Cellular glycosphingolipid imbalance modulates EMT in cancer cells

Sphingolipids are key components of the plasma membrane and are regulators of complex biological processes often altered in cancer cells. In human tumors, genes of key enzymes that regulate levels of glucosylceramide and lactosylceramide are often amplified. However, it is unknown why these traits are positively selected in transformed cells. In this work, we used CRISPR-Cas9 to knockout two key enzymes amplified in tumors in HeLa and H1703 tumor-derived cell-lines. As expected, the knockout lines had dramatic accumulation of GlcCer and LacCer. However, unexpectedly, they showed significantly decreased in-vitro wound-healing capacity and an almost complete loss of in-vitro extra-cellular matrix invasion. Based on these results, we probed for protein markers of the epithelial-to-mesenchymal transition (EMT). Data showed a significant increase of the levels of E-cadherin and a decrease of N-cadherin, suggesting that knockout cells acquired a more epithelial-like phenotype. Knockout lines also had significant changes in SNAIL levels, an important regulator of E-cadherin expression and EMT marker. As SNAIL can be regulated by growth factor receptors such as EGFR, we probed for global changes in growth factor receptor tyrosine kinase (RTK) activation. Results showed that, compared to their otherwise isogenic wild-type counterparts, knockout lines had broad changes in growth factor RTK activation patterns. The knockout cells also had significant changes in their responses to cytotoxic chemotherapeutic agents. Our work suggests that increased expression of key glycosphingolipid regulating enzymes in transformed cells are critical to promote malignant phenotypes by impacting EMT, sustained activity of growth factor RTKs, and responses to therapy.https://scholarscompass.vcu.edu/uresposters/1430/thumbnail.jp

Budgett's frog (Lepidobatrachus laevis): A new amphibian embryo for developmental biology

AbstractThe large size and rapid development of amphibian embryos has facilitated ground-breaking discoveries in developmental biology. Here, we describe the embryogenesis of the Budgett's frog (Lepidobatrachus laevis), an unusual species with eggs that are over twice the diameter of laboratory Xenopus, and embryos that can tolerate higher temperatures to develop into a tadpole four times more rapidly. In addition to detailing their early development, we demonstrate that, like Xenopus, these embryos are amenable to explant culture assays and can express exogenous transcripts in a tissue-specific manner. Moreover, the steep developmental trajectory and large scale of Lepidobatrachus make it exceptionally well-suited for morphogenesis research. For example, the developing organs of the Budgett's frog are massive compared to those of most model species, and are composed of larger individual cells, thereby affording increased subcellular resolution of early vertebrate organogenesis. Furthermore, we found that complete limb regeneration, which typically requires months to achieve in most vertebrate models, occurs in a matter of days in the Budgett's tadpole, which substantially accelerates the pace of experimentation. Thus, the unusual combination of the greater size and speed of the Budgett's frog model provides inimitable advantages for developmental studies—and a novel inroad to address the mechanisms of spatiotemporal scaling during evolution

Insights from the protein interaction Universe of the multifunctional “Goldilocks” kinase DYRK1A

Human Dual specificity tyrosine (Y)-Regulated Kinase 1A (DYRK1A) is encoded by a dosage-dependent gene located in the Down syndrome critical region of human chromosome 21. The known substrates of DYRK1A include proteins involved in transcription, cell cycle control, DNA repair and other processes. However, the function and regulation of this kinase is not fully understood, and the current knowledge does not fully explain the dosage-dependent function of this kinase. Several recent proteomic studies identified DYRK1A interacting proteins in several human cell lines. Interestingly, several of known protein substrates of DYRK1A were undetectable in these studies, likely due to a transient nature of the kinase-substrate interaction. It is possible that the stronger-binding DYRK1A interacting proteins, many of which are poorly characterized, are involved in regulatory functions by recruiting DYRK1A to the specific subcellular compartments or distinct signaling pathways. Better understanding of these DYRK1A-interacting proteins could help to decode the cellular processes regulated by this important protein kinase during embryonic development and in the adult organism. Here, we review the current knowledge of the biochemical and functional characterization of the DYRK1A protein-protein interaction network and discuss its involvement in human disease

