Counting absolute number of molecules using unique molecular identifiers

Advances in molecular biology have made it easy to identify different DNA or RNA species and to copy them. Identification of nucleic acid species can be accomplished by reading the DNA sequence; currently millions of molecules can be sequenced in a single day using massively parallel sequencing. Efficient copying of DNA-molecules of arbitrary sequence was made possible by molecular cloning, and the polymerase chain reaction. Differences in the relative abundance of a large number of different sequences between two or more samples can in turn be measured using microarray hybridization and/or tag sequencing. However, determining the relative abundance of two different species and/or the absolute number of molecules present in a single sample has proven much more challenging. This is because it is hard to detect individual molecules without copying them, and even harder to make defined number of copies of molecules. We show here that this limitation can be overcome by using unique molecular identifiers (umis), which make each molecule in the sample distinct

Tracing the fate of microplastic carbon in the aquatic food web by compound-specific isotope analysis

Increasing abundance of microplastics (MP) in marine and freshwaters is currently one of the greatest environmental concerns. Since plastics are fairly resistant to chemical decomposition, breakdown and reutilization of MP carbon complexes requires microbial activity. Currently, only a few microbial isolates have been shown to degrade MPs, and direct measurements of the fate of the MP carbon are still lacking. We used compound-specific isotope analysis to track the fate of fully labelled 13C-polyethylene (PE) MP carbon across the aquatic microbial-animal interface. Isotopic values of respired CO2 and membrane lipids showed that MP carbon was partly mineralized and partly used for cell growth. Microbial mineralization and assimilation of PE-MP carbon was most active when inoculated microbes were obtained from highly humic waters, which contain recalcitrant substrate sources. Mixotrophic algae (Cryptomonas sp.) and herbivorous zooplankton (Daphnia magna) used microbial mediated PE-MP carbon in their cell membrane fatty acids. Moreover, heteronanoflagellates and mixotrophic algae sequestered MP carbon for synthesizing essential ω-6 and ω-3 polyunsaturated fatty acids. Thus, this study demonstrates that aquatic micro-organisms can produce, biochemically upgrade, and trophically transfer nutritionally important biomolecules from PE-MP.Increasing abundance of microplastics (MP) in marine and freshwaters is currently one of the greatest environmental concerns. Since plastics are fairly resistant to chemical decomposition, breakdown and reutilization of MP carbon complexes requires microbial activity. Currently, only a few microbial isolates have been shown to degrade MPs, and direct measurements of the fate of the MP carbon are still lacking. We used compound-specific isotope analysis to track the fate of fully labelled C-13-polyethylene (PE) MP carbon across the aquatic microbial-animal interface. Isotopic values of respired CO2 and membrane lipids showed that MP carbon was partly mineralized and partly used for cell growth. Microbial mineralization and assimilation of PE-MP carbon was most active when inoculated microbes were obtained from highly humic waters, which contain recalcitrant substrate sources. Mixotrophic algae (Cryptomonas sp.) and herbivorous zooplankton (Daphnia magna) used microbial mediated PE-MP carbon in their cell membrane fatty acids. Moreover, heteronanoflagellates and mixotrophic algae sequestered MP carbon for synthesizing essential omega-6 and omega-3 polyunsaturated fatty acids. Thus, this study demonstrates that aquatic micro-organisms can produce, biochemically upgrade, and trophically transfer nutritionally important biomolecules from PE-MP.Peer reviewe

Functional divergence in the role of N-linked glycosylation in smoothened signaling

