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1.
Am J Forensic Med Pathol ; 40(4): 304-311, 2019 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-31687979

RESUMEN

Semen is crucial evidence for some sex crimes, with its sole confirmation being sperm detection. The success of sperm detection is dependent on all levels of preanalytic and analytic procedures. Specimen collection must be performed by well-trained and competent forensic physicians as well as forensic nurses, with preservation done properly before laboratory transfer. Laboratory procedures should consider archival sperm identification, by visualization, with adequate amounts separated from other cells to obtain male DNA profiles. Differential extraction is robust and accepted as the forensic standard but is time consuming and may result in male DNA loss. Thus, alternative methods and microdevices have been developed. Challenges in sperm isolation from vaginal or buccal epithelium mixes and discrimination in multiperpetrator cases have been overcome by single-cell profiling; however, problems inherent in identical twin discrimination and azoospermia have yet to be solved. Epigenetics and future molecular biomarkers may hold the key; therefore, all laboratory processes must consider DNA and RNA protection. Long-term specimen preservation should be done when possible in light of future confirmatory tests.


Asunto(s)
Ciencias Forenses/métodos , Manejo de Especímenes , Espermatozoides/citología , Separación Celular , Dermatoglifia del ADN , Metilación de ADN , Femenino , Humanos , Captura por Microdisección con Láser , Masculino , Repeticiones de Microsatélite , Reacción en Cadena de la Polimerasa/métodos , Antígeno Prostático Específico/aislamiento & purificación , ARN Mensajero/aislamiento & purificación , Proteínas de Secreción de la Vesícula Seminal/aislamiento & purificación , Delitos Sexuales , Coloración y Etiquetado , Factores de Tiempo
2.
Exp Parasitol ; 206: 107767, 2019 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-31520603

RESUMEN

Schistosoma mansoni eggs can influence immune responses directed at them, and the mechanisms by which this is achieved are being unravelled. Going towards, developing effective tools for the study of how S. mansoni influences naïve T cells, we have developed S. mansoni eggs expressing chicken ovalbumin (OVA), using a lentiviral transduction system. Indeed, such a parasite could be used in conjunction with cells from OT-II transgenic mice as a source of naïve, antigen-specific T cells. The expression of the transgenic protein was confirmed by real-time RT-PCR of OVA-specific mRNA and western blotting using polyclonal antibodies specific for OVA. T cells from OT-II transgenic mice expressing a T cell receptor specific for the OVA323-339 peptide recognised the OVA-transduced S. mansoni eggs. Using flow cytometry on CFSE-labelled OT-II splenocytes, we demonstrated that OVA-transduced eggs elicit higher OT-II proliferative responses than untransduced eggs. The OT-II T cells also produced TNF-α and IFN-γ following exposure to OVA-transduced eggs. In addition, moderate amounts of IL-6 and IL-17A were also detected. In contrast, no IL-10, IL-4 and IL-2 were detected in cultures, whether the cells were stimulated with transduced or untransduced eggs. Thus, the cytokine signatures showed the transfected eggs induced predominantly a Th1 response, with a small amount of IL-6 and IL-17.


Asunto(s)
Ovalbúmina/análisis , Receptores de Antígenos de Linfocitos T/inmunología , Schistosoma mansoni/metabolismo , Linfocitos T/inmunología , Animales , Western Blotting , Pollos , Citocinas/análisis , Citocinas/metabolismo , Electroforesis en Gel de Agar , Femenino , Citometría de Flujo , Interleucina-17/análisis , Interleucina-17/metabolismo , Interleucina-2/análisis , Interleucina-2/metabolismo , Interleucina-6/análisis , Interleucina-6/metabolismo , Hígado/parasitología , Ganglios Linfáticos/citología , Ratones , Ratones Endogámicos BALB C , Ratones Transgénicos , Ovalbúmina/genética , Ovalbúmina/inmunología , Ovalbúmina/metabolismo , Óvulo/metabolismo , ARN Mensajero/análisis , ARN Mensajero/aislamiento & purificación , Reacción en Cadena en Tiempo Real de la Polimerasa , Receptores de Antígenos de Linfocitos T/genética , Transcripción Reversa , Schistosoma mansoni/genética , Schistosoma mansoni/crecimiento & desarrollo , Bazo/citología , Linfocitos T/citología
3.
Int J Radiat Oncol Biol Phys ; 105(4): 834-842, 2019 11 15.
Artículo en Inglés | MEDLINE | ID: mdl-31419511

