Search Results (75)

Nearly four decades have passed since fluorescence in situ hybridisation was first applied in plants to support molecular cytogenetic analyses across a wide range of species. Subsequent advances in DNA sequencing, bioinformatic analysis, and microscopy, together with the immunolocalisation of various nuclear components, have provided unprecedented insights into the cytomolecular organisation of the nuclear genome in both model and non-model plants, with crop species being perhaps the most significant. The ready availability of sequenced genomes is now facilitating the application of state-of-the-art cytomolecular techniques across diverse plant species. However, these same advances in genomics also pose a challenge to the future of plant molecular cytogenetics, as DNA sequence analysis is increasingly perceived as offering comparable insights into genome organisation. This perception persists despite the continued relevance of FISH-based approaches for the physical anchoring of genome assemblies to chromosomes. Furthermore, cytogenetic approaches cannot currently rival purely genomic methods in terms of throughput, standardisation, and automation. This review highlights the latest key topics in plant cytomolecular research, with particular emphasis on chromosome identification and karyotype evolution, chromatin and interphase nuclear organisation, chromosome structure, hybridisation and polyploidy, and cytogenetics-assisted crop improvement. In doing so, it underscores the distinctive contributions that cytogenetic techniques continue to offer in genomic research. Additionally, we critically assess future directions and emerging opportunities in the field, including those related to CRISPR/Cas-based live-cell imaging and chromosome engineering, as well as AI-assisted image analysis and karyotyping. Full article

Muehlenbeckia tuggeranong is an endangered subshrub with an estimated seven individuals remaining in its native habitat, and twelve held in an ex situ living collection in the Australian National Botanic Gardens, Canberra. We conducted a genetic analysis on all known individuals of the species both in situ and ex situ to inform the conservation management of one of the rarest plants in Australia, certainly the rarest in the Australian Capital Territory. We found recent seedlings did not result from hybridisation with M. axillaris but resulted from sexual reproduction within the ex situ collection, leading to greater genetic diversity ex situ than in situ. However, low genetic diversity across the species indicates a high risk of extinction. Through simulations we identified the optimal breeding pairs to minimise further genetic diversity loss and increase the number of available genotypes for future reintroduction. Our work highlights the need to incorporate genetically informed breeding programs into living collections management of endangered plant species, particular those with unique life history traits. Full article

Microbiological quality control in cosmetic and pharmaceutical products is crucial for consumer safety. Traditional culture-based detection methods, such as plating on selective media, are time-consuming and may lack sensitivity. Fluorescence In Situ Hybridisation (FISH), a molecular and culture-independent technique, enables rapid and precise microbial identification by targeting specific RNA or DNA sequences with fluorescent probes. In this study, a novel DNA-FISH probe was developed for the detection of Candida albicans in cosmetic formulations. The probe’s specificity was assessed in silico and experimentally using flow cytometry (flow-FISH) on C. albicans and non-target microorganisms, including Pichia kudriavzevii, commonly known as Candida krusei, Saccharomyces cerevisiae, Wickerhamomyces anomalus, Escherichia coli, and Staphylococcus aureus. The probe exhibited 98.9% hybridization efficiency with C. albicans, yielding a fluorescence intensity (FI) of 25,000 (a.u.), while non-target yeasts demonstrated minimal hybridization (4.7%, 2.3%, and 1.9% for C. krusei, S. cerevisiae, and W. anomalus, respectively) and bacteria showed negligible FI. Additionally, the probe’s applicability was evaluated in a tonic formulation, where C. albicans’ hybridization efficiency was slightly reduced to 88.4%, suggesting that formulation components may influence probe performance. Nevertheless, the probe maintained high specificity and efficiency without formamide, a toxic reagent commonly used in FISH. These findings highlight the potential of FISH probes for rapid, accurate, and safe microbial detection, offering a valuable tool for microbiological quality control in the cosmetics industry. Full article

