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1.
Diagn Cytopathol ; 50(3): 112-122, 2022 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-34984861

RESUMO

BACKGROUND: Adipocytic tumors are the most common soft tissue tumors, with lipomas and atypical lipomatous tumor/well-differentiated liposarcomas (ALT/WDL), which comprise most cases. Preoperative differential diagnosis of lipoma or ALT/WDL can provide important information for decisions regarding treatment. We evaluated the cytological findings of 20 cases of lipoma and ALT/WDL. METHODS: Fluorescence in situ hybridization (FISH) was performed on formalin-fixed paraffin-embedded specimens (FFPE) to examine mouse double minute 2 homolog (MDM2) amplification in all cases. Tissue samples were collected from the center of the surgical materials, stained with Pap, and evaluated for 12 cytological parameters by six cytotechnologists. RESULTS: The findings regarding large atypical cells, multinucleated cells, and nuclear pleomorphism were highly concordant among the cytotechnologists and were associated with MDM2 amplification. Large atypical cells, considered a highly specific feature of ALT/WDL, were not observed in lipoma cases. However, the sensitivity of the large atypical cell findings was not high (67%); therefore, comprehensive evaluation of multinucleated cells and pleomorphism is crucial for predicting ALT/WDL diagnosis. FISH of MDM2 on Pap-stained specimens was performed in four cases. In two, the results were similar to those of MDM2 FISH performed on FFPE sections and were reproducible, whereas in the other two, the signal could not be evaluated because of the strong background coloration. CONCLUSIONS: Cytology specimens may be useful for the preoperative diagnosis of adipocytic tumors, particularly if the FISH conditions for Pap-stained specimens and the detection accuracy of MDM2 amplification can be improved.


Assuntos
Lipoma , Lipossarcoma , Animais , Diagnóstico Diferencial , Humanos , Hibridização in Situ Fluorescente/métodos , Lipoma/diagnóstico , Lipoma/genética , Lipoma/patologia , Lipossarcoma/diagnóstico , Lipossarcoma/genética , Lipossarcoma/patologia , Camundongos , Proteínas Proto-Oncogênicas c-mdm2/genética
2.
J Egypt Natl Canc Inst ; 34(1): 23, 2022 May 30.
Artigo em Inglês | MEDLINE | ID: mdl-35644823

RESUMO

BACKGROUND: Programmed death ligand-1 (PD-L1), anaplastic lymphoma kinase (ALK), and c-ros oncogene1 (ROS1) expression may influence the prognosis of non-small cell lung carcinoma (NSCLC). We aimed to investigate the prognostic and predictive significance of PD-1/PD-L1 along with c-ros ROS1 and ALK in NSCLC patients. METHODS: Immunohistochemistry used to identify ALK, ROS1, PD-1, and PD-L1 proteins expression as well as ROS1 rearrangement via fluorescence in situ hybridization, in 70 NSCLC patients. Results were related to clinicopathological feature, survival, and treatment response. RESULTS: Expression of ROS1, ALK, PD-1, and PD-L1 and ROS1-rearrangement were detected in 18.57%, 54.29%, 84.29%, 87.14%, and 15.71% of the cases, respectively. No association was found between ROS1, PD-1, and PD-L1 and any clinicopathological features, survival, or treatment outcome. ALK expression significantly associated with stage-IV and left-sided tumors. Epidermal growth factor receptor (EGFR) mutation and ALK-positive patients had significantly reduced progression-free survival than patients with wild type EGFR [HR: 1.99, 95% CI: 1.37-2.93, p < 0.001] and negative-ALK expression [HR: 1.46, 95% CI: 1.03-2.07, p = 0.03]. In multivariate analysis, lymph node metastasis, EGFR-mutations, and ALK were independent predictors of NSCLC. PD-L1 expression was significantly correlated with PD-1 but not with ROS1, ALK, or EGFR-mutation. CONCLUSION: Positive ALK expression and EGFR-mutations are independent adverse predictors of NSCLC. Overexpression of PD-1/PD-L1 is not a significant prognostic marker in NSCLC patients receiving chemotherapy, making them susceptible to immunotherapy. Since PD-1/PD-L1 expression is independent to oncogenic driver mutations, future studies into specific immune checkpoint inhibitors combined with targeted therapies for individualized treatment of NSCLC is warranted. Positive ALK expression and EGFR mutations are independent risk factors for NSCLC. Overexpression of PD-1/PD-L1 is not a significant prognostic factor in patients with NSCLC who are receiving chemotherapy, making them immunotherapy susceptible. Given that PD-1/PD-L1 expression is not dependent on oncogenic driver mutations, additional research into specific immune checkpoint inhibitors in combination with targeted therapies for the treatment of NSCLC on an individual basis is warranted.