RESUMEN

PURPOSE: Although preoperative chemoradiotherapy (PCRT) is regarded as a standard treatment for locally advanced rectal cancer, there is no reliable biomarker for predicting responsiveness to PCRT. We aimed to develop a biomarker model for predicting response to PCRT. METHODS AND MATERIALS: We included 184 patients who received PCRT followed by surgical resection and categorized them as good responders (complete or near-complete regression) or poor responders (all other patients). Candidate gene mRNAs were isolated from formalin-fixed paraffin-embedded tumor specimens and analyzed using the NanoString nCounter gene expression assay. Stepwise logistic regression analysis was used to select genes in discovery and training phases. A quantitative radio-responsiveness prediction model was developed and validated using internal cross-validation groups, and the model's predictive value was assessed based on the area under the receiver operating characteristic curve (AUC). RESULTS: By comparing the gene expressions between good and poor responders, we created a multigene mRNA model using FZD9, HRAS, ITGA7, MECOM, MMP3, NKD1, PIK3CD, and PRKCB. This panel showed good ability to predict treatment response (AUC: 0.846 for the whole data set). Internal cross-validation was performed to evaluate the model's predictive stability among 3 cohorts, which provided AUC values of 0.808-0.909. The satisfactory diagnostic performance of the radio-response prediction index persisted regardless of other clinicopathologic features such as clinical T or N stage, interval between radiation and surgery, and pretreatment carcinoembryonic antigen levels (P = .001, 95% CI, 0.686-0.905). CONCLUSIONS: We developed a multigene mRNA-based biomarker model that allows prediction of rectal cancer response to PCRT, which may help identify patients who will benefit most from PCRT.


Asunto(s)
Quimioradioterapia , Perfilación de la Expresión Génica/métodos , Neoplasias del Recto/genética , Neoplasias del Recto/terapia , Adulto , Anciano , Anciano de 80 o más Años , Área Bajo la Curva , Quimioradioterapia/métodos , Esquema de Medicación , Femenino , Marcadores Genéticos , Humanos , Masculino , Persona de Mediana Edad , Cuidados Preoperatorios , ARN Mensajero/aislamiento & purificación , Curva ROC , Neoplasias del Recto/patología , Análisis de Regresión , Estudios Retrospectivos , Resultado del Tratamiento
4.
Exp Parasitol ; 204: 107732, 2019 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-31374184

RESUMEN

In the present study, the cytotoxic effects of amitraz, an octopamine receptor agonist on the reproductive system of engorged adult females of Rhipicephalus (Boophilus) annulatus were assessed using histology, electron microscopy and octopamine beta (OCTß) receptor transcriptional expression analysis. Adult immersion test (AIT) was performed by immersing the fully engorged female ticks for 2 min in different concentrations of amitraz (200, 250, 300, 350 ppm). Amitraz at the dose of 300 ppm, caused an adult tick mortality of 16.66 ±â€¯6.80 per cent, inhibition of fecundity of 75.80 per cent and hatching of 50 per cent of ova laid by treated ticks. Histological changes in the ovaries of ticks collected after 24 h of treatment with amitraz (300 ppm), in comparison with controls (distilled water/methanol) were identified by microscopical examination of sections (4  µm) stained using haematoxylin and eosin. These changes included reduction in size and basophilia of stage I oocytes, presence of cytoplasmic vacuoles of various sizes around germinal vesicle of stage II oocytes, wavy basement membrane of stage III oocytes and reduction in size and number of mature stage IV and V oocytes. Electron microscopy was employed for understanding the structural changes in the ultrathin sections (60 nm) of ovaries. Ticks treated with amitraz showed major ultrastructural changes such as irregular nuclear membrane, crystolysis of mitochondria and detachment of external and internal layers of basal lamina of oocytes. The cDNA synthesized from the total RNA of whole ticks and ovaries of ticks treated with amitraz along with controls were used for relative quantification of Octopamine ß receptor (OCTß-R) expression based on the 2-ΔΔCT method by quantitative real time PCR (qRT PCR). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as endogenous control. Down regulation of expression of OCTß-R mRNA in the ovaries of amitraz treated ticks was observed compared to controls. Thus, the inhibition of fecundity observed in the ticks treated with amitraz can be attributed to the major structural changes and decreased expression of OCT ß receptor mRNA induced by it in the ovary.


Asunto(s)
Insecticidas/farmacología , Rhipicephalus/efectos de los fármacos , Toluidinas/farmacología , Análisis de Varianza , Animales , Membrana Basal/efectos de los fármacos , Membrana Basal/ultraestructura , Regulación hacia Abajo , Femenino , Fertilidad/efectos de los fármacos , Expresión Génica , Microscopía Electrónica de Transmisión , Mitocondrias/efectos de los fármacos , Mitocondrias/ultraestructura , Oocitos/efectos de los fármacos , Oocitos/ultraestructura , Ovario/anatomía & histología , Ovario/efectos de los fármacos , Ovario/ultraestructura , Oviposición/efectos de los fármacos , ARN Mensajero/aislamiento & purificación , ARN Mensajero/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Receptores de Amina Biogénica/agonistas , Receptores de Amina Biogénica/efectos de los fármacos , Rhipicephalus/anatomía & histología , Rhipicephalus/genética , Rhipicephalus/ultraestructura , Espectrofotometría , Control de Ácaros y Garrapatas/métodos , Vacuolas/efectos de los fármacos , Vacuolas/ultraestructura
5.
Nat Commun ; 10(1): 3544, 2019 08 07.
Artículo en Inglés | MEDLINE | ID: mdl-31391463

