Pregnancy establishment in mammals requires a complex sequence of events, including bi-lateral embryo-maternal communication, leading up to implantation. This is the time when most pregnancy loss occurs in mammals (including humans and food production species) and dysregulation in embryo-maternal communication contributes to pregnancy loss. Embryo-derived factors modify the function of the endometrium for pregnancy success. We hypothesise that these previously unexplored conceptus-derived proteins may be involved in altering the function of the endometrium to facilitate early pregnancy events in mammals with different early pregnancy phenotypes. Here, we show that protein disulphide-isomerase (PDI) is a highly conserved protein among mammals and provide evidence for a species-specific role for PDI in endometrial function in mammals with different implantation strategies. We show how PDI alters the endometrial transcriptome in human and bovine in vitro in a species-specific manner and using a microfluidic approach we demonstrate that it alters the secretome capability of the endometrium. We also provide evidence from in vitro assays using human-derived cells that MNS1, a transcript commonly downregulated in response to PDI in human and bovine endometrial epithelial cells, may be involved in the attachment phase of implantation. We propose that the trophoblast-derived protein PDI, is involved in supporting the modulation of the uterine luminal fluid (ULF) secreted by the endometrium to support conceptus nourishment, and in the process of embryo attachment to the uterine lumen for pregnancy success in mammals.

ABSTRACT Pregnancy establishment in mammals requires a complex sequence of events, including bi-lateral embryo-maternal communication, leading up to implantation. This is the time when most pregnancy loss occurs in mammals (including humans and food production species) and dysregulation in embryo-maternal communication contributes to pregnancy loss. Embryo-derived factors modify the function of the endometrium for pregnancy success. We hypothesise that these previously unexplored conceptus-derived proteins may be involved in altering the function of the endometrium to facilitate early pregnancy events in mammals with different early pregnancy phenotypes. Here, we show that protein disulphide-isomerase (PDI) is a highly conserved protein among mammals, and provide evidence for a species-specific roles for PDI in endometrial function in mammals with different implantation strategies. We show how PDI alters the endometrial transcriptome in human and bovine in vitro in a species-specific manner, and using a microfluidic approach we demonstrate that it alters the secretome capability of the endometrium. We also provide evidence from in vitro assays using human-derived cells that MNS1, a transcript commonly downregulated in response to PDI in human and bovine endometrial epithelial cells, may be involved in the attachment (but not invasion) phase of implantation. We propose that the trophoblast-derived protein PDI, is involved in supporting the modulation of the uterine luminal fluid secreted by the endometrium to support conceptus nourishment, and also in the process of embryo attachment to the uterine lumen for pregnancy success in mammals. SIGNIFICANCE STATEMENT We provide evidence that a highly conserved protein (PDI) alters the endometrial transcriptome in a species- and cell-specific manner. Exposure of endometrial epithelia to PDI altered genes belonging to immune modulatory, pro-inflammatory, and adhesion-pathways. One transcript, MNS1, was commonly downregulated in endometrial epithelia from species with superficial (bovine) and invasive (human) implantation morphologies. Knockdown of MNS1 expression in humans epithelia altered the ability of human trophoblast BeWo spheroids to attach suggesting a mechanism by which PDI affects implantation in human and bovine. In addition, using a microfluidics approach we have shown that PDI alters the secretome in a species-specific manner demonstrating PDI alters a key function of the endometrium in mammals.

Systematic quantification of phosphoprotein within cell signaling networks in solid tissues remains challenging and precise quantification in large scale samples has great potential for biomarker identification and validation. We developed a reverse phase protein array (RPPA) based phosphor-antibody characterization approach by taking advantage of the lysis buffer compatible with alkaline phosphatase (AP) treatment that differs from the conventional RPPA antibody validation procedure and applied it onto fresh frozen (FF) and formalin-fixed and paraffin-embedded tissue (FFPE) to test its applicability. By screening 106 phospho-antibodies using RPPA, we demonstrated that AP treatment could serve as an independent factor to be adopted for rapid phospho-antibody selection. We also showed desirable reproducibility and specificity in clincical specimens indicating its potential for tissue-based phospho-protein profiling. Of further clinical significance, using the same approach, based on melanoma and lung cancer FFPE samples, we showed great interexperimental reproducibility and significant correlation with pathological markers in both tissues generating meaningful data that match clinical features. Our findings set a benchmark of an efficient workflow for phospho-antibody characterization that is compatible with high-plex clinical proteomics in precison oncology.

