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Mayaro fever, caused by Mayaro virus (MAYV) is a sub-lethal disease with symptoms that are easily confused with those of dengue fever, except for polyarthralgia, which may culminate in physical incapacitation. Recently, outbreaks of MAYV have been documented in metropolitan areas, and to date, there is no therapy or vaccine available. Moreover, there is no information regarding the three-dimensional structure of the viral proteins of MAYV, which is important in the search for antivirals. In this work, we constructed a three-dimensional model of protein C of MAYV by homology modelling, and this was employed in a manner similar to that of receptors in virtual screening studies to evaluate 590 molecules as prospective antiviral agents. In vitro bioassays were utilized to confirm the potential antiviral activity of the flavonoid epicatechin isolated from Salacia crassifolia (Celastraceae). The virtual screening showed that six flavonoids were promising ligands for protein C. The bioassays showed potent antiviral action of epicatechin, which protected the cells from almost all of the effects of viral infection. An effective concentration (EC50) of 0.247 µmol/mL was observed with a selectivity index (SI) of 7. The cytotoxicity assay showed that epicatechin has low toxicity, with a 50% cytotoxic concentration (CC50) greater than 1.723 µmol/mL. Epicatechin was found to be twice as potent as the reference antiviral ribavirin. Furthermore, a replication kinetics assay showed a strong inhibitory effect of epicatechin on MAYV growth, with a reduction of at least four logs in virus production. Our results indicate that epicatechin is a promising candidate for further testing as an antiviral agent against Mayaro virus and other alphaviruses.
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Alphavirus/química , Antígenos Virales/química , Antivirales/farmacología , Catequina/farmacología , Salacia/química , Proteínas Virales/química , Alphavirus/metabolismo , Animales , Antígenos Virales/metabolismo , Antivirales/química , Antivirales/aislamiento & purificación , Sitios de Unión , Catequina/química , Catequina/aislamiento & purificación , Chlorocebus aethiops , Ensayos Analíticos de Alto Rendimiento , Humanos , Concentración 50 Inhibidora , Simulación del Acoplamiento Molecular , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Ribavirina/química , Ribavirina/farmacología , Homología Estructural de Proteína , Interfaz Usuario-Computador , Células Vero , Proteínas Virales/antagonistas & inhibidores , Proteínas Virales/metabolismo , Replicación Viral/efectos de los fármacosRESUMEN
The strawberry (Fragaria x ananassa Dutch.), is the only vegetable belonging to the rosacea family. All strawberry species have now emerged from wild species and belong to the genus Fragaria, being that this genus presents more than 45 described species, and only 11 are considered natural species. Due to the octoploid nature of strawberry and its variability after hybridization, selecting one or more characters may result in unfavorable genotypes and even the exclusion of promising ones, because negative genetic correlations have been observed among them that cause inefficient selection. Therefore, the objective of this study was to verify the efficiency of selection indices in selecting experimental strawberry hybrids for in natura consumption and processing. Seven commercial cultivars and 103 hybrids were used, which were obtained from populations derived from their crossings. The experiment was conducted in augmented blocks, in which four agronomical traits (total mass, amount of commercial fruit, amount of noncommercial fruit, and average fruit mass) and seven physical-chemical traits (soluble solids, soluble solids:titratable acidity ratio, total sugars, total pectin, vigor, and internal and external coloration) were evaluated. For hybrid selection, the following indices were used: Mulamba and Mock (1978), Smith (1936), Hazel (1943), and genotype-ideotype, which selected 20% of the genotypes evaluated. The three indices selected about 9% of the hybrids. The selection of two experimental hybrids (89 and 495) and the use of selection indices resulted in larger estimates of selection gains. The Mulamba and Mock (1978), Smith (1936), and Hazel (1943) indices had the highest percentage of gains on selection, and are therefore recommended for the selection of strawberry clones.