Assuntos
Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , Quinase do Linfoma Anaplásico/genética , Antígeno B7-H1/genética , Antígeno B7-H1/metabolismo , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/patologia , Egito/epidemiologia , Receptores ErbB/genética , Humanos , Inibidores de Checkpoint Imunológico , Hibridização in Situ Fluorescente , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Prognóstico , Receptor de Morte Celular Programada 1/genética , Proteínas Tirosina Quinases/genética , Proteínas Tirosina Quinases/metabolismo , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo
3.
Front Immunol ; 13: 919012, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35686130

RESUMO

High-grade B-cell lymphoma (HGBL) is a newly introduced category of rare and heterogeneous invasive B-cell lymphoma (BCL), which is diagnosed depending on fluorescence in situ hybridization (FISH), an expensive and laborious analysis. In order to identify HGBL with minimal workup and costs, a total of 187 newly diagnosed BCL patients were enrolled in a cohort study. As a result, the overall survival (OS) and progression-free survival (PFS) of the HGBL group were inferior to those of the non-HGBL group. HGBL (n = 35) was more likely to have a high-grade histomorphology appearance, extranodal involvement, bone marrow involvement, and whole-body maximum standardized uptake (SUVmax). The machine learning classification models indicated that histomorphology appearance, Ann Arbor stage, lactate dehydrogenase (LDH), and International Prognostic Index (IPI) risk group were independent risk factors for diagnosing HGBL. Patients in the high IPI risk group, who are CD10 positive, and who have extranodal involvement, high LDH, high white blood cell (WBC), bone marrow involvement, old age, advanced Ann Arbor stage, and high SUVmax had a higher risk of death within 1 year. In addition, these models prompt the clinical features with which the patients should be recommended to undergo a FISH test. Furthermore, this study supports that first-line treatment with R-CHOP has dismal efficacy in HGBL. A novel induction therapeutic regimen is still urgently needed to ameliorate the poor outcome of HGBL patients.


Assuntos
Linfoma de Células B , Aprendizado de Máquina , Estudos de Coortes , Humanos , Hibridização in Situ Fluorescente , L-Lactato Desidrogenase , Linfoma de Células B/diagnóstico , Prognóstico
4.
PLoS One ; 17(6): e0269521, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35679311

RESUMO

The efficacy of salicylic acid paste (SA) in the treatment of ulcerative bovine digital dermatitis (BDD) was assessed by combining clinical and histopathological analyses with molecular biological techniques. The latter were conducted in a blinded manner to reach maximum objectivity. Prior to treatment, M2-stage BDD lesions (n = 26, diagnosed in 21 dairy cows) exhibited ulceration, with severe perivascular, chronic, lymphoplasmacytic dermatitis and extensive keratinolysis being noted in most cases. Pretreatment biopsy samples (n = 12) followed by povidone-iodine ointment under bandage for one week before administration of SA paste were tested positive for Treponema spp. by blinded PCR and fluorescent in situ hybridization (FISH). Subsequent treatment consisted of application of SA and bandaging at weekly intervals until lesions had completely resolved. The treatment duration ranged between 2 and 4 weeks. Complete healing was achieved in 100% of cases, with 2/21 animals requiring a second round of treatment upon disease reoccurrence. Importantly, only 3/26 biopsies taken from previously affected sites still tested positive by Treponema PCR, and in another biopsy, the outermost layers of the stratum corneum scored weakly positive by Treponema-specific FISH. None of these Treponema DNA-positive biopsies showed signs of ulceration. One case exhibited focal keratinolysis. Positive PCR or FISH in these cases may have arisen from DNA traces of dead bacteria or environmental contamination during biopsy harvesting. To our knowledge, this is the first study on blinded molecular biological monitoring of the therapeutic efficacy of SA with respect to treponemal infection, and on complete BDD M2-stage remission in all animals achieved by SA treatment according to an optimized protocol. Although the etiology of BDD is considered as multifactorial, our data further support the concept that treponemes have a decisive role in BDD pathogenesis.


Assuntos
Doenças dos Bovinos , Dermatite , Dermatite Digital , Infecções por Treponema , Animais , Bovinos , Doenças dos Bovinos/microbiologia , Dermatite Digital/microbiologia , Feminino , Hibridização in Situ Fluorescente , Ácido Salicílico/uso terapêutico , Treponema/genética , Infecções por Treponema/microbiologia
5.
Zhonghua Xue Ye Xue Za Zhi ; 43(5): 408-413, 2022 May 14.
Artigo em Chinês | MEDLINE | ID: mdl-35680599

RESUMO

Objective: To investigate the influence of the number of high-risk cytogenetic abnormalities (HRCA) on the clinical characteristics and prognosis of patients with newly diagnosed multiple myeloma (MM) . Methods: A total of 360 patients with newly diagnosed MM admitted to Jiangsu Province Hospital between November 2013 and September 2020 were included in this study. Cytoplasmic light chain immunofluorescence with fluorescence in situ hybridization (cIg-FISH) was used to detect HRCA. Cytogenetic abnormalities were combined with clinical characteristics and outcomes for further analysis. Results: Among the 360 patients, 120 patients (33.3%) presented with no HRCAs, and 175 (48.6%) , 61 (16.9%) , and four (1.1%) patients had one, two, and three HRCA (s) , respectively. Patients were divided into three groups, including the no-HRCA group, one-HRCA group, and ≥two-HRCA group, according to the number of HRCAs. There were significant differences in the R-ISS stage, hemoglobin level, albumin level, and the proportion of bone marrow plasma cells among the three groups (P<0.05) . The COX proportional-hazards model identified extramedullary disease (P=0.018) , HRCA ≥ 2 (P=0.001) , and absence of autologous hematopoietic stem cell transplantation (P<0.001) as independent risk factors for progression free survival (PFS) and identified lactate dehydrogenase (LDH) level ≥ 220 U/L (P<0.001) , HRCA ≥2 (P=0.001) , and absence of autologous hematopoietic stem cell transplantation (P=0.005) as independent risk factors for overall survival (OS) . The median PFS was 28 months, 22 months, and 14 months (P=0.005) for the three cohorts, and their OS was not reached,60 months, and 30 months (P=0.001) , respectively. Conclusions: HRCA ≥ 2 is an independent risk factor for decreased survival in patients with newly diagnosed MM. More HRCAs result in heavier tumor burden, as well as a higher risk of disease progression and death.


