The development of next-generation sequencing (NGS) technologies for HLA and KIR genotyping is rapidly advancing knowledge of genetic variation of these highly polymorphic loci. NGS genotyping is poised to replace older methods for clinical use, but standard methods for reporting and exchanging these new, high quality genotype data are needed. The Immunogenomic NGS Consortium, a broad collaboration of histocompatibility and immunogenetics clinicians, researchers, instrument manufacturers and software developers, has developed the Minimum Information for Reporting Immunogenomic NGS Genotyping (MIRING) reporting guidelines. MIRING is a checklist that specifies the content of NGS genotyping results as well as a set of messaging guidelines for reporting the results. A MIRING message includes five categories of structured information – message annotation, reference context, full genotype, consensus sequence and novel polymorphism – and references to three categories of accessory information – NGS platform documentation, read processing documentation and primary data. These eight categories of information ensure the long-term portability and broad application of this NGS data for all current histocompatibility and immunogenetics use cases. In addition, MIRING can be extended to allow the reporting of genotype data generated using pre-NGS technologies. Because genotyping results reported using MIRING are easily updated in accordance with reference and nomenclature databases, MIRING represents a bold departure from previous methods of reporting HLA and KIR genotyping results, which have provided static and less-portable data. More information about MIRING can be found online at miring.immunogenomics.org.
NGS; HLA; KIR; MIRING; genotyping; data standards
We conducted a phase I clinical trial (clinicaltrials.gov identifier, NCT00641017) of the experimental live-attenuated human parainfluenza virus type 1 (HPIV-1) vaccine rHPIV-1/84/del 170/942A sequentially in 3 groups: adults, HPIV-1–seropositive children, and HPIV-1–seronegative children, the target population for vaccination. rHPIV-1/84/del 170/942A was appropriately restricted in replication in adults and HPIV-1–seropositive children but was overattenuated (ie, insufficiently infectious and immunogenic) for HPIV-1–seronegative children.
children; HPIV-1 vaccine; lower respiratory tract illness
Centrioles are essential for the formation of centrosomes and cilia. While numerical and/or structural centrosomes aberrations are implicated in cancer, mutations in centriolar and centrosomal proteins are genetically linked to ciliopathies, microcephaly, and dwarfism. The evolutionarily conserved mechanisms underlying centrosome biogenesis are centered on a set of key proteins, including Plk4, Sas‐6, and STIL, whose exact levels are critical to ensure accurate reproduction of centrioles during cell cycle progression. However, neither the intracellular levels of centrosomal proteins nor their stoichiometry within centrosomes is presently known. Here, we have used two complementary approaches, targeted proteomics and EGFP‐tagging of centrosomal proteins at endogenous loci, to measure protein abundance in cultured human cells and purified centrosomes. Our results provide a first assessment of the absolute and relative amounts of major components of the human centrosome. Specifically, they predict that human centriolar cartwheels comprise up to 16 stacked hubs and 1 molecule of STIL for every dimer of Sas‐6. This type of quantitative information will help guide future studies of the molecular basis of centrosome assembly and function.
centriole; centrosome; fluorescence imaging; proteomics; selected reaction monitoring; Cell Adhesion, Polarity & Cytoskeleton; Cell Cycle; Post-translational Modifications, Proteolysis & Proteomics
Measuring precise concentrations of proteins can provide insights into biological processes. Here, we use efficient protein extraction and sample fractionation and state-of-the-art quantitative mass spectrometry techniques to generate a comprehensive, condition-dependent protein abundance map of Escherichia coli. We measure cellular protein concentrations for 55% of predicted E. coli genes (>2300 proteins) under 22 different experimental conditions and identify methylation and N-terminal protein acetylations previously not known to be prevalent in bacteria. We uncover system-wide proteome allocation, expression regulation, and post-translational adaptations. These data provide a valuable resource for the systems biology and broader E. coli research communities.
