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
Human parainfluenza virus type 3 (HPIV3) is a common cause of upper and lower respiratory tract illness in infants and young children. Live-attenuated cold-adapted HPIV3 vaccines have been evaluated in infants but a suitable interval for administration of a second dose of vaccine has not been defined.
HPIV3-seronegative children between the ages of 6 and 36 months were randomized 2:1 in a blinded study to receive two doses of 105 TCID50 (50% tissue culture infectious dose) of live-attenuated, recombinant cold-passaged human PIV3 vaccine (rHPIV3cp45) or placebo 6 months apart. Serum antibody levels were assessed prior to and approximately 4-6 weeks after each dose. Vaccine virus infectivity, defined as detection of vaccine-HPIV3 in nasal wash and/or a ≥ 4-fold rise in serum antibody titer, and reactogenicity were assessed on days 3, 7, and 14 following immunization.
Forty HPIV3-seronegative children (median age 13 months; range 6-35 months) were enrolled; 27 (68%) received vaccine and 13 (32%) received placebo. Infectivity was detected in 25 (96%) of 26 evaluable vaccinees following dose 1 and 9 of 26 subject (35%) following dose 2. Among those who shed virus, the median duration of viral shedding was 12 days (range, 6-15 days) after dose 1 and 6 days (range 3-8 days) after dose 2, with a mean peak log10 viral titer of 3.4 PFU/mL (SD: 1.0) after dose 1 compared to1.5 PFU/mL (SD: 0.92) after dose 2. Overall, reactogenicity was mild, with no difference in rates of fever and upper respiratory infection symptoms between vaccine and placebo groups.
rHPIV3cp45 was immunogenic and well-tolerated in seronegative young children. A second dose administered 6 months after the initial dose was restricted in those previously infected with vaccine virus; however, the second dose boosted antibody responses and induced antibody responses in two previously uninfected children.
Parainfluenza; Live-attenuated vaccine; Recombinant virus vaccine; Pediatric vaccine
Mutations in the GNAL gene have recently been shown to cause primary torsion dystonia. The GNAL-encoded protein (Gαolf) is important for dopamine D1 receptor function and odorant signal transduction. We sequenced all 12 exons of GNAL in 461 patients from Germany, Serbia, and Japan, including 318 patients with dystonia (190 with cervical dystonia), 51 with hyposmia and Parkinson disease, and 92 with tardive dyskinesia or acute dystonic reactions.
We identified the following two novel heterozygous putative mutations in GNAL: p.Gly213Ser in a German patient and p.Ala353Thr in a Japanese patient. These variants were predicted to be pathogenic in silico, were absent in ethnically matched control individuals, and impaired Gαolf coupling to D1 receptors in a bioluminescence energy transfer (BRET) assay. Two additional variants appeared to be benign because they behaved like wild-type samples in the BRET assay (p.Ala311Thr) or were detected in ethnically matched controls (p.Thr92Ala). Both patients with likely pathogenic mutations had craniocervical dystonia with onset in the fifth decade of life. No pathogenic mutations were detected in the patients with hyposmia and Parkinson disease, tardive dyskinesias, or acute dystonic reactions.
Conclusions and Relevance
Mutations in GNAL can cause craniocervical dystonia in different ethnicities. The BRET assay may be a useful tool to support the pathogenicity of identified variants in the GNAL gene.
Background. Human metapneumovirus (HMPV) causes lower respiratory tract infections in young children. rHMPV-SHs is a recombinant HMPV (rHMPV) based on a biologically derived wild-type HMPV strain. We characterized its infectivity and immunogenicity in healthy adults to determine whether it would be suitable for use as the parent virus for the development of live attenuated rHMPV vaccines.
Methods. Twenty-one healthy adults were inoculated intranasally with 106 plaque-forming units of rHMPV-SHs. Respiratory symptoms and shedding of challenge virus were assessed. Neutralizing antibody responses, serum immunoglobulin G and A, and nasal wash specimen immunoglobulin A antibody responses to the HMPV F protein were also measured. Induction of nasal cytokines was assessed with electrochemiluminescence assays.
