RESUMO
Acetoacetate (AcAc) and D-beta-hydroxybutyrate (D-ßOHB), the two major ketone bodies found in circulation, are linked to multiple physiological and pathophysiological states. Therefore, analytical methodologies surrounding the quantification of total ketone body (TKB) concentrations in biological matrices are paramount. Traditional methods to quantify TKBs relied on indirect spectrophotometric assays with narrow dynamic ranges, which have been significantly improved upon by modern mass spectrometry (MS)-based approaches. However, the lack of stable isotope-labeled internal standards (ISs) for AcAc and the need to distinguish D-ßOHB from its closely related structural and enantiomeric isomers pose significant obstacles. Here, we provide a protocol to synthesize and quantify a [13C] stable isotope-labeled IS for AcAc, which, in conjunction with a commercially available [2H] stable isotope-labeled IS for ßOHB, allows TKBs to be measured across multiple biological matrices. This rapid (7 min) analysis employs reverse phase ultra-high performance liquid chromatography (RP-UHPLC) coupled to tandem MS (MS/MS) to distinguish ßOHB from three structural isomers using parallel reaction monitoring (PRM), providing excellent specificity and selectivity. Finally, a method is provided that distinguishes D-ßOHB from L-ßOHB using a simple one-step derivatization to produce the corresponding diastereomers, which can be chromatographically resolved using the same rapid RP-UHPLC separation with new PRM transitions. In summary, this method provides a rigorous analytical pipeline for the analysis of TKBs in biological matrices via leveraging two authentic stable isotope-labeled ISs and RP-UHPLC-MS/MS.
Assuntos
Isótopos de Carbono , Marcação por Isótopo , Espectrometria de Massas em Tandem , Espectrometria de Massas em Tandem/métodos , Marcação por Isótopo/métodos , Cromatografia Líquida de Alta Pressão/métodos , Humanos , Isótopos de Carbono/química , Corpos Cetônicos/química , Acetoacetatos/química , Cromatografia de Fase Reversa/métodos , Padrões de Referência , Ácido 3-Hidroxibutírico/química , Ácido 3-Hidroxibutírico/análise , AnimaisRESUMO
BACKGROUND: The goal of this study was to develop and validate a UPLC-MS/MS method for simultaneous mea-surement of 13 AEDs, including carbamazepine, oxcarbazepine, lamotrigine, levetiracetam, topiramate, primidone, zonisamide, gabapentin, lacosamide, perampanel, pregabalin, rufinamide, and vigabatrin, in whole blood samples. METHODS: A UPLC-MS/MS method for simultaneous determination of 13 AEDs in whole blood was developed, and validation was conducted for accuracy, precision, limit of quantification (LOQ), matrix effect, and stability. Our method was compared to two different hospitals using UPLC-MS/MS. RESULTS: All AEDs exhibited linearity across the AMR (analytical measurement range), with R2 values ranging from 0.994 to 1.000. The imprecision and inaccuracy for low and high quality control (QC) levels were within an acceptable range, with the coefficient of variation (CV) < 15%. The LOQ was 0.62 µg/mL for carbamazepine, 1.61 µg/mL for oxcarbazepine, 1.30 µg/mL for lamotrigine, 13.20 µg/mL for levetiracetam, 1.26 µg/mL for topira-mate, 1.01 µg/mL for primidone, 1.59 µg/mL for zonisamide, 1.09 µg/mL for lacosamide, 1.61 µg/mL for gabapentin, 0.50 µg/mL for pregabalin, 0.07 ng/mL for perampanel, 3.00 µg/mL for rufinamide, and 2.06 µg/mL for vigabatrin. All AEDs demonstrated acceptable assay parameters for carryover, stability, and matrix effects. Moreover, the assay showed satisfactory results compared to two different hospitals with a bias of less than 15%. CONCLUSIONS: We successfully developed and validated a fast and robust UPLC-MS/MS method for routine therapeutic drug monitoring of thirteen antiepileptic drugs simultaneously.