Cambay amber originates from the warmest period of the Eocene, which is also well known for the appearance of early angiosperm-dominated megathermal forests. The humid climate of these forests may have triggered the evolution of epiphytic lineages of bryophytes; however, early Eocene fossils of bryophytes are rare. Here, we present evidence for lejeuneoid liverworts and pleurocarpous mosses in Cambay amber. The preserved morphology of the moss fossil is inconclusive for a detailed taxonomic treatment. The liverwort fossil is, however, distinctive; its zig-zagged stems, suberect complicate-bilobed leaves, large leaf lobules, and small, deeply bifid underleaves suggest a member of Lejeuneaceae subtribe Lejeuneinae (Harpalejeunea, Lejeunea, Microlejeunea). We tested alternative classification possibilities by conducting divergence time estimates based on DNA sequence variation of Lejeuneinae using the age of the fossil for corresponding age constraints. Consideration of the fossil as a stem group member of Microlejeunea or Lejeunea resulted in an Eocene to Late Cretaceous age of the Lejeuneinae crown group. This reconstruction is in good accordance with published divergence time estimates generated without the newly presented fossil evidence. Balancing available evidence, we describe the liverwort fossil as the extinct species Microlejeunea nyiahae, representing the oldest crown group fossil of Lejeuneaceae.
Protein delivery based on bacterial type III secretion allows controllable injection of bacterial, viral, and human proteins into target cells, functional interaction studies, targeting of proteins to subcellular locations, and systems-level signaling analysis of a given translocated protein.
Methods enabling the delivery of proteins into eukaryotic cells are essential to address protein functions. Here we propose broad applications to cell biology for a protein delivery tool based on bacterial type III secretion (T3S). We show that bacterial, viral, and human proteins, fused to the N-terminal fragment of the Yersinia enterocolitica T3S substrate YopE, are effectively delivered into target cells in a fast and controllable manner via the injectisome of extracellular bacteria. This method enables functional interaction studies by the simultaneous injection of multiple proteins and allows the targeting of proteins to different subcellular locations by use of nanobody-fusion proteins. After delivery, proteins can be freed from the YopE fragment by a T3S-translocated viral protease or fusion to ubiquitin and cleavage by endogenous ubiquitin proteases. Finally, we show that this delivery tool is suitable to inject proteins in living animals and combine it with phosphoproteomics to characterize the systems-level impact of proapoptotic human truncated BID on the cellular network.
The characterization of the ABO blood group status is vital for blood transfusion and solid organ transplantation. Several methods for the molecular characterization of the ABO gene, which encodes the alleles that give rise to the different ABO blood groups, have been described. However, the application of those methods has so far been restricted to selected samples and not been applied to population-scale analysis.
We describe a cost-effective method for high-throughput genotyping of the ABO system by next generation sequencing. Sample specific barcodes and sequencing adaptors are introduced during PCR, rendering the products suitable for direct sequencing on Illumina MiSeq or HiSeq instruments. Complete sequence coverage of exons 6 and 7 enables molecular discrimination of the ABO subgroups and many alleles. The workflow was applied to ABO genotype more than a million samples. We report the allele group frequencies calculated on a subset of more than 110,000 sampled individuals of German origin. Further we discuss the potential of the workflow for high resolution genotyping taking the observed allele group frequencies into account. Finally, sequence analysis revealed 287 distinct so far not described alleles of which the most abundant one was identified in 174 samples.
The described workflow delivers high resolution ABO genotyping at low cost enabling population-scale molecular ABO characterization.
Electronic supplementary material
The online version of this article (doi:10.1186/s12864-016-2687-1) contains supplementary material, which is available to authorized users.
Blood group; ABO; NGS; Genotyping; Illumina; Amplicon
Pre-hospital paediatric airway management is complex. A variety of pitfalls need prompt response to establish and maintain adequate ventilation and oxygenation. Anatomical disparity render laryngoscopy different compared to the adult. The correct choice of endotracheal tube size and depth of insertion is not trivial and often challenged due to the initially unknown age of child.
