bluuebunny/crossref_metadata_2025_split huggingface dataset:
Cas9-generated auxotrophs ofPhaeodactylum tricornutumare characterized by small and large deletions that can be complemented by plasmid-based genes
Authors: Slattery Samuel S. , Wang Helen , Giguere Daniel J. , Kocsis Csanad , Urquhart Bradley L. , Karas Bogumil J. , Edgell David R. 
Published: April 2020

ABSTRACTThe model diatomPhaeodactylum tricornutumis an attractive candidate for synthetic biology applications. Development of auxotrophic strains ofP. tricornutumwould provide alternative selective markers to commonly used antibiotic resistance genes. Here, using CRISPR/Cas9, we show successful editing of genes in the uracil, histidine, and tryptophan biosynthetic pathways. Editing events are characterized by loss of heterozygosity and by the occurrence of large deletions of up to ~2.7-kb centred on the editing site. The uracil and histidine-requiring phenotypes can be complemented by plasmid-based copies of the intact genes after curing of the Cas9-editing plasmid. Growth of uracil auxotrophs on media supplemented with 5-FOA and uracil results in loss of the complementing plasmid, providing a facile method for plasmid curing with potential applications in strain engineering and CRISPR editing. Metabolomic characterization of uracil auxotrophs revealed changes in cellular orotate concentrations consistent with partial or complete loss of orotate phosphoribosyl transferase activity in knockout strains. Our results expand the range ofP. tricornutumauxotrophic strains and demonstrate that auxotrophic complementation markers provide a viable alternative to traditionally used antibiotic selection markers. Plasmid-based auxotrophic markers should expand the range of genome engineering applications and provide a means for biocontainment of engineeredP. tricornutumstrains.
Deletion of immune evasion genes provides an effective vaccine design for tumor-associated herpesviruses
Authors: Brar Gurpreet , Farhat Nisar A. , Sukhina Alisa , Lam Alex K. , Kim Yong Hoon , Hsu Tiffany , Tong Leming , Lin Wai Wai , Ware Carl F. , Blackman Marcia A. , Sun Ren , Wu Ting-Ting 
Published: April 2020

AbstractVaccines based on live attenuated viruses often induce broad, multifaceted immune responses. However, they also usually sacrifice immunogenicity for attenuation. It is particularly difficult to elicit an effective vaccine for herpesviruses due to an armament of immune evasion genes and a latent phase. Here, to overcome the limitation of attenuation, we developed a rational herpesvirus vaccine in which viral immune evasion genes were deleted to enhance immunogenicity while also attaining safety. To test this vaccine strategy, we utilized murine gammaherpesvirus-68 (MHV-68) as a proof-of-concept model for the cancer-associated human γ-herpesviruses, Epstein-Barr virus and Kaposi sarcoma-associated herpesvirus. We engineered a recombinant MHV-68 virus by targeted inactivation of viral antagonists of type I interferon (IFN-I) pathway and deletion of the latency locus responsible for persistent infection. This recombinant virus is highly attenuated with no measurable capacity for replication, latency, or persistence in immunocompetent hosts. It stimulates robust innate immunity, differentiates virus-specific memory T cells, and elicits neutralizing antibodies. A single vaccination affords durable protection that blocks the establishment of latency following challenge with the wild type MHV-68 for at least six months post-vaccination. These results provide a novel approach to effective vaccination against cancer-associated herpesviruses through the elimination of latency and key immune evasion mechanisms from the pathogen.
Doxycycline Significantly Enhances Induction of iPSCs to Endoderm by Enhancing survival via AKT Phosphorylation
Authors: Esteva-Font Cristina , Su Tao , Peaslee Caitlin , Duwaerts Caroline , Liu Ke , Medina Marisa , Maher Jacquelyn J. , Mattis Aras N. 
Published: April 2020

AbstractInduced pluripotent stem cells (iPSCs) provide an important tool for the generation patient-derived cells including hepatocyte-like cells via developmental cues through an endoderm intermediate. However, most iPSCs fail to differentiate into endoderm, with induction resulting in apoptosis. To address this issue, we built upon published methods to develop an improved protocol with our discovery that doxycycline dramatically enhances the iPSC to endoderm differentiation efficiency by inhibiting apoptosis and promoting proliferation via the AKT pathway. We tested this new protocol in more than 70 iPSC lines with consistent formation of complete sheets of endoderm in 90%. Endoderm generated by our method achieves similar transcriptomic profiles, including FOXA2, HNF1β, CXCR4, and SOX17 positive cells, and the ability to be further differentiated. Furthermore this method achieves a four-fold increase in endoderm cell number and will accelerate studies of human diseasesin vitroand facilitate the expansion of iPSC-derived cells for transplantation studies.
Single nucleus sequencing fails to detect microglial activation in human tissue
Authors: Thrupp N. , Frigerio C. Sala , Wolfs L. , Skene N. G. , Poovathingal S. , Fourne Y. , Matthews P. M. , Theys T. , Mancuso R. , de Strooper B. , Fiers M. 
Published: April 2020

