RESUMO
Pseudomonas aeruginosa is one of the most important nosocomial pathogens that possess the ability to produce multiple antibiotic resistance and virulence factors. Elastase B (LasB) is the major factor implicated in tissue invasion and damage during P. aeruginosa infections, whose synthesis is regulated by the quorum sensing (QS) system. Anti-virulence approach is now considered as potential therapeutic alternative and/or adjuvant to current antibiotics' failure. The aim of this study is primarily to find out the impact of the efflux pump inhibitor (EPI) phenylalanine arginyl ß-naphthylamide (PAßN) on the production of elastase B and the gene expression of lasI quorum sensing and lasB virulence factor in clinical isolates of P. aeruginosa. Five P. aeruginosa isolates recovered from patients with respiratory tract infections were examined in this study. Antimicrobial susceptibility of isolates was performed by the disk agar diffusion method. Effect of the PAßN on imipenem susceptibility, bacterial viability, and elastase production was evaluated. The expression of lasB and lasI genes was measured by quantitative real-time PCR in the presence of PAßN. All isolates were identified as multidrug-resistant (MDR) and showed resistance to carbapenem (MIC = 64-256 µg/mL). Susceptibility of isolates to imipenem was highly increased in the presence of efflux inhibitor. PAßN significantly reduced elastase activity in three isolates tested without affecting bacterial growth. In addition, the relative expression of both lasB and lasI genes was diminished in all isolates in the presence of inhibitor. Efflux inhibition by using the EPI PAßN could be a potential target for controlling the P. aeruginosa virulence and pathogenesis. Furthermore, impairment of drug efflux by PAßN indicates its capability to be used as antimicrobial adjuvant that can decrease the resistance and lower the effective doses of current drugs.
Assuntos
Antibacterianos , Proteínas de Bactérias , Dipeptídeos , Imipenem , Testes de Sensibilidade Microbiana , Elastase Pancreática , Infecções por Pseudomonas , Pseudomonas aeruginosa , Percepção de Quorum , Fatores de Virulência , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/isolamento & purificação , Percepção de Quorum/efeitos dos fármacos , Fatores de Virulência/genética , Fatores de Virulência/metabolismo , Elastase Pancreática/genética , Elastase Pancreática/metabolismo , Antibacterianos/farmacologia , Humanos , Infecções por Pseudomonas/microbiologia , Dipeptídeos/farmacologia , Imipenem/farmacologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Farmacorresistência Bacteriana , MetaloendopeptidasesRESUMO
The majority of the world's natural rubber comes from the rubber tree (Hevea brasiliensis). As a key enzyme for synthesizing phenylpropanoid compounds, phenylalanine ammonia-lyase (PAL) has a critical role in plant satisfactory growth and environmental adaptation. To clarify the characteristics of rubber tree PAL family genes, a genome-wide characterization of rubber tree PALs was conducted in this study. Eight PAL genes (HbPAL1-HbPAL8), which spread over chromosomes 3, 7, 8, 10, 12, 13, 14, 16, and 18, were found to be present in the genome of H. brasiliensis. Phylogenetic analysis classified HbPALs into groups I and II, and the group I HbPALs (HbPAL1-HbPAL6) displayed similar conserved motif compositions and gene architectures. Tissue expression patterns of HbPALs quantified by quantitative real-time PCR (qPCR) proved that distinct HbPALs exhibited varying tissue expression patterns. The HbPAL promoters contained a plethora of cis-acting elements that responded to hormones and stress, and the qPCR analysis demonstrated that abiotic stressors like cold, drought, salt, and H2O2-induced oxidative stress, as well as hormones like salicylic acid, abscisic acid, ethylene, and methyl jasmonate, controlled the expression of HbPALs. The majority of HbPALs were also regulated by powdery mildew, anthracnose, and Corynespora leaf fall disease infection. In addition, HbPAL1, HbPAL4, and HbPAL7 were significantly up-regulated in the bark of tapping panel dryness rubber trees relative to that of healthy trees. Our results provide a thorough comprehension of the characteristics of HbPAL genes and set the groundwork for further investigation of the biological functions of HbPALs in rubber trees.
