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Plants thrive in diverse environments, where root-microbe interactions play a pivotal role. Date palm (Phoenix dactylifera L.), with its genetic diversity and resilience, is an ideal model for studying microbial adaptation to different genotypes and stresses. This study aimed to analyze the bacterial and fungal communities associated with traditional date palm cultivars and the widely cultivated "Deglet Nour" were explored using metabarcoding approaches. The microbial diversity analysis identified a rich community with 13,189 bacterial and 6442 fungal Amplicon Sequence Variants (ASVs). Actinobacteriota, Proteobacteria, and Bacteroidota dominated bacterial communities, while Ascomycota dominated fungal communities. Analysis of the microbial community revealed the emergence of two distinct clusters correlating with specific date palm cultivars, but fungal communities showed higher sensitivity to date palm genotype variations compared to bacterial communities. The commercial cultivar "Deglet Nour" exhibited a unique microbial composition enriched in pathogenic fungal taxa, which was correlated with its genetic distance. Overall, our study contributes to understanding the complex interactions between date palm genotypes and soil microbiota, highlighting the genotype role in microbial community structure, particularly among fungi. These findings suggest correlations between date palm genotype, stress tolerance, and microbial assembly, with implications for plant health and resilience. Further research is needed to elucidate genotype-specific microbial interactions and their role in enhancing plant resilience to environmental stresses.
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Bacterias , Hongos , Microbiota , Phoeniceae , Microbiología del Suelo , Phoeniceae/microbiología , Phoeniceae/genética , Bacterias/clasificación , Bacterias/genética , Bacterias/aislamiento & purificación , Hongos/genética , Hongos/clasificación , Hongos/aislamiento & purificación , Hongos/fisiología , Genotipo , Raíces de Plantas/microbiología , Suelo/químicaRESUMEN
Pulmonary arterial hypertension (PAH) is a chronic disorder characterized by excessive pulmonary vascular remodeling, leading to elevated pulmonary vascular resistance and right ventricle (RV) overload and failure. MicroRNA-146a (miR-146a) promotes vascular smooth muscle cell proliferation and vascular neointimal hyperplasia, both hallmarks of PAH. This study aimed to investigate the effects of miR-146a through pharmacological or genetic inhibition on experimental PAH and RV pressure overload animal models. Additionally, we examined the overexpression of miR-146a on human pulmonary artery smooth muscle cells (hPASMCs). Here, we showed that miR-146a genic expression was increased in the lungs of patients with PAH and the plasma of monocrotaline (MCT) rats. Interestingly, genetic ablation of miR-146a improved RV hypertrophy and systolic pressures in Sugen 5415/hypoxia (SuHx) and pulmonary arterial banding (PAB) mice. Pharmacological inhibition of miR-146a improved RV remodeling in PAB-wild type mice and MCT rats, and enhanced exercise capacity in MCT rats. However, overexpression of miR-146a did not affect proliferation, migration, and apoptosis in control-hPASMCs. Our findings show that miR-146a may play a significant role in RV function and remodeling, representing a promising therapeutic target for RV hypertrophy and, consequently, PAH.
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MicroARNs , Hipertensión Arterial Pulmonar , Arteria Pulmonar , Función Ventricular Derecha , Animales , MicroARNs/genética , MicroARNs/metabolismo , Ratas , Humanos , Ratones , Masculino , Hipertensión Arterial Pulmonar/genética , Hipertensión Arterial Pulmonar/metabolismo , Arteria Pulmonar/metabolismo , Arteria Pulmonar/patología , Modelos Animales de Enfermedad , Monocrotalina , Proliferación Celular/genética , Miocitos del Músculo Liso/metabolismo , Hipertensión Pulmonar/genética , Hipertensión Pulmonar/metabolismo , Hipertensión Pulmonar/fisiopatología , Hipertrofia Ventricular Derecha/genética , Hipertrofia Ventricular Derecha/fisiopatología , Hipertrofia Ventricular Derecha/metabolismo , Remodelación Vascular/genética , Ratas Sprague-DawleyRESUMEN
Successful plant reproduction depends on the adequate development of flower organs controlled by cell proliferation and other processes. The SCI1 gene regulates cell proliferation and affects the final size of the female reproductive organ. To unravel the molecular mechanism exerted by SCI1 in cell proliferation control, we searched for its interaction partners through semi-in vivo pulldown experiments, uncovering a cyclin-dependent kinase, NtCDKG;2. Bimolecular fluorescence complementation (BiFC) and co-localization experiments showed that SCI1 interacts with NtCDKG;2 and its cognate NtCyclin L in nucleoli and splicing speckles. The screening of a yeast two-hybrid (Y2H) cDNA library using SCI1 as bait revealed a novel DEAD-box RNA helicase (NtRH35). The interaction between the NtCDKG;2-NtCyclin L complex, and NtRH35 was also shown. Subcellular localization experiments showed that SCI1, NtRH35, and the NtCDKG;2-NtCyclin L complex associate with each other within splicing speckles. The Y2H screening of NtCDKG;2 and NtRH35 identified the conserved spliceosome components U2a', NKAP, and CACTIN. This work presents SCI1 and its interactors NtCDKG;2-NtCyclin L complex, and NtRH35 as new spliceosome-associated proteins. Our findings reveal a network of interactions and suggest that SCI1 may regulate cell proliferation through the splicing process. This study provides new valuable insights into the intricate molecular pathways governing plant development.
