Increasing anthropogenic CO2 emissions to the atmosphere are partially sequestered into the global oceans through the air-sea exchange of CO2 and its subsequent movement to depth, and this collective large-scale absorption is commonly referred to as the global ocean carbon sink. Quantifying this ocean carbon sink provides a key component for closing the global carbon budget which is used to inform and guide policy decisions. These estimates are typically accompanied by an uncertainty budget built by selecting what are perceived as critical uncertainty components based on selective experimentation. However, there is a growing realisation that these budgets are incomplete and may be underestimated, which limits their power as a constraint within global budgets. In this study, we present a methodology for quantifying spatially and temporally varying uncertainties in the air-sea CO2 flux calculations and data that allows an exhaustive assessment of all known sources of uncertainties, including decorrelation length scales between gridded measurements, and the approach follows standard uncertainty propagation methodologies. The resulting standard uncertainties are higher than previously suggested budgets, but the components are consistent with previous work, and they identify how the significance and importance of key uncertainty components change in space and time. For an exemplar method (the UEP-FNN-U method) the work identifies that we can currently estimate the annual ocean carbon sink to an accuracy of ±0.72PgCyr-1 (1 standard deviation uncertainty). Due to this method having been built on established uncertainty propagation and approaches, it appears applicable to all data-product assessments of the ocean carbon sink.

The ShcA adapter protein is necessary for early embryonic development. The role of ShcA in development is primarily attributed to its 52 and 46 kDa isoforms that transduce receptor tyrosine kinase signaling through the extracellular signal regulated kinase (ERK). During embryogenesis, ERK acts as the primary signaling effector, driving fate acquisition and germ layer specification. P66Shc, the largest of the ShcA isoforms, has been observed to antagonize ERK in several contexts; however, its role during embryonic development remains poorly understood. We hypothesized that p66Shc could act as a negative regulator of ERK activity during embryonic development, antagonizing early lineage commitment. To explore the role of p66Shc in stem cell self-renewal and differentiation, we created a p66Shc knockout murine embryonic stem cell (mESC) line. Deletion of p66Shc enhanced basal ERK activity, but surprisingly, instead of inducing mESC differentiation, loss of p66Shc enhanced the expression of core and naive pluripotency markers. Using pharmacologic inhibitors to interrogate potential signaling mechanisms, we discovered that p66Shc deletion permits the self-renewal of naive mESCs in the absence of conventional growth factors, by increasing their responsiveness to leukemia inhibitory factor (LIF). We discovered that loss of p66Shc enhanced not only increased ERK phosphorylation but also increased phosphorylation of Signal transducer and activator of transcription in mESCs, which may be acting to stabilize their naive-like identity, desensitizing them to ERK-mediated differentiation cues. These findings identify p66Shc as a regulator of both LIF-mediated ESC pluripotency and of signaling cascades that initiate postimplantation embryonic development and ESC commitment.

Normalizing RT-qPCR miRNA datasets that encompass numerous preimplantation embryo stages requires the identification of miRNAs that may be used as stable reference genes. A need has also arisen for the normalization of the accompanying conditioned culture media as extracellular miRNAs may serve as biomarkers of embryo developmental competence. Here, we evaluate the stability of six commonly used miRNA normalization candidates, as well as small nuclear U6, using five different means of evaluation (BestKeeper, NormFinder, geNorm, the comparative Delta Ct method and RefFinder comprehensive analysis) to assess their stability throughout murine preimplantation embryo development from the oocyte to the late blastocyst stages, both in whole embryos and the associated conditioned culture media. In descending order of effectiveness, miR-16, miR-191 and miR-106 were identified as the most stable individual reference miRNAs for developing whole CD1 murine preimplantation embryos, while miR-16, miR-106 and miR-103 were ideal for the conditioned culture media. Notably, the widely used U6 reference was among the least appropriate for normalizing both whole embryo and conditioned media miRNA datasets. Incorporating multiple reference miRNAs into the normalization basis via a geometric mean was deemed beneficial, and combinations of each set of stable miRNAs are further recommended, pending validation on a per experiment basis.