Assuntos
Regulação da Expressão Gênica de Plantas , Hevea , Família Multigênica , Fenilalanina Amônia-Liase , Proteínas de Plantas , Perfilação da Expressão Gênica , Genoma de Planta , Hevea/genética , Hevea/enzimologia , Hevea/metabolismo , Fenilalanina Amônia-Liase/genética , Fenilalanina Amônia-Liase/metabolismo , Filogenia , Doenças das Plantas/genética , Doenças das Plantas/microbiologia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Regiões Promotoras Genéticas/genética , Estresse Fisiológico/genéticaRESUMO
The interaction of L-Phe with the membrane components, i.e., lipids and proteins, has been discussed in the current literature due to the interest to understand the effect of single amino acids in relation to the formation of amyloid aggregates. In the present work, it is shown that L-Phe interacts with 9:1 DMPC (1,2-dimyristoyl-sn-glycero-3 phosphocholine)/DPPC (1,2-dipalmitoyl-sn-glycero-3 phosphocholine) mixtures but not in the 1:9 one. An important observation is that the interaction disappears when DPPC is replaced by diether PC (2-di-O-hexadecyl-sn-glycero-3-phosphocholine) a lipid lacking carbonyl groups (CO). This denotes that CO groups may interact specifically with L-Phe in accordance with the appearance of a new peak observed by Infrared spectroscopy (FTIR-ATR). The interaction of L-Phe affects the compressibility pattern of the 9:1 DMPC/DPPC mixture which is congruent with the changes observed by Raman spectra. The specific interaction of L-Phe with CO, propagates to phosphate and choline groups in this particular mixture as analyzed by FTIR-ATR spectroscopy and is absent when DMPC is dopped with diether PC.
Assuntos
Dimiristoilfosfatidilcolina , Fenilalanina , Fenilalanina/química , Fenilalanina/metabolismo , Dimiristoilfosfatidilcolina/química , Espectroscopia de Infravermelho com Transformada de Fourier , Bicamadas Lipídicas/química , Bicamadas Lipídicas/metabolismo , 1,2-Dipalmitoilfosfatidilcolina/química , 1,2-Dipalmitoilfosfatidilcolina/metabolismo , Lipídeos de Membrana/química , Lipídeos de Membrana/metabolismoRESUMO
Status epilepticus (SE) is described as continuous and self-sustaining seizures, which triggers hippocampal neurodegeneration, inflammation, and gliosis. N-formyl peptide receptor (FPR) has been associated with inflammatory process. N-formyl-methionyl-leucyl-phenylalanine (fMLP) peptide plays an anti-inflammatory role, mediated by the activation of G-protein-coupled FPR. Here, we evaluated the influence of fMLP peptides on the behavior of limbic seizures, memory consolidation, and hippocampal neurodegeneration process. Male Wistar rats (Rattus norvegicus) received microinjections of pilocarpine in hippocampus (H-PILO, 1.2 mg/µL, 1 µL) followed by fMLP (1 mg/mL, 1 µL) or vehicle (VEH, saline 0.9%, 1 µL). During the 90 min of SE, epileptic seizures were analyzed according to the Racine's Scale. After 24 h of SE, memory impairment was assessed by the inhibitory avoidance test and the neurodegeneration process was evaluated in hippocampal areas. There was no change in latency and number of wet dog shake (WDS) after administration of fMLP. However, our results showed that the intrahippocampal infusion of fMLP reduced the severity of seizures, as well as the number of limbic seizures. In addition, fMLP infusion protected memory dysfunction followed by SE. Finally, the intrahippocampal administration of fMLP attenuated the process of neurodegeneration in both hippocampi. Taken together, our data suggest a new insight into the functional role of fMLP peptides, with important implications for their potential use as a therapeutic agent for the treatment of brain disorders, such as epilepsy. Schematic drawing on the neuroprotective and anticonvulsant role of fMLP during status epilepticus. Initially, a cannula was implanted in hippocampus and pilocarpine/saline was administered into the hippocampus followed by fMLP/saline (A-C). fMLP reduced seizure severity and neuronal death in the hippocampus, as well as protecting against memory deficit (D).
Assuntos
Epilepsia , Estado Epiléptico , Ratos , Masculino , Animais , Anticonvulsivantes/farmacologia , Anticonvulsivantes/uso terapêutico , N-Formilmetionina Leucil-Fenilalanina/farmacologia , N-Formilmetionina Leucil-Fenilalanina/uso terapêutico , Pilocarpina/uso terapêutico , Ratos Wistar , Estado Epiléptico/tratamento farmacológico , Estado Epiléptico/complicações , Convulsões/tratamento farmacológico , Epilepsia/tratamento farmacológico , Peptídeos/uso terapêuticoRESUMO
BACKGROUND: Measuring resting energy expenditure (REE) in individuals living with phenylketonuria (PKU) using indirect calorimetry (IC) is unusual in healthcare facilities because it requires specific protocols and expensive equipment. Considering that determining REE is crucial for devising nutritional strategies for the management of PKU, the aim of this study was to identify the predictive equations that provide the best estimates of REE in children and adolescents living with PKU and to propose a predictive equation for determining REE in this population. METHODS: An REE concordance study was conducted with children and adolescents living with PKU. Anthropometric and body composition assessments using bioimpedance and REE assessment using IC were performed. The results were compared to 29 predictive equations. RESULTS: Fifty-four children and adolescents were evaluated. The REE obtained using IC differed from all estimated REE, except Henry's equation for male children (p = 0.058). Only this equation showed good agreement (0.900) with IC. Eight variables were associated with the REE obtained using IC with emphasis on fat-free mass (kg) (r = 0.786), weight (r = 0.775), height (r = 0.759) and blood phenylalanine (r = 0.503). With these variables, three REE equations were suggested, with R2 = 0.660, 0.635 and 0.618, respectively, and the third equation, which involves weight and height, showed adequate sample size for a statistical power of 0.942. CONCLUSION: Most equations, not specific for individuals living with PKU, overestimate the REE of this population. We propose a predictive equation for assessing REE for children and adolescents living with PKU to be used in settings where IC is not available.
