Reorganisation of human sperm nuclear architecture during formation of pronuclei in a model system

2009 ◽  
Vol 21 (5) ◽  
pp. 665 ◽  
Author(s):  
Olga Mudrak ◽  
Rajeev Chandra ◽  
Estella Jones ◽  
Earl Godfrey ◽  
Andrei Zalensky
Keyword(s):  
In Situ Hybridisation ◽  
Nuclear Architecture ◽  
Human Sperm ◽  
Model Systems ◽  
Sperm Chromatin ◽  
Human Embryos ◽  
Fluorescence In Situ Hybridisation ◽  
Paternal Genome ◽  
Adequate Model

By fertilisation, two terminally differentiated cells, namely the egg and spermatozoon, are combined to create a totipotent zygote. During this process, the inactive sperm nucleus is transformed into a functional male pronucleus. Recent studies demonstrate that human sperm chromatin has an elaborate multilevel organisation, but almost nothing is known about how sperm chromosomes are transformed during fertilisation. Because of ethical reasons and technical complications, experimentation with human embryos is generally unworkable and adequate model systems are necessary to study the formation of male pronuclei. Here, we analyse remodelling of human sperm chromatin and chromosome architecture in Xenopus egg extracts using immunofluorescent localisation of protamines and centromere protein A, as well as fluorescence in situ hybridisation localisation of major α-satellite DNA and whole chromosome territory (CT). We demonstrate noticeable relocalisation of centromeres and remodelling of CT during the decondensation–recondensation cycle, mimicking cellular events that occur in the paternal genome in vivo during fertilisation.

scholarly journals A novel long non-coding RNA GK-IT1 promotes the carcinogenesis and progression of esophageal squamous cell carcinoma via mediating the phosphorylation of MAPK1

2020 ◽  
Author(s):  
Xin Yang ◽  
Tian Yang Zeng ◽  
Zi Yang Liu ◽  
Wan Lun He ◽  
Meng Ting Hu ◽  
...  
Keyword(s):  
Squamous Cell Carcinoma ◽  
Esophageal Squamous Cell Carcinoma ◽  
Cell Carcinoma ◽  
Squamous Cell ◽  
Mapk Pathway ◽  
Statistical Significance ◽  
In Situ Hybridisation ◽  
Fluorescence In Situ Hybridisation ◽  
Normal Tissues

Abstract Background: Recent studies have shown that Long non-coding RNAs (lncRNAs) are crucial in the invasion, angiogenesis, progression, and metastasis of esophageal squamous cell carcinoma (ESCC). However, the biological functions and potential molecular mechanism of LncRNA GK-IT1 in esophageal squamous cell carcinoma has not been reported.Methods: We analysed the expression of GK-IT1 in ESCC and their adjacent normal tissues in the TCGA database. The quantitative real-time-PCR (qRT-PCR) was used to detect the expression of GK-IT1 in Clinical specimens. The Kaplan-Meier method was employed to draw the survival curve and then the statistical significance was calculated using the logarithmic rank test. a range of functional experiments in vivo and in vitro were used to explore the role of GK-IT1 in the carcinogenesis and development of ESCC. RNA pull down assay, RNA immunoprecipitation (RIP), fluorescence in situ hybridisation (FISH), agarose gel electrophoresis and immunofluorescence were all employed to explore the interaction mechanism between GK-IT1 and MAPK1 (mitogen activated protein kinase 1).Results: The expression of GK-IT1 was higher in ESCC than adjacent normal tissues, which was positively correlated with the clinical stage and shorter survival time. The knockout of the GK-IT1 gene significantly attenuated the abilities of ESCC cell proliferation, invasion and migration, induced apoptosis and autophagy in ESCC cells and inhibited tumour growth and tumour metastasis in vivo. on the contrary, the upregulation of GK-IT1 had the opposite effect. Further studies have shown that GK-IT1 can regulate the biological process of ESCC by regulating the phosphorylation of MAPK1.Conclusion: Our study reveals that GK-IT1 mediated the phosphorylation of MAPK1 improve the carcinogenesis and development of esophageal squamous cell carcinoma through ERK/MAPK pathway which indicates that GK-IT1 possesses substantial potential as a novel biomarker for ESCC prognosis and therapy.

scholarly journals CircSERPINE2 protects against osteoarthritis by targeting miR-1271 and ETS-related gene