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Fragaria/crecimiento & desarrollo , Fragaria/genética , Quimera , Frutas/genética , Genotipo , Fenotipo , Fitomejoramiento/métodos , Poliploidía , Selección ArtificialRESUMEN
The processing and combustion of coal in thermal power plants release anthropogenic chemicals into the environment. Baccharis trimera is a common plant used in folk medicine that grows readily in soils degraded by coal mining activities. This shrub bioaccumulates metals released into the environment, and thus its consumption may be harmful to health. The purpose of this study was to investigate the phytochemical profile, antioxidant capacity (DPPH), genotoxic (comet assay) and mutagenic potential (CBMN-cyt) in V79 cells of B. trimera aqueous extracts in the coal-mining region of Candiota (Bt-AEC), and in Bagé, a city that does not experience the effects of exposure to coal (Bt-AEB, a reference site). In the comet assay, only Bt-AEC was genotoxic at the highest doses (0.8mg/mL and 1.6mg/mL), compared to the control. For extracts from both areas, mutagenic effects were observed at higher concentrations compared to the control. The cell damage parameters were significantly high in both extracts; however, more striking values were observed for Bt-AEC, up to the dose of 0.8mg/mL. In chemical analysis, no variation was observed in the contents of flavonoids and phenolic compounds, neither the antioxidant activity, which may suggest that DNA damage observed in V79 cells was induced by the presence of coal contaminants absorbed by the plant.
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Antioxidantes/farmacología , Baccharis , Carbón Mineral/toxicidad , Daño del ADN , Mutágenos/toxicidad , Extractos Vegetales/toxicidad , Animales , Antioxidantes/aislamiento & purificación , Baccharis/química , Baccharis/crecimiento & desarrollo , Línea Celular , Supervivencia Celular/efectos de los fármacos , Minas de Carbón , Ensayo Cometa , Cricetinae , Cricetulus , Flavonoides/análisis , Metales , Mutágenos/aislamiento & purificación , Fenoles/análisis , Extractos Vegetales/aislamiento & purificación , Centrales EléctricasRESUMEN
Single nucleotide polymorphisms (SNPs) were screened in FABP3 and FABP4 by automatic sequencing of pools of DNA from crossbred animals whose phenotypes belonged to the upper and lower extremes for back fat and marbling, as well as of a pool of DNA from sires used for crossbreeding. Five SNPs were identified in FABP3 and another nine SNPs were identified in FAPB4. Of these, only one SNP had no previous registry in the SNAP database (dbSNP). Three polymorphisms were selected for further evaluation of their association with production traits using restriction fragment length polymorphism-PCR (RFLP-PCR) or real-time PCR genotyping. All 3 markers were in Hardy-Weinberg equilibrium at the 5% significance level for all 7 genetic groups analyzed. Significant association was observed between FABP3-G/A with rib eye area (P = 0.035) and the rib eye area/hot carcass weight ratio (P = 0.025) and between FABP4/TasI with marbling (P = 0.052) and meat texture (P = 0.053). No significant association was observed between the FABP4-G/C polymorphism and any of the observed traits. Previous association studies with allelic variants in these genes have shown mixed results, probably because of the small effect of the genes for these traits, which suggests that results should be replicated in other populations.
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Proteínas de Unión a Ácidos Grasos/genética , Polimorfismo de Nucleótido Simple , Alelos , Animales , Bovinos , Femenino , Frecuencia de los Genes , Estudios de Asociación Genética , Genotipo , Masculino , Fenotipo , Carácter Cuantitativo Heredable , Carne Roja/normasRESUMEN
A self-consistent analysis of proton and deuterium nuclear spin relaxation times in the smectic phases of a partially deuterated smectogen is presented here. Proton spin-lattice relaxation times T(1Z) were measured as a function of Larmor frequency over a range of 1 kHz to 300 MHz at selected temperatures. Deuterium spin relaxation times T(1Z) and T(1Q) were measured as a function of temperature at two different magnetic fields in the smectic A phase. The deuterium data provide dynamic parameters such as rotational diffusion constants and internal jump rates as well as the nematic order parameter S. The proton data are analyzed using a number of relaxation mechanisms, one of which is the molecular reorientation. It is found helpful in these latter analyses to use the nematic order parameter and to fix the contribution from molecular reorientations determined by the deuterium spin relaxation. The fits to the proton T(1) frequency and temperature dispersions by the remaining relaxation mechanisms such as layer undulations and translational self-diffusion will be discussed for the smectic A and chiral smectic C phases.
