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RESEARCH QUESTION: Does double blastocyst vitrification and warming affect pregnancy, miscarriage or live birth rates, or birth outcomes, from embryos that have undergone preimplantation genetic testing for aneuploidies (PGT-A) testing? DESIGN: This retrospective observational analysis of embryo transfers was performed at a single centre between January 2017 and August 2022. The double-vitrification group included frozen blastocysts that were vitrified after 5-7 days of culture, warmed, biopsied (either once or twice) and re-vitrified. The single vitrification (SV) group included fresh blastocysts that were biopsied at 5-7 days and then vitrified. RESULTS: A comparison of the 84 double-vitrification blastocysts and 729 control single-vitrification blastocysts indicated that the double-vitrification embryos were frozen later in development and had expanded more than the single-vitrification embryos. Of the 813 embryo transfer procedures reported, 452 resulted in the successful delivery of healthy infants (56%). There were no significant differences between double-vitrification and single-vitrification embryos in the pregnancy, miscarriage or live birth rates achieved after single-embryo transfer (55% versus 56%). Logistic regression indicated that while reduced live birth rates were associated with increasing maternal age at oocyte collection, longer culture prior to freezing and lower embryo quality, double vitrification was not a significant predictor of live birth rate. CONCLUSIONS: Blastocyst double vitrification was not shown to impact pregnancy, miscarriage or live birth rates. Although caution is necessary due to the study size, no effects of double vitrification on miscarriage rates, birthweight or gestation period were noted. These data offer reassurance given the absence of the influence of double vitrification on all outcomes after PGT-A.
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Aborto Espontâneo , Coeficiente de Natalidade , Blastocisto , Criopreservação , Transferência Embrionária , Vitrificação , Humanos , Feminino , Gravidez , Estudos Retrospectivos , Adulto , Aborto Espontâneo/epidemiologia , Transferência Embrionária/métodos , Taxa de Gravidez , Nascido Vivo , Resultado da GravidezRESUMO
The study of pig preimplantation embryo development has several potential uses: from agriculture to the production of medically relevant genetically modified organisms and from rare breed conservation to acting as a physiologically relevant model for progressing human and other (e.g., endangered) species' in vitro fertilisation technology. Despite this, barriers to the widespread adoption of pig embryo in vitro production include lipid-laden cells that are hard to visualise, slow adoption of contemporary technologies such as the use of time-lapse incubators or artificial intelligence, poor blastulation and high polyspermy rates. Here, we employ a commercially available time-lapse incubator to provide a comprehensive overview of the morphokinetics of pig preimplantation development for the first time. We tested the hypotheses that (a) there are differences in developmental timings between blastulating and non-blastulating embryos and (b) embryo developmental morphokinetic features can be used to predict the likelihood of blastulation. The abattoir-derived oocytes fertilised by commercial extended semen produced presumptive zygotes were split into two groups: cavitating/blastulating 144 h post gamete co-incubation and those that were not. The blastulating group reached the 2-cell and morula stages significantly earlier, and the time taken to reach the 2-cell stage was identified to be a predictive marker for blastocyst formation. Reverse cleavage was also associated with poor blastulation. These data demonstrate the potential of morphokinetic analysis in automating and upscaling pig in vitro production through effective embryo selection.
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The high-elevation peatlands of the páramos of the northern Andes constitute a diverse environment that harbors large numbers of species and several types of plant communities along altitudinal, latitudinal, and environmental gradients. However, little is known about the structure and functioning of these ecosystems, including peatland vegetation types and their relative contribution to the production and accumulation of peat soils. In this paper we characterized the structure of peatland plant communities of the humid páramos of northern Ecuador by describing the distribution of plant growth-forms and their aboveground biomass patterns. Along an elevation gradient of 640 m we sampled vegetation in 16 peatlands and aboveground biomass in four peatlands. Three distinct peatland vegetation types were identified: High elevation Cushion peatlands, dominated by Plantago rigida and Distichia muscoides, Sedge and rush peatlands dominated by Carex spp. and Juncus spp., and Herbaceous and shrubby peatlands, with a more heterogenous and structurally complex vegetation. In terms of aboveground biomass, we found an 8-fold reduction in the higher peatlands compared to the lower sites, suggesting that the steep elevational gradients characteristic of Andean environments might be crucial in structuring the physiognomy and composition of peatland vegetation, either through its effects on temperature and other environmental factors, or through its effects on the age and development of soils. Additional studies are needed to evaluate the potential effects of temperature, hydrology, micro-topography, geological setting, and land-use, which are likely to influence vegetation patters in these peatlands.
