Cloning of a Multi-Tissue Tumour Suppressor/Replcative Senescence Gene on Human Chromosome 7q31

The q31 region of human chromosome 7 is frequently deleted in a broad spectrum of human cancers, and is believed to harbour a multi-tissue tumour suppressor gene. I found loss of heterozygosity at one or more microsatellite markers from 7q31 in 36% of breast carcinomas. The smallest common deleted region was between two CA.GT microsatellite markers, D7S522 and 17TA-5/17B-RE3 in the CFTR locus. Loss of 7q31 markers in two non-tumourigenic, human fibroblast cell lines, SUSM-1 and KMST-6 (both immortality complementation group D), has been associated with the emergence of an immortal phenotype. This phenotype can be suppressed by re-introduction of an intact copy of chromosome 7. We conjectured that the multi-tissue tumour suppressor and immortality complementation group D gene, which we named SEND, are one and the same. A physical and functional cloning strategy was adopted to isolate SEND. Intact copies of a hygromycin-resistance tagged human chromosome 7 were introduced into SUSM-1 cells by microcell-mediated monochromosome transfer. This induced replicative senescence in a significant proportion of the hygromycin-resistant colonies recovered. Occasional immortal segregants also arose, most likely as a result of inactivating SEND on the introduced chromosome. The sites of inactivation were mapped by analysing polymorphic microsatellite markers that differed between donor and recipient chromosomes. This also entailed my generating novel polymorphic markers. Using this strategy, I defined three 'hot spots' of allele loss on chromosome 7 in immortal segregants. One, an approximately 500Kbp interval between 724CA and 786CA, two novel CA.GT dinucleotide repeats, was nested within the smallest common region of allele loss determined for breast cancers. The putative tumour suppressor gene/SEND may reside in this interval. I assembled a YAC, cosmid, and PAC clone contig for the smallest region of allele loss in breast tumours and mapped a number of genes to it by a combination of approaches, including exon-trapping, sequencing, and EST-content mapping. Certain candidate genes were further characterised. This analysis included cloning of full length cDNAs, determining the genomic (exon-intron) structure, determining expression in tumour cell lines by northern and western blot analysis, and looking for mutations by SSCP analysis. Perhaps the most interesting candidate, CAVEOLIN-1, although not mutated in human cancers, was found to be transcriptionally silenced in a number of tumour-derived cell lines by methylation of 5'-sequences

Do item-dependent context representations underlie serial order in cognition?

Logan (2021) presented an impressive unification of serial order tasks including whole report, typing, and serial recall in the form of the context retrieval and updating (CRU) model. Despite the wide breadth of the model’s coverage, its reliance on encoding and retrieving context representations that consist of the previous items may prevent it from being able to address a number of critical benchmark findings in the serial order literature that have shaped and constrained existing theories. In this commentary, we highlight three major challenges that motivated the development of a rival class of models of serial order, namely positional models. These challenges include the mixed-list phonological similarity effect, the protrusion effect, and interposition errors in temporal grouping. Simulations indicated that CRU can address the mixed list phonological similarity effect if phonological confusions can occur during its output stage, suggesting that the serial position curves from this paradigm do not rule out models that rely on inter-item associations, as has been previously been suggested. The other two challenges are more consequential for the model’s representations, and simulations indicated the model was not able to provide a complete account of them. We highlight and discuss how revisions to CRU’s representations or retrieval mechanisms can address these phenomena and emphasize that a fruitful direction forward would be to either incorporate positional representations or approximate them with its existing representations

Desmosomal cadherins in zebrafish epiboly and gastrulation

<p>Abstract</p> <p>Background</p> <p>The desmosomal cadherins (DCs), desmocollin (Dsc) and desmoglein (Dsg), are the adhesion molecules of desmosomes, intercellular adhesive junctions of epithelia and cardiac muscle. Both the DCs and desmosomes have demonstrably essential roles in mammalian development. In order to initiate their study in a more tractable developmental system we have characterised zebrafish DCs and examined their roles in early zebrafish development.</p> <p>Results</p> <p>We find that zebrafish possess one Dsc, the orthologue of mammalian Dsc1, which we designate zfDsc. Unlike mammalian Dscs, zfDsc exists only as the "a" form since it lacks the alternatively-spliced mini-exon that shortens the cytoplasmic domain to produce the "b" form. Zebrafish possess two Dsgs, designated zfDsgα and zfDsgβ, orthologues of mammalian Dsg2. They show 43.8% amino acid identity and the α form has a 43 amino acid glycine-rich sequence of unknown function in its extracellular domain. Both zfDsc and zfDsgα were present as maternal and zygotic transcripts whereas zfDsgβ was first expressed from 8 hours post-fertilisation (hpf). All three transcripts were present throughout subsequent stages of development. Morpholino knockdown of both zfDsc and zfDsgα expression produced similar defects in epiboly, axis elongation and somite formation, associated with abnormal desmosomes or reduced desmosome numbers.</p> <p>Conclusions</p> <p>These results demonstrate an important role for DCs and desmosomes in the early morphogenesis of the zebrafish embryo, provide a basis for more detailed analysis of their role and raise interesting questions relating to the evolution and functional significance of DC isoforms.</p