Assuntos
Transplante de Células-Tronco Hematopoéticas , Mieloma Múltiplo , Aberrações Cromossômicas , Humanos , Hibridização in Situ Fluorescente , Mieloma Múltiplo/diagnóstico , Mieloma Múltiplo/genética , Prognóstico , Estudos Retrospectivos , Transplante Autólogo
6.
Cell Mol Life Sci ; 79(7): 357, 2022 Jun 09.
Artigo em Inglês | MEDLINE | ID: mdl-35680727

RESUMO

BACKGROUND: Radiation is currently used to be a mainstay of salvage therapy for nasopharyngeal carcinoma (NPC), however, development of radioresistance largely limits the radiation efficacy. Circular RNAs (circRNAs) have been shown to affect NPC progression, but its role in radioresistance remain unclear. METHODS: The circular structure of circFIP1L1(circ_0069740) was verified by RNA-sequencing, RT-PCR based on gDNA or cDNA, RNase R treatment, and actinomycin D treatment. Cellular localization of circFIP1L1 and miR-1253 was detected by nucleoplasmic separation and/or fluorescence in situ hybridization. Expression of non-coding RNAs and mRNAs was detected by qRT-PCR, protein expression was detected by Western blot. Functionally, EdU, CCK-8, and colony formation experiments were employed to assess cell proliferation, flow cytometry was adopted to estimate cell cycle and apoptosis. Xenograft tumor growth was performed to detect the role of circFIP1L1 in vivo. Mechanistically, we examined the interplay between miR-1253 and circFIP1L1 or EIF4A3 through dual-luciferase reporter assay. The potential regulatory impacts of EIF4A3 on circFIP1L1 or PTEN was examined by RNA immunoprecipitation and RNA pull-down assays. RESULTS: CircFIP1L1 overexpression and miR-1253 knockdown repressed NPC cell proliferation, facilitated NPC cell apoptosis, and enhanced NPC radiosensitivity. Mechanistically, circFIP1L1 was revealed to repress miR-1253 by binding to it, and EIF4A3 is a target gene of miR-1253. CircFIP1L1 regulated NPC proliferation, apoptosis, and radiosensitivity through miR-1253/EIF4A3. Moreover, we found that EIF4A3 bound to FIP1L1 mRNA transcript and induced circFIP1L1 formation, and thus stabilizing PTEN mRNA. CONCLUSION: Our findings suggested that EIF4A3-induced circFIP1L1 repressed NPC cell proliferation, facilitated NPC cell apoptosis, and enhanced NPC radiosensitivity by miR-1253.


Assuntos
MicroRNAs , Neoplasias Nasofaríngeas , Linhagem Celular Tumoral , Proliferação de Células/genética , RNA Helicases DEAD-box/genética , Fator de Iniciação 4A em Eucariotos/metabolismo , Regulação Neoplásica da Expressão Gênica , Humanos , Hibridização in Situ Fluorescente , MicroRNAs/genética , MicroRNAs/metabolismo , Carcinoma Nasofaríngeo/genética , Carcinoma Nasofaríngeo/radioterapia , Neoplasias Nasofaríngeas/genética , Neoplasias Nasofaríngeas/metabolismo , Neoplasias Nasofaríngeas/radioterapia , RNA Mensageiro , Tolerância a Radiação/genética
7.
Zhongguo Shi Yan Xue Ye Xue Za Zhi ; 30(3): 897-907, 2022 Jun.
Artigo em Chinês | MEDLINE | ID: mdl-35680824

RESUMO

OBJECTIVE: To establish the technique that take the advantages of flow cytometry combined fluorescence in situ hybridization (Flow-FISH) to identify the Epstein-Barr virus(EBV) infected lymphocyte subtypies in patients' peripheral blood sample. METHODS: Peripheral Blood monocyte from 9 patients with EBV infection enrolled at Children's Hospital in Chongqing Medical University were isolated by Ficoll-paque centrifugal separation. The expressions of EBER1, EBER2 in cell were detected by qRT-PCR. The surface markers of cell were detected by Flow cytometry after staining with their antibodies. The cell was treated Fix-Permeabilization Buffer before hybridization with fluorescent labeled probe at 37 ℃ overnight. The cell status, surface markers and targeted mRNA are detected by flow cytometry and fluorescence microscope. RESULTS: It was optimized that the Fix-Permeabilization Buffer and recipe with 0.2% Tween-20 were picked out as providing a good cell integrity and high resolution of surface markers. Hybridization with 20% formamide and 7% dextran sulfate at 37 ℃ overnight is the optimal hybridization condition as a good hybridization effect, a detectable cell integrity and a high resolution of cell markers under flow cytometry detection. Finally, upon the established Flow-FISH method, lymphocyte subpopulations of the EBV+ cells from cell lines and blood samples of patients were identified successfully. CONCLUSION: A Flow-FISH technology is established, which can be applied in the identification of EBV infected cell subtypes. This research provides a foundmental for its application in clinical test in EBV+ related proliferative diseases.