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In porcine in vitro production (IVP) systems, the use of oocytes derived from prepubertal gilts, whilst being commercially attractive, remains challenging due to their poor developmental competence following in vitro maturation (IVM). Follicular fluid contains important growth factors and plays a key role during oocyte maturation; therefore, it is a common supplementation for porcine IVM medium. However, follicular fluid contains many poorly characterized components, is batch variable, and its use raises biosecurity concerns. In an effort to design a defined IVM system, growth factors such as cytokines have been previously tested. These include leukaemia inhibitory factor (LIF), fibroblast growth factor 2 (FGF2), and insulin-like growth factor 1 (IGF1), the combination of which is termed 'FLI'. Here, using abattoir-derived oocytes in a well established porcine IVP system, we compared follicular fluid and FLI supplementation during both IVM and embryo culture to test the hypothesis that FLI can substitute for follicular fluid without compromising oocyte nuclear and cytoplasmic maturation. We demonstrate that in oocytes derived from prepubertal gilts, FLI supplementation enhances oocyte meiotic maturation and has a positive effect on the quality and developmental competence of embryos. Moreover, for the first time, we studied the effects of follicular fluid and FLI combined showing no synergistic effects.
Assuntos
Fator 2 de Crescimento de Fibroblastos , Fator de Crescimento Insulin-Like I , Suínos , Animais , Feminino , Fator 2 de Crescimento de Fibroblastos/farmacologia , Fator 2 de Crescimento de Fibroblastos/metabolismo , Fator Inibidor de Leucemia/farmacologia , Fator Inibidor de Leucemia/metabolismo , Fator de Crescimento Insulin-Like I/farmacologia , Oócitos , Sus scrofa , Suplementos Nutricionais , Técnicas de Maturação in Vitro de Oócitos , Fertilização in vitroRESUMO
Hyperaldosteronism causes cardiovascular disease as well as hypomagnesemia. Mechanisms are ill-defined but dysregulation of TRPM7, a Mg2+-permeable channel/α-kinase, may be important. We examined the role of TRPM7 in aldosterone-dependent cardiovascular and renal injury by studying aldosterone-salt treated TRPM7-deficient (TRPM7+/Δkinase) mice. Plasma/tissue [Mg2+] and TRPM7 phosphorylation were reduced in vehicle-treated TRPM7+/Δkinase mice, effects recapitulated in aldosterone-salt-treated wild-type mice. Aldosterone-salt treatment exaggerated vascular dysfunction and amplified cardiovascular and renal fibrosis, with associated increased blood pressure in TRPM7+/Δkinase mice. Tissue expression of Mg2+-regulated phosphatases (PPM1A, PTEN) was downregulated and phosphorylation of Smad3, ERK1/2, and Stat1 was upregulated in aldosterone-salt TRPM7-deficient mice. Aldosterone-induced phosphorylation of pro-fibrotic signaling was increased in TRPM7+/Δkinase fibroblasts, effects ameliorated by Mg2+ supplementation. TRPM7 deficiency amplifies aldosterone-salt-induced cardiovascular remodeling and damage. We identify TRPM7 downregulation and associated hypomagnesemia as putative molecular mechanisms underlying deleterious cardiovascular and renal effects of hyperaldosteronism.
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Hiperaldosteronismo , Canais de Cátion TRPM , Aldosterona/farmacologia , Animais , Fibrose , Hiperaldosteronismo/genética , Hiperaldosteronismo/metabolismo , Rim/metabolismo , Magnésio/metabolismo , Camundongos , Proteína Fosfatase 2C/metabolismo , Cloreto de Sódio , Canais de Cátion TRPM/deficiência , Canais de Cátion TRPM/genética , Canais de Cátion TRPM/metabolismoRESUMO
Osteogenic factors, such as osteoprotegerin (OPG), are protective against vascular calcification. However, OPG is also positively associated with cardiovascular damage, particularly in pulmonary hypertension, possibly through processes beyond effects on calcification. In the present study, we focused on calcification-independent vascular effects of OPG through activation of syndecan-1 and NADPH oxidases (Noxs) 1 and 4. Isolated resistance arteries from Wistar-Kyoto (WKY) rats, exposed to exogenous OPG, studied by myography exhibited endothelial and smooth muscle dysfunction. OPG decreased nitric oxide (NO) production, eNOS activation and increased reactive oxygen species (ROS) production in endothelial cells. In VSMCs, OPG increased ROS production, H2O2/peroxynitrite levels and activation of Rho kinase and myosin light chain. OPG vascular and redox effects were also inhibited by the syndecan-1 inhibitor synstatin (SSNT). Additionally, heparinase and chondroitinase abolished OPG effects on VSMCs-ROS production, confirming syndecan-1 as OPG molecular partner and suggesting that OPG binds to heparan/chondroitin sulphate chains of syndecan-1. OPG-induced ROS production was abrogated by NoxA1ds (Nox1 inhibitor) and GKT137831 (dual Nox1/Nox4 inhibitor). Tempol (SOD mimetic) inhibited vascular dysfunction induced by OPG. In addition, we studied arteries from Nox1 and Nox4 knockout (KO) mice. Nox1 and Nox4 KO abrogated OPG-induced vascular dysfunction. Vascular dysfunction elicited by OPG is mediated by a complex signalling cascade involving syndecan-1, Nox1 and Nox4. Our data identify novel molecular mechanisms beyond calcification for OPG, which may underlie vascular injurious effects of osteogenic factors in conditions such as hypertension and/or diabetes.