Results. Nine subjects (43%) were infected with challenge virus as determined by virus detection and/or ≥4-fold rise in serum antibody titers. Peak viral shedding occurred on days 7–9 after infection. Four weeks after inoculation, 35% of subjects had any antibody response. Six of 9 infected subjects had respiratory symptoms, and 3 had headache after inoculation. Cytokine patterns differed considerably between subjects with similar illness severity and viral shedding.
Conclusions. The rHMPV-SHs virus is infectious and is a suitable parent virus for development of live-attenuated HMPV vaccine candidates.
Clinical Trials Registration. NCT01109329.
human metapneumovirus; HMPV; challenge
Recent studies have provided evidence for pulses in the diversification of angiosperms, ferns, gymnosperms, and mosses as well as various groups of animals during the Cretaceous revolution of terrestrial ecosystems. However, evidence for such pulses has not been reported so far for liverworts. Here we provide new insight into liverwort evolution by integrating a comprehensive molecular dataset with a set of 20 fossil age constraints. We found evidence for a relative constant diversification rate of generalistic liverworts (Jungermanniales) since the Palaeozoic, whereas epiphytic liverworts (Porellales) show a sudden increase of lineage accumulation in the Cretaceous. This difference is likely caused by the pronounced response of Porellales to the ecological opportunities provided by humid, megathermal forests, which were increasingly available as a result of the rise of the angiosperms.
Tight spatio-temporal signaling of cytoskeletal and adhesion dynamics is required for localized membrane protrusion that drives directed cell migration. Different ensembles of proteins are therefore likely to get recruited and phosphorylated in membrane protrusions in response to specific cues.
Here, we use an assay that allows to biochemically purify extending protrusions of cells migrating in response to three prototypical receptors: integrins, recepor tyrosine kinases and G-coupled protein receptors. Using quantitative proteomics and phospho-proteomics approaches, we provide evidence for the existence of cue-specific, spatially distinct protein networks in the different cell migration modes.
The integrated analysis of the large-scale experimental data with protein information from databases allows us to understand some emergent properties of spatial regulation of signaling during cell migration. This provides the cell migration community with a large-scale view of the distribution of proteins and phospho-proteins regulating directed cell migration.
Fibroblast; Directional cell migration; Signaling; Proteomics; Phosphorylation
Complete reference maps or datasets, like the genomic map of an organism, are highly beneficial tools for biological and biomedical research. Attempts to generate such reference datasets for a proteome so far failed to reach complete proteome coverage, with saturation apparent at approximately two thirds of the proteomes tested, even for the most thoroughly characterized proteomes. Here, we used a strategy based on high-throughput peptide synthesis and mass spectrometry to generate a close to complete reference map (97% of the genome-predicted proteins) of the S. cerevisiae proteome. We generated two versions of this mass spectrometric map one supporting discovery- (shotgun) and the other hypothesis-driven (targeted) proteomic measurements. The two versions of the map, therefore, constitute a complete set of proteomic assays to support most studies performed with contemporary proteomic technologies. The reference libraries can be browsed via a web-based repository and associated navigation tools. To demonstrate the utility of the reference libraries we applied them to a protein quantitative trait locus (pQTL) analysis, which requires measurement of the same peptides over a large number of samples with high precision. Protein measurements over a set of 78 S. cerevisiae strains revealed a complex relationship between independent genetic loci, impacting on the levels of related proteins. Our results suggest that selective pressure favors the acquisition of sets of polymorphisms that maintain the stoichiometry of protein complexes and pathways.