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Biometric evaluations are essential to determine the growth characteristics related to the weight and length of fish. This study aimed to determine the growth patterns of juvenile piraputangas (Brycon hilarii) produced in hapas within an excavated pond. The piraputangas were anesthetized and micro-chipped and their biometric characteristics were measured. Subsequently, the fish were distributed in six hapas of eight m3 at the density of 20 fish/hapa, totaling 120 animals. During the experimental period six months, the fish were fed twice (5% of the biomass) a day. Every 30 days, all fish were sampled to measure the biometric characteristics of body weight (g); standard length; total length; head height; head length; body height and body width (cm). The calculations of the weight ratio with the biometric characteristics were determined using allometric equation and estimated by linear regression according to the equation log Y = log a + b log X. All tested relationships were significant by the Student t-test (p < 0.05). Allometric growth was positive for: weight x total length; standard weight x length; weight x head height; weight x head length and weight x body height. The relative condition factor of piraputangas observed in this study was 1.00. The study provided information on the allometric parameters of juvenile Brycon hilarii produced in hapas, and the relative condition factor indicated good growth conditions for piraputangas with positive allometric growth.
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Characiformes , Animales , Biomasa , EstanquesRESUMEN
Using a first-principles theoretical model the adsorption of a methyl radical on different sized silver nanoparticles is compared to the adsorption of the same radical on model surfaces. Calculations of our structural, dynamical and electronic properties indicated that small changes in the local environment will lead to small changes in infrared (IR) wavenumbers, but in dramatic changes in the IR signal. Our calculations indicate the lower the adsorption site coordination, the higher is the signal strength, suggesting that small changes in the electronic charge distribution will result in bigger changes in the polarizability and hence in the spectroscopic signal intensity. This effect explains, among others, the signal magnification observed for nanoparticles in surface enhanced Raman spectroscopic (SERS) experiments.
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INTRODUCTION: Hip replacement is one of the most successful operations in orthopaedic surgery. Periprosthetic hip fractures (PPHF) have very serious consequences for the patient, and they also entail a very important economic impact on healthcare systems. The aim of the study was to provide the first detailed cost analysis of PPHF in a traumatology and orthopaedics service in a third level hospital in Spain. METHODS: The study included all patients admitted between 2009 and 2019 with a diagnosis of "PPHF". We assessed hospital stay cost, total cost of the operating theatre, cost of the implants used, analyses, consultations with other specialists, rehabilitation sessions, radiological tests, microbiology, blood transfusions and other surgical operations during the same admission. RESULTS: 78 patients were included in the study, 49 women and 29 men, with a mean age of 78.74 years (R 45-92). 69 patients received surgical treatment, 75% had open reduction and internal fixation (ORIF), and 25% had revision surgery. The total cost was 1,139,650.17. The average cost was 14,610.90. Significantly higher costs were incurred for revision compared to ORIF treatments, admissions that lasted more than 30 days, and patients who required more than one operation during admission. The most costly factors were the hospital stay (46%), the cost of the surgery itself (35%), and the implants (24%). CONCLUSIONS: Revision arthroplasty versus ORIF treatment, admissions lasting more than 30 days, and patients requiring more than one operation on admission incurred significantly higher costs. The average cost, from a hospital perspective, generated by a PPHF was 14,610.90. The most costly factors were, in descending order, the hospital stay, the cost of the surgery itself, and the implants. It is necessary to establish protocols and updated therapeutic algorithms in the perioperative management of PPHF in order to reduce both morbidity rates and associated costs.
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INTRODUCTION: Hip replacement is one of the most successful operations in orthopaedic surgery. Periprosthetic hip fractures (PPHF) have very serious consequences for the patient, and they also entail a very important economic impact on healthcare systems. The aim of the study was to provide the first detailed cost analysis of PPHF in a traumatology and orthopaedics service in a third level hospital in Spain. METHODS: The study included all patients admitted between 2009 and 2019 with a diagnosis of «PPHF¼. We assessed hospital stay cost, total cost of the operating theatre, cost of the implants used, analyses, consultations with other specialists, rehabilitation sessions, radiological tests, microbiology, blood transfusions and other surgical operations during the same admission. RESULTS: Seventy-eight patients were included in the study, 49 women and 29 men, with a mean age of 78.74 years (R 45-92). Sixty-nine patients received surgical treatment, 75% had open reduction and internal fixation (ORIF), and 25% had revision surgery. The total cost was 1 139 650.17. The average cost was 14 610.90. Significantly higher costs were incurred for revision compared to ORIF treatments, admissions that lasted more than 30 days, and patients who required more than one operation during admission. The most costly factors were the hospital stay (46%), the cost of the surgery itself (35%), and the implants (24%). CONCLUSIONS: Revision arthroplasty versus ORIF treatment, admissions lasting more than 30 days, and patients requiring more than one operation on admission incurred significantly higher costs. The average cost, from a hospital perspective, generated by a PPHF was 14 610.90. The most costly factors were, in descending order, the hospital stay, the cost of the surgery itself, and the implants. It is necessary to establish protocols and updated therapeutic algorithms in the perioperative management of PPHF in order to reduce both morbidity rates and associated costs.
