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1.
Nat Biotechnol ; 38(2): 233-244, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31907405

RESUMO

Despite the global rapid increase in the number of clinical trials employing chimeric antigen receptors (CARs), no comprehensive survey of their scope, targets and design exists. In this study, we present an interactive CAR clinical trial database, spanning 64 targets deployed in T cells (CAR-T), natural killer cells (CAR-NK) or mixtures (CAR-NK/T) from over 500 clinical trials in 20 countries, encompassing >20,000 patients. By combining these data with transcriptional and proteomic data, we create a 'targetable landscape' for CAR cell therapies based on 13,206 proteins and RNAs across 78 tissues, 124 cell types and 20 cancer types. These data suggest a landscape of over 100 single targets and over 100,000 target pairs using logical switches for CAR cell engineering. Our analysis of the CAR cellular therapeutic landscape may aid the design of future therapies, improve target-to-patient matching, and ultimately help inform our understanding of CAR therapy's safety and efficacy.

2.
J Biomol Tech ; 2020 Jan 13.
Artigo em Inglês | MEDLINE | ID: mdl-31969795

RESUMO

Shared research resource facilities, also known as core laboratories (Cores), are responsible for generating a significant and growing portion of the research data in academic biomedical research institutions. Cores represent a central repository for institutional knowledge management, with deep expertise in the strengths and limitations of technology and its applications. They inherently support transparency and scientific reproducibility by protecting against cognitive bias in research design and data analysis, and thedy have institutional responsibility for the conduct of research (research ethics, regulatory compliance, and financial accountability) performed in their Cores. The Association of Biomolecular Resource Facilities (ABRF) is a FASEB-member scientific society whose members are scientists and administrators that manage or support Cores. The ABRF Research Groups (RGs), representing expertise for an array of cutting-edge and established technology platforms, perform multicenter research studies to determine and communicate best practices and community-based standards. This review provides a summary of the contributions of the ABRF RGs to promote scientific rigor and reproducibility in Cores from the published literature, ABRF meetings, and ABRF RGs communications.

3.
Transl Res ; 215: 31-40, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31520587

RESUMO

Precision medicine has generated diagnoses for many patients with challenging undiagnosed disorders. Some individuals remain without a diagnosis despite comprehensive testing, and this impedes their treatment. This report addresses the role of personalized medicine in identifying effective therapy for an undiagnosed disease. A 22-year-old woman presented with chronic severe recurrent trismus, facial pain, progressive multicentric inflammatory and fibrotic masses, and high C-reactive protein. Sites of disease included the pterygomaxillary region, masseter muscles, mandible, lung, pericardium, intrabdominal cavity, and retroperitoneum. A diagnosis was not established after an extensive assessment, including multiple biopsies. The patient was subsequently evaluated under the Undiagnosed Diseases Program at the National Institutes of Health. Large scale genotyping, proteomic studies, and in vitro and gene expression analyses of fibroblasts obtained from a major disease locus were performed. Germline genetic testing did not identify strong candidate genes; proteomic studies of the patient's serum and bronchoalveolar lavage fluid and gene expression analyses of her cells were consistent with dysregulation of the tumor necrosis factor-alpha pathway. The patient's cultured fibroblasts were incubated with selected drugs, and cell proliferation was inhibited by hydroxychloroquine. Treatment of the patient with hydroxychloroquine conferred prolonged beneficial clinical effects, including stabilization of trismus and reduction of corticosteroid dose, C-reactive protein, and size of masses. This case represents an example of precision medicine applied to discover effective treatments for individuals with enigmatic undiagnosed disorders.

4.
Mol Cell ; 2019 Nov 25.
Artigo em Inglês | MEDLINE | ID: mdl-31810760

RESUMO

The RNA modification N6-methyladenosine (m6A) modulates mRNA fate and thus affects many biological processes. We analyzed m6A across the transcriptome following infection by dengue virus (DENV), Zika virus (ZIKV), West Nile virus (WNV), and hepatitis C virus (HCV). We found that infection by these viruses in the Flaviviridae family alters m6A modification of specific cellular transcripts, including RIOK3 and CIRBP. During viral infection, the addition of m6A to RIOK3 promotes its translation, while loss of m6A in CIRBP promotes alternative splicing. Importantly, viral activation of innate immune sensing or the endoplasmic reticulum (ER) stress response contributes to the changes in m6A in RIOK3 or CIRBP, respectively. Further, several transcripts with infection-altered m6A profiles, including RIOK3 and CIRBP, encode proteins that influence DENV, ZIKV, and HCV infection. Overall, this work reveals that cellular signaling pathways activated during viral infection lead to alterations in m6A modification of host mRNAs to regulate infection.