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Hemodinâmica/efeitos dos fármacos , Músculo Liso Vascular/efeitos dos fármacos , Miócitos de Músculo Liso/efeitos dos fármacos , NADPH Oxidases/metabolismo , Osteoprotegerina/toxicidade , Estresse Oxidativo , Espécies Reativas de Oxigênio/metabolismo , Sindecana-1/metabolismo , Animais , Células Cultivadas , Masculino , Artérias Mesentéricas/efeitos dos fármacos , Artérias Mesentéricas/enzimologia , Artérias Mesentéricas/fisiopatologia , Camundongos Endogâmicos C57BL , Músculo Liso Vascular/enzimologia , Músculo Liso Vascular/fisiopatologia , Miócitos de Músculo Liso/enzimologia , NADPH Oxidase 1/genética , NADPH Oxidase 1/metabolismo , NADPH Oxidase 4/genética , NADPH Oxidase 4/metabolismo , NADPH Oxidases/genética , Ratos Endogâmicos WKY , Transdução de SinaisRESUMO
Cryopreservation of both gametes and embryos, both for storage and for the preservation of their developmental capacity is a critical aspect of assisted reproductive technology. The survival of reproductive material following cryopreservation protocols is not only vital to clinical applications in the human in vitro fertilisation clinic, but is also important in the in vitro production of livestock embryos. The ability to routinely cryopreserve oocytes and embryos of livestock species has the potential to improve animal welfare, reduce environmental impact, and reduce the associated costs for breeding companies through the reduction of live animal transportation. Unfortunately, frozen oocytes and embryos are regularly documented to contain a higher proportion of apoptotic cells compared to their non-frozen counterparts, with freezing procedures thought to trigger apoptotic pathways of cell death. Comparisons between frozen and non-frozen samples also show changes in the gene expression of apoptotic factors such as Bcl-2 and Bax in response to cryopreservation. Apoptotic inhibition has the potential to improve cryosurvival, and how to achieve this is subject to debate. Here, we review how exposure to low temperatures during cryopreservation may be responsible for the abnormal activation of apoptotic pathways in mammalian oocytes and embryos, and discuss the ways in which they can be influenced to improve cryopreservation protocols, particularly in agriculturally important species.
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Criopreservação , Embrião de Mamíferos , Animais , Apoptose , Criopreservação/veterinária , Fertilização in vitro/veterinária , OócitosRESUMO
Pathogenic variants in the gene HGSNAT (heparan-α-glucosaminide N-acetyltransferase) have been reported to underlie two distinct recessive conditions, depending on the specific genotype, mucopolysaccharidosis type IIIC (MPSIIIC)-a severe childhood-onset lysosomal storage disorder, and adult-onset nonsyndromic retinitis pigmentosa (RP). Here we describe the largest cohort to-date of HGSNAT-associated nonsyndromic RP patients, and describe their retinal phenotype, leukocyte enzymatic activity, and likely pathogenic genotypes. We identified biallelic HGSNAT variants in 17 individuals (15 families) as the likely cause of their RP. None showed any other symptoms of MPSIIIC. All had a mild but significant reduction of HGSNAT enzyme activity in leukocytes. The retinal condition was generally of late-onset, showing progressive degeneration of a concentric area of paramacular retina, with preservation but reduced electroretinogram responses. Symptoms, electrophysiology, and imaging suggest the rod photoreceptor to be the cell initially compromised. HGSNAT enzymatic testing was useful in resolving diagnostic dilemmas in compatible patients. We identified seven novel sequence variants [p.(Arg239Cys); p.(Ser296Leu); p.(Phe428Cys); p.(Gly248Ala); p.(Gly418Arg), c.1543-2A>C; c.1708delA], three of which were considered to be retina-disease-specific alleles. The most prevalent retina-disease-specific allele p.(Ala615Thr) was observed heterozygously or homozygously in 8 and 5 individuals respectively (7 and 4 families). Two siblings in one family, while identical for the HGSNAT locus, but discordant for retinal disease, suggest the influence of trans-acting genetic or environmental modifying factors.