Diffuse large B-cell lymphoma is a prevalent subtype of adult non-Hodgkin lymphoma; noted for its biological and clinical variability. Background and Objectives: This study seeks to assess the expression and prognostic implications of Ki-67, MYC, and BCL2 utilising immunohistochemistry on a cohort of Romanian patients diagnosed with DLBCL while also addressing the limitations imposed by the absence of fluorescence in situ hybridisation testing in resource-constrained settings. Materials and Methods: A single-centre, retrospective study involved 66 cases of formalin-fixed, paraffin-embedded tissue specimens obtained from patients with this lymphoma. Results: The median age at diagnosis was 61.81 years, with most individuals being 60 years or older; 59.1% of the patients were male. Our study identified that 65.2% of the cases belonged to the non-GCB subtype (ABC). MYC-positive expression was observed in 5 out of 66 cases (7.6%), and BCL2 protein expression exhibited a trend toward statistical significance, indicating a lower overall survival for BCL-2-positive patients. The expression of Ki-67 demonstrated a significant correlation with variations in overall survival (OS) (p < 0.001). Patients with low Ki-67 expression had an average survival duration of 76.39 months, contrasting with individuals exhibiting high Ki-67 expression, with a mean survival of 38.98 months. In conclusion, MYC, BCL2, and Ki-67 may be valuable prognostic indicator biomarkers. Conclusions: The prognostic significance of each biomarker varies based on the established cut-off point value. Future research should examine the relationship between protein biomarkers, morphological characteristics, and clinical outcomes in Romanian patients diagnosed with DLBCL, aiming to elucidate clinical ramifications and foster effective management. Full article

Acute lymphoblastic leukaemia is the most common childhood malignancy that remains a leading cause of death in childhood. It may be characterised by multiple known recurrent genetic aberrations that inform prognosis, the most common being hyperdiploidy and t(12;21) ETV6::RUNX1. We aimed to assess the applicability of a new imaging flow cytometry methodology that incorporates cell morphology, immunophenotype, and fluorescence in situ hybridisation (FISH) to identify aneuploidy of chromosomes 4 and 21 and the translocation ETV6::RUNX1. We evaluated this new “immuno-flowFISH” platform on 39 cases of paediatric ALL of B-lineage known to have aneuploidy of chromosomes 4 and 21 and the translocation ETV6::RUNX1. After identifying the leukaemic population based on immunophenotype (i.e., expression of CD34, CD10, and CD19 antigens), we assessed for copy numbers of loci for the centromeres of chromosomes 4 and 21 and the ETV6 and RUNX1 regions using fluorophore-labelled DNA probes in more than 1000 cells per sample. Trisomy 4 and 21, tetrasomy 21, and translocations of ETV6::RUNX1, as well as gains and losses of ETV6 and RUNX1, could all be identified based on FISH spot counts and digital imagery. There was variability in clonal makeup in individual cases, suggesting the presence of sub-clones. Copy number alterations and translocations could be detected even when the cell population comprised less than 1% of cells and included cells with a mature B-cell phenotype, i.e., CD19-positive, lacking CD34 and CD10. In this proof-of-principle study of 39 cases, this sensitive and specific semi-automated high-throughput imaging flow cytometric immuno-flowFISH method has been able to show that alterations in ploidy and ETV6::RUNX1 could be detected in the 39 cases of paediatric ALL. This imaging flow cytometric FISH method has potential applications for diagnosis and monitoring disease and marrow regeneration (i.e., distinguishing residual ALL from regenerating haematogones) following chemotherapy. Full article

Metaproteomic analysis of microbiome post-translation modifications (PTMm) is challenging, and little is known about the effects of inflammation on the bacterial PTM landscape in IBD. Here, we adapted and optimised fluorescence in situ hybridisation–flow cytometry (FISH-FC) to study microbiome-wide tyrosine phosphorylation (p-Tyr) in children with and without inflammatory bowel disease (IBD). Microbial p-Tyr signal was significantly higher in children with IBD, compared to those without. Faecalibacterium prausnitzii, Bacteroidota, Gammaproteobacteria and Bifidobacteria tended to be more abundant in IBD than in non-IBD control children but there were only minor differences in p-Tyr among these bacterial communities in those with and without IBD. p-Tyr was significantly lower in non-IBD children older than 9 yrs compared with those less than 9 yrs, and the effect was seen in all four bacterial subgroups studied. The opposite trend was seen in patients with IBD. p-Tyr overall is higher in children with IBD but the effects of inflammation on p-Tyr vary according to the bacterial community. The overall microbiome p-Tyr signal changes with age in healthy children. FISH-FC can be used to study the microbiome-wide PTM landscape. Full article

