Project description:Functional characterisation of noncoding variants linked to human congenital disorders remains challenging due to the lack of efficient in vivo models. Here we introduce dual-enSERT, a robust two-color fluorescent reporter system which enables rapid comparative assessment of human enhancer variant activities in live mice. We use this new technology to examine the gain- and loss-of-function effects of enhancer variants previously linked to limb polydactyly, autism, and craniofacial malformation. By combining dual-enSERT with single-cell transcriptomics, we characterize variant enhancer allele activity at cellular resolution, revealing candidate molecular pathways implicated in pathogenic enhancer misregulation. We also use dual-enSERT to show that independent polydactyly-linked enhancer variants lead to ectopic expression in the same cell populations, indicating shared genetic mechanisms underlying noncoding variant pathogenesis. Finally, we streamline dual-enSERT for F0 analysis by placing both reporters on the same allele separated by an insulator. Our F0 dual-enSERT cuts the experimental time of noncoding variant selection to corresponding comparative in vivo activity in live embryos from three months to two weeks.
Project description:Functional analysis of non-coding variants associated with human congenital disorders remains challenging due to the lack of efficient in vivo models. Here we introduce dual-enSERT, a robust Cas9-based two-color fluorescent reporter system which enables rapid, quantitative comparison of enhancer allele activities in live mice of any genetic background. We use this new technology to examine and measure the gain- and loss-of-function effects of enhancer variants linked to limb polydactyly, autism, and craniofacial malformation. By combining dual-enSERT with single-cell transcriptomics, we characterize variant enhancer alleles at cellular resolution, thereby implicating candidate molecular pathways in pathogenic enhancer misregulation. We further show that independent, polydactyly-linked enhancer variants lead to ectopic expression in the same cell populations, indicating shared genetic mechanisms underlying non-coding variant pathogenesis. Finally, we streamline dual-enSERT for analysis in F0 animals by placing both reporters on the same transgene separated by a synthetic insulator. Dual-enSERT allows researchers to go from identifying candidate enhancer variants to analysis of comparative enhancer activity in live embryos in under two weeks.
Project description:Complete data set for "Molecular and immunological interrogation of a live-attenuated enterotoxigenic Escherichia coli vaccine highlights features unique to wild type infection"
Project description:O-GlcNAc is a nutritionally and metabolically relevant post-translational modification on thousands of nucleocytoplas-mic proteins. O-GlcNAcylation level dynamically responds to environmental cues in temporal and spatial dimensions, leading to discrete signaling transductions and physiological effects. Spatiotemporal regulation of O-GlcNAcylation level is essential for O-GlcNAc functional interrogation and manipulation of cell behaviors for desired outcomes, which is yet challenging owing to limited approaches. Here we achieved spatiotemporal O-GlcNAc reduction in live cells by designing a 4-hydroxytamoxifen (4-HT)-triggered O-GlcNAcase (OGA) activation strategy. After rational engineering and optimiza-tion, OGA variants bearing an intein located to different subcellular regions were generated and validated, whose deglyco-sidase activity can be turned on by 4-HT in a time- and dose-dependent manner. Finally, we demonstrated the dual func-tionality of 4-HT on breast cancer cells expressing the engineered OGA, which accelerated cell death with a lower dosage and thus mitigated the likelihood of acquiring drug resistance. Altogether, our strategy facilitates the precise regulation and functional understandings of O-GlcNAcylation in live cells.
Project description:Enterotoxigenic Escherichia coli (ETEC) infections are a common cause of diarrheal illness in low- and middle-income countries. The live-attenuated ACE527 vaccine, adjuvanted with double mutant LT (dmLT), affords clear but partial protection against ETEC challenge inhuman volunteers. Comparatively, initial wild-type ETEC challenge completely protects against severe diarrhea on homologous re-challenge...To investigate molecular determinants of protection, vaccine antigen content was compared to wild-type ETEC, and proteome microarrays were used to assess immune responses following vaccination and ETEC challenge... Although molecular interrogation of the vaccine confirmed expression of targeted canonical antigens, relative to wild-type ETEC, vaccine strains were deficient in production of flagellar antigens, immotile, and lacked production of the EtpA adhesin. Similarly, vaccination ± dmLT elicited responses to targeted canonical antigens, but relative to wild-type challenge, vaccine responses to some potentially protective non-canonical antigens including EtpA were diminished or absent...These studies highlight important differences in vaccine and wild-type ETEC antigen content and call attention to distinct immunologic signatures that could inform investigation of correlates of protection, and guide vaccine antigen selection for these pathogens of global importance.
Project description:The SARS-CoV-2 virus is continuously evolving, with appearance of new variants characterized by multiple genomic mutations, some of which can affect functional properties, including infectivity, interactions with host immunity, and disease severity. The rapid spread of new SARS-CoV-2 variants has highlighted the urgency to trace the virus evolution, to help limit its diffusion, and to assess effectiveness of containment strategies. We propose here a PCR-based rapid, sensitive and low-cost allelic discrimination assay panel for the identification of SARS-CoV-2 genotypes, useful for detection in different sample types, such as nasopharyngeal swabs and wastewater. The tests carried out demonstrate that this in-house assay, whose results were confirmed by SARS-CoV-2 whole-genome sequencing, can detect variations in up to 10 viral genome positions at once and is specific and highly sensitive for identification of all tested SARS-CoV-2 clades, even in the case of samples very diluted and of poor quality, particularly difficult to analyze.
Project description:Novel TCR sequencing and cloning methods for sensitive and quantitative interrogation of repertoires and rapid isolation of tumor-reactive TCRs
Project description:Molecular and immunological interrogation of a live-attenuated enterotoxigenic Escherichia coli vaccine highlights features unique to wild type infection