fluorescence in situ hybridisation
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2022 ◽  
Vol 8 (1) ◽  
Dianbo Long ◽  
Yiyang Xu ◽  
Guping Mao ◽  
Ruobing Xin ◽  
Zengfa Deng ◽  

AbstracttRNA-derived fragments (tRFs) are new noncoding RNAs, and recent studies have shown that tRNAs and tRFs have important functions in cell metabolism via posttranscriptional regulation of gene expression. However, whether tRFs regulate cellular metabolism of the anterior cruciate ligament (ACL) remains elusive. The aim of this study was to investigate the role and action mechanism of tRFs in ACL cell metabolism. A tRF array was used to determine tRF expression profiles in different human ACL cells, and quantitative real-time polymerase chain reaction and fluorescence in situ hybridisation were used to determine TRF365 expression. ACL cells were transfected with a TRF365 mimic or a TRF365 inhibitor to determine whether TRF365 regulates IKBKB expression. A rescue experiment and dual-luciferase reporter assay were conducted to determine whether the 3′-untranslated region (UTR) of IKBKB has a TRF365-binding site. TRF365 was weakly expressed in osteoarthritis (OA) ACL and interleukin-1β-treated ACL cells. IKBKB was highly expressed in OA ACL and interleukin-1β-treated ACL cells; transfection with the TRF365 mimic suppressed IKBKB expression, whereas transfection with the TRF365 inhibitor had the opposite effect. A dual-luciferase reporter assay showed that TRF365 silenced the expression of IKBKB by binding to its 3′-UTR. Thus, TRF365 regulates the metabolism of ACL cells by targeting IKBKB. In summary, TRF365 may provide a new direction for the study of ACL degeneration and on the pathophysiological process of OA.

Nutrients ◽  
2021 ◽  
Vol 13 (12) ◽  
pp. 4447
Litai Liu ◽  
Carlos Poveda ◽  
Paul E. Jenkins ◽  
Gemma E. Walton

Individuals with anorexia nervosa (AN) often suffer psychological and gastrointestinal problems consistent with a dysregulated gut microbial community. Psychobiotics have been postulated to modify microbiota and improve mental well-being and gut symptoms, but there is currently a lack of evidence for such approaches in AN. The aim of this study was to use an in vitro colonic model to evaluate the impact of dietary restrictions associated with AN on the intestinal ecosystem and to assess the impact of pre and probiotic intervention. Bacteriology was quantified using flow cytometry combined with fluorescence in situ hybridisation and metabolic end products (including neurotransmitters) by gas chromatography and liquid chromatography mass spectrometry Consistent with previous research, the nutritional changes significantly reduced total microbiota and metabolites compared with healthy conditions. Pre and probiotic supplementation on restricted conditions enhanced the microbial community and modulated metabolic activity to resemble that of a healthy diet. The model system indicates that nutritional changes associated with AN can impact the microbial community, and that these changes can, at least in part, be restored through the use of pre and probiotic interventions.

2021 ◽  
Vol 8 (1) ◽  
pp. e001127
John A Mackintosh ◽  
Maria Pietsch ◽  
Viviana Lutzky ◽  
Debra Enever ◽  
Sandra Bancroft ◽  

IntroductionRecent discoveries have identified shortened telomeres and related mutations in people with pulmonary fibrosis (PF). There is evidence to suggest that androgens, including danazol, may be effective in lengthening telomeres in peripheral blood cells. This study aims to assess the safety and efficacy of danazol in adults and children with PF associated with telomere shortening.Methods and analysisA multi-centre, double-blind, placebo-controlled, randomised trial of danazol will be conducted in subjects aged >5 years with PF associated with age-adjusted telomere length ≤10th centile measured by flow fluorescence in situ hybridisation; or in children, a diagnosis of dyskeratosis congenita. Adult participants will receive danazol 800 mg daily in two divided doses or identical placebo capsules orally for 12 months, in addition to standard of care (including pirfenidone or nintedanib). Paediatric participants will receive danazol 2 mg/kg/day orally in two divided doses or identical placebo for 6 months. If no side effects are encountered, the dose will be escalated to 4 mg/kg/day (maximum 800 mg daily) orally in two divided doses for a further 6 months. The primary outcome is change in absolute telomere length in base pairs, measured using the telomere shortest length assay (TeSLA), at 12 months in the intention to treat population.Ethics and disseminationEthics approval has been granted in Australia by the Metro South Human Research Ethics Committee (HREC/2020/QMS/66385). The study will be conducted and reported according to Standard Protocol Items: Recommendations for Interventional Trials guidelines. Results will be published in peer-reviewed journals and presented at international and national conferences.Trial registration numbersNCT04638517; Australian New Zealand Clinical Trials Registry (ACTRN12620001363976p).