5.
Nat Commun ; 10(1): 4155, 2019 09 13.
Artigo em Inglês | MEDLINE | ID: mdl-31519912

RESUMO

Zika virus (ZIKV) infection results in an increased risk of spontaneous abortion and poor intrauterine growth although the underlying mechanisms remain undetermined. Little is known about the impact of ZIKV infection during the earliest stages of pregnancy, at pre- and peri-implantation, because most current ZIKV pregnancy studies have focused on post-implantation stages. Here, we demonstrate that trophectoderm cells of pre-implantation human and mouse embryos can be infected with ZIKV, and propagate virus causing neural progenitor cell death. These findings are corroborated by the dose-dependent nature of ZIKV susceptibility of hESC-derived trophectoderm cells. Single blastocyst RNA-seq reveals key transcriptional changes upon ZIKV infection, including nervous system development, prior to commitment to the neural lineage. The pregnancy rate of mice is >50% lower in pre-implantation infection than infection at E4.5, demonstrating that pre-implantation ZIKV infection leads to miscarriage. Cumulatively, these data elucidate a previously unappreciated association of pre- and peri-implantation ZIKV infection and microcephaly.


Assuntos
Complicações Infecciosas na Gravidez/metabolismo , Infecção por Zika virus/complicações , Infecção por Zika virus/metabolismo , Zika virus/patogenicidade , Aborto Espontâneo/metabolismo , Aborto Espontâneo/fisiopatologia , Animais , Blastocisto/citologia , Blastocisto/metabolismo , Implantação do Embrião/fisiologia , Feminino , Desenvolvimento Fetal/genética , Desenvolvimento Fetal/fisiologia , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Gravidez , RNA Viral/genética , Pesquisa Médica Translacional/métodos , Trofoblastos/citologia , Trofoblastos/metabolismo
6.
Nat Commun ; 10(1): 4079, 2019 09 09.
Artigo em Inglês | MEDLINE | ID: mdl-31501426

RESUMO

The epitranscriptomics field has undergone an enormous expansion in the last few years; however, a major limitation is the lack of generic methods to map RNA modifications transcriptome-wide. Here, we show that using direct RNA sequencing, N6-methyladenosine (m6A) RNA modifications can be detected with high accuracy, in the form of systematic errors and decreased base-calling qualities. Specifically, we find that our algorithm, trained with m6A-modified and unmodified synthetic sequences, can predict m6A RNA modifications with ~90% accuracy. We then extend our findings to yeast data sets, finding that our method can identify m6A RNA modifications in vivo with an accuracy of 87%. Moreover, we further validate our method by showing that these 'errors' are typically not observed in yeast ime4-knockout strains, which lack m6A modifications. Our results open avenues to investigate the biological roles of RNA modifications in their native RNA context.


Assuntos
Adenosina/análogos & derivados , RNA/genética , RNA/metabolismo , Adenosina/metabolismo , Sequência de Bases , Eletricidade , Saccharomyces cerevisiae/genética , Análise de Sequência de RNA , Máquina de Vetores de Suporte
8.
Sci China Life Sci ; 62(7): 937-946, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31124003

RESUMO

RNA sequencing (RNA-seq) has greatly facilitated the exploring of transcriptome landscape for diverse organisms. However, transcriptome reconstruction is still challenging due to various limitations of current tools and sequencing technologies. Here, we introduce an efficient tool, QuaPra (Quadratic Programming combined with Apriori), for accurate transcriptome assembly and quantification. QuaPra could detect at least 26.5% more low abundance (0.1-1 FPKM) transcripts with over 2.1% increase of sensitivity and precision on simulated data compared to other currently popular tools. Moreover, around one-quarter more known transcripts were correctly assembled by QuaPra than other assemblers on real sequencing data. QuaPra is freely available at https://doi.org/www.megabionet.org/QuaPra/ .

