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BACKGROUND: Access to an on-demand pericoital oral contraceptive pill - used to prevent pregnancy within a defined window around sexual intercourse - could offer women more reproductive agency. A contraceptive with this indication is not currently available in any market. This review aims to understand international user appeal for an on-demand pericoital oral contraceptive pill. METHODS: Systematic scoping review, comprising 30 peer-reviewed papers published between 2014-2023. RESULTS: Data from 30 papers reporting on research from 16 countries across five World Health Organisation regions suggests widespread user appeal for on-demand oral contraceptive pills that can be used peri- or post-coitally, especially among women who are younger, more educated or who have less frequent sex. Women of varying age, wealth, employment or relationship status, and with different prior experience of using modern contraceptives, were also interested. Women identified clear rationale for use and preference of these types of product: close alignment with women's sexual lives that comprised unplanned, spontaneous or occasional sex; perceived convenience and effectiveness; discreet use of pills to negotiate contextual circumstances that constrained their reproductive agency. Factors inhibiting use included knowledge barriers and attitudes of service providers, a lack of knowledge and misinformation among end-users, women's dislike of menstrual side effects and myths related to the effects of hormone content on future fertility. CONCLUSIONS: Introduction of an on-demand pericoital oral contraceptive pill could expand contraceptive choice for diverse women experiencing unmet need for modern contraception and constrained sexual and reproductive agency. Priorities for future research include: broadening the geographical scope of evidence to include SE Asia and the Pacific, and international rural and peri-urban settings; documenting the perspectives of adolescents and unmarried young people; identifying opportunities for innovation in the supply channels to enhance appropriate, affordable access to on-demand oral contraceptives; and unpacking how to bring new pericoital contraceptives to the market in a variety of international settings.
Access to an oral contraceptive pill that is used as needed to prevent pregnancy and taken within a defined window around sexual intercourse (i.e. an on-demand pericoital oral contraceptive pill) could offer women more reproductive agency. Though not currently available in any market, our analysis from this review of international literature reveals widespread appeal among women for using this type of contraceptive product. Clear rationale supporting use and preference included: (1) closer alignment with women's sexual lives that comprised desired but unplanned, spontaneous or occasional sex than other contraceptives; (2) perceived convenience and effectiveness, offering benefits over other modern contraceptives; and (3) women feeling able to overcome social values and beliefs that constrained their reproductive agency. There were also barriers to use of this type of product, including knowledge gaps and attitudes of service providers, a lack of knowledge and misinformation among end-users, women's dislike of the side effects, and myths and misconceptions about the impact of the hormone content in pills on future fertility. Introduction of an on-demand pericoital oral contraceptive pill could expand contraceptive choice for diverse women experiencing unmet need for modern contraception and constrained sexual and reproductive agency. Priorities for future research include: broadening the geographical scope of evidence to include SE Asia and the Pacific, and rural and peri-urban settings; documenting the perspectives of adolescents and unmarried young people; identifying opportunities for innovation in the supply channels to enhance appropriate, affordable access to this type of contraceptive; and unpacking how to bring this new contraceptive to the market in a variety of international settings.