Data from 425 paediatric patients (<17 years of age) with any airway manipulation treated by a Swiss Air-Ambulance crew between June 2010 and December 2013 were retrospectively analysed. Endpoints were: 1) Endotracheal intubation success rate and incidence of difficult airway management in primary missions. 2) Correlation of endotracheal tube size and depth of insertion with patient’s age in all (primary and secondary) missions.
In primary missions, the first laryngoscopy-guided endotracheal intubation attempt was successful in 95.3% of cases, with an overall success rate of 98.6%. Difficult airway management was reported in 10 (4.7%) patients. Endotracheal tube size was frequently chosen inadequately large (overall 50 of 343 patients: 14.6%), especially and statistically significant in the age group below 1 year (19 of 33 patients; p < 0.001). Tubes were frequently and distinctively more deeply inserted (38.9%) than recommended by current formulae.
Difficult airway management, including cannot intubate and cannot ventilate situations during pre-hospital paediatric emergency treatment was rare. In contrast, the success rate of endotracheal intubation at the first attempt was very high. High numbers of inadequate endotracheal tube size and deep placement according to patient age require further analysis. Practical algorithms need to be found to prevent potentially harmful treatment.
Paediatric airway; Pre-hospital airway; Emergency airway; Endotracheal tube size and depth; HEMS
The heterogeneous nature of HLA information in real-life stem cell donor registries may hamper unrelated donor searches. It is even possible that fully HLA-matched donors with incomplete HLA information are not identified. In our simulation study, we estimated the probability of these unnecessarily failed donor searches. For that purpose, we carried out donor searches in several virtual donor registries. The registries differed by size, composition with respect to HLA typing levels, and genetic diversity. When up to three virtual HLA typing requests were allowed within donor searches, the share of unnecessarily failed donor searches ranged from 1.19% to 4.13%, thus indicating that non-identification of completely HLA-matched stem cell donors is a problem of practical relevance. The following donor registry characteristics were positively correlated with the share of unnecessarily failed donor searches: large registry size, high genetic diversity, and, most strongly correlated, large fraction of registered donors with incomplete HLA typing. Increasing the number of virtual HLA typing requests within donor searches up to ten had a smaller effect. It follows that the problem of donor non-identification can be substantially reduced by complete high-resolution HLA typing of potential donors.
An integrated account of the molecular changes occurring during the process of cellular aging is crucial towards understanding the underlying mechanisms. Here, using novel culturing and computational methods as well as latest analytical techniques, we mapped the proteome and transcriptome during the replicative lifespan of budding yeast. With age, we found primarily proteins involved in protein biogenesis to increase relative to their transcript levels. Exploiting the dynamic nature of our data, we reconstructed high-level directional networks, where we found the same protein biogenesis-related genes to have the strongest ability to predict the behavior of other genes in the system. We identified metabolic shifts and the loss of stoichiometry in protein complexes as being consequences of aging. We propose a model whereby the uncoupling of protein levels of biogenesis-related genes from their transcript levels is causal for the changes occurring in aging yeast. Our model explains why targeting protein synthesis, or repairing the downstream consequences, can serve as interventions in aging.
Aging is a complex process, and so many scientists use baker’s yeast as a simpler model to understand it. Although many genes that influence aging have been found, all the generated knowledge is still rather fragmented. It also remains difficult to disentangle cause and consequence. That is to say, sometimes a gene that looks like it might cause aging could simply be a gene that responds to an age related phenomenon. To unravel this puzzle of cause and effect, it is necessary to first get an idea on a system level of everything that changes as an organism ages.
Now, Janssens, Meinema et al. have managed to map many of the molecular changes that occur as baker’s yeast ages; this is something that has yet to be achieved for any other organism. The work first involved developing a new way of growing baker’s yeast to keep and generate large cohorts of aging yeast cells in a constant environment. It also required the use of a mathematical ‘un-mixing’ tool to separate the data obtained from the aging cohort from the data from the young offspring that the yeast produce while they age.