S. cerevisiae; selected reaction monitoring; SRM; MRM; spectral library; peptide library; mass spectrometric map; protein QTL
Pyrococcus Furiosus (Pfu) is an excellent organism to generate reference samples for proteomics labs because of its moderately sized genome and very little sequence duplication within the genome. We demonstrated a stable and consistent method to prepare proteins in bulk that eliminates growth and preparation as a source of uncertainty in the standard. We performed several proteomic studies in different laboratories using each laboratory's specific workflow as well as separate and integrated data analysis. This study demonstrated that a Pfu whole cell lysate provides suitable protein sample complexity to not only validate proteomic methods, work flows, and benchmark new instruments, but also to facilitate comparison of experimental data generated over time and across instruments or labs.
Pyrococcus furiosus (Pfu); Proteomics; Protein Complex Standard; MudPIT; OFFGEL electrophoresis; Directed LC-MS/MS
The goal of the IDAWG is to facilitate the consistent analysis of HLA and KIR data, and the sharing of those data among the immunogenomic and larger genomic communities. However, the data-management approaches currently applied by immunogenomic researchers are not widely discussed or reported in the literature, and the effect of different approaches on data-analyses is not known.
With ASHI’s support, the IDAWG developed a forty-five question survey on HLA and KIR data-generation, data-management, and data-analysis practices. Survey questions detailed the loci genotyped, typing systems used, nomenclature versions reported, computer operating systems and software used to manage and transmit data, the approaches applied to resolve HLA ambiguity, and the methods used for basic population-level analyses. Respondents were invited to demonstrate their HLA ambiguity resolution approaches in simulated data sets.By May 2012, 156 respondents from 35 nations had completed the survey . These survey respondents represent a broad sampling of the Immunogenomic community; 52% were European, 30% North American, 10% Asian, 4% South American, and 4% from the Pacific.
The project will continue in conjunction with the 17th Workshop, with the aim of developing community data-sharing standards, ambiguity resolution documentation formats, single-task data-Management tools, and, novel data-analysis methods and applications. While additional project details and plans for the 17th IHIW will be forthcoming, we welcome the input and participation in these projects from the histocompatibility and immunogenetics community.
Meta-analysis; Statistics; HLA; Genetics
Population-specific matching probabilities (MP) are a key parameter to assess the benefits of unrelated stem cell donor registries and the need for further donor recruitment efforts. In this study, we describe a general framework for MP estimations of specific and mixed patient populations under consideration of international stem cell donor exchange. Calculations were based on population-specific 4-locus (HLA-A, -B, -C, -DRB1) high-resolution haplotype frequencies (HF) of up to 21 populations. In various scenarios, we calculated several quantities of high practical relevance, including the maximal MP that can be reached by recruiting a fixed number of donors, the corresponding optimal composition by population of new registrants, and the minimal number of donors who need to be recruited to reach a defined MP. Starting at current donor numbers, the largest MP increases due to n = 500,000 additional same-population donors were observed for patients from Bosnia-Herzegovina (+0.25), Greece (+0.21) and Romania (+0.20). Especially small MP increases occurred for European Americans (+0.004), Germans (+0.01) and Hispanic Americans (+0.01). Due to the large Chinese population, the optimal distribution of n = 5,000,000 new donors worldwide included 3.9 million Chinese donors. As a general result of our calculations, we observed a need for same-population donor recruitment in order to increase population-specific MP efficiently. This result was robust despite limitations of our input data, including the use of HF derived from relatively small samples ranging from n = 1028 (Bosnia-Herzegovina) to n = 33,083 (Turkey) individuals. National strategies that neglect domestic donor recruitment should therefore be critically re-assessed, especially if only few donors have been recruited so far.
A close match of the HLA alleles between donor and recipient is an important prerequisite for successful unrelated hematopoietic stem cell transplantation. To increase the chances of finding an unrelated donor, registries recruit many hundred thousands of volunteers each year. Many registries with limited resources have had to find a trade-off between cost and resolution and extent of typing for newly recruited donors in the past. Therefore, we have taken advantage of recent improvements in NGS to develop a workflow for low-cost, high-resolution HLA typing.