The culture media recommended for the isolation and enumeration of the Fusarium spp. lack selectivity for Fusarium graminearum. Five fungicides—Amistar® (250 g·L−1 azoxystrobin), Filan® (500 g·kg−1 boscalid), Comet® 200 (200 g·L−1 pyraclostrobin), Imtrex® (62.5 g·L−1 fluxapyroxad), Poraz® (450 g·L−1 prochloraz)—were investigated for their potential as selective inhibitors in culture media for the isolation of F. graminearum from soil and plant material. Based on the screening, fluxapyroxad was further tested for selective inhibition for the isolation of F. graminearum from soil. Additionally, selective media were compared for the isolation of F. graminearum from plant material. The fungicides tested did not prove to be effective inhibitors for the development of selective media. For the detection of F. graminearum in plant material, Czapek Dox propiconazole dichloran agar was found to be a better medium than Komada’s media, as the former resulted in colonies with darker pigmentation over a shorter incubation time and appeared to have a less inhibitory effect on F. graminearum growth.

As obese and overweight patients commonly display hyperlipidemia and are increasingly accessing fertility clinics for their conception needs, our studies are directed at understanding the effects of hyperlipidemia on early pregnancy. We have focused on investigating palmitic acid (PA) and oleic acid (OA) treatment alone and in combination from the mouse two-cell stage embryos as a model for understanding their effects on the mammalian preimplantation embryo. We recently reported that PA exerts a negative effect on mouse two-cell progression to the blastocyst stage, whereas OA co-treatment reverses that negative effect. In the present study, we hypothesized that PA treatment of mouse embryos would disrupt proper localization of cell fate determining and blastocyst formation gene products and that co-treatment with OA would reverse these effects. Our results demonstrate that PA treatment significantly (P < 0.05) reduces blastocyst development and cell number but did not prevent nuclear localization of YAP in outer cells. PA treatment significantly reduced the number of OCT4+ and CDX2+ nuclei. PA-treated embryos had lower expression of blastocyst formation proteins (E-cadherin, ZO-1 and Na/K-ATPase alpha1 subunit). Importantly, co-treatment of embryos with OA reversed PA-induced effects on blastocyst development and increased inner cell mass (ICM) and trophectoderm (TE) cell numbers and expression of blastocyst formation proteins. Our findings demonstrate that PA treatment does not impede cell fate gene localization but does disrupt proper blastocyst formation gene localization during mouse preimplantation development. OA treatment is protective and reverses PA's detrimental effects. The results advance our understanding of the impact of FFA exposure on mammalian preimplantation development.

Obese women experience greater incidence of infertility, with reproductive tracts exposing preimplantation embryos to elevated free fatty acids (FFA) such as palmitic acid (PA) and oleic acid (OA). PA treatment impairs mouse preimplantation development in vitro, while OA co-treatment rescues blastocyst development of PA treated embryos. In the present study, we investigated the effects of PA and OA treatment on NRF2/Keap1 localization, and relative antioxidant enzyme (Glutathione peroxidase; Gpx1, Catalase; Cat, Superoxide dismutase; Sod1 and γ-Glutamylcysteine ligase catalytic unit; Gclc) mRNA levels, during in vitro mouse preimplantation embryo development. Female mice were superovulated, mated, and embryos cultured in the presence of bovine Serum albumin (BSA) control or PA, or OA, alone (each at 100 μM) or PA + OA combined (each at 100 μM) treatment. NRF2 displayed nuclear localization at all developmental stages, whereas Keap1 primarily displayed cytoplasmic localization throughout control mouse preimplantation development in vitro. Relative transcript levels of Nrf2, Keap1, and downstream antioxidants significantly increased throughout control mouse preimplantation development in vitro. PA treatment significantly decreased blastocyst development and the levels of nuclear NRF2, while OA and PA + OA treatments did not. PA and OA treatments did not impact relative mRNA levels of Nrf2, Keap1, Gpx1, Cat, Sod1 or Gclc. Our outcomes demonstrate that cultured mouse embryos display nuclear NRF2, but that PA treatment reduces nuclear NRF2 and thus likely impacts NRF2/KEAP1 stress response mechanisms. Further studies should investigate whether free fatty acid effects on NRF2/KEAP1 contribute to the reduced fertility displayed by obese patients.