Janssens, Meinema et al. measured both the majority of the transcriptome and much of the proteome of baker’s yeast throughout its reproductive lifespan. The “transcriptome” refers to the collection of RNA molecules in the cell, which are produced whenever a gene is expressed. The “proteome” refers to all the proteins in the cell, which are translated from the RNA transcripts by the cell’s so-called “translational machinery”. These experiments revealed that this yeast’s proteome reflects its transcriptome less and less as it ages. In particular, this ‘uncoupling’ of the proteome from the transcriptome was seen most strongly for the proteins related to the cell’s translational machinery; these proteins accumulated with age relative to their transcripts.
Janssens, Meinema et al. then conducted a computational network-based analysis of the data. This indicated that the uncoupling is the driving force behind the aging process. Many of the other molecular changes that occur with aging were predicted to be consequences of this uncoupling.
These findings give a framework for many observations in the existing literature. However, it remains unclear why proteins related to translational machinery are overrepresented in aging yeast in the first place. This question should be explored in future work.
replicative aging; proteome; transcriptome; S. cerevisiae
Cord blood is an important source of stem cells. However, nearly 90% of public cord blood banks have declared that they are struggling to maintain their financial sustainability and avoid bankruptcy. The objective of this study is to evaluate how characteristics of cord blood units influence their utilization, then use this information to model the economic viability and therapeutic value of different banking strategies.
Retrospective analysis of cord blood data registered between January 1st, 2009 and December 31st, 2011 in Bone Marrow Donor Worldwide. Data were collected from four public banks in France, Germany and the USA. Samples were eligible for inclusion in the analysis if data on cord blood and maternal HLA typing and biological characteristics after processing were available (total nucleated and CD34+ cell counts). 9,396 banked cord blood units were analyzed, of which 5,815 were Caucasian in origin. A multivariate logistic regression model assessed the influence of three parameters on the CBU utilization rate: ethnic background, total nucleated and CD34+ cell counts. From this model, we elaborated a Utilization Score reflecting the probability of transplantation for each cord blood unit. We stratified three Utilization Score thresholds representing four different banking strategies, from the least selective (scenario A) to the most selective (scenario D). We measured the cost-effectiveness ratio for each strategy by comparing performance in terms of number of transplanted cord blood units and level of financial deficit.
When comparing inputs and outputs over three years, Scenario A represented the most extreme case as it delivered the highest therapeutic value for patients (284 CBUs transplanted) along with the highest financial deficit (USD 5.89 million). We found that scenario C resulted in 219 CBUs transplanted with a limited deficit (USD 0.98 million) that charities and public health could realistically finance over the long term. We also found that using a pre-freezing level of 18 x 108 TNC would be the most cost-effective strategy for a public bank.
Our study shows that a swift transition from strategy A to C can play a vital role in preventing public cord blood banks worldwide from collapsing.
Preservation of liverworts in amber, a fossilized tree resin, is often exquisite. Twenty-three fossil species of liverworts have been described to date from Eocene (35–50 Ma) Baltic amber. In addition, two inclusions have been assigned to the extant species Ptilidium pulcherrimum (Ptilidiales or Porellales). However, the presence of the boreal P. pulcherrimum in the subtropical or warm-temperate Baltic amber forest challenges the phytogeographical interpretation of the Eocene flora. A re-investigation of one of the fossils believed to be P. pulcherrimum reveals that this specimen in fact represents the first fossil evidence of the genus Tetralophozia, and thus is re-described here as Tetralophozia groehnii sp. nov. A second fossil initially assigned to P. pulcherrimum is apparently lost, and can be reassessed only based on the original description and illustrations. This fossil is morphologically similar to the extant North Pacific endemic Ptilidium californicum, rather than P. pulcherrimum. Divergence time estimates based on chloroplast DNA sequences provide evidence of a Miocene origin of P. pulcherrimum, and thus also argue against the presence of this taxon in the Eocene. Ptilidium californicum originated 25–43 Ma ago. As a result, we cannot rule out that the Eocene fossil belongs to P. californicum. Alternatively, the fossil might represent a stem lineage element of Ptilidium or an early crown group species with morphological similarities to P. californicum.