We have established a straightforward three-step workflow for high-throughput HLA typing: Exons 2 and 3 of HLA-A, -B, -C, -DRB1, -DQB1 and -DPB1 are amplified by PCR on Fluidigm Access Array microfluidic chips. Illumina sequencing adapters and sample specific tags are directly incorporated during PCR. Upon pooling and cleanup, 384 samples are sequenced in a single Illumina MiSeq run. We developed “neXtype” for streamlined data analysis and HLA allele assignment. The workflow was validated with 1140 samples typed at 6 loci. All neXtype results were concordant with the Sanger sequences, demonstrating error-free typing of more than 6000 HLA loci. Current capacity in routine operation is 12,000 samples per week.
The workflow presented proved to be a cost-efficient alternative to Sanger sequencing for high-throughput HLA typing. Despite the focus on cost efficiency, resolution exceeds the current standards of Sanger typing for donor registration.
Human leukocyte antigen; HLA typing; NGS; Dual indexing; 4-primer approach; Amplicon-based sequencing; Fluidigm Access Array; Illumina MiSeq
Disturbances of fetal autonomic brain development can be evaluated from fetal heart rate patterns (HRP) reflecting the activity of the autonomic nervous system. Although HRP analysis from cardiotocographic (CTG) recordings is established for fetal surveillance, temporal resolution is low. Fetal magnetocardiography (MCG), however, provides stable continuous recordings at a higher temporal resolution combined with a more precise heart rate variability (HRV) analysis. A direct comparison of CTG and MCG based HRV analysis is pending. The aims of the present study are: (i) to compare the fetal maturation age predicting value of the MCG based fetal Autonomic Brain Age Score (fABAS) approach with that of CTG based Dawes-Redman methodology; and (ii) to elaborate fABAS methodology by segmentation according to fetal behavioral states and HRP. We investigated MCG recordings from 418 normal fetuses, aged between 21 and 40 weeks of gestation. In linear regression models we obtained an age predicting value of CTG compatible short term variability (STV) of R2 = 0.200 (coefficient of determination) in contrast to MCG/fABAS related multivariate models with R2 = 0.648 in 30 min recordings, R2 = 0.610 in active sleep segments of 10 min, and R2 = 0.626 in quiet sleep segments of 10 min. Additionally segmented analysis under particular exclusion of accelerations (AC) and decelerations (DC) in quiet sleep resulted in a novel multivariate model with R2 = 0.706. According to our results, fMCG based fABAS may provide a promising tool for the estimation of fetal autonomic brain age. Beside other traditional and novel HRV indices as possible indicators of developmental disturbances, the establishment of a fABAS score normogram may represent a specific reference. The present results are intended to contribute to further exploration and validation using independent data sets and multicenter research structures.
prenatal diagnosis; fetal autonomic brain age; magnetocardiography; cardiotocography
Lejeunea is a largely epiphytic, subcosmopolitan liverwort genus with a complex taxonomic history. Species circumscriptions and their relationships are subject to controversy; biogeographic history and diversification through time are largely unknown.
Methodology and Results
We employed sequences of two chloroplast regions (trnL-trnF, rbcL) and the nuclear ribosomal ITS region of 332 accessions to explore the phylogeny of the Harpalejeunea-Lejeunea-Microlejeunea complex. Lejeunea forms a well-supported clade that splits into two main lineages corresponding to L. subg. Lejeunea and L. subg. Crossotolejeunea. Neotropical accessions dominate early diverging lineages of both main clades of Lejeunea. This pattern suggests an origin in the Neotropics followed by several colonizations from the Neotropics into the Paleotropics and vice versa. Most Afro-Madagascan clades are related to Asian clades. Several temperate Lejeunea radiations were detected. Eighty two of the 91 investigated Lejeunea species could be identified to species level. Of these 82 species, 54 were represented by multiple accessions (25 para- or polyphyletic, 29 monophyletic). Twenty nine of the 36 investigated species of L. subg. Lejeunea were monoicous and 7 dioicous. Within L. subg. Crossotolejeunea, 15 of the 46 investigated species were monoicous and 31 dioicous. Some dioicous as well as some monoicous species have disjunct ranges.
