An ostrich-derived phage display library
Ontology highlight
ABSTRACT:
PROVIDER: PRJEB67560 | ENA |
REPOSITORIES: ENA
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Project description:We engineered a new m6A editing system consisting of GCN4 (SunTag)-dCas13b, anti-GCN4 single chain variable fragment (scFv)-FTO or ALKBH5-GBI and sgRNA to provide an applicable method to remove m6A modification at specific loci.
2020-10-09 | GSE139647 | GEO
Project description:Aminoacyl tRNA synthetases (aaRSs) have long been viewed as mere housekeeping proteins and have therefore often been overlooked in drug discovery. However, recent findings have revealed that many aaRSs have non-canonical functions and several of them have been linked to autoimmune diseases, cancer and neurological disorders. In order to study these proteins further, recombinant high-affinity antibodies have been generated to a selection of thirteen cytoplasmic and one mitochondrial aaRSs. Selected domains of these proteins were produced recombinantly in Escherichia coli and used as antigens in phage display selections using a synthetic human single-chain fragment variable (scFv) library. All targets yielded large sets of antibody candidates that were validated through a panel of binding assays against the purified antigen. Furthermore, the top performing binders were tested in immunoprecipitation followed by mass spectrometry (IP-MS) for their ability to capture the endogenous protein from mammalian cell lysates. Interestingly, for antibodies targeting individual members of the multi-tRNA synthetase complex (MSC), we were able to detect all members of the complex, co-immunoprecipitating with the target, in several cell types. The functionality of a sub-set of binders for each target was also confirmed in immunofluorescence. To conclude, we have created an open source resource, in the form of high quality recombinant proteins and antibodies to accelerate and empower future research of the role of aaRSs in health and disease.
2020-08-13 | PXD020104 | Pride
Project description:AS TCR related antibody Fab fragment was selected on a HLA-A2 peptide library displayed on yeast for cross-reactivity check
2022-10-09 | GSE215018 | GEO
Project description:Background. Food can affect the microbial balance in the human intestine, and the ingestion of probiotics may play a role in the current obesity pandemic. The objective of our study was to determine if increased Lactobacillus spp. in the intestinal microflora of mice can promote growth and if changes in the intestinal microflora are associated with modifications in metabolism. Methodology. Female BALBc mice were divided between one control and two experimental groups and inoculated either once or twice with 4×1010 Lactobacillus per animal in PBS or with PBS alone. Fecal samples were collected and tested using qPCR to detect and quantify Lactobacillus spp., Bacteroidetes and Firmicutes. Gene expression by microarray and RT-PCR was studied in liver and adipose tissue. Finally, metabolic parameters in the plasma were tested. Principal Findings. In three independent experiments, we observed an increase in both weight gain and liver weight in mice inoculated with 4×1010 Lactobacillus. Inoculation with Lactobacillus sp. (ostrich) increased the Lactobacillus spp. and Firmicutes DNA copy number in feces. The transcriptional profile of liver tissue from mice inoculated with Lactobacillus sp. (ostrich) was enriched for Gene Ontology terms related to the immune response and metabolic modifications. The mRNA levels of fatty acyl synthase (Fas), sterol regulatory element binding factor 1 (Srebp1c), tumor necrosis factor alpha (Tnf), cytochrome P450 2E1 (Cyp2e1) and 3-phosphoinositide-dependent protein kinase-1 (Pdpk1) were significantly elevated in liver tissue in experimental group animals. In gonadal adipose tissue, the expression of leptin, peroxisome proliferator-activated receptor gamma (Pparg and Srebp1c was significantly higher in experimental group animals, whereas the expression of adiponectin was significantly lower. Conclusions. Alterations in the intestinal microbiota resulted in increased weight gain. Furthermore, increased Lactobacillus spp. in the intestinal microflora of mice inoculated with Lactobacillus sp. (ostrich) resulted in accelerated weight gain, liver enlargement and metabolic changes in the plasma, liver and adipose tissue.