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Acetiltransferases/genética , Mucopolissacaridose III/genética , Doenças Retinianas/genética , Retinose Pigmentar/genética , Adolescente , Adulto , Criança , Feminino , Genótipo , Humanos , Leucócitos/metabolismo , Masculino , Pessoa de Meia-Idade , Mucopolissacaridose III/complicações , Mucopolissacaridose III/patologia , Linhagem , Retina/patologia , Doenças Retinianas/complicações , Doenças Retinianas/patologia , Retinose Pigmentar/complicações , Retinose Pigmentar/patologia , Adulto JovemRESUMO
Eukaryotic cells respond to hypothermic stress through a series of regulatory mechanisms that preserve energy resources and prolong cell survival. These mechanisms include alterations in gene expression, attenuated global protein synthesis and changes in the lipid composition of the phospholipid bilayer. Cellular responses to hyperthermia, hypoxia, nutrient deprivation and oxidative stress have been comprehensively investigated, but studies of the cellular response to cold stress are more limited. Responses to cold stress are however of great importance both in the wild, where exposure to low and fluctuating environmental temperatures is common, and in medical and biotechnology settings where cells and tissues are frequently exposed to hypothermic stress and cryopreservation. This means that it is vitally important to understand how cells are impacted by low temperatures and by the decreases and subsequent increases in temperature associated with cold stress. Here, we review the ways in which eukaryotic cells respond to hypothermic stress and how these compare to the well-described and highly integrated stress response systems that govern the cellular response to other types of stress.
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Eucariotos/fisiologia , Hipotermia/fisiopatologia , Estresse Fisiológico , Animais , Membrana Celular/metabolismo , Humanos , Hipóxia/fisiopatologia , Estresse OxidativoRESUMO
Increased risk and severity of idiopathic pulmonary arterial hypertension (iPAH) is associated with elevated estradiol in men and postmenopausal women. Pulmonary arteries synthesise estradiol via aromatase and metabolise it via CYP1B1 to mitogenic metabolites; SNPs in aromatase and CYP1B1 have been associated with PAH. This suggests that estradiol metabolism could be altered in iPAH. This proof-of-concept study profiles estradiol and several metabolites of estradiol simultaneously in serum from iPAH patients and controls. We show that the estradiol and metabolite profile is altered in iPAH and that 16-hydroxyestrone and 16-hydroxyestradiol accumulate in iPAH patients with 16-hydroxyestrone levels relating to disease severity.
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AIMS: Transient Receptor Potential Melastatin 7 (TRPM7) cation channel is a chanzyme (channel + kinase) that influences cellular Mg2+ homeostasis and vascular signalling. However, the pathophysiological significance of TRPM7 in the cardiovascular system is unclear. The aim of this study was to investigate the role of this chanzyme in the cardiovascular system focusing on inflammation and fibrosis. METHODS AND RESULTS: TRPM7-deficient mice with deletion of the kinase domain (TRPM7+/Δkinase) were studied and molecular mechanisms investigated in TRPM7+/Δkinase bone marrow-derived macrophages (BMDM) and co-culture systems with cardiac fibroblasts. TRPM7-deficient mice had significant cardiac hypertrophy, fibrosis, and inflammation. Cardiac collagen and fibronectin content, expression of pro-inflammatory mediators (SMAD3, TGFß) and cytokines [interleukin (IL)-6, IL-10, IL-12, tumour necrosis factor-α] and phosphorylation of the pro-inflammatory signalling molecule Stat1, were increased in TRPM7+/Δkinase mice. These processes were associated with infiltration of inflammatory cells (F4/80+CD206+ cardiac macrophages) and increased galectin-3 expression. Cardiac [Mg2+]i, but not [Ca2+]i, was reduced in TRPM7+/Δkinase mice. Calpain, a downstream TRPM7 target, was upregulated (increased expression and activation) in TRPM7+/Δkinase hearts. Vascular functional and inflammatory responses, assessed in vivo by intra-vital microscopy, demonstrated impaired neutrophil rolling, increased neutrophil: endothelial attachment and transmigration of leucocytes in TRPM7+/Δkinase mice. TRPM7+/Δkinase BMDMs had increased levels of galectin-3, IL-10, and IL-6. In co-culture systems, TRPM7+/Δkinase macrophages increased expression of fibronectin, proliferating cell nuclear antigen, and TGFß in cardiac fibroblasts from wild-type mice, effects ameliorated by MgCl2 treatment. CONCLUSIONS: We identify a novel anti-inflammatory and anti-fibrotic role for TRPM7 and suggest that its protective effects are mediated, in part, through Mg2+-sensitive processes.