2019 ◽  
Vol 78 (6) ◽  
pp. 826-836 ◽  
Author(s):  
Shuying Shen ◽  
Yizheng Wu ◽  
Junxin Chen ◽  
Ziang Xie ◽  
Kangmao Huang ◽  
...  
Keyword(s):  
In Situ Hybridisation ◽  
Rabbit Model ◽  
Pathological Process ◽  
Luciferase Reporter ◽  
Circular Rnas ◽  
Fluorescence In Situ Hybridisation ◽  
Related Gene ◽  
Downstream Target

ObjectivesCircular RNAs (circRNA) expression aberration has been identified in various human diseases. In this study, we investigated whether circRNAs could act as competing endogenous RNAs to regulate the pathological process of osteoarthritis (OA).MethodsCircRNA deep sequencing was performed to the expression of circRNAs between OA and control cartilage tissues. The regulatory and functional role of CircSERPINE2 upregulation was examined in OA and was validated in vitro and in vivo, downstream target of CircSERPINE2 was explored. RNA pull down, a luciferase reporter assay, biotin-coupled microRNA capture and fluorescence in situ hybridisation were used to evaluate the interaction between CircSERPINE2 and miR-1271-5 p, as well as the target mRNA, E26 transformation-specific-related gene (ERG). The role and mechanism of CircSERPINE2 in OA was also explored in rabbit models.ResultsThe decreased expression of CircSERPINE2 in the OA cartilage tissues was directly associated with excessive apoptosis and imbalance between anabolic and catabolic factors of extracellular matrix (ECM). Mechanistically, CircSERPINE2 acted as a sponge of miR-1271-5 p and functioned in human chondrocytes (HCs) through targeting miR-1271-5 p and ERG. Intra-articular injection of adeno-associated virus-CircSERPINE2-wt alleviated OA in the rabbit model.ConclusionsOur results reveal an important role for a novel circRNA-CircSERPINE2 in OA progression. CircSERPINE2 overexpression could alleviate HCs apoptosis and promote anabolism of ECM through miR-1271-ERG pathway. It provides a potentially effective therapeutic strategy for OA progression.

Liquid biopsies to track trastuzumab resistance in metastatic HER2-positive gastric cancer

Gut ◽  
2018 ◽  
Vol 68 (7) ◽  
pp. 1152-1161 ◽  
Author(s):  
De-Shen Wang ◽  
Ze-Xian Liu ◽  
Yun-Xin Lu ◽  
Hua Bao ◽  
Xue Wu ◽  
...  
Keyword(s):  
Gastric Cancer ◽  
In Situ Hybridisation ◽  
Targeted Sequencing ◽  
In Vivo Studies ◽  
Fluorescence In Situ Hybridisation ◽  
Trastuzumab Resistance ◽  
Liquid Biopsies ◽  
Her2 Positive

ObjectiveTo monitor trastuzumab resistance and determine the underlying mechanisms for the limited response rate and rapid emergence of resistance of HER2+ metastatic gastric cancer (mGC).DesignTargeted sequencing of 416 clinically relevant genes was performed in 78 paired plasma and tissue biopsy samples to determine plasma-tissue concordance. Then, we performed longitudinal analyses of 97 serial plasma samples collected from 24 patients who were HER2+  to track the resistance during trastuzumab treatment and validated the identified candidate resistance genes.ResultsThe results from targeted sequencing-based detection of somatic copy number alterations (SCNA) of HER2 gene were highly consistent with fluorescence in situ hybridisation data, and the detected HER2 SCNA was better than plasma carcinoembryonic antigen levels at predicting tumour shrinkage and progression. Furthermore, most patients with innate trastuzumab resistance presented high HER2 SCNA during progression compared with baseline, while HER2 SCNA decreased in patients with acquired resistance. PIK3CA mutations were significantly enriched in patients with innate resistance, and ERBB2/4 genes were the most mutated genes, accounting for trastuzumab resistance in six (35.3%) and five (29.4%) patients in baseline and progression plasma, respectively. Patients with PIK3CA/R1/C3 or ERBB2/4 mutations in the baseline plasma had significantly worse progression-free survival. Additionally, mutations in NF1 contributed to trastuzumab resistance, which was further confirmed through in vitro and in vivo studies, while combined HER2 and MEK/ERK blockade overcame trastuzumab resistance.ConclusionLongitudinal circulating tumour DNA sequencing provides novel insights into gene alterations underlying trastuzumab resistance in HER2+mGC.