Assuntos
Metabolismo Basal , Fenilcetonúrias , Humanos , Masculino , Adolescente , Criança , Reprodutibilidade dos Testes , Metabolismo Energético , Índice de Massa Corporal , Calorimetria Indireta/métodos , Valor Preditivo dos TestesRESUMO
There are concerns about muscle and bone health in patients with Phenylketonuria (PKU). Our aim was to compare muscle mass, function, and bone health among young adults with PKU who maintained or suspended dietary treatment. METHODS: Three groups were considered-PKU-1: 10 patients who used a protein substitute (PS) without phenylalanine (Phe); PKU-2: 14 patients who used the PS without Phe until eighteen years old and then practiced mostly a vegan diet; and 24 matched healthy controls. A 24 h recall survey, blood parameters, body composition and bone mineral density through DEXA, rectus femoris thickness by ultrasound, hand grip strength, submaximal exercise test, and walking speed were assessed. RESULTS: PKU-1 patients had lower hand grip strength than their matched controls, but no other differences. Compared to controls, the PKU-2 group had lower fat-free mass (p = 0.01), less spine and femoral bone mineral density (p = 0.04 and p < 0.01, respectively), and peak workload on the incremental test (p = 0.03). When comparing PKU groups, blood Phe levels were significantly lower in the PKU-1 group (p = 0.02). CONCLUSIONS: Among PKU patients, abandoning the dietary treatment and maintaining high blood Phe concentrations could be deleterious for muscles and bones. However, we cannot discard other causes of bone and muscle damage in these patients.
Assuntos
Fenilalanina , Fenilcetonúrias , Adulto Jovem , Humanos , Adolescente , Densidade Óssea , Chile , Força da Mão , Dieta , Músculos/metabolismoRESUMO
OBJECTIVE: To evaluate effects of sapropterin dihydrochloride on blood phenylalanine (Phe) and symptoms of neuropsychiatric impairment in children and adolescents with phenylketonuria (PKU). STUDY DESIGN: PKU subjects 8-17 years of age (n = 86) were randomized to double-blind treatment with sapropterin (n = 43) or placebo (n = 43) for 13 weeks, then all received open-label sapropterin therapy for an additional 13 weeks. Blood Phe and symptoms of inattention, hyperactivity/impulsivity (Attention-Deficit/Hyperactivity Disorder Rating Scale IV [ADHD RS-IV]), executive functioning (Behavior Rating Inventory of Executive Function), depression (Hamilton Rating Scale for Depression), and anxiety (Hamilton Rating Scale for Anxiety) were assessed. RESULTS: Following the 13-week randomization phase, the sapropterin and placebo groups had mean changes in blood Phe of -20.9% and +2.9%, respectively. Corresponding least square mean differences in ADHD RS-IV scores were significantly greater for the sapropterin vs the placebo group: Total (-3.2 points, P = .02), Inattention subscale (-1.8 points, P = .04), and Hyperactivity/Impulsivity subscale (-1.6 points, P = .02). Forest plots favored sapropterin treatment over placebo for all ADHD RS-IV and Behavior Rating Inventory of Executive Function indices. There were no significant differences in reported problems with attention or executive function between the 2 groups at baseline or at week 26 following the 13-week open-label treatment period. Anxiety and depression scores did not differ significantly between cohorts at any time. Sapropterin was well tolerated, with a favorable safety profile. CONCLUSIONS: Sapropterin reduced blood Phe and was associated with significant improvement in parent-reported symptoms of inattention, hyperactivity/impulsivity, and executive functioning in children and adolescents with PKU. TRIAL REGISTRATION: ClinicalTrials.gov, NCT01114737. Registered 27 April 2010, https://clinicaltrials.gov/ct2/show/NCT01114737.