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Anticonceptivos Orales , Humanos , Femenino , Anticonceptivos Orales/administración & dosificación , Conducta Anticonceptiva/estadística & datos numéricos , Coito , Conocimientos, Actitudes y Práctica en Salud , Embarazo , Conducta Sexual , Aceptación de la Atención de Salud , Anticoncepción/métodosRESUMEN
In the budding yeast Saccharomyces cerevisiae, exit from mitosis is coupled to spindle position to ensure successful genome partitioning between mother and daughter cell. This coupling occurs through a GTPase signaling cascade known as the mitotic exit network (MEN). The MEN senses spindle position via a Ras-like GTPase Tem1 which localizes to the spindle pole bodies (SPBs, yeast equivalent of centrosomes) during anaphase and signals to its effector protein kinase Cdc15. How Tem1 couples the status of spindle position to MEN activation is not fully understood. Here, we show that Cdc15 has a relatively weak preference for Tem 1 GTP and Tem1's nucleotide state does not change upon MEN activation. Instead, we find that Tem1's nucleotide cycle establishes a localization-based concentration difference in the cell where only Tem 1 GTP is recruited to the SPB, and spindle position regulates the MEN by controlling Tem1 localization. SPB localization of Tem1 primarily functions to promote Tem1-Cdc15 interaction for MEN activation by increasing the effective concentration of Tem1. Consistent with this model, we demonstrate that artificially tethering Tem1 to the SPB or concentrating Tem1 in the cytoplasm with genetically encoded multimeric nanoparticles could bypass the requirement of Tem 1 GTP and correct spindle position for MEN activation. This localization/concentration-based GTPase signaling mechanism for Tem1 differs from the canonical Ras-like GTPase signaling paradigm and is likely relevant to other localization-based signaling scenarios.
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Respiratory syncytial virus (RSV) causes severe infections in infants, immunocompromised or elderly individuals resulting in annual epidemics of respiratory disease. Currently, limited clinical surveillance and the lack of predictable seasonal dynamics limits the public health response. Wastewater-based epidemiology (WBE) has recently been used globally as a key metric in determining prevalence of SARS-CoV-2 in the community but its application to other respiratory viruses is limited. In this study, we present an integrated genomic WBE approach, applying RT-qPCR and partial G-gene sequencing to track RSV levels and variants in the community. We report increasing detection of RSV in wastewater concomitant with increasing numbers of positive clinical cases. Analysis of wastewater-derived RSV sequences permitted identification of distinct circulating lineages within and between seasons. Altogether, our genomic WBE platform has the potential to complement ongoing global surveillance and aid the management of RSV by informing the timely deployment of pharmaceutical and non-pharmaceutical interventions.
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Multi-site randomized controlled trials (RCTs) provide unbiased estimates of the average impact in the study sample. However, their ability to accurately predict the impact for individual sites outside the study sample, to inform local policy decisions, is largely unknown. To extend prior research on this question, we analyzed six multi-site RCTs and tested modern prediction methods-lasso regression and Bayesian Additive Regression Trees (BART)-using a wide range of moderator variables. The main study findings are that: (1) all of the methods yielded accurate impact predictions when the variation in impacts across sites was close to zero (as expected); (2) none of the methods yielded accurate impact predictions when the variation in impacts across sites was substantial; and (3) BART typically produced "less inaccurate" predictions than lasso regression or than the Sample Average Treatment Effect. These results raise concerns that when the impact of an intervention varies considerably across sites, statistical modelling using the data commonly collected by multi-site RCTs will be insufficient to explain the variation in impacts across sites and accurately predict impacts for individual sites.
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Mycotoxins are known environmental pollutants that may contaminate food and feed chains. Some mycotoxins are regulated in many countries to limit the trading of contaminated and harmful commodities. However, the so-called emerging mycotoxins are poorly understood and need to be investigated further. Fusaric acid is an emerging mycotoxin, noxious to plants and animals, but is known to be less toxic to plants when hydroxylated. The detoxification routes effective in animals have not been elucidated yet. In this context, this study integrated in silico and in vitro techniques to discover potential bioremediation routes to turn fusaric acid to its less toxic metabolites. The toxicodynamics of these forms in humans have also been addressed. An in silico screening process, followed by molecular docking and dynamics studies, identified CYP199A4 from the bacterium Rhodopseudomonas palustris HaA2 as a potential fusaric acid biotransforming enzyme. Its activity was confirmed in vitro. However, the effect of hydroxylation seemed to have a limited impact on the modelled toxicodynamics against human targets. This study represents a starting point to develop a hybrid in silico/in vitro pipeline to find bioremediation agents for other food, feed and environmental contaminants.
