Mass spectrometry-based proteomics enables the global identification and quantification of proteins and their posttranslational modifications in complex biological samples. However, proteomic ...analysis requires a complete and accurate reference set of proteins and is therefore largely restricted to model organisms with sequenced genomes.
Here, we demonstrate the feasibility of deep genome-free proteomics by using a reference proteome derived from heterogeneous mRNA data. We identify more than 11,000 proteins with 99% confidence from the unfertilized Xenopus laevis egg and estimate protein abundance with approximately 2-fold precision. Our reference database outperforms the provisional gene models based on genomic DNA sequencing and references generated by other methods. Surprisingly, we find that many proteins in the egg lack mRNA support and that many of these proteins are found in blood or liver, suggesting that they are taken up from the blood plasma, together with yolk, during oocyte growth and maturation, potentially contributing to early embryogenesis.
To facilitate proteomics in nonmodel organisms, we make our platform available as an online resource that converts heterogeneous mRNA data into a protein reference set. Thus, we demonstrate the feasibility and power of genome-free proteomics while shedding new light on embryogenesis in vertebrates.
•Genome-free proteomics identifies more than 11,000 proteins in the Xenopus laevis egg•Each protein’s expression level is predicted with approximately 2-fold precision•Many blood plasma proteins are taken up from oocyte during growth in the ovary•Web tool generates proteomic reference sets from mRNA data for any organism
Wühr et al. demonstrate the feasibility of deep proteomics, without the use of a sequenced genome, using the example of the egg of the African clawed frog Xenopus laevis. The authors identify more than 11,000 proteins and can predict each protein’s expression level with approximately 2-fold precision.
A biochemical explanation of development from the fertilized egg to the adult requires an understanding of the proteins and RNAs expressed over time during embryogenesis. We present a comprehensive ...characterization of protein and mRNA dynamics across early development in Xenopus. Surprisingly, we find that most protein levels change little and duplicated genes are expressed similarly. While the correlation between protein and mRNA levels is poor, a mass action kinetics model parameterized using protein synthesis and degradation rates regresses protein dynamics to RNA dynamics, corrected for initial protein concentration. This study provides detailed data for absolute levels of ∼10,000 proteins and ∼28,000 transcripts via a convenient web portal, a rich resource for developmental biologists. It underscores the lasting impact of maternal dowry, finds surprisingly few cases where degradation alone drives a change in protein level, and highlights the importance of transcription in shaping the dynamics of the embryonic proteome.
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•A genome-scale resource of mRNA and protein expression for vertebrate embryogenesis•Temporal patterns of change in mRNA and protein abundance are poorly correlated•A simple kinetic model explains protein expression as a function of mRNA levels•Embryogenesis is driven by maternal protein dowry and tissue-specific transcription
Embryos express proteins at the correct time during development, by balancing the maternal contribution with protein synthesis and degradation. Peshkin et al. determine the absolute concentrations of ∼10,000 proteins and ∼28,000 transcripts across Xenopus development, uncovering the relative roles of these three processes across the proteome and revealing global trends.
Mammals have extremely limited regenerative capabilities; however, axolotls are profoundly regenerative and can replace entire limbs. The mechanisms underlying limb regeneration remain poorly ...understood, partly because the enormous and incompletely sequenced genomes of axolotls have hindered the study of genes facilitating regeneration. We assembled and annotated a de novo transcriptome using RNA-sequencing profiles for a broad spectrum of tissues that is estimated to have near-complete sequence information for 88% of axolotl genes. We devised expression analyses that identified the axolotl orthologs of cirbp and kazald1 as highly expressed and enriched in blastemas. Using morpholino anti-sense oligonucleotides, we find evidence that cirbp plays a cytoprotective role during limb regeneration whereas manipulation of kazald1 expression disrupts regeneration. Our transcriptome and annotation resources greatly complement previous transcriptomic studies and will be a valuable resource for future research in regenerative biology.
