Existing methods for paired antibody heavy- and light-chain repertoire sequencing rely on specialized equipment and are limited by their commercial availability and high costs. Here, we report a ...novel simple and cost-effective emulsion-based single-cell paired antibody repertoire sequencing method that employs only basic laboratory equipment. We performed a proof-of-concept using mixed mouse hybridoma cells and we also showed that our method can be used for discovery of novel antigen-specific monoclonal antibodies by sequencing human CD19
B cell IgM and IgG repertoires isolated from peripheral whole blood before and seven days after Td (Tetanus toxoid/Diphtheria toxoid) booster immunization. We anticipate broad applicability of our method for providing insights into adaptive immune responses associated with various diseases, vaccinations, and cancer immunotherapies.
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RNA interference (RNAi) is an evolutionarily conserved defence mechanism whereby genes are specifically silenced through degradation of messenger RNAs; this process is mediated by homologous ...double-stranded (ds)RNA molecules. In invertebrates, long dsRNAs have been used for genome-wide screens and have provided insights into gene functions. Because long dsRNA triggers a nonspecific interferon response in many vertebrates, short interfering (si)RNA or short hairpin (sh)RNAs must be used for these organisms to ensure specific gene silencing. Here we report the generation of a genome-scale library of endoribonuclease-prepared short interfering (esi)RNAs from a sequence-verified complementary DNA collection representing 15,497 human genes. We used 5,305 esiRNAs from this library to screen for genes required for cell division in HeLa cells. Using a primary high-throughput cell viability screen followed by a secondary high content videomicroscopy assay, we identified 37 genes required for cell division. These include several splicing factors for which knockdown generates mitotic spindle defects. In addition, a putative nuclear-export terminator was found to speed up cell proliferation and mitotic progression after knockdown. Thus, our study uncovers new aspects of cell division and establishes esiRNA as a versatile approach for genomic RNAi screens in mammalian cells.
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Immune checkpoint inhibitors (ICIs) are increasingly being used in non-small-cell lung cancer (NSCLC), yet biomarkers predicting their benefit are lacking. We evaluated if on-treatment changes of ...circulating tumor DNA (ctDNA) from ICI start (t
) to after two cycles (t
) assessed with a commercial panel could identify patients with NSCLC who would benefit from ICI.
The molecular ctDNA response was evaluated as a predictor of radiographic tumor response and long-term survival benefit of ICI. To maximize the yield of ctDNA detection, de novo mutation calling was performed. Furthermore, the impact of clonal hematopoiesis (CH)-related variants as a source of biologic noise was investigated.
After correction for CH-related variants, which were detected in 75 patients (44.9%), ctDNA was detected in 152 of 167 (91.0%) patients. We observed only a fair agreement of the molecular and radiographic response, which was even more impaired by the inclusion of CH-related variants. After exclusion of those, a ≥ 50% molecular response improved progression-free survival (10
2 months; hazard ratio HR, 0.55; 95% CI, 0.39 to 0.77;
.0011) and overall survival (18.4
5.9 months; HR, 0.44; 95% CI, 0.31 to 0.62;
< .0001) compared with patients not achieving this end point. After adjusting for clinical variables, ctDNA response and
/
mutations (HR, 2.08; 95% CI, 1.4 to 3.0;
< .001) remained independent predictors for overall survival, irrespective of programmed death ligand-1 expression. A landmark survival analysis at 2 months (n = 129) provided similar results.
On-treatment changes of ctDNA in plasma reveal predictive information for long-term clinical benefit in ICI-treated patients with NSCLC. A broader NSCLC patient coverage through de novo mutation calling and the use of a variant call set excluding CH-related variants improved the classification of molecular responders, but had no significant impact on survival.
Global investigation of histone marks in acute myeloid leukemia (AML) remains limited. Analyses of 38 AML samples through integrated transcriptional and chromatin mark analysis exposes 2 major ...subtypes. One subtype is dominated by patients with NPM1 mutations or MLL-fusion genes, shows activation of the regulatory pathways involving HOX-family genes as targets, and displays high self-renewal capacity and stemness. The second subtype is enriched for RUNX1 or spliceosome mutations, suggesting potential interplay between the 2 aberrations, and mainly depends on IRF family regulators. Cellular consequences in prognosis predict a relatively worse outcome for the first subtype. Our integrated profiling establishes a rich resource to probe AML subtypes on the basis of expression and chromatin data.