Assuntos
Transtorno do Deficit de Atenção com Hiperatividade , Fenilcetonúrias , Adolescente , Humanos , Criança , Lactente , Fenilcetonúrias/tratamento farmacológico , Transtorno do Deficit de Atenção com Hiperatividade/tratamento farmacológico , Função Executiva , Cognição , Método Duplo-Cego , Fenilalanina , Resultado do TratamentoRESUMO
Natural pigments are components very important in the dye industry. The betalains are pigments found in plants from Caryophyllales order and are relevant in the food manufacturing. The main source of betalains is beetroot, which has unfavorable aftertaste. Therefore, the demand for alternative species producing betalains has increased. Elicitor molecules such as methyl jasmonate (MeJA) induce metabolic reprogramming acting in the biosynthesis of specialized metabolites and can enhance pigment concentrations. Here, we used this strategy to identify if treatment with MeJA at 100 µM can promote the accumulation of betalains and other bioactive compounds in Alternanthera philoxeroides and Alternanthera sessilis. We performed the gene expression, concentration of betalains, phenols, flavonoids, amino acids (phenylalanine and tyrosine), and antioxidant activity. The results showed that MeJA treatment increased betalains and other bioactive compounds in the two Alternanthera species but A. sessilis had a better performance. One key factor in this pathway is related to the phenylalanine and tyrosine concentration. However, the species have distinct metabolic regulation: in A. philoxeroides, high concentrations of betalain pigments increase the tyrosine concentration and gene expression (include ADH) under MeJA and in A. sessilis, high concentrations of betalain pigments reduce the gene expression and tyrosine concentration after 2 days under MeJA. This study brings new questions about betalain biosynthesis and sheds light on the evolution of this pathway in Caryophyllales.
Assuntos
Amaranthaceae , Betalaínas , Pigmentos Biológicos , Amaranthaceae/genética , Amaranthaceae/metabolismo , Betalaínas/biossíntese , Pigmentos Biológicos/análise , Fenilalanina , Tirosina , Redes e Vias Metabólicas , Regulação da Expressão Gênica de Plantas , Flavonoides/análise , Fenóis/análise , Antioxidantes/análiseRESUMO
Enzyme immobilization has been reported as a promising approach to improving parameters such as thermal stability, pH and reusability. In this study, a polyacrylamide cryogel functionalized with L-phenylalanine was prepared to be used in the adsorption of ß-glucosidase from Thermoascus aurantiacus, aiming at its separation and also its immobilization on the cryogel matrix. The enzyme was produced by solid state fermentation. First, the adsorption was studied as a function of the pH and the resulting yield (Y, %) and purification factor (PF, dimensionless) were determined (1.57-5.13 and 64.19-91.20, respectively). The PF and yield from eluate samples obtained at pH 3.0 were the highest (5.13 and 91.20, respectively). Then, ß-glucosidase was immobilized on the hydrophobic cryogel and the recovery activities (%) were determined as a function of temperature and in the presence of different saline solutions. The values ranged from 14.45 to 45.97. As expected, salt type and ionic strength affected the activity remained in the immobilized ß-glucosidase. The average bioreactor activity was 39.9 U/g of dry cryogel and its operational stability was measured, with no decrease in activity being observed during seven cycles. Kinetic parameters of free and immobilized enzyme were determined according to different models.
Assuntos
Criogéis , Thermoascus , Criogéis/química , Adsorção , beta-Glucosidase/química , Enzimas Imobilizadas/química , Interações Hidrofóbicas e Hidrofílicas , Concentração de Íons de HidrogênioRESUMO
Severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) infection triggers inflammatory clinical stages that affect the outcome of patients with coronavirus disease 2019 (COVID-19). Disease severity may be associated with a metabolic imbalance related to amino acids, lipids, and energy-generating pathways. The aim of this study was to characterize the profile of amino acids and acylcarnitines in COVID-19 patients. A multicenter, cross-sectional study was carried out. A total of 453 individuals were classified by disease severity. Levels of 11 amino acids, 31 acylcarnitines, and succinylacetone in serum samples were analyzed by electrospray ionization-triple quadrupole tandem mass spectrometry. Different clusters were observed in partial least squares discriminant analysis, with phenylalanine, alanine, citrulline, proline, and succinylacetone providing the major contribution to the variability in each cluster (variable importance in the projection >1.5). In logistic models adjusted by age, sex, type 2 diabetes mellitus, hypertension, and nutritional status, phenylalanine was associated with critical outcomes (odds ratio=5.3 (95% CI 3.16-9.2) in the severe vs. critical model, with an area under the curve of 0.84 (95% CI 0.77-0.90). In conclusion the metabolic imbalance in COVID-19 patients might affect disease progression. This work shows an association of phenylalanine with critical outcomes in COVID-19 patients, highlighting phenylalanine as a potential metabolic biomarker of disease severity.