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We argue that near a Kondo breakdown critical point, a spin liquid with spatial modulations can form. Unlike its uniform counterpart, we find that this occurs via a second order phase transition. The amount of entropy quenched when ordering is of the same magnitude as for an antiferromagnet. Moreover, the two states are competitive, and at low temperatures are separated by a first order phase transition. The modulated spin liquid we find breaks Z4 symmetry, as recently seen in the hidden order phase of URu2Si2. Based on this, we suggest that the modulated spin liquid is a viable candidate for this unique phase of matter.
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In this work a systematic study of the dependence of the structural, electronic, and vibrational properties on nanoparticle size is performed. Based on our total energy calculations we identified three characteristic regimes associated with the nanoparticle's dimensions: (i) below 1.5 nm (100 atoms) where remarkable molecular aspects are observed; (ii) between 1.5 and 2.0 nm (100 and 300 atoms) where the molecular behavior is influenced by the inner core crystal properties; and (iii) above 2.0 nm (more than 300 atoms) where the crystal properties are preponderant. In all considered regimes the nanoparticle's surface modulates its properties. This modulation decreases with the increasing of the nanoparticle's size.
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In this work, a 2.0 nm nanoparticle (low limit synthesized system) is compared to possible simplified models: passivated clusters, small (1.3 nm) nanoparticles and sets of plane surfaces. Our density functional theory results suggest that even when geometric aspects are properly described by the simplifications considered, electronic properties might be very different, especially when edge atoms are not properly taken into account in the nanoparticle's modeling. In addition, we propose a protocol that might help future theoretical descriptions of nanoparticles.
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An upflow anaerobic sludge bed (UASB)-submerged aerated biofilter (SAB) system that treats effluents from a jeans factory was evaluated. The 210-day operational period was divided into three phases (PI, PII and PIII), each with a different hydraulic retention time (HRT in h) and organic loading rate (OLR in kg COD/m3.d). In PI, the best performance was achieved using the UASB (HRT 24, OLR 1.3) with COD and color removal efficiencies of 59 and 64%, respectively; the corresponding values were 77 and 86% for the final effluent. In PII, the efficiencies were 50 and 55% using the UASB (HRT 16, OLR 1.2), respectively, and 69 and 81% for the final system effluent, respectively. In PIII, the UASB (HRT 12 and ORL 3.2) showed the poorest performance; the efficiencies decreased to 48 and 50%, respectively. The same phenomenon occurred in the system with corresponding efficiencies decreasing to 69 and 61%. Throughout the experiment, the system removal efficiencies were between 57 and 88% for nitrogen and between 14 and 63% for sulfate. The final effluent showed relatively non-toxicity or moderate toxicity using Daphnia magna as an indicator. Therefore, the overall results showed that the use of a sequential anaerobic-aerobic system is promising for treatment of textile industrial wastewater.