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 2619-2619
Kathy Fuller ◽  
Henry Hui ◽  
Jason Stanley ◽  
Wendy N. Erber

Abstract Chronic lymphocytic leukaemia is a genetically heterogeneous disease with treatment and prognosis varying based on chromosomal abnormalities. These are detectable in up to 80% of cases when tested on the nuclei of interphase cells by fluorescence in situ hybridisation (FISH). Despite the clinical importance of FISH in management, as only up to 200 nuclei are generally assessed, it is not suitable for minimal residual disease (MRD) assessment. Since clinical decisions are based on detection thresholds of 10 -4, MRD assays are restricted to flow cytometry and molecular based assessment. Here we have explored the utility of a cutting-edge automated imaging flow cytometry method that incorporates cell immunophenotype and FISH ("immuno-flowFISH") to detect chromosomal abnormalities in CLL. Aims: Our aim was to determine the capability of immuno-flowFISH using imaging flow cytometry to detect del(17p) and +12 in CLL, and, the lowest limit of detection. We hypothesized that this integrated automated immuno-flowFISH method would be suitable for MRD assessment of CLL. Methods: Blood from 75 patients with CLL, at diagnosis or on therapy, was analysed. For MRD studies, cells from the CI cell line were spiked into normal blood at concentrations of 0.001 - 10%. After red cell lysis, samples were incubated with CD3, CD5 and CD19 fluorophore-conjugated antibodies (fluorophores: BV480, BV605, AF647). Following fixation and membrane permeabilization, DNA was denatured at 78 oC for 5 mins. FISH probes to 17p12 and centromeres of chromosomes 12 and 17 were added and hybridized for 24 hours at 42 oC. Nuclei were then stained with SYTOX AADvanced and up to 600,000 cells acquired on the Amnis ® ImageStream ®XMk II imaging flow cytometer. Digital images (x60 objective) and quantitative data derived from computational algorithms (IDEAS software) were used to assess FISH signals overlying cell nuclei. IDEAS was then used to assess the number and percent CD19/CD5-positive CLL cells with FISH abnormalities. Results: Between 10,000 and 600,000 cells (mean 60,000) were acquired. CLL (CD19/CD5-positive) and T- (CD3/CD5-positive) cells could be clearly identified by their immunophenotype and assessed individually for probe signals. FISH signals were identifiable on the digital images as specific "spots" overlying the SYTOX AADvanced nuclear stain. The IDEAS software could enumerate the number of FISH spots per cell and this was confirmed by quantitative mean channel fluorescence intensity for each probe. A chromosome 12 or 17 abnormality was detected in 23 of the 75 CLL cases. Of these, 10 cases had only one 17p signal (but 2 for the centromere of chromosome 17), indicative of del(17p). Del(17p) was detected in 2-35% of CD19/CD5-positive cells (i.e. 0.4-23% or 270-35,441 of all cells), the lowest seen in a patient on cytoreductive therapy. In 13/75 cases, there were 3 FISH signals for CEP12, consistent with trisomy 12 (+12) in 0.1-46% of all cells analysed; the lowest number of 0.1% was when 26 out of 26,000 cells analysed were CD19/CD5-positive and had +12. We also performed multi-FISH, incorporating CEP12, CEP17 and 17p probes together with the CD3, CD5 and CD19 antibodies. This required 7-fluorophores (antibodies, probes and nucleus) and confirmed the ability to detect del(17p) and chromosome 12 copy number simultaneously in a single analysis. Spiking of CI CLL cells into normal blood demonstrated that +12 could be detected to a lowest limit of 10 -5. In all analyses, CLL cells had normal diploid spots for the control CEP17 probe, and the CD3/CD5-positive T cells had dual signals for CEP12, CEP17 and 17p12 probes on numerical analysis and on digital imagery. Conclusion: This study of confirms that high-throughput automated imaging flow cytometry, integrating FISH and immunophenotyping, can detect chromosomal defects in CLL. The lowest limit of detection for a FISH-detectable abnormality was 10 -5. This high sensitivity and specificity exceeds current clinical practice (10 -4), and was achieved through the analysis of many thousands of cells and positive identification of CLL cells based on their phenotype. This immuno-flowFISH method does not require any prior cell separation and is automated. It adds a new dimension to chromosomal analysis in CLL, both at diagnosis and for MRD monitoring. The high precision and specificity of immuno-flowFISH illustrates that this has a real place as a new MRD assessment tool for CLL. Disclosures No relevant conflicts of interest to declare.