Assuntos
Infecções por Vírus Epstein-Barr , Citometria de Fluxo/métodos , Herpesvirus Humano 4 , Humanos , Hibridização in Situ Fluorescente/métodos , Subpopulações de Linfócitos
8.
Int J Mol Sci ; 23(11)2022 May 31.
Artigo em Inglês | MEDLINE | ID: mdl-35682858

RESUMO

The hollow trait is crucial for commercial quality of cucumber (Cucumis sativus L.) fruit, and its molecular regulatory mechanism is poorly understood due to its environmental sensitivity. In the previous research, we obtained the hollow and the non-hollow materials of ecotype cucumbers of South China, which were not easily affected by the external environment through a systematic breeding method. In this study, first, we proposed to use the percentage of the hollow area as the criterion to compare the hollow characteristics between two materials, and to analyze the formation mechanism of early hollow trait from the perspective of cytology. The results showed that the hollow trait occurred in the early stage of fruit development, and formed with the opening of carpel ventral zipped bi-cell layer, which formed rapidly from 2 to 4 days, and then slowed to a constant rate from 14 to 16 days. Meanwhile, the different genetic populations were constructed using these materials, and fine mapping was performed by bulked segregant analysis (BSA) and kompetitive allele specific PCR (KASP) method. The Csa1G630860 (CsALMT2), encoding protein ALMT2, was determined as a candidate gene for regulating the hollow trait in fruit. Furthermore, the expression profile of CsALMT2 was analyzed by qRT-PCR and fluorescence in situ hybridization. The expression of CsALMT2 had obvious tissue specificity, and it was abundantly expressed in the ovule development zone inside the fruit. In the hollow material of cucumber fruit, the expression of CsALMT2 was significantly downregulated. The subcellular localization in tobacco leaves indicated that CsALMT2 was distributed on the plasma membrane. In conclusion, in this study, for the first time, we found the regulatory gene of hollow trait in cucumber fruit, which laid the foundation for subsequent research on the molecular mechanism of hollow trait formation in cucumber fruit, and made it possible to apply this gene in cucumber breeding.


Assuntos
Cucumis sativus , Cucumis sativus/genética , Frutas/genética , Hibridização in Situ Fluorescente , Fenótipo , Melhoramento Vegetal
9.
Arch Pathol Lab Med ; 146(5): 0, 2022 Jun 10.
Artigo em Inglês | MEDLINE | ID: mdl-35687792

RESUMO

CONTEXT.­: Targeted anti-human epidermal growth factor receptor 2 (HER2) therapy has recently become the standard for advanced-stage and recurrent HER2-positive endometrial serous carcinoma (ESC) in the United States, and an endometrial carcinoma-specific HER2 testing algorithm has been proposed. However, comprehensive studies on the specific features of HER2 gene amplification in these tumors are lacking. OBJECTIVE.­: To evaluate the characteristics of HER2 amplification in ESC in the context of breast and gastric HER2 fluorescence in situ hybridization (FISH) guidelines. DESIGN.­: Ninety-four ESCs with available HER2 immunohistochemistry (IHC) and FISH were included. HER2 IHC was scored according to the proposed endometrial carcinoma-specific algorithm and FISH was evaluated by using the 2018 ESC clinical trial criteria, the American Society of Clinical Oncology/College of American Pathologists (ASCO/CAP) 2016 gastric criteria, and ASCO/CAP 2013 and 2018 breast criteria. RESULTS.­: Most tumors (90.4%; 85 of 94) had a 2+ HER2 IHC score. Polysomy of chromosome 17 was present in 16% (15 of 94) and monosomy 17 was seen in 2% (2 of 94) of tumors. HER2 FISH interpretation per the clinical trial criteria (HER2/CEP17 ratio ≥ 2.0) showed 99% concordance with the current gastric and breast HER2 FISH interpretations. CONCLUSIONS.­: Our results support the clinical trial criteria for HER2 FISH in ESC with a modification to include HER2 IHC 2+ and HER2/CEP17 ratio less than 2.0 and average HER2 copy number of 6.0 or greater in the HER2-positive category. Future prospective clinical investigations are necessary to assess the correlation between specific HER2 FISH result categories and therapeutic response.


Assuntos
Neoplasias da Mama , Cistadenocarcinoma Seroso , Neoplasias do Endométrio , Biomarcadores Tumorais/genética , Neoplasias da Mama/genética , Cistadenocarcinoma Seroso/genética , Neoplasias do Endométrio/genética , Feminino , Amplificação de Genes , Humanos , Hibridização in Situ Fluorescente/métodos , Recidiva Local de Neoplasia , Receptor ErbB-2/genética , Receptor ErbB-2/metabolismo
10.
Sci Rep ; 12(1): 9585, 2022 Jun 10.
Artigo em Inglês | MEDLINE | ID: mdl-35688861