Desmosomal cadherins in zebrafish epiboly and gastrulation

<p>Abstract</p> <p>Background</p> <p>The desmosomal cadherins (DCs), desmocollin (Dsc) and desmoglein (Dsg), are the adhesion molecules of desmosomes, intercellular adhesive junctions of epithelia and cardiac muscle. Both the DCs and desmosomes have demonstrably essential roles in mammalian development. In order to initiate their study in a more tractable developmental system we have characterised zebrafish DCs and examined their roles in early zebrafish development.</p> <p>Results</p> <p>We find that zebrafish possess one Dsc, the orthologue of mammalian Dsc1, which we designate zfDsc. Unlike mammalian Dscs, zfDsc exists only as the "a" form since it lacks the alternatively-spliced mini-exon that shortens the cytoplasmic domain to produce the "b" form. Zebrafish possess two Dsgs, designated zfDsgα and zfDsgβ, orthologues of mammalian Dsg2. They show 43.8% amino acid identity and the α form has a 43 amino acid glycine-rich sequence of unknown function in its extracellular domain. Both zfDsc and zfDsgα were present as maternal and zygotic transcripts whereas zfDsgβ was first expressed from 8 hours post-fertilisation (hpf). All three transcripts were present throughout subsequent stages of development. Morpholino knockdown of both zfDsc and zfDsgα expression produced similar defects in epiboly, axis elongation and somite formation, associated with abnormal desmosomes or reduced desmosome numbers.</p> <p>Conclusions</p> <p>These results demonstrate an important role for DCs and desmosomes in the early morphogenesis of the zebrafish embryo, provide a basis for more detailed analysis of their role and raise interesting questions relating to the evolution and functional significance of DC isoforms.</p

Minimal contribution of ERK1/2-MAPK signalling towards the maintenance of oncogenic GNAQ(Q209P)-driven uveal melanomas in zebrafish

Mutations affecting Gαq proteins are pervasive in uveal melanoma (UM), suggesting they ‘drive’ UM pathogenesis. The ERK1/2-MAPK pathway is critical for cutaneous melanoma development and consequently an important therapeutic target. Defining the contribution of ERK1/2-MAPK signalling to UM development has been hampered by the lack of an informative animal model that spontaneously develops UM. Towards this end, we engineered transgenic zebrafish to express oncogenic GNAQ(Q209P) in the melanocyte lineage. This resulted in hyperplasia of uveal melanocytes, but with no evidence of malignant progression, nor perturbation of skin melanocytes. Combining expression of oncogenic GNAQ(Q209P) with p53 inactivation resulted in earlier onset and even more extensive hyperplasia of uveal melanocytes that progressed to UM. Immunohistochemistry revealed only weak immunoreactivity to phosphorylated (p)ERK1/2 in established uveal tumours—in contrast to strong immunoreactivity in oncogenic RAS-driven skin lesions—but ubiquitous positive staining for nuclear Yes-associated protein (YAP). Moreover, no changes were observed in pERK1/2 levels upon transient knockdown of GNAQ or phospholipase C-beta (PLC-β) inhibition in the majority of human UM cell lines we tested harbouring GNAQ mutations. In summary, our findings demonstrate a weak correlation between oncogenic GNAQ(Q209P) mutation and sustained ERK1/2-MAPK activation, implying that ERK1/2 signalling is unlikely to be instrumental in the maintenance of GNAQ(Q209P)-driven UMs

Do item-dependent context representations underlie serial order in cognition?:Commentary on Logan (2021)

Logan (2021) presented an impressive unification of serial order tasks including whole report, typing, and serial recall in the form of the context retrieval and updating (CRU) model. Despite the wide breadth of the model's coverage, its reliance on encoding and retrieving context representations that consist of the previous items may prevent it from being able to address a number of critical benchmark findings in the serial order literature that have shaped and constrained existing theories. In this commentary, we highlight three major challenges that motivated the development of a rival class of models of serial order, namely positional models. These challenges include the mixed-list phonological similarity effect, the protrusion effect, and interposition errors in temporal grouping. Simulations indicated that CRU can address the mixed-list phonological similarity effect if phonological confusions can occur during its output stage, suggesting that the serial position curves from this paradigm do not rule out models that rely on interitem associations, as has been previously been suggested. The other two challenges are more consequential for the model's representations, and simulations indicated the model was not able to provide a complete account of them. We highlight and discuss how revisions to CRU's representations or retrieval mechanisms can address these phenomena and emphasize that a fruitful direction forward would be to either incorporate positional representations or approximate them with its existing representations

6A3-5/Osa2 is an Early Activated Gene Implicated in the Control of Vascular Smooth Muscle Cell Functions

