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1.
Rinsho Ketsueki ; 62(4): 245-250, 2021.
Artículo en Japonés | MEDLINE | ID: mdl-33967147

RESUMEN

Acute myeloid leukemia (AML) associated with double-minute chromosomes (dmin) is a rare condition and has a poor prognosis. A 68-year-old man with leukocytosis and thrombocytopenia was admitted to our hospital. Bone marrow aspiration showed that 79.5% of myeloblasts were positive for myeloperoxidase. The patient was diagnosed with acute myeloid leukemia (French-American-British classification: M2, World Health Organization classification: AML, not otherwise specified, AML with maturation). Chromosomal analysis revealed the presence of dmin: 45, X, -Y, 5-33 dmin. Fluorescence in situ hybridization revealed multiple MYC signals, and spectral karyotyping showed that dmin was derived from chromosome 8. These findings indicated resistance to chemotherapy alone. After the standard induction therapy with daunorubicin and cytarabine, the number of myeloblasts in the bone marrow decreased, and the amplified MYC signals disappeared. Then, the patient achieved complete remission. Reportedly, most patients with AML correlated with dmin have a complex karyotype, except for this case. Owing to the absence of a complex karyotype, the patient had good sensitivity to chemotherapy. Further studies with a larger population of patients with AML associated with dmin, but without complex karyotypes, should be conducted to accurately predict prognosis in such cases.


Asunto(s)
Genes myc , Leucemia Mieloide Aguda , Anciano , Cromosomas , Amplificación de Genes , Humanos , Hibridación Fluorescente in Situ , Quimioterapia de Inducción , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/genética , Masculino , Inducción de Remisión
2.
BMC Plant Biol ; 21(1): 167, 2021 Apr 06.
Artículo en Inglés | MEDLINE | ID: mdl-33823797

RESUMEN

BACKGROUND: DNA sequence composition affects meiotic recombination. However, the correlation between tandem repeat composition and meiotic recombination in common wheat (Triticum aestivum L.) is unclear. RESULTS: Non-denaturing fluorescent in situ hybridization (ND-FISH) with oligonucleotide (oligo) probes derived from tandem repeats and single-copy FISH were used to investigate recombination in three kinds of the long arm of wheat 5A chromosome (5AL). 5AL535-18/275 arm carries the tandem repeats pTa-535, Oligo-18, and pTa-275, 5AL119.2-18/275 arm carries the tandem repeats pSc119.2, Oligo-18 and pTa-275, and 5AL119.2 arm carries the tandem repeats pSc119.2. In the progeny of 5AL535-18/275 × 5AL119.2, double recombination occurred between pSc119.2 and pTa-535 clusters (119-535 interval), and between pTa-535 and Oligo-18/pTa-275 clusters (535-18 interval). The recombination rate in the 119-535 interval in the progeny of 5AL535-18/275 × 5AL119.2-18/275 was higher than that in the progeny of 5AL535-18/275 × 5AL119.2. Recombination in the 119-535 interval produced 5AL119 + 535 segments with pTa-535 and pSc119.2 tandem repeats and 5ALNo segments without these repeats. The 5AL119 + 535 and 5ALNo segments were localized between the signal sites of the single-copy probes SC5A-479 and SC5A-527. The segment between SC5A-479 and SC5A-527 in the metaphase 5ALNo was significantly longer than that in the metaphase 5AL119 + 535. CONCLUSION: The structural variations caused by tandem repeats might be one of the factors affecting meiotic recombination in wheat. Meiotic recombination aggregated two kinds of tandemly repeated clusters into the same chromosome, making the metaphase chromosome more condensed. To conclude, our study provides a robust tool to measure meiotic recombination and select parents for wheat breeding programs.


Asunto(s)
Cromosomas de las Plantas , Recombinación Homóloga , Meiosis , Triticum/genética , Hibridación Fluorescente in Situ , Sondas de Oligonucleótidos
3.
Hua Xi Kou Qiang Yi Xue Za Zhi ; 39(2): 188-194, 2021 Apr 01.
Artículo en Chino | MEDLINE | ID: mdl-33834674