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Aims: We hypothesize that miRs are key players in the dynamics of the hypertrophy phenotype in aortic stenosis (AS) patients. In our study, we aimed to identify the transcriptional patterns (protein-coding transcripts and miRs) from myocardial sample biopsies that could be associated with the absence of left ventricular (LV) mass regression after aortic valve replacement (AVR) in patients with severe AS and LV hypertrophy. Methods and results: We prospectively included 40 patients with severe AS, LV hypertrophy, and preserved ejection fraction undergoing AVR. Myocardial biopsies obtained during surgery were analysed for transcriptomic analysis performed by next-generation sequencing. At a 1-year follow-up, no hypertrophy reversal was observed in about half of the patients in the absence of patient-prosthesis mismatch and prosthesis dysfunction of uncontrolled hypertension. Predictors of mass regression were assessed from clinical, echocardiographic, and biochemical variables as well as from 300 miRs obtained from myocardial specimens, allowing the identification 29 differentially expressed. miR-4709-3p was found as a positive independent predictor of hypertrophy regression together with high-sensitivity troponin T (cTNT-hs) as a negative predictor. Gene transcripts RFX1, SIX5, MAPK8IF3, and PKD1 were predicted as simultaneous targets of five upregulated miRs suggesting its importance in LV hypertrophy. Conclusion: In our cohort, tissue miR-4709-3p and cTNT-hs were independent predictors of hypertrophy regression. The hypertrophy reversal process will likely depend from a complex network where miRNAs may have an important role, allowing a potential opportunity for therapy.
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INTRODUCTION: Some studies show that participants do not always fully understand the informed consent form (ICF), which is one of the reasons for dropouts. This study aimed to adapt the Quality of Informed Consent (QuIC) questionnaire into a valid instrument to be applied to the Portuguese population and to measure its reliability and validity in the Portuguese population, by applying it to a sample of participants in controlled trials. METHODS: The QuIC questionnaire was developed to assess the quality of informed consent in clinical trials and consists of two parts, addressing both the objective (part A) and the subjective (part B) understanding. After being translated and validated into Portuguese, it was implemented in 100 cardiac participants of phase III clinical trials in a University Hospital Center. RESULTS: The QuIC-PT questionnaire showed excellent stability over time and good validity. All patients evaluated their participation and their health positively and recognized the main purpose of the clinical trial. Almost all participants understood their role in helping future patients and the purpose of the trial and realized that, by signing the ICF, they were participating in a clinical trial. However, none of them knew that their experimental treatment was not proven to be the best alternative for their condition. CONCLUSION: The QuIC-PT questionnaire seems to be a valid and useful instrument to evaluate the participants' understanding of the ICF. In this study, we found that some concepts, like 'study protocol' or 'randomization', were not well understood by participants when signing the ICF, especially by participants with lower education levels. They also believed that the experimental intervention would solve their health condition. Greater awareness about the importance of the informed consent process and ICF is necessary so that participants can fully understand the protocol, especially the risks involved, and their rights as participants.
