14308 results for ""Pennsylvania+German"+ACS+study+'Socius'+open+access"

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• DynaTag for efficient profiling of transcription factors in small samples and single cells

  Systematic discovery of transcription factor (TF) landscapes in low-input samples and at single cell level is a major challenge in the fields of molecular biology, genetics, and epigenetics. To address this, we present here cleavage under Dynamic targets and Tagmentation (DynaTag), which allows robust mapping of specific TF-DNA interactions via the use of a physiological salt solution during sample preparation. Using this method, we revealed occupancy alterations for 15 different TFs in stem cell and cancer tissue models. Of note, we showcased for NANOG, MYC, and OCT4 differentially occupied genomic loci during stem cell differentiation, both in bulk and at single-cell resolution. DynaTag demonstrated superior signal-to-background ratio and occupancy resolution in comparison to CUT&RUN and ChIP-seq. In addition, a model for small cell lung cancer chemotherapy treatment in vivo showed increased chromatin occupancies of the gain-of-function mutant P53 p.R248Q, FOXA1, and MYC at altered gene pathways, such as epithelial-mesenchymal-transition. Altogether, these results highlight the technical advancement of DynaTag to uncover TF landscapes in cell models and in tissues of a complex disease model.

  Study EGAS50000001074

• TDP43 proteinopathy leads to divergent cryptic splicing in the cortex and spinal cord

  Mislocalization of the nuclear TAR DNA-binding protein 43 (TDP43) is a hallmark of ALS and FTD which leads to de-repression and inclusion of cryptic exons, promising biomarkers of TDP43 pathology in a spectrum of neurodegenerative diseases. However, most cryptic exons to date have been identified from in vitro models or a single cortical FTD dataset, and little is known about cryptic splicing in the spinal cord, or within different neuronal subtypes. We meta-analyzed published bulk RNAseq datasets representing 1,778 RNAseq profiles of ALS and FTD post-mortem tissue, and in vitro models with experimentally depleted TDP43. We identified 142 cryptic splices, including 76 novel events. We found a novel pattern of spinal cord cryptic splicing, validated in an independent cohort by qPCR, which differed significantly from cortical and in vitro splicing. Finally, leveraging multiple public single-nucleus RNAseq datasets of ALS and FTD motor and frontal cortex, we confirmed the elevation of cortical-enriched splices in disease and localized them to layer-specific neuronal populations. This catalog of cryptic splices could inform efforts to develop biomarkers for tissue-specific and cell type-specific TDP43 pathology.

  Study EGAS50000000575

• Vaccine-Expanded Plasmablast-like B Cells Are Associated with Response to Dendritic Cell Therapy in Metastatic Melanoma

  Dendritic cells (DCs), either monocyte-derived (moDCs) or naturally circulating, have been employed in cancer treatment across both metastatic and adjuvant settings. DC vaccines (DC Vax) are capable of inducing tumor-specific immune responses, yet clinical outcomes have thus far fallen short of expectations. Here, we performed a multi-omics analysis of the apheresis product, including CD14⁺ monocytes, and the final DCVax formulation. Among all the components analyzed, the most striking finding was the unexpected presence of CD19⁺ B cells within the DC Vax formulation, which distinguished Responders from Non-Responders to moDC vaccination in metastatic melanoma. The abundance of B cells in DC Vax correlated with their peripheral frequency; however, within the vaccine, these cells acquired a CD20lowCD27hiCD38hi plasmablast-like phenotype, distinct from their autologous circulating B-cell precursors. Notably, a parallel observation was made in Responders, whose pre-treatment lesions were enriched in B cells organized as Tertiary Lymphoid Structures. These results underscore the importance of B cells in both baseline immunity and DCVax formulation, hinting at unexplored mechanisms by which they may influence vaccine effectiveness.