RESUMEN

Simultaneous measurement of proteins and mRNA in single cells enables quantitative understanding and modeling of cellular functions. Here, we present an automated microfluidic system for multi-parameter and ultra-sensitive protein/mRNA measurements in single cells. Our technology improves the sensitivity of digital proximity ligation assay by up to 55-fold, with a detection limit of 2277 proteins per cell and with detection efficiency of as few as 29 protein molecules. Our measurements using this system reveal higher mRNA/protein correlation in single mammalian cells than previous estimates. Furthermore, time-lapse imaging of herpes simplex virus 1 infected epithelial cells enabled by our device shows that expression of ICP4 -a major transcription factor regulating hundreds of viral genes- is only partially correlated with viral protein counts, suggesting that many cells go through abortive infection. These results highlight the importance of high-sensitivity protein/mRNA quantification for understanding fundamental molecular mechanisms in individual cells.


Asunto(s)
Proteínas/aislamiento & purificación , ARN Mensajero/aislamiento & purificación , Análisis de la Célula Individual/métodos , Animales , Dosificación de Gen , Humanos , Microscopía Intravital/instrumentación , Microscopía Intravital/métodos , Dispositivos Laboratorio en un Chip , Límite de Detección , Microfluídica/instrumentación , Microfluídica/métodos , Análisis de la Célula Individual/instrumentación , Imagen de Lapso de Tiempo/instrumentación , Imagen de Lapso de Tiempo/métodos , Células Vero
6.
Nucleic Acids Res ; 47(16): 8755-8769, 2019 09 19.
Artículo en Inglés | MEDLINE | ID: mdl-31269210

RESUMEN

Thousands of eukaryotic protein-coding genes generate circular RNAs that have covalently linked ends and are resistant to degradation by exonucleases. To prove their circularity as well as biochemically enrich these transcripts, it has become standard in the field to use the 3'-5' exonuclease RNase R. Here, we demonstrate that standard protocols involving RNase R can fail to digest >20% of all highly expressed linear RNAs, but these shortcomings can largely be overcome. RNAs with highly structured 3' ends, including snRNAs and histone mRNAs, are naturally resistant to RNase R, but can be efficiently degraded once a poly(A) tail has been added to their ends. In addition, RNase R stalls in the body of many polyadenylated mRNAs, especially at G-rich sequences that have been previously annotated as G-quadruplex (G4) structures. Upon replacing K+ (which stabilizes G4s) with Li+ in the reaction buffer, we find that RNase R is now able to proceed through these sequences and fully degrade the mRNAs in their entirety. In total, our results provide important improvements to the current methods used to isolate circular RNAs as well as a way to reveal RNA structures that may naturally inhibit degradation by cellular exonucleases.


Asunto(s)
Exorribonucleasas/química , G-Cuádruplex , ARN Mensajero/aislamiento & purificación , ARN Nuclear Pequeño/genética , ARN/aislamiento & purificación , Región de Flanqueo 3' , Células HeLa , Humanos , Litio/farmacología , Poliadenilación , Potasio/farmacología , ARN/química , ARN/genética , ARN/metabolismo , División del ARN/efectos de los fármacos , ARN Mensajero/química , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Nuclear Pequeño/metabolismo , Análisis de Secuencia de ARN
7.
Int J Med Sci ; 16(4): 537-547, 2019.
Artículo en Inglés | MEDLINE | ID: mdl-31171905

RESUMEN

Objective: Retinal neovascularization is a severe complication of many ocular diseases. To clarify the possible functions and therapeutic potential of long non-coding RNAs (lncRNAs) and messenger RNAs (mRNAs) in retinal neovascularization, we assessed their expression profile in a mouse model of oxygen-induced retinopathy (OIR). Methods: Microarray analysis was performed to identify altered lncRNA and mRNA expressions between OIR and control mice. The microarray results were validated by qRT-PCR. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were conducted to determine biological functions and signaling pathways of the altered or interacted mRNAs. A coding-non-coding gene co-expression (CNC) network was constructed to identify the interaction of lncRNAs and mRNAs. Results: We identified 198 up-regulated and 175 down-regulated lncRNAs (fold change≥2.0, P<0.05), respectively in OIR mice compared to control mice. We also identified 412 up-regulated and 127 down-regulated mRNAs (fold change≥2.0, P<0.05), respectively in OIR mice compared to control mice. GO and KEGG analyses suggested that altered mRNAs were enriched in immune system process, exopeptidase activity, ECM-receptor interaction and protein digestion and absorption. Four validated lncRNAs (ENSMUST00000165968, ENSMUST00000153785, ENSMUST00000134409, and ENSMUST00000154285) and the nearby coding gene pairs were analyzed. A CNC network profile based on those validated altered lncRNAs as well as 410 interacted mRNAs was composed of 509 connections. Moreover, the GO and KEGG analyses demonstrated that these interacted mRNAs mainly enriched in blood vessel development, angiogenesis, cell adhesion molecules and leukocyte transendothelial migration pathways. Conclusion: Our data highlight the utility of altered lncRNA and mRNA profiling in understanding the pathogenesis of ischemia-induced retinal neovascularization and further suggest that therapeutic potential of altered lncRNA for retinal neovascularization.