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Fire plays a key role in grasslands, determining the distribution and evolution of species and boundaries with neighboring ecosystems. Evidence of community-wide responses to fire is largely based on taxonomic and functional descriptors, while the phylogenetic dimension is overlooked. Here we evaluated how the taxonomic and phylogenetic structure of grassland plant communities responded to a time since fire (TSF) gradient. We sampled 12 communities in Southern Brazil under varying TSF and calculated taxonomic species richness (S) and dominance (D), phylogenetic diversity (PD), and mean phylogenetic distances (MPD). We used Structural Equation Models to test the relationships between the environmental gradient and community descriptors. Communities with longer TSF presented higher PD and MPD but lower species richness and increased taxonomic dominance. These sites were dominated by monocots, specifically C4 grasses, but also presented exclusive clades, whereas recently-burned sites presented lower taxonomic dominance and more species distributed in a wider variety of clades. Our results indicate that these scenarios are interchangeable and dependent on fire management. Fire adaptation was not constrained by phylogenetic relatedness, contrasting with previous findings for tropical savannahs and indicating that temperate and tropical non-forest ecosystems from South America respond differently to fire, possibly due to different evolutionary histories.
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Biodiversidad , Incendios , Pradera , Filogenia , Brasil , Plantas/clasificación , Plantas/genética , Poaceae/genética , Poaceae/clasificación , EcosistemaRESUMEN
BACKGROUND: The primary genetic risk factor for heritable pulmonary arterial hypertension is the presence of monoallelic mutations in the BMPR2 gene. The incomplete penetrance of BMPR2 mutations implies that additional triggers are necessary for pulmonary arterial hypertension occurrence. Pulmonary artery stenosis directly raises pulmonary artery pressure, and the redirection of blood flow to unobstructed arteries leads to endothelial dysfunction and vascular remodeling. We hypothesized that right pulmonary artery occlusion (RPAO) triggers pulmonary hypertension (PH) in rats with Bmpr2 mutations. METHODS AND RESULTS: Male and female rats with a 71 bp monoallelic deletion in exon 1 of Bmpr2 and their wild-type siblings underwent acute and chronic RPAO. They were subjected to full high-fidelity hemodynamic characterization. We also examined how chronic RPAO can mimic the pulmonary gene expression pattern associated with installed PH in unobstructed territories. RPAO induced precapillary PH in male and female rats, both acutely and chronically. Bmpr2 mutant and male rats manifested more severe PH compared with their counterparts. Although wild-type rats adapted to RPAO, Bmpr2 mutant rats experienced heightened mortality. RPAO induced a decline in cardiac contractility index, particularly pronounced in male Bmpr2 rats. Chronic RPAO resulted in elevated pulmonary IL-6 (interleukin-6) expression and decreased Gdf2 expression (corrected P value<0.05 and log2 fold change>1). In this context, male rats expressed higher pulmonary levels of endothelin-1 and IL-6 than females. CONCLUSIONS: Our novel 2-hit rat model presents a promising avenue to explore the adaptation of the right ventricle and pulmonary vasculature to PH, shedding light on pertinent sex- and gene-related effects.
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Receptores de Proteínas Morfogenéticas Óseas de Tipo II , Modelos Animales de Enfermedad , Hemodinámica , Mutación , Arteria Pulmonar , Animales , Receptores de Proteínas Morfogenéticas Óseas de Tipo II/genética , Receptores de Proteínas Morfogenéticas Óseas de Tipo II/metabolismo , Femenino , Masculino , Arteria Pulmonar/fisiopatología , Arteria Pulmonar/metabolismo , Hipertensión Pulmonar/fisiopatología , Hipertensión Pulmonar/genética , Hipertensión Pulmonar/etiología , Hipertensión Pulmonar/metabolismo , Ratas , Ratas Sprague-Dawley , Remodelación Vascular/genética , Hipertensión Arterial Pulmonar/fisiopatología , Hipertensión Arterial Pulmonar/genética , Hipertensión Arterial Pulmonar/metabolismo , Hipertensión Arterial Pulmonar/etiología , Estenosis de Arteria Pulmonar/genética , Estenosis de Arteria Pulmonar/fisiopatología , Estenosis de Arteria Pulmonar/metabolismo , Presión Arterial , Contracción Miocárdica/fisiologíaRESUMEN
BACKGROUND: Obstructive sleep apnea (OSA), due to its high prevalence, has been associated with a number of comorbidities, frequently impacting the overall course of these other diseases if left untreated. Recent studies highlight a potential association between OSA and cancer. This study investigates how OSA severity and hypoxia affect cancer prognosis, aiming to elucidate how they interplay. METHODS: Retrospective study including patients with a diagnosis of OSA after any cancer type followed up in a tertiary center during a 10-year period. OSA was mainly diagnosed after level III polysomnographic studies. RESULTS: Nocturnal hypoxia was significantly more prevalent in patients presenting lung cancer versus other malignancies and was associated with higher rates of oncologic disease progression. Overall survival was significantly lower in severe OSA patients and also in patients presenting nocturnal hypoxia. A composite hypoxia score considering both OSA severity and significant hypoxia was an independent predictor of mortality regardless of clinical cancer staging and treatment. Shorter time between cancer and OSA diagnosis was also associated with worse prognosis. CONCLUSION: This study suggests an association between OSA severity and nocturnal hypoxia and increased cancer mortality independently from possible confounding factors such as age, cancer clinical staging at diagnosis, treatment modality and also progression. Neoplastic patients with severe OSA and/or complex hypoxia seem to have lower overall survival rates than those with less severe OSA and nocturnal hypoxia.