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•Creation of a transcriptome with near-complete sequence data for 88% of axolotl genes•Expression analyses identify tissue-enriched transcripts for key tissues•The RNA-binding protein cirbp plays a cytoprotective role in limb regeneration•Knockdown and overexpression of kazald1 in blastema cells impair limb regeneration
Discovery of genes driving axolotl limb regeneration has been challenging, due to limited genomic resources. Bryant et al. have created a transcriptome with near-complete sequence information for most axolotl genes, identified transcriptional profiles that distinguish blastemas from differentiated limb tissues, and uncovered functional roles for cirbp and kazald1 in limb regeneration.
Objective
Coronavirus disease 2019 (COVID‐19) continues to spread, and younger patients are also being critically affected. This study analyzed obesity as an independent risk factor for mortality in ...hospitalized patients younger than 50.
Methods
This study retrospectively analyzed data of patients with COVID‐19 who were hospitalized to a large academic hospital system in New York City between March 1, 2020, and May 17, 2020. Data included demographics, comorbidities, BMI, and smoking status. Obesity groups included the following: BMI of 30 to < 40 kg/m2 and BMI ≥ 40 kg/m2. Multivariable logistic regression models identified variables independently associated with mortality in patients younger and older than 50.
Results
Overall, 3,406 patients were included; 572 (17.0%) patients were younger than 50. In the younger age group, 60 (10.5%) patients died. In the older age group, 1,076 (38.0%) patients died. For the younger population, BMI ≥ 40 was independently associated with mortality (adjusted odds ratio 5.1; 95% CI: 2.3‐11.1). For the older population, BMI ≥ 40 was also independently associated with mortality to a lesser extent (adjusted odds ratio 1.6; 95% CI: 1.2‐2.3).
Conclusions
This study demonstrates that hospitalized patients younger than 50 with severe obesity are more likely to die of COVID‐19. This is particularly relevant in the Western world, where obesity rates are high.
Summary Background The use of transvaginal mesh and biological graft material in prolapse surgery is controversial and has led to a number of enquiries into their safety and efficacy. Existing trials ...of these augmentations are individually too small to be conclusive. We aimed to compare the outcomes of prolapse repair involving either synthetic mesh inlays or biological grafts against standard repair in women. Methods We did two pragmatic, parallel-group, multicentre, randomised controlled trials for our study (PROSPECT PROlapse Surgery: Pragmatic Evaluation and randomised Controlled Trials) in 35 centres (a mix of secondary and tertiary referral hospitals) in the UK. We recruited women undergoing primary transvaginal anterior or posterior compartment prolapse surgery by 65 gynaecological surgeons in these centres. We randomly assigned participants by a remote web-based randomisation system to one of the two trials: comparing standard (native tissue) repair alone with standard repair augmented with either synthetic mesh (the mesh trial) or biological graft (the graft trial). We assigned women (1:1:1 or 1:1) within three strata: assigned to one of the three treatment options, comparison of standard repair with mesh, and comparison of standard repair with graft. Participants, ward staff, and outcome assessors were masked to randomisation where possible; masking was obviously not possible for the surgeon. Follow-up was for 2 years after the surgery; the primary outcomes, measured at 1 year and 2 years, were participant-reported prolapse symptoms (i.e. the Pelvic Organ Prolapse Symptom Score POP-SS) and condition-specific (ie, prolapse-related) quality-of-life scores, analysed in the modified intention-to-treat population. This trial is registered as an International Standard Randomised Controlled Trial, number ISRCTN60695184. Findings Between Jan 8, 2010, and Aug 30, 2013, we randomly allocated 1352 women to treatment, of whom 1348 were included in the analysis. 865 women were included in the mesh trial (430 to standard repair alone, 435 to mesh augmentation) and 735 were included in the graft trial (367 to standard repair alone, 368 to graft augmentation). Because the analyses were carried out separately for each trial (mesh trial and graft trial) some women in the standard repair arm assigned to all treatment options were included in the standard repair group of both trials. 