Assuntos
COVID-19 , Diabetes Mellitus Tipo 2 , Humanos , SARS-CoV-2 , Estudos Transversais , Aminoácidos , FenilalaninaRESUMO
Phenylketonuria (PKU) is a rare genetic disease that causes brain toxicity due to the inability of the body to convert dietary phenylalanine to tyrosine by the action of phenylalanine hydroxylase. The only treatment for PKU so far is lifelong dietary intervention to ensure normal human growth and neurodevelopment. However, in adults, low long-term adherence to this type of dietary intervention has been observed. Given the important role of the intestinal microbiota in the process of digestion and disease prevention, probiotics could be a therapeutic strategy to help degrade dietary phenylalanine, reducing its levels before ingestion. Genetically modified probiotics designed as live biotherapeutic agents for the treatment of specific diseases are sophisticated alternative therapeutic strategies. In this review, the focus is on demonstrating what has been elucidated so far about the use of next-generation probiotics as a therapeutic strategy in the treatment of individuals with PKU. The results described in the literature are encouraging and use genetically modified engineered probiotics showing efficacy both in vitro and in vivo. These probiotics appear to be suitable for meeting the unmet need for new drugs for PKU.
Assuntos
Fenilalanina Hidroxilase , Fenilcetonúrias , Probióticos , Adulto , Dieta , Humanos , Fenilalanina/metabolismo , Fenilalanina/uso terapêutico , Fenilalanina Hidroxilase/genética , Fenilalanina Hidroxilase/metabolismo , Fenilalanina Hidroxilase/uso terapêutico , Fenilcetonúrias/tratamento farmacológico , Fenilcetonúrias/genética , Probióticos/uso terapêuticoRESUMO
Phenylalanine (Phe) is an amino acid that has been identified in carbonaceous meteorites; its formation mechanism in space is unknown, and its radioresistance has been the subject of investigation. This work aims at studying, in the laboratory, the Phe radiolysis by cosmic analogues. The Phe destruction rate, at 300 K, is measured for H, He, and N ion beam irradiation in the 0.5 to 2 kinetic MeV range. Fourier transform infrared (FTIR) spectroscopy was employed to monitor the molecular degradation as a function of fluence. The Phe apparent destruction cross-section, σapd, which includes radiolysis and sputtering processes, is determined to be proportional to the electronic stopping power, Se. The measured parameter D0 = 14.3 ± 2.2 eV/molec in the relationship, and σdap = Se/D0 is interpreted as the mean absorbed dose necessary to dissociate or eject a Phe molecule. The Phe half-life in the interstellar medium is predicted to be about 10 million years, H+ ions the main destructive cosmic ray constituent.
Assuntos
Meteoroides , Fenilalanina , Íons , Cinética , Espectroscopia de Infravermelho com Transformada de FourierRESUMO
Introducción: la fenilcetonuria (PKU) es el error congénito del metabolismo de las proteínas más frecuente. El tratamiento dietético consiste en un plan de alimentación con una ingesta de proteínas naturales restringida, un sustituto proteico libre o de bajo contenido en fenilalanina (Phe) y el aporte de alimentos muy bajos en proteínas. El objetivo principal de este trabajo fue investigar si es posible aumentar la ingesta de proteína natural (PN) que se indica a los pacientes con PKU manteniendo los dosajes de Phe en sangre en rangos de seguridad. Materiales y método: se buscaron en 6 bases de datos electrónicas artículos publicados. Se identificaron un total de 154 artículos de Pub Med por intervalo de años desde 1999 a 2020. Se eligieron 15 artículos que se adaptaron a los criterios de inclusión y exclusión y respondían al objeto de estudio de esta revisión bibliográfica. Resultados: hay varios factores que pueden influenciar la estimación de la tolerancia de Phe como la severidad del fenotipo del paciente, la edad, el rango de seguridad de Phe en sangre, la prescripción de Phe y la adherencia al sustituto proteico. Si los niveles de Phe en sangre se mantienen en forma constante dentro del rango adecuado y por un período determinado, se debería considerar un incremento de la ingesta de Phe. El aumento de la ingesta de PN deberá ser realizado de manera controlada, individual y evaluando en forma constante el impacto en los dosajes de Phe en sangre. Conclusión: optimizar la ingesta de PN ofrece una mejora en la calidad de vida de pacientes con PKU, facilita la capacidad del paciente para socializar y contribuye a una mejor adherencia a la dieta(AU).
Introduction: phenylketonuria (PKU) is the most frequent inborn error of protein metabolism. The dietary treatment consists of a diet with a restricted natural protein intake, a free or low phenylalanine (Phe) protein substitute, and the intake of low protein food. The main objective of this work is to analyze if it is possible to increase the natural protein (NP) intake prescribed to PKU patients while maintaining blood Phe dosages within safe range. Materials and method: studies published were searched in 6 electronic data- basis. A total of 154 Pub Med articles were identified by range of years from 1999 to 2020. Fifteen articles which met the inclusion and exclusion criteria and responded to the objective of this bibliographic review were chosen. Results: several factors may influence Phe tolerance, such as severity of the patient´s phenotype, age, blood Phe safe range, Phe prescription and adherence to protein substitute. If Phe blood levels remain constantly within safe range and for a certain period, an increase of Phe intake should be considered. Increase of NP intake must be carried out in a controlled manner, individually and constantly evaluating blood Phe levels. Conclusion: optimizing NP intake offers the PKU patient an improvement in quality of life, facilitates the patient´s ability to socialize and contributes to a better adherence to the diet(AU).