The G protein-coupled receptor (GPCR) Smoothened (Smo) is the requisite signal transducer of the evolutionarily conserved Hedgehog (Hh) pathway. Although aspects of Smo signaling are conserved from Drosophila to vertebrates, significant differences have evolved. These include changes in its active sub-cellular localization, and the ability of vertebrate Smo to induce distinct G protein-dependent and independent signals in response to ligand. Whereas the canonical Smo signal to Gli transcriptional effectors occurs in a G protein-independent manner, its non-canonical signal employs Gαi. Whether vertebrate Smo can selectively bias its signal between these routes is not yet known. N-linked glycosylation is a post-translational modification that can influence GPCR trafficking, ligand responsiveness and signal output. Smo proteins in Drosophila and vertebrate systems harbor N-linked glycans, but their role in Smo signaling has not been established. Herein, we present a comprehensive analysis of Drosophila and murine Smo glycosylation that supports a functional divergence in the contribution of N-linked glycans to signaling. Of the seven predicted glycan acceptor sites in Drosophila Smo, one is essential. Loss of N-glycosylation at this site disrupted Smo trafficking and attenuated its signaling capability. In stark contrast, we found that all four predicted N-glycosylation sites on murine Smo were dispensable for proper trafficking, agonist binding and canonical signal induction. However, the under-glycosylated protein was compromised in its ability to induce a non-canonical signal through Gαi, providing for the first time evidence that Smo can bias its signal and that a post-translational modification can impact this process. As such, we postulate a profound shift in N-glycan function from affecting Smo ER exit in flies to influencing its signal output in mice

Dynamic Interpretation of Hedgehog Signaling in the Drosophila Wing Disc

Morphogens are classically defined as molecules that control patterning by acting at a distance to regulate gene expression in a concentration-dependent manner. In the Drosophila wing imaginal disc, secreted Hedgehog (Hh) forms an extracellular gradient that organizes patterning along the anterior–posterior axis and specifies at least three different domains of gene expression. Although the prevailing view is that Hh functions in the Drosophila wing disc as a classical morphogen, a direct correspondence between the borders of these patterns and Hh concentration thresholds has not been demonstrated. Here, we provide evidence that the interpretation of Hh signaling depends on the history of exposure to Hh and propose that a single concentration threshold is sufficient to support multiple outputs. Using mathematical modeling, we predict that at steady state, only two domains can be defined in response to Hh, suggesting that the boundaries of two or more gene expression patterns cannot be specified by a static Hh gradient. Computer simulations suggest that a spatial “overshoot” of the Hh gradient occurs, i.e., a transient state in which the Hh profile is expanded compared to the Hh steady-state gradient. Through a temporal examination of Hh target gene expression, we observe that the patterns initially expand anteriorly and then refine, providing in vivo evidence for the overshoot. The Hh gene network architecture suggests this overshoot results from the Hh-dependent up-regulation of the receptor, Patched (Ptc). In fact, when the network structure was altered such that the ptc gene is no longer up-regulated in response to Hh-signaling activation, we found that the patterns of gene expression, which have distinct borders in wild-type discs, now overlap. Our results support a model in which Hh gradient dynamics, resulting from Ptc up-regulation, play an instructional role in the establishment of patterns of gene expression

Mapping differential interactomes by affinity purification coupled with data independent mass spectrometry acquisition

Characterizing changes in protein-protein interactions associated with sequence variants (e.g. disease-associated mutations or splice forms) or following exposure to drugs, growth factors or hormones is critical to understanding how protein complexes are built, localized and regulated. Affinity purification (AP) coupled with mass spectrometry permits the analysis of protein interactions under near-physiological conditions, yet monitoring interaction changes requires the development of a robust and sensitive quantitative approach, especially for large-scale studies where cost and time are major considerations. To this end, we have coupled AP to data-independent mass spectrometric acquisition (SWATH), and implemented an automated data extraction and statistical analysis pipeline to score modulated interactions. Here, we use AP-SWATH to characterize changes in protein-protein interactions imparted by the HSP90 inhibitor NVP-AUY922 or melanoma-associated mutations in the human kinase CDK4. We show that AP-SWATH is a robust label-free approach to characterize such changes, and propose a scalable pipeline for systems biology studies

Mapping interactions with the chaperone network reveals factors that protect against tau aggregation.