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Ácido Fusárico , Micotoxinas , Animales , Humanos , Ácido Fusárico/toxicidad , Simulación del Acoplamiento Molecular , Micotoxinas/toxicidad , Alimentación Animal/análisis , Sistema Enzimático del Citocromo P-450RESUMEN
The cytochrome P450 family of heme metalloenzymes (CYPs) catalyse important biological monooxygenation reactions. Mycobacterium marinum contains a gene encoding a CYP105Q4 enzyme of unknown function. Other members of the CYP105 CYP family have key roles in bacterial metabolism including the synthesis of secondary metabolites. We produced and purified the cytochrome P450 enzyme CYP105Q4 to enable its characterization. Several nitrogen-donor atom-containing ligands were found to bind to CYP105Q4 generating type II changes in the UV-vis absorbance spectrum. Based on the UV-vis absorbance spectra none of the potential substrate ligands we tested with CYP105Q4 were able to displace the sixth distal aqua ligand from the heme, though there was evidence for binding of oleic acid and amphotericin B. The crystal structure of CYP105Q4 in the substrate-free form was determined in an open conformation. A computational structural similarity search (Dali) was used to find the most closely related characterized relatives within the CYP105 family. The structure of CYP105Q4 enzyme was compared to the GfsF CYP enzyme from Streptomyces graminofaciens which is involved in the biosynthesis of a macrolide polyketide. This structural comparison to GfsF revealed conformational changes in the helices and loops near the entrance to the substrate access channel. A disordered B/C loop region, usually involved in substrate recognition, was also observed.
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Mycobacterium marinum , Mycobacterium marinum/genética , Mycobacterium marinum/metabolismo , Sistema Enzimático del Citocromo P-450/metabolismo , Estructura Secundaria de Proteína , Macrólidos/química , Macrólidos/metabolismo , Hemo/química , Cristalografía por Rayos XRESUMEN
Millions of people require custom ocular prostheses due to eye loss or congenital defects. The current fully manual manufacturing processes used by highly skilled ocularists are time-consuming with varying quality. Additive manufacturing technology has the potential to simplify the manufacture of ocular prosthetics, but existing approaches just replace to various degrees craftsmanship by manual digital design and still require substantial expertise and time. Here we present an automatic digital end-to-end process for producing custom ocular prostheses that uses image data from an anterior segment optical coherence tomography device and considers both shape and appearance. Our approach uses a statistical shape model to predict, based on incomplete surface information of the eye socket, a best fitting prosthesis shape. We use a colour characterized image of the healthy fellow eye to determine and procedurally generate the prosthesis's appearance that matches the fellow eye. The prosthesis is manufactured using a multi-material full-colour 3D printer and postprocessed to satisfy regulatory compliance. We demonstrate the effectiveness of our approach by presenting results for 10 clinic patients who received a 3D printed prosthesis. Compared to a current manual process, our approach requires five times less labour of the ocularist and produces reproducible output.
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Miembros Artificiales , Ojo Artificial , Humanos , Diseño de Prótesis , Impresión Tridimensional , Implantación de PrótesisRESUMEN
Oxidised derivatives of cholesterol have been shown to inhibit the growth of Mycobacterium tuberculosis (Mtb). The bacteriostatic activity of these compounds has been attributed to their inhibition of CYP125A1 and CYP142A1, two metabolically critical cytochromes P450 that initiate degradation of the sterol side chain. Here, we synthesise and characterise an extensive library of 28 cholesterol derivatives to develop a structure-activity relationship for this class of inhibitors. The candidate compounds were evaluated for MIC with virulent Mtb and in binding studies with CYP125A1 and CYP142A1 from Mtb.
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Mycobacterium tuberculosis , Sistema Enzimático del Citocromo P-450/metabolismo , Colesterol/metabolismo , Relación Estructura-ActividadRESUMEN
While there is a considerable body of knowledge regarding the molecular and structural biology and biochemistry of archaeal information processing machineries, far less is known about the nature of the substrate for these machineries-the archaeal nucleoid. In this article, we will describe recent advances in our understanding of the three-dimensional organization of the chromosomes of model organisms in the crenarchaeal phylum.