scholarly journals Novel lncRNA UPLA1 mediates tumorigenesis and prognosis in lung adenocarcinoma

2020 ◽  
Vol 11 (11) ◽  
Author(s):  
Xiaoyang Han ◽  
Hua Jiang ◽  
Jianni Qi ◽  
Jiamei Li ◽  
Jinghan Yang ◽  
...  
Keyword(s):  
Lung Adenocarcinoma ◽  
In Situ Hybridisation ◽  
Vital Role ◽  
Luciferase Reporter ◽  
Fluorescence In Situ Hybridisation ◽  
Biological Functions ◽  
Pulldown Assay

AbstractWith the development of molecular biotechnology and sequencing techniques, long non-coding RNAs (lncRNAs) have been shown to play a vital role in a variety of cancers including lung cancer. In our previous study, we used RNA sequencing and high-content screening proliferation screening data to identify lncRNAs that were significantly associated with tumour biological functions such as LINC01426. Herein, based on previous work, we report a novel lncRNA UPLA1 (upregulation promoting LUAD-associated transcript-1), which has not been explored or reported in any previous studies. Our results showed that UPLA1 is highly expressed and regulates important biological functions in lung adenocarcinoma. In vitro experiments revealed that UPLA1 promoted the migration, invasion, and proliferation abilities, and is related to cell cycle arrest, in lung adenocarcinoma cells. Moreover, the upregulation of UPLA1 significantly improved the growth of tumours in vivo. We identified that UPLA1 was mainly located in the nucleus using fluorescence in situ hybridisation, and that it promoted Wnt/β-catenin signalling by binding to desmoplakin using RNA pulldown assay and mass spectrometry. Additionally, luciferase reporter assay revealed that YY1 is the transcription factor of UPLA1 and suppressed the expression of UPLA1 as a transcriptional inhibitor. This finding provides important evidence regarding the two roles of YY1 in cancer. Furthermore, in situ hybridisation assay results showed that UPLA1 was closely related to the prognosis and tumour, node, metastasis (TNM) stage of lung adenocarcinoma. In summary, our results suggest that the novel lncRNA UPLA1 promotes the progression of lung adenocarcinoma and may be used as a prognostic marker, and thus, has considerable clinical significance.

scholarly journals Critical assessment and integration of separate lines of evidence for risk assessment of chemical mixtures

2019 ◽  
Vol 93 (10) ◽  
pp. 2741-2757 ◽  
Author(s):  
Antonio F. Hernandez ◽  
Aleksandra Buha ◽  
Carolina Constantin ◽  
David R. Wallace ◽  
Dimosthenis Sarigiannis ◽  
...  
Keyword(s):  
In Silico ◽  
Mixture Toxicity ◽  
Chemical Exposure ◽  
Model Systems ◽  
Chemical Mixtures ◽  
Adequate Model ◽  
Chemical Mixture ◽  
Single Chemical

Abstract Humans are exposed to multiple chemicals on a daily basis instead of to just a single chemical, yet the majority of existing toxicity data comes from single-chemical exposure. Multiple factors must be considered such as the route, concentration, duration, and the timing of exposure when determining toxicity to the organism. The need for adequate model systems (in vivo, in vitro, in silico and mathematical) is paramount for better understanding of chemical mixture toxicity. Currently, shortcomings plague each model system as investigators struggle to find the appropriate balance of rigor, reproducibility and appropriateness in mixture toxicity studies. Significant questions exist when comparing single-to mixture-chemical toxicity concerning additivity, synergism, potentiation, or antagonism. Dose/concentration relevance is a major consideration and should be subthreshold for better accuracy in toxicity assessment. Previous work was limited by the technology and methodology of the time, but recent advances have resulted in significant progress in the study of mixture toxicology. Novel technologies have added insight to data obtained from in vivo studies for predictive toxicity testing. These include new in vitro models: omics-related tools, organs-on-a-chip and 3D cell culture, and in silico methods. Taken together, all these modern methodologies improve the understanding of the multiple toxicity pathways associated with adverse outcomes (e.g., adverse outcome pathways), thus allowing investigators to better predict risks linked to exposure to chemical mixtures. As technology and knowledge advance, our ability to harness and integrate separate streams of evidence regarding outcomes associated with chemical mixture exposure improves. As many national and international organizations are currently stressing, studies on chemical mixture toxicity are of primary importance.