10.
Anal Chem ; 91(10): 6746-6753, 2019 May 21.
Artigo em Inglês | MEDLINE | ID: mdl-31002238

RESUMO

Recent studies have indicated that circulating noncoding RNAs (ncRNAs) such as miRNAs are stable biomarkers for the diagnosis and prognosis of human diseases. However, due to low concentrations of circulating ncRNAs in blood, data normalization in plasma/serum ncRNA experiments using next-generation sequencing and quantitative real time RT-qPCR is a challenge. We found that the current normalization methods based on synthetic external spiked-in controls or published endogenous miRNA controls are inappropriate as they are not stably expressed and therefore fail to reliably detect differentially expressed ncRNAs. Using the alternative of individual ncRNAs as biomarkers, we considered a ratio-based normalization method calculated taking the ratio of any two ncRNAs in the same sample and used the resulting ratios as biomarkers. We mathematically verified the method to be independent of spiked-in and internal controls, and more robust than existing reference control based normalization methods to identify differentially expressed ncRNAs as potential biomarkers for human diseases. Thus, the ratio-based method can solve the difficult normalization problem for circuiting ncRNA data to identify reliable biomarkers to meet real clinical practice.

11.
Nat Biotechnol ; 37(5): 567, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30899106

RESUMO

In the version of this article initially published online, two pairs of headings were switched with each other in Table 4: "Recall (PCR free)" was switched with "Recall (with PCR)," and "Precision (PCR free)" was switched with "Precision (with PCR)." The error has been corrected in the print, PDF and HTML versions of this article.

12.
Front Genet ; 10: 8, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30881372

RESUMO

The importance of diversity and cellular specialization is clear for many reasons, from population-level diversification, to improved resiliency to unforeseen stresses, to unique functions within metazoan organisms during development and differentiation. However, the level of cellular heterogeneity is just now becoming clear through the integration of genome-wide analyses and more cost effective Next Generation Sequencing (NGS). With easy access to single-cell NGS (scNGS), new opportunities exist to examine different levels of gene expression and somatic mutational heterogeneity, but these assays can generate yottabyte scale data. Here, we model the importance of heterogeneity for large-scale analysis of scNGS data, with a focus on the utilization in oncology and other diseases, providing a guide to aid in sample size and experimental design.

13.
Front Genet ; 10: 133, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30881380

RESUMO

Leukemia, specifically acute myeloid leukemia (AML), is a common malignancy that can be differentiated into multiple subtypes based on leukemogenic history and etiology. Although genetic aberrations, particularly cytogenetic abnormalities and mutations in known oncogenes, play an integral role in AML development, epigenetic processes have been shown as a significant and sometimes independent dynamic in AML pathophysiology. Here, we summarize how tumors evolve and describe AML through an epigenetic lens, including discussions on recent discoveries that include prognostics from epialleles, changes in RNA function for hematopoietic stem cells and the epitranscriptome, and novel epigenetic treatment options. We further describe the limitations of treatment in the context of the high degree of heterogeneity that characterizes acute myeloid leukemia.

14.
Nat Biotechnol ; 37(5): 555-560, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30858580

RESUMO

Standardized benchmarking approaches are required to assess the accuracy of variants called from sequence data. Although variant-calling tools and the metrics used to assess their performance continue to improve, important challenges remain. Here, as part of the Global Alliance for Genomics and Health (GA4GH), we present a benchmarking framework for variant calling. We provide guidance on how to match variant calls with different representations, define standard performance metrics, and stratify performance by variant type and genome context. We describe limitations of high-confidence calls and regions that can be used as truth sets (for example, single-nucleotide variant concordance of two methods is 99.7% inside versus 76.5% outside high-confidence regions). Our web-based app enables comparison of variant calls against truth sets to obtain a standardized performance report. Our approach has been piloted in the PrecisionFDA variant-calling challenges to identify the best-in-class variant-calling methods within high-confidence regions. Finally, we recommend a set of best practices for using our tools and evaluating the results.