23 of these women did not receive any surgery (15 in the mesh trial, 13 in the graft trial; five were included in both trials) and were included in the baseline analyses only. Mean POP-SS at 1 year did not differ substantially between comparisons (standard 5·4 SD 5·5 vs mesh 5·5 5·1, mean difference 0·00, 95% CI −0·70 to 0·71; p=0·99; standard 5·5 SD 5·6 vs graft 5·6 5·6; mean difference −0·15, −0·93 to 0·63; p=0·71). Mean prolapse-related quality-of-life scores also did not differ between groups at 1 year (standard 2·0 SD 2·7 vs mesh 2·2 2·7, mean difference 0·13, 95% CI −0·25 to 0·51; p=0·50; standard 2·2 SD 2·8 vs graft 2·4 2·9; mean difference 0·13, −0·30 to 0·56; p=0·54). Mean POP-SS at 2 years were: standard 4·9 (SD 5·1) versus mesh 5·3 (5·1), mean difference 0·32, 95% CI −0·39 to 1·03; p=0·37; standard 4·9 (SD 5·1) versus graft 5·5 (5·7); mean difference 0·32, −0·48 to 1·12; p=0·43. Prolapse-related quality-of-life scores at 2 years were: standard 1·9 (SD 2·5) versus mesh 2·2 (2·6), mean difference 0·15, 95% CI −0·23 to 0·54; p=0·44; standard 2·0 (2·5) versus graft 2·2 (2·8); mean difference 0·10, −0·33 to 0·52; p=0·66. Serious adverse events such as infection, urinary retention, or dyspareunia or other pain, excluding mesh complications, occurred with similar frequency in the groups over 1 year (mesh trial: 31/430 7% with standard repair vs 34/435 8% with mesh, risk ratio RR 1·08, 95% CI 0·68 to 1·72; p=0·73; graft trial: 23/367 6% with standard repair vs 36/368 10% with graft, RR 1·57, 0·95 to 2·59; p=0·08). The cumulative number of women with a mesh complication over 2 years in women actually exposed to synthetic mesh was 51 (12%) of 434. Interpretation Augmentation of a vaginal repair with mesh or graft material did not improve women's outcomes in terms of effectiveness, quality of life, adverse effects, or any other outcome in the short term, but more than one in ten women had a mesh complication. Therefore, follow-up is vital to identify any longer-term potential benefits and serious adverse effects of mesh or graft reinforcement in vaginal prolapse surgery. Funding UK National Institute of Health Research.
We have compared the dorsoventral development of hemichordates and chordates to deduce the organization of their common ancestor, and hence to identify the evolutionary modifications of the chordate ...body axis after the lineages split. In the hemichordate embryo, genes encoding bone morphogenetic proteins (Bmp) 2/4 and 5/8, as well as several genes for modulators of Bmp activity, are expressed in a thin stripe of ectoderm on one midline, historically called "dorsal." On the opposite midline, the genes encoding Chordin and Anti-dorsalizing morphogenetic protein (Admp) are expressed. Thus, we find a Bmp-Chordin developmental axis preceding and underlying the anatomical dorsoventral axis of hemichordates, adding to the evidence from Drosophila and chordates that this axis may be at least as ancient as the first bilateral animals. Numerous genes encoding transcription factors and signaling ligands are expressed in the three germ layers of hemichordate embryos in distinct dorsoventral domains, such as pox neuro, pituitary homeobox, distalless, and tbx2/3 on the Bmp side and netrin, mnx, mox, and single-minded on the Chordin-Admp side. When we expose the embryo to excess Bmp protein, or when we deplete endogenous Bmp by small interfering RNA injections, these expression domains expand or contract, reflecting their activation or repression by Bmp, and the embryos develop as dorsalized or ventralized limit forms. Dorsoventral patterning is independent of anterior/posterior patterning, as in Drosophila but not chordates. Unlike both chordates and Drosophila, neural gene expression in hemichordates is not repressed by high Bmp levels, consistent with their development of a diffuse rather than centralized nervous system. We suggest that the common ancestor of hemichordates and chordates did not use its Bmp-Chordin axis to segregate epidermal and neural ectoderm but to pattern many other dorsoventral aspects of the germ layers, including neural cell fates within a diffuse nervous system. Accordingly, centralization was added in the chordate line by neural-epidermal segregation, mediated by the pre-existing Bmp-Chordin axis. Finally, since hemichordates develop the mouth on the non-Bmp side, like arthropods but opposite to chordates, the mouth and Bmp-Chordin axis may have rearranged in the chordate line, one relative to the other.