Assuntos
Anticonvulsivantes , Limite de Detecção , Espectrometria de Massas em Tandem , Anticonvulsivantes/sangue , Anticonvulsivantes/análise , Espectrometria de Massas em Tandem/métodos , Espectrometria de Massas em Tandem/normas , Humanos , Cromatografia Líquida de Alta Pressão/métodos , Cromatografia Líquida de Alta Pressão/normas , Reprodutibilidade dos Testes , Monitoramento de Medicamentos/métodos , Monitoramento de Medicamentos/normas , Padrões de Referência , Espectrometria de Massa com Cromatografia LíquidaRESUMO
BACKGROUND: B chromosomes are extra non-essential elements present in several eukaryotes. Unlike A chromosomes which are essential and present in all individuals of a species, B chromosomes are not necessary for normal functioning of an organism. Formerly regarded as genetically inactive, B chromosomes have been discovered to not only express their own genes, but also to exert influence on gene expression in A chromosomes. Recent studies have shown that, in some Psalidodon (Characiformes, Characidae) species, B chromosomes might be associated with phenotypic effects, such as changes in the reproductive cycle and gene expression. METHODS AND RESULTS: In this study, we aimed to establish stable reference genes for RT-qPCR experiments conducted on gonads of three fish species within Psalidodon genus, both in the presence and absence of B chromosomes. The stability of five selected reference genes was assessed using NormFinder, geNorm, BestKeeper, and RefFinder algorithms. We determined ppiaa and pgk1 as the most stable genes in P. fasciatus, whereas ppiaa and hmbsa showed the highest stability in P. bockmanni. For P. paranae, tbp and hprt1 were the most stable genes in females, and ppiaa and hprt1 were the most stable in males. CONCLUSIONS: We determined the most stable reference genes in gonads of three Psalidodon species considering the presence of B chromosomes. This is the first report of reference gene stability in the genus and provides valuable tools to better understand the effects of B chromosomes at gene expression level.
Assuntos
Cromossomos , Animais , Masculino , Feminino , Cromossomos/genética , Reação em Cadeia da Polimerase em Tempo Real/métodos , Reação em Cadeia da Polimerase em Tempo Real/normas , Padrões de Referência , Perfilação da Expressão Gênica/métodos , Perfilação da Expressão Gênica/normas , Gônadas/metabolismo , Characidae/genética , Caraciformes/genéticaRESUMO
Gene expression through RT-qPCR can be performed by the relative quantification method, which requires the expression normalization through reference genes. Therefore, it is essential to validate, experimentally, the candidate reference genes. Thus, although there are several studies that are performed to identify the most stable reference genes, most them validate genes for very specific conditions, not exploring the whole potential of the research since not all possible combinations of treatments and/or conditions of the study are explored. For this reason, new experiments must be conducted by researchers that have interest in analyzing gene expression of treatments and/or conditions present, but not explored, in these studies. Here, we present the RGeasy tool, which aims to facilitate the selection of reference genes, allowing the user to choose genes for a greater number of combinations of treatments/conditions, compared to the ones present in the original articles, through just a few clicks. RGeasy was validated with RT-qPCR data from gene expression studies performed in two coffee species, Coffea arabica and Coffea canephora, and it can be used for any animal, plant or microorganism species. In addition to displaying a rank of the most stable reference genes for each condition or treatment, the user also has access to the primer pairs for the selected reference genes.
Assuntos
Perfilação da Expressão Gênica , Reação em Cadeia da Polimerase em Tempo Real , Padrões de Referência , Software , Reação em Cadeia da Polimerase em Tempo Real/normas , Reação em Cadeia da Polimerase em Tempo Real/métodos , Perfilação da Expressão Gênica/métodos , Perfilação da Expressão Gênica/normas , Genes de Plantas , Coffea/genética , Regulação da Expressão Gênica de PlantasRESUMO
An international collaborative study was organised by the European Directorate for the Quality of Medicines & HealthCare (EDQM, Council of Europe) to calibrate replacement batches for the current European Pharmacopoeia (Ph. Eur.) Prekallikrein Activator (PKA) in albumin Biological Reference Preparation (BRP) whose stocks were dwindling. The study was run in the framework of the Biological Standardisation Programme (BSP) of the Council of Europe and the European Union (EU) Commission. Twenty-four laboratories from official medicines control authorities and manufacturers in Europe and outside Europe took part in the study. Three candidate replacement batches were produced with albumin solutions artificially spiked with a PKA concentrate to increase their PKA level. Participants were requested to evaluate the candidate batches against the 3rd World Health Organization (WHO) International Standard (IS) for Prekallikrein activator in albumin (16/364) using their routine assay method. The Ph. Eur. PKA in albumin BRP batch 7 (BRP7) was also included in the test panel to ensure the continuity of the consecutive BRP batches. The 3 candidate replacement batches were considered suitable for their intended use as BRPs. The study confirmed the stability of the PKA content of the current BRP7. Thermal stress study on the candidate batches confirmed the stability of their PKA activity. In December 2023, the Ph. Eur. Commission officially adopted the 3 candidate batches as Ph. Eur. PKA in albumin BRP batches 8, 9 and 10 with assigned potencies of 37 IU/vial, 33 IU/vial and 34 IU/vial, respectively. The activity of the 3 new batches of Ph. Eur. PKA in albumin BRP will be regularly monitored.