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AIM: Hospital-acquired infections (HAIs) caused by antimicrobial-resistant ESKAPE pathogens are a significant concern for the healthcare industry, with an estimated cost of up to ${\$}$45 billion per year in the US alone. Clostridioides difficile is an additional opportunistic pathogen that also poses a serious threat to immunocompromised patients in hospitals. Infections caused by these pathogens lead to increased hospital stays and repeated readmission, resulting in a significant economic burden. Disinfectants and sporicidals are essential to reduce the risk of these pathogens in hospitals, but commercially available products can have a number of disadvantages including inefficacy, long contact times, short shelf lives, and operator health hazards. In this study we evaluated the effectiveness of Rosin (a natural substance secreted by coniferous trees as a defence mechanism against wounds in tree bark) and its commercial derivative Rosetax-21 as disinfectants and sporicidal against the six ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) and spore preparations from Clostridioides difficile. METHODS AND RESULTS: Both Rosin and Rosetax-21 were tested under simulated clean and dirty conditions (with BSA) against the ESKAPE pathogens, and C. difficile spore preparations. In clean conditions, Rosin (5% weight/volume: w/v) demonstrated significant efficacy against five of the ESKAPE pathogens, with A. baumannii and E. faecium being the most susceptible, and K. pneumoniae the most resistant, showing only a one-log reduction after a 5 min treatment. However, in dirty conditions, all pathogens including K. pneumoniae exhibited at least a 3-log reduction to Rosin within 5 min. Rosetax-21 (5% w/v) was found to be less effective than Rosin in clean conditions, a trend that was exacerbated in the presence of BSA. Additionally, both Rosin and Rosetax-21 at 2.5% (w/v) achieved complete eradication of C. difficile spores when combined with 0.5% glutaraldehyde, though their standalone sporicidal activity was limited. CONCLUSIONS: The findings from this study highlight the potential of Rosin and Rosetax-21 as both bactericidal and sporicidal disinfectants, with their efficacy varying based on the conditions and the pathogens tested. This presents an avenue for the development of novel healthcare disinfection strategies, especially against HAIs caused by antimicrobial-resistant ESKAPE pathogens and C. difficile.
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HIV prevention programs focus on global "key populations" and more localized "priority populations" to ensure effective targeting of interventions. These HIV population categories have been subject to considerable scholarly scrutiny, particularly key populations, with less attention given to critically unpacking priority populations at local levels, for example "serodiscordant couples" (one partner has HIV, but not the other). We examine this population in the context of Papua New Guinea to consider how local configurations, relational pathways, and lived realities of serodiscordant relationships strain the boundaries of this population category and raise intriguing questions about its intersection with contemporary biomedical agendas.
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Infecciones por VIH , Parejas Sexuales , Humanos , Infecciones por VIH/prevención & control , Papúa Nueva Guinea , Antropología MédicaRESUMEN
The cytochrome P450 monooxygenases (CYPs) are a class of heme-thiolate enzymes that insert oxygen into unactivated C-H bonds. These enzymes can be converted into peroxygenases via protein engineering, which enables their activity to occur using hydrogen peroxide (H2 O2 ) without the requirement for additional nicotinamide co-factors or partner proteins. Here, we demonstrate that soaking crystals of an engineered P450 peroxygenase with H2 O2 enables the enzymatic reaction to occur within the crystal. Crystals of the designed P450 peroxygenase, the T252E mutant of CYP199A4, in complex with 4-methoxybenzoic acid were soaked with different concentrations of H2 O2 for varying times to initiate the in crystallo O-demethylation reaction. Crystal structures of T252E-CYP199A4 showed a distinct loss of electron density that was consistent with the O-demethylated metabolite, 4-hydroxybenzoic acid. A new X-ray crystal structure of this enzyme with the 4-hydroxybenzoic acid product was obtained to enable comparison alongside the existing substrate-bound structure. The visualisation of enzymatic catalysis in action is challenging in structural biology and the ability to initiate the reactions of P450 enzymes, in crystallo by simply soaking crystals with H2 O2 will enable new structural biology methods and techniques to be applied to study their mechanism of action.
