RESUMEN
Tissue-intrinsic error correction enables epithelial cells to detect abnormal neighboring cells and facilitate their removal from the tissue. One of these pathways, "interface surveillance," is triggered by cells with aberrant developmental and cell-fate-patterning pathways. It remains unknown which molecular mechanisms provide cells with the ability to compare fate between neighboring cells. We demonstrate that Drosophila imaginal discs express an array of cell surface molecules previously implicated in neuronal axon guidance processes. They include members of the Robo, Teneurin, Ephrin, Toll-like, or atypical cadherin families. Importantly, a mismatch in expression levels of these cell surface molecules between adjacent cells is sufficient to induce interface surveillance, indicating that differences in expression levels between neighboring cells, rather than their absolute expression levels, are crucial. Specifically, a mismatch in Robo2 and Robo3, but not Robo1, induces enrichment of actin, myosin II, and Ena/Vasp, as well as activation of JNK and apoptosis at clonal interfaces. Moreover, Robo2 can induce interface surveillance independently of its cytosolic domain and without the need for the Robo-ligand Slit. The expression of Robo2 and other cell surface molecules, such as Teneurins or the Ephrin receptor is regulated by fate-patterning pathways intrinsic and extrinsic to the wing disc, as well as by expression of oncogenic RasV12. Combined, we demonstrate that neighboring cells respond to a mismatch in surface code patterns mediated by specific transmembrane proteins and reveal a novel function for these cell surface proteins in cell fate recognition and removal of aberrant cells during development and homeostasis of epithelial tissues.
Asunto(s)
Proteínas de Drosophila , Receptores Inmunológicos , Humanos , Animales , Receptores Inmunológicos/metabolismo , Proteínas Roundabout , Drosophila/fisiología , Axones/fisiología , Proteínas de Drosophila/metabolismo , Efrinas/metabolismoRESUMEN
COVID has expedited and expanded the already precarious smart city development in India with the multitude of 'smart' COVID responses-broadly known as CovTech-introduced since the early days of the global pandemic. This paper offers an analysis of the scope and scale of smart responses to COVID in the first 20 cities prioritized for smart city implementation-as part of the Smart Cities Mission in India. The analysis unravels the diversity within the smart COVID response, as 125 COVID applications, 14 COVID-War-Rooms, and numerous examples of smart public place initiatives are discussed. The findings include a typology of COVID applications and shed light on the operations of COVID-War-Rooms throughout the nation. The learnings point toward a mostly top-down approach to smart COVID response. Yet, early evidence indicates the existence of an alternative subaltern smart COVID response to provide bottom-up support to the most vulnerable groups, filling the gaps in the top-down approach. More research is required to thoroughly understand the scope and scale of the subaltern smart response to COVID.
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COVID-19 , Humanos , Ciudades/epidemiología , COVID-19/epidemiología , India/epidemiología , Ambiente , Impulso (Psicología)RESUMEN
Tissue-intrinsic defense mechanisms eliminate aberrant cells from epithelia and thereby maintain the health of developing tissues or adult organisms. 'Interface surveillance' comprises one such distinct mechanism that specifically guards against aberrant cells which undergo inappropriate cell fate and differentiation programs. The cellular mechanisms which facilitate detection and elimination of these aberrant cells are currently unknown. We find that in Drosophila imaginal discs, clones of cells with inappropriate activation of cell fate programs induce bilateral JNK activation at clonal interfaces, where wild type and aberrant cells make contact. JNK activation is required to drive apoptotic elimination of interface cells. Importantly, JNK activity and apoptosis are highest in interface cells within small aberrant clones, which likely supports the successful elimination of aberrant cells when they arise. Our findings are consistent with a model where clone size affects the topology of interface contacts and thereby the strength of JNK activation in wild type and aberrant interface cells. Bilateral JNK activation is unique to 'interface surveillance' and is not observed in other tissue-intrinsic defense mechanisms, such as classical 'cell-cell competition'. Thus, bilateral JNK interface signaling provides an independent tissue-level mechanism to eliminate cells with inappropriate developmental fate but normal cellular fitness. Finally, oncogenic Ras-expressing clones activate 'interface surveillance' but evade elimination by bilateral JNK activation. Combined, our work establishes bilateral JNK interface signaling and interface apoptosis as a new hallmark of interface surveillance and highlights how oncogenic mutations evade tumor suppressor function encoded by this tissue-intrinsic surveillance system.