The giant, single-celled organism Stentor coeruleus has a long history as a model system for studying pattern formation and regeneration in single cells. Stentor 1, 2 is a heterotrichous ciliate ...distantly related to familiar ciliate models, such as Tetrahymena or Paramecium. The primary distinguishing feature of Stentor is its incredible size: a single cell is 1 mm long. Early developmental biologists, including T.H. Morgan 3, were attracted to the system because of its regenerative abilities—if large portions of a cell are surgically removed, the remnant reorganizes into a normal-looking but smaller cell with correct proportionality 2, 3. These biologists were also drawn to Stentor because it exhibits a rich repertoire of behaviors, including light avoidance, mechanosensitive contraction, food selection, and even the ability to habituate to touch, a simple form of learning usually seen in higher organisms 4. While early microsurgical approaches demonstrated a startling array of regenerative and morphogenetic processes in this single-celled organism, Stentor was never developed as a molecular model system. We report the sequencing of the Stentor coeruleus macronuclear genome and reveal key features of the genome. First, we find that Stentor uses the standard genetic code, suggesting that ciliate-specific genetic codes arose after Stentor branched from other ciliates. We also discover that ploidy correlates with Stentor’s cell size. Finally, in the Stentor genome, we discover the smallest spliceosomal introns reported for any species. The sequenced genome opens the door to molecular analysis of single-cell regeneration in Stentor.
•The introns of Stentor coeruleus, a giant ciliate, are 15–16 nt long•The short introns of Stentor are the shortest spliceosomal introns yet reported•Stentor uses a standard genetic code, unlike other characterized ciliates•The ploidy of the Stentor macronucleus is proportional to the volume of the cell
Stentor coeruleus is a giant single-celled organism that can regenerate after being cut in half. Slabodnick et al. describe the Stentor genome, a key tool for future experiments to understand regeneration in a single cell. The genome is unusual in that it contains extremely small introns.
Introduction and hypothesis
Next to existing terminology of the lower urinary tract, due to its increasing complexity, the terminology for pelvic floor dysfunction in women may be better updated by a ...female-specific approach and clinically based consensus report.
Methods
This report combines the input of members of the Standardization and Terminology Committees of two International Organizations, the International Urogynecological Association (IUGA) and the International Continence Society (ICS), assisted at intervals by many external referees. Appropriate core clinical categories and a subclassification were developed to give an alphanumeric coding to each definition. An extensive process of 15 rounds of internal and external review was developed to exhaustively examine each definition, with decision-making by collective opinion (consensus).
Results
A terminology report for female pelvic floor dysfunction, encompassing over 250 separate definitions, has been developed. It is clinically based with the six most common diagnoses defined. Clarity and user-friendliness have been key aims to make it interpretable by practitioners and trainees in all the different specialty groups involved in female pelvic floor dysfunction. Female-specific imaging (ultrasound, radiology, and MRI) has been a major addition while appropriate figures have been included to supplement and help clarify the text. Ongoing review is not only anticipated but will be required to keep the document updated and as widely acceptable as possible.
Conclusions
A consensus-based terminology report for female pelvic floor dysfunction has been produced aimed at being a significant aid to clinical practice and a stimulus for research.
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