A network of molecular chaperones is known to bind proteins ('clients') and balance their folding, function and turnover. However, it is often unclear which chaperones are critical for selective recognition of individual clients. It is also not clear why these key chaperones might fail in protein-aggregation diseases. Here, we utilized human microtubule-associated protein tau (MAPT or tau) as a model client to survey interactions between ~30 purified chaperones and ~20 disease-associated tau variants (~600 combinations). From this large-scale analysis, we identified human DnaJA2 as an unexpected, but potent, inhibitor of tau aggregation. DnaJA2 levels were correlated with tau pathology in human brains, supporting the idea that it is an important regulator of tau homeostasis. Of note, we found that some disease-associated tau variants were relatively immune to interactions with chaperones, suggesting a model in which avoiding physical recognition by chaperone networks may contribute to disease

Development of actionable targets of multi-kinase inhibitors (AToMI) screening platform to dissect kinase targets of staurosporines in glioblastoma cells

Therapeutic resistance to kinase inhibitors constitutes a major unresolved clinical challenge in cancer and especially in glioblastoma. Multi-kinase inhibitors may be used for simultaneous targeting of multiple target kinases and thereby potentially overcome kinase inhibitor resistance. However, in most cases the identification of the target kinases mediating therapeutic effects of multi-kinase inhibitors has been challenging. To tackle this important problem, we developed an actionable targets of multi-kinase inhibitors (AToMI) strategy and used it for characterization of glioblastoma target kinases of staurosporine derivatives displaying synergy with protein phosphatase 2A (PP2A) reactivation. AToMI consists of interchangeable modules combining drug-kinase interaction assay, siRNA high-throughput screening, bioinformatics analysis, and validation screening with more selective target kinase inhibitors. As a result, AToMI analysis revealed AKT and mitochondrial pyruvate dehydrogenase kinase PDK1 and PDK4 as kinase targets of staurosporine derivatives UCN-01, CEP-701, and K252a that synergized with PP2A activation across heterogeneous glioblastoma cells. Based on these proof-of-principle results, we propose that the application and further development of AToMI for clinically applicable multi-kinase inhibitors could provide significant benefits in overcoming the challenge of lack of knowledge of the target specificity of multi-kinase inhibitors

Cost-effectiveness analysis of pemetrexed versus docetaxel in the second-line treatment of non-small cell lung cancer in Spain: results for the non-squamous histology population

BackgroundThe objective of this study was to conduct a cost-effectiveness evaluation of pemetrexed compared to docetaxel in the treatment of advanced or metastatic non-small cell lung cancer (NSCLC) for patients with predominantly non-squamous histology in the Spanish healthcare setting.MethodsA Markov model was designed consisting of stable, responsive, progressive disease and death states. Patients could also experience adverse events as long as they received chemotherapy. Clinical inputs were based on an analysis of a phase III clinical trial that identified a statistically significant improvement in overall survival for non-squamous patients treated with pemetrexed compared with docetaxel. Costs were collected from the Spanish healthcare perspective.ResultsOutcomes of the model included total costs, total quality-adjusted life years (QALYs), total life years gained (LYG) and total progression-free survival (PFS). Mean survival was 1.03 years for the pemetrexed arm and 0.89 years in the docetaxel arm; QALYs were 0.52 compared to 0.42. Per-patient lifetime costs were € 34677 and € 32343, respectively. Incremental cost-effectiveness ratios were € 23967 per QALY gained and € 17225 per LYG.ConclusionsPemetrexed as a second-line treatment option for patients with a predominantly non-squamous histology in NSCLC is a cost-effective alternative to docetaxel according to the € 30000/QALY threshold commonly accepted in Spain

Fish Consumption and Omega-3 Polyunsaturated Fatty Acids in Relation to Depressive Episodes: A Cross-Sectional Analysis

High fish consumption and omega-3 polyunsaturated fatty acid (PUFA) intake are suggested to benefit mental well-being but the current evidence is conflicting. Our aim was to evaluate whether a higher level of fish consumption, a higher intake of omega-3 PUFAs, and a higher serum concentration of omega-3 PUFAs link to a lower 12-month prevalence of depressive episodes