Assuntos
Benchmarking , Exoma/genética , Genoma Humano/genética , Sequenciamento de Nucleotídeos em Larga Escala , Algoritmos , Genômica/tendências , Células Germinativas , Humanos , Polimorfismo de Nucleotídeo Único/genética , Software
15.
Nat Commun ; 10(1): 821, 2019 02 18.
Artigo em Inglês | MEDLINE | ID: mdl-30778059

RESUMO

lncRNAs make up a majority of the human transcriptome and have key regulatory functions. Here we perform unbiased de novo annotation of transcripts expressed during the human humoral immune response to find 30% of the human genome transcribed during this process, yet 58% of these transcripts manifest striking differential expression, indicating an lncRNA phylogenetic relationship among cell types that is more robust than that of coding genes. We provide an atlas of lncRNAs in naive and GC B-cells that indicates their partition into ten functionally categories based on chromatin features, DNase hypersensitivity and transcription factor localization, defining lncRNAs classes such as enhancer-RNAs (eRNA), bivalent-lncRNAs, and CTCF-associated, among others. Specifically, eRNAs are transcribed in 8.6% of regular enhancers and 36.5% of super enhancers, and are associated with coding genes that participate in critical immune regulatory pathways, while plasma cells have uniquely high levels of circular-RNAs accounted for by and reflecting the combinatorial clonal state of the Immunoglobulin loci.


Assuntos
Linfócitos B/fisiologia , Imunidade Humoral/genética , RNA Longo não Codificante/imunologia , Linfócitos B/imunologia , Elementos Facilitadores Genéticos , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/imunologia , Redes Reguladoras de Genes/imunologia , Genoma Humano , Humanos , RNA
16.
Nat Commun ; 10(1): 579, 2019 02 04.
Artigo em Inglês | MEDLINE | ID: mdl-30718479

RESUMO

The DNA base modification N6-methyladenine (m6A) is involved in many pathways related to the survival of bacteria and their interactions with hosts. Nanopore sequencing offers a new, portable method to detect base modifications. Here, we show that a neural network can improve m6A detection at trained sequence contexts compared to previously published methods using deviations between measured and expected current values as each adenine travels through a pore. The model, implemented as the mCaller software package, can be extended to detect known or confirm suspected methyltransferase target motifs based on predictions of methylation at untrained contexts. We use PacBio, Oxford Nanopore, methylated DNA immunoprecipitation sequencing (MeDIP-seq), and whole-genome bisulfite sequencing data to generate and orthogonally validate methylomes for eight microbial reference species. These well-characterized microbial references can serve as controls in the development and evaluation of future methods for the identification of base modifications from single-molecule sequencing data.


Assuntos
Adenosina/análogos & derivados , Metilação de DNA/fisiologia , Análise de Sequência de DNA/métodos , Adenosina/análise , Algoritmos , Metilação de DNA/genética , Imunoprecipitação , Software
18.
J Biomol Tech ; 30(Suppl): S47, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31896984

RESUMO

The majority of the world's population lives in cities, yet our understanding of the dynamics and distribution of bacteria, viruses, and antimicrobial resistance (AMR) genes in urban built environments is limited. Cities and urban transport hubs like metro stations and airports represent the sites of highest human population density, and are thus hotspot areas for microbial and genetic exchange in both developing and developed countries. Moreover, most inhabitants, visitors and passenger carry a mobile phone, which serves as a molecular echo of the travel history of a person as well as a representation of his or her personal microbiome. Through leveraging next-generation sequencing (NGS) and metagenome sequencing methods, combined with rapid computational analysis, we can reveal the microbial and genetic profiles, AMR gene content, localization, and movement patterns imprinted on phones and city surfaces, as readily as for clinical samples. International Consortium for Metagenomics of Subways and Urban Biomes (MetaSUB) at Weill Cornell Medicine, which is funded by the NIH, WorldQuant, and the Bill and Melinda Gates Foundation. We are currently analyzing >5.000 whole-genome shotgun metagenomes using samples collected in the subways (15000 samples total of more than 85 cities in 21 different countries). In parallel we analyzed for interrogating microbiomes obtained from > 2000 mobile phone surfaces, which we showcased during several international conferences. To our knowledge this is the first study investigating a comprehensive and worldwide view of urban microbiomes and AMR exchange. To complement our knowledge, we are comparing the generated metagenomes with data collected by the Extreme Microbiome Project and microbial isolates collected at the ISS by NASA during the last 11 years. Furthermore, to evaluate our findings and improve the accuracy of microbiome analyses, we developed novel protocols for metagenomic linked-DNA genomic sequencing (using 10X Genomics Chromium) and long-read sequencing (using Oxford Nanopore MinION and PromethION sequencers).