RESUMEN

OBJECTIVES: To evaluate the effects of antimicrobial peptide GH12 designed de novo on the structure, morphology, and composition of a cariogenic three-species biofilm. METHODS: The cariogenic three-species biofilm consis-ted of the cariogenic Streptococcus mutans (S. mutans) and commensal bacteria Streptococcus sanguinis (S. sanguinis) and Streptococcus gordonii (S. gordonii). The biofilm was treated using GH12 (2, 4, and 8 mg·L-1), and untreated biofilm was used as the control. Changes in the morphology and structure of the three-species biofilm were evaluated through crystal violet staining, scanning electron microscopy (SEM), and fluorescent in situ hybridization (FISH). Moreover, S. mutans in the biofilm was selectively cultured, and its colony-forming units were counted. RESULTS: The biomass and density of the cariogenic three-species biofilm treated with GH12 decreased compared with those of the control. The number of S. mutans decreased gradually and eventually became undetectable, whereas the number of S. gordonii and S. sanguinis increased and became predominant in the biofilm. CONCLUSIONS: GH12 can reduce the number of S. mutans within the cariogenic three-species biofilm, destroys its integrity, and consequently makes the biofilm easy to remove.


Asunto(s)
Caries Dental , Biopelículas , Humanos , Hibridación Fluorescente in Situ , Proteínas Citotóxicas Formadoras de Poros , Streptococcus mutans
4.
Science ; 372(6538)2021 04 09.
Artículo en Inglés | MEDLINE | ID: mdl-33833095

RESUMEN

During multicellular development, spatial position and lineage history play powerful roles in controlling cell fate decisions. Using a serine integrase-based recording system, we engineered cells to record lineage information in a format that can be read out in situ. The system, termed integrase-editable memory by engineered mutagenesis with optical in situ readout (intMEMOIR), allowed in situ reconstruction of lineage relationships in cultured mouse cells and flies. intMEMOIR uses an array of independent three-state genetic memory elements that can recombine stochastically and irreversibly, allowing up to 59,049 distinct digital states. It reconstructed lineage trees in stem cells and enabled simultaneous analysis of single-cell clonal history, spatial position, and gene expression in Drosophila brain sections. These results establish a foundation for microscopy-readable lineage recording and analysis in diverse systems.


Asunto(s)
Linaje de la Célula , Expresión Génica , Células Madre Embrionarias de Ratones/citología , Neuronas/citología , Análisis de la Célula Individual , Animales , Encéfalo/citología , Línea Celular , Células Clonales/citología , Drosophila melanogaster/citología , Drosophila melanogaster/embriología , Perfilación de la Expresión Génica , Respuesta al Choque Térmico , Hibridación Fluorescente in Situ , Integrasas/metabolismo , Ratones , Mutagénesis , Análisis Espacial , Imagen de Lapso de Tiempo , Transcripción Genética
6.
Klin Lab Diagn ; 66(3): 154-159, 2021 Mar 30.
Artículo en Inglés | MEDLINE | ID: mdl-33793114

RESUMEN

Telomere length can be measured by polymerase chain reaction (PCR), allowing to obtain the absolute length of telomeres (ALT) in base pair, and by flow cytometry, which can only estimate the relative telomere length. The aim of the study was to compare the results of the two methods and to develop an accurate and reliable way of converting the relative telomere length to absolute. The peripheral blood from 21 donors was analyzed. Measurement of leukocyte telomere length by flow cytometry was carried out using a commercial Telomere PNA Kit / FITC (Dako, Denmark) with two CytoFLEX flow cytometers (Beckman Coulter, China) and BD FACSCanto II (Becton Dickinson, USA), obtaining the molecular equivalent of fluorescence (MEF). To measure telomere length by real-time PCR, calibrators with a known number of telomeric repeats were prepared. Two quantitative PCRs were carried out: one for telomeric repeats, the other for determining the number of genome-equivalents of DNA, three times for each sample, which made it possible to calculate ALT. A strong direct relationship was found between the MEF obtained with BD FACSCanto II and CytoFLEX (r = 0.97). Analysis of PCR and flow cytometry results showed a significant correlation between ALT and MEF. We calculated the regression equations of ALT and MEF for CytoFLEX - y = 0.0043x (r = 0.84) and for BD FACSCanto II - y = 0.0051x (r = 0.82). Correlation analysis showed a high comparability of telomere lengths measured by two methods. The obtained regression equations allow converting the results of flow cytometry into absolute values, allowing the comparison of the results of different research groups and the use of this method in clinical trials.