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Sistema Enzimático del Citocromo P-450 , Oxigenasas de Función Mixta , Parabenos , Sistema Enzimático del Citocromo P-450/metabolismo , CatálisisRESUMEN
Rhodococcus globerulus (R. globerulus) was isolated from the soil beneath a Eucalypt tree. Metabolic growth studies revealed that R. globerulus was capable of living on certain monoterpenes, including 1,8-cineole and p-cymene, as sole sources of carbon and energy. Multiple P450 genes were identified in the R. globerulus genome that shared homology to known bacterial, monoterpene hydroxylating P450s. To date, two of these P450s have been expressed and characterised as 1,8-cineole (CYP176A1) and p-cymene (CYP108N12) monooxygenases that are believed to initiate the biodegradation of these terpenes. In this work, another putative P450 gene (CYP108N14) was identified in R. globerulus genome. Given its amino acid sequence identity to other monoterpene hydroxylating P450s it was hypothesised to catalyse monoterpene hydroxylation. These include CYP108A1 from Pseudomonas sp. (47 % identity, 68 % similarity) which hydroxylates α-terpineol, and CYP108N12 also from R. globerulus (62 % identity, 77 % similarity). Also present in the operon containing CYP108N14 were putative ferredoxin and ferredoxin reductase genes, suggesting a typical Class I P450 system. CYP108N14 was successfully over-expressed heterologously and purified, resulting in a good yield of CYP108N14 holoprotein. However, neither the ferredoxin nor ferredoxin reductase could be produced heterologously. Binding studies with CYP108N14 revealed a preference for the monoterpenes p-cymene, (R)-limonene, (S)-limonene, (S)-α-terpineol and (S)-4-terpineol. An active catalytic system was reconstituted with the non-native redox partners cymredoxin (from the CYP108N12 system) and putidaredoxin reductase (from the CYP101A1 system). CYP108N14 when supported by these redox partners was able to catalyse the hydroxylation of the five aforementioned substrates selectively at the methyl benzylic/allylic positions.
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Monoterpenos Ciclohexánicos , Cimenos , Sistema Enzimático del Citocromo P-450 , Monoterpenos , Rhodococcus , Monoterpenos/metabolismo , Eucaliptol , Sistema Enzimático del Citocromo P-450/metabolismo , Ferredoxinas , LimonenoRESUMEN
Proteins in the structural maintenance of chromosomes (SMC) superfamily play key roles in chromosome organization and are ubiquitous across all domains of life. However, SMC proteins are notably absent in the Desulfurococcales of phylum Crenarchaeota. Intrigued by this observation, we performed chromosome conformation capture experiments in the model Desulfurococcales species Aeropyrum pernix. As in other archaea, we observe chromosomal interaction domains across the chromosome. The boundaries between chromosomal interaction domains show a dependence on transcription and translation for their definition. Importantly, however, we reveal an additional higher-order, bipartite organization of the chromosome-with a small high-gene-expression and self-interacting domain that is defined by transcriptional activity and loop structures. Viewing these data in the context of the distribution of SMC superfamily proteins in the Crenarchaeota, we suggest that the organization of the Aeropyrum genome represents an evolutionary antecedent of the compartmentalized architecture observed in the Sulfolobus lineage.
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Crenarchaeota , Sulfolobus , Archaea/genética , Crenarchaeota/genética , Expresión Génica , Sulfolobus/genética , CromosomasRESUMEN
Norcarane is a mechanistic probe of monooxygenase enzymes that is able to detect the presence of cationic or radical intermediates. The addition of substituents around the bicycloheptane ring of the norcarane scaffold can assist in improving enzyme binding affinity and thus improve the regioselectivity of oxidation. Here we prepare in three-step, diastereoselective syntheses, ten norcaranes monosubstituted α to the cyclopropane as advanced probes. Four of these compounds were examined in enzyme binding experiments to evaluate their potential as probe substrates. Additionally, 19 potential products of enzymatic oxidation were generated via two additional synthetic steps for use as product standards in future studies.