Asunto(s)
ARN Largo no Codificante/genética , ARN Mensajero/genética , Neovascularización Retiniana/genética , Animales , Modelos Animales de Enfermedad , Regulación de la Expresión Génica/genética , Ontología de Genes , Humanos , Ratones , Ratones Endogámicos C57BL , Oxígeno/toxicidad , ARN Largo no Codificante/aislamiento & purificación , ARN Mensajero/aislamiento & purificación , Neovascularización Retiniana/inducido químicamente , Neovascularización Retiniana/patología , Transducción de Señal/genética
8.
Genome Res ; 29(7): 1188-1197, 2019 07.
Artículo en Inglés | MEDLINE | ID: mdl-31235656

RESUMEN

Control of metazoan embryogenesis shifts from maternal to zygotic gene products as the zygotic genome becomes transcriptionally activated. In Drosophila, zygotic genome activation (ZGA) has been thought to occur in two phases, starting with a minor wave, in which a small number of genes become expressed, and progressing to the major wave, in which many more genes are activated. However, technical challenges have hampered the identification of early transcripts or obscured the onset of their transcription. Here, we develop an approach to isolate transcribed mRNAs and apply it over the course of Drosophila early genome activation. Our results increase by 10-fold the genes reported to be activated during what has been thought of as the minor wave and show that early genome activation is continuous and gradual. Transposable-element mRNAs are also produced, but discontinuously. Genes transcribed in the early and middle part of ZGA are short with few if any introns, and their transcripts are frequently aborted and tend to have retained introns, suggesting that inefficient splicing as well as rapid cell divisions constrain the lengths of early transcripts.


Asunto(s)
Drosophila/genética , Regulación del Desarrollo de la Expresión Génica , Genoma de los Insectos , ARN Mensajero/aislamiento & purificación , Animales , Drosophila/embriología , Femenino , Intrones , Transcripción Genética , Cigoto
9.
World J Gastroenterol ; 25(15): 1828-1839, 2019 Apr 21.
Artículo en Inglés | MEDLINE | ID: mdl-31057297

RESUMEN

BACKGROUND: Gastric cancer (GC) is one of the main causes of cancer mortality worldwide. Recent studies on tumor microenvironments have shown that tumor metabolism exerts a vital role in cancer progression. AIM: To investigate whether lysyl oxidase (LOX) and hypoxia-inducible factor 1α (HIF1α) are prognostic and predictive biomarkers in GC. METHODS: A total of 80 tissue and blood samples were collected from 140 patients admitted to our hospital between August 2008 and March 2012. Immunohistochemical staining was performed to measure the expression of LOX and HIF1α in tumor and adjacent tissues collected from patients with GC. Real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis was used to detect the mRNA expression levels of LOX and HIF1α in patients with GC. In addition, single-factor analysis was applied to analyze the relationship between LOX, HIF1α and prognosis of GC. RESULTS: Immunohistochemical staining suggested that the expression levels of LOX and HIF1α increased in tumor tissues from patients with GC. QRT-PCR analysis indicated that mRNA expression of LOX and HIF1α was also upregulated in tumor tissues, which was in accordance with the above results. We also detected expression of these two genes in blood samples. The expression level of LOX and HIF1α was higher in patients with GC than in healthy controls. Additional analysis showed that the expression level of LOX and HIF1α was related to the clinicopathological characteristics of GC. Expression of LOX and HIF1α increased with the number of lymph node metastases , deeper infiltration depth and later tumor-node-metastasis stages. Single-factor analysis showed that high expression of LOX and HIF1α led to poor prognosis of patients with GC. CONCLUSION: LOX and HIF1α can be used as prognostic and predictive biomarkers for GC.


Asunto(s)
Biomarcadores de Tumor/metabolismo , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Metástasis Linfática/patología , Proteína-Lisina 6-Oxidasa/metabolismo , Neoplasias Gástricas/patología , Biomarcadores de Tumor/análisis , Carcinogénesis/patología , Progresión de la Enfermedad , Supervivencia sin Enfermedad , Femenino , Humanos , Masculino , Persona de Mediana Edad , Pronóstico , Proteína-Lisina 6-Oxidasa/análisis , ARN Mensajero/aislamiento & purificación , ARN Mensajero/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Estómago/patología , Neoplasias Gástricas/sangre , Neoplasias Gástricas/mortalidad , Tasa de Supervivencia , Microambiente Tumoral
10.
Biosens Bioelectron ; 137: 58-71, 2019 Jul 15.
Artículo en Inglés | MEDLINE | ID: mdl-31078841