RESUMO

Most cases of acute leukemia (AL) with KMT2A rearrangement (KMT2A-r) have a dismal prognosis. Detection of this aberration in Chinese adult patients relies on reverse transcription polymerase chain reaction (RT-PCR) and chromosome banding analysis (CBA). The fluorescence in situ hybridization (FISH) probe for KMT2A detects KMT2A-r and copy number variation (CNV) but is not routinely used as a detection technique. This study investigated the potential value of FISH in the treatment of AL by performing FISH along with CBA and RT-PCR in 269 de novo cases of AL. The three detection techniques were compared in identification of KMT2A-r, and the applicability of FISH for detecting KMT2A CNV was evaluated. Twenty-three samples were identified as positive for KMT2A-r (20 using FISH, 15 using RT-PCR, 16 using CBA, and eight according to all three). FISH also identified 17 KMT2A CNV, 15 with gains and two with deletions. Ten patients with acute myeloid leukemia (AML) harboring KMT2A CNV had a complex karyotype, a negative prognostic factor in AML. Adding FISH of KMT2A to routine detection leads to more accurate detection of KMT2A-r and improved identification of KMT2A CNV, which would benefit patients by improving the risk stratification in AL.


Assuntos
Variações do Número de Cópias de DNA , Leucemia Mieloide Aguda , Rearranjo Gênico , Humanos , Hibridização in Situ Fluorescente , Leucemia Mieloide Aguda/diagnóstico , Leucemia Mieloide Aguda/genética , Proteína de Leucina Linfoide-Mieloide/genética , Medição de Risco
11.
J Int Med Res ; 50(6): 3000605221104181, 2022 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-35689392

RESUMO

OBJECTIVE: Anaplastic lymphoma kinase (ALK) rearrangement is an important oncogenic driver in some non-small cell lung cancers (NSCLC). Treatment with ALK tyrosine kinase inhibitors improves survival. The availability of diagnostic immunohistochemistry (IHC) has led to a paradigm shift in ALK testing. This study examined the prevalence of ALK rearrangement in Jordanian patients with NSCLC and compared the results of IHC and fluorescence in situ hybridization (FISH) for detecting ALK rearrangement. METHODS: This retrospective study on 449 patients with NSCLC treated at the King Hussein Cancer Center in Jordan tested biopsy samples for ALK rearrangement using FISH and/or IHC (D5F3) between 2018 and 2020. RESULTS: Eighteen patients (4%) had ALK-positive NSCLC. The calculated sensitivity and specificity of ALK immunostaining compared with FISH were 87.5% and 96%, respectively. ALK-positive patients were significantly younger than their ALK-negative counterparts, and women were three times more likely to carry ALK rearrangement than men. ALK rearrangement was significantly associated with smoking history, with most ALK-positive patients being non-smokers, former smokers, or light smokers. CONCLUSIONS: IHC is a reasonable alternative to FISH for ALK testing with advantages in terms of robustness, turnaround times, and cost-effectiveness.


Assuntos
Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , Quinase do Linfoma Anaplásico/genética , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/patologia , Feminino , Humanos , Hibridização in Situ Fluorescente/métodos , Jordânia/epidemiologia , Neoplasias Pulmonares/patologia , Inibidores de Proteínas Quinases , Receptores Proteína Tirosina Quinases/genética , Estudos Retrospectivos
12.
Artigo em Inglês | MEDLINE | ID: mdl-35649669

RESUMO

We examined frequencies of radiation-induced chromosomal aberrations, using classical cytological methods, and DNA damage in interphase and metaphase cells, using a combination of FISH, CO-FISH, TIF (telomere dysfunction induced assay) and simultaneous detection of DNA damage and telomeric sequences in metaphase chromosomes, in Chinese hamster cells defective in BRCA2 and control cells. Given that the Chinese hamster genome contains large blocks of interstitial telomeric sites, our results allow us to examine the role of BRCA2 in the potential fragility of these sites, but also whether BRCA2 affects DNA repair within terminal telomeric sequences. BRCA2 defective cells exhibited greater frequencies of DNA damage within interstitial telomeric sites, as well as within terminal telomeric sites, relative to control cells. Therefore, BRCA2 deficiency contributes to the telomere dysfunction phenotype in Chinese hamster cells.


Assuntos
Aberrações Cromossômicas , Telômero , Animais , Cricetinae , Cricetulus , Dano ao DNA , Hibridização in Situ Fluorescente/métodos , Telômero/genética
13.
Int Heart J ; 63(3): 466-475, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35650148

RESUMO

Almost 40% of medical radiation exposure is related to cardiac imaging or intervention. However, the biological effects of low-dose radiation from medical imaging remain largely unknown. This study aimed to evaluate the effects of ionized radiation from cardiac catheterization on genomic DNA integrity and inflammatory cytokines in patients and operators.Peripheral mononuclear cells (MNCs) were isolated from patients (n = 51) and operators (n = 35) before and after coronary angiography and/or percutaneous coronary intervention. The expression of γH2AX, a marker for DNA double-strand breaks, was measured by immunofluorescence. Dicentric chromosomes (DICs), a form of chromosome aberrations, were assayed using a fluorescent in situ hybridization technique.In the patient MNCs, the numbers of γH2AX foci and DICs increased after cardiac catheterization by 4.5 ± 9.4-fold and 71 ± 122%, respectively (P < 0.05 for both). The mRNA expressions of interleukin (IL)-1α, IL-1ß, leukemia inhibitory factor, and caspase-1 were significantly increased by radiation exposure from cardiac catheterization. The increase in IL-1ß was significantly correlated with that of γH2AX, but not with the dose area product. In the operators, neither γH2AX foci nor the DIC level was changed, but IL-1ß mRNA was significantly increased. The protein expression of IκBα was significantly decreased in both groups.DNA damage was increased in the MNCs of patients, but not of operators, who underwent cardiac catheterization. Inflammatory cytokines were increased in both the patients and operators, presumably through NF-κB activation. Further efforts to reduce radiation exposure from cardiac catheterization are necessary for both patients and operators.