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Hipoxia , Neoplasias , Polisomnografía , Apnea Obstructiva del Sueño , Humanos , Apnea Obstructiva del Sueño/mortalidad , Apnea Obstructiva del Sueño/complicaciones , Masculino , Femenino , Estudios Retrospectivos , Hipoxia/mortalidad , Hipoxia/complicaciones , Persona de Mediana Edad , Neoplasias/mortalidad , Neoplasias/complicaciones , Anciano , Pronóstico , Índice de Severidad de la Enfermedad , Comorbilidad , Prevalencia , Progresión de la EnfermedadRESUMEN
Neotropical fishes exhibit remarkable karyotype diversity, whose evolution is poorly understood. Here, we studied genetic differences in 60 individuals, from 11 localities of one species, the wolf fish Hoplias malabaricus, from populations that include six different "karyomorphs". These differ in Y-X chromosome differentiation, and, in several cases, by fusions with autosomes that have resulted in multiple sex chromosomes. Other differences are also observed in diploid chromosome numbers and morphologies. In an attempt to start understanding how this diversity was generated, we analyzed within- and between-population differences in a genome-wide sequence data set. We detect clear genotype differences between karyomorphs. Even in sympatry, samples with different karyomorphs differ more in sequence than samples from allopatric populations of the same karyomorph, suggesting that they represent populations that are to some degree reproductively isolated. However, sequence divergence between populations with different karyomorphs is remarkably low, suggesting that chromosome rearrangements may have evolved during a brief evolutionary time. We suggest that the karyotypic differences probably evolved in allopatry, in small populations that would have allowed rapid fixation of rearrangements, and that they became sympatric after their differentiation. Further studies are needed to test whether the karyotype differences contribute to reproductive isolation detected between some H. malabaricus karyomorphs.
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BACKGROUND: In-vivo diffusion tensor cardiovascular magnetic resonance (DT-CMR) is an emerging technique for microstructural tissue characterization in the myocardium. Most studies are performed at 3T, where higher signal-to-noise ratio (SNR) should benefit this signal-starved method. However, a few studies have suggested that DT-CMR is possible at 1.5T, where echo planar imaging artifacts may be less severe and 1.5T hardware is more widely available. METHODS: We recruited 20 healthy volunteers and performed mid-ventricular short-axis DT-CMR at 1.5T and 3T. Acquisitions were performed at peak systole and end-diastole using both stimulated echo acquisition mode (STEAM) and motion-compensated spin-echo (MCSE) sequences at matched spatial resolutions. DT-CMR parameters were averaged over the left ventricle and compared between 1.5T and 3T sequences using both datasets with and without the blow reference data included. RESULTS: Eleven (1.5T) and 12 (3T) diastolic MCSE acquisitions were rejected as the helix angle (HA) demonstrated <50% normal appearance circumferentially or the acquisition was abandoned due to poor image quality; a maximum of one acquisition was rejected for other datasets. Subjective HA map quality was significantly better at 3T than 1.5T for STEAM (p < 0.05), but not for MCSE and other DT-CMR quality measures were consistent with improvements in STEAM at 3T over 1.5T. When blow data were excluded, no significant differences in mean diffusivity were observed between field strengths, but fractional anisotropy was significantly higher at 1.5T than 3T for STEAM systole (p < 0.05). Absolute second eigenvector orientation (E2A, sheetlet angle) was significantly higher at 1.5T than 3T for MCSE systole and STEAM diastole, but significantly lower for STEAM systole (all p < 0.05). Transmural HA distribution was less steep at 1.5T than 3T for STEAM diastole data (p < 0.05). SNR was higher at 3T than 1.5T for all acquisitions (p < 0.05). CONCLUSION: While 3T provides benefits in terms of SNR, both STEAM and MCSE can be performed at 1.5T. However, MCSE is unreliable in diastole at both field strengths and STEAM benefits from the improved SNR at 3T over 1.5T. Future clinical research studies may be able to leverage the wider availability of 1.5T CMR hardware where MCSE acquisitions are desirable.