Pathogens ◽  
2021 ◽  
Vol 10 (11) ◽  
pp. 1401
Maude Dagenais ◽  
Jared Q. Gerlach ◽  
George R. Wendt ◽  
James J. Collins ◽  
Louise E. Atkinson ◽  

Parasitic helminths are master manipulators of host immunity. Their strategy is complex and involves the release of excreted/secreted products, including extracellular vesicles (EVs). The protein and miRNA contents of EVs have been characterised for many parasitic helminths but, despite reports suggesting the importance of EV surface carbohydrate structures (glycans) in the interactions with target cells and thus subsequent effector functions, little is known about parasite EV glycomics. Using lectin microarrays, we identified several lectins that exhibit strong adhesion to Schistosoma mansoni EVs, suggesting the presence of multiple glycan structures on these vesicles. Interestingly, SNA-I, a lectin that recognises structures with terminal sialic acid, displayed strong affinity for S. mansoni EVs, which was completely abolished by neuraminidase treatment, suggesting sialylation in the EV sample. This finding is of interest, as sialic acids play important roles in the context of infection by aiding immune evasion, affecting target recognition, cell entry, etc., but are not thought to be synthesised by helminths. These data were validated by quantitative analysis of free sialic acid released from EVs following treatment with neuraminidase. Lectin histochemistry and fluorescence in situ hybridisation analyses on whole adult worms suggest the involvement of sub-tegumental cell bodies, as well as the digestive and excretory systems, in the release of EVs. These results support previous reports of EV biogenesis diversity in trematodes and potentially highlight new means of immune modulation and evasion employed by schistosomes.

2021 ◽  
pp. bjophthalmol-2021-319580
Marina Knudsen Kirkegaard ◽  
Marthe Minderman ◽  
Lene Dissing Sjö ◽  
Steven T Pals ◽  
Patrick R G Eriksen ◽  