Asunto(s)
Leucocitos , Telómero , ADN , Citometría de Flujo , Humanos , Hibridación Fluorescente in Situ , Telómero/genética
7.
Mol Syst Biol ; 17(4): e10232, 2021 04.
Artículo en Inglés | MEDLINE | ID: mdl-33904651

RESUMEN

Exacerbated pro-inflammatory immune response contributes to COVID-19 pathology. However, despite the mounting evidence about SARS-CoV-2 infecting the human gut, little is known about the antiviral programs triggered in this organ. To address this gap, we performed single-cell transcriptomics of SARS-CoV-2-infected intestinal organoids. We identified a subpopulation of enterocytes as the prime target of SARS-CoV-2 and, interestingly, found the lack of positive correlation between susceptibility to infection and the expression of ACE2. Infected cells activated strong pro-inflammatory programs and produced interferon, while expression of interferon-stimulated genes was limited to bystander cells due to SARS-CoV-2 suppressing the autocrine action of interferon. These findings reveal that SARS-CoV-2 curtails the immune response and highlights the gut as a pro-inflammatory reservoir that should be considered to fully understand SARS-CoV-2 pathogenesis.


Asunto(s)
Intestinos/inmunología , Análisis de la Célula Individual , /virología , Microbioma Gastrointestinal , Humanos , Hibridación Fluorescente in Situ , Organoides/metabolismo , Análisis de Secuencia de ARN
8.
Rinsho Ketsueki ; 62(3): 180-185, 2021.
Artículo en Japonés | MEDLINE | ID: mdl-33828011

RESUMEN

We report the case of a 26-year-old male patient with chronic myelogenous leukemia in the chronic phase with the e13a3 (b2a3) variant of BCR-ABL1 fusion. Despite the presence of Philadelphia chromosome and fluorescence in situ hybridization-detectable BCR-ABL1 fusion signals, quantitative measurement of BCR-ABL1 on the ABL1 using a reverse primer in exon 2 of ABL1 failed to detect the fusion transcripts. PCR direct sequencing analysis with a sense primer for exon 13 of BCR and an antisense primer for exon 3 of ABL1 revealed the e13a3 variant of BCR-ABL1 fusion. The variant fusion transcript level was successfully monitored by the TaqMan assay using a forward primer and probe both in exon 13 of BCR and a reverse primer in exon 3 of ABL1. The patient responded extremely well to imatinib treatment, similar to previously reported e13a3 cases. The patient achieved a molecular response (undetectable e13a3 transcripts) after 12 months of treatment.


Asunto(s)
Proteínas de Fusión bcr-abl , Leucemia Mielógena Crónica BCR-ABL Positiva , Adulto , Proteínas de Fusión bcr-abl/genética , Humanos , Mesilato de Imatinib/uso terapéutico , Hibridación Fluorescente in Situ , Leucemia Mielógena Crónica BCR-ABL Positiva/tratamiento farmacológico , Leucemia Mielógena Crónica BCR-ABL Positiva/genética , Masculino , Inhibidores de Proteínas Quinasas/uso terapéutico
9.
Sichuan Da Xue Xue Bao Yi Xue Ban ; 52(2): 319-325, 2021 Mar.
Artículo en Chino | MEDLINE | ID: mdl-33829709

RESUMEN

Objective: To explore the application of array-based comparative genomic hybridization (a-CGH) technology in the prenatal diagnostic assessment of abnormal serological prenatal screening results of Down's syndrome (DS). Methods: A total of 3 578 amniotic fluid samples from pregnant women who underwent amniocentesis for prenatal diagnosis solely due to abnormal serological prenatal screening results were selected. The samples were categorized into 3 groups, 2 624 in the high-risk group, 662 in the borderline-risk group, and 292 in the abnormal multiple of median (MoM) group. a-CGH was performed on the Agilent CGX ™ (8×60K) platform and the data were analyzed by the Genoglyphix ® software. Results: The overall detection rate of chromosomal abnormalities was 3.38% (121/3 578). Among the chromosomal abnormalities, 49.59% (60/121) was aneuploidies, 42.15% (51/121) was pathogenic copy number variants (pCNVs), and 8.26% (10/121) was likely pathogenic CNVs (lpCNVs). The detection rate of copy number variant of uncertain significance (VUS) was 1.03% (37/3 578). In the high-risk, the borderline-risk and the abnormal MoM groups, the detection rate of chromosomal abnormalities was 3.54% (93/2 624), 2.87% (19/662) and 3.08% (9/292), respectively; the detection rate of p/lp CNVs was 1.64% (43/2 624), 1.81% (12/662) and 2.05% (6/292), respectively; the detection rate of trisomy 21 and trisomy 18 was 1.37% (36/2 624), 0.76% (5/662) and 0.34% (1/292) in the three groups, respectively. There were no significant differences in all the detection rate among these groups ( P>0.05). One sample with X(51)/XYY(49) confirmed by fluorescence in situ hybridization (FISH) was misdiagnosed by a-CGH. Conclusion: Prenatal diagnosis with a-CGH is of great significance for reducing birth defects in pregnancies with abnormal serological prenatal screening results of DS. It can also be used to detect CNVs of microdeletion/microduplication syndromes.