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BACKGROUND: Adverse Childhood Experiences (ACEs) have been associated with health-risk behaviors and several chronic diseases in adulthood. However, the relationship between exposure to ACEs and dietary patterns at school age is unknown. OBJECTIVES: To investigate the association between ACEs and dietary patterns of 10-year-olds. METHODS: The study included 5034 children from the Generation XXI cohort, recruited in 2005/2006 in Porto, Portugal. ACEs were assessed through a self-administered questionnaire covering the first 10 years (y) of life, quantified and grouped into 5 dimensions: "abuse," "school problems," "death/severe disease," "life changes," and "household dysfunction." Dietary patterns were identified by latent class analysis using data collected with a validated food frequency questionnaire. Five dietary patterns were studied: "low consumption," "energy-dense foods," "snacking," "intermediate consumption," and "healthier" (used as reference). Multinomial regression analyses were conducted, adjusted for the child's sex, household income, family structure, and mother's age [odds ratio (OR) and 99% confidence intervals (CIs)]. RESULTS: Most children were exposed to ≥1 ACE (96%), and â¼27% had reported 6 or more ACEs throughout life. Those reporting 4-5 and ≥6 ACEs were more likely to follow the "Energy-dense foods" dietary pattern compared with those with no ACEs (OR: 2.41; 99% CI: 1.00, 5.77 and OR: 2.65; 99% CI: 1.10, 6.39, respectively). Children exposed to "abuse" in the first 10 y showed 28% higher odds of following the "low consumption" dietary pattern when compared to children with no reported ACEs and using the "healthier" dietary pattern as a reference (OR: 1.28; 99% CI: 1.00, 1.63). CONCLUSIONS: Exposure to ACEs was associated with less healthy dietary patterns in school-aged children. Results suggest a cumulative effect of the adverse experiences resulting in a dietary pattern higher in energy-dense foods. Children with ACEs reported under the dimension of "abuse" seemed to have reduced food consumption.
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Experiencias Adversas de la Infancia , Patrones Dietéticos , Niño , Femenino , Humanos , Masculino , Experiencias Adversas de la Infancia/estadística & datos numéricos , Cohorte de Nacimiento , Estudios de Cohortes , Portugal , Encuestas y CuestionariosRESUMEN
Hypertension is a globally prevalent condition, and low adherence to antihypertensive therapy is considered one of the main causes of poor blood pressure (BP) control. Non-adherence to antihypertensive treatment is a complex issue that can arise from various factors; however, gaining an understanding of this provides key targets for intervention strategies. This study aimed to provide an overview of the current status and recent developments regarding our understanding of the determinants of patients' adherence to antihypertensives. A systematic review was performed using the electronic databases MEDLINE/PubMed, Web of Science, Scientific Electronic Library Online (SciELO), and "Índex das Revistas Médicas Portuguesas", which included studies published between 2017 and 2021 following the PICOS model: (P) Adult patients with the diagnosis of primary hypertension, using at least one antihypertensive agent; (I) all interventions on both pharmacological and non-pharmacological level; (C) patient's adherence against their non-adherence; (O) changes in adherence to the therapeutic plan; and (S) any study design (except review articles) written in English, French, Spanish or Portuguese. Articles were reviewed by two researchers and their quality was assessed. Subsequently, determinants were classified according to their consistent or inconsistent association with adherence or non-adherence. Only 45 of the 635 reports identified met the inclusion criteria. Adherence was consistently associated with patient satisfaction with communication, patient-provider relationship, their treatment, and use of eHealth and mHealth strategies; a patient's mental and physical health, including depression, cognitive impairment, frailty, and disability, previous hospitalization, occurrence of vital events; drug treatment type and appearance; and unwillingness due to health literacy, self-efficacy, and both implicit and explicit attitudes towards treatment. There were discrepancies regarding the association of other factors to adherence, but these inconsistent factors should also be taken into account. In conclusion, the barriers to adherence are varied and often interconnected between socioeconomic, patient, therapy, condition, and healthcare system levels. Healthcare teams should invest in studying patients' non-adherence motives and tailoring interventions to individual levels, by using a multifaceted approach to assess adherence. Further research is needed to analyze the impact of implicit attitudes, the use of new technological approaches, and the influence of factors that are inconsistently associated with non-adherence, to understand their potential in implementing adherence strategies.