AimsTo (1) reclassify ocular adnexal large B-cell lymphomas (OA-LBCLs) per 2016 WHO lymphoma classification and (2) determine the prevalence of MYD88 and CD79B mutations and their association with clinical parameters among OA-LBCLs.MethodsThis study is a retrospective analysis of all OA-LBCLs diagnosed in Denmark between 1980 and 2018. Medical records and tissue samples were retrieved. Thirty-four OA-LBCLs were included. Fluorescence in situ hybridisation and Epstein-Barr-encoded RNA in situ hybridisation were used for the reclassification. Mutational status was established by allele-specific PCR and confirmed by Sanger sequencing. Primary endpoints were overall survival, disease-specific survival (DSS) and progression-free survival (PFS).ResultsTwo LBCL subtypes were identified: diffuse large B-cell lymphoma (DLBCL) (27 of 32; 84%) and high-grade B-cell lymphoma (HGBL) with MYC and BCL2 and/or BCL6 rearrangements (5 of 32; 16%). cMYC/BCL2 double-expressor DLBCLs had a poorer DSS than non-double-expressor DLBCLs (5-year DSS, 25% vs 78%) (HR 0.23; 95% CI 0.06 to 0.85; p=0.014). MYD88 mutations were present in 10 (29%) of 34 lymphomas and carried a poorer PFS than wild-type cases (5-year PFS, 0% vs 43%) (HR 0.78; 95% CI 0.61 to 0.98; p=0.039). CD79B mutations were present in 3 (9%) of 34 cases.ConclusionOA-LBCL consists mainly of two subtypes: DLBCL and HGBL with MYC and BCL2 and/or BCL6 rearrangements. MYD88 mutations are important drivers of OA-LBCL. MYD88 mutations, as well as cMYC/BCL2 double-expressor DLBCL, appear to be associated with a poor prognosis. Implementing MYD88 mutational analysis in routine diagnostics may improve OA-LBCL prognostication.

Plants ◽  
2021 ◽  
Vol 10 (11) ◽  
pp. 2292
Pilar Prieto ◽  
Carmen Palomino ◽  
Zuny Cifuentes ◽  
Adoración Cabrera

Crested wheatgrass (Agropyron cristatum L. Gaertn., genome P), included in the Triticeae tribe (family Poaceae), is one of the most important grasses in temperate regions. It has been valued as a donor of important agronomic traits for wheat improvement, including tolerance to cold, drought, and high salinity, as well as resistance to leaf rust, stripe rust, and powdery mildew. For successful incorporation of beneficial alleles into wheat, it is essential that recombination between wheat and A. cristatum chromosomes occurs. In this work, we analysed chromosome associations during meiosis in wheat lines carrying chromosome introgressions from A. cristatum chromosomes 5P and 6P in the presence and absence of Ph1 locus using fluorescence in situ hybridisation. The results showed that the Ph1 locus does not affect chromosome associations between A. cristatum and wheat chromosomes because there were no interspecific chromosome associations; therefore, no recombination between chromosomes from wheat and Agropyron were observed in the absence of the Ph1 locus. The 5P and 6P A. cristatum chromosomes do not have a suppressor effect on the Ph1 locus. Wheat univalents in metaphase I suggest that Agropyron chromosomes might carry genes having a role in wheat homologous chromosome associations. Putative effect of the Agropyron genes on wheat chromosome associations does not interact with the Ph1 locus.

DNA ◽  
2021 ◽  
Vol 1 (2) ◽  
pp. 68-76
Nicole M. Lewis ◽  
Claudia C. Rathje ◽  
Carla Canedo-Ribeiro ◽  
Lisa M. Bosman ◽  
Lucas G. Kiazim ◽  

Pigs (Sus scrofa) have vast economic importance, with pork accounting for over 30% of the global meat consumption. Chromosomal abnormalities, and in particular reciprocal translocations (RTs), are an important cause of hypoprolificacy (litter size reduction) in pigs. However, these do not necessarily present with a recognizable phenotype and may cause significant economic losses for breeders when undetected. Here, we present a reappraisal of the incidence of RTs across several European pig herds, using contemporary methodology, as well as an analysis modelling the economic impact of these abnormalities. Molecular cytogenetic investigation was completed by karyotyping and/or multiprobe FISH (fluorescence in situ hybridisation) between 2016–2021, testing 2673 animals. We identified 19 types of chromosome abnormalities, the prevalence of these errors in the database was 9.1%, and the estimated incidence of de novo errors was 0.90%. Financial modelling across different scenarios revealed the potential economic impact of an undetected RT, ranging from £69,802 for an individual affected terminal boar in a commercial farm selling weaned pigs, to £51,215,378 for a genetics company with an undetected RT in a dam line boar used in a nucleus farm. Moreover, the added benefits of screening by FISH instead of karyotyping were estimated, providing a strong case for proactive screening by this approach.

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