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Cell spheroids are an important three-dimensional (3D) model for in vitro testing and are gaining interest for their use in clinical applications. More natural 3D cell culture environments that support cell-cell interactions have been created for cancer drug discovery and therapy applications, such as the scaffold-free 3D Petri Dish® technology. This technology uses reusable and autoclavable silicone micro-molds with different topographies, and it conventionally uses gelled agarose for hydrogel formation to preserve the topography of the selected micro-mold. The present study investigated the feasibility of using a patterned Poly(vinyl alcohol) hydrogel using the circular topography 12-81 (9 × 9 wells) micro-mold to form HeLa cancer cell spheroids and compare them with the formed spheroids using agarose hydrogels. PVA hydrogels showed a slightly softer, springier, and stickier texture than agarose hydrogels. After preparation, Fourier transform infrared (FTIR) spectra showed chemical interactions through hydrogen bonding in the PVA and agarose hydrogels. Both types of hydrogels favor the formation of large HeLa spheroids with an average diameter of around 700-800 µm after 72 h. However, the PVA spheroids are more compact than those from agarose, suggesting a potential influence of micro-mold surface chemistry on cell behavior and spheroid formation. This was additionally confirmed by evaluating the spheroid size, morphology, integrity, as well as E-cadherin and Ki67 expression. The results suggest that PVA promotes stronger cell-to-cell interactions in the spheroids. Even the integrity of PVA spheroids was maintained after exposure to the drug cisplatin. In conclusion, the patterned PVA hydrogels were successfully prepared using the 3D Petri Dish® micro-molds, and they could be used as suitable platforms for studying cell-cell interactions in cancer drug therapy.
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Breast cancer (BC) affects millions of women worldwide, causing over 500,000 deaths annually. It is the leading cause of cancer mortality in women, with 70% of deaths occurring in developing countries. Elastography, which evaluates tissue stiffness, is a promising real-time minimally invasive technique for BC diagnosis. This study assessed strain elastography (SE) and the fat-to-lesion (F/L) index for BC diagnosis. This prospective study included 216 women who underwent SE, ultrasound, mammography, and breast biopsy (108 malignant, 108 benign). Three expert radiologists performed imaging and biopsies. Mean F/L index was 3.70 ± 2.57 for benign biopsies and 18.10 ± 17.01 for malignant. We developed two predictive models: a logistic regression model with AUC 0.893, 79.63% sensitivity, 87.62% specificity, 86.9% positive predictive value (+PV), and 80.7% negative predictive value (-PV); and a neural network with AUC 0.902, 80.56% sensitivity, 88.57% specificity, 87.9% +PV, and 81.6% -PV. The optimal Youden F/L index cutoff was >5.76, with 84.26% sensitivity and specificity. The F/L index positively correlated with BI-RADS (Spearman's r = 0.073, p < 0.001) and differed among molecular subtypes (Kruskal-Wallis, p = 0.002). SE complements mammography for BC diagnosis. With adequate predictive capacity, SE is fast, minimally invasive, and useful when mammography is contraindicated.
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Neoplasias Encefálicas , Neoplasias da Mama , Técnicas de Imagem por Elasticidade , Feminino , Humanos , Neoplasias da Mama/diagnóstico por imagem , Neoplasias da Mama/patologia , Técnicas de Imagem por Elasticidade/métodos , Estudos Prospectivos , Ultrassonografia Mamária/métodos , Mamografia , Biópsia , Sensibilidade e EspecificidadeRESUMO
Pregnancy is a challenging metabolic and physiological condition. The aim of this study was to include a second demanding situation as a low protein/high carbohydrate diet (LPHCD) to characterize the histological and functional responses of the maternal liver. It is unknown how the maternal liver responds during early and late pregnancy to LPHCD intake. We explored early pregnancy (3 and 8 gestational age, G) and late pregnancy (15 and 20 G). The results indicated that pregnant rats under control diet showed an evident presence of ballooned hepatocytes, lipid vesicles and edema at late pregnancy (15G); in contrast, pregnant rats under LPHCD showed similar pattern of histological modification but at early pregnancy (3G). Unexpectedly, the serum biomarkers didn't display functional alterations in either group, despite of the evident histological changes no liver malfunction was detected. We conclude that pregnant rats fed with control diet and experimental LPHCD, are subjected to metabolic and physiological conditions that impact the histopathological condition of the maternal liver. Control diet promoted the histological modifications during late pregnancy whereas LPCHCD advanced the onset of these changes. Further experiments are needed to explore the biochemical mechanisms that underlie these histological modifications. Our results are also an example of the resilience associated with the pregnancy: since no functional hepatic alterations accompanied the histopathological changes, another conclusion is that no evident pathological condition was detected in this nutritional protocol.