We present the first global phylogeny of Lejeunea and the first example of a Neotropical origin of a Pantropical liverwort genus. Furthermore, we provide evidence for the Neotropics as a cradle of Lejeunea lineages and detect post-colonization radiations in Asia, Australasia, Afro-Madagascar and Europe. Dioicy/monoicy shifts are likely non-randomly distributed. The presented phylogeny points to the need of integrative taxonomical studies to clarify many Lejeunea binomials. Most importantly, it provides a framework for future studies on the diversification of this lineage in space and time, especially in the context of sexual systems in Lejeuneaceae.
Human parainfluenza viruses (PIVs) cause acute respiratory illness in children, the elderly, and immunocompromised patients. PIV3 is a common cause of bronchiolitis and pneumonia, whereas PIV1 and 2 are frequent causes of upper respiratory tract illness and croup. To assess how PIV1, 2, and 3 differ with regard to replication and induction of type I interferons, interleukin-6, and relevant chemokines, we infected primary human airway epithelium (HAE) cultures from the same tissue donors and examined replication kinetics and cytokine secretion. PIV1 replicated to high titer yet did not induce cytokine secretion until late in infection, while PIV2 replicated less efficiently but induced an early cytokine peak. PIV3 replicated to high titer but induced a slower rise in cytokine secretion. The T cell chemoattractants CXCL10 and CXCL11 were the most abundant chemokines induced. Differences in replication and cytokine secretion might explain some of the differences in PIV serotype-specific pathogenesis and epidemiology.
Human parainfluenza virus; Human airway epithelium; cytokines; chemokines; interferon; pathogenesis
Regional HLA frequency differences are of potential relevance for the optimization of stem cell donor recruitment. We analyzed a very large sample (n = 123,749) of registered Polish stem cell donors. Donor figures by 1-digit postal code regions ranged from n = 5,243 (region 9) to n = 19,661 (region 8). Simulations based on region-specific haplotype frequencies showed that donor recruitment in regions 0, 2, 3 and 4 (mainly located in the south-eastern part of Poland) resulted in an above-average increase of matching probabilities for Polish patients. Regions 1, 7, 8, 9 (mainly located in the northern part of Poland) showed an opposite behavior. However, HLA frequency differences between regions were generally small. A strong indication for regionally focused donor recruitment efforts can, therefore, not be derived from our analyses. Results of haplotype frequency estimations showed sample size effects even for sizes between n≈5,000 and n≈20,000. This observation deserves further attention as most published haplotype frequency estimations are based on much smaller samples.
Human parainfluenza viruses (HPIVs) are a common cause of acute respiratory illness throughout life. Infants, children, and the immunocompromised are the most likely to develop severe disease. HPIV1 and HPIV2 are best known to cause croup while HPIV3 is a common cause of bronchiolitis and pneumonia. HPIVs replicate productively in respiratory epithelial cells and do not spread systemically unless the host is severely immunocompromised. Molecular studies have delineated how HPIVs evade and block cellular innate immune responses to permit efficient replication, local spread, and host-to-host transmission. Studies using ex vivo human airway epithelium have focused on virus tropism, cellular pathology and the epithelial inflammatory response, elucidating how events early in infection shape the adaptive immune response and disease outcome.
