Differentially expressed Transcribed ultraconserved regions (T-UCRs) in Crohn's disease (CD) comparing to normal controls (NC)
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ABSTRACT: To date, 481 transcribed ultraconserved regions (T-UCRs) have been discovered in human genome. We aimed to investigate their characteristics in Crohnâs disease (CD) with conparison to healthy normal controls, to reveal differentially expressed T-UCRs. 3 CD patients and 3 NC volunteers were recruited in this study. With colonoscopy, colon mocosa pinch biopsy samles were got at inflammed site in CD patients and normal sites in NC volunteers respectively.
Project description:To date, 481 transcribed ultraconserved regions (T-UCRs) have been discovered in human genome. We aimed to investigate their characteristics in Crohn’s disease (CD) with conparison to healthy normal controls, to reveal differentially expressed T-UCRs.
Project description:Recent studies in non-human model systems have shown therapeutic potential of modified mRNA (modRNA) treatments for lysosomal storage diseases. Here, we assessed the efficacy of a modRNA treatment to restore the expression of the α-galactosidase (GLA) gene in a human cardiac model generated from induced-pluripotent stem cell-derived from two patients with Fabry disease. In line with the clinical phenotype, cardiomyocytes from Fabry patient’s induced pluripotent stem cells show accumulation of the glycosphinolipid Globotriaosylceramide (GB3), which is an α-galactosidase substrate. Further, the patient-specific cardiomyocytes have significant upregulation of lysosomal associated proteins. Upon modRNA treatment, a subset of lysosomal proteins were partially restored to wildtype levels, implying the rescue of the molecular phenotype associated with the Fabry genotype. Importantly, a significant reduction of GB3 levels was observed in GLA modRNA treated cardiomyocytes demonstrating that α-galactosidase enzymatic activity was restored. Together, our results validate the utility of patient IPSC-derived cardiomyocytes as a model to study disease processes in Fabry disease and the therapeutic potential of GLA modRNA treatment to reduce GB3 accumulation in the heart.
Project description:In this project we have applied palaeoproteomics to study archaeological shell beads dating back to the beginning of the 7th mill. cal BCE and recovered from a prehistoric site in Jordan (Ba'ja). These beads were made of mollusc shells and were of unknown origin, but suspected to be made of larger bivalves such as Tridacna or Spondylus sp. The aim of the study was to analyse ancient proteins extracted from these beads in order to infer their biological origin. Tiny amounts of ancient samples were used and the extracted proteins were analysed by high resolution liquid chromatography coupled with tandem mass spectrometry (HPLC-MS/MS). In order to identify obtained peptides, we have created an ‘in-house’ molluscan protein database, by gathering molluscan sequences (proteins and transcriptomes), which were downloaded from publicly available databases in NCBI. Searching against this comprehensive database, we were able to identify a number of shell proteins from Tridacna sp., the giant clam shells. Our study reports the oldest molluscan protein sequences ever recovered that come from a very warm environment.
Project description:HeLa cells were labelled with lysosomally localized photocrosslinkable and clickable probes for sphingosine and cholesterol. Upon photocrosslinking and cell lysis, the protein-lipid complexes were clicked to biotin azide and enriched via Streptavidin-immunoprecipitation. Enriched protein-lipid complexes were then analyzed by LC-MS/MS to identify proteins interacting with sphingosine and cholesterol, respectively.
Project description:Ubash3b, also known as suppressor of T-cell receptor signaling or Sts-1, is an ill-studied atypical tyrosine phosphatase with ubiquitin binding ability. In our previous study, we hypothesized that Ubash3b plays an inhibitory role in BCR-ABL signaling through binding and dephosphorylating BCR-ABL and its interactors. The Hantschel lab recently solved the crystal structures of the p210 PH and DH domains, which are absent in the p190 variant, and demonstrated that loss-of-function mutations in the PH domain altered BCR-ABL localization, thereby reducing the interaction between Ubash3b and p2104. Taken together, this suggests differential subcellular localization of Ubash3b as a mechanism by which it interacts more strongly with p201 as compared to p190. To better understand the global impact Ubash3b has on p210, its direct kinase substrates and proteins in its phosphotyrosine signaling network, we have taken an integrative approach by combining global phosphotyrosine profiling, proximity-dependent biotinylation (BioID) and total protein analysis to investigate p210 signaling upon Ubash3b knockdown (KD). The BioID system was used to characterize Ubash3b function in p210 signaling by examining its interactome. Importantly, in all of our BioID experiments, we employed a newly technique that we have recently developed, Biotinylation Site Identification Technology (BioSITe), which directly identifies biotinylated peptides thereby increasing the reliability of the identified interactors. Here, we additionally used short hairpin RNA (shRNA) interference and generated Ubash3b knock-down (KD) and non-targeting control shRNA lines in Ba/F3 BirA*-p210 cells. Ubash3b expression was reduced >90 % in the KD cells and had a substantial effect on global tyrosine phosphorylation and on the interactome of p210. Of the 1,421 unique tyrosine phosphorylation sites identified from 830 proteins, 379 sites (from 286 proteins) exhibited a substantial increase (≥2-fold) in tyrosine phosphorylation upon Ubash3b KD cells compared to control cells. To date, the interactome of Ubash3b has not been extensively investigated, however, some examination of Ubash3b in the context p210 signaling has been undertaken. We designed constructs of C-terminal BirA* tagged full length Ubash3b and a deletion mutant lacking the UBA and SH3 domains leaving only the phosphatase domain tethered to BirA*. A comparative analysis of the core interactors of p210 from previous studies and Ubash3b interactome from the current study revealed 36 proteins that interact with both p210 and Ubash3b.