Assuntos
Fenilcetonúrias , Fenilcetonúrias/dietoterapia , Proteínas , Ingestão de Alimentos , MetabolismoRESUMO
Establishing the genotypes of patients with hyperphenylalaninemia (HPA)/phenylketonuria (PKU, MIM#261600) has been considered a cornerstone for rational medical management. However, knowledge of the phenylalanine hydroxylase gene (PAH) mutational spectrum in Latin American populations is still limited. Herein, we aim to update the mutational PAH spectrum in the largest cohort of HPA/PKU Mexican patients (N = 124) reported to date. The biallelic PAH genotype was investigated by Sanger automated sequencing, and genotypes were correlated with documented biochemical phenotypes and theoretical tetrahydrobiopterin (BH4) responsiveness. Patients were biochemically classified as having classic PKU (50%, 62/124), mild PKU (20.2%, 25/124) and mild HPA (29.8%, 37/124). Furthermore, 78.2% of the included patients (97/124) were identified by newborn screening. A total of 60 different pathogenic variants were identified, including three novel ones (c. 23del, c. 625_626insC and c. 1315 + 5_1315 + 6insGTGTAACAG), the main categories being missense changes (58%, 35/60) and those affecting the catalytic domain (56.6%, 34/60), and c. 60 + 5G > T was the most frequent variant (14.5%, 36/248) mainly restricted (69.2%) to patients from the central and western parts of Mexico. These 60 types of variants constituted 100 different biallelic PAH genotypes, with the predominance of compound-heterozygous ones (96/124, 77%). The expected BH4 responsiveness based on the PAH genotype was estimated in 52% of patients (65/124), mainly due to the p. (Val388Met) (rs62516101) allele. Instead, our study identified 27 null variants with an allelic phenotype value of zero, with a predominance of c. 60 + 5G > T, which predicts the absence of BH4 responsiveness. An identical genotype reported in BIOPKUdb was found in 92/124 (74%) of our patients, leading to a genotype-phenotype concordance in 80/92 (86.9%) of them. The high number of variants found confirms the heterogeneous and complex mutational landscape of HPA/PKU in Mexico.
Assuntos
Mutação , Fenilalanina Hidroxilase/química , Fenilalanina Hidroxilase/genética , Fenilcetonúrias/genética , Análise de Sequência de DNA/métodos , Substituição de Aminoácidos , Domínio Catalítico , Feminino , Técnicas de Genotipagem , Humanos , Recém-Nascido , Mutação com Perda de Função , Masculino , México , Modelos Moleculares , Mutação de Sentido Incorreto , Triagem Neonatal , Conformação ProteicaRESUMO
BACKGROUND: Although there are several severity predictors for COVID-19, none are specific. Serum levels of phenylalanine were recently associated with increased inflammation, higher SOFA scores, ICU admission, and mortality rates among non-COVID-19 patients. Here, we investigated the relationship between phenylalanine and inflammatory markers in adults with COVID-19. METHODS: We assessed adults with COVID-19 at hospital admission for clinical and laboratory data. Nuclear magnetic resonance spectroscopy measured serum levels of phenylalanine and other amino acids of its metabolomic pathway. Flow Cytometry measured serum levels of IL-2, IL-4, IL-6, Il-10, TNF-α, and IFN-γ. Linear regression models adjusted for potential confounders assessed the relationship between serum levels of phenylalanine and inflammatory cytokines. RESULTS: Phenylalanine and tyrosine were significantly lower in mild disease as compared to moderate and severe groups. Linear regression models showed that phenylalanine is independently and positively associated with disease severity regardless of the cytokine analyzed and after adjustment for potential confounders. In addition, mild cases showed consistently lower serum phenylalanine levels within the first ten days from disease onset to hospital admission. CONCLUSIONS: Phenylalanine is a marker of disease severity. This association is independent of the time between the onset of symptoms and the magnitude of the inflammatory state.