Assuntos
Cardiomegalia/metabolismo , Cardiomiopatias/metabolismo , Mediadores da Inflamação/metabolismo , Inflamação/metabolismo , Miocárdio/metabolismo , Canais de Cátion TRPM/metabolismo , Remodelação Ventricular , Animais , Cardiomegalia/genética , Cardiomegalia/patologia , Cardiomegalia/fisiopatologia , Cardiomiopatias/genética , Cardiomiopatias/patologia , Cardiomiopatias/fisiopatologia , Proliferação de Células , Células Cultivadas , Técnicas de Cocultura , Fibroblastos/metabolismo , Fibroblastos/patologia , Fibrose , Inflamação/genética , Inflamação/patologia , Inflamação/fisiopatologia , Migração e Rolagem de Leucócitos , Macrófagos/metabolismo , Macrófagos/patologia , Magnésio/metabolismo , Masculino , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Miocárdio/patologia , Transdução de Sinais , Canais de Cátion TRPM/deficiência , Canais de Cátion TRPM/genética , Migração Transendotelial e TransepitelialRESUMO
Obesity is a common comorbidity for pulmonary arterial hypertension (PAH). Additionally, oestrogen and its metabolites are risk factors for the development of PAH. Visceral adipose tissue (VAT) is a major site of oestrogen production; however, the influence of obesity-induced changes in oestrogen synthesis and metabolism on the development of PAH is unclear. To address this we investigated the effects of inhibiting oestrogen synthesis and metabolism on the development of pulmonary hypertension in male and female obese mice.We depleted endogenous oestrogen in leptin-deficient (ob/ob) mice with the oestrogen inhibitor anastrozole (ANA) and determined the effects on the development of pulmonary hypertension, plasma oestradiol and urinary 16α-hydroxyestrone (16αOHE1). Oestrogen metabolism through cytochrome P450 1B1 (CYP1B1) was inhibited with 2,2',4,6'-tetramethoxystilbene (TMS).ob/ob mice spontaneously develop pulmonary hypertension, pulmonary vascular remodelling and increased reactive oxygen species production in the lung; these effects were attenuated by ANA. Oestradiol levels were decreased in obese male mice; however, VAT CYP1B1 and 16αOHE1 levels were increased. TMS also attenuated pulmonary hypertension in male ob/ob mice. Intra-thoracic fat from ob/ob mice and VAT conditioned media produce 16αOHE1 and can contribute to oxidative stress, effects that are attenuated by both ANA and TMS.Obesity can induce pulmonary hypertension and changes in oestrogen metabolism, resulting in increased production of 16αOHE1 from VAT that contributes to oxidative stress. Oestrogen inhibitors are now in clinical trials for PAH. This study has translational consequences as it suggests that oestrogen inhibitors may be especially beneficial in treating obese individuals with PAH.
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Estrogênios/metabolismo , Gordura Intra-Abdominal/metabolismo , Obesidade/metabolismo , Hipertensão Arterial Pulmonar/enzimologia , Artéria Pulmonar/enzimologia , Anastrozol , Animais , Citocromo P-450 CYP1B1/genética , Estradiol/sangue , Feminino , Hidroxiestronas/urina , Hipóxia/complicações , Leptina/metabolismo , Pulmão/enzimologia , Pulmão/patologia , Masculino , Camundongos , Camundongos Knockout , Miócitos de Músculo Liso/metabolismo , Miócitos de Músculo Liso/patologia , Obesidade/genética , Hipertensão Arterial Pulmonar/induzido quimicamente , Hipertensão Arterial Pulmonar/patologia , Artéria Pulmonar/patologia , EstilbenosRESUMO
Chemerin and its G protein-coupled receptor [chemerin receptor 23 (ChemR23)] have been associated with endothelial dysfunction, inflammation, and insulin resistance. However, the role of chemerin on insulin signaling in the vasculature is still unknown. We aimed to determine whether chemerin reduces vascular insulin signaling and whether there is interplay between chemerin/ChemR23, insulin resistance, and vascular complications associated with type 2 diabetes (T2D). Molecular and vascular mechanisms were probed in mesenteric arteries and cultured vascular smooth muscle cells (VSMCs) from C57BL/6J, nondiabetic lean db/m, and diabetic obese db/db mice as well as in human microvascular endothelial cells (HMECs). Chemerin decreased insulin-induced vasodilatation in C57BL/6J mice, an effect prevented by CCX832 (ChemR23 antagonist) treatment. In VSMCs, chemerin, via oxidative stress- and ChemR23-dependent mechanisms, decreased insulin-induced Akt phosphorylation, glucose transporter 4 translocation to the membrane, and glucose uptake. In HMECs, chemerin decreased insulin-activated nitric oxide signaling. AMP-activated protein kinase phosphorylation was reduced by chemerin in both HMECs and VSMCs. CCX832 treatment of db/db mice decreased body weight, insulin, and glucose levels as well as vascular oxidative stress. CCX832 also partially restored vascular insulin responses in db/db and high-fat diet-fed mice. Our novel in vivo findings highlight chemerin/ChemR23 as a promising therapeutic target to limit insulin resistance and vascular complications associated with obesity-related diabetes. NEW & NOTEWORTHY Our novel findings show that the chemerin/chemerin receptor 23 axis plays a critical role in diabetes-associated vascular oxidative stress and altered insulin signaling. Targeting chemerin/chemerin receptor 23 may be an attractive strategy to improve insulin signaling and vascular function in obesity-associated diabetes.