Assuntos
Exposição à Radiação , Cateterismo Cardíaco/efeitos adversos , Cateterismo Cardíaco/métodos , Citocinas , Dano ao DNA , Humanos , Hibridização in Situ Fluorescente , RNA Mensageiro , Exposição à Radiação/efeitos adversos
14.
Methods Mol Biol ; 2472: 131-149, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35674897

RESUMO

Notch signaling is crucial to animal development and homeostasis. Notch triggers the transcription of its target genes, which produce diverse outcomes depending on context. The high resolution and spatially precise assessment of Notch-dependent transcription is essential for understanding how Notch operates normally in its native context in vivo and how Notch defects lead to pathogenesis. Here we present biological and computational methods to assess Notch-dependent transcriptional activation in stem cells within their niche, focusing on germline stem cells in the nematode Caenorhabditis elegans. Specifically, we describe visualization of single RNAs in fixed gonads using single-molecule RNA fluorescence in situ hybridization (smFISH), live imaging of transcriptional bursting in the intact organism using the MS2 system, and custom-made MATLAB codes, implementing new image processing algorithms to capture the spatiotemporal patterns of Notch-dependent transcriptional activation. These methods allow a powerful analysis of in vivo transcriptional activation and its dynamics in a whole tissue. Our methods can be adapted to essentially any tissue or cell type for any transcript.


Assuntos
Proteínas de Caenorhabditis elegans , Animais , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Células Germinativas/metabolismo , Hibridização in Situ Fluorescente/métodos , RNA/metabolismo , Receptores Notch/genética , Receptores Notch/metabolismo
15.
Zhonghua Bing Li Xue Za Zhi ; 51(6): 494-499, 2022 Jun 08.
Artigo em Chinês | MEDLINE | ID: mdl-35673719

RESUMO

Objective: To investigate the clinical, histologic, immunohistochemical (IHC) and molecular genetic features of clear cell carcinoma (CCC) of salivary gland in the head and neck regions. Methods: Seven cases of CCC diagnosed in the Department of Pathology, the First Affiliated Hospital of Nanjing Medical University from 2018 to 2021 were included. The clinical and pathologic data, HE sections and IHC staining were reviewed, and EWSR1 gene translocation was detected by fluorescence in situ hybridization (FISH). The relevant literature was also reviewed. Results: There were five males and two females, with an age range of 32 to 71 years (mean 50 years). The tumors were located in the palate, base of tongue, subglottic, right submaxillary and nasopharynx. Histologically the tumors were composed of sheets, nests, and trabecular of large, monomorphic cells which possessed abundant clear and eosinophilic cytoplasm. The stroma was characterized by abundant hyalinized fibrous strands admixed with cellular fibrous (desmoplastic) tissue. The tumor growth was infiltrative. IHC staining revealed positivity for CKpan and squamous cell immunophenotypic markers (CK5/6, p63 and p40), but negativity for myoepithelial markers (SMA, calponin, GFAP and CD10). The EWSR1 gene translocation was detected by FISH. The prognosis was excellent, with the follow-up periods ranging from 8 months to 33 months. During this period, six patients survived without tumor, only one patient with cervical lymph node metastasis. Conclusions: CCC of salivary gland is rare and needs to be differentiated from various other types of tumors containing clear cells. Awareness of the histopathologic characteristics, and combined with IHC and molecular genetic examination can avoid misdiagnosis. The biological behavior of the tumor is indolent with a good overall prognosis.


Assuntos
Adenocarcinoma de Células Claras , Neoplasias das Glândulas Salivares , Adenocarcinoma de Células Claras/patologia , Biomarcadores Tumorais/genética , Feminino , Humanos , Imuno-Histoquímica , Hibridização in Situ Fluorescente , Masculino , Neoplasias das Glândulas Salivares/diagnóstico , Neoplasias das Glândulas Salivares/genética , Glândulas Salivares/patologia
16.
Zhonghua Bing Li Xue Za Zhi ; 51(6): 524-529, 2022 Jun 08.
Artigo em Chinês | MEDLINE | ID: mdl-35673724

RESUMO

Objective: To study the clinicopathological, immunophenotypic and molecular genetic characteristics of nodular fasciitis (NF) in unusual sites. Methods: A total of 50 cases of NF diagnosed between January 2015 and January 2021 were reviewed in the Department of Pathology, Henan Provincial People's Hospital, and the clinical and pathologic data were analyzed. Among them, 14 cases from unusual sites were included in this study. Immunohistochemical (IHC) staining was used to detect the expression of related proteins, and fluorescence in situ hybridization (FISH) was used to detect the breakage of the USP6 gene. Results: There were seven males and seven females in the 14 NF respectively. The lesions were located in the extremities, perineum, breast, wrist joints, the gap between lumbar vertebra 4/5, and in eight cases there was involvement of unusual tissues (six cases in skeletal muscle, one case in nerve root, and one case was intravascular). The tumor boundary was unclear with infiltrating growth. Spindle-shaped myofibroblasts were arranged in bundles or chaotically, with mild pleomorphic, small nucleoli and various mitotic figures. The tumor stroma showed collagenization to myxoid degeneration with erythrocyte extravasation and infiltration of inflammatory cells. IHC staining showed that the spindle cells expressed SMA focally or partially, and p16 diffusely and strongly. FISH showed that 12 of 14 cases had USP6 gene breakage, and two of them occurred in the intrathoracic skeletal muscle with the red signal amplification of USP6 gene. Conclusions: NF in unusual sites shows similar clinicopathological and genetic characteristics to classic NF, but the tumor mostly has infiltrating borders, non-specific and strong expression of p16, and USP6 red signal amplification. The pathological diagnosis of NF in rare sites should be highly vigilant.