RESUMEN

The development of biosensors for cancer biomarkers has recently been expanding rapidly, offering promising biomedical applications of these sensors as highly sensitive, selective, and inexpensive bioanalytical tools that can provide alternative methodology to that afforded by the advanced hyphenated-instrumental techniques. In this review, we focus particularly on the detection of a member of the inhibitor of apoptosis proteins (IAP) family, protein survivin (Sur), a ubiquitous re-organizer of the cell life cycle with the ability to inhibit the apoptosis and induce an enhanced proliferation leading to the unimpeded cancer growth and metastasis. Herein, we critically evaluate the progress in the development of novel biosensing systems and biosensors for the detection of two survivin (Sur) biomarkers: the Sur protein and its messenger RNA (Sur mRNA), including immunosensors, electrochemical piezo- and impedance-sensors, electrochemi-luminescence biosensors, genosensors based on oligonucleotide molecular beacons (MBs) with fluorescent or electrochemical transduction, as well as the microfluidic and related analytical platforms based on solution chemistry. The in-situ applications of survivin biomarkers' detection technologies to equip nanocarriers of the controlled drug delivery systems with MB-based fluorescence imaging capability, apoptosis control, and mitigation of the acquired drug resistance are also presented and critically evaluated. Finally, we turn the attention to the application of biosensors for the analysis of Sur biomarkers in exosomes and circulating tumor cells for a non-invasive liquid biopsy. The prospect of a widespread screening for early cancers, based on inexpensive point-of-care testing using biosensors and multiplex biosensor arrays, as a means of reducing the high cancer fatality rate, is discussed.


Asunto(s)
Proteínas Reguladoras de la Apoptosis/aislamiento & purificación , Técnicas Biosensibles , Neoplasias/diagnóstico , Survivin/aislamiento & purificación , Apoptosis , Proteínas Reguladoras de la Apoptosis/genética , Detección Precóz del Cáncer , Humanos , Neoplasias/genética , ARN Mensajero/genética , ARN Mensajero/aislamiento & purificación , Survivin/genética
11.
Biosens Bioelectron ; 137: 199-206, 2019 Jul 15.
Artículo en Inglés | MEDLINE | ID: mdl-31100599

RESUMEN

The use of mRNA in biotechnology has expanded with novel applications such as vaccines and therapeutic mRNA delivery recently demonstrated. For mRNA to be used in patients, quality control assays will need to be routinely established. Currently, there is a gap between the highly sophisticated RNA integrity tests available and broader application of mRNA-based products by non-specialist users, e.g. in mass vaccination campaigns. Therefore, the aim of this work was to develop a low-cost biosensor able to test the integrity of a mRNA molecule with low technological requirements and easy end-user application. The biosensor is based on a bi-functional fusion protein, composed by the λN peptide that recognizes its cognate aptamer encoded on the 5' end of the RNA under study and ß-lactamase, which is able to produce a colorimetric response through a simple test. We propose two different mechanisms for signal processing adapted to two levels of technological sophistication, one based on spectrophotometric measurements and other on visual inspection. We show that the proposed λN-ßLac chimeric protein specifically targets its cognate RNA aptamer, boxB, using both gel shift and biolayer interferometry assays. More importantly, the results presented confirm the biosensor performs reliably, with a wide dynamic range and a proportional response at different percentages of full-length RNA, even when gene-sized mRNAs were used. Thus, the features of the proposed biosensor would allow to end-users of products such as mRNA vaccines to test the integrity of the product before its application in a low-cost fashion, enabling a more reliable application of these products.


Asunto(s)
Aptámeros de Nucleótidos/química , Técnicas Biosensibles , Estabilidad del ARN/genética , ARN Mensajero/aislamiento & purificación , Aptámeros de Nucleótidos/genética , Humanos , Interferometría , ARN Mensajero/química , ARN Mensajero/genética
12.
Nat Commun ; 10(1): 1823, 2019 04 23.
Artículo en Inglés | MEDLINE | ID: mdl-31015452

RESUMEN

Granulomas are the pathological hallmark of tuberculosis (TB) and the niche where bacilli can grow and disseminate or the immunological microenvironment in which host cells interact to prevent bacterial dissemination. Here we show 34 immune transcripts align to the morphology of lung sections from Mycobacterium tuberculosis-infected mice at cellular resolution. Colocalizing transcript networks at <10 µm in C57BL/6 mouse granulomas increase complexity with time after infection. B-cell clusters develop late after infection. Transcripts from activated macrophages are enriched at subcellular distances from M. tuberculosis. Encapsulated C3HeB/FeJ granulomas show necrotic centers with transcripts associated with immunosuppression (Foxp3, Il10), whereas those in the granuloma rims associate with activated T cells and macrophages. We see highly diverse networks with common interactors in similar lesions. Different immune landscapes of M. tuberculosis granulomas depending on the time after infection, the histopathological features of the lesion, and the proximity to bacteria are here defined.