Assuntos
Diabetes Mellitus/metabolismo , Artérias Mesentéricas/metabolismo , Receptores Acoplados a Proteínas G/antagonistas & inibidores , Transdução de Sinais , Vasodilatação , Animais , Antioxidantes/farmacologia , Células Cultivadas , Diabetes Mellitus/fisiopatologia , Endotélio Vascular/metabolismo , Humanos , Insulina/metabolismo , Masculino , Artérias Mesentéricas/efeitos dos fármacos , Artérias Mesentéricas/fisiopatologia , Camundongos , Camundongos Endogâmicos C57BL , Músculo Liso Vascular/metabolismo , Óxido Nítrico/metabolismo , Compostos Orgânicos/farmacologia , Estresse Oxidativo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Vasodilatadores/farmacologiaRESUMO
Pulmonary arterial hypertension (PAH) is a progressive disease characterised by increased pulmonary vascular resistance and pulmonary artery remodelling as result of increased vascular tone and vascular cell proliferation, respectively. Eventually, this leads to right heart failure. Heritable PAH is caused by a mutation in the bone morphogenetic protein receptor-II (BMPR-II). Female susceptibility to PAH has been known for some time, and most recent figures show a female-to-male ratio of 4:1. Variations in the female sex hormone estrogen and estrogen metabolism modify FPAH risk, and penetrance of the disease in BMPR-II mutation carriers is increased in females. Several lines of evidence point towards estrogen being pathogenic in the pulmonary circulation, and thus increasing the risk of females developing PAH. Recent studies have also suggested that estrogen metabolism may be crucial in the development and progression of PAH with studies indicating that downstream metabolites such as 16α-hydroxyestrone are upregulated in several forms of experimental pulmonary hypertension (PH) and can cause pulmonary artery smooth muscle cell proliferation and subsequent vascular remodelling. Conversely, other estrogen metabolites such as 2-methoxyestradiol have been shown to be protective in the context of PAH. Estrogen may also upregulate the signalling pathways of other key mediators of PAH such as serotonin.
Assuntos
Pressão Arterial , Hipertensão Pulmonar/fisiopatologia , Artéria Pulmonar/fisiopatologia , Remodelação Vascular , Fatores Etários , Animais , Anti-Hipertensivos/uso terapêutico , Pressão Arterial/efeitos dos fármacos , Receptores de Proteínas Morfogenéticas Ósseas Tipo II/genética , Receptores de Proteínas Morfogenéticas Ósseas Tipo II/metabolismo , Feminino , Predisposição Genética para Doença , Hormônios Esteroides Gonadais/metabolismo , Humanos , Hipertensão Pulmonar/tratamento farmacológico , Hipertensão Pulmonar/epidemiologia , Hipertensão Pulmonar/metabolismo , Masculino , Artéria Pulmonar/efeitos dos fármacos , Artéria Pulmonar/metabolismo , Receptores de Estrogênio/metabolismo , Fatores de Risco , Caracteres Sexuais , Fatores Sexuais , Transdução de Sinais , Resultado do Tratamento , Remodelação Vascular/efeitos dos fármacosRESUMO
Rats dosed with the vascular endothelial growth factor inhibitor Sugen 5416 (Su), subjected to hypoxia, and then restored to normoxia have become a widely used model of pulmonary arterial hypertension (PAH). However, the mechanism by which Su exacerbates pulmonary hypertension is unclear. We investigated Su activation of the aryl hydrocarbon receptor (AhR) in human pulmonary artery smooth muscle cells (hPASMCs) and blood outgrowth endothelial cells (BOECs) from female patients with PAH. We also examined the effect of AhR on aromatase and estrogen levels in the lung. Protein and mRNA analyses demonstrated that CYP1A1 was very highly induced in the lungs of Su/hypoxic (Su/Hx) rats. The AhR antagonist CH223191 (8 mg/kg/day) reversed the development of PAH in this model in vivo and normalized lung CYP1A1 expression. Increased lung aromatase and estrogen levels in Su/Hx rats were also normalized by CH223191, as was AhR nuclear translocator (ARNT [HIF-1ß]), which is shared by HIF-1α and AhR. Su reduced HIF-1α expression in hPASMCs. Su induced proliferation in BOECs and increased apoptosis in human pulmonary microvascular ECs and also induced translocation of AhR to the nucleus in hPASMCs. Under normoxic conditions, hPASMCs did not proliferate to Su. However, when grown in hypoxia (1%), Su induced hPASMC proliferation. In combination with hypoxia, Su is proliferative in hPASMCs and BOECs from patients with PAH, and Su/Hx-induced PAH in rats may be facilitated by AhR-induced CYP1A1, ARNT, and aromatase. Inhibition of AhR may be a novel approach to the treatment of pulmonary hypertension.