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Oxigenasas de Función Mixta , Terpenos , Oxidación-Reducción , Terpenos/química , Oxigenasas de Función Mixta/metabolismo , HidroxilaciónRESUMEN
The members of the bacterial cytochrome P450 (CYP) monooxygenase family CYP125, catalyze the oxidation of steroid derivatives including cholesterol and phytosterols, as the initial activating step in their catabolism. However, several bacterial species contain multiple genes encoding CYP125 enzymes and other CYP enzymes which catalyze cholesterol/cholest-4-en-3-one hydroxylation. An important question is why these bacterium have more than one enzyme with overlapping substrate ranges capable of catalyzing the terminal oxidation of the alkyl chain of these sterols. To further understand the role of these enzymes we investigated CYP125A6 and CYP125A7 from Mycobacterium marinum with various cholesterol analogues. These have modifications on the A and B rings of the steroid and we assessed the substrate binding and catalytic activity of these with each enzyme. CYP125A7 gave similar results to those reported for the CYP125A1 enzyme from M. tuberculosis. Differences in the substrate binding and catalytic activity with the cholesterol analogues were observed with CYP125A6. For example, while cholesteryl sulfate could bind to both enzymes it was only oxidized by CYP125A6 and not by CYP125A7. CYP125A6 generated higher levels of metabolites with the majority of C-3 and C-7 substituted cholesterol analogues such 7-ketocholesterol. However, 5α-cholestan-3ß-ol was only oxidized by CYP125A7 enzyme. The cholest-4-en-3-one and 7-ketocholesterol-bound forms of the CYP125A6 and CYP125A7 enzymes were modelled using AlphaFold. The structural models highlighted differences in the binding modes of the steroid derivatives within the same enzyme. Significant changes in the binding mode of the steroids between these CYP125 enzymes and other bacterial cholesterol oxidizing enzymes, CYP142A3 and CYP124A1, were also seen. Despite this, all these models predicted the selectivity for terminal methyl hydroxylation, in agreement with the experimental data.
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Mycobacterium marinum , Mycobacterium tuberculosis , Oxidación-Reducción , Sistema Enzimático del Citocromo P-450/metabolismo , Esteroides , EsterolesRESUMEN
A highly sought after reaction in chemical synthesis is the activation of unactivated carbon-hydrogen bonds. We demonstrate the hydroxylation of fatty acids using an engineered thermostable archaeal cytochrome P450 enzyme. By replacing a seven amino acid section of the I-helix, the nicotinamide cofactor-dependent monooxygenase was converted into a hydrogen peroxide using peroxygenase, enabling the efficient biocatalytic oxidation of C-H bonds at room temperature to 90 °C.
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Sistema Enzimático del Citocromo P-450 , Hemo , Sistema Enzimático del Citocromo P-450/metabolismo , Oxidación-Reducción , Biocatálisis , Hidroxilación , Hemo/químicaRESUMEN
The cytochrome P450 enzymes (CYPs) are heme-thiolate monooxygenases that catalyse the insertion of an oxygen atom into the C-H bonds of organic molecules. In most CYPs, the activation of dioxygen by the heme is aided by an acid-alcohol pair of residues located in the I-helix of the enzyme. Mutation of the threonine residue of this acid-alcohol pair of CYP199A4, from the bacterium Rhodospeudomonas palustris HaA2, to a glutamate residue induces peroxygenase activity. In the X-ray crystal structures of this variant an interaction of the glutamate side chain and the distal aqua ligand of the heme was observed and this results in this ligand not being readily displaced in the peroxygenase mutant on the addition of substrate. Here we use a range of bulky hydrophobic and nitrogen donor containing ligands in an attempt to displace the distal aqua ligand of the T252E mutant of CYP199A4. Ligand binding was assessed by UV-visible absorbance spectroscopy, native mass spectrometry, electron paramagnetic resonance and X-ray crystallography. None of the ligands tested, even the nitrogen donor ligands which bind directly to the iron in the wild-type enzyme, resulted in displacement of the aqua ligand. Therefore, modification of the I-helix threonine residue to a glutamate residue results in a significant strengthening of the ferric distal aqua ligand. This ligand was not displaced using any of the ligands during this study and this provides a rationale as to why this mutant can shutdown the monooxygenase pathway of this enzyme and switch to peroxygenase activity.