  Study EGAS50000001177

• M116 Proteome Extracellular Vesicle Profiling

  For extracellular vesicles (ECV) extraction from plasma, 1M ammonium acetate was added to precipitate ECVs on ice for 45 min. Then, 100 mM ammonium acetate was added to the mixture, and ECVs were precipitated by centrifugation at 20,000g for 30 min. ECVs were washed with 50 mM ammonium bicarbonate (Sigma-Aldrich, Cat. 1066-33-7). Then, 600 µl of 1% ammonium deoxycholate (Sigma Aldrich, Cat. K2755-1MG) were added. The concentration of protein in each sample was measured using a bicinchoninic acid assay (BCA assay). Ammonium bicarbonate was used to dilute 500 µg of protein into a final volume of 500 µl. Next, dithiothreitol (Sigma-Aldrich, Cat. D9779) was added to obtain a final concentration of 20 mM, followed by iodoacetamide (Sigma-Aldrich, Cat. I6125) to a final concentration of 40 mM. Next, trypsin (Roche, Cat. RTRYP-RO) was added to the sample in a 1:25 protein ratio and incubated at 37°C overnight. Next day, formic acid (ThermoFisher Scientific, Cat. 28905) in a final concentration of 0.1% was added and extraction of proteins was done using Empore™ Solid Phase Extraction Cartridges (3M), following manufacturer’s instructions. The eluted samples were then centrifuged for 90 min. using a speed vacuum centrifuge (Thermo, RC1010), followed by snap freezing in liquid nitrogen. Then, the samples were kept in a freeze dryer (LyoDry Compact Benchtop, MechaTech) overnight. Next, the samples were reconstituted in 30 µl of 0.1% formic acid (FA) and an o-Phthaladehyde (Oparil) assay was performed to determine the concentration of each sample. After that, the sample was prepared in a concentration of 0.5 µg/µl using 0.1% FA and alcohol dehydrogenase (ADH). The samples were prepared in glass mass spectrometry vials for proteomic analysis using a Waters Synapt G2Si High-Definition Mass Spectrometry (Waters Corporation) operated by the MassLynx 4.1., 110 min. running time with 2 µl of an injection containing 1 µg of peptide. Quality controls were also run along with samples to guarantee consistency. Pooled quality controls were made from all samples, in which the samples were run at the beginning, middle and end of the mass spectrometry run. Samples were randomized before running the experiment. The proteomic data was then imported into Progenesis software 4.2 (Nonlinear Dynamic, UK) to identify and quantify peptides and proteins.

  Dataset EGAD50000001679

• Kids First: The Genomic Architecture of Hirschsprung Disease (HSCR)

  Hirschsprung disease (HSCR) is a male biased developmental disorder associated with a lack of innervation of the gastrointestinal tract. Genetic studies have been instrumental in understanding its multifactorial inheritance, high heritability, syndromic associations, and genetic heterogeneity with variable penetrance and expressivity. At least 24 genes and 9 loci with pathogenic alleles (PAs) are known to underlie HSCR pathogenesis and explain 62% of its population attributable risk (PAR). Despite this heterogeneity, there is functional unity in HSCR: ~53% of HSCR PAs disrupt RET and EDNRB signaling in the developing enteric nervous system (ENS) in which 11 genes comprising a gene regulatory network control RET and EDNRB gene expression. We propose to identify the remaining 30% PAR by studying 857 unrelated HSCR cases, their 125 affected and 1,446 unaffected firstdegree relatives by whole genome sequencing to increase statistical power of gene discovery through improved detection of all types of coding and regulatory PAs. HSCR arises from cell autonomous defects in enteric neural crest cell precursors (ENCCs) affecting their proliferation, differentiation and migration in the ENS, functional studies that will guide our detection of novel genes.

  Study phs003662

• Error-corrected flow-based sequencing at whole genome scale and its application to circulating cell-free DNA profiling

  Differentiating sequencing errors from true variants is a central genomics challenge calling for error suppression strategies that balance costs when sparse material or ultra-rare variants require high-accuracy methods. For example, circulating cell-free DNA (ccfDNA) sequencing for cancer monitoring is limited by sparsity of circulating tumor DNA (ctDNA), abundance of genomic material in samples, library preparation error rates and sequencing errors. Whole-genome sequencing (WGS) can overcome the low abundance of ccfDNA by integrating signal across the mutation landscape, but higher cost limits adoption compared with targeted panels, where ccfDNA abundance limits maximal coverage depths. Here we applied deep (~120x) lower-cost WGS (Ultima Genomics) with analytic error correction for tumor-informed ctDNA detection within the part-per-million range. We further developed high-coverage duplex error-corrected WGS of ccfDNA, achieving 7.7x10-8 error rates, to assess disease burden in melanoma and urothelial cancer patients without matched tumor sequencing. Together, we show that deeply sequenced error-corrected WGS accurately calls somatic variants, demonstrating the feasibility of WGS for tumor-informed and tumor-agnostic ctDNA detection.