AbstractSingle nucleus RNA-Seq (snRNA-Seq) methods are used as an alternative to single cell RNA-Seq methods, as they allow transcriptomic profiling of frozen tissue. However, it is unclear whether snRNA-Seq is able to detect cellular state in human tissue. Indeed, snRNA-Seq analyses of human brain samples have failed to detect a consistent microglial activation signature in Alzheimer’s Disease. A comparison of microglia from single cells and single nuclei of four human subjects reveals that ~1% of genes is depleted in nuclei compared to whole cells. This small population contains 18% of genes previously implicated in microglial activation, includingAPOE, CST3, FTL, SPP1, andCD74. We confirm our findings across multiple previous single nucleus and single cell studies. Given the low sensitivity of snRNA-Seq to this population of activation genes, we conclude that snRNA-Seq is not suited to detecting cellular activation in microglia in human disease.
A truncated form of HpARI stabilises IL-33, amplifying responses to the cytokine
Authors: Chauché Caroline , Vacca Francesco , Chia Shin Li , Richards Josh , Gregory William F , Ogunkanbi Adefunke , Wear Martin , McSorley Henry J 
Published: April 2020

1AbstractThe murine intestinal nematodeHeligmosomoides polygyrusreleases theH. polygyrusAlarmin Release Inhibitor (HpARI) - a protein which binds to IL-33 and to DNA, effectively tethering the cytokine in the nucleus of necrotic cells. Previous work showed that a non-natural truncation consisting of the first 2 domains of HpARI (HpARI_CCP1/2) retains binding to both DNA and IL-33, and inhibited IL-33 releasein vivo. Here, we show that the affinity of HpARI_CCP1/2 for IL-33 is significantly lower than that of the full-length protein, and that HpARI_CCP1/2 lacks the ability to prevent interaction of IL-33 with its receptor. When HpARI_CCP1/2 was appliedin vivoit potently amplified IL-33-dependent immune responses toAlternaria alternataallergen,Nippostrongylus brasiliensisinfection and recombinant IL-33 injection. Mechanistically, we found that HpARI_CCP1/2 is able to bind to and stabilise IL-33, preventing its degradation and maintaining the cytokine in its active form. This study highlights the importance of IL-33 inactivation, the potential for IL-33 stabilisationin vivo, and describes a new tool for IL-33 research.
Systematic identification of a nuclear receptor-enriched predictive signature for erastin-induced ferroptosis
Authors: Kwon Ok-Seon , Kwon Eun-Ji , Kong Hyeon-Joon , Choi Jeong-Yoon , Kim Yun-Jeong , Lee Eun-Woo , Kim Wankyu , Lee Haeseung , Cha Hyuk-Jin 
Published: April 2020

AbstractErastin, which has been initially identified as a synthetic lethal compound against cancer expressing an RAS oncogene, inhibits cystine/glutamate antiporters and causes ferroptic cell death in various cell types, including therapy-resistant mesenchymal cancer cells. However, despite recent emerging evidence for the mechanisms underlying ferroptosis, molecular biomarkers associated with erastin-dependent ferroptosis have not yet been identified. In the present study, we employed isogenic lung cancer cell models with therapy-resistant mesenchymal properties to show that a redox imbalance leads to glutathione depletion and ferroptotic cell death. Subsequent gene expression analysis of pan-cancer cell lines revealed that the activity of transcription factors, including nuclear factor erythroid 2-related factor 2 (NRF2) and aryl hydrocarbon receptor (AhR), serve as important markers of erastin resistance. Based on the integrated expression of genes in the nuclear receptor meta-pathway (NRM), we constructed an NRM model and validated its robustness using an independent pharmacogenomics dataset. The NRM model was further evaluated by employing it in the sensitivity testing of nine cancer cell lines for which erastin sensitivities had not yet been undetermined. Our pharmacogenomics approach has the potential to pave the way for the efficient classification of patients for therapeutic intervention using erastin or erastin analogs.
CBL mutations promote activation of PI3K/AKT signaling via LYN kinase
Authors: Belizaire Roger , Koochaki Sebastian H.J. , Udeshi Namrata D. , Vedder Alexis , Sun Lei , Svinkina Tanya , Hartigan Christina , Stanclift Caroline , Schenone Monica , Carr Steven A. , Padron Eric , Ebert Benjamin L. 
Published: April 2020