Assuntos
Fasciite , Fibroma , Fasciite/diagnóstico , Fasciite/genética , Fasciite/patologia , Feminino , Humanos , Hibridização in Situ Fluorescente , Masculino , Biologia Molecular , Ubiquitina Tiolesterase/genética
17.
Microbiome ; 10(1): 84, 2022 Jun 04.
Artigo em Inglês | MEDLINE | ID: mdl-35659305

RESUMO

BACKGROUND: The increased use of metagenomics and single-cell genomics led to the discovery of organisms from phyla with no cultivated representatives and proposed new microbial lineages such as the candidate phyla radiation (CPR or Patescibacteria). These bacteria have peculiar ribosomal structures, reduced metabolic capacities, small genome, and cell sizes, and a general host-associated lifestyle was proposed for the radiation. So far, most CPR genomes were obtained from groundwaters; however, their diversity, abundance, and role in surface freshwaters is largely unexplored. Here, we attempt to close these knowledge gaps by deep metagenomic sequencing of 119 samples of 17 different freshwater lakes located in Europe and Asia. Moreover, we applied Fluorescence in situ Hybridization followed by Catalyzed Reporter Deposition (CARD-FISH) for a first visualization of distinct CPR lineages in freshwater samples. RESULTS: A total of 174 dereplicated metagenome-assembled genomes (MAGs) of diverse CPR lineages were recovered from the investigated lakes, with a higher prevalence from hypolimnion samples (162 MAGs). They have reduced genomes (median size 1 Mbp) and were generally found in low abundances (0.02-14.36 coverage/Gb) and with estimated slow replication rates. The analysis of genomic traits and CARD-FISH results showed that the radiation is an eclectic group in terms of metabolic capabilities and potential lifestyles, ranging from what appear to be free-living lineages to host- or particle-associated groups. Although some complexes of the electron transport chain were present in the CPR MAGs, together with ion-pumping rhodopsins and heliorhodopsins, we believe that they most probably adopt a fermentative metabolism. Terminal oxidases might function in O2 scavenging, while heliorhodopsins could be involved in mitigation against oxidative stress. CONCLUSIONS: A high diversity of CPR MAGs was recovered, and distinct CPR lineages did not seem to be limited to lakes with specific trophic states. Their reduced metabolic capacities resemble the ones described for genomes in groundwater and animal-associated samples, apart from Gracilibacteria that possesses more complete metabolic pathways. Even though this radiation is mostly host-associated, we also observed organisms from different clades (ABY1, Paceibacteria, Saccharimonadia) that appear to be unattached to any other organisms or were associated with 'lake snow' particles (ABY1, Gracilibacteria), suggesting a broad range of potential life-strategies in this phylum. Video Abstract.


Assuntos
Reanimação Cardiopulmonar , Metagenoma , Animais , Bactérias , Hibridização in Situ Fluorescente , Lagos/microbiologia , Metagenoma/genética , Metagenômica , Filogenia
18.
Mol Med Rep ; 26(1)2022 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-35642665

RESUMO

Airway fibrosis (AF) is a common disease that can severely affect patient prognosis. Epithelial­mesenchymal transition (EMT) participates in the pathophysiological development of AF and several studies have demonstrated that some microRNAs (miRNAs) contribute to the development of EMT. The aim of this study was to investigate the function of miR­423­5p in the EMT process and its possible underlying mechanism in BEAS­2B cells. The present study utilized the BEAS­2B cell line to model EMT in AF. Online tools, fluorescence in situ hybridization analysis and an RNA pull­down assay were used to identify potential target genes of miR­423­5p. In addition, immunohistochemistry, wound healing assays, Transwell migration assays, flow cytometry, enzyme­linked immunosorbent assay, reverse transcription­quantitative PCR, western blot analysis and immunofluorescence staining were used to determine the function of miR­423­5p and its target gene in the EMT process in AF. The results indicated that the miR­423­5p expression in AF tissues and BEAS­2B cells stimulated with 10 ng/ml TGF­ß1 for 24 h was significantly increased compared with that in the control group. Overexpression of miR­423­5p facilitated TGF­ß1­induced EMT in BEAS­2B cells; by contrast, downregulation of miR­423­5p suppressed TGF­ß1­induced EMT in BEAS­2B cells. Furthermore, forkhead box p4 (FOXP4) was identified as a potential target gene of miR­423­5p and changes in the miR­423­5p and FOXP4 expression were shown to significantly affect the expression of PI3K/AKT/mTOR pathway members. In summary, overexpression of miR­423­5P promoted the EMT process in AF by downregulating FOXP4 expression and the underlying mechanism may partly involve activation of the PI3K/AKT/mTOR pathway.