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Sistema Enzimático del Citocromo P-450 , Hemo , Hemo/química , Ligandos , Sistema Enzimático del Citocromo P-450/metabolismo , Hierro/química , Nitrógeno , Treonina , GlutamatosRESUMEN
PURPOSE: Growth hormone receptor knockout (GHR-KO) pigs have recently been developed, which serve as a large animal model of Laron syndrome (LS). GHR-KO pigs, like individuals with LS, are obese but lack some comorbidities of obesity. The purpose of this study was to examine the histological and transcriptomic phenotype of adipose tissue (AT) in GHR-KO pigs and humans with LS. METHODS: Intraabdominal (IA) and subcutaneous (SubQ) AT was collected from GHR-KO pigs and examined histologically for adipocyte size and collagen content. RNA was isolated and cDNA sequenced, and the results were analyzed to determine differentially expressed genes that were used for enrichment and pathway analysis in pig samples. For comparison, we also performed limited analyses on human AT collected from a single individual with and without LS. RESULTS: GHR-KO pigs have increased adipocyte size, while the LS AT had a trend towards an increase. Transcriptome analysis revealed 55 differentially expressed genes present in both depots of pig GHR-KO AT. Many significant terms in the enrichment analysis of the SubQ depot were associated with metabolism, while in the IA depot, IGF and longevity pathways were negatively enriched. In pathway analysis, multiple expected and novel pathways were significantly affected by genotype, i.e. KO vs. controls. When GH related gene expression was analyzed, SOCS3 and CISH showed species-specific changes. CONCLUSION: AT of GHR-KO pigs has several similarities to that of humans with LS in terms of adipocyte size and gene expression profile that help describe the depot-specific adipose phenotype of both groups.
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Obesidad , Receptores de Somatotropina , Humanos , Animales , Porcinos , Obesidad/genética , Receptores de Somatotropina/genética , Receptores de Somatotropina/metabolismo , Tejido Adiposo/metabolismo , Hormona del Crecimiento/metabolismo , Perfilación de la Expresión Génica , Factor I del Crecimiento Similar a la Insulina/metabolismoRESUMEN
Replication protein A (RPA) binds single-stranded DNA (ssDNA) and serves critical functions in eukaryotic DNA replication, the DNA damage response, and DNA repair. During DNA replication, RPA is required for extended origin DNA unwinding and DNA synthesis. To determine the requirements for RPA during these processes, we tested ssDNA-binding proteins (SSBs) from different domains of life in reconstituted Saccharomyces cerevisiae origin unwinding and DNA replication reactions. Interestingly, Escherichia coli SSB, but not T4 bacteriophage Gp32, fully substitutes for RPA in promoting origin DNA unwinding. Using RPA mutants, we demonstrated that specific ssDNA-binding properties of RPA are required for origin unwinding but that its protein-interaction domains are dispensable. In contrast, we found that each of these auxiliary RPA domains have distinct functions at the eukaryotic replication fork. The Rfa1 OB-F domain negatively regulates lagging-strand synthesis, while the Rfa2 winged-helix domain stimulates nascent strand initiation. Together, our findings reveal a requirement for specific modes of ssDNA binding in the transition to extensive origin DNA unwinding and identify RPA domains that differentially impact replication fork function.