Pathogen access to host nutrients in infected tissues is fundamental for pathogen growth and virulence, disease progression, and infection control. However, our understanding of this crucial process is still rather limited because of experimental and conceptual challenges. Here, we used proteomics, microbial genetics, competitive infections, and computational approaches to obtain a comprehensive overview of Salmonella nutrition and growth in a mouse typhoid fever model. The data revealed that Salmonella accessed an unexpectedly diverse set of at least 31 different host nutrients in infected tissues but the individual nutrients were available in only scarce amounts. Salmonella adapted to this situation by expressing versatile catabolic pathways to simultaneously exploit multiple host nutrients. A genome-scale computational model of Salmonella in vivo metabolism based on these data was fully consistent with independent large-scale experimental data on Salmonella enzyme quantities, and correctly predicted 92% of 738 reported experimental mutant virulence phenotypes, suggesting that our analysis provided a comprehensive overview of host nutrient supply, Salmonella metabolism, and Salmonella growth during infection. Comparison of metabolic networks of other pathogens suggested that complex host/pathogen nutritional interfaces are a common feature underlying many infectious diseases.
Infectious diseases are a major health problem worldwide. To cause disease, pathogens need to acquire host nutrients for growth in infected tissues and for the expression of virulence factors. In this study, we investigated Salmonella nutrition and growth in a well-characterized mouse model of human typhoid fever. We used a panel of Salmonella mutants with metabolic defects to assess the importance of various nutrient utilization pathways for Salmonella growth. We derived from these experimental data a computational model that predicts nutrient uptake rates, activity of metabolic pathways, and the effects of Salmonella enzyme defects on in vivo growth. The vast majority of these predictions were in close agreement with independent experimental data suggesting the model provided a consistent overview of Salmonella metabolism during infection. The data showed that Salmonella depend on a highly complex diet with many different host nutrients, but each of these nutrients is available in only scarce amounts. To grow and cause disease, Salmonella must simultaneously exploit these various nutrients with versatile degradation pathways. Similar complex pathogen diets might also drive many other infectious diseases.
The cellular ESCRT (endosomal sorting complex required for transport) system functions in cargo-sorting, in the formation of intraluminal vesicles that comprise multivesicular bodies (MVB), and in cytokinesis, and this system can be hijacked by a number of enveloped viruses to promote budding. The respiratory pathogen human parainfluenza virus type I (HPIV1) encodes a nested set of accessory C proteins that play important roles in down-regulating viral transcription and replication, in suppressing the type I interferon (IFN) response, and in suppressing apoptosis. Deletion or mutation of the C proteins attenuates HPIV1 in vivo, and such mutants are being evaluated preclinically and clinically as vaccines. We show here that the C proteins interact and co-localize with the cellular protein Alix, which is a member of the class E vacuolar protein sorting (Vps) proteins that assemble at endosomal membranes into ESCRT complexes. The HPIV1 C proteins interact with the Bro1 domain of Alix at a site that is also required for the interaction between Alix and Chmp4b, a subunit of ESCRT-III. The C proteins are ubiquitinated and subjected to proteasome-mediated degradation, but the interaction with AlixBro1 protects the C proteins from degradation. Neither over-expression nor knock-down of Alix expression had an effect on HPIV1 replication, although this might be due to the large redundancy of Alix-like proteins. In contrast, knocking down the expression of Chmp4 led to an approximately 100-fold reduction in viral titer during infection with wild-type (WT) HPIV1. This level of reduction was similar to that observed for the viral mutant, P(C-) HPIV1, in which expression of the C proteins were knocked out. Chmp4 is capable of out-competing the HPIV1 C proteins for binding Alix. Together, this suggests a possible model in which Chmp4, through Alix, recruits the C proteins to a common site on intracellular membranes and facilitates budding.