Assuntos
Apoptose/efeitos dos fármacos , Hipóxia Celular/fisiologia , Citocromo P-450 CYP1A1/biossíntese , Hipertensão Pulmonar/patologia , Indóis/toxicidade , Pirróis/toxicidade , Receptores de Hidrocarboneto Arílico/metabolismo , Transporte Ativo do Núcleo Celular/fisiologia , Animais , Aromatase/metabolismo , Translocador Nuclear Receptor Aril Hidrocarboneto/metabolismo , Translocador Nuclear Receptor Aril Hidrocarboneto/farmacologia , Compostos Azo/farmacologia , Proliferação de Células/efeitos dos fármacos , Citocromo P-450 CYP1A1/genética , Células Endoteliais/metabolismo , Estrogênios/metabolismo , Feminino , Humanos , Hipertensão Pulmonar/induzido quimicamente , Hipertensão Pulmonar/tratamento farmacológico , Subunidade alfa do Fator 1 Induzível por Hipóxia/biossíntese , Pulmão/patologia , Músculo Liso Vascular/metabolismo , Pirazóis/farmacologia , RNA Mensageiro/biossíntese , Ratos , Ratos Wistar , Receptores de Hidrocarboneto Arílico/antagonistas & inibidores , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidoresRESUMO
Preveraison treatment of Shiraz berries with either 1-naphthaleneacetic acid (NAA) or Ethrel delayed the onset of ripening and harvest. NAA was more effective than Ethrel, delaying harvest by 23 days, compared to 6 days for Ethrel. Sensory analysis of wines from NAA-treated fruit showed significant differences in 10 attributes, including higher "pepper" flavor and aroma compared to those of the control wines. A nontargeted analysis of headspace volatiles revealed modest differences between wines made from control and NAA- or Ethrel-treated berries. However, the concentration of rotundone, the metabolite responsible for the pepper character, was below the level of detection by solid phase microextraction-gas chromatography-mass spectrometry in control wines, low in Ethrel wines (2 ng/L), and much higher in NAA wines (29 ng/L). Thus, NAA, and to a lesser extent Ethrel, treatment of grapes during the preveraison period can delay ripening and enhance rotundone concentrations in Shiraz fruit, thereby enhancing wine "peppery" attributes.
Assuntos
Aromatizantes/química , Ácidos Naftalenoacéticos/farmacologia , Reguladores de Crescimento de Plantas/farmacologia , Vitis/química , Vinho/análise , Adulto , Feminino , Frutas/química , Frutas/efeitos dos fármacos , Frutas/crescimento & desenvolvimento , Cromatografia Gasosa-Espectrometria de Massas , Humanos , Masculino , Pessoa de Meia-Idade , Estrutura Molecular , Odorantes/análise , Olfato , Paladar , Vitis/efeitos dos fármacos , Vitis/crescimento & desenvolvimento , Adulto JovemRESUMO
PURPOSE: Severe Combined Immunodeficiency (SCID) is considered to be a paediatric emergency and unless identified promptly can be life-threatening. Frequently, infants are not diagnosed with SCID until they have become seriously ill with infection leading to treatment complications and a poorer prognosis. We aimed to test a newly available commercial duplex assay to measure T cell receptor excision circles (TRECs) to establish if this would be suitable for newborn screening for SCID in the UK. METHODS: Over 5000 anonymous retrospective dried blood spots (DBS) were used alongside 18 confirmed SCID positive DBS with a newly available duplex assay to measure TRECs levels and control gene levels. We also included testing of premature babies and babies from neonatal intensive care units (NICU) as these have been shown to have high false positive rates in other TREC screening assays. RESULTS: All 18 SCID DBS samples were successfully identified as SCID positives in the study. The number of presumptive positives detected was dependent on the TREC cut-off threshold settings. When analysed with five different TRECs cut-off values (20, 25, 30, 35 and 40 TREC copies/µl blood) the presumptive positive rate ranged from 0.04 to 1.00 % of samples tested. Premature infants and neonates from NICU did not show high presumed false positive rates in this assay. CONCLUSIONS: The study demonstrated that this duplex assay kit will identify all newborns with SCID as presumptive positives. The data also shows that with suitable TREC cut-off settings the number of presumptive positives from non-SCID newborns will be manageable in the context of a national screening service.