Asunto(s)
Linfocitos B/inmunología , Macrófagos/inmunología , Mycobacterium tuberculosis/inmunología , Tuberculoma/inmunología , Tuberculosis Pulmonar/inmunología , Animales , Linfocitos B/metabolismo , Modelos Animales de Enfermedad , Factores de Transcripción Forkhead/genética , Factores de Transcripción Forkhead/inmunología , Factores de Transcripción Forkhead/metabolismo , Humanos , Interleucina-10/genética , Interleucina-10/inmunología , Interleucina-10/metabolismo , Pulmón/inmunología , Pulmón/microbiología , Pulmón/patología , Macrófagos/metabolismo , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Mycobacterium tuberculosis/aislamiento & purificación , ARN Mensajero/aislamiento & purificación , Factores de Tiempo , Tuberculoma/microbiología , Tuberculoma/patología , Tuberculosis Pulmonar/microbiología , Tuberculosis Pulmonar/patología
13.
BMC Cancer ; 19(1): 331, 2019 Apr 08.
Artículo en Inglés | MEDLINE | ID: mdl-30961575

RESUMEN

BACKGROUND: Non-muscular invasive bladder cancer (NMIBC) has a high risk of recurrence. As androgen receptor (AR) reportedly affects bladder cancer, we assessed the correlation between NMIBC recurrence and tumor AR expression in Japanese patients. METHODS: We retrospectively reviewed 53 specimens of non-metastatic NMIBC, with recurrence-free survival (RFS) as the primary endpoint. We used real-time quantitative polymerase chain reaction to quantify AR mRNA expression. Kaplan-Meier product-limit estimators were used to assess RFS distribution, log-rank tests to analyze differences in RFS between high- and low-risk groups; and multivariate analyses of AR mRNA expression and other clinicopathological factors to predict independent factors for RFS. RESULTS: The high AR mRNA-expressing group (n = 43) tended to have a longer median RFS (not reached) than did the low-AR group (n = 10; 9.04 months; P = 0.112). Multivariate analysis showed female sex (hazard ratio [HR]: 7.360, 95% CI: 1.649-32.856, P = 0.009), tumor size ≥3 cm (HR: 23.697, 95% CI: 4.383-128.117, P < 0.001) and low AR mRNA expression (HR: 0.202, 95% CI: 0.048-0.841, P = 0.028) to be independent predictors of shorter RFS. CONCLUSION: Our study showed that low AR mRNA expression level is an independent risk factor for RFS in Japanese patients with NMIBC. Further studies are necessary but AR expression might be a new indicator of recurrence of NMIBC.


Asunto(s)
Biomarcadores de Tumor/metabolismo , ARN Mensajero/metabolismo , Receptores Androgénicos/metabolismo , Neoplasias de la Vejiga Urinaria/patología , Adulto , Anciano , Anciano de 80 o más Años , Biomarcadores de Tumor/genética , Cistectomía/métodos , Supervivencia sin Enfermedad , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Japón , Estimación de Kaplan-Meier , Masculino , Persona de Mediana Edad , Recurrencia Local de Neoplasia , Valor Predictivo de las Pruebas , Pronóstico , ARN Mensajero/aislamiento & purificación , Reacción en Cadena en Tiempo Real de la Polimerasa , Receptores Androgénicos/genética , Estudios Retrospectivos , Factores de Riesgo , Vejiga Urinaria/patología , Vejiga Urinaria/cirugía , Neoplasias de la Vejiga Urinaria/genética , Neoplasias de la Vejiga Urinaria/mortalidad , Neoplasias de la Vejiga Urinaria/cirugía
14.
Nat Commun ; 10(1): 1626, 2019 04 09.
Artículo en Inglés | MEDLINE | ID: mdl-30967537

RESUMEN

MicroRNAs (miRNAs) are key mediators of post-transcriptional gene expression silencing. So far, no comprehensive experimental annotation of functional miRNA target sites exists in Drosophila. Here, we generated a transcriptome-wide in vivo map of miRNA-mRNA interactions in Drosophila melanogaster, making use of single nucleotide resolution in Argonaute1 (AGO1) crosslinking and immunoprecipitation (CLIP) data. Absolute quantification of cellular miRNA levels presents the miRNA pool in Drosophila cell lines to be more diverse than previously reported. Benchmarking two CLIP approaches, we identify a similar predictive potential to unambiguously assign thousands of miRNA-mRNA pairs from AGO1 interaction data at unprecedented depth, achieving higher signal-to-noise ratios than with computational methods alone. Quantitative RNA-seq and sub-codon resolution ribosomal footprinting data upon AGO1 depletion enabled the determination of miRNA-mediated effects on target expression and translation. We thus provide the first comprehensive resource of miRNA target sites and their quantitative functional impact in Drosophila.