We report a high quality and system-wide proteome catalogue covering 71% (3,542 proteins) of the predicted genes of fission yeast, Schizosaccharomyces pombe, presenting the largest protein dataset to date for this important model organism. We obtained this high proteome and peptide (11.4 peptides/protein) coverage by a combination of extensive sample fractionation, high resolution Orbitrap mass spectrometry, and combined database searching using the iProphet software as part of the Trans-Proteomics Pipeline. All raw and processed data are made accessible in the S. pombe PeptideAtlas. The identified proteins showed no biases in functional properties and allowed global estimation of protein abundances. The high coverage of the PeptideAtlas allowed correlation with transcriptomic data in a system-wide manner indicating that post-transcriptional processes control the levels of at least half of all identified proteins. Interestingly, the correlation was not equally tight for all functional categories ranging from rs >0.80 for proteins involved in translation to rs <0.45 for signal transduction proteins. Moreover, many proteins involved in DNA damage repair could not be detected in the PeptideAtlas despite their high mRNA levels, strengthening the translation-on-demand hypothesis for members of this protein class. In summary, the extensive and publicly available S. pombe PeptideAtlas together with the generated proteotypic peptide spectral library will be a useful resource for future targeted, in-depth, and quantitative proteomic studies on this microorganism.
In syntrophic conversion of butyrate to methane and CO2, butyrate is oxidized to acetate by secondary fermenting bacteria such as Syntrophomonas wolfei in close cooperation with methanogenic partner organisms, e.g., Methanospirillum hungatei. This process involves an energetically unfavourable shift of electrons from the level of butyryl-CoA oxidation to the substantially lower redox potential of proton and/or CO2 reduction, in order to transfer these electrons to the methanogenic partner via hydrogen and/or formate.
In the present study, all prominent membrane-bound and soluble proteins expressed in S. wolfei specifically during syntrophic growth with butyrate, in comparison to pure-culture growth with crotonate, were examined by one- and two-dimensional gel electrophoresis, and identified by peptide fingerprinting-mass spectrometry. A membrane-bound, externally oriented, quinone-linked formate dehydrogenase complex was expressed at high level specifically during syntrophic butyrate oxidation, comprising a selenocystein-linked catalytic subunit with a membrane-translocation pathway signal (TAT), a membrane-bound iron-sulfur subunit, and a membrane-bound cytochrome. Soluble hydrogenases were expressed at high levels specifically during growth with crotonate. The results were confirmed by native protein gel electrophoresis, by formate dehydrogenase and hydrogenase-activity staining, and by analysis of formate dehydrogenase and hydrogenase activities in intact cells and cell extracts. Furthermore, constitutive expression of a membrane-bound, internally oriented iron-sulfur oxidoreductase (DUF224) was confirmed, together with expression of soluble electron-transfer flavoproteins (EtfAB) and two previously identified butyryl-CoA dehydrogenases.
Our findings allow to depict an electron flow scheme for syntrophic butyrate oxidation in S. wolfei. Electrons derived from butyryl-CoA are transferred through a membrane-bound EtfAB:quinone oxidoreductase (DUF224) to a menaquinone cycle and further via a b-type cytochrome to an externally oriented formate dehydrogenase. Hence, an ATP hydrolysis-driven proton-motive force across the cytoplasmatic membrane would provide the energy input for the electron potential shift necessary for formate formation.
Mutations in THAP1 have been associated with dystonia 6 (DYT6). THAP1 encodes a transcription factor that represses the expression of DYT1. To further evaluate the mutational spectrum of THAP1 and its associated phenotype, we sequenced THAP1 in 567 patients with focal (n=461), segmental (n=68), or generalized dystonia (n=38). We identified 10 novel variants, including six missense substitutions within the DNA-binding Thanatos-associated protein domain (Arg13His, Lys16Glu, His23Pro, Lys24Glu, Pro26Leu, Ile80Val), a 1bp-deletion downstream of the nuclear localization signal (Asp191Thrfs*9), and three alterations in the untranslated regions. The effect of the missense variants was assessed using prediction tools and luciferase reporter gene assays. This indicated the Ile80Val substitution as a benign variant. The subcellular localization of Asp191Thrfs*9 suggests a disturbed nuclear import for this mutation. Thus, we consider six of the 10 novel variants as pathogenic mutations accounting for a mutation frequency of 1.1%. Mutation carriers presented mainly with early onset dystonia (<12 years in five of six patients). Symptoms started in an arm or neck and spread to become generalized in three patients or segmental in two patients. Speech was affected in four mutation carriers. In conclusion, THAP1 mutations are rare in unselected dystonia patients and functional analysis is necessary to distinguish between benign variants and pathogenic mutations.