Cellular metabolism plays both an active and passive role in embryonic development, pluripotency, and cell-fate decisions. However, little is known regarding the role of metabolism in regulating the recently described "formative" pluripotent state. The pluripotent developmental continuum features a metabolic switch from a bivalent metabolism (both glycolysis and oxidative phosphorylation) in naive cells, to predominantly glycolysis in primed cells. We investigated the role of pyruvate kinase muscle isoforms 1/2 (PKM1/2) in naive, formative, and primed mouse embryonic stem cells through modulation of PKM1/2 messenger RNA transcripts using steric blocking morpholinos that downregulate PKM2 and upregulate PKM1. We have examined these effects in naive, formative, and primed cells by quantifying the effects of PKM1/2 modulation on pluripotent and metabolic transcripts and by measuring shifts in the population frequencies of cells expressing naive and primed cell surface markers by flow cytometry. Our results demonstrate that modulating PKM1 and PKM2 levels alters the transition from the naive state into a primed pluripotent state by enhancing the proportion of the affected cells seen in the "formative" state. Therefore, we conclude that PKM1/2 actively contributes to mechanisms that oversee early stem pluripotency and their progression toward a primed pluripotent state.

This chapter details 3D morphological topography of colony architecture optimization and nuclear protein localization by co-immunofluorescent confocal microscopy analysis. Colocalization assessment of nuclear and cytoplasmic cell regions is detailed to demonstrate nuclear and cytoplasmic localization in mEpiSCs by confocal microscopy and orthogonal colocalization assessment. Protein colocalization within mESCs, mEpiLCs, and mEpiSCs can be efficiently completed using these optimized protocols.

SLC6A14 (ATB(0,+)) is unique among SLC proteins in its ability to transport 18 of the 20 proteinogenic (dipolar and cationic) amino acids and naturally occurring and synthetic analogues (including anti-viral prodrugs and nitric oxide synthase (NOS) inhibitors). SLC6A14 mediates amino acid uptake in multiple cell types where increased expression is associated with pathophysiological conditions including some cancers. Here, we investigated how a key position within the core LeuT-fold structure of SLC6A14 influences substrate specificity. Homology modelling and sequence analysis identified the transmembrane domain 3 residue V128 as equivalent to a position known to influence substrate specificity in distantly related SLC36 and SLC38 amino acid transporters. SLC6A14, with and without V128 mutations, was heterologously expressed and function determined by radiotracer solute uptake and electrophysiological measurement of transporter-associated current. Substituting the amino acid residue occupying the SLC6A14 128 position modified the binding pocket environment and selectively disrupted transport of cationic (but not dipolar) amino acids and related NOS inhibitors. By understanding the molecular basis of amino acid transporter substrate specificity we can improve knowledge of how this multi-functional transporter can be targeted and how the LeuT-fold facilitates such diversity in function among the SLC6 family and other SLC amino acid transporters.

Treatment of mouse preimplantation embryos with elevated palmitic acid (PA) reduces blastocyst development, whereas cotreatment with PA and oleic acid (OA) together rescues blastocyst development to control frequencies. To understand the mechanistic effects of PA and OA treatment on early mouse embryos, we investigated the effects of PA and OA, alone and in combination, on autophagy during preimplantation development in vitro. We hypothesized that PA would alter autophagic processes and that OA cotreatment would restore control levels of autophagy. Two-cell stage mouse embryos were placed into culture medium supplemented with 100 μM PA, 250 μM OA, 100 μM PA and 250 μM OA, or potassium simplex optimization media with amino acid (KSOMaa) medium alone (control) for 18–48 h. The results demonstrated that OA cotreatment slowed developmental progression after 30 h of cotreatment but restored control blastocyst frequencies by 48 h. PA treatment elevated light chain 3 (LC3)-II puncta and p62 levels per cell whereas OA cotreatment returned to control levels of autophagy by 48 h. Autophagic mechanisms are altered by nonesterified fatty acid (NEFA) treatments during mouse preimplantation development in vitro, where PA elevates autophagosome formation and reduces autophagosome degradation levels, whereas cotreatment with OA reversed these PA effects. Autophagosome-lysosome colocalization only differed between PA and OA alone treatment groups. These findings advance our understanding of the effects of free fatty acid exposure on preimplantation development, and they uncover principles that may underlie the associations between elevated fatty acid levels and overall declines in reproductive fertility.