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Fígado Gorduroso , Falência Hepática , Feminino , Gravidez , Ratos , Animais , Fígado Gorduroso/patologia , Fígado/metabolismo , Hepatócitos/metabolismo , CarboidratosRESUMO
Multiple sclerosis (MS) is a chronic autoimmune inflammatory disease that affects the nervous system. Peripheral blood leukocyte telomere length (LTL) and mitochondrial DNA copy number (mtDNA-CN) are potential biomarkers of neurological disability and neural damage. Our objective was to assess the LTL and mtDNA-CN in relapsing-remitting MS (RRMS). We included 10 healthy controls, 75 patients with RRMS, 50 of whom had an Expanded Disability Status Scale (EDSS) from 0 to 3 (mild to moderate disability), and 25 had an EDSS of 3.5 to 7 (severe disability). We use the Real-Time Polymerase Chain Reaction (qPCR) technique to quantify absolute LTL and absolute mtDNA-CN. ANOVA test show differences between healthy control vs. severe disability RRMS and mild-moderate RRMS vs. severe disability RRMS (p = 0.0130). LTL and mtDNA-CN showed a linear correlation in mild-moderate disability RRMS (r = 0.378, p = 0.007). Furthermore, we analyzed LTL between RRMS groups with a ROC curve, and LTL can predict severe disability (AUC = 0.702, p = 0.0018, cut-off < 3.0875 Kb, sensitivity = 75%, specificity = 62%), whereas the prediction is improved with a logistic regression model including LTL plus age (AUC = 0.762, p = 0.0001, sensitivity = 79.17%, specificity = 80%). These results show that LTL is a biomarker of disability in RRMS and is correlated with mtDNA-CN in mild-moderate RRMS patients.
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Esclerose Múltipla Recidivante-Remitente , Esclerose Múltipla , Humanos , Esclerose Múltipla Recidivante-Remitente/genética , Esclerose Múltipla/genética , DNA Mitocondrial/genética , Variações do Número de Cópias de DNA , Leucócitos , Telômero/genéticaRESUMO
BACKGROUND: Dyskeratosis congenita is a rare disease characterized by bone marrow failure and a clinical triad of oral leukoplakia, nail dystrophy, and abnormal skin pigmentation. The genetics of dyskeratosis congenita include mutations in genes involved in telomere maintenance, including TINF2. CASE SUMMARY: Here, we report a female patient who presented thrombocytopenia, anemia, reticulate hyperpigmentation, dystrophy in fingernails and toenails, and leukoplakia on the tongue. A histopathological study of the skin showed dyskeratocytes; however, a bone marrow biopsy revealed normal cell morphology. The patient was diagnosed with dyskeratosis congenita, but her family history did not reveal significant antecedents. Whole-exome sequencing showed a novel heterozygous punctual mutation in exon 6 from the TINF2 gene, namely, NM_001099274.1:c.854delp.(Val285Alafs*32). An analysis of telomere length showed short telomeres relative to the patient's age. CONCLUSION: The disease in this patient was caused by a germline novel mutation of TINF2 in one of her parents.
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ABSTRACT This work analyzes the electrical impedance (EI) measurement of cervical mucus (CM) using a device to determine the fertile window. In this prospective and longitudinal study, fourteen healthy women aged 18 to 44 were enrolled to evaluate three menstrual cycles. EI was measured through a medical device inserted into the vagina for two minutes daily. Patients were monitored by urine luteinizing hormone (LH) strip, blood collection, and vaginal ultrasound to visualize the dominant follicle. Finally, the predictive EI capacity was validated by the receiver operating characteristic (ROC) of anovulatory vs. ovulatory impedances. The peak of LH was 35.7 (±4.5) mUI/ml and the dominant follicle size was 15.45 mm (±0.559). There were statistical differences in EI measurements between the follicular and luteal phases vs. the ovulation phase (p<0.0361 and p<0.0160). After data normalization, an area under the ROC curve (AUC) of 0.713 (P value= 0.0253), a Youden J index of 0.4545Ω, a sensitivity of 63.6%, and a specificity of 81.8% were found. Low EI in the ovulatory period belongs to the LH ovulatory peak and follicular release. EI can be used for ovulation monitoring, birth control, or promoting pregnancy as a safe and innocuous method.