Assuntos
Padrões de Referência , Calibragem , Farmacopeias como Assunto/normas , Europa (Continente) , Humanos , Cooperação Internacional , Albuminas/normas , Albuminas/análiseRESUMO
Background: RT-qPCR is a powerful strategy for recognizing the most appropriate reference genes, which can successfully minimize experimental mistakes through accurate normalization. Ludisia discolor, recognized for its ornamental value, features little, distinctive blossoms with twisted lips and gynostemium showing chiral asymmetry, together with striking blood-red fallen leaves periodically marked with golden blood vessels. Methods and Results: To ensure the accuracy of qRT-PCR, selecting appropriate reference genes for quantifying target gene expression levels is essential. This study aims to identify stable reference genes during the development of L. discolor. In this study, the entire floral buds, including the lips and gynostemium from different development stages, were taken as materials. Based upon the transcriptome information of L. discolor, nine housekeeping genes, ACT, HIS, EF1-α1, EF1-α2, PP2A, UBQ1, UBQ2, UBQ3, and TUB, were selected in this research study as prospect interior referral genes. The expression of these nine genes were found by RT-qPCR and afterwards comprehensively examined by four software options: geNorm, NormFinder, BestKeeper, and ΔCt. The outcomes of the analysis showed that ACT was the most steady gene, which could be the most effective inner referral gene for the expression evaluation of flower advancement in L. discolor. Conclusions: The results of this study will contribute to the molecular biology research of flower development in L. discolor and closely related species.
Assuntos
Flores , Regulação da Expressão Gênica de Plantas , Flores/genética , Flores/crescimento & desenvolvimento , Genes de Plantas , Perfilação da Expressão Gênica/métodos , Genes Essenciais/genética , Padrões de Referência , Reação em Cadeia da Polimerase em Tempo Real/normas , Reação em Cadeia da Polimerase em Tempo Real/métodos , Transcriptoma/genética , Proteínas de Plantas/genéticaRESUMO
Background: The physical health of adolescents is crucial for the prosperity and sustainable development of a nation. Developing specific growth standards is essential for prioritizing the wellbeing of the youth of Pakistan. This study aimed to establish normative standards for height, weight, and body mass index (BMI) among 12- to 16-year-olds in South Punjab, facilitating accurate health assessments and tailored interventions. Method: This study utilized a cross-sectional design and stratified random sampling to select 2,970 adolescents (49.73% boys and 50.26% girls) aged 12-16 years from South Punjab, Pakistan. Anthropometric measurements, including height, weight, and BMI, were collected. The data were stratified by age and sex, and smoothed percentile curves were computed using the LMS method, which incorporates the L (γ-lambda), M (µ-mu), and S (δ-sigma) parameters. The results were compared to international references to provide a comprehensive analysis. Results: The results highlight sex-specific trends in anthropometric indicators among adolescents. Boys exhibited higher mean values in height (160.50 ± 11.50 cm), weight (45.02 ± 9.78 kg), and BMI (17.30 ± 2.41) than girls (158.57 ± 9.34 cm, 41.00 ± 7.89 kg, and 16.29 ± 2.82, respectively). Growth patterns indicate boys grow faster in height and weight between ages 12 and 14, whereas girls show slower annual increases. Comparative analysis with international standards reveals that boys' height and weight were generally lower than international medians (P50th), whereas girls' height was comparable or higher. BMI values for both sexes were lower than international norms, reflecting unique regional growth patterns. Conclusion: This research establishes updated age- and sex-specific normative reference standards for adolescents in South Punjab, Pakistan. The study revealed that Pakistani adolescent boys exhibit higher mean values in height, weight, and BMI than girls, with faster growth rates between ages 12 and 14. Compared to international standards, Pakistani adolescents show lower BMI values, highlighting unique regional growth patterns. These standards have practical applications in screening, monitoring, and health strategy planning, contributing to efforts to promote a healthier future for the population. Future studies are recommended to utilize these local growth references for health surveillance and treatment in the local population.