The oxygen-limiting (hypoxic) microenvironment of tumors induces metabolic reprogramming and cell survival, but the underlying mechanisms involving mitochondria remain poorly understood. We previously demonstrated that hypoxia-inducible factor 1 mediates the hyperfusion of mitochondria by inducing Bcl-2/adenovirus E1B 19-kDa interacting protein 3 and posttranslational truncation of the mitochondrial ATP transporter outer membrane voltage-dependent anion channel 1 in hypoxic cells. In addition, we showed that truncation is associated with increased resistance to drug-induced apoptosis and is indicative of increased patient chemoresistance. We now show that silencing of the tumor suppressor TP53 decreases truncation and increases drug-induced apoptosis. We also show that TP53 regulates truncation through induction of the mitochondrial protein Mieap. While we found that truncation was independent of mitophagy, we observed local microfusion between mitochondria and endolysosomes in hypoxic cells in culture and in patients' tumor tissues. Since we found that the endolysosomal asparagine endopeptidase was responsible for truncation, we propose that it is a readout of mitochondrial-endolysosomal microfusion in hypoxia. These novel findings provide the framework for a better understanding of hypoxic cell metabolism and cell survival through mitochondrial-endolysosomal microfusion regulated by hypoxia-inducible factor 1 and TP53.
sexual objectification; person perception; object perception; artifact; inversion effect; gender
The data described here provide a systematic performance evaluation of popular data-dependent (DDA) and independent (DIA) mass spectrometric (MS) workflows currently used in quantitative proteomics. We assessed the limits of identification, quantification and detection for each method by analyzing a dilution series of 20 unmodified and 10 phosphorylated synthetic heavy labeled reference peptides, respectively, covering six orders of magnitude in peptide concentration with and without a complex human cell digest background. We found that all methods performed very similarly in the absence of background proteins, however, when analyzing whole cell lysates, targeted methods were at least 5–10 times more sensitive than directed or DDA methods. In particular, higher stage fragmentation (MS3) of the neutral loss peak using a linear ion trap increased dynamic quantification range of some phosphopeptides up to 100-fold. We illustrate the power of this targeted MS3 approach for phosphopeptide monitoring by successfully quantifying 9 phosphorylation sites of the kinetochore and spindle assembly checkpoint component Mad1 over different cell cycle states from non-enriched pull-down samples. The data are associated to the research article ‘Evaluation of data-dependent and data-independent mass spectrometric workflows for sensitive quantification of proteins and phosphorylation sites׳ (Bauer et al., 2014) . The mass spectrometry and the analysis dataset have been deposited to the ProteomeXchange Consortium (http://proteomecentral.proteomexchange.org) via the PRIDE partner repository with the dataset identifier PXD000964.
Viral respiratory infections cause significant morbidity and mortality in infants and young children as well as in at-risk adults and the elderly. Although many viral pathogens are capable of causing respiratory disease, vaccine development has to focus on a limited number of pathogens, such as those that commonly cause serious lower respiratory illness (LRI). Whereas influenza virus vaccines have been available for some time (see the review by Clark and Lynch in this issue), vaccines against other medically important viruses such as respiratory syncytial virus (RSV), the parainfluenza viruses (PIVs), and metapneumovirus (MPVs) are not available. This review aims to provide a brief update on investigational vaccines against RSV, the PIVs, and MPV that have been evaluated in clinical trials or are currently in clinical development.