Identification of a BMP inhibitor-responsive promoter module required for expression of the early neural gene zic1

AbstractExpression of the transcription factor zic1 at the onset of gastrulation is one of the earliest molecular indicators of neural fate determination in Xenopus. Inhibition of bone morphogenetic protein (BMP) signaling is critical for activation of zic1 expression and fundamental for establishing neural identity in both vertebrates and invertebrates. The mechanism by which interruption of BMP signaling activates neural-specific gene expression is not understood. Here, we report identification of a 215 bp genomic module that is both necessary and sufficient to activate Xenopus zic1 transcription upon interruption of BMP signaling. Transgenic analyses demonstrate that this BMP inhibitory response module (BIRM) is required for expression in the whole embryo. Multiple consensus binding sites for specific transcription factor families within the BIRM are required for its activity and some of these regions are phylogenetically conserved between orthologous vertebrate zic1 genes. These data suggest that interruption of BMP signaling facilitates neural determination via a complex mechanism, involving multiple regulatory factors that cooperate to control zic1 expression

Effectiveness of interventions to indirectly support food and drink intake in people with dementia : Eating and Drinking Well IN dementiA (EDWINA) systematic review

© 2016 Bunn et al. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.BACKGROUND: Risks and prevalence of malnutrition and dehydration are high in older people but even higher in older people with dementia. In the EDWINA (Eating and Drinking Well IN dementiA) systematic review we aimed to assess effectiveness of interventions aiming to improve, maintain or facilitate food/drink intake indirectly, through food service or dining environment modification, education, exercise or behavioural interventions in people with cognitive impairment or dementia (across all settings, levels of care and support, types and degrees of dementia). METHODS: We comprehensively searched Medline and twelve further databases, plus bibliographies, for intervention studies with ≥3 cognitively impaired adult participants (any type/stage). The review was conducted with service user input in accordance with Cochrane Collaboration's guidelines. We duplicated assessment of inclusion, data extraction, and validity assessment, tabulating data. Meta-analysis (statistical pooling) was not appropriate so data were tabulated and synthesised narratively. RESULTS: We included 56 interventions (reported in 51 studies). Studies were small and there were no clearly effective, or clearly ineffective, interventions. Promising interventions included: eating meals with care-givers, family style meals, soothing mealtime music, constantly accessible snacks and longer mealtimes, education and support for formal and informal care-givers, spaced retrieval and Montessori activities, facilitated breakfast clubs, multisensory exercise and multicomponent interventions. CONCLUSIONS: We found no definitive evidence on effectiveness, or lack of effectiveness, of specific interventions but studies were small and short term. A variety of promising indirect interventions need to be tested in large, high-quality RCTs, and may be approaches that people with dementia and their formal or informal care-givers would wish to try. TRIAL REGISTRATION: The systematic review protocol was registered (CRD42014007611) and is published, with the full MEDLINE search strategy, on Prospero (http://www.crd.york.ac.uk/PROSPERO/display_record.asp?ID=CRD42014007611).Peer reviewedFinal Published versio

Circadian signaling in Homarus americanus: Region-specific de novo assembled transcriptomes show that both the brain and eyestalk ganglia possess the molecular components of a putative clock system