Asunto(s)
Síndrome de Down , Aberraciones Cromosómicas , Hibridación Genómica Comparativa , Síndrome de Down/diagnóstico , Síndrome de Down/genética , Femenino , Humanos , Hibridación Fluorescente in Situ , Embarazo , Diagnóstico Prenatal
10.
Cesk Patol ; 57(1): 12-18, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33910344

RESUMEN

There is no universal immunohistochemical panel which would be useful for the diagnosis of soft tissue tumors in all circumstances. Nevertheless, especially when faced with an uncharacteristic spindle cell neoplasm, a basic immunohistochemical panel can be recommended consisting of CD34, desmin, epithelial membrane antigen, broad-spectrum cytokeratins, S100 protein and smooth muscle actin. This review will address the utility and pitfalls of this panel. The use of MDM2 immunohistochemistry and fluorescence in situ hybridization in the diagnosis of lipomatous tumors will be discussed as well.


Asunto(s)
Neoplasias de los Tejidos Blandos , Biomarcadores de Tumor , Humanos , Inmunohistoquímica , Hibridación Fluorescente in Situ , Proteínas S100 , Neoplasias de los Tejidos Blandos/diagnóstico
11.
Zhonghua Xue Ye Xue Za Zhi ; 42(2): 124-128, 2021 Feb 14.
Artículo en Chino | MEDLINE | ID: mdl-33858042

RESUMEN

Objective: To investigate the incidence of high-grade B-cell lymphoma with MYC and BCL2 and/or BCL6 rearrangement in Chinese diffuse large B-cell lymphoma (DLBCL) . Methods: From January 2013 to August 2020, 922 DLBCL cases were collected. C-MYC and BCL2 protein expression levels were analyzed by immunohistochemistry staining. Fluorescence in situ hybridization was used to detect the structural abnormalities of MYC, BCL2, and BCL6, including gene breaks and copy number changes. Results: MYC and BCL2 and/or BCL6 gene breaks were found in 29 out of 922 DLBCL cases (3.15%) , including 25 cases of double-hit lymphoma (DHL; 14 cases involving MYC and BCL2 rearrangements and 11 cases involving MYC and BCL6 rearrangements) and four cases involving MYC, BCL2, and BCL6 rearrangements, referring to triple-hit lymphoma. According to the threshold of C-MYC ≥40% and BCL2 ≥50%, 541 cases (58.68%) overexpressed C-MYC and BCL2 proteins, including 22 DHL cases. Moreover, according to the threshold of C-MYC ≥70% and BCL2 ≥50%, 52 cases (5.64%) overexpressed C-MYC and BCL2 proteins, including nine DHL cases. The P53 protein expression was detected by immunohistochemistry staining. The mutant P53 expression pattern was shown in 101 out of 709 cases (14.25%) , whereas 13 cases (1.83%) were negative, likely indicating P53 gene fragment deletion. Conclusion: The incidence of high-grade B-cell lymphoma with MYC and BCL2 and/or BCL6 rearrangements was low in DLBCLs, and no significant correlation between gene abnormality and protein overexpression was shown. The correct diagnosis of DHL depends on molecular genetic detection.


Asunto(s)
Linfoma de Células B Grandes Difuso , Proteínas Proto-Oncogénicas c-myc , Reordenamiento Génico , Humanos , Hibridación Fluorescente in Situ , Incidencia , Linfoma de Células B Grandes Difuso/epidemiología , Linfoma de Células B Grandes Difuso/genética , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteínas Proto-Oncogénicas c-bcl-6/genética , Proteínas Proto-Oncogénicas c-myc/genética
12.
Zhonghua Bing Li Xue Za Zhi ; 50(5): 476-481, 2021 May 08.
Artículo en Chino | MEDLINE | ID: mdl-33915654