RESUMEN Este trabajo analiza la medición de la impedancia eléctrica (IE) del moco cervical (MC) mediante un dispositivo para determinar la ventana fértil. En este estudio prospectivo y longitudinal, se incluyeron 14 mujeres sanas de 18 a 44 años para evaluar tres ciclos menstruales. La IE se midió a través de un dispositivo médico colocado en la vagina durante dos minutos diarios. Las pacientes fueron monitoreadas con una tira de hormona luteinizante (LH) en orina, recolección de sangre y ultrasonido vaginal para visualizar el folículo dominante. Finalmente, la capacidad predictiva de IE fue validada por la curva ROC (receiver operating characteristic) de impedancias anovulatorias vs. ovulatorias. El pico de LH fue de 35.7(±4.5) mUI/ml; el folículo de tamaño dominante fue de 15.45 mm (±0.559). Se encontraron diferencias estadísticas para la medición de la IE de las fases folicular y lútea versus la fase de ovulación (p<0.0361 y p<0.0160). Después de la normalización de los datos, se encontró un área bajo la curva ROC (AUC) de 0.713 (valor de P = 0.0253), un índice de Youden J de 0.4545 Ω, sensibilidad del 63.6 % y especificidad del 81.8 %. La IE baja en el período ovulatorio que pertenece al pico ovulatorio de LH y liberación folicular. La IE se puede utilizar para el control de la ovulación, el control de la natalidad o la promoción del embarazo como método seguro e inocuo.
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Background: Several studies have shown that patients with cancer have antibodies in serum that react with cellular autoantigens, known as Tumor-Associated Antigens (TAA). The present work aimed to determine whether a mini-array comprising four recombinant TAA increases the detection of specific serum antibodies for the diagnosis of early-stage breast cancer. Methods: The mini-array included Alpha 1-AntiTrypsin (A1AT), TriosePhosphate Isomerase 1 (TPI1), Peptidyl-Prolyl cis-trans Isomerase A (PPIA), and PeroxiReDoXin 2 (PRDX2) full-length recombinant proteins. The proteins were produced after gene cloning, expression, and purification, and were verified by Western blot assays. Then, Dot-Blot was performed to find antibodies against the four TAA in 12 sera from women with early-stage breast cancer (stage II) and 12 sera from healthy women. Results: Antibody detection against individual TAA in early-stage breast cancer sera ranged from 58.3% to 83.3%. However, evaluation of the four TAA showed that there was a positive antibody reaction reaching a sensitivity of 100% and a specificity of 85% in early-stage breast cancer, suggesting that this mini-array must be evaluated as a clinical diagnostic tool for early-stage breast cancer in a larger sample size. Conclusion: Our results suggest that TAA mini-arrays may provide a promising and powerful method for improving the detection of breast cancer in Mexican women.
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Biomarcadores Tumorais/análise , Técnicas Biossensoriais , Neoplasias da Mama/diagnóstico , Soro/química , Adulto , Antígenos de Neoplasias , Neoplasias da Mama/sangue , Feminino , Testes Hematológicos , Humanos , Pessoa de Meia-IdadeRESUMO
The objective of the present study was to identify volatile prints from exhaled breath, termed breath-print, from breast cancer (BC) patients and healthy women by means of an electronic nose and to evaluate its potential use as a screening method. A cross-sectional study was performed on 443 exhaled breath samples from women, of whom 262 had been diagnosed with BC by biopsy and 181 were healthy women (control group). Breath-print analysis was performed utilizing the Cyranose 320 electronic nose. Group data were evaluated by principal component analysis (PCA), canonical discriminant analysis (CDA), and support vector machine (SVM), and the test's diagnostic power was evaluated by means of receiver operating characteristic (ROC) curves. The results obtained using the model generated from the CDA, which best describes the behavior of the assessed groups, indicated that the breath-print of BC patients was different from that of healthy women and that they presented with a variability of up to 98.8% and a correct classification of 98%. The sensitivity, specificity, negative predictive value, and positive predictive value reached 100% according to the ROC curve. The present study demonstrates the capability of the electronic nose to separate between healthy subjects and BC patients. This research could have a beneficial impact on clinical practice as we consider that this test could probably be used at the first point before the application of established gold tests (mammography, ultrasound, and biopsy) and substantially improve screening tests in the general population.