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For dark matter to be detectable with gravitational waves from binary black holes, it must reach higher than average densities in their vicinity. In the case of light (wavelike) dark matter, the density of dark matter between the binary can be significantly enhanced by accretion from the surrounding environment. Here we show that the resulting dephasing effect on the last ten orbits of an equal mass binary is maximized when the Compton wavelength of the scalar particle is comparable to the orbital separation, 2π/µâ¼d. The phenomenology of the effect is different from the channels that are usually discussed, where dynamical friction (along the orbital path) and radiation of energy and angular momentum drive the dephasing, and is rather dominated by the radial force (the spacetime curvature in the radial direction) towards the overdensity between the black holes. While our numerical studies limit us to scales of the same order, this effect may persist at larger separations and/or particle masses, playing a significant role in the merger history of binaries.
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OBJECTIVES: to evaluate the morphological and functional characteristics of the peri-implant bone tissue that was formed during the healing process by the placement implants using two different surface treatments: hydrophilic Acqua™ (ACQ) and rough NeoPoros™ (NEO), in spontaneously hypertensive (SHR) and normotensive rats (Wistar) whether or not treated with losartan. METHODOLOGY: In total, 96 male rats (48 Wistar and 48 SHR) were divided into eight subgroups: absolute control rough (COA NEO), absolute control hydrophilic (COA ACQ), losartan control rough (COL NEO), losartan control hydrophilic (COL ACQ), SHR absolute rough (SHR NEO), SHR absolute hydrophilic (SHR ACQ), SHR losartan rough (SHRL NEO), and SHR losartan hydrophilic (SHRL ACQ). The rats medicated with losartan received daily doses of the medication. NeoPoros™ and Acqua™ implants were installed in the tibiae of the rats. After 14 and 42 days of the surgery, the fluorochromes calcein and alizarin were injected in the rats. The animals were euthanized 67 days after treatment. The collected samples were analyzed by immunohistochemistry, biomechanics, microcomputerized tomography, and laser confocal scanning microscopy analysis. RESULTS: The osteocalcin (OC) and vascular endothelium growth factor (VEGF) proteins had moderate expression in the SHRL ACQ subgroup. The same subgroup also had the highest implant removal torque. Regarding microarchitectural characteristics, a greater number of trabeculae was noted in the control animals that were treated with losartan. In the bone mineralization activity, it was observed that the Acqua™ surface triggered higher values of MAR (mineral apposition rate) in the COA, COL, and SHRL groups (p<0.05). CONCLUSION: the two implant surface types showed similar responses regarding the characteristics of the peri-implant bone tissue, even though the ACQ surface seems to improve the early stages of osseointegration.
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Implantes Dentales , Losartán , Ratas Endogámicas SHR , Ratas Wistar , Propiedades de Superficie , Microtomografía por Rayos X , Animales , Losartán/farmacología , Masculino , Propiedades de Superficie/efectos de los fármacos , Factores de Tiempo , Reproducibilidad de los Resultados , Inmunohistoquímica , Interacciones Hidrofóbicas e Hidrofílicas , Oseointegración/efectos de los fármacos , Resultado del Tratamiento , Implantación Dental Endoósea/métodos , Microscopía Confocal , Tibia/efectos de los fármacos , Tibia/cirugía , Análisis de Varianza , Fenómenos Biomecánicos , Valores de Referencia , Osteocalcina/análisisRESUMEN
Secreted phospholipase A2 (sPLA2) is a Ca2+-dependent, widely distributed enzyme superfamily in almost all mammalian tissues and bacteria. It is also a critical component of the venom of nearly all snakes, as well as many invertebrate species. In non-venomous contexts, sPLA2 assumes significance in cellular signaling pathways by binding cell membranes and catalyzing ester bond hydrolysis at the sn-2 position of phospholipids. Elevated levels of GIIA sPLA2 have been detected in the synovial fluid of arthritis patients, where it exhibits a pro-inflammatory function. Consequently, identifying sPLA2 inhibitors holds promise for creating highly effective pharmaceutical treatments. Beyond arthritis, the similarities among GIIA sPLA2s offer an opportunity for developing treatments against snakebite envenoming, the deadliest neglected tropical disease. Despite decades of study, the details of PLA2 membrane-binding, substrate-binding, and reaction mechanism remain elusive, demanding a comprehensive understanding of the sPLA2 catalytic mechanism. This study explores two reaction mechanism hypotheses, involving one or two water molecules, and distinct roles for the Ca2+ cofactor. Our research focuses on the human synovial sPLA2 enzyme bound to lipid bilayers of varying phospholipid compositions, and employing adiabatic QM/MM and QM/MM MD umbrella sampling methods to energetically and geometrically characterize the structures found along both reaction pathways. Our studies demonstrate the higher frequency of productive conformations within the single-water pathway, also revealing a lower free energy barrier for hydrolyzing POPC. Furthermore, we observe that the TS of this concerted one-step reaction closely resembles transition state geometries observed in X-ray crystallography complexes featuring high-affinity transition state analogue inhibitors.