RESUMEN

Objective: To investigate the clinicopathological and genetic features of nodular fasciitis of the breast (NFB). Methods: The clinical and histologic features of seven NFBs were retrospectively reviewed. Immunohistochemistry, fluorescence in situ hybridization (FISH) and reverse transcription-polymerase chain reaction (RT-PCR) were performed. Results: All the seven patients were female, with a mean age of 36 years (range from 15 to 51 years). The duration of the lesion ranged from 10 days to 2 years. There was no history of trauma for all patients. The lesions occurred in the upper quadrant (4 cases), the lower quadrant (2 cases) and the axillary tail region (1 case). The maximum diameter was 1.0-3.5 cm. All cases showed similar morphology as nodular fasciitis occurring elsewhere in the body. They were composed of plump spindle cells arranged in short bundles or fascicles within a loose collagenous/myxoid stroma. Erythrocyte extravasation, mixed chronic inflammatory cells infiltration and microcystic changes were typically seen. Mitoses were present, with no atypical mitoses observed. The spindle cells were positive for smooth muscleactin(SMA, 6/6), CD10(2/3), and negative for desmin, ß-catenin, CD34, CKpan, EMA, S-100, p63 and ALK-1.The Ki-67 index were 5%-15%. USP6 gene rearrangement was found in six cases and MYH9-USP6 gene fusion in two cases. Local resection was performed in six cases. Spontaneous regression was observed in one case. Follow-up of all seven cases revealed no recurrence or metastasis. Conclusions: Although rare, NFB can mimic breast cancer clinically, radiologically and histologically. It should be always considered in the differential diagnosis for the spindle cell proliferations of the breast. A diagnosis of NFB can be achieved basing on the typical clinicopathological presentation. FISH detection of USP6 gene rearrangement in challenging cases is of great value.


Asunto(s)
Fascitis , Fibroma , Adolescente , Adulto , Fascitis/genética , Femenino , Humanos , Hibridación Fluorescente in Situ , Masculino , Persona de Mediana Edad , Estudios Retrospectivos , Ubiquitina Tiolesterasa , Adulto Joven
13.
Water Res ; 194: 116963, 2021 Apr 15.
Artículo en Inglés | MEDLINE | ID: mdl-33652229

RESUMEN

Granular sludge exhibits unique features, including rapid settling velocity, high loading rate and relative insensitivity against inhibitors, thus being a favorable platform for the cultivation of slow-growing and vulnerable microorganisms, such as anaerobic ammonium oxidation (anammox) bacteria and nitrite/nitrate-dependent anaerobic methane oxidation (n-DAMO) microorganisms. While anammox granules have been widely applied, little is known about how to speed up the granulation process of n-DAMO microorganisms, which grow even slower than anammox bacteria. In this study, we used mature anammox granules as biotic carriers to embed n-DAMO microorganisms, which obtained combined anammox + n-DAMO granules within 6 months. The results of whole-granule 16S rRNA gene amplicon sequencing showed the coexistence of anammox bacteria, n-DAMO bacteria and n-DAMO archaea. The microbial stratification along granule radius was further elucidated by cryosection-16S rRNA gene amplicon sequencing, showing the dominance of n-DAMO archaea and anammox bacteria at inner and outer layers, respectively. Moreover, the images of cryosection-fluorescence in situ hybridization (FISH) verified this stratification and also indicated a shift in microbial stratification. Specifically, n-DAMO bacteria and n-DAMO archaea attached to the anammox granule surface initially, which moved to the inner layer after 4-months operation. On the basis of combined anammox + n-DAMO granules, a practically useful nitrogen removal rate (1.0 kg N/m3/d) was obtained from sidestream wastewater, which provides new avenue to remove nitrogen from wastewater using methane as carbon source.


Asunto(s)
Compuestos de Amonio , Metano , Anaerobiosis , Reactores Biológicos , Desnitrificación , Hibridación Fluorescente in Situ , Nitratos , Nitrógeno , Oxidación-Reducción , ARN Ribosómico 16S/genética
14.
Zhonghua Bing Li Xue Za Zhi ; 50(3): 194-200, 2021 Mar 08.
Artículo en Chino | MEDLINE | ID: mdl-33677881