Anorexia nervosa (AN) is a psychiatric illness with harmful physical consequences. Studies have observed differences in the faecal microbiota of patients with AN compared to healthy controls. Diet has an impact on the gut microbiota, facilitating an altered community, such changes could impact the gut–brain axis. In this study, a three-stage gut model system that mimics the luminal microbiology of the large intestine was conducted to identify relationships between diet and gut microbiota. A microbial medium was developed to provide nutrients more appropriate to restricting subtype AN (R-AN). The model was inoculated with faeces and samples were taken to compare differences in the microbiota and end products following the fermentation of healthy control medium (HC) compared to R-AN medium. Then, 16S amplicon sequencing along with flow cytometry–fluorescence in situ hybridisation were used to ascertain changes in the microbiota. Gas chromatography (GC) was used to assess changes in microbial metabolites. There were reduced levels of SCFA following the fermentation of R-AN medium. The fermentation of R-AN media led to fewer total bacteria numbers, along with less bifidobacteria and Rumincoccus proximally, but more Clostridium and Enterobacteriaceae. Nutrient-deficient medium resulted in reduced neurotransmitter-producing bacteria, reduced butyrate-producing bacteria, and increased protein-utilising bacteria, all of which could be maintaining factors in AN. The model system provides a novel tool for exploring how extreme dietary changes impact the microbiota and could therefore could be useful for assessing appropriate gut–brain targeted treatments. Full article

The changing notion of “companion animals” and their increasing global status as family members underscores the dynamic interaction between gut microbiota and host health. This review provides a comprehensive understanding of the intricate microbial ecology within companion animals required to maintain overall health and prevent disease. Exploration of specific diseases and syndromes linked to gut microbiome alterations (dysbiosis), such as inflammatory bowel disease, obesity, and neurological conditions like epilepsy, are highlighted. In addition, this review provides an analysis of the various factors that impact the abundance of the gut microbiome like age, breed, habitual diet, and microbe-targeted interventions, such as probiotics. Detection methods including PCR-based algorithms, fluorescence in situ hybridisation, and 16S rRNA gene sequencing are reviewed, along with their limitations and the need for future advancements. Prospects for longitudinal investigations, functional dynamics exploration, and accurate identification of microbial signatures associated with specific health problems offer promising directions for future research. In summary, it is an attempt to provide a deeper insight into the orchestration of multiple microbial species shaping the health of companion animals and possible species-specific differences. Full article

Sunitinib has greatly improved the survival of clear cell renal cell carcinoma (ccRCC) patients in recent years. However, 20–30% of treated patients do not respond. To identify miRNAs and genes associated with a response, comparisons were made between biopsies from responder and non-responder ccRCC patients. Using integrated transcriptomic analyses, we identified 37 miRNAs and 60 respective target genes, which were significantly associated with the NF-kappa B, PI3K-Akt and MAPK pathways. We validated expression of the miRNAs (miR-223, miR-155, miR-200b, miR-130b) and target genes (FLT1, PRDM1 and SAV1) in 35 ccRCC patients. High levels of miR-223 and low levels of FLT1, SAV1 and PRDM1 were associated with worse overall survival (OS), and combined miR-223 + SAV1 levels distinguished responders from non-responders (AUC = 0.92). Using immunohistochemical staining of 170 ccRCC patients, VEGFR1 (FLT1) expression was associated with treatment response, histological grade and RECIST (Response Evaluation Criteria in Solid Tumors) score, whereas SAV1 and BLIMP1 (PRDM1) were associated with metachronous metastatic disease. Using in situ hybridisation (ISH) to detect miR-155 we observed higher tumoural cell expression in non-responders, and non-tumoural cell expression with increased histological grade. In summary, our preliminary analysis using integrated miRNA-target gene analyses identified several novel biomarkers in ccRCC patients that surely warrant further investigation. Full article