Assuntos
COVID-19/sangue , Fenilalanina/sangue , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores/sangue , COVID-19/complicações , Comorbidade , Estudos Transversais , Citocinas/sangue , Feminino , Humanos , Inflamação/complicações , Inflamação/metabolismo , Modelos Lineares , Masculino , Pessoa de Meia-Idade , Índice de Gravidade de Doença , Adulto JovemRESUMO
Prenatal screening in pregnant women between 35 and 37 weeks of gestation and intrapartum antibiotic prophylaxis has successfully reduced the incidence of neonatal morbidity and mortality related to Streptococcus agalactiae. However, the contamination rates of newborns are still considerable. In traditional and folk medicines, it has been observed that garlic has been effective in treating S. agalactiae infection. The aim of this study was to isolate and identify the active compounds from garlic that have antimicrobial activity against S. agalactiae. In order to do this, SP80 (Sep-Pak 80%) obtained from crude garlic extract (CGE) was fractionated by reverse-phase ultrafast liquid chromatography with UV (RP-UFLC-UV) using a Shim-pack PREP-ODS column. All fractions obtained were tested using a microbial growth inhibition test against the S. agalactiae strain (ATCC 12386). Five clinical isolates were used to confirm the action of the fractions with antimicrobial activity, and the bacterial growth curve was determined. Identification of the antimicrobial compounds was carried out through liquid chromatography coupled with mass spectrometry (LC/MS) and nuclear magnetic resonance (NMR). The active compounds found to exhibit antimicrobial activity were Ƴ-glutamyl-S-allyl-cysteine (fraction 18), Ƴ-glutamyl-phenylalanine (fraction 20), and the two stereoisomers (E and Z) of ajoene (fraction 42). The MICs of these fractions were 5.41 mg/ml, 4.60 mg/ml, and 0.16 mg/ml, respectively, and they inhibited the growth of the clinical isolates tested. Antimicrobial compounds from garlic may be a promising source in the search for new drugs against S. agalactiae. IMPORTANCE Invasive disease due to group B streptococcal (GBS) infection results in a wide spectrum of clinical disease in neonates. Maternal colonization by GBS is the primary risk factor for disease. The strategy recommended by the Centers for Disease Control to reduce neonatal GBS infection is the culture-based screening of all pregnant women at 35 to 37 weeks of gestation and intrapartum antibiotic prophylaxis (IAP). However, indiscriminate use of antibiotics favors the selection and spread of resistant bacteria. The global scenario of antibacterial resistance has been of great concern for public health, and natural products can be a source of new substances to help us grapple with this problem.
Assuntos
Antibacterianos/farmacologia , Alho/química , Extratos Vegetais/farmacologia , Infecções Estreptocócicas/microbiologia , Streptococcus agalactiae/efeitos dos fármacos , Antibacterianos/química , Cromatografia Líquida de Alta Pressão , Avaliação Pré-Clínica de Medicamentos , Humanos , Testes de Sensibilidade Microbiana , Extratos Vegetais/química , Infecções Estreptocócicas/tratamento farmacológico , Streptococcus agalactiae/genética , Streptococcus agalactiae/fisiologiaRESUMO
Neurotoxic effects caused by high phenylalanine (Phe) in patients with phenylketonuria (PKU) can be avoided through dietary treatment. However, achieving the recommended Phe levels has been a challenge. This study aimed to investigate factors associated with adherence to PKU treatment among patients followed at a medical genetics public service in southern Brazil. Twenty-nine patients (early diagnosed, n = 20; late-diagnosed, n = 9) with classical (n = 16) or mild PKU (n = 13) aged 6-34 years (16.4 ± 7.5) and 16 caregivers were included. Blood Phe levels were recorded, and assessment tools measuring barriers to treatment, IQ, knowledge about disease, treatment, and perceived adherence were collected. Classical PKU patients showed higher current blood Phe levels than mild PKU patients (U = 37.000, p = 0.003). Lifetime and childhood Phe levels were associated with recent metabolic control (τ = 0.76, p = 0.000; τ = 0.70, p = 0.000, respectively). The perception of barriers to treatment was associated with a higher blood Phe level (τ = 0.39, p = 0.003). Tolerance to Phe, metabolic control throughout childhood, and perceived difficulty in living with demands of treatment are important factors of greater vulnerability to poor adherence in PKU patients.
Assuntos
Dieta , Fenilalanina/sangue , Fenilcetonúrias/dietoterapia , Fenilcetonúrias/genética , Adolescente , Brasil/epidemiologia , Criança , Feminino , Humanos , Masculino , Fenilalanina/efeitos adversos , Fenilcetonúrias/sangue , Fenilcetonúrias/patologia , Adulto JovemRESUMO
Phenylketonuria (PKU) is an inborn error of metabolism caused by phenylalanine hydroxylase (PAH) deficiency and characterized by elevated plasma levels of phenylalanine (hyperphenylalaninemia-HPA). In severe cases, PKU patients present with neurological dysfunction and hepatic damage, but the underlying mechanisms are not fully elucidated. Other forms of HPA also characterized by neurological symptoms occur in rare instances due to defects in the metabolism of the PAH cofactor tetrahydrobiopterin. This review aims to gather the knowledge acquired on the phenylalanine-induced toxicity focusing on findings obtained from pre-clinical studies. Mounting evidence obtained from PKU genetic mice, rats submitted to different HPA models, and cell cultures exposed to phenylalanine has shown that high levels of this amino acid impair mitochondrial bioenergetics, provoke changes in oxidative and inflammatory status, and induce apoptosis. Noteworthy, some data demonstrated that phenylalanine-induced oxidative stress occurs specifically in mitochondria. Further studies have shown that the metabolites derived from phenylalanine, namely phenylpyruvate, phenyllactate, and phenylacetate, also disturb oxidative status. Therefore, it may be presumed that mitochondrial damage is one of the most important mechanisms responsible for phenylalanine toxicity. It is expected that the findings reviewed here may contribute to the understanding of PKU and HPA pathophysiology and to the development of novel therapeutic strategies for these disorders.