RESUMEN

Objective: To investigate the clinicopathological diagnosis and differential diagnosis of inflammatory myofibroblastic tumor (IMT). Methods: Thirty-two cases of IMT collected at the People's Hospital of Jiangsu Province from May 2010 to May 2020 were evaluated for their clinical, histologic, immunohistochemical and genomic features, and relevant literature was reviewed. Results: There were 19 male and 13 female patients, with age ranging from 5 to 65 years (mean, 37 years). The tumors were located in the lung and mediastinum (10 cases), gastrointestinal tract and mesentery/omentum (12 cases), urinary bladder (5 cases), head and neck (3 cases), somatic soft tissue (1 case), and retroperitoneum (1 case). Four cases of epithelioid inflammatory myofibroblastic sarcoma (EIMS) were all located intra-abdominally. Histologically, the tumor cells were myofibroblasts and fibroblasts arranged in predominantly fusiform pattern, with variably edematous to myxoid background or sclerotic collagenized stroma, and variably mixed chronic or acute inflammatory cells infiltration. EIMS were composed mainly of epithelioid tumor cells, with myxoid stroma and numerous neutrophils. Immunohistochemically, the tumor cells expressed cytoplasmic ALK (25/32, 78%), whereas the four EIMS showed nuclear membrane ALK staining pattern. The tumor cells also expressed CKpan (8/19), SMA (24/32, 75%) and desmin (12/32, 38%); all four EIMS also showed strong positivity for desmin. Fluorescence in situ hybridization (FISH) for ALK gene rearrangement showed split apart signals in 12 of 15 cases, most commonly with atypical signals. Next-generation sequencing (NGS) was performed in three tumors and showed that one case of lower leg IMT harbored a novel CLIP2-ALK fusion, and two cases of EIMS harbored RANBP2-ALK fusion. Follow-up data were available in 29 patients. Twenty-two patients were alive with no evidence of tumor, four patients had tumor recurrences (three patients were treated with crizotinib and were alive with tumor), and three patients died of the disease (including two patients with EIMS). Conclusions: IMTs show a wide morphologic spectrum, and should be differentiated form a variety of benign or malignant tumors. Immunohistochemistry (ALKp80, ALKD5F3) and FISH (ALK break-apart probe) could assist the diagnosis of IMT, with NGS recommended for the atypical cases.


Asunto(s)
Granuloma de Células Plasmáticas , Sarcoma , Adolescente , Adulto , Anciano , Quinasa de Linfoma Anaplásico/genética , Biomarcadores de Tumor , Niño , Preescolar , Femenino , Granuloma de Células Plasmáticas/diagnóstico por imagen , Granuloma de Células Plasmáticas/genética , Humanos , Hibridación Fluorescente in Situ , Masculino , Persona de Mediana Edad , Adulto Joven
15.
Mol Cell ; 81(7): 1566-1577.e8, 2021 04 01.
Artículo en Inglés | MEDLINE | ID: mdl-33657402

RESUMEN

Cas9 in complex with a programmable guide RNA targets specific double-stranded DNA for cleavage. By harnessing Cas9 as a programmable loader of superhelicase to genomic DNA, we report a physiological-temperature DNA fluorescence in situ hybridization (FISH) method termed genome oligopaint via local denaturation (GOLD) FISH. Instead of global denaturation as in conventional DNA FISH, loading a superhelicase at a Cas9-generated nick allows for local DNA denaturation, reducing nonspecific binding of probes and avoiding harsh treatments such as heat denaturation. GOLD FISH relies on Cas9 cleaving target DNA sequences and avoids the high nuclear background associated with other genome labeling methods that rely on Cas9 binding. The excellent signal brightness and specificity enable us to image nonrepetitive genomic DNA loci and analyze the conformational differences between active and inactive X chromosomes. Finally, GOLD FISH could be used for rapid identification of HER2 gene amplification in patient tissue.


Asunto(s)
Proteína 9 Asociada a CRISPR/química , Sistemas CRISPR-Cas , Calor , Hibridación Fluorescente in Situ , Desnaturalización de Ácido Nucleico , ARN Guia/química , Línea Celular , Femenino , Fibroblastos/química , Fibroblastos/metabolismo , Humanos
16.
Nat Commun ; 12(1): 1832, 2021 03 23.
Artículo en Inglés | MEDLINE | ID: mdl-33758201

RESUMEN

Synthetic glucocorticoids (GCs), one of the most effective treatments for chronic inflammatory and autoimmune conditions in children, have adverse effects on the growing skeleton. GCs inhibit angiogenesis in growing bone, but the underlying mechanisms remain unclear. Here, we show that GC treatment in young mice induces vascular endothelial cell senescence in metaphysis of long bone, and that inhibition of endothelial cell senescence improves GC-impaired bone angiogenesis with coupled osteogenesis. We identify angiogenin (ANG), a ribonuclease with pro-angiogenic activity, secreted by osteoclasts as a key factor for protecting the neighboring vascular cells against senescence. ANG maintains the proliferative activity of endothelial cells through plexin-B2 (PLXNB2)-mediated transcription of ribosomal RNA (rRNA). GC treatment inhibits ANG production by suppressing osteoclast formation in metaphysis, resulting in impaired endothelial cell rRNA transcription and subsequent cellular senescence. These findings reveal the role of metaphyseal blood vessel senescence in mediating the action of GCs on growing skeleton and establish the ANG/PLXNB2 axis as a molecular basis for the osteoclast-vascular interplay in skeletal angiogenesis.