dystonia; THAP1; mutation; DNA binding; phenotype–genotype
Human parainfluenza virus type 3 (HPIV3) is an important cause of lower respiratory tract illness in children, yet a licensed vaccine or antiviral drug is not available. We evaluated the safety, tolerability, infectivity, and immunogenicity of two intranasal, live-attenuated HPIV3 vaccines, designated rHPIV3-NB and rB/HPIV3, that were cDNA-derived chimeras of HPIV3 and bovine PIV3 (BPIV3). These were evaluated in adults, HPIV3 seropositive children, and HPIV3 seronegative children. A total of 112 subjects participated in these studies. Both rB/HPIV3 and rHPIV3-NB were highly restricted in replication in adults and seropositive children but readily infected seronegative children, who shed mean peak virus titers of 102.8 vs. 103.7 pfu/mL, respectively. Although rB/HPIV3 was more restricted in replication in seronegative children than rHPIV3-NB, it induced significantly higher titers of hemagglutination inhibition (HAI) antibodies against HPIV3. Taken together, these data suggest that the rB/HPIV3 vaccine is the preferred candidate for further clinical development.
parainfluenza; live-attenuated vaccine; clinical trial
In children under 5 years of age, human parainfluenza viruses (HPIVs) as a group are the second most common etiology of acute respiratory illness leading to hospitalization, surpassed only by respiratory syncytial virus but ahead of influenza viruses. Using reverse genetics systems for HPIV serotypes 1, 2 and 3 (HPIV1, 2 and 3), several live-attenuated HPIVs have been generated and evaluated as intranasal vaccines in adults and in children. Two vaccines against HPIV3 were found to be well tolerated, infectious and immunogenic in Phase I trials in HPIV3-seronegative infants and children and should progress to proof-of-concept trials. Vaccines against HPIV1 and HPIV2 are less advanced and have just entered pediatric trials.
acute respiratory illness; clinical trial; intranasal; live-attenuated; parainfluenza virus vaccine; pediatric; vaccine
Data on absolute molecule numbers will empower the modeling, understanding, and comparison of cellular functions and biological systems. We quantified transcriptomes and proteomes in fission yeast during cellular proliferation and quiescence. This rich resource provides the first comprehensive reference for all RNA and most protein concentrations in a eukaryote under two key physiological conditions. The integrated data set supports quantitative biology and affords unique insights into cell regulation. Although mRNAs are typically expressed in a narrow range above 1 copy/cell, most long, noncoding RNAs, except for a distinct subset, are tightly repressed below 1 copy/cell. Cell-cycle-regulated transcription tunes mRNA numbers to phase-specific requirements but can also bring about more switch-like expression. Proteins greatly exceed mRNAs in abundance and dynamic range, and concentrations are regulated to functional demands. Upon transition to quiescence, the proteome changes substantially, but, in stark contrast to mRNAs, proteins do not uniformly decrease but scale with cell volume.
► Cellular numbers for all RNAs and most proteins during proliferation and quiescence ► Cells contain 1-10 copies of most mRNAs and ∼100–1 million copies of most proteins ► Distinct subset of long noncoding RNAs is expressed above 1 copy/cell ► Quiescent cells show ∼4-fold lower RNA concentrations and highly remodeled proteome
Quantitative RNA-seq and mass spectrometry in two cellular states are used to show that proteins greatly exceed mRNAs in abundance and dynamic range in yeast, and concentrations are regulated to functional demands. Upon transition to quiescence, the proteome changes substantially, but in contrast to mRNAs, proteins do not uniformly decrease but scale with cell volume.