Assuntos
Antropometria , Estatura , Índice de Massa Corporal , Peso Corporal , Humanos , Adolescente , Paquistão , Masculino , Feminino , Estudos Transversais , Criança , Fatores Sexuais , Fatores Etários , Valores de Referência , Padrões de ReferênciaRESUMO
AIMS: To assess the diagnostic accuracy of dobutamine stress echocardiography (DSE) in symptomatic patients with a low to intermediate pretest probability of obstructive coronary artery disease (CAD) and a positive coronary CT angiography (CCTA). METHODS: We prospectively enrolled 104 consecutive patients undergoing coronary angiography for symptoms of stable CAD and a CCTA indicative of obstructive CAD. The diagnostic performance of DSE was evaluated against two intracoronary pressure indices: (a) fractional flow reserve (FFR) with a cut-off of ≤0.80 and (b) instantaneous wave-free ratio (iFR) with a cut-off of ≤0.89, indicating haemodynamically significant stenoses. RESULTS: Of 102 patients, 46 (45%) had at least one significant lesion as defined by FFR, as did 37 (36%) as defined by iFR. DSE showed positive results in 33% (34/102) of cases. The discriminative power of DSE for detecting significant CAD was moderate, with areas under the curve of 0.63 (p=0.024) compared with FFR and 0.64 (p=0.025) compared with iFR. The accuracy, sensitivity and specificity of DSE were, respectively, 61%, 43%, and 75% against FFR, and 64%, 46% and 74% against iFR. The diagnostic accuracy of DSE did not differ significantly between FFR and iFR as a reference (p=0.549). CONCLUSION: In patients with positive CCTA, DSE has a moderate ability to identify haemodynamically significant CAD, with low sensitivity and moderate specificity. When assessed against FFR and iFR criteria, its additive diagnostic value is limited in patients with low to intermediate pretest probability of obstructive CAD. TRIAL REGISTRATION NUMBER: NCT03045601.
Assuntos
Angiografia por Tomografia Computadorizada , Angiografia Coronária , Doença da Artéria Coronariana , Ecocardiografia sob Estresse , Reserva Fracionada de Fluxo Miocárdico , Valor Preditivo dos Testes , Humanos , Reserva Fracionada de Fluxo Miocárdico/fisiologia , Masculino , Feminino , Ecocardiografia sob Estresse/métodos , Ecocardiografia sob Estresse/normas , Estudos Prospectivos , Angiografia Coronária/métodos , Pessoa de Meia-Idade , Idoso , Doença da Artéria Coronariana/fisiopatologia , Doença da Artéria Coronariana/diagnóstico por imagem , Doença da Artéria Coronariana/diagnóstico , Reprodutibilidade dos Testes , Angiografia por Tomografia Computadorizada/métodos , Angiografia por Tomografia Computadorizada/normas , Estenose Coronária/fisiopatologia , Estenose Coronária/diagnóstico por imagem , Estenose Coronária/diagnóstico , Vasos Coronários/diagnóstico por imagem , Vasos Coronários/fisiopatologia , Dobutamina/administração & dosagem , Padrões de ReferênciaRESUMO
We conducted a comprehensive examination of liquid mycotoxin reference standards. A total of 30 different standards were tested, each containing 10 samples of three distinct substances: Aflatoxin B1, Deoxynivalenol, and Zearalenone. The standards were sourced from 10 different global market leading manufacturers. To facilitate comparison, all the standard sets were adjusted to the same concentration level. The standards were analyzed using the techniques LC-MS/MS, HPLC-DAD, and LC-HRMS to assess their quality attributes. Regarding the validation of the reference values, it was observed that 30% of the suppliers provided reference standards that were either below the lower acceptance limit or above the higher acceptance limit, confirmed by both the LC-MS/MS and HPLC-DAD methods. Furthermore, a total of 12 impurities were found in the DON standards, 10 in the AFB1 standards, and 8 in the ZON standards, distributed across all the suppliers. Therefore, this study suggests relevant adjustments to the ISO 17034 standard, proposing that the purity of a raw material should be uniformly based on q-NMR analysis, as most manufacturers state the purity of their certificates is determined using HPLC-UV or LC-MS/MS. Liquid standards with a shelf life of ≤1 year should not exceed an uncertainty of 3%. Standards that have a longer shelf life should not have more than 5% uncertainty. This study also emphasizes the importance of stability. The standards should undergo continuous long-term monitoring; otherwise, products may exhibit a target value of only 80%, as seen in one instance. It is also recommended to include proof of HPLC and LC-MS/MS analyses on the certificate of each released batch of a final product.