19.
J Biomol Tech ; 30(Suppl): S56, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31897012

RESUMO

Previous studies have reported that various stimuli, including viral infection, cancer, and heat shock, lead to changes to m6A methylation of mRNAs from global increases or decreases in m6A to shifts in the distribution of methylation over transcripts or changes at specific loci. Reported estimates of m6A gains and losses range from zero to thousands of sites, depending on the stimulus and analysis methods. Most studies map m6A using methylated RNA immunoprecipitation sequencing (MeRIP-seq). Methylated regions are identified through peaks in transcript coverage from the immunoprecipitated fraction compared to an input fraction. However, for any two given studies looking at similar conditions, we found peak overlap varied from ∼30 to 60%, independent of the cell lines studied. Although we verified appropriate statistical methods to detect changes in peaks using positive and negative controls, we were unable to validate consistent changes across studies of similar experimental interventions. Eventually, single-molecule methods may help in these challenges, and as such, we also present evidence of m6A within nature RNA nanopore molecules with a new tool (EpinNano).

20.
BMC Microbiol ; 18(1): 175, 2018 11 23.
Artigo em Inglês | MEDLINE | ID: mdl-30466389

RESUMO

BACKGROUND: The antimicrobial resistance (AMR) phenotypic properties, multiple drug resistance (MDR) gene profiles, and genes related to potential virulence and pathogenic properties of five Enterobacter bugandensis strains isolated from the International Space Station (ISS) were carried out and compared with genomes of three clinical strains. Whole genome sequences of ISS strains were characterized using the hybrid de novo assembly of Nanopore and Illumina reads. In addition to traditional microbial taxonomic approaches, multilocus sequence typing (MLST) analysis was performed to classify the phylogenetic lineage. Agar diffusion discs assay was performed to test antibiotics susceptibility. The draft genomes after assembly and scaffolding were annotated with the Rapid Annotations using Subsystems Technology and RNAmmer servers for downstream analysis. RESULTS: Molecular phylogeny and whole genome analysis of the ISS strains with all publicly available Enterobacter genomes revealed that ISS strains were E. bugandensis and similar to the type strain EB-247T and two clinical isolates (153_ECLO and MBRL 1077). Comparative genomic analyses of all eight E. bungandensis strains showed, a total of 4733 genes were associated with carbohydrate metabolism (635 genes), amino acid and derivatives (496 genes), protein metabolism (291 genes), cofactors, vitamins, prosthetic groups, pigments (275 genes), membrane transport (247 genes), and RNA metabolism (239 genes). In addition, 112 genes identified in the ISS strains were involved in virulence, disease, and defense. Genes associated with resistance to antibiotics and toxic compounds, including the MDR tripartite system were also identified in the ISS strains. A multiple antibiotic resistance (MAR) locus or MAR operon encoding MarA, MarB, MarC, and MarR, which regulate more than 60 genes, including upregulation of drug efflux systems that have been reported in Escherichia coli K12, was also observed in the ISS strains. CONCLUSION: Given the MDR results for these ISS Enterobacter genomes and increased chance of pathogenicity (PathogenFinder algorithm with > 79% probability), these species pose important health considerations for future missions. Thorough genomic characterization of the strains isolated from ISS can help to understand the pathogenic potential, and inform future missions, but analyzing them in in-vivo systems is required to discern the influence of microgravity on their pathogenicity.


Assuntos
Farmacorresistência Bacteriana Múltipla , Enterobacter/efeitos dos fármacos , Enterobacter/genética , Infecções por Enterobacteriaceae/microbiologia , Astronave , Antibacterianos/farmacologia , Enterobacter/classificação , Enterobacter/isolamento & purificação , Genoma Bacteriano , Genômica , Humanos , Testes de Sensibilidade Microbiana , Tipagem de Sequências Multilocus , Filogenia , Astronave/estatística & dados numéricos , Sequenciamento Completo do Genoma
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