2010-09-21 | GSE24219 | GEO
Project description:We leverage the extraordinary molecular diversity of modified heparan sulfate (HS) glycans 8 to establish cellular glycotypes, defined by binding patterns of a panel of flow-cytometry compatible single-chain variable fragment antibodies (scFvs) specific for differentially modified HS. We find distinct glycotypes between closely related hematopoietic progenitors and lineages. The glycotypes of murine and human hematopoietic stem and progenitor cells (HSPCs) reveal dynamic yet similar HS modification patterns in vivo and in vitro, including along megakaryocyte and erythrocyte differentiation. Prospective HS scFv-based sorting identifies new cellular subtypes from both immunophenotypic megakaryocyte-erythrocyte progenitors and heterogeneous pools of HSPCs, thus offering additional discriminative power beyond conventional CD markers. Mechanistically, single-cell RNAseq revealed that a heptad of HS-related genes participate in megakaryocyte-erythrocyte fate determination and are reflective of the HS epitope recognized by specific HS scFvs. In summary, HS glycotyping establishes a role for HS modification patterns in hematopoietic lineage differentiation in mouse and human, and provides an orthogonal approach to define and isolate viable cell types across different cell lineages and species at unprecedented resolution.
2022-08-24 | GSE206671 | GEO
Project description:We leverage the extraordinary molecular diversity of modified heparan sulfate (HS) glycans to establish cellular glycotypes, defined by binding patterns of a panel of flow-cytometry compatible single-chain variable fragment antibodies (scFvs) specific for differentially modified HS. We find distinct glycotypes between closely related hematopoietic progenitors and lineages. The glycotypes of murine and human hematopoietic stem and progenitor cells (HSPCs) reveal dynamic yet similar HS modification patterns in vivo and in vitro, including along megakaryocyte and erythrocyte differentiation. Prospective HS scFv-based sorting identifies new cellular subtypes from both immunophenotypic megakaryocyte-erythrocyte progenitors and heterogeneous pools of HSPCs, thus offering additional discriminative power beyond conventional CD markers. Mechanistically, single-cell RNAseq revealed that a heptad of HS-related genes participate in megakaryocyte-erythrocyte fate determination and are reflective of the HS epitope recognized by specific HS scFvs. In summary, HS glycotyping establishes a role for HS modification patterns in hematopoietic lineage differentiation in mouse and human, and provides an orthogonal approach to define and isolate viable cell types across different cell lineages and species at unprecedented resolution.
2022-08-24 | GSE206670 | GEO
Project description:The oldest authenticated peptide sequences to date were reported in 2016 from 3.8 Ma old ostrich eggshell (OES) from the site of Laetoli, Tanzania (Demarchi et al., 2016). Here we demonstrate survival of the same sequences in 6.5-9Ma OES recovered from a palaeosteppe setting in northwestern China. The eggshell is thicker than those observed in extant species and consistent with the Liushu Struthio sp. ootaxon. These findings push the preservation of ancient proteins back to the Miocene and highlight their potential for paleontology, paleoecology and evolutionary biology.
2022-12-29 | PXD035872 | Pride
Project description:Illumina sequencing of one ostrich female
| PRJEB24106 | ENA
Project description:Background. Food can affect the microbial balance in the human intestine, and the ingestion of probiotics may play a role in the current obesity pandemic. The objective of our study was to determine if increased Lactobacillus spp. in the intestinal microflora of mice can promote growth and if changes in the intestinal microflora are associated with modifications in metabolism. Methodology. Female BALBc mice were divided between one control and two experimental groups and inoculated either once or twice with 4×1010 Lactobacillus per animal in PBS or with PBS alone. Fecal samples were collected and tested using qPCR to detect and quantify Lactobacillus spp., Bacteroidetes and Firmicutes. Gene expression by microarray and RT-PCR was studied in liver and adipose tissue. Finally, metabolic parameters in the plasma were tested. Principal Findings. In three independent experiments, we observed an increase in both weight gain and liver weight in mice inoculated with 4×1010 Lactobacillus. Inoculation with Lactobacillus sp. (ostrich) increased the Lactobacillus spp. and Firmicutes