Asunto(s)
Proteínas Argonauta/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Regulación de la Expresión Génica , MicroARNs/metabolismo , ARN Mensajero/metabolismo , Animales , MicroARNs/genética , MicroARNs/aislamiento & purificación , ARN Mensajero/genética , ARN Mensajero/aislamiento & purificación , Análisis de Secuencia de ARN , Transcriptoma/genética
15.
J Vis Exp ; (145)2019 03 17.
Artículo en Inglés | MEDLINE | ID: mdl-30933064

RESUMEN

A single biological sample holds a plethora of information, and it is now common practice to simultaneously investigate several macromolecules to capture a full picture of the multiple levels of molecular processing and changes between different conditions. This protocol presents the method of isolating DNA, RNA, and protein from the same sample of the nematode Caenorhabditis elegans to remove the variation introduced when these biomolecules are isolated from replicates of similarly treated but ultimately different samples. Nucleic acids and protein are extracted from the nematode using the acid guanidinium thiocyanate-phenol-chloroform extraction method, with subsequent precipitation, washing, and solubilization of each. We show the successful isolation of RNA, DNA, and protein from a single sample from three strains of nematode and HeLa cells, with better protein isolation results in adult animals. Additionally, guanidinium thiocyanate-phenol-chloroform-extracted protein from nematodes improves the resolution of larger proteins, with enhanced detectable levels as observed by immunoblotting, compared to the traditional RIPA extraction of protein. The method presented here is useful when investigating samples using a multiomic approach, specifically for the exploration of the proteome and transcriptome. Techniques that simultaneously assess multiomics are appealing because molecular signaling underlying complex biological phenomena is thought to occur at complementary levels; however, it has become increasingly common to see that changes in mRNA levels do not always reflect the same change in protein levels and that the time of collection is relevant in the context of circadian regulations. This method removes any intersample variation when assaying different contents within the same sample (intrasample.).


Asunto(s)
Proteínas de Caenorhabditis elegans/aislamiento & purificación , Caenorhabditis elegans/metabolismo , Nucleótidos/aislamiento & purificación , Animales , ADN/aislamiento & purificación , Regulación de la Expresión Génica , Células HeLa , Humanos , ARN Mensajero/genética , ARN Mensajero/aislamiento & purificación , ARN Mensajero/metabolismo
16.
Artículo en Inglés | MEDLINE | ID: mdl-30735812

RESUMEN

INTRODUCTION: FFPE samples represent a rich pool of tissue samples for retrospective analyses of mRNA and miRNA analyses. However, the initial formalin fixation introduces a chemical modification of RNA and causes its degradation, therefore, a longer storage of tissue in formalin is predicted to render ribonucleic acids lost for isolation and subsequent analyses. METHODS: Herein, we tested the impact of several factors on isolation of total RNA and detection with RT-qPCR from mouse liver tissue stored for over two years in formalin at room temperature. RESULTS: The incubation of tissue in TAE buffer before RNA isolation yielded higher concentration and purity of RNA and also improved subsequent analyses. Using gene-specific primers for cDNA synthesis and shorter PCR products (under 70 bp) significantly improved RT-qPCR analyses. We also compared the level of expression and differential expression of mRNA and miRNA with matched fresh frozen liver tissue, and observed similar changes in differential expression of selected mRNA and miRNA as in matched fresh frozen tissue. DISCUSSION: Conclusion is that adjustments of the protocol can substantially improve analyses of mRNA and miRNA from tissues stored in formalin for longer time.


Asunto(s)
Formaldehído , MicroARNs/análisis , MicroARNs/aislamiento & purificación , ARN Mensajero/análisis , ARN Mensajero/aislamiento & purificación , Fijación del Tejido/métodos , Animales , Secciones por Congelación , Hígado , Ratones , Ratones Noqueados , Reacción en Cadena en Tiempo Real de la Polimerasa , Reproducibilidad de los Resultados , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Esterol 14-Desmetilasa/deficiencia
17.
Methods Mol Biol ; 1914: 169-196, 2019.
Artículo en Inglés | MEDLINE | ID: mdl-30729465
18.
Mol Biol Rep ; 46(1): 1257-1262, 2019 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-30788763

RESUMEN

MicroRNAs (miRNAs) are small non-coding RNAs ~ 19-25 nucleotides long that are involved in the regulation of gene expression. They negatively regulate the gene expression via inhibition or complete degradation of mRNAs by binding the complementary target sequences in 3' untranslated region. The present investigation was aimed at profiling of miRNAs expressed in the Bubaline mammary tissue at dry stage of lactation cycle. Small RNAs were isolated from freshly collected mammary tissues and T4 RNA ligase was used to ligate the enriched miRNAs with 3' and 5' linker sequences in two separate reactions. cDNA copies were synthesized from linkered small RNAs followed by the PCR amplification. The PCR products were resolved on 15% non-denaturing polyacrylamide gel electrophoresis by gelstar staining. The PCR products were cloned using pGEM®-T easy vector system and the desired clones (with linkered small RNA sequences) were confirmed using restriction digestion of plasmids with EcoRI. Out of 15 Bubaline small RNA sequences, eight sequences (Seq. ID I-VIII) matched the size range of miRNA molecules i.e., 18-26 nucleotides. The Bubaline small RNA sequences II and III showed partial alignment with various mammalian and non-mammalian miRNAs. The small RNA sequences obtained in the present study did not show any perfect match with already reported mRNA, rRNA or tRNA sequences in different databases. Hence, only the Bubaline small RNA sequences that showed partial homology with miRNAs were considered as putative Bubaline miRNAs. The present study established the basic repertoire of miRNAs expressed at dry stage of lactation in Bubaline mammary gland.