Assuntos
Transição Epitelial-Mesenquimal , Fatores de Transcrição Forkhead , MicroRNAs , Fibrose Pulmonar , Regulação para Baixo , Transição Epitelial-Mesenquimal/genética , Fibrose , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/metabolismo , Regulação Neoplásica da Expressão Gênica , Humanos , Hibridização in Situ Fluorescente , MicroRNAs/genética , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Fibrose Pulmonar/genética , Serina-Treonina Quinases TOR/metabolismo , Fator de Crescimento Transformador beta1/farmacologia
19.
J Contemp Dent Pract ; 23(1): 123-131, 2022 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-35656669

RESUMO

AIM: To evaluate the application of cytogenetic techniques in determining the diagnosis, prognosis, and therapeutics in oral cancer. BACKGROUND: Genetic aberrations that play an important role in oral oncogenesis demand substantial research for in-depth characterization of the tumor. Cytogenetic techniques have the potential to detect these aberrations. This review highlights about various cytogenetic approaches in cancer and how these findings support its application in the field of oral oncology. METHODS: Google scholar search was done for articles on cancer cytogenetics, and in particular, PubMed database was queried for articles published from 2015 to 2020 using keywords cytogenetics, chromosomal aberrations, conventional cytogenetics, karyotyping, banding techniques, molecular cytogenetics, fluorescent in situ hybridization, spectral karyotyping, comparative genomic hybridization, multiplex ligation probe analysis, and next-generation sequencing (NGS) in oral cancer. Abstracts were reviewed, and relevant full text was accessed to extract the cytogenetic findings in oral cancer. RESULTS: Data regarding various cytogenetic approaches from conventional to molecular techniques have been published in oral cancer. They convey a highly complex cytogenetic finding from gross chromosomal aberrations to specific gene mutations in oral cancer. CONCLUSION: Crucial information in the development and progression of oral cancer is achieved through cytogenetic findings in particular with the molecular cytogenetic techniques. Novel technologies like NGS have emerged in recent years that hold promise in the detection of these alterations more efficiently. CLINICAL SIGNIFICANCE: An appraisal of cytogenetic analysis in oral cancer helps to determine the diagnosis and the most important prognosticators. It assists in building targeted therapies for patient benefit.


Assuntos
Neoplasias Bucais , Aberrações Cromossômicas , Hibridização Genômica Comparativa , Análise Citogenética/métodos , Humanos , Hibridização in Situ Fluorescente/métodos , Neoplasias Bucais/diagnóstico , Neoplasias Bucais/genética
20.
Front Endocrinol (Lausanne) ; 13: 859381, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35707464

RESUMO

We collected 61 craniopharyngioma (CP) specimens to investigate the expression of TrkA, ß-catenin, BRAF gene mutation, and NTRK1 fusion in CP. There were 37 male and 24 female individuals with a median age of 34 years (range, 4-75 years). Histologically, there were 46 cases of adamantinomatous craniopharyngioma (ACP), 14 cases of papillary craniopharyngioma (PCP), and 1 case with a mixed adamantinomatous and papillary pattern. By immunohistochemistry, we found that moderate/high TrkA expression was detected in 47% (28/60) CP and was significantly higher in adult patients (p = 0.018). Interestingly, TrkA is more expressed in "whorled epithelium" cells in ACP, similar to the localization of abnormal ß-catenin. The abnormal expression rate of ß-catenin was 70% (43/61), and the medium/high cyclin D1 expression rate was 73% (44/60), both of which were significantly higher in ACP than in PCP. Of the CP, 41% (21/51) had a moderate/strong P16-positive signal; 58% (34/59) showed a high Ki-67 expression, and there was a significant correlation between high Ki-67 L.I. and high tumor recurrence (p = 0.021). NTRK1 fusion was not found in CP by fluorescence in situ hybridization (FISH). By PCR, 26% (15/58) CP showed BRAF V600E gene mutation, which mainly occurred in PCP (100%, 14/14) except one case of mixed CP. Moreover, TrkA expression was negatively correlated with Ki-67 index and positively correlated with P16 expression. There was a significantly negative correlation between BRAF V600E mutation and abnormal ß-catenin expression. Our results demonstrate for the first time that TrkA expression might occur in CP, especially in adult CP patients, and suggest that cyclin D1 could be used for ACP histological classification in addition to ß-catenin and BRAF V600E mutation, while Ki-67 could be used as a marker to predict CP recurrence.


Assuntos
Craniofaringioma , Neoplasias Hipofisárias , Proteínas Proto-Oncogênicas B-raf , Receptor trkA , beta Catenina , Adolescente , Adulto , Idoso , Ciclo Celular , Criança , Pré-Escolar , Craniofaringioma/genética , Ciclina D1/genética , Feminino , Humanos , Hibridização in Situ Fluorescente , Antígeno Ki-67/genética , Masculino , Pessoa de Meia-Idade , Mutação , Neoplasias Hipofisárias/diagnóstico , Neoplasias Hipofisárias/genética , Neoplasias Hipofisárias/metabolismo , Prognóstico , Proteínas Proto-Oncogênicas B-raf/genética , Receptor trkA/genética , Adulto Jovem , beta Catenina/genética
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