Coral reefs are facing unprecedented mass bleaching and mortality events due to marine heatwaves and climate change. To avoid extirpation, corals must adapt. Individual variation in heat tolerance and its heritability underpin the potential for coral adaptation. However, the magnitude of heat tolerance variability within coral populations is largely unresolved. We address this knowledge gap by exposing corals from a single reef to an experimental marine heatwave. We found that double the heat stress dosage was required to induce bleaching in the most-tolerant 10%, compared to the least-tolerant 10% of the population. By the end of the heat stress exposure, all of the least-tolerant corals were dead, whereas the most-tolerant remained alive. To contextualize the scale of this result over the coming century, we show that under an ambitious future emissions scenario, such differences in coral heat tolerance thresholds equate to up to 17 years delay until the onset of annual bleaching and mortality conditions. However, this delay is limited to only 10 years under a high emissions scenario. Our results show substantial variability in coral heat tolerance which suggests scope for natural or assisted evolution to limit the impacts of climate change in the short-term. For coral reefs to persist through the coming century, coral adaptation must keep pace with ocean warming, and ambitious emissions reductions must be realized.

Extracellular DNA (eDNA) is a key component of many microbial biofilms including dental plaque. However, the roles of extracellular deoxyribonuclease (DNase) enzymes within biofilms are poorly understood. Streptococcus gordonii is a pioneer colonizer of dental plaque. Here, we identified and characterised SsnA, a cell wall-associated protein responsible for extracellular DNase activity of S. gordonii . The SsnA-mediated extracellular DNase activity of S. gordonii was suppressed following growth in sugars. SsnA was purified as a recombinant protein and shown to be inactive below pH 6.5. SsnA inhibited biofilm formation by Streptococcus mutans in a pH-dependent manner. Further, SsnA inhibited the growth of oral microcosm biofilms in human saliva. However, inhibition was ameliorated by the addition of sucrose. Together, these data indicate that S. gordonii SsnA plays a key role in interspecies competition within oral biofilms. Acidification of the medium through sugar catabolism could be a strategy for cariogenic species such as S. mutans to prevent SsnA-mediated exclusion from biofilms.

Coral cover on tropical reefs has declined during the last three decades due to the combined effects of climate change, destructive fishing, pollution, and land use change. Drastic reductions in greenhouse gas emissions combined with effective coastal management and conservation strategies are essential to slow this decline. Innovative approaches, such as selective breeding for adaptive traits combined with large-scale sexual propagation, are being developed with the aim of pre-adapting reefs to increased ocean warming. However, there are still major gaps in our understanding of the technical and methodological constraints to producing corals for such restoration interventions. Here we propose a framework for selectively breeding corals and rearing them from eggs to 2.5-year old colonies using the coral Acropora digitifera as a model species. We present methods for choosing colonies for selective crossing, enhancing early survivorship in ex situ and in situ nurseries, and outplanting and monitoring colonies on natal reefs. We used a short-term (7-day) temperature stress assay to select parental colonies based on heat tolerance of excised branches. From six parental colonies, we produced 12 distinct crosses, and compared survivorship and growth of colonies transferred to in situ nurseries or outplanted to the reef at different ages. We demonstrate that selectively breeding and rearing coral colonies is technically feasible at small scales and could be upscaled as part of restorative assisted evolution initiatives. Nonetheless, there are still challenges to overcome before selective breeding can be implemented as a viable conservation tool, especially at the post-settlement and outplanting phases. Although interdisciplinary approaches will be needed to overcome many of the challenges identified in this study, selective breeding has the potential to be a viable tool within a reef managers toolbox to support the persistence of selected reefs in the face of climate change.