Vaccine; parainfluenza virus; metapneumovirus; respiratory syncytial virus; clinical trial; clinical development
Viral vaccines and the cell substrates used to manufacture them are subjected to tests for adventitious agents, including viruses, which might contaminant them. Some of the compendial methods (in vivo and in vitro in cell culture) were established in the mid-20th century. These methods have not been subjected to current assay validation, as new methods would need to be. This study was undertaken to provide insight into the breadth (selectivity) and sensitivity (limit of detection) of the routine methods, two such validation parameters. Sixteen viral stocks were prepared and characterized. These stocks were tested in serial dilutions by the routine methods to establish which viruses were detected by which methods and above what limit of detection. Sixteen out of sixteen viruses were detected in vitro, though one (bovine viral diarrhea virus) required special conditions to detect and another (rubella virus) was detected with low sensitivity. Many were detected at levels below 1 TCID50 or PFU (titers were established on the production cell line in most cases). In contrast, in vivo, only 6/11 viruses were detected, and 4 of these were detected only at amounts one or more logs above 1 TCID50 or PFU. Only influenza virus and vesicular stomatitis virus were detected at lower amounts in vivo than in vitro. Given the call to reduce, refine, or replace (3 R's) the use of animals in product safety testing and the emergence of new technologies for the detection of viruses, a re-examination of the current adventitious virus testing strategies seems warranted. Suggested pathways forward are offered.
Vaccines; Viral Safety; In Vitro; In Vivo; Adventitious Agents; 3Rs
Although much is currently known about hypersexuality (in the form of excessive sexual behavior) among sexual offenders, the degree to which hypersexual behavior is linked to paraphilic and especially pedophilic interests in non-forensic populations has not been established. The purpose of the present study was to elucidate the associations between total sexual outlets (TSO) and other sex drive indicators, antisocial behavior, pedophilic interests, and sexual offending behavior in a large population-based community sample of males. The sample included 8,718 German men who participated in an online study. Hypersexual behavior as measured by self-reported TSO, self-reported sex drive, criminal history, and pedophilic interests were assessed. In moderated hierarchical logistic regression analyses self-reported contact sexual offending against children was linked to sexual fantasizing about children and antisociality. There was no association between aggregated sex drive, and sexual abusive behaviour in the multivariate analyses. In contrast, self-reported child pornography consumption was associated with sex drive, sexual fantasies involving children, and antisociality. Nevertheless, in clinical practice an assessment of criminal history and pedophilic interests in hypersexual individuals and vice versa hypersexuality in antisocial or pedophilic men should be considered as particularly antisociality and pedophilic interest are important predictors of sexual offending against prepubescent children.
Some higher plants, both angiosperms and gymnosperms, can produce resins and some of these resins can polymerize and fossilize to form ambers. Various physical and chemical techniques have been used to identify and profile different plant resins and have then been applied to fossilized resins (ambers), to try to detect their parent plant affinities and understand the process of polymerization, with varying levels of success. Here we focus on resins produced from today’s most resinous conifer family, the Araucariaceae, which are thought to be the parent plants of some of the Southern Hemisphere’s fossil resin deposits. Fourier transform infrared (FTIR) spectra of the resins of closely related Araucariaceae species were examined to test whether they could be distinguished at genus and species level and whether the results could then be used to infer the parent plant of a New Zealand amber. The resin FTIR spectra are distinguishable from each other, and the three Araucaria species sampled produced similar FTIR spectra, to which Wollemia resin is most similar. Interspecific variability of the FTIR spectra is greatest in the three Agathis species tested. The New Zealand amber sample is similar in key shared features with the resin samples, but it does differ from the extant resin samples in key distinguishing features, nonetheless it is most similar to the resin of Agathis australis in this dataset. However on comparison with previously published FTIR spectra of similar aged amber and older (Eocene) resinites both found in coals from New Zealand and fresh Agathis australis resin, our amber has some features that imply a relatively immature resin, which was not expected from an amber of the Miocene age.