AbstractCBLencodes an E3 ubiquitin ligase and signaling adaptor that acts downstream of cytokine receptors. RecurrentCBLmutations occur in myeloid malignancies, but the mechanism by which these mutations drive oncogenesis remains incompletely understood. Here we performed a series of studies to define the phosphoproteome, CBL interactome and molecular mechanisms of signaling activation in cells expressing an allelic series ofCBLmutants. Our analyses revealed that increased LYN activation and interaction with mutant CBL are key drivers of enhanced PIK3R1 recruitment and downstream PI3K/AKT signaling inCBL-mutant cells. Furthermore, we demonstratedin vitroandin vivoefficacy of LYN inhibition by dasatinib inCBL-mutant cell lines and primary chronic myelomonocytic leukemia cells. Overall, our data provide rationale for exploring the therapeutic potential of LYN inhibition in patients withCBL-mutated myeloid malignancies.Statement of SignificanceWe investigated the oncogenic mechanisms of myeloid malignancy-associatedCBLmutations by mass spectrometry-based proteomics and interactomics. Our findings indicate that increased LYN kinase activity inCBL-mutant cells stimulates PI3K/AKT signaling, revealing opportunities for the use of targeted inhibitors inCBL-mutated myeloid malignancies.
Bioinformatic characterization of angiotensin-converting enzyme 2, the entry receptor for SARS-CoV-2
Authors: Barker Harlan , Parkkila Seppo 
Published: April 2020

ABSTRACTThe World Health Organization declared the COVID-19 epidemic a public health emergency of international concern on March 11th, 2020, and the pandemic is rapidly spreading worldwide. COVID-19 is caused by a novel coronavirus SARS-CoV-2, which enters human target cells via angiotensin converting enzyme 2 (ACE2). We used a number of bioinformatics tools to computationally characterize ACE2 by determining its cell-specific expression in trachea, lung, and small intestine, derive its putative functions, and predict transcriptional regulation. The small intestine expressed higher levels of ACE2 than any other organ. The large intestine, kidney and testis showed moderate signals, whereas the signal was weak in the lung. Single cell RNA-Seq data from trachea indicated positive signals along the respiratory tract in key protective cell types including club, goblet, proliferating, and ciliary epithelial cells; while in lung the ratio of ACE2-expressing cells was low in all cell types (<2.6%), but was highest in vascular endothelial and goblet cells. Gene ontology analysis suggested that, besides its classical role in renin-angiotensin system, ACE2 may be functionally associated with angiogenesis/blood vessel morphogenesis. Using a novel tool for the prediction of transcription factor binding sites we identified several putative binding sites within two tissue-specific promoters of theACE2gene. Our results also confirmed that age and gender play no significant role in the regulation of ACE2 mRNA expression in the lung.IMPORTANCEVaccines and new medicines are urgently needed to prevent spread of COVID-19 pandemic, reduce the symptoms, shorten the duration of disease, prevent virus spread in the body, and most importantly to save lives. One of the key drug targets could be angiotensin-converting enzyme 2 (ACE2), which is a crucial receptor for the corona virus (SARS-CoV-2). It is known that SARS coronavirus infections lead to worse outcome in the elderly and in males. Therefore, one aim of the present study was to investigate whether age or sex could contribute to the regulation of ACE2 expression. We also decided to explore the transcriptional regulation ofACE2gene expression. Since data on ACE2 distribution is still conflicting, we aimed to get a more comprehensive view of the cell types expressing the receptor of SARS-CoV-2. Finally, we studied the coexpression of ACE2 with other genes and explored its putative functions using gene ontology enrichment analysis.
A high-throughput genomic screen identifies a role for the plasmid-borne Type II secretion system ofEscherichiacoli O157:H7 (Sakai) in plant-microbe interactions
Authors: Holmes Ashleigh , Pritchard Leighton , Hedley Peter , Morris Jenny , McAteer Sean P. , Gally David L. , Holden Nicola J. 
Published: April 2020