Asunto(s)
Senescencia Celular/efectos de los fármacos , Células Endoteliales/metabolismo , Glucocorticoides/farmacología , Neovascularización Fisiológica/efectos de los fármacos , Proteínas del Tejido Nervioso/metabolismo , Osteoclastos/metabolismo , Ribonucleasa Pancreática/metabolismo , Animales , Apoptosis/efectos de los fármacos , Desarrollo Óseo/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Senescencia Celular/genética , Células Endoteliales/efectos de los fármacos , Células Endoteliales de la Vena Umbilical Humana , Humanos , Inmunohistoquímica , Hibridación Fluorescente in Situ , Metilprednisolona/farmacología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Neovascularización Patológica , Proteínas del Tejido Nervioso/genética , Osteoclastos/efectos de los fármacos , Osteoclastos/enzimología , Osteogénesis/efectos de los fármacos , ARN Ribosómico/biosíntesis , ARN Interferente Pequeño , Proteínas Recombinantes , Ribonucleasa Pancreática/genética , Ribonucleasa Pancreática/farmacología , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Tomógrafos Computarizados por Rayos X
17.
Arch Oral Biol ; 125: 105107, 2021 May.
Artículo en Inglés | MEDLINE | ID: mdl-33735629

RESUMEN

OBJECTIVE: The current study aimed to assess the antimicrobial activity of ursolic acid (UA) against multi-species biofilms formed by Streptococcus mutans, Streptococcus sanguinis, and Streptococcus gordonii, as well as to measure its biocompatibility. METHODS: Crystal violet staining, CFU counting, CCK-8 assays and scanning electron microscopy (SEM) were applied to investigate the effect of UA on multi-species biofilms. UA's effect on exopolysaccharides (EPS) production was measured using confocal laser scanning microscopy (CLSM) and the anthrone-sulfuric acid method. Fluorescent in situ hybridization (FISH) was applied to visualize and quantify the microbial composition of multi-species biofilms. Quantitative real-time PCR (qRT-PCR) was used to measure the expression of virulence genes of S. mutans, S. sanguinis, and S. gordonii under UA treatment. Moreover, CCK-8 assays were performed to evaluate its cytotoxicity against human oral keratinocytes (HOKs) and human gingival epithelial cells (HGEs). RESULTS: The results showed that UA had significant antimicrobial activity against common oral streptococci. UA also reduced the EPS synthesis of oral streptococci and suppressed gtf genes' expression. In addition, UA reduced the proportion of S. mutans in multi-species biofilms. Besides, UA had low cytotoxicity against HOKs and HGEs. CONCLUSIONS: UA exhibited antibiofilm activity against oral pathogenic bacteria and had the potential to be used in dental caries treatment.


Asunto(s)
Caries Dental , Streptococcus mutans , Biopelículas , Caries Dental/tratamiento farmacológico , Humanos , Hibridación Fluorescente in Situ , Streptococcus gordonii , Streptococcus mutans/genética , Streptococcus sanguis , Triterpenos
18.
J Vis Exp ; (169)2021 03 04.
Artículo en Inglés | MEDLINE | ID: mdl-33749670

RESUMEN

The genome is associated with several structures inside cell nuclei, in order to regulate its activity and anchor it in specific locations. These structures are collectively known as the nucleoskeleton and include the nuclear lamina, the nucleoli, and nuclear bodies. Although many variants of fluorescence in situ hybridization (FISH) exist to study the genome and its organization, these are often limited by resolution and provide insufficient information on the genome's association with nuclear structures. The DNA halo method uses high salt concentrations and nonionic detergents to generate DNA loops that remain anchored to structures within nuclei through attachment regions within the genome. Here, soluble nuclear proteins, such as histones, lipids, and DNA not tightly bound to the nuclear matrix, are extracted. This leads to the formation of a halo of unattached DNA surrounding a residual nucleus which itself contains DNA closely associated with internal nuclear structures and extraction-resistant proteins. These extended DNA strands enable increased resolution and can facilitate physical mapping. In combination with FISH, this method has the added advantage of studying genomic interactions with all the structures that the genome is anchored by. This technique, termed HALO-FISH, is highly versatile whereby DNA halos can be coupled with nucleic acid probes to reveal gene loci, whole chromosomes, alpha satellite, telomeres and even RNA. This technique provides an insight into nuclear organization and function in normal cells and in disease progression such as with cancer.