Assuntos
Aflatoxina B1 , Padrões de Referência , Espectrometria de Massas em Tandem , Tricotecenos , Zearalenona , Tricotecenos/análise , Zearalenona/análise , Aflatoxina B1/análise , Cromatografia Líquida de Alta Pressão , Contaminação de Alimentos/análise , Micotoxinas/análise , Cromatografia LíquidaRESUMO
Accurate measurement of gene expression levels is vital for advancing plant biology research. This study explores the identification and validation of stable reference genes (RGs) for gene expression analysis in Spinacia oleracea. Leveraging transcriptome data from various developmental stages, we employed rigorous statistical analyses to identify potential RGs. A total of 1196 candidate genes were initially screened based on expression variability, with subsequent refinement using criteria such as low variance and stability. Among 12 commonly used candidate RGs, EF1α and H3 emerged as the most stable across diverse experimental conditions, while GRP and PPR exhibited lower stability. These findings were further validated through qRT-PCR assays and comprehensive statistical analyses, including geNorm, NormFinder, BestKeeper, and RefFinder. Our study underscores the importance of systematic RG selection to ensure accurate normalization in gene expression studies, particularly in the context of S. oleracea developmental stages and physiological processes like flowering. These validated RGs provide a robust foundation for future gene expression analysis in S. oleracea and contribute to the advancement of molecular research in plant biology.
Assuntos
Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Spinacia oleracea , Transcriptoma , Spinacia oleracea/genética , Perfilação da Expressão Gênica/métodos , Perfilação da Expressão Gênica/normas , Padrões de Referência , Genes de Plantas , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismoAssuntos
Sequenciamento de Nucleotídeos em Larga Escala , Inclusão em Parafina , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Indicadores e Reagentes , Biblioteca Gênica , Controle de Qualidade , Padrões de Referência , Sensibilidade e Especificidade , Neoplasias/genética , Neoplasias/diagnóstico , DNA de Neoplasias/genética , DNA de Neoplasias/análiseRESUMO
Rodents are commonly used as animal models in studies investigating various experimental conditions, often requiring gene expression analysis. Quantitative real-time reverse transcription PCR (RT-qPCR) is the most widely used tool to quantify target gene expression levels under different experimental conditions in various biological samples. Relative normalization with reference genes is a crucial step in RT-qPCR to obtain reliable quantification results. In this work, the main reference genes used in gene expression studies among the three rodents commonly employed in scientific research-hamster, rat, and mouse-are analyzed and described. An individual literature search for each rodent was conducted using specific search terms in three databases: PubMed, Scopus, and Web of Science. A total of 157 articles were selected (rats = 73, mice = 79, and hamsters = 5), identifying various reference genes. The most commonly used reference genes were analyzed according to each rodent, sample type, and experimental condition evaluated, revealing a great variability in the stability of each gene across different samples and conditions. Classic genes, which are expected to be stably expressed in both samples and conditions analyzed, demonstrated greater variability, corroborating existing concerns about the use of these genes. Therefore, this review provides important insights for researchers seeking to identify suitable reference genes for their validation studies in rodents.