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Neoplasias da Mama/diagnóstico , Testes Respiratórios/métodos , Nariz Eletrônico , Expiração , Compostos Orgânicos Voláteis/análise , Adulto , Estudos de Casos e Controles , Estudos Transversais , Análise Discriminante , Feminino , Humanos , Pessoa de Meia-Idade , Análise de Componente Principal , Curva ROC , Reprodutibilidade dos TestesRESUMO
The present study aimed to analyze the methylation pattern of the MIR200 family in the colorectal tissues and peripheral blood of colorectal cancer (CRC) patients. Previous informed consent, 102 samples of colorectal tissues (tumor and adjacent normal tissues) and 40 peripheral blood samples were collected from CRC patients. Additionally, we included a reference group of 40 blood samples. DNA extraction was done for colorectal tissues and peripheral blood. For methylation-specific PCR, we used bisulfite-treated DNA and controls for methylated and unmethylated DNA were included to each assay. PCR fragments were separated by 6% polyacrylamide gel electrophoresis. Methylation-positive and methylation-negative results were confirmed by bisulfite genomic sequencing technique. We analyzed 102 colorectal tissues and 40 blood samples from 51 CRC patients. MIR200B/MIR200A/MIR429 methylation analysis discloses no differences among tissues (p>0.05). However, MIR200C/MIR141 methylation showed differences between colorectal tissues and peripheral blood of CRC patients (p<0.0001) and mainly methylated alleles were observed in peripheral blood. These findings suggest a tissue-specific methylation pattern for the MIR200C/MIR141 promoter.
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Neoplasias Colorretais/genética , Metilação de DNA , MicroRNAs/metabolismo , Adulto , Idoso , Neoplasias Colorretais/sangue , Neoplasias Colorretais/metabolismo , DNA/análise , Feminino , Humanos , Masculino , México , MicroRNAs/sangue , MicroRNAs/genética , Pessoa de Meia-IdadeRESUMO
BACKGROUND: Diffuse gastric cancer (DGC) is associated with the reduction or absence of the expression of the cell adhesion protein E-cadherin (encoded by the CDH1 gene). Molecular characteristics are less well described for mixed gastric cancer (MGC). The main somatic alterations that have been described in the CDH1 gene are mutations, loss of heterozygosity (LOH) and promoter methylation. The aim was to analyze CDH1 somatic alterations in Mexican patients with diffuse and mixed gastric cancer. METHODS: We searched for mutations in the CDH1 gene in tumor DNA from DGC (n = 13) and MGC (n = 7) patients by next generation sequencing (NGS). Validation of findings was performed using Sanger sequencing. LOH was analyzed using dinucleotide repeat markers surrounding the CDH1 gene, and methylation was investigated by DNA bisulfite conversion and sequencing. E-cadherin protein deficiency was analyzed by immunohistochemistry. RESULTS: Seventeen point variants were identified by NGS, 13 of them were validated by Sanger sequencing. Only 1/13 had not been previously reported (c.-137C > A), and 12/13 were already reported as polymorphisms. Two DGC cases presented LOH at the locus 16q22.1 (13.3%). CDH1 promoter methylation was positive in (7/11) 63.6% and (4/6) 66.6% of the cases with DGC and MGC, respectively. E-cadherin protein deficiency was observed in 58.3% of DGC cases while 100% in MGC cases. CONCLUSIONS: While no pathogenic somatic mutations were found that could explain the diffuse histology of gastric cancer in DGC and MGC, methylation was the most common somatic inactivation event of the CDH1 gene, and LOH was rare. The previously unreported c.-137C > A variant modify the CDH1 gene expression since it alters the binding sites for transcription factors.