DNA copy number in feces. The transcriptional profile of liver tissue from mice inoculated with Lactobacillus sp. (ostrich) was enriched for Gene Ontology terms related to the immune response and metabolic modifications. The mRNA levels of fatty acyl synthase (Fas), sterol regulatory element binding factor 1 (Srebp1c), tumor necrosis factor alpha (Tnf), cytochrome P450 2E1 (Cyp2e1) and 3-phosphoinositide-dependent protein kinase-1 (Pdpk1) were significantly elevated in liver tissue in experimental group animals. In gonadal adipose tissue, the expression of leptin, peroxisome proliferator-activated receptor gamma (Pparg and Srebp1c was significantly higher in experimental group animals, whereas the expression of adiponectin was significantly lower. Conclusions. Alterations in the intestinal microbiota resulted in increased weight gain. Furthermore, increased Lactobacillus spp. in the intestinal microflora of mice inoculated with Lactobacillus sp. (ostrich) resulted in accelerated weight gain, liver enlargement and metabolic changes in the plasma, liver and adipose tissue. For microarray analysis, we used the livers from two LB1 and two control mice that had been euthanized 20 days after inoculation. We used a whole mouse genome oligomicroarray 4x44K kit (44,000 60-mer oligonucleotides) and performed a one-color microarray-based gene expression analysis, as previously described. Labeled RNAs (Low RNA Input Fluorescent Amplification Kit, Perkin Elmer) were deposited on slides and hybridized using an in situ Hybridization Plus Kit (Agilent Technologies) for 17 h. The arrays were scanned using a DNA Microarray Scanner G2505B (Agilent Technologies), and the image analysis and correction of intra-array signals were performed using Feature Extraction Software A.9.1.3 (Agilent Technologies). Microarray data analysis was performed using GeneSpring 10.01 with the default setting for inter-array normalization and inter-replicate corrections. To identify genes that were differentially expressed, we used a Student’s t-test with p<0.05, and an absolute fold change (FC) greater than 2.0 considered significant. An analysis of Gene Ontology (GO) terms was performed to identify any altered biological processes. Additional data analysis was performed using R software, version 2.8.1. RNA extraction and cDNA synthesis were performed as described above. Primers and probes to target the mouse genes for cytochrome P450 2E1 (Cyp2e1), 3-phosphoinositide-dependent protein kinase-1 (Pdpk1) and glyceraldehyde 3-phosphate dehydrogenase (Gapdh, used as a housekeeping gene) were purchased from Applied Biosystems. qPCR reactions were performed as described above. The relative amount of each examined mRNA was normalized to the Gapdh mRNA expression level and is expressed relative to the mean amount of the same mRNA in the control group.
2010-09-21 | E-GEOD-24219 | biostudies-arrayexpress
Project description:Chimeric Antigen Receptor T cells express an extracellular domain consisting of a single-chain fragment variable (scFv) targeting a surface tumor-associated antigen (TAA), it is important to select scFv regarding safety profile through evaluation of the efficacy/toxicity balance, especially in cases where the target antigen is also expressed on healthy cells. Here, we developed five CAR T cells targeting CD123 (CD123 CAR-T) by substituting only the scFv, looking for differences in terms of efficacy and on-target/off-tumor effects. Using in vitro models, we found that three of the five CD123 CAR-T were more cytotoxic against leukemic cells, without increasing lysis of monocytes or endothelial cells. Using the Incucyte system, we confirmed the low cytotoxicity of CD123 CAR-T on endothelial cells. Hematotoxicity evaluated by progenitor culture and CD34 cell lysis showed that two of the five CD123 CAR-T were less cytotoxic against HSC, and we confirmed in a humanized mouse model that CD123- cells were not eliminated by the CD123 CAR-T. These two CD123 CAR-T reduced tumor infiltration and increased overall survival of mice in three in vivo models of BPDCN. Moreover, in BPDCN aggressive model, bulk RNA-sequencing analysis showed that these CD123 CAR-T upregulated genes associated with cytotoxicity and activation/exhaustion a few days after the injection. Finally, we were able to produce CD123 CAR-T selected previously from patient T cells. Together, these results emphasize the importance of screening different scFv for the development of CAR-T constructs, to select the one with the optimal risk-benefit ratio for clinical development.
2024-06-03 | GSE253738 | GEO