Assuntos
Inflamação/patologia , Mitocôndrias/patologia , Estresse Oxidativo , Fenilcetonúrias/patologia , Fenilcetonúrias/fisiopatologia , Animais , Modelos Animais de Doenças , Inflamação/complicações , Oxirredução , Fenilcetonúrias/complicaçõesRESUMO
Aluminum oxide (Al2O3) nanoparticles (NPs) are among the nanoparticles most used industrially, but their impacts on living organisms are widely unknown. We evaluated the effects of 50-1000 mg L-1 Al2O3 NPs on the growth, metabolism of lignin and its monomeric composition in soybean plants. Al2O3 NPs did not affect the length of roots and stems. However, at the microscopic level, Al2O3 NPs altered the root surface inducing the formation of cracks near to root apexes and damage to the root cap. The results suggest that Al2O3 NPs were internalized and accumulated into the cytosol and cell wall of roots, probably interacting with organelles such as mitochondria. At the metabolic level, Al2O3 NPs increased soluble and cell wall-bound peroxidase activities in roots and stems but reduced phenylalanine ammonia-lyase activity in stems. Increased lignin contents were also detected in roots and stems. The Al2O3 NPs increased the p-hydroxyphenyl monomer levels in stems but reduced them in roots. The total phenolic content increased in roots and stems; cell wall-esterified p-coumaric and ferulic acids increased in roots, while the content of p-coumaric acid decreased in stems. In roots, the content of ionic aluminum (Al+3) was extremely low, corresponding to 0.0000252% of the aluminum applied in the nanoparticulate form. This finding suggests that all adverse effects observed were due to the Al2O3 NPs only. Altogether, these findings suggest that the structure and properties of the soybean cell wall were altered by the Al2O3 NPs, probably to reduce its uptake and phytotoxicity.
Assuntos
Óxido de Alumínio , Parede Celular , Glycine max , Lignina , Nanopartículas , Óxido de Alumínio/toxicidade , Parede Celular/efeitos dos fármacos , Lignina/química , Lignina/metabolismo , Nanopartículas/toxicidade , Glycine max/efeitos dos fármacosRESUMO
The membrane-active nature of phospholipase A2-derived peptides makes them potential candidates for antineoplastic and antibacterial therapies. Two short 13-mer C-terminal fragments taken from snake venom Lys49-PLA2 toxins (p-AppK and p-Acl), differing by a leucine/phenylalanine substitution, were synthesized and their bioactivity was evaluated. Their capacity to interfere with the survival of Gram-positive and Gram-negative bacteria as well as with solid and liquid tumors was assessed in vitro. Toxicity to red blood cells was investigated via in silico and in vitro techniques. The mode of action was mainly studied by molecular dynamics simulations and membrane permeabilization assays. Briefly, both peptides have dual activity, i.e., they act against both bacteria, including multidrug-resistant strains and tumor cells. All tested bacteria were susceptible to both peptides, Pseudomonas aeruginosa being the most affected. RAMOS, K562, NB4, and CEM cells were the main leukemic targets of the peptides. In general, p-Acl showed more significant activity, suggesting that phenylalanine confers advantages to the antibacterial and antitumor mechanism, particularly for osteosarcoma lines (HOS and MG63). Peptide-based treatment increased the uptake of a DNA-intercalating dye by bacteria, suggesting membrane damage. Indeed, p-AppK and p-Acl did not disrupt erythrocyte membranes, in agreement with in silico predictions. The latter revealed that the peptides deform the membrane and increase its permeability by facilitating solvent penetration. This phenomenon is expected to catalyze the permeation of solutes that otherwise could not cross the hydrophobic membrane core. In conclusion, the present study highlights the role of a single amino acid substitution present in natural sequences towards the development of dual-action agents. In other words, dissecting and fine-tuning biomembrane remodeling proteins, such as snake venom phospholipase A2 isoforms, is again demonstrated as a valuable source of therapeutic peptides.