  Study EGAS50000000844

• A Therapeutic Vaccine for Fibrolamellar Hepatocellular Carcinoma

  Fibrolamellar hepatocellular carcinoma (FLC) is a rare form of liver cancer affecting 33 children and young adults that is driven by a chimeric protein, DNAJ-PKAc. We conducted a phase 1 clinical trial of a therapeutic vaccine targeting DNAJ-PKAc 38 (FLC-Vac), in combination with nivolumab and ipilimumab, in children and adults with advanced FLC. Among 16 patients enrolled, 12 completed the vaccine priming phase and were evaluable for both immunological and clinical endpoints. Grade 3 treatment-related adverse events were reported by six patients (37.5%). DNAJ43 PKAc-specific T cell responses were detected in 9/12 patients after treatment. In the subset of patients who completed the initial priming phase the disease control rate (DCR) was 75% (9/12), with three partial responses (25%). All patients with clinical responses also had DNAJ-PKAc specific T cell responses, from which we identified multiple class II-restricted T cell receptors (TCRs) with specificity for DNAJ-PKAc. Correlates of response included both functional neoantigen reactivity and changes in TCR repertoire features over time. Immune escape in two patients corresponded with immune exhaustion rather than neoantigen escape or HLA loss.

  Study phs003970

• Whole exome sequencing of bladder tumors

  Bladder carcinomas are immunogenic, and patients with bladder cancer benefit from immune checkpoint therapy. This is correlated to a high tumor mutation burden, which provides a higher number of neoepitopes that can be recognized by tumor-specific CD8+ T cells. Intravesical Bacillus Calmette-Guérin (BCG) is used to treat non-muscle invasive bladder cancer (NMIBC), but its mechanism of action remains elusive. Most lymphocytes appearing in the urine of BCG-treated patients are CD4+ T cells though preclinical studies showed that CD8+ T cells are also necessary for BCG treatment efficacy. It is currently unknown which proportion of patients with non-metastatic bladder cancer develop a spontaneous antitumor CD8+ response, and if BCG treatment influences this response. were prepared using the xGen Dual Index UMI Adapters (IDT), and genomic DNA (gDNA) extracted (DNeasy kit, Qiagen) from formalin-fixed paraffin- embedded (FFPE) or snap-frozen tumor tissue, as well as autologous lymphocytes. Libraries were subjected to paired-end sequencing using Illumina HiSeq4000 with a coverage depth of 250X. Library preparation was carried out with the AllType FASTplex NGS 11 Loci Flex Kit (One Lambda), and sequencing was conducted on the Illumina MiSeq platform.

  Study EGAS50000001248

• Establishing stable brain tumor stem cell lines and translational research for new treatments

  Choroid plexus carcinomas (CPCs) have dismal prognoses with limited clinical options. A lack of characterized cell lines has hindered the development of new treatment strategies. Here, we perform whole genome sequencing analyses of two cell lines established from CPCs. Whole genome sequencing (WGS),was performed in CCHE-45 and NGT131 cell lines. Both cell lines harbored pathogenic TP53 point mutations; CCHE-45 additionally displayed TP53 loss. Furthermore, alterations of the NOTCH and WNT pathways were also detected in both cell lines. Two protein-coding gene fusions, BZW2-URGCP, and CTTNBP2-ERBB4, mutations of two oncodrivers, GBP-4 and KRTAP-12-2, and several copy number alterations were observed in CCHE-45, but not NGT131. The discovered difference's importance and implications highlight the possible diversity of choroid plexus carcinoma and call for additional research to fully understand disease pathogenesis. Whole genome sequencing revealed that the two choroid plexus carcinoma cell lines shared TP53 mutations and other common pathway alterations and activation of NOTCH and WNT pathways. Noticeable differences were also observed. These cell lines can serve as valuable models to propose new treatments in these rare but deadly brain tumors.

  Study JGAS000657

• Effective screening strategy for deafness using a new-genetation sequencing platform: a consecutive analysis

  The diagnosis of the genetic etiology of deafness contributes to the clinical management of patients. We performed the following four genetic tests in three stages for 52 consecutive deafness subjects in one facility. We used the Invade assay and Sanger sequencing for the GJB2 gene or SLC26A4 gene in the first stage test, TaqMan genotyping assay in second stage test, and targeted exon sequencing using the massively parallel DNA sequencing in third stage test. Overall, we identified the genetic cause in 40% (21/52) of patients. The diagnostic rates of the first-, second- and third-stage genetic testing were 17%(9/52), 9%(4/43) and 21% (8/39), respectively. The combination approach using these genetic tests appears to be useful as a diagnostic tool for deafness patients. We recommended that genetic testing for the screening of common mutations in deafness genes using the Invader assay or TaqMan genotyping assay be performed as the initial evaluation. For the remaining undiagnosed cases, targeted exon sequencing using MPS is clinically and economically beneficial. This data set was MPS reads data (fastq) for 39 sensorineural hearing loss patients.

  Study JGAS000032