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Adenoma , Neoplasias del Oído , Oído Medio , Humanos , Neoplasias del Oído/cirugía , Neoplasias del Oído/patología , Neoplasias del Oído/diagnóstico por imagen , Oído Medio/diagnóstico por imagen , Oído Medio/cirugía , Oído Medio/patología , Adenoma/cirugía , Adenoma/diagnóstico por imagen , Adenoma/patología , Endoscopía , Tumores Neuroendocrinos/cirugía , Tumores Neuroendocrinos/diagnóstico por imagen , Tumores Neuroendocrinos/patología , Femenino , Tomografía Computarizada por Rayos X , Masculino , Persona de Mediana EdadRESUMEN
This chapter describes how to test different amyloid preparations for catalytic properties. We describe how to express, purify, prepare and test two types of pathological amyloid (tau and α-synuclein) and two functional amyloid proteins, namely CsgA from Escherichia coli and FapC from Pseudomonas. We therefore preface the methods section with an introduction to these two examples of functional amyloid and their remarkable structural and kinetic properties and high physical stability, which renders them very attractive for a range of nanotechnological designs, both for structural, medical and catalytic purposes. The simplicity and high surface exposure of the CsgA amyloid is particularly useful for the introduction of new functional properties and we therefore provide a computational protocol to graft active sites from an enzyme of interest into the amyloid structure. We hope that the methods described will inspire other researchers to explore the remarkable opportunities provided by bacterial functional amyloid in biotechnology.
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Amiloide , Proteínas de Escherichia coli , Escherichia coli , Ingeniería de Proteínas , alfa-Sinucleína , Proteínas tau , Amiloide/química , Amiloide/metabolismo , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , alfa-Sinucleína/química , alfa-Sinucleína/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Ingeniería de Proteínas/métodos , Proteínas tau/metabolismo , Proteínas tau/química , Humanos , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Pseudomonas/metabolismo , Pseudomonas/química , Catálisis , Dominio CatalíticoRESUMEN
PURPOSE: This study aims to evaluate whether the clinical outcomes of cycles with frozen embryo transfer (FET) in hormonal replacement treatment supplemented with dydrogesterone (DYD) following detection of low circulating levels of progesterone (P4) were comparable to the results of cycles with otherwise normal serum P4 values. METHODS: Extended analyses of a retrospective cohort that included FET cycles performed between July 2019 and March 2022 after a cycle of artificial endometrial preparation using valerate-estradiol and micronized vaginal P4 (400 mg twice daily). Whenever the serum P4 value was considered low on the morning of the planned transfer, 10 mg of DYD three times a day was added as a supplement. Only single-embryo transfers of a blastocyst were considered. The primary endpoint was live birth rate. RESULTS: Five-hundred thirty-five FET cycles were analyzed, of which 136 (25.4%) underwent treatment with DYD. There were 337 pregnancies (63%), 207 live births (38.6%), and 130 miscarriages (38.5%). The P4 values could be modeled by a gamma distribution, with a mean of 14.5 ng/ml and a standard deviation of 1.95 ng/ml. The variables female age on the day of FET, ethnicity, and weight were associated with a variation in the serum P4 values. There were no differences in the results between cycles with or without the indication for DYD supplementation. CONCLUSIONS: Live birth rate did not vary significantly in females with low and normal serum P4 levels on the day of FET when DYD was used as rescue therapy.