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Reactores Biológicos , Filtración/métodos , Residuos Industriales/análisis , Industria Textil , Eliminación de Residuos Líquidos/métodos , Colorantes/química , Ácidos Grasos Volátiles/química , Filtración/instrumentación , Concentración de Iones de Hidrógeno , Nitrógeno/química , Oxígeno , Azufre , Contaminantes Químicos del Agua/químicaRESUMEN
This study investigated the use of melatonin to arrest the effects of apoptosis in vitrified zebrafish (D. rerio) embryos. Dechorionated embryos at 22-24 somite-stage were divided (n = 60/treatment) into a non-vitrified (Control Group, 0 M melatonin) and vitrified treatments with 0 M (T1), 1 µM (T2) and 1 mM of melatonin (T3). For vitrified treatments, a solution methanol/propylene glycol based was used and the embryos stored in -196 °C for a week. After thaw, survival rate, scanning electron microscopy, expression of anti (bcl-2) and pro-apoptotic (bax/caspase-3) genes, reactive oxygen species (ROS) formation and DNA fragmentation analyses were performed. No live embryos were obtained from vitrified treatments, observing a rapid degeneration immediately after thawing, with the vitelline layer rupture and leakage of its content, followed by breakdown of epithelial cells and melanisation of the tissue. Regarding the apoptotic process, T3 had the highest relative gene expression, for the three genes (P < 0.05) furthermore, T2 had similar expression of pro-apoptotic genes to CG (P < 0.05). ROS formation revealed that CG presented lower percentage of embryo surface area affected (3.80 ± 0.40%) (P < 0.05), in contrast, no differences were found among the other groups. T1 was most significantly (P < 0.05) damaged by DNA fragmentation. The vitrified groups with melatonin had similar damage levels of CG (P > 0.05). The inclusion of 1 µM of melatonin in the vitrifying solution, countered the effects of apoptotic process in post-thaw embryos, suggesting its utility in cryopreserving fish embryos.
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Melatonina , Pez Cebra , Animales , Apoptosis , Criopreservación , Melatonina/farmacologíaRESUMEN
PURPOSE: The PAINESD risk score was developed in 2015 as a tool to stratify the risk of acute hemodynamic decompensation during ventricular tachycardia (VT) ablation in structural heart disease patients and further then used for post procedure 30-day mortality prediction. The original cohort however did not include Chagas disease (ChD) patients. We aim to evaluate the relevance of the score in a ChD population. METHODS: The PAINESD risk score gives weighted values for specific characteristics (chronic obstructive pulmonary disease, age > 60 years, ischemic cardiomyopathy, New York Heart Association [NYHA] functional class 3 or 4, ejection fraction less than 25%, VT storm, and diabetes). The score was applied in a retrospective cohort of ChD VT ablations in a single tertiary center in Brazil. Data were collected by VT study reports and patient record analysis at baseline and on follow-up. RESULTS: Between January 2013 and December 2018, 157 VT catheter ablation procedures in 121 ChD patients were analyzed. Overall, 30-day mortality was 9.0%. Multivariate analysis correlated NYHA functional class (HR 1.78, 95% CI 1.03-3.08, P 0.038) and the need for urgent surgery (HR 31.5, 95% CI 5.38-184.98, P < 0.001), as well as a tendency for VT storm at presentation (HR 2.72, 95% CI 0.87-8.50, P 0.084) as risk factors for the primary endpoint. The median PAINESD risk score in this population was 3 (3-8). The area under the receiver operating characteristic (ROC) curve was 0.64 (95% CI 0.479-0.814). CONCLUSIONS: The PAINESD risk score did not perform well in predicting 30-day mortality in ChD patients. Pre-procedure NYHA functional class and the need for urgent surgery due to refractory pericardial bleeding were independently associated with increased 30-day mortality. Prospective studies are needed to take final conclusions in Chagas disease when using PAINESD score.
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Ablación por Catéter , Enfermedad de Chagas , Taquicardia Ventricular , Humanos , Persona de Mediana Edad , Estudios Retrospectivos , Factores de Riesgo , Taquicardia Ventricular/cirugía , Resultado del TratamientoRESUMEN
We report a rare case of myiasis caused simultaneously by three dipterous species. A 54 yr-old indigent patient was admitted to Andaraí Hospital with painful eruptions on the scalp. The parieto-occipital sulcus showed two lesions caused by scratching associated with deep, odoriferous and exudative pediculosis. Larvae removed with the help of forceps and vaseline produced 153 adults, identified in the laboratory as 114 specimens of Chrysomya megacephala (F., 1794), 38 of Sarcophaga (Liopygia) ruficornis (F., 1794), and one of Musca domestica (L., 1758).