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•Systematic exploration of the chromatin landscape and regulatory basis of AMLs•Identification of 2 main AML subtypes based on chromatin states•Distinct genetic mutations converge at the chromatin level•Chromatin signatures reveal diverse stemness phenotypes and cellular consequences
Yi et al. find that despite pronounced genetic heterogeneity, integrative chromatin profiling converges acute myeloid leukemia (AML) patients with stemness-attributed similarity into the same subtype. The study provides a comprehensive resource for investigating the molecular basis and underpinnings of AML progression in primary samples.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Non-alcoholic fatty liver disease (NAFLD) has a broad spectrum of disease states ranging from mild steatosis characterized by an abnormal retention of lipids within liver cells to steatohepatitis ...(NASH) showing fat accumulation, inflammation, ballooning and degradation of hepatocytes, and fibrosis. Ultimately, steatohepatitis can result in liver cirrhosis and hepatocellular carcinoma.
In this study we have analyzed three different mouse strains, A/J, C57BL/6J, and PWD/PhJ, that show different degrees of steatohepatitis when administered a 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) containing diet. RNA-Seq gene expression analysis, protein analysis and metabolic profiling were applied to identify differentially expressed genes/proteins and perturbed metabolite levels of mouse liver samples upon DDC-treatment. Pathway analysis revealed alteration of arachidonic acid (AA) and S-adenosylmethionine (SAMe) metabolism upon other pathways. To understand metabolic changes of arachidonic acid metabolism in the light of disease expression profiles a kinetic model of this pathway was developed and optimized according to metabolite levels. Subsequently, the model was used to study in silico effects of potential drug targets for steatohepatitis.
We identified AA/eicosanoid metabolism as highly perturbed in DDC-induced mice using a combination of an experimental and in silico approach. Our analysis of the AA/eicosanoid metabolic pathway suggests that 5-hydroxyeicosatetraenoic acid (5-HETE), 15-hydroxyeicosatetraenoic acid (15-HETE) and prostaglandin D2 (PGD2) are perturbed in DDC mice. We further demonstrate that a dynamic model can be used for qualitative prediction of metabolic changes based on transcriptomics data in a disease-related context. Furthermore, SAMe metabolism was identified as being perturbed due to DDC treatment. Several genes as well as some metabolites of this module show differences between A/J and C57BL/6J on the one hand and PWD/PhJ on the other.
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The recently released human genome sequences provide us with reference data to conduct comparative genomic research on primates, which will be important to understand what genetic information makes ...us human. Here we present a first-generation human-chimpanzee comparative genome map and its initial analysis. The map was constructed through paired alignment of 77,461 chimpanzee bacterial artificial chromosome end sequences with publicly available human genome sequences. We detected candidate positions, including two clusters on human chromosome 21 that suggest large, nonrandom regions of difference between the two genomes.
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Metastasis-Associated in Colon Cancer 1 (
) is a strong prognostic biomarker inducing proliferation, migration, invasiveness, and metastasis of cancer cells. The context of
dysregulation in cancers ...is, however, still poorly understood. Here, we investigated whether chromosomal instability and somatic copy number alterations (SCNA) frequently occurring in CRC contribute to
dysregulation, with prognostic and predictive impacts. Using the Oncotrack and Charité CRC cohorts of CRC patients, we showed that elevated
mRNA expression was tightly dependent on increased
gene SCNA and was associated with metastasis and shorter metastasis free survival. Deep analysis of the COAD-READ TCGA cohort revealed elevated
expression due to SCNA for advanced tumors exhibiting high chromosomal instability (CIN), and predominantly classified as CMS2 and CMS4 transcriptomic subtypes. For that cohort, we validated that elevated
mRNA expression correlated with reduced disease-free and overall survival. In conclusion, this study gives insights into the context of
expression in CRC. Increased
expression is largely driven by CIN, SCNA gains, and molecular subtypes, potentially determining the molecular risk for metastasis that might serve as a basis for patient-tailored treatment decisions.
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58.
Cancer Precision Medicine Schütte, Moritz; Ogilvie, Lesley A.; Rieke, Damian T. ...