Assuntos
Perfilação da Expressão Gênica , Padrões de Referência , Roedores , Animais , Ratos , Perfilação da Expressão Gênica/métodos , Perfilação da Expressão Gênica/normas , Camundongos , Roedores/genética , Reação em Cadeia da Polimerase em Tempo Real/normas , Reação em Cadeia da Polimerase em Tempo Real/métodos , Expressão Gênica/genéticaRESUMO
El objetivo de la ruta es establecer las pautas formales, respecto a la atención integral en salud en las niñas, niños y adolescentes, ayudando a optimizar las acciones, disminuir costos, incrementar la satisfacción de los usuarios, así como mejorar la productividad y competitividad del personal de salud. En razón de lo anterior presentamos la ruta de trabajo, para la adecuación de la normativa técnica, producto del trabajo de las diferentes dependencias vinculadas directa e indirectamente a la atención en salud a niñas, niños y adolescentes
The objective of the route is to establish formal guidelines regarding comprehensive health care in the girls, boys and adolescents, helping to optimize actions, reduce costs, increase user satisfaction, as well as improving the productivity and competitiveness of health personnel. Due to the above, we present the work route, for the adaptation of the technical regulations, product of the work of the different agencies linked directly and indirectly to care in health for girls, boys and adolescents
Assuntos
Padrões de Referência , Jurisprudência , El SalvadorRESUMO
Tendons are one of the major load-bearing tissues in the body; subjected to enormous peak stresses, and thus vulnerable to injury. Cellular responses to tendon injury are complex, involving inflammatory and repair components, with the latter employing both resident and recruited exogenous cell populations. Gene expression analyses are valuable tools for investigating tendon injury, allowing assessment of repair processes and pathological responses such as fibrosis, and permitting evaluation of therapeutic pharmacological interventions. Quantitative polymerase chain reaction (qPCR) is a commonly used approach for such studies, but data obtained by this method must be normalised to reference genes: genes known to be stably expressed between the experimental conditions investigated. Establishing suitable tendon injury reference genes is thus essential. Accordingly we investigated mRNA expression stability in a rat model of tendon injury, comparing both injured and uninjured tendons, and the effects of rapamycin treatment, at 1 and 3 weeks post injury. We used 11 candidate genes (18S, ACTB, AP3D1, B2M, CSNK2A2, GAPDH, HPRT1, PAK1IP1, RPL13a, SDHA, UBC) and assessed stability via four complementary algorithms (Bestkeeper, deltaCt, geNorm, Normfinder). Our results suggests that ACTB, CSNK2A2, HPRT1 and PAK1IP1 are all stably expressed in tendon, regardless of injury or drug treatment: any three of these would serve as universally suitable reference gene panel for normalizing qPCR expression data in the rat tendon injury model. We also reveal 18S, UBC, GAPDH, and SDHA as consistently poor scoring candidates (with the latter two exhibiting rapamycin- and injury-associated changes, respectively): these genes should be avoided.
Assuntos
Tendão do Calcâneo , Padrões de Referência , Traumatismos dos Tendões , Animais , Tendão do Calcâneo/lesões , Tendão do Calcâneo/patologia , Tendão do Calcâneo/metabolismo , Ratos , Traumatismos dos Tendões/genética , Reação em Cadeia da Polimerase em Tempo Real/métodos , Reação em Cadeia da Polimerase em Tempo Real/normas , Modelos Animais de Doenças , Perfilação da Expressão Gênica/métodos , Masculino , RNA Mensageiro/genética , Sirolimo/farmacologia , Ratos Sprague-DawleyRESUMO
In up to two thirds of prostate-specific membrane antigen (PSMA) PET scans, unspecific bone uptake has been described. The aim of this study was to estimate the diagnostic accuracy of [68Ga]Ga-PSMA-11 PET/CT for bone metastases and the occurrence of equivocal lesions. Methods: We analyzed retrospectively 118 patients who underwent a [68Ga]Ga-PSMA-11 PET/CT for initial staging or recurrence evaluation. Lesions were interpreted according to the PSMA reporting and data system (PSMA-RADS) and the prostate cancer molecular imaging standardized evaluation (PROMISE) criteria. The SUVmax and the localization of each lesion were recorded. A combination of prior or follow-up examinations was used as a reference standard to categorize benign and malignant lesions. Correlation between the final diagnosis and imaging or clinicobiochemical parameters was tested. The diagnostic accuracy was calculated for different cutoffs of PSMA-RADS criteria, for PROMISE criteria, and the sequential combination of both. Results: In total, 265 bone abnormalities were identified in 70 of 118 patients. Among these, 148 (55.8%) lesions in 50 (42.4%) patients were classified as PSMA-RADS-3B. There were no PSMA-RADS-3D lesions in our cohort. Equivocal lesions were more frequent on the ribs (30.6%) followed by the pelvis (26.5%), but in the ribs, such an uptake was malignant in 33.3% of cases versus 66.7% in the pelvis. A significant association was found between the final diagnosis and the SUVmax, prostate-specific antigen (PSA), PSA doubling time, International Society of Urological Pathology score, and the number of foci. The sensitivity and specificity were 100% and 63.6% for the PSMA-RADS-3B cutoff, respectively; 40.5% and 100% for the PSMA-RADS-4 cutoff, respectively; and 89.3% and 96.6% for both the PROMISE criteria and the sequential PSMA-RADS/PROMISE strategy, respectively. In the sequential method, the number of equivocal lesions was reduced from 147 to 2. We found that 53% of PSMA-RADS-3B lesions were malignant; 95.5% of lesions classified positive by the sequential method were true positives, whereas 32.6% were false negatives. Conclusion: [68Ga]Ga-PSMA-11 PET/CT has high accuracy for the diagnosis of bone metastases. Equivocal lesions constitute nearly half of the lesions seen on PSMA PET. The sequential combination of PSMA-RADS and PROMISE criteria reduces the number of lesions classified as equivocal. PSMA-RADS-3B lesions which are positive according to the PROMISE criteria should be considered highly suggestive of malignancy.