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Dípteros/patogenicidad , Exudados y Transudados/metabolismo , Moscas Domésticas/patogenicidad , Miasis/parasitología , Cuero Cabelludo/parasitología , Animales , Concienciación , Exudados y Transudados/parasitología , Humanos , Higiene , Larva/crecimiento & desarrollo , Masculino , Persona de Mediana EdadRESUMEN
In this work we employ the state of the art pseudopotential method, within a generalized gradient approximation to the density functional theory, to investigate the adsorption process of furan on the silicon (001) surface. A direct comparison of different adsorption structures with x-ray photoelectron spectroscopy (XPS), ultra-violet photoelectron spectroscopy (UPS), high resolution electron energy loss spectroscopy (HREELS), near edge x-ray absorption fine structure (NEXAFS), and high resolution spectroscopy experimental data allows us to identify the [4+2 ] cycloaddition reaction as the most probable adsorbate. In addition, theoretical scanning tunnelling microscopy (STM) images are presented, with a view to contributing to further experimental investigations.
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Within the recently proposed doped-carrier representation of the projected lattice electron operators we derive a full Ising version of the t-J model. This model possesses the global discrete Z(2) symmetry as a maximal spin symmetry of the Hamiltonian at any values of the coupling constants, t and J. In contrast, in the spin anisotropic limit of the t-J model, usually referred to as the t-J(z) model, the global SU(2) invariance is fully restored at J(z) = 0, so that only the spin-spin interaction has in this model the true Ising form. We discuss a relationship between these two models and the standard isotropic t-J model. We show that the low-energy quasiparticles in all three models share qualitatively similar properties at low doping and small values of J/t. The main advantage of the proposed Ising t-J model over the t-J(z) one is that the former allows for the unbiased Monte Carlo calculations on large clusters of up to 10(3) sites. Within this model we discuss in detail the destruction of the antiferromagnetic (AF) order by doping as well as the interplay between the AF order and hole mobility. We also discuss the effect of the exchange interaction and that of the next-nearest-neighbour hoppings on the destruction of the AF order at finite doping. We show that the short-range AF order is observed in a wide range of temperatures and dopings, much beyond the boundaries of the AF phase. We explicitly demonstrate that the local no-double-occupancy constraint plays the dominant role in destroying the magnetic order at finite doping. Finally, a role of inhomogeneities is discussed.
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The aim of this study was to evaluate transcriptome changes in the muscle tissue of Bos taurus indicus cull cows subjected to recovery weight gain under grazing conditions. In all, 38 Nellore cull cows were divided randomly into two different management groups: (1) Maintenance (MA) and (2) Recovery gain (RG) from weight loss by moderate growth under high forage availability. After slaughter, RNA analysis was performed on the Longissimus thoracis muscle. Semaphorin 4A, solute carrier family 11 member 1, and Ficolin-2 were expressed in the RG, which may indicate an inflammatory response during tissue regrowth. Signaling factors, such as Myostatin, related to fibroblast activation, negative control of satellite cell proliferation in adults and muscle protein synthesis were less abundant in the RG group. The only gene related to anabolic processes that were more abundant in the MA group was related to fat deposition. The genes that were differentially expressed in the experiment showed muscle repair-related changes during RG based on the greater expression of genes involved in inflammatory responses and the lower expression of negative regulators of muscle cell proliferation and hypertrophy.
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Bovinos/genética , Matriz Extracelular/genética , Expresión Génica , Músculo Esquelético/metabolismo , Transcriptoma/fisiología , Aumento de Peso , Animales , Bovinos/fisiología , Dieta , Femenino , Distribución AleatoriaRESUMEN
The purpose of this work was to quantify glucose in aqueous solutions containing chitosan by high-performance liquid chromatography (HPLC) with evaporative light scattering detection (ELSD). Chitosan is a natural compound that is used alone or as an additive in several formulations. Microencapsulation of bioactive compounds such as glucose, by means of chitosan, is being explored, but difficulties arise when glucose needs to be determined in the presence of chitosan. HPLC is the technique most commonly used for glucose analysis, and ELSD may offer advantages (e.g. sensitivity and the possibility of operating in gradient mode) compared with other detectors. The influence of chitosan in the analysis of glucose by HPLC with ELSD was investigated at different pH values of the aqueous solutions. Isocratic elution with an acetonitrile/water mixture (80:20, v/v) and water washing between runs were the best options to avoid the mucoadhesive properties of chitosan, which are responsible for column degradation and variability of the retention time of glucose. The developed methodology was considered completely adequate for rapid glucose analysis in aqueous solutions with low pH (< 3), in the presence of chitosan.