Asunto(s)
Cromosomas/metabolismo , ADN/metabolismo , Sitios Genéticos , Hibridación Fluorescente in Situ , Telómero/metabolismo , Núcleo Celular/metabolismo , Células Cultivadas , Cromosomas Artificiales Bacterianos/metabolismo , Dermis/citología , Fibroblastos/metabolismo , Humanos , Procesamiento de Imagen Asistido por Computador
19.
Nat Commun ; 12(1): 1836, 2021 03 23.
Artículo en Inglés | MEDLINE | ID: mdl-33758175

RESUMEN

To prevent damage to the host or its commensal microbiota, epithelial tissues must match the intensity of the immune response to the severity of a biological threat. Toll-like receptors allow epithelial cells to identify microbe associated molecular patterns. However, the mechanisms that mitigate biological noise in single cells to ensure quantitatively appropriate responses remain unclear. Here we address this question using single cell and single molecule approaches in mammary epithelial cells and primary organoids. We find that epithelial tissues respond to bacterial microbe associated molecular patterns by activating a subset of cells in an all-or-nothing (i.e. digital) manner. The maximum fraction of responsive cells is regulated by a bimodal epigenetic switch that licenses the TLR2 promoter for transcription across multiple generations. This mechanism confers a flexible memory of inflammatory events as well as unique spatio-temporal control of epithelial tissue-level immune responses. We propose that epigenetic licensing in individual cells allows for long-term, quantitative fine-tuning of population-level responses.


Asunto(s)
Bacterias/inmunología , Células Epiteliales/inmunología , Inmunidad Innata , Lipopéptidos/inmunología , FN-kappa B/metabolismo , Receptor Toll-Like 2/metabolismo , Animales , Bacterias/metabolismo , Línea Celular , Citocinas/metabolismo , Citocinas/farmacología , Metilación de ADN/efectos de los fármacos , Epigénesis Genética/efectos de los fármacos , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Flagelina/farmacología , Regulación de la Expresión Génica/genética , Regulación de la Expresión Génica/inmunología , Humanos , Procesamiento de Imagen Asistido por Computador , Inmunidad Innata/efectos de los fármacos , Inmunidad Innata/genética , Hibridación Fluorescente in Situ , Glándulas Mamarias Animales , Ratones , Organoides/efectos de los fármacos , Organoides/inmunología , Organoides/metabolismo , Regiones Promotoras Genéticas , RNA-Seq , Transducción de Señal/inmunología , Análisis de la Célula Individual , Receptor Toll-Like 2/agonistas , Receptor Toll-Like 2/genética , Receptores Toll-Like/agonistas , Receptores Toll-Like/metabolismo
20.
Int J Mol Sci ; 22(5)2021 Feb 26.
Artículo en Inglés | MEDLINE | ID: mdl-33652823

RESUMEN

Fluorescence in situ hybridization (FISH) and Hi-C methods are largely used to investigate the three-dimensional organization of the genome in the cell nucleus and are applied here to study the organization of genes (LMBR1, NOM1, MNX1, UBE3C, PTPRN2) localized in the human 7q36.3 band. This region contains the MNX1 gene, which is normally not expressed in human lymphocytes beyond embryonic development. However, this homeobox gene is frequently activated in leukemic cells and its expression is associated with an altered gene positioning in the leukemia cell nuclei. In this study, we used FISH on 3D-preserved nuclei to investigate the nuclear positioning of MNX1 in the leukemia-derived cell line K562. Of the five copies of the MNX1 gene present in K562, four alleles were positioned in the nuclear periphery and only one in the nuclear interior. Using the Juicebox's Hi-C dataset, we identified five chromatin loops in the 7q36.3 band, with different extensions related to the size and orientation of the genes located here, and independent from their expression levels. We identified similar loops in 11 human and three mouse cell lines, showing that these loops are highly conserved in different human cell lines and during evolution. Moreover, the chromatin loop organization is well conserved also during neuronal cell differentiation, showing consistency in genomic organization of this region in development. In this report, we show that FISH and Hi-C are two different approaches that complement one another and together give complete information on the nuclear organization of specific chromosomal regions in different conditions, including cellular differentiation and genetic diseases.


Asunto(s)
Cromatina/genética , Cromosomas Humanos/genética , Proteínas de Homeodominio/genética , Leucemia/genética , Familia de Multigenes , Factores de Transcripción/genética , Animales , Línea Celular Tumoral , Núcleo Celular/genética , Humanos , Hibridación Fluorescente in Situ , Ratones
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