BACKGROUND:It is not enough to optimize proteomics assays. It is critical those assays are robust to operating conditions. Without robust assays, proteomic biomarkers are unlikely to translate readily into the clinic. This study outlines a structured approach to the identification of a robust operating window for proteomics assays and applies that method to Sequential Window Acquisition of all Theoretical Spectra Mass Spectroscopy (SWATH-MS).METHODS:We used a sequential quality by design approach exploiting a fractional screening design to first identify critical SWATH-MS parameters, then using response surface methods to identify a robust operating window with good reproducibility, before validating those settings in a separate validation study.RESULTS:The screening experiment identified two critical SWATH-MS parameters. We modelled the number of proteins and reproducibility as a function of those parameters identifying an operating window permitting robust maximization of the number of proteins quantified in human serum. In a separate validation study, these settings were shown to give good proteome-wide coverage and high quantification reproducibility.CONCLUSIONS:Using design of experiments permits identification of a robust operating window for SWATH-MS. The method gives a good understanding of proteomics assays and greater data-driven confidence in SWATH-MS performance.

Coral cover on tropical reefs has declined during the last three decades due to the combined effects of climate change, destructive fishing, pollution, and land use change. Drastic reductions in greenhouse gas emissions combined with effective coastal management and conservation strategies are essential to slow this decline. Innovative approaches, such as selective breeding for adaptive traits combined with large-scale sexual propagation, are being developed with the aim of pre-adapting reefs to increased ocean warming. However, there are still major gaps in our understanding of the technical and methodological constraints to producing corals for such restoration interventions. Here we propose a framework for selectively breeding corals and rearing them from eggs to 2.5-year old colonies using the coral Acropora digitifera as a model species. We present methods for choosing colonies for selective crossing, enhancing early survivorship in ex situ and in situ nurseries, and outplanting and monitoring colonies on natal reefs. We used a short-term (7-day) temperature stress assay to select parental colonies based on heat tolerance of excised branches. From six parental colonies, we produced 12 distinct crosses, and compared survivorship and growth of colonies transferred to in situ nurseries or outplanted to the reef at different ages. We demonstrate that selectively breeding and rearing coral colonies is technically feasible at small scales and could be upscaled as part of restorative assisted evolution initiatives. Nonetheless, there are still challenges to overcome before selective breeding can be implemented as a viable conservation tool, especially at the post-settlement and outplanting phases. Although interdisciplinary approaches will be needed to overcome many of the challenges identified in this study, selective breeding has the potential to be a viable tool within reef managers’ toolbox to support the persistence of selected reefs in the face of climate change.

During the preimplantation period of pregnancy in eutherian mammals, transcriptional and proteomic changes in the uterine endometrium are required to facilitate receptivity to an implanting blastocyst. These changes are mediated, in part, by proteins produced by the developing conceptus (inner cell mass and extraembryonic membranes). We hypothesized that this common process in early pregnancy in eutheria may be facilitated by highly conserved conceptus-derived proteins such as macrophage capping protein (CAPG). We propose that CAPG may share functionality in modifying the transcriptome of the endometrial epithelial cells to facilitate receptivity to implantation in species with different implantation strategies. A recombinant bovine form of CAPG (91% sequence identity between bovine and human) was produced and bovine endometrial epithelial (bEECs) and stromal (bESCs) and human endometrial epithelial cells (hEECs) were cultured for 24 hours with and without recombinant bovine CAPG (rbCAPG). RNA sequencing and quantitative real-time PCR analysis were used to assess the transcriptional response to rbCAPG (Control, vehicle, CAPG 10, 100, 1000 ng/mL: n = 3 biological replicates per treatment per species). Treatment of bEECs with CAPG resulted in alterations in the abundance of 1052 transcripts (629 increased and 423 decreased) compared to vehicle controls. Treatment of hEECs with bovine CAPG increased expression of transcripts previously known to interact with CAPG in different systems (CAPZB, CAPZA2, ADD1,andADK) compared with vehicle controls (P < .05). In conclusion, we have demonstrated that CAPG, a highly conserved protein in eutherian mammals, elicits a transcriptional response in the endometrial epithelium in species with different implantation strategies that may contribute to pregnancy success.

Liquid chromatography coupled with mass spectrometric detection is one of the major technologies used for protein sequencing, identification, and quantification. This review provides an introduction of the current state-of-the-art technology in peptide profiling using nano-liquid chromatography–mass spectrometry applied to large-scale protein or proteome analysis. In particular, different aspects of the bottom-up proteomics workflow are covered, including aspects of sample preparation such as protein digestion, nanoflow gradient reversed-phase chromatographic separation, LC–MS interfacing via electrospray ionization, tandem mass spectrometry of digests, protein identification via database searches, and finally peptide quantitation.