Assessment of aneuploidy and developmental potential of human embryos by fluorescence in situ hybridisation and amino acid profiling

2002 ◽  
Vol 78 ◽  
pp. S185
Author(s):  
Julie M Clyde ◽  
Franchesca D Houghton ◽  
Anthony J Rutherford ◽  
Jan E Hogg ◽  
Henry J Leese ◽  
...  
Keyword(s):  
Amino Acid ◽  
In Situ Hybridisation ◽  
Human Embryos ◽  
Fluorescence In Situ Hybridisation ◽  
Developmental Potential ◽  
Amino Acid Profiling

scholarly journals Human Sperm Chromosomes: To Form Hairpin-Loops, Or Not to Form Hairpin-Loops, That Is the Question

Genes ◽  
2019 ◽  
Vol 10 (7) ◽  
pp. 504 ◽  
Author(s):  
Ioannou ◽  
Tempest
Keyword(s):  
Human Sperm ◽  
Sperm Nucleus ◽  
Sperm Chromatin ◽  
Wide Field ◽  
Loop Model ◽  
Hairpin Loop ◽  
Paternal Genome ◽  
Hairpin Loops ◽  
Sperm Chromosomes ◽  
Radial Organization

Background: Genomes are non-randomly organized within the interphase nucleus; and spermatozoa are proposed to have a unique hairpin-loop configuration, which has been hypothesized to be critical for the ordered exodus of the paternal genome following fertilization. Recent studies suggest that the hairpin-loop model of sperm chromatin organization is more segmentally organized. The purpose of this study is to examine the 3D organization and hairpin-loop configurations of chromosomes in human spermatozoa. Methods: Three-color sperm-fluorescence in-situ hybridization was utilized against the centromeres, and chromosome p- and q-arms of eight chromosomes from five normozoospermic donors. Wide-field fluorescence microscopy and 3D modelling established the radial organization and hairpin-loop chromosome configurations in spermatozoa. Results: All chromosomes possessed reproducible non-random radial organization (p < 0.05) and formed discrete hairpin-loop configurations. However, chromosomes preferentially formed narrow or wide hairpin-loops. We did not find evidence to support the existence of a centralized chromocenter(s) with centromeres being more peripherally localized than one or both of their respective chromosome arms. Conclusion: This provides further evidence to support a more segmental organization of chromatin in the human sperm nucleus. This may be of significance for fertilization and early embryogenesis as specific genomic regions are likely to be exposed, remodeled, and activated first, following fertilization.

scholarly journals In vitro and in silico modeling of cellular and matrix-related changes during the early phase of osteoarthritis

2019 ◽  
Author(s):  
Marie-Christin Weber ◽  
Lisa Fischer ◽  
Alexandra Damerau ◽  
Igor Ponomarev ◽  
Moritz Pfeiffenberger ◽  
...  
Keyword(s):  
In Silico ◽  
Early Phase ◽  
Cell Number ◽  
Model Systems ◽  
Adequate Model ◽  
In Silico Modeling ◽  
Efficient Alternative ◽  
Factor Α

AbstractObjectiveUnderstanding the pathophysiological processes of osteoarthritis (OA) require adequate model systems. Although different in vitro or in vivo models have been described, further comprehensive approaches are needed to study specific parts of the disease. This study aimed to combine in vitro and in silico modeling to describe cellular and matrix-related changes during the early phase of OA. We developed an in vitro OA model based on scaffold-free cartilage-like constructs (SFCCs), which was mathematically modeled using a partial differential equation (PDE) system to resemble the processes during the onset of OA.DesignSFCCs were produced from mesenchymal stromal cells and analyzed weekly by histology and qPCR to characterize the cellular and matrix-related composition. To simulate the early phase of OA, SFCCs were treated with interleukin-1β (IL-1β), tumor necrosis factor α (TNFα) and examined after 3 weeks or cultivated another 3 weeks without inflammatory cytokines to validate the regeneration potential. Mathematical modeling was performed in parallel to the in vitro experiments.ResultsSFCCs expressed cartilage-specific markers, and after stimulation an increased expression of inflammatory markers, matrix degrading enzymes, a loss of collagen II (Col-2) and a reduced cell density was observed which could be partially reversed by retraction of stimulation. Based on the PDEs, the distribution processes within the SFCCs, including those of IL-1β, Col-2 degradation and cell number reduction was simulated.ConclusionsBy combining in vitro and in silico methods, we aimed to develop a valid, efficient alternative approach to examine and predict disease progression and new therapeutic strategies.

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