Essentially all organisms exhibit recurring patterns of physiology/behavior that oscillate with a period of ~24-h and are synchronized to the solar day. Crustaceans are no exception, with robust circadian rhythms having been documented in many members of this arthropod subphylum. However, little is known about the molecular underpinnings of their circadian rhythmicity. Moreover, the location of the crustacean central clock has not been firmly established, although both the brain and eyestalk ganglia have been hypothesized as loci. The American lobster, Homarus americanus, is known to exhibit multiple circadian rhythms, and immunodetection data suggest that its central clock is located within the eyestalk ganglia rather than in the brain. Here, brain- and eyestalk ganglia-specific transcriptomes were generated and used to assess the presence/absence of transcripts encoding the commonly recognized protein components of arthropod circadian signaling systems in these two regions of the lobster central nervous system. Transcripts encoding putative homologs of the core clock proteins clock, cryptochrome 2, cycle, period and timeless were found in both the brain and eyestalk ganglia assemblies, as were transcripts encoding similar complements of putative clock-associated, clock input pathway and clock output pathway proteins. The presence and identity of transcripts encoding core clock proteins in both regions were confirmed using PCR. These findings suggest that both the brain and eyestalk ganglia possess all of the molecular components needed for the establishment of a circadian signaling system. Whether the brain and eyestalk clocks are independent of one another or represent a single timekeeping system remains to be determined. Interestingly, while most of the proteins deduced from the identified transcripts are shared by both the brain and eyestalk ganglia, assembly-specific isoforms were also identified, e.g., several period variants, suggesting the possibility of region-specific variation in clock function, especially if the brain and eyestalk clocks represent independent oscillators

MRCK-1 Drives Apical Constriction in C. elegans by Linking Developmental Patterning to Force Generation

Apical constriction is a change in cell shape that drives key morphogenetic events including gastrulation and neural tube formation. Apical force-producing actomyosin networks drive apical constriction by contracting while connected to cell-cell junctions. The mechanisms by which developmental patterning regulates these actomyosin networks and associated junctions with spatial precision are not fully understood. Here, we identify a myosin light chain kinase MRCK-1 as a key regulator of C. elegans gastrulation that integrates spatial and developmental patterning information. We show that MRCK-1 is required for activation of contractile actomyosin dynamics and elevated cortical tension in the apical cell cortex of endodermal precursor cells. MRCK-1 is apically localized by active Cdc42 at the external, cell-cell contact-free surfaces of apically constricting cells, downstream of cell fate determination mechanisms. We establish that the junctional components α-catenin, β-catenin, and cadherin become highly enriched at the apical junctions of apically-constricting cells, and that MRCK-1 and myosin activity are required in vivo for this enrichment. Taken together, our results define mechanisms that position a myosin activator to a specific cell surface where it both locally increases cortical tension and locally enriches junctional components to facilitate apical constriction. These results reveal crucial links that can tie spatial information to local force generation to drive morphogenesis

Clinical evaluation of magnetic resonance imaging in coronary heart disease: The CE-MARC study

<p>Abstract</p> <p>Background</p> <p>Several investigations are currently available to establish the diagnosis of coronary heart disease (CHD). Of these, cardiovascular magnetic resonance (CMR) offers the greatest information from a single test, allowing the assessment of myocardial function, perfusion, viability and coronary artery anatomy. However, data from large scale studies that prospectively evaluate the diagnostic accuracy of multi-parametric CMR for the detection of CHD in unselected populations are lacking, and there are few data on the performance of CMR compared with current diagnostic tests, its prognostic value and cost-effectiveness.</p> <p>Methods/design</p> <p>This is a prospective diagnostic accuracy cohort study of 750 patients referred to a cardiologist with suspected CHD. Exercise tolerance testing (ETT) will be preformed if patients are physically able. Recruited patients will then undergo CMR and single photon emission tomography (SPECT) followed in all patients by invasive X-ray coronary angiography. The order of the CMR and SPECT tests will be randomised. The CMR study will comprise rest and adenosine stress perfusion, cine imaging, late gadolinium enhancement and whole-heart MR coronary angiography. SPECT will use a gated stress/rest protocol. The primary objective of the study is to determine the diagnostic accuracy of CMR in detecting significant coronary stenosis, as defined by X-ray coronary angiography. Secondary objectives include an assessment of the prognostic value of CMR imaging, a comparison of its diagnostic accuracy against SPECT and ETT, and an assessment of cost-effectiveness.</p> <p>Discussion</p> <p>The CE-MARC study is a prospective, diagnostic accuracy cohort study of 750 patients assessing the performance of a multi-parametric CMR study in detecting CHD using invasive X-ray coronary angiography as the reference standard and comparing it with ETT and SPECT.</p> <p>Trial Registration</p> <p>Current Controlled Trials ISRCTN77246133</p