Araucariaceae; Amber; New Caledonia; FTIR; New Zealand
One of the most important issues in molecular dating studies concerns the incorporation of reliable fossil taxa into the phylogenies reconstructed from DNA sequence variation in extant taxa. Lichens are symbiotic associations between fungi and algae and/or cyanobacteria. Several lichen fossils have been used as minimum age constraints in recent studies concerning the diversification of the Ascomycota. Recent evolutionary studies of Lecanoromycetes, an almost exclusively lichen-forming class in the Ascomycota, have utilized the Eocene amber inclusion Alectoria succinic as a minimum age constraint. However, a re-investigation of the type material revealed that this inclusion in fact represents poorly preserved plant remains, most probably of a root. Consequently, this fossil cannot be used as evidence of the presence of the genus Alectoria (Parmeliaceae, Lecanorales) or any other lichens in the Paleogene. However, newly discovered inclusions from Paleogene Baltic and Bitterfeld amber verify that alectorioid morphologies in lichens were in existence by the Paleogene. The new fossils represent either a lineage within the alectorioid group or belong to the genus Oropogon.
Complex biological systems rely on cell surface cues that govern cellular self-recognition and selective interactions with appropriate partners. Molecular diversification of cell surface recognition molecules through DNA recombination and complex alternative splicing has emerged as an important principle for encoding such interactions. However, the lack of tools to specifically detect and quantify receptor protein isoforms is a major impediment to functional studies. We here developed a workflow for targeted mass spectrometry by selected reaction monitoring that permits quantitative assessment of highly diversified protein families. We apply this workflow to dissecting the molecular diversity of the neuronal neurexin receptors and uncover an alternative splicing-dependent recognition code for synaptic ligands.
To create a protein, a gene is first copied to form an RNA molecule that contains regions known as introns and exons. Splicing removes the introns and joins the exons together to form a molecule of ‘messenger RNA’, which is translated into a protein.
Over the course of evolution, many groups—or families—of proteins have expanded and diversified their roles. One way in which this can occur is through a process known as alternative splicing, in which different exons can be included or excluded to generate the final messenger RNA. In this way, a single gene can produce a number of different proteins. These closely related proteins are known as isoforms.
The brain contains billions of neurons that communicate with one another across connections known as synapses. A family of proteins called neurexins helps neurons to form these synapses. Humans have three neurexin genes, which undergo extensive alternative splicing to produce thousands of protein isoforms. However, it is not known whether all of these isoforms are produced in neurons, as existing experimental techniques were not sensitive enough to easily distinguish one isoform from another.
A technique known as ‘selected reaction monitoring’ (or SRM for short) has recently emerged as a promising way to identify proteins. This allows proteins containing specific sequences to be separated out for analysis, in contrast to existing techniques that test randomly selected protein samples, which will result in most isoforms being missed. Schreiner, Simicevic et al. have now developed SRM further and show that this technique can detect the identity and amount of the neurexin isoforms present at synapses, including those that are only produced in very small quantities. Using SRM, Schreiner, Simicevic et al. demonstrate that neurexin isoforms differ in how they interact with synaptic receptors. Thus, alternative splicing of neurexins underlies a ‘recognition code’ at neuronal synapses.
In the future, this newly developed SRM method could be used to investigate isoforms in other protein families and tissues, and so may prove valuable for understanding how a wide range of cellular recognition processes work.
alternative splicing; SRM; MRM; synapse; neuroligin; recognition; mouse
Cell surface proteins are major targets of biomedical research due to their utility as cellular markers and their extracellular accessibility for pharmacological intervention. However, information about the cell surface protein repertoire (the surfaceome) of individual cells is only sparsely available. Here, we applied the Cell Surface Capture (CSC) technology to 41 human and 31 mouse cell types to generate a mass-spectrometry derived Cell Surface Protein Atlas (CSPA) providing cellular surfaceome snapshots at high resolution. The CSPA is presented in form of an easy-to-navigate interactive database, a downloadable data matrix and with tools for targeted surfaceome rediscovery (http://wlab.ethz.ch/cspa). The cellular surfaceome snapshots of different cell types, including cancer cells, resulted in a combined dataset of 1492 human and 1296 mouse cell surface glycoproteins, providing experimental evidence for their cell surface expression on different cell types, including 136 G-protein coupled receptors and 75 membrane receptor tyrosine-protein kinases. Integrated analysis of the CSPA reveals that the concerted biological function of individual cell types is mainly guided by quantitative rather than qualitative surfaceome differences. The CSPA will be useful for the evaluation of drug targets, for the improved classification of cell types and for a better understanding of the surfaceome and its concerted biological functions in complex signaling microenvironments.