Assuntos
Neoplasias Ósseas , Ácido Edético , Isótopos de Gálio , Radioisótopos de Gálio , Oligopeptídeos , Tomografia por Emissão de Pósitrons combinada à Tomografia Computadorizada , Neoplasias da Próstata , Humanos , Masculino , Neoplasias da Próstata/diagnóstico por imagem , Neoplasias da Próstata/patologia , Neoplasias Ósseas/secundário , Neoplasias Ósseas/diagnóstico por imagem , Idoso , Tomografia por Emissão de Pósitrons combinada à Tomografia Computadorizada/métodos , Ácido Edético/análogos & derivados , Pessoa de Meia-Idade , Estudos Retrospectivos , Idoso de 80 Anos ou mais , Padrões de ReferênciaRESUMO
The liver plays a vital role in lipid synthesis and metabolism in poultry. To study the functional genes more effectively, it is essential to screen of reliable reference genes in the chicken liver, including females, males, embryos, as well as the Leghorn Male Hepatoma (LMH) cell line. Traditional reference gene screening involves selecting commonly used housekeeping genes (HKGs) for RT-qPCR experiments and using different algorithms to identify the most stable ones. However, this approach is limited in selecting the best reference gene from a small pool of HKGs. High-throughput sequencing technology may offer a solution to this limitation. This study aimed to identify the most consistently expressed genes by utilizing multiple published RNA-seq data of chicken liver and LMH cells. Subsequently, the stability of the newly identified reference genes was assessed in comparison to previously validated stable poultry liver expressed reference genes and the commonly employed HKGs using RT-qPCR. The findings indicated that there is a higher degree of similarity in stable expression genes between female and male liver (such as LSM14A and CDC40). In embryonic liver, the optimal new reference genes were SUDS3, TRIM33, and ERAL1. For LMH cells, the optimal new reference genes were ALDH9A1, UGGT1, and C21H1orf174. However, it is noteworthy that most HKGs did not exhibit stable expression across multiple samples, indicating potential instability under diverse conditions. Furthermore, RT-qPCR experiments proved that the stable expression genes identified from RNA-seq data outperformed commonly used HKGs and certain validated reference genes specific to poultry liver. Over all, this study successfully identified new stable reference genes in chicken liver and LMH cells using RNA-seq data, offering researchers a wider range of reference gene options for RT-qPCR in diverse situations.
Assuntos
Galinhas , Genes Essenciais , Fígado , Reação em Cadeia da Polimerase em Tempo Real , Padrões de Referência , Animais , Galinhas/genética , Fígado/metabolismo , Masculino , Feminino , Reação em Cadeia da Polimerase em Tempo Real/normas , Reação em Cadeia da Polimerase em Tempo Real/métodos , Perfilação da Expressão Gênica/normas , Perfilação da Expressão Gênica/métodos , Linhagem Celular Tumoral , Embrião de GalinhaRESUMO
This study introduces a new NMR-based methodology for identification (ID) and quantification (purity, strength) assays of widely used amino acids. A detailed analysis of four amino acids and their available salts was performed with both a high-field (600â¯MHz) and a benchtop (60â¯MHz) NMR instrument. To assess sensitivity constraints, samples for 1H NMR analysis were initially prepared using only 10â¯mg of analyte and 1â¯mg of maleic acid (MA) as an internal calibrant (IC) and secondary chemical shift reference. The characteristic dispersion of the peak patterns indicating the presence or absence of a counterion (mostly chloride) was conserved at both high and low-field strength instruments, showing that the underlying NMR spectroscopic parameters, i.e., chemical shifts and coupling constants, are independent of the magnetic field strength. However, as the verbal descriptions of 1H NMR spectra are challenging in the context of reference materials and pharmaceutical monographs, an alternative method for the identification (ID) of amino acids is proposed that uses 13C NMR patterns from multiplicity-edited HSQC (ed-HSQC), which are both compound-specific and straightforward to document. For ed-HSQC measurements, the sample amount was increased to 30â¯mg of the analyte and several acquisition parameters were tested, including t1 increments used in the pulse program, number of scans, and repetition time. Excellent congruence with deviations <0.1â¯ppm was achieved for the 13C chemical shifts from 1D 13C NMR spectra (150â¯MHz) vs. those extracted from ed-HSQC (15â¯MHz traces). Finally, all samples of amino acid candidate reference materials were quantified by 1H qNMR (abs-qHNMR) at both 600 and 60â¯MHz. At high field, both IC and relative quantitations were performed, however, with the low-field instrument, only the IC method was used. The results showed that the analyzed reference material candidates were generally highly pure compounds. To achieve adequately low levels of uncertainty for such high-purity materials, the sample amounts were increased to 100â¯mg of analytes and 10â¯mg of the IC and replicates were analyzed for selected amino acids.