Advances in liquid chromatography-mass spectrometry have facilitated the incorporation of proteomic studies to many biology experimental workflows. In particular, the introduction of Data-Independent Acquisition platforms, such as SWATH, offers several advantages for label-free quantitative assessment of complex proteomes over Data-Dependent Acquisition (DDA) approaches. However, SWATH data interpretation requires spectral libraries as a reference resource. This is often not available for many species of experimental models. The guinea pig (cavia porcellus) is an excellent experimental model for translation to many aspects of human physiology and disease yet there is limited experimental information regarding its proteome. In an effort to overcome this knowledge gap, we generated a comprehensive spectral library of the guinea pig proteome. Homogenates and tryptic peptide digests were prepared from 16 tissues and subjected to >200 DDA runs. Analysis of >250,000 peptide-spectrum matches resulted in the construction of a library of 73594 peptides corresponding to 7667 proteins. This spectral library furnishes the research community with the first comprehensive guinea pig proteomic resource that will facilitate future molecular-phenotypic studies using (re-engaging) the guinea pig as an experimental model of relevance to human biology. The guinea pig spectral library and MS data are freely accessible in the MassIVE repository (MSV000083199).

Buskin, Adriana and Zhu, Lili and Chichagova, Valeria and Basu, Basudha and Moza ari-Jovin, Sina and Dolan, David and Droop, Alastair and Collin, Joseph and Bronstein, Revital and Mehrotra, Sudeep and Farkas, Michael and Hilgen, Gerrit and White, Kathryn and Pan, Kuan-Ting and Treumann, Achim and Hallam, Dean and Bialas, Katarzyna and Chung, Git and Mellough, Carla and Ding, Yuchun and Krasnogor, Natalio and Przyborski, Stefan and Zwolinski, Simon and Al-Aama, Jumana and Alharthi, Sameer and Xu, Yaobo and Wheway, Gabrielle and Szymanska, Katarzyna and McKibbin, Martin and Inglehearn, Chris F. and Elliott, David J. and Lindsay, Susan and Ali, Robin R. and Steel, David H. and Armstrong, Lyle and Sernagor, Evelyne and Urlaub, Henning and Pierce, Eric and L uhrmann, Reinhard and Grellscheid, Sushma-Nagaraja and Johnson, Colin A. and Lako, Majlinda (2018) 'Disrupted alternative splicing for genes implicated in splicing and ciliogenesis causes PRPF31 retinitis pigmentosa.', Nature communications., 9 (1). p. 4234.

The availability of in vitro models of the human retina in which to perform pharmacological and toxicological studies is an urgent and unmet need. An essential step for developing in vitro models of human retina is the ability to generate laminated, physiologically functional, and light-responsive retinal organoids from renewable and patient specific sources. We investigated five different human-induced pluripotent stem cell (iPSC) lines and showed a significant variability in their efficiency to generate retinal organoids. Despite this variability, by month 5 of differentiation, all iPSC-derived retinal organoids were able to generate light responses, albeit immature, comparable to the earliest light responses recorded from the neonatal mouse retina, close to the period of eye opening. All iPSC-derived retinal organoids exhibited at this time a well-formed outer nuclear like layer containing photoreceptors with inner segments, connecting cilium, and outer like segments. The differentiation process was highly dependent on seeding cell density and nutrient availability determined by factorial experimental design. We adopted the differentiation protocol to a multiwell plate format, which enhanced generation of retinal organoids with retinal-pigmented epithelium (RPE) and improved ganglion cell development and the response to physiological stimuli. We tested the response of iPSC-derived retinal organoids to Moxifloxacin and showed that similarly to in vivo adult mouse retina, the primary affected cell types were photoreceptors. Together our data indicate that light responsive retinal organoids derived from carefully selected and differentiation efficient iPSC lines can be generated at the scale needed for pharmacology and drug screening purposes. Stem Cells 2018;36:1535-1551.

The original version of this Article omitted the following from the Acknowledgements: 'This work was support by EPSRC grant EP/K504336/1 and Leverhulme Trust grant RPG-2016-017.' This has been corrected in both the PDF and HTML versions of the Article.