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Antígenos CD/genética , Caderinas/genética , Mutação , Neoplasias Gástricas/genética , Neoplasias Gástricas/patologia , Alelos , Metilação de DNA , Análise Mutacional de DNA , Feminino , Regulação Neoplásica da Expressão Gênica , Predisposição Genética para Doença , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Perda de Heterozigosidade , Masculino , México , Polimorfismo Genético , Regiões Promotoras GenéticasRESUMO
BACKGROUND: Human breath contains volatile organic compounds (VOCs) that are biomarkers of breast cancer. We investigated the positive and negative predictive values (PPV and NPV) of breath VOC biomarkers as indicators of breast cancer risk. METHODS: We employed ultra-clean breath collection balloons to collect breath samples from 54 women with biopsy-proven breast cancer and 124 cancer-free controls. Breath VOCs were analyzed with gas chromatography (GC) combined with either mass spectrometry (GC MS) or surface acoustic wave detection (GC SAW). Chromatograms were randomly assigned to a training set or a validation set. Monte Carlo analysis identified significant breath VOC biomarkers of breast cancer in the training set, and these biomarkers were incorporated into a multivariate algorithm to predict disease in the validation set. In the unsplit dataset, the predictive algorithms generated discriminant function (DF) values that varied with sensitivity, specificity, PPV and NPV. RESULTS: Using GC MS, test accuracy = 90% (area under curve of receiver operating characteristic in unsplit dataset) and cross-validated accuracy = 77%. Using GC SAW, test accuracy = 86% and cross-validated accuracy = 74%. With both assays, a low DF value was associated with a low risk of breast cancer (NPV > 99.9%). A high DF value was associated with a high risk of breast cancer and PPV rising to 100%. CONCLUSION: Analysis of breath VOC samples collected with ultra-clean balloons detected biomarkers that accurately predicted risk of breast cancer.
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Biomarcadores , Neoplasias da Mama/diagnóstico , Neoplasias da Mama/metabolismo , Compostos Orgânicos Voláteis/metabolismo , Fatores Etários , Algoritmos , Biópsia , Neoplasias da Mama/epidemiologia , Feminino , Cromatografia Gasosa-Espectrometria de Massas , Humanos , Prognóstico , Curva ROC , Reprodutibilidade dos Testes , Medição de Risco , Fatores de Risco , Sensibilidade e Especificidade , Compostos Orgânicos Voláteis/análiseRESUMO
Bone fractures are a worldwide public health concern. Therefore, improving understanding of the bone healing process at a molecular level, which could lead to the discovery of potential therapeutic targets, is important. In the present study, a model of open tibial fractures with hematoma disruption, periosteal rupture and internal fixation in 6-month-old male Wistar rats was established, in order to identify expression patterns of key genes and their protein products throughout the bone healing process. A tibial shaft fracture was produced using the three-point bending technique, the hematoma was drained through a 4-mm incision on the medial aspect of the tibia and the fracture stabilized by inserting a needle into the medullary canal. Radiographs confirmed that the induced fractures were diaphyseal and this model was highly reproducible (kappa inter-rater reliability, 0.82). Rats were sacrificed 5, 14, 21, 28 and 35 days post-fracture to obtain samples for histological, immunohistochemical and molecular analysis. Expression of interleukin-1ß (Il-1ß), transforming growth factor-ß2 (Tgf-ß2), bone morphogenetic protein-6 (Bmp-6), bone morphogenetic protein-7 (Bmp-7) and bone γ-carboxyglutamic acid-containing protein (Bglap) genes was determined by reverse transcription quantitative polymerase chain reaction and protein expression was evaluated by immunohistochemistry, while histological examination allowed characterization of the bone repair process. Il-1ß showed a biphasic expression, peaking 5 and 28 days post-fracture. Expression of Tgf-ß2, Bmp-6 and Bmp-7 was restricted to the period 21 days post-fracture. Bglap expression increased gradually, peaking 21 days post-fracture, although it was expressed in all evaluated stages. Protein expression corresponded with the increased expression of their corresponding genes. In conclusion, a clear and well-defined expression pattern of the evaluated genes and proteins was observed, where their maximal expression correlated with their known participation in each stage of the bone healing process.