The immunogenicity and protective efficacy of a candidate tetravalent dengue virus purified inactivated vaccine (TDENV PIV) formulated with alum or an Adjuvant System (AS01, AS03 tested at three different dose levels, or AS04) was evaluated in a 0, 1-month vaccination schedule in rhesus macaques. One month after dose 2, all adjuvanted formulations elicited robust and persisting neutralizing antibody titers against all four dengue virus serotypes. Most of the formulations tested prevented viremia after challenge, with the dengue serotype 1 and 2 virus strains administered at 40 and 32 weeks post-dose 2, respectively. This study shows that inactivated dengue vaccines, when formulated with alum or an Adjuvant System, are candidates for further development.
Pelobacter carbinolicus and P. acetylenicus oxidize ethanol in syntrophic cooperation with methanogens. Cocultures with Methanospirillum hungatei served as model systems for the elucidation of syntrophic ethanol oxidation previously done with the lost “Methanobacillus omelianskii” coculture. During growth on ethanol, both Pelobacter species exhibited NAD+-dependent alcohol dehydrogenase activity. Two different acetaldehyde-oxidizing activities were found: a benzyl viologen-reducing enzyme forming acetate, and a NAD+-reducing enzyme forming acetyl-CoA. Both species synthesized ATP from acetyl-CoA via acetyl phosphate. Comparative 2D-PAGE of ethanol-grown P. carbinolicus revealed enhanced expression of tungsten-dependent acetaldehyde: ferredoxin oxidoreductases and formate dehydrogenase. Tungsten limitation resulted in slower growth and the expression of a molybdenum-dependent isoenzyme. Putative comproportionating hydrogenases and formate dehydrogenase were expressed constitutively and are probably involved in interspecies electron transfer. In ethanol-grown cocultures, the maximum hydrogen partial pressure was about 1,000 Pa (1 mM) while 2 mM formate was produced. The redox potentials of hydrogen and formate released during ethanol oxidation were calculated to be EH2 = -358±12 mV and EHCOOH = -366±19 mV, respectively. Hydrogen and formate formation and degradation further proved that both carriers contributed to interspecies electron transfer. The maximum Gibbs free energy that the Pelobacter species could exploit during growth on ethanol was −35 to −28 kJ per mol ethanol. Both species could be cultivated axenically on acetaldehyde, yielding energy from its disproportionation to ethanol and acetate. Syntrophic cocultures grown on acetoin revealed a two-phase degradation: first acetoin degradation to acetate and ethanol without involvement of the methanogenic partner, and subsequent syntrophic ethanol oxidation. Protein expression and activity patterns of both Pelobacter spp. grown with the named substrates were highly similar suggesting that both share the same steps in ethanol and acetalydehyde metabolism. The early assumption that acetaldehyde is a central intermediate in Pelobacter metabolism was now proven biochemically.
Our current understanding of how natural genetic variation affects gene expression beyond
well-annotated coding genes is still limited. The use of deep sequencing technologies for the study
of expression quantitative trait loci (eQTLs) has the potential to close this gap. Here, we
generated the first recombinant strain library for fission yeast and conducted an RNA-seq-based QTL
study of the coding, non-coding, and antisense transcriptomes. We show that the frequency of distal
effects (trans-eQTLs) greatly exceeds the number of local effects
(cis-eQTLs) and that non-coding RNAs are as likely to be affected by eQTLs as
protein-coding RNAs. We identified a genetic variation of swc5 that modifies the
levels of 871 RNAs, with effects on both sense and antisense transcription, and show that this
effect most likely goes through a compromised deposition of the histone variant H2A.Z. The strains,
methods, and datasets generated here provide a rich resource for future studies.
antisense transcription; histone variant; non-coding RNA; QTL; Schizosaccharomyces pombe