Type II DNA topoisomerases (EC 5.99.1.3) are enzymes that catalyse topological changes in DNA in an ATP dependent manner. Strand passage reactions involve passing one double stranded DNA duplex (transported helix) through a transient enzyme-bridged break in another (gated helix). This activity is required for a range of cellular processes including transcription. Vertebrates have two isoforms: topoisomerase IIα and β. Topoisomerase IIβ was first reported in 1987. Here we review the research on DNA topoisomerase IIβ over the 30 years since its discovery.

Mutations in pre-mRNA processing factors (PRPFs) cause autosomal-dominant retinitis pigmentosa (RP), but it is unclear why mutations in ubiquitously expressed genes cause non-syndromic retinal disease. Here, we generate transcriptome profiles from RP11 (PRPF31-mutated) patient-derived retinal organoids and retinal pigment epithelium (RPE), as well as Prpf31+/- mouse tissues, which revealed that disrupted alternative splicing occurred for specific splicing programmes. Mis-splicing of genes encoding pre-mRNA splicing proteins was limited to patient-specific retinal cells and Prpf31+/- mouse retinae and RPE. Mis-splicing of genes implicated in ciliogenesis and cellular adhesion was associated with severe RPE defects that include disrupted apical - basal polarity, reduced trans-epithelial resistance and phagocytic capacity, and decreased cilia length and incidence. Disrupted cilia morphology also occurred in patient-derived photoreceptors, associated with progressive degeneration and cellular stress. In situ gene editing of a pathogenic mutation rescued protein expression and key cellular phenotypes in RPE and photoreceptors, providing proof of concept for future therapeutic strategies.

Background and aims: Elevated urinary 11-dehydro thromboxane B-2 (TxB(2)), a measure of thromboxane A(2) formation in vivo, predicts future atherothrombotic events. To further understand this relationship, the genetic determinants of 11-dehydro TxB(2) and their associations with cardiovascular morbidity were investigated in this study. Methods: Genome-wide and targeted genetic association studies of urinary 11-dehydro TxB2 were conducted in 806 Anglo-Scandinavian Cardiac Outcomes Trial (ASCOT) participants. Results: The strongest associations were in PPARGC1B (rs4235745, rs32582, rs10515638) and CNTN4 (rs10510230, rs4684343), these 5 single nucleotide polymorphisms (SNPs) were independently associated with 11-dehydro TxB(2) formation. Haplotypes of 11-dehydro TxB(2) increasing alleles for both PPARGC1B and CNTN4 were significantly associated with 11-dehydro TxB(2), explaining 5.2% and 4.5% of the variation in the whole cohort, and 8.8% and 7.9% in participants not taking aspirin, respectively. In a second ASCOT population (n = 6199), addition of these 5 SNPs significantly improved the covariate-only Cox proportional hazards model for cardiovascular events (chisq = 14.7, p = 0.01). Two of the risk alleles associated with increased urinary 11-dehydro TxB(2) were individually associated with greater incidences of cardiovascular events -rs10515638 (HR = 1.31, p = 0.01) and rs10510230 (HR = 1.25, p = 0.007); effect sizes were larger in those not taking aspirin. Conclusions: PPARGC1B and CNTN4 genotypes are associated with elevated thromboxane A2 formation and with an excess of cardiovascular events. Aspirin appears to blunt these associations. If specific protection of PPARGC1B and CNTN4 variant carriers by aspirin is confirmed by additional studies, PPARGC1B and CNTN4 genotyping could potentially assist in clinical decision making regarding the use of aspirin in primary prevention. (c) 2017 Elsevier B.V. All rights reserved.

Muscle tissue poses a particular challenge to proteomic analysis due to a very wide range of protein abundances arising from the dominant expression of myofilament-related proteins. We address this issue by describing proteomic analysis with liquid chromatography-mass spectrometry (LC-MS) and sequential window acquisition of all theoretical mass spectra (SWATH), of guinea pig cardiac tissue prepared in two homogenization buffers: (1) An SDS-based buffer designed to extract "all" tissue proteins and (2) a longestablished EDTA-containing buffer thought to preferentially extract non-myofibril-related proteins. We use gene ontology (GO) annotation-based assessment of subcellular localization to indicate if these enriched proteins congregate in the cytoplasm or in organellar lumens. This technique results in the preferential quantitation of less abundant non-myofibrillar proteins and, for future studies, offers the opportunity for more complete analyses of changes in heart tissue protein expression with biological circumstance.