Vascular smooth muscle cells (VSMC) growth plays a key role in the pathophysiology of vascular diseases. However, the molecular mechanisms controlling gene transcription in VSMC remain poorly understood. We previously identified, by differential display, a new gene (6A3-5) overexpressed in proliferating rat VSMC. In this study, we have cloned the full-length cDNA by screening a rat foetal brain cDNA library and investigated its functions. The 6A3-5 protein shows 4 putative conserved functional motifs: a DNA binding domain called ARID (AT-rich interaction domain), two recently described motifs (Osa Homology Domain), and a nuclear localization signal. The deduced protein sequence was observed to be 85% identical to the recently described human Osa2 gene. Immunolabelling, using an anti-6A3-5/Osa2 monoclonal antibody, showed a nuclear localization of the 6A3-5/Osa2 protein. In addition, PDGF upregulated 6A3-5/Osa2 expression at both the transcript and protein levels in a dose and time-dependent fashion. The pattern of upregulation by PDGF was reminiscent of the early responsive gene c-fos. The PDGF-induced upregulation of 6A3-5/Osa2 and proliferation of VSMC were significantly inhibited in a dose and sequence-dependent fashion by an antisense, but not by sense, scrambled or mismatched oligonucleotides directed against 6A3-5/Osa2. In VSMC of aortas derived from hypertensive (LH) rats, 6A3-5/Osa2 is overexpressed as compared to that in normotensive (LL) rats. The 6A3-5/Osa2-gene expression is downregulated by an ACE inhibitor and upregulated by exogenous AngiotensinII in LH rats. In summary, these results indicate that 6A3-5/Osa2 is an early activated gene that belongs to a new family of proteins involved in the control of VSMC growth

CD4-Transgenic Zebrafish Reveal Tissue-Resident Th2- and Regulatory T Cell-like Populations and Diverse Mononuclear Phagocytes.

CD4+ T cells are at the nexus of the innate and adaptive arms of the immune system. However, little is known about the evolutionary history of CD4+ T cells, and it is unclear whether their differentiation into specialized subsets is conserved in early vertebrates. In this study, we have created transgenic zebrafish with vibrantly labeled CD4+ cells allowing us to scrutinize the development and specialization of teleost CD4+ leukocytes in vivo. We provide further evidence that CD4+ macrophages have an ancient origin and had already emerged in bony fish. We demonstrate the utility of this zebrafish resource for interrogating the complex behavior of immune cells at cellular resolution by the imaging of intimate contacts between teleost CD4+ T cells and mononuclear phagocytes. Most importantly, we reveal the conserved subspecialization of teleost CD4+ T cells in vivo. We demonstrate that the ancient and specialized tissues of the gills contain a resident population of il-4/13b-expressing Th2-like cells, which do not coexpress il-4/13a Additionally, we identify a contrasting population of regulatory T cell-like cells resident in the zebrafish gut mucosa, in marked similarity to that found in the intestine of mammals. Finally, we show that, as in mammals, zebrafish CD4+ T cells will infiltrate melanoma tumors and obtain a phenotype consistent with a type 2 immune microenvironment. We anticipate that this unique resource will prove invaluable for future investigation of T cell function in biomedical research, the development of vaccination and health management in aquaculture, and for further research into the evolution of adaptive immunity.European Research Council (Grant IDs: ERC-2011-StG-282059 (PROMINENT), 677501 (ZF_Blood)), Biotechnology and Biological Sciences Research Council (Grant ID: BB/L007401/1), Dowager Countess Eleanor Peel Trust (Grant ID: TH-PRCL.FID2228), Medical Research Council, Department for International Development (Career Development Award Fellowship MR/J009156/1), Medical Research Foundation (Grant ID: R/140419), Cancer Research UK (Grant ID: C45041/A14953), Wellcome Trust and Medical Research Council to the Wellcome Trust–Medical Research Council Cambridge Stem Cell Institute (core support grant)This is the final version of the article. It first appeared from The American Association of Immunologists via https://doi.org/10.4049/​jimmunol.160095

Systematic Roadmap for Cancer Drug Screening Using Zebrafish Embryo Xenograft Cancer Models: Melanoma Cell Line as a Case Study

From MDPI via Jisc Publications RouterHistory: accepted 2021-07-19, pub-electronic 2021-07-23Publication status: PublishedFunder: Gobierno de Navarra; Grant(s): 0011-1408-2016-000004, 0011-1365-2020-000292Zebrafish embryo tumor transplant models are widely utilized in cancer research. Compared with traditional murine models, the small size and transparency of zebrafish embryos combined with large clutch sizes that increase statistical power and cheap husbandry make them a cost-effective and versatile tool for in vivo drug discovery. However, the lack of a comprehensive analysis of key factors impacting the successful use of these models impedes the establishment of basic guidelines for systematic screening campaigns. Thus, we explored the following crucial factors: (i) user-independent inclusion criteria, focusing on sample homogeneity; (ii) metric definition for data analysis; (iii) tumor engraftment criteria; (iv) image analysis versus quantification of human cancer cells using qPCR (RNA and gDNA); (v) tumor implantation sites; (vi) compound distribution (intratumoral administration versus alternative inoculation sites); and (vii) efficacy (intratumoral microinjection versus compound solution in media). Based on these analyses and corresponding assessments, we propose the first roadmap for systematic drug discovery screening in zebrafish xenograft cancer models using a melanoma cell line as a case study. This study aims to help the wider cancer research community to consider the adoption of this versatile model for cancer drug screening projects