Mycetoma is a neglected tropical chronic granulomatous inflammatory disease of the skin and subcutaneous tissues. More than 70 species with a broad taxonomic diversity have been implicated as agents of mycetoma. Understanding the full range of causative organisms and their antibiotic sensitivity profiles are essential for the appropriate treatment of infections. The present study focuses on the analysis of full genome sequences and antibiotic inhibitory concentration profiles of actinomycetoma strains from patients seen at the Mycetoma Research Centre in Sudan with a view to developing rapid diagnostic tests. Seventeen pathogenic isolates obtained by surgical biopsies were sequenced using MinION and Illumina methods, and their antibiotic inhibitory concentration profiles determined. The results highlight an unexpected diversity of actinomycetoma causing pathogens, including three Streptomyces isolates assigned to species not previously associated with human actinomycetoma and one new Streptomyces species. Thus, current approaches for clinical and histopathological classification of mycetoma may need to be updated. The standard treatment for actinomycetoma is a combination of sulfamethoxazole/trimethoprim and amoxicillin/clavulanic acid. Most tested isolates had a high IC (inhibitory concentration) to sulfamethoxazole/trimethoprim or to amoxicillin alone. However, the addition of the β-lactamase inhibitor clavulanic acid to amoxicillin increased susceptibility, particularly for Streptomyces somaliensis and Streptomyces sudanensis . Actinomadura madurae isolates appear to have a particularly high IC under laboratory conditions, suggesting that alternative agents, such as amikacin, could be considered for more effective treatment. The results obtained will inform future diagnostic methods for the identification of actinomycetoma and treatment.

Autophagy is an essential cellular degradation pathway in the mammalian preimplantation embryo. In this review, the process of autophagy is outlined and its function pertaining to preimplantation embryos is discussed. The current methods of measuring autophagy in preimplantation embryos are also summarized, with highlights of advances and challenges that one may encounter when examining preimplantation embryo autophagy. This article identifies the techniques, assays, and available models that are viable but infrequently used in the context of examining autophagy in preimplantation embryos. We aim to bring attention to alternative assays that could contribute to future analysis of autophagic pathways in preimplantation embryos.Abbreviations: AMBRA1: Activating molecule in Beclin-1-regulated autophagy 1; ATG: Autophagy-related protein; AL: Autolysosome; BCL-2: B-cell lymphoma 2; BECN1: Beclin-1; CQ: Chloroquine; ER: endoplasmic reticulum; ES cells: Embryonic stem cells; GFP: Green fluorescent protein; ICM: Inner cell mass; IF: Immunofluorescence; LC3: Light chain 3; LC3-II: Light chain 3 (lipid-conjugated); MAPK: mitogen-activating protein kinase; mTOR: Mammalian target of rapamycin; p62/SQSTM1: Sequestome 1; PA: Palmitic acid; PG: Phagophore; PtdIns3K: Phosphatidylinositol 3-kinase; PtdIns3P: phosphatidylinositol-3 phosphate; qPCR: Quantitative polymerase chain reaction; RFP: Red fluorescent protein; ROS: Reactive oxygen species; SNARE: Soluble N-ethylmaleimide-sensitive factor attachment protein receptor; TS cells: Trophoblast stem cells; ULK1: UNC-51-like kinase 1; WB: Western blot

Fusarium graminearum is a globally important cereal pathogen, causing head blight in wheat, resulting in yield losses and mycotoxin contamination. Currently, triazole fungicides are used to suppress Fusarium graminearum, however, the declining effectiveness of triazoles and concerns over the safety of pesticides have led to the pursuit of safe alternative crop protection strategies such as biofumigation. In the present study, species belonging to Brassicaceae (Brassica juncea, Raphanus sativus, Eruca sativa) were assessed for their biofumigation potential against F. graminearum and the glucosinolate profile of the brassicas was determined. In Petri dishes, mycelial plugs of Fusarium graminearum were exposed to frozen/defrosted leaf discs of brassicas collected at early-leaf, stem-extension, and early-bud stages. Additionally, F. graminearum inoculum was incubated in soil amended with chopped tissues of brassicas in a closed jar experiment. Glucosinolate analysis of the leaf tissue of brassicas revealed that the total glucosinolate concentration of B. juncea ‘Brons’ increased with advancing growth stage (24.5–51.9 µmol g−1). Brassica juncea leaf discs were effective against mycelial growth, while the sinigrin content in the leaf tissue corresponded to the level of suppression. At the stem-extension and early-bud stages, B. juncea ‘Brons’ showed 87–90% suppression with four leaf discs, and 100% suppression with eight leaf discs. Brassica juncea ‘Caliente Rojo’ leaf discs collected at the stem-extension stage showed 94% inhibition with eight discs. In the closed jar experiment, each brassica species significantly suppressed F. graminearum inoculum by 41–55%. The findings suggest that the brassica species investigated in the present study could be effective in reducing the inoculum of F. graminearum in soil prior to cereal production.