Project description:People with HIV (PWH) experience an increased vulnerability to premature aging and inflammation-associated comorbidities, even when HIV replication is suppressed by antiretroviral therapy (ART). However, the factors that contribute to or are associated with this vulnerability remain uncertain. In the general population, alterations in the glycomes of circulating IgGs trigger inflammation and precede the onset of aging-associated diseases. Here, we investigate the IgG glycomes of cross-sectional and longitudinal samples from 1,216 women and men, both living with virally suppressed HIV and those without HIV. Our glycan-based machine learning models indicate that living with chronic HIV significantly accelerates the accumulation of pro-aging associated glycomic alterations. Consistently, PWH exhibit heightened expression of senescence associated glycan-degrading enzymes compared to their controls. These glycomic alterations correlate with elevated markers of inflammatory aging and the severity of comorbidities, potentially preceding the development of such comorbidities. Mechanistically, HIV-specific antibodies glycoengineered with these alterations exhibit reduced anti-HIV IgG-mediated innate immune functions. These findings hold significant potential for the development of glycomic based biomarkers and tools to identify and prevent premature aging and comorbidities in people living with chronic viral infections.
Project description:T cell recognition of human leukocyte antigen (HLA)-presented tumor-associated peptides is central for cancer immune surveillance. Mass spectrometry (MS)-based immunopeptidomics represents the only unbiased method for directly identifying and characterizing naturally presented tumor-associated peptides, which represent a key prerequisite for the development of T cell-based immunotherapies. This study reports on the de novo implementation of ion mobility separation-based timsTOF MS for next-generation immunopeptidomics, enabling high-speed and sensitive detection of HLA peptides. A direct comparison of timsTOF-based with state-of-the-art immunopeptidomics using orbitrap technology showed significantly increased HLA peptide identifications from benign and malignant primary samples of solid tissue and hematological origin. First application of timsTOF immunopeptidomics for tumor antigen discovery enabled (i) the expansion of benign reference immunopeptidome databases with more than 150,000 HLA peptides from 94 primary benign tissue samples, (ii) the refinement of previously described tumor antigens, and (iii) the identification of a vast array of novel tumor antigens comprising low abundant neoepitopes that might serve as targets for future cancer immunotherapy development.
Project description:Patient-derived bone tumor (osteosarcoma and giant cell tumor of bone) cells, and the normal mesenchymal stem cells and osteoblasts were cultured and subjected to UV crosslinking (UV) at 254 nm or without crosslinking (noUV) as negative controls. Subsequently, RNA-binding proteins (RBPs) were identified by eRIC.
Project description:For identification of proteins that associate with Makorin1 (MKRN1) in RNA-dependent and RNA-independent manners, we affinity purified FLAG-tagged Makorin1 (MKRN1) from mouse embryonic stem cells constitutively expressing FLAG:MKRN1. Anti-FLAG control immunoprecipitations were performed from a FLAG vectrol control (FLAG:Ctrl) mouse embryonic stem cell line that did not express FLAG:MKRN1. Following FLAG immunoprecipitation, anti-FLAG beads from FLAG:MKRN1 and FLAG:Ctrl immunoprecipitations were split into separate tubes such that half of the beads were digested with 200Ã∞â≈ Ã≠µg/mL RNase A while the other half of the beads were undigested. RNase A-digested and undigested immunoprecipitates were subjected to LC-MS/MS analysis. Of the 48 RNA-related proteins previously identified to associate with FLAG:MKRN1, L1TD1, PABPC1, PABPC4, YBX1, IGF2BP1 and UPF1 were found to remain associated with FLAG:MKRN1 in the presence of RNase A.
Project description:The[Q3] gelatinases, matrix metalloproteinase 2 (MMP-2) and MMP-9, are key for leukocyte penetration of the brain parenchymal border in neuroinflammation and the functional integrity of this barrier; however, it is unclear which MMP substrates are involved. Using a tailored, sensitive, label-free mass spectrometry–based secretome approach, not previously applied to nonimmune cells, we identified 119 MMP-9 and 21 MMP-2 potential substrates at the cell surface of primary astrocytes, including known substrates (β-dystroglycan) and a broad spectrum of previously unknown MMP-dependent events involved in cell-cell and cell-matrix interactions. Using neuroinflammation as a model of assessing compromised astroglial barrier function, a selection of the potential MMP substrates were confirmed in vivo and verified in human samples, including vascular cell adhesion molecule–1 and neuronal cell adhesion molecule. We provide a unique resource of potential MMP-2/MMP-9 substrates specific for the astroglia barrier. Our data support a role for the gelatinases in the formation and maintenance of this barrier but also in astrocyte-neuron interactions.