Assuntos
Aminoácidos , Espectroscopia de Ressonância Magnética , Aminoácidos/análise , Aminoácidos/química , Espectroscopia de Ressonância Magnética/métodos , Padrões de Referência , Calibragem , Espectroscopia de Prótons por Ressonância Magnética/métodos , Maleatos/química , Maleatos/análiseRESUMO
Analysis of gene expression is a pivotal method at the core of biomarkers studies and cancer research. Currently, RT-qPCR is the most commonly used technique to investigate the expression of certain genes. The accurate and reliable result relies on an effective normalization step using suitable reference genes. The present study was designed to evaluate the eligibility of a set of candidate mRNAs and snoRNA as reference genes in the most common human thyroid neoplasms. We tested the expression levels of eleven mRNA and small RNA housekeeping genes in thyroid samples. The stability of the candidate genes was examined in different thyroid lesions and under different experimental conditions. Results were compared to the reported data in the TCGA database. Our results suggested HPRT1 and ACTB as the best mRNA reference genes, SNORD96A, and SNORD95 as the best miRNA reference genes in thyroid tissues. These genes showed the most stable expression pattern among different thyroid lesions as well as different experimental conditions. The findings in this study highlight the effect of reference genes selection on data interpretation and emphasize the importance of testing for suitable reference genes to be used in specific types of cells and experimental conditions to ensure the validity and accuracy of results.
Assuntos
Perfilação da Expressão Gênica , MicroRNAs , RNA Mensageiro , Neoplasias da Glândula Tireoide , Humanos , Neoplasias da Glândula Tireoide/genética , Neoplasias da Glândula Tireoide/patologia , MicroRNAs/genética , RNA Mensageiro/genética , Perfilação da Expressão Gênica/métodos , Perfilação da Expressão Gênica/normas , Padrões de Referência , Regulação Neoplásica da Expressão Gênica/genética , Reação em Cadeia da Polimerase em Tempo Real/métodos , Biomarcadores Tumorais/genéticaRESUMO
Mass spectrometry (MS) is a widely used analytical technique including medical diagnostics, forensic toxicology, food and water analysis. The gold standard for quantifying compounds involves using stable isotope-labeled internal standards (SIL-IS). However, when these standards are not commercially available, are prohibitively expensive, or are extremely difficult to synthesize, alternative external quantification techniques are employed. We hereby present a novel, convenient and cheap quantification approach-quantification via post column infusion (PCI). As a proof of concept, we demonstrated PCI quantification for the immunosuppressant tacrolimus in whole blood using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The validation results met the criteria according to the guideline on bioanalytical method validation of the European Medicine Agency (EMA), achieving imprecisions and inaccuracies with coefficient of variation and relative bias below 15%. Anonymized and leftover whole blood samples from immunosuppressed patients receiving tacrolimus were used for method comparison (PCI quantification vs. conventional internal standard (IS) quantification). Both methods showed strong agreement with a Pearson correlation coefficient of r = 0.9532. This novel PCI quantification technique (using the target analyte itself) expands the quantification options available in MS, providing reliable results, particularly when internal standards are unavailable or unaffordable. With the current paper, we aim to demonstrate that our innovative PCI technique has great potential to overcome practical issues in quantification and to provide guidance on how to incorporate PCI in existing or new LC-MS methods. Moreover, this study demonstrated a more convenient method for correcting matrix effects in comparison to alternative PCI techniques.