Asunto(s)
Búfalos/genética , MicroARNs/genética , MicroARNs/aislamiento & purificación , Animales , Secuencia de Bases/genética , Simulación por Computador , Femenino , Glándulas Mamarias Animales/metabolismo , Reacción en Cadena de la Polimerasa , ARN Mensajero/genética , ARN Mensajero/aislamiento & purificación
19.
Acta Cytol ; 63(6): 479-488, 2019.
Artículo en Inglés | MEDLINE | ID: mdl-30783027

RESUMEN

Liquid biopsy represents the analysis of tumor-derived material in the blood and other body fluids of cancer patients. This portrays a minimally invasive detection tool for molecular biomarkers. Liquid biopsy has emerged as a complementary or alternative method to surgical biopsy. This non-invasive detection tool overcomes the recurrent problems in the clinical assessment of tumors that stem from the lack of accessibility to the tumor tissue and its clonal heterogeneity. Moreover, body fluid-derived components have shown to reflect the genetic profile of both primary and metastatic lesions and provide a real-time monitoring of tumor dynamics, representing a great promise for personalized medicine. This review will highlight the latest breakthroughs and the current applications of several tumor-derived biomarkers that can be found in body fluids. The authors will focus on tumor-derived exosomes, tumor-educated platelets, and circulating tumor miRNAs and mRNAs, and how these can be used for tumor detection.


Asunto(s)
Biomarcadores de Tumor/sangre , ADN Tumoral Circulante/sangre , Recurrencia Local de Neoplasia/diagnóstico , Neoplasias/diagnóstico , Células Neoplásicas Circulantes/química , Antineoplásicos/uso terapéutico , Biomarcadores de Tumor/aislamiento & purificación , Ácidos Nucleicos Libres de Células/sangre , Ácidos Nucleicos Libres de Células/aislamiento & purificación , ADN Tumoral Circulante/aislamiento & purificación , Exosomas/química , Exosomas/patología , Humanos , Biopsia Líquida/métodos , MicroARNs/sangre , MicroARNs/aislamiento & purificación , Monitoreo Fisiológico , Mutación , Recurrencia Local de Neoplasia/sangre , Recurrencia Local de Neoplasia/tratamiento farmacológico , Recurrencia Local de Neoplasia/patología , Neoplasias/sangre , Neoplasias/tratamiento farmacológico , Neoplasias/patología , Células Neoplásicas Circulantes/patología , Medicina de Precisión/métodos , Pronóstico , ARN Mensajero/sangre , ARN Mensajero/aislamiento & purificación
20.
PLoS One ; 14(2): e0212297, 2019.
Artículo en Inglés | MEDLINE | ID: mdl-30779773

RESUMEN

Polysome profiling is a widely used method to monitor the translation status of mRNAs. Although it is theoretically a simple technique, it is labor intensive. Repetitive polysome fractionation rapidly generates a large number of samples to be handled in the downstream processes of protein elimination, RNA extraction and quantification. Here, we propose a multiplex polysome profiling experiment in which distinct cellular extracts are pooled before loading on the sucrose gradient for fractionation. We used the multiplexing method to study translation in E. coli. Multiplexing polysome profiling experiments provided similar mRNA translation status to that obtained with the non-multiplex method with comparable distribution of mRNA copies between the polysome profiling fractions, similar ribosome occupancy and ribosome density. The multiplexing method was used for parallel characterization of gene translational responses to changing mRNA levels. When the mRNA level of two native genes, cysZ and lacZ was increased by transcription induction, their global translational response was similar, with a higher ribosome load leading to increased ribosome occupancy and ribosome densities. However the pattern and the magnitude of the translational response were gene specific. By reducing the number of polysome profiling experiments, the multiplexing method saved time and effort and reduced cost and technical bias. This method would be useful to study the translational effect of mRNA sequence-dependent parameters that often require testing multiple samples and conditions in parallel.


Asunto(s)
Escherichia coli/genética , Polirribosomas/genética , ARN Mensajero/metabolismo , Regiones no Traducidas 3' , Escherichia coli/crecimiento & desarrollo , Proteínas de Escherichia coli/genética , Polirribosomas/metabolismo , Biosíntesis de Proteínas , ARN Mensajero/aislamiento & purificación , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos
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