Asunto(s)
Proteínas de Drosophila , Drosophila melanogaster , Células Epiteliales , Proteínas Quinasas JNK Activadas por Mitógenos , Animales , Apoptosis , Drosophila melanogaster/citología , Drosophila melanogaster/metabolismo , Proteínas de Drosophila/metabolismo , Epitelio/metabolismo , Genes Supresores de Tumor , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Sistema de Señalización de MAP Quinasas , Células Epiteliales/citología , Células Epiteliales/metabolismoRESUMEN
This critical commentary reflects on a rapidly mobilised international podcast project, in which 25 urban scholars from around the world provided audio recordings about their cities during COVID-19. New digital tools are increasing the speeds, formats and breadth of the research and communication mediums available to researchers. Voice recorders on mobile phones and digital audio editing on laptops allows researchers to collaborate in new ways, and this podcast project pushed at the boundaries of what a research method and community might be. Many of those who provided short audio 'reports from the field' recorded on their mobile phones were struggling to make sense of their experience in their city during COVID-19. The substantive sections of this commentary discuss the digital methodology opportunities that podcasting affords geographical scholarship. In this case the methodology includes the curated production of the podcast and critical reflection on the podcast process through collaborative writing. Then putting this methodology into action some limited reflections on cities under COVID-19 lockdown and social distancing initiatives around the world are provided to demonstrate the utility and limitations of this method.
RESUMEN
BACKGROUND: Gene knockout technologies involving programmable nucleases have been used to create knockouts in several applications. Gene editing using Zinc-finger nucleases (ZFNs), Transcription activator like effectors (TALEs) and CRISPR/Cas systems has been used to create changes in the genome in order to make it non-functional. In the present study, we have looked into the possibility of using six fingered CompoZr ZFN pair to target the E6 gene of HPV 16 genome. METHODS: HPV 16+ve cell lines; SiHa and CaSki were used for experiments. CompoZr ZFNs targeting E6 gene were designed and constructed by Sigma-Aldrich. TALENs targeting E6 and E7 genes were made using TALEN assembly kit. Gene editing was monitored by T7E1 mismatch nuclease and Nuclease resistance assays. Levels of E6 and E7 were further analyzed by RT-PCR, western blot as well as immunoflourescence analyses. To check if there is any interference due to methylation, cell lines were treated with sodium butyrate, and Nocodazole. RESULTS: Although ZFN editing activity in yeast based MEL-I assay was high, it yielded very low activity in tumor cell lines; only 6% editing in CaSki and negligible activity in SiHa cell lines. Though editing efficiency was better in CaSki, no significant reduction in E6 protein levels was observed in immunocytochemical analysis. Further, in silico analysis of DNA binding prediction revealed that some of the ZFN modules bound to sequence that did not match the target sequence. Hence, alternate ZFN pairs for E6 and E7 were not synthesized since no further active sites could be identified by in silico analyses. Then we designed TALENs to target E6 and E7 gene. TALENs designed to target E7 gene led to reduction of E7 levels in CaSki and SiHa cervical cancer cell lines. However, TALEN designed to target E6 gene did not yield any editing activity. CONCLUSIONS: Our study highlights that designed nucleases intended to obtain bulk effect should have a reasonable editing activity which reflects phenotypically as well. Nucleases with low editing efficiency, intended for generation of knockout cell lines nucleases could be obtained by single cell cloning. This could serve as a criterion for designing ZFNs and TALENs.
RESUMEN
Human Papillomavirus E7 and E6 oncoproteins have been considered as suitable candidate anti-viral targets since they cause malignant conversion in cervical cancers. Transcription Activator-Like Effector Nucleases (TALENs) are recent editing tools to knockout genes by inducing double stranded breaks at specific sites in the genome. In here, we have designed specific TALENs to target E7 and analyzed their efficiency in inducing cell death in cervical cancer cells. We found that designed TALENs could yield about 10-12% editing activity as observed from T7E1 and nuclease resistance assays. Down-regulation of E7 and E6 was further evident at the transcript as well as proteins levels indicating that the selected TALENs were effective. TALEN-mediated E7 editing led to cell death as ascertained by cell cycle and Annexin V assays. Annexin profiling suggested that cell death could be due to necrosis as observed by upregulation of necrotic markers such as LDH A, Rip-1, and Cyclophilin A. Necrosis appears to be a better therapeutic response as it could further activate pro-inflammatory cytokines to attract immune cells to eliminate HPV-integrated cells and therefore TALEN editing strategy has the potential to be a promising tool as an adjuvant therapy in cervical cancer along with surgery.