Assuntos
Testes Genéticos/métodos , Triagem Neonatal/métodos , Imunodeficiência Combinada Severa/diagnóstico , Algoritmos , Pré-Escolar , Idade Gestacional , Humanos , Lactente , Recém-Nascido , Kit de Reagentes para Diagnóstico , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
BACKGROUND: The currently recommended technologies of HPLC and isoelectric focusing for newborn blood spot screening for sickle cell disease (SCD) identify both the disease and carrier states, resulting in large numbers of infants being followed up unnecessarily. Analysis of blood spot tryptic peptides performed by using tandem mass spectrometry (MS/MS) is an alternative technology to detect hemoglobin (Hb) variant disorders. METHODS: We analyzed 2154 residual newborn blood spots and 675 newborn blood spots from infants with Hb variants by using MS/MS after trypsin digestion. Screening cutoffs were developed by using the ratio between the variant peptide-to-wild-type peptide abundance for HbS, C, D(Punjab), O(Arab), Lepore, and E peptides. A postanalytical data analysis protocol was developed using these cutoffs to detect only the disease states of SCD and not to identify carrier states. A parallel study of 13 249 newborn blood spots from a high-prevalence SCD area were analyzed by both MS/MS and HPLC. RESULTS: Screening cutoffs developed distinguished the infants with the disease states of SCD, infants who were carriers of SCD, and infants with normal Hb. In the parallel study no false-negative results were identified, and all clinically relevant cases were correctly identified using the MS/MS protocol. Unblinding the data revealed a total of 328 carrier infants that were successfully excluded by the protocol. CONCLUSIONS: The screening protocol developed correctly identified infants with the disease states of SCD. Furthermore, large numbers of sickle cell carrier infants were successfully not identified, thereby avoiding unnecessary follow-up testing and referral for genetic counseling.
Assuntos
Anemia Falciforme/sangue , Testes Genéticos/métodos , Hemoglobina Falciforme/isolamento & purificação , Triagem Neonatal/métodos , Espectrometria de Massas em Tandem/métodos , Anemia Falciforme/epidemiologia , Anemia Falciforme/genética , Coleta de Amostras Sanguíneas , Cromatografia Líquida de Alta Pressão/métodos , Variação Genética , Hemoglobina Falciforme/genética , Hemoglobinas Anormais/genética , Hemoglobinas Anormais/isolamento & purificação , Humanos , Recém-Nascido , Focalização Isoelétrica , Fragmentos de Peptídeos/análise , Sensibilidade e Especificidade , Traço Falciforme/sangue , Traço Falciforme/epidemiologia , Traço Falciforme/genética , Tripsina/químicaRESUMO
Mucopolysaccharidosis IVA (MPS IVA; Morquio A syndrome) is an autosomal recessive lysosomal storage disorder resulting from a deficiency of N-acetylgalactosamine-6-sulfate sulfatase (GALNS) activity. Diagnosis can be challenging and requires agreement of clinical, radiographic, and laboratory findings. A group of biochemical genetics laboratory directors and clinicians involved in the diagnosis of MPS IVA, convened by BioMarin Pharmaceutical Inc., met to develop recommendations for diagnosis. The following conclusions were reached. Due to the wide variation and subtleties of radiographic findings, imaging of multiple body regions is recommended. Urinary glycosaminoglycan analysis is particularly problematic for MPS IVA and it is strongly recommended to proceed to enzyme activity testing even if urine appears normal when there is clinical suspicion of MPS IVA. Enzyme activity testing of GALNS is essential in diagnosing MPS IVA. Additional analyses to confirm sample integrity and rule out MPS IVB, multiple sulfatase deficiency, and mucolipidoses types II/III are critical as part of enzyme activity testing. Leukocytes or cultured dermal fibroblasts are strongly recommended for enzyme activity testing to confirm screening results. Molecular testing may also be used to confirm the diagnosis in many patients. However, two known or probable causative mutations may not be identified in all cases of MPS IVA. A diagnostic testing algorithm is presented which attempts to streamline this complex testing process.
Assuntos
Glicosaminoglicanos/urina , Mucopolissacaridose IV/diagnóstico , Mucopolissacaridose IV/enzimologia , Algoritmos , Fibroblastos/enzimologia , Humanos , Leucócitos/enzimologia , Mucolipidoses/diagnóstico , Mucopolissacaridose IV/genética , Mucopolissacaridose IV/urina , Doença da Deficiência de Múltiplas Sulfatases/diagnóstico , Mutação , Patologia Molecular/métodosRESUMO
Grape (Vitis vinifera L.) berries are considered to be nonclimacteric fruit as they do not exhibit a large rise in ethylene production or respiration rate at the onset of ripening (veraison). However, ethylene may still play a role in berry development and in ripening in particular. (2-Chloroethyl)phosphonic acid (CEPA), an ethylene-releasing reagent, delayed ripening when applied early in berry development. In agreement with a role for ethylene in controlling the timing of ripening, the application of an inhibitor of ethylene biosynthesis, aminoethoxyvinylglycine (AVG), advanced ripening, as did abscisic acid, when applied during the preveraison period. Applications of CEPA nearer to the time of veraison enhanced berry colouration. Changes in the expression of ethylene biosynthesis and receptor genes were observed throughout berry development. Transcript levels of some of these genes were increased by CEPA and decreased by AVG, suggesting changes in ethylene synthesis and perception during the preveraison period that might contribute to the biphasic response to CEPA (ethylene). The significant delay of ripening in field-grown grapes through the application of CEPA also indicates that this may be useful in controlling the timing of veraison, and therefore harvest date, in warmer climates.