The APIS Breast Cancer Subtyping Kit is an mRNA-based assessment of the seven parameters including three biomarkers routinely assessed in all the newly diagnosed breast cancers (BC), oestrogen receptor (ER), progesterone receptor (PR) and HER-2 and an additional four genes that create a novel proliferation signature, MKI67, PCNA, CCNA2 and KIF23. Taken together, the data are used to produce a molecular subtype for every sample. The kit was evaluated against the current standard protocol of immunohistochemistry (IHC) and/or in situ hybridisation (ISH) in breast cancer patients. The data were presented at the weekly breast multidisciplinary team (MDT) meeting. A total of 98 consecutive cases of pre-operative breast cancer core biopsies and two core biopsies of nodal metastases yielding 100 cases were assessed. IHC and APIS results were available for 100 and 99 cases. ER was concordant in 97% cases, PR was concordant in 89% and HER-2 results were concordant with IHC/ISH in 100% of the cases. Ki-67 IHC was discordant in 3% of cases when compared with MK167 alone but discordant in 24% when compared with the four-gene proliferation signature. In conclusion, our study indicates that the APIS Breast Cancer Subtyping Kit is highly concordant when compared to the results produced for ER/PR/HER-2 by IHC and/or ISH. The assay could play a role in the routine assessment of newly diagnosed breast cancer (BC) specimens. Full article

Increased intestinal permeability is linked to low-grade systemic inflammation associated with chronic diseases. Undigested dietary proteins reach the colon, where they are fermented by components of the gut microbiota to produce metabolites shown to increase intestinal permeability in vitro. As evidence for sex differences in the microbiota grows, we hypothesised that the effects of the microbial fermentation of protein would also be sex-dependent. Thus, our objective was to determine whether there were sexual dimorphisms in microbial composition and metabolic output following the fermentation of different proteins using in vitro human gut model systems. Faeces from healthy male (n = 5) and female (n = 5) donors were used to inoculate gut fermentation systems supplemented with non-hydrolysed proteins (0.9 g) derived from whey, fish, milk, soya, mycoprotein, egg or pea. At 0, 8, 24 and 48 h, the microbiota composition was quantified using fluorescence in situ hybridisation coupled with flow cytometry, while bacterial-derived metabolite production was assessed via gas chromatography/mass spectroscopy and an ELISA. Increased protein availability resulted in significant increases in proteolytic Bacteroides spp. (p < 0.01) and Clostridium coccoides (p < 0.01) and significant increases in the production of potentially detrimental metabolites including phenol (p < 0.01), p-cresol (p < 0.01), indole (p = 0.018) and ammonia (p < 0.01), all of which were highly dependent on protein type. Furthermore, we showed higher abundances of Clostridium cluster IX (p = 0.03) and concentrations of p-cresol (p = 0.025) at 24 h in males, while females produced more ammonia (p = 0.02) irrespective of the protein source. The fermentation of mycoprotein resulted in significantly higher abundances of Clostridium cluster IX in males at 8 and 24 h compared to females (p < 0.01). There were also significant interactions between sex, protein source, bacterial populations and bacterial-derived metabolic-end-product concentrations. Our study provides new evidence that the effects of the microbial fermentation of dietary proteins in vitro are highly dependent on the source of the protein and the sex of the donor. Consequently, we suggest that different proteins are likely to have differential impacts on intestinal barrier function in vivo, and these effects may be different in males and females. If corroborated in human studies, our results would have important implications for dietary recommendations to limit chronic diseases. Full article