Public health genomics,
01/2017, Volume:
20, Issue:
2
Journal Article
Peer reviewed
Open access
Every tumour is different. They arise in patients with different genomes, from cells with different epigenetic modifications, and by random processes affecting the genome and/or epigenome of a ...somatic cell, allowing it to escape the usual controls on its growth. Tumours and patients therefore often respond very differently to the drugs they receive. Cancer precision medicine aims to characterise the tumour (and often also the patient) to be able to predict, with high accuracy, its response to different treatments, with options ranging from the selective characterisation of a few genomic variants considered particularly important to predict the response of the tumour to specific drugs, to deep genome analysis of both tumour and patient, combined with deep transcriptome analysis of the tumour. Here, we compare the expected results of carrying out such analyses at different levels, from different size panels to a comprehensive analysis incorporating both patient and tumour at the DNA and RNA levels. In doing so, we illustrate the additional power gained by this unusually deep analysis strategy, a potential basis for a future precision medicine first strategy in cancer drug therapy. However, this is only a step along the way of increasingly detailed molecular characterisation, which in our view will, in the future, introduce additional molecular characterisation techniques, including systematic analysis of proteins and protein modification states and different types of metabolites in the tumour, systematic analysis of circulating tumour cells and nucleic acids, the use of spatially resolved analysis techniques to address the problem of tumour heterogeneity as well as the deep analyses of the immune system of the patient to, e.g., predict the response of the patient to different types of immunotherapy. Such analyses will generate data sets of even greater complexity, requiring mechanistic modelling approaches to capture enough of the complex situation in the real patient to be able to accurately predict his/her responses to all available therapies.
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Mutation is a fundamental process in tumorigenesis. However, the degree to which the rate of somatic mutation varies across the human genome and the mechanistic basis underlying this variation remain ...to be fully elucidated. Here, we performed a cross-cancer comparison of 402 whole genomes comprising a diverse set of childhood and adult tumors, including both solid and hematopoietic malignancies. Surprisingly, we found that the inactive X chromosome of many female cancer genomes accumulates on average twice and up to four times as many somatic mutations per megabase, as compared to the individual autosomes. Whole-genome sequencing of clonally expanded hematopoietic stem/progenitor cells (HSPCs) from healthy individuals and a premalignant myelodysplastic syndrome (MDS) sample revealed no X chromosome hypermutation. Our data suggest that hypermutation of the inactive X chromosome is an early and frequent feature of tumorigenesis resulting from DNA replication stress in aberrantly proliferating cells.
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•X chromosome has up to 4× more mutations than the autosomes in female cancer genomes•Hypermutations only affect the inactive X chromosome•X hypermutation involves somatic point mutations and indels, but not germline mutations•No X hypermutation is found in clonal expansions of normal or premalignant cells
A comparison of 402 cancer genomes identifies a surprisingly high level of somatic mutations in the inactive X chromosome of female cancer genomes. As hypermutability of the inactive X was not observed in clonal hematopoietic progenitor or preleukemic samples, it is likely that it may be a contributing factor to tumorigenesis.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
We investigated parent-of-origin and allele-specific expression effects on obesity and hepatic gene expression in reciprocal crosses between the Berlin Fat Mouse Inbred line (BFMI) and C57Bl/6NCrl ...(B6N). We found that F1-males with a BFMI mother developed 1.8 times more fat mass on a high fat diet at 10 weeks than F1-males of a BFMI father. The phenotype was detectable from six weeks on and was preserved after cross-fostering. RNA-seq data of liver provided evidence for higher biosynthesis and elongation of fatty acids (p = 0.00635) in obese male offspring of a BFMI mother versus lean offspring of a BFMI father. Furthermore, fatty acid degradation (p = 0.00198) and the peroxisome pathway were impaired (p = 0.00094). The circadian rhythm was affected as well (p = 0.00087). Among the highest up-regulated protein coding genes in obese males were Acot4 (1.82 fold, p = 0.022), Cyp4a10 (1.35 fold, p = 0.026) and Cyp4a14 (1.32 fold, p = 0.012), which hydroxylize fatty acids and which are known to be increased in liver steatosis. Obese males showed lower expression of the genetically imprinted and paternally expressed 3 (Peg3) gene (0.31 fold, p = 0.046) and higher expression of the androgen receptor (Ar) gene (2.38 fold, p = 0.068). Allelic imbalance was found for expression of ATP-binding cassette transporter gene Abca8b. Several of the differentially expressed genes contain estrogen response elements. Parent-of-origin effects during gametogenesis and/or fetal development in an obese mother epigenetically modify the transcription of genes that lead to enhanced fatty acid synthesis and impair beta-oxidation in the liver of male, but not female F1 offspring. Down-regulation of Peg3 could contribute to trigger this metabolic setting. At puberty, higher amounts of the androgen receptor and altered access to estrogen response elements in affected genes are likely responsible for male specific expression of genes that were epigenetically triggered. A suggestive lack of estrogen binding motifs was found for highly down-regulated genes in adult hepatocytes of obese F1 males (p = 0.074).
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