Fusarium graminearum is the most important causal agent of head blight in wheat, and stalk and ear rot in maize. A field experiment was conducted to investigate the effect of incorporation of Brassicaceae cover crops on Fusarium graminearum in a wheat-maize rotation. Five species belonging to Brassicaceae (Brassica juncea, Eruca sativa, Raphanus sativus, B. carinata, B. oleracea var. caulorapa L.) were used in the field experiment to investigate their potential to suppress F. graminearum inoculum in soil, disease incidence in maize and to reduce subsequent mycotoxin contamination in maize. Brassica juncea was found to contain the highest glucosinolate concentration in shoots (31 µmol g−1). Severity of ear rot and stalk rot in maize was not significantly reduced in the amended plots. Incorporation of R. sativus ‘Terranova’ significantly decreased the amount of F. graminearum DNA by 58% compared with the cultivated fallow treatment, however the DNA concentration was not significantly different to fallow uncultivated. Fusarium graminearum DNA and deoxynivalenol in maize was 50% lower after incorporation of B. oleracea var. caulorapa L. compared to after fallow treatment but the difference was not significant. The brassica crops used in the present field experiment were not effective in suppressing F. graminearum, therefore further studies to optimise the current approach are recommended.

Isothiocyanates are biotoxic degradation products formed as a result of enzymatic hydrolysis of glucosinolates present in Brassica species. The application of biofumigant Brassica crops, as an alternative crop protection method for soilborne pathogens and pests is increasingly gaining interest. However, little is known of the potential of biofumigation to reduce the inoculum of Fusarium species affecting cereals. The aim of this study was to evaluate the antifungal activity of five isothiocyanates, namely allyl, benzyl, ethyl, 2-phenylethyl and methyl isothiocyanates, against germination and growth of Fusarium graminearum under in vitro conditions. Aromatic isothiocyanates were more inhibitory than the aliphatic isothiocyanates against mycelial growth, whereas the reverse was observed for conidial germination. Among the tested isothiocyanates, allyl and methyl isothiocyanates were more efficient overall, showing lower ED50 values (35-150 mg/L) for conidial germination and mycelial radial growth. The findings suggest that Brassica plants containing allyl and methyl glucosinolates could have a suppressive effect, reducing the inoculum of F. graminearum in soil prior to cereal production.

Reproduction, Fertility and Development is an international journal publishing original research , review and comment in the fields of reproduction and developmental biology in humans, domestic animals and wildlife

All genomes include gene families with very limited taxonomic distributions that potentially represent new genes and innovations in protein-coding sequence, raising questions on the origins of such genes. Some of these genes are hypothesized to have formed de novo, from noncoding sequences, and recent work has begun to elucidate the processes by which de novo gene formation can occur. A special case of de novo gene formation, overprinting, describes the origin of new genes from noncoding alternative reading frames of existing open reading frames (ORFs). We argue that additionally, out-of-frame gene fission/fusion events of alternative reading frames of ORFs and out-of-frame lateral gene transfers could contribute to the origin of new gene families. To demonstrate this, we developed an original pattern-search in sequence similarity networks, enhancing the use of these graphs, commonly used to detect in-frame remodeled genes. We applied this approach to gene families in 524 complete genomes of Escherichia coli. We identified 767 gene families whose evolutionary history likely included at least one out-of-frame remodeling event. These genes with out-of-frame components represent ∼2.5% of all genes in the E. coli pangenome, suggesting that alternative reading frames of existing ORFs can contribute to a significant proportion of de novo genes in bacteria.

Here we describe methodologies to characterize, delineate, and quantify pluripotent cells between naïve, formative, and primed pluripotent state mouse embryonic stem cell (mESCs) populations using flow cytometric analysis. This methodology can validate pluripotent states, sort individual cells of interest, and determine the efficiency of transitioning naïve mESCs to a primed-like state as mouse epiblast-like cells (mEpiLCs) and onto fully primed mouse epiblast stem cells (mEpiSCs). Quantification of the cell surface markers; SSEA1(CD15) and CD24 introduces an effective method of distinguishing individual cells from a population by their respective positioning in the pluripotent spectrum. Additionally, this protocol can be used to demarcate and sort cells via fluorescently activated cell sorting for downstream applications. Flow cytometric analysis within mESCs, mEpiLCs, and mEpiSCs can be efficiently completed using these optimized protocols.