Background: Neuroblastoma is the most common extracranial solid tumour in children, accounting for 15% of paediatric cancer deaths. Multiple genetic abnormalities have been identified as prognostically significant in neuroblastoma patients. Optical genome mapping (OGM) is a novel cytogenetic technique used to detect structural variants, which has not previously been tested in neuroblastoma. We used OGM to identify copy number and structural variants (SVs) in neuroblastoma which may have been missed by standard cytogenetic techniques. Methods: Five neuroblastoma cell lines (SH-SY5Y, NBLW, GI-ME-N, NB1691 and SK-N-BE2(C)) and two neuroblastoma tumours were analysed using OGM with the Bionano Saphyr^® instrument. The results were analysed using Bionano Access software and compared to previous genetic analyses including G-band karyotyping, FISH (fluorescent in situ hybridisation), single-nucleotide polymorphism (SNP) array and RNA fusion panels for cell lines, and SNP arrays and whole genome sequencing (WGS) for tumours. Results: OGM detected copy number abnormalities found using previous methods and provided estimates for absolute copy numbers of amplified genes. OGM identified novel SVs, including fusion genes in two cell lines of potential clinical significance. Conclusions: OGM can reliably detect clinically significant structural and copy number variations in a single test. OGM may prove to be more time- and cost-effective than current standard cytogenetic techniques for neuroblastoma. Full article

The enzootic abortion of ewes, caused by the bacterium Chlamydia abortus (C. abortus), is one of the main causes of abortion in sheep. There are multiple contributory factors, including chlamydial growth, host immune response, and hormonal balance, that result in different pregnancy outcomes, such as abortion, the birth of weak lambs that may die, or healthy lambs. This study aimed to determine the relationship between phenotypical patterns of immune cell infiltration and different pregnancy outcomes in twin-bearing sheep (both lambs born dead; one alive and one dead; both alive) when experimentally infected with C. abortus. Both the sheep uteri and placentae were collected after parturition. All samples were analysed for specific immune cell features, including cell surface antigens and the T-regulatory (Treg) cell-associated transcription factor and cytokines, by immunohistochemistry and in situ hybridisation. Some of these immunological antigens were evaluated in ovine reproductive tissues for the first time. Differential patterns of T helper/Treg cells revealed significant group effects in the placentae. It suggests the potential role that the balance of lymphocyte subsets may play in affecting different pregnancy outcomes in C. abortus-infected sheep. The present study provides novel detailed information about the immune responses observed at the maternofoetal interface in sheep at the time of pre-term abortion or lambing. Full article

Probiotic supplements are increasingly being used to target the gut microbiome with a view to improving cognitive and psychological function via the gut-brain axis. One possible mechanism behind the effect of probiotics is through alterations to microbially-derived metabolites including short-chain fatty acids (SCFA) and neurotransmitters. However, research to date has largely been conducted in animal models or under conditions irrelevant to the human gastrointestinal tract (GIT). The aim of the current work was therefore to use anaerobic, pH controlled in vitro batch cultures to (a) assess the production of neuroactive metabolites in human faecal microbiota under conditions relevant to the human GIT, and (b) to explore how several pre-selected probiotic strains may affect bacterial composition and metabolite production. Enumeration of bacteria was assessed using fluorescence in situ hybridisation with flow cytometry, and concentrations of SCFAs and neurotransmitters were measured using gas chromatography and liquid chromatography mass spectroscopy, respectively. GABA, serotonin, tryptophan, and dopamine were successfully detected, suggesting some level of microbial derivation. The addition of Lactococcus lactis W58 and Lactobacillus rhamnosus W198 resulted in a significant increase in lactate after 8 h of fermentation, while no significant effect of probiotics on bacterial composition or neurotransmitter production was found. Full article

The MyoD gene was duplicated during the teleost whole genome duplication and, while a second MyoD gene (MyoD2) was subsequently lost from the genomes of some lineages (including zebrafish), many fish lineages (including Alcolapia species) have retained both MyoD paralogues. Here we reveal the expression patterns of the two MyoD genes in Oreochromis (Alcolapia) alcalica using in situ hybridisation. We report our analysis of MyoD1 and MyoD2 protein sequences from 54 teleost species, and show that O. alcalica, along with some other teleosts, include a polyserine repeat between the amino terminal transactivation domains (TAD) and the cysteine-histidine rich region (H/C) in MyoD1. The evolutionary history of MyoD1 and MyoD2 is compared to the presence of this polyserine region using phylogenetics, and its functional relevance is tested using overexpression in a heterologous system to investigate subcellular localisation, stability, and activity of MyoD proteins that include and do not include the polyserine region. Full article