivo phosphorylation sites on the MEN proteins associated with centrosomes and identifying when they occur during the cell cycle.We combined centrosome enrichment with mass spectrometry analysis to examine phosphorylation from asynchronously growing cells.¹² Centrosomes were also prepared from cells arrested in G₁ and mitosis¹² to monitor potentially cell cycle-regulated sites. We obtained significant coverage of a number of the MEN proteins, several of which have human homologs (​and33, column 1), of which eight sites lie within Cdk/Cdc28 motifs [S/T(P)], (23 Mob1 and Dbf2 are known phosphoproteins²⁴ for which we observed peptide coverage but no phosphorylation. Surprisingly, we did not detect phosphorylation on Bub2 despite the high peptide coverage; it is possible that the mitotic centrosome preparations (using a Cdc20 depletion protocol) affect the phosphorylation state of Bub2, as Bub2 is required for mitotic exit arrest in cdc20 mutants.²⁵ Additionally, specific phosphorylation sites have not been mapped on Bub2, suggesting that modifications on this protein may be difficult to observe by mass spectrometry. Lte1 does not localize to the centrosome, and we did not recover Lte1 peptides in our preparations. Many phosphorylation events on MEN proteins were observed in mitotic centrosomal preparations, most likely in preparation for their subsequent role in exit from mitosis (

Evidence against the use of surrogates for biomonitoring of Neotropical floodplains

1. Community concordance measures the level of association between the compositional patterns shown by two groups of organisms. If strong community concordance occurs, one group could be used as a surrogate for another in conservation planning and biodiversity monitoring. In this study, we evaluated the variability in the strength of community concordance, the likely mechanisms underlying community concordance and the degree to which one community can predict another in a set of Neotropical floodplain lakes (Upper Paraná River floodplain, Brazil). 2. We used a data set including six aquatic communities: fish, macrophytes, benthic macroinvertebrates, zooplankton, phytoplankton and periphyton. We used Mantel and PROTEST approaches to evaluate the levels of community concordance in up to four sampling periods. Also, we used partial Mantel test and information about biotic interactions to investigate reasons for observed patterns of concordance. Finally, we used co‐correspondence analysis to evaluate the performance of one taxonomic group in predicting the structures of other communities. 3. The levels of community concordance varied over time for almost all cross‐taxa comparisons. Concordance between phytoplankton and periphyton probably resulted from similar responses to environmental gradients, whereas other patterns of concordance were likely generated by interactions among groups. However, the levels of predictability were low, and no particular taxonomic group significantly predicted all other groups. 4. The low and temporally variable levels of community concordance cast doubts on the use of surrogate groups for biodiversity management in Neotropical floodplains.