AbstractFood-borne illness arising from Shiga-toxigenicEscherichia coli(STEC) is often linked to consumption of fruit and vegetables as the bacteria have the ability to interact with plants and use them as alternative or secondary hosts. The initial stages of the interaction involve chemotaxis, attachment and potentially, responding to the early stages of microbe perception by the plant host. We used a high-throughput positive-selection approach to identify early interaction factors ofE. coliO157:H7 isolate Sakai to spinach. A bacterial artificial chromosome (BAC) clone library was quantified by microarray hybridisation, and gene loci enrichment measured using a Bayesian hierarchical model. The screen of four successive rounds of short-term (2 hour) interaction with spinach roots produced in 115 CDS credible candidates, comprising seven contiguous genomic regions. Two candidate regions were selected for functional assessment: a chaperone-usher fimbrial gene cluster (loc6) and the pO157 plasmid-encoded type two secretion system (T2SS). Interaction of bacteria with spinach tissue was reduced in the absence of the pO157 plasmid, which was appeared to involve the T2SS EtpD secretin protein, whereas loss ofloc6did not impact interactions. The T2SS genes,etpDandetpC, were expressed at a plant-relevant temperature of 18 °C, andetpDexpressedin plantabyE. coliSakai on spinach plants. Thus, a whole genome screening approach using a combination of computational modelling and functional assays has identified a novel function for STEC T2SS in interactions with plant tissue.
CNV-PG: a machine-learning framework for accurate copy number variation predicting and genotyping
Authors: Wang Taifu , Sun Jinghua , Zhang Xiuqing , Wang Wen-Jing , Zhou Qing 
Published: April 2020

AbstractMotivationCopy-number variants (CNVs) are one of the major causes of genetic disorders. However, current methods for CNV calling have high false-positive rates and low concordance, and a few of them can accurately genotype CNVs.ResultsHere we propose CNV-PG (CNV Predicting and Genotyping), a machine-learning framework for accurately predicting and genotyping CNVs from paired-end sequencing data. CNV-PG can efficiently remove false positive CNVs from existing CNV discovery algorithms, and integrate CNVs from multiple CNV callers into a unified call set with high genotyping accuracy.AvailabilityCNV-PG is available athttps://github.com/wonderful1/CNV-PG
Proteome analysis of human visceral and subcutaneous adipocytes identifies depot-specific heterogeneity at metabolic control points
Authors: Raajendiran Arthe , Krisp Christoph , Souza David P. De , Ooi Geraldine , Burton Paul R. , Taylor Renea A. , Molloy Mark P. , Watt Matthew J. 
Published: April 2020

AbstractAdipose tissue is a primary regulator of energy balance and metabolism. The distribution of adipose tissue depots is of clinical interest because the accumulation of upper-body subcutaneous (ASAT) and visceral adipose tissue (VAT) is associated with cardiometabolic diseases, whereas lower-body gluteal-femoral adipose tissue (GFAT) appears to be protective. There is heterogeneity in morphology and metabolism of adipocytes obtained from different regions of the body, but detailed knowledge of the constituent proteins in each depot is lacking. Here, we determined the human adipocyte proteome from ASAT, VAT and GFAT using high-resolution SWATH mass spectrometry proteomics. We quantified 4220 proteins in adipocytes, and 2329 proteins were expressed in all three adipose depots. Comparative analysis revealed significant differences between adipocytes from different regions (6 and 8% when comparing VATvs.ASAT and GFAT, 3% when comparing ASATvs.GFAT), with marked differences in proteins that regulate metabolic functions. The VAT adipocyte proteome was overrepresented with proteins of glycolysis, lipogenesis, oxidative stress and mitochondrial dysfunction. The GFAT adipocyte proteome predicted activation of PPARα, fatty acid and BCAA oxidation, enhanced TCA cycle flux and oxidative phosphorylation, which was supported by metabolomic data obtained from adipocytes from the same patient donors. Together, this proteomic analysis provides an important resource and novel insights that enhance the understanding of metabolic heterogeneity in the regional adipocytes of humans.
Whole genome comparisons of ergot fungi reveals the divergence and evolution of species within the genus Claviceps are the result of varying mechanisms driving genome evolution and host range expansion
Authors: Wyka Stephen A. , Mondo Stephen J. , Liu Miao , Dettman Jeremy , Nalam Vamsi , Broders Kirk D. 
Published: April 2020

AbstractThe genus Claviceps has been known for centuries as an economically important fungal genera for pharmacology and agricultural research. Only recently have researchers begun to unravel the evolutionary history of the genus, with origins in South America and classification of four distinct sections through ecological, morphological, and metabolic features (Claviceps sects. Citrinae, Paspalorum, Pusillae, and Claviceps). The first three sections are additionally characterized by narrow host range, while sect. Claviceps is considered evolutionarily more successful and adaptable as it has the largest host range and biogeographical distribution. However, the reasons for this success and adaptability remain unclear. Our study elucidates factors influencing adaptability by sequencing and annotating 50 Claviceps genomes, representing 21 species, for a comprehensive comparison of genome architecture and plasticity in relation to host range potential. Our results show the trajectory from specialized one-speed genomes (sects. Citrinae and Paspalorum) towards adaptive two-speed genomes (sects. Pusillae and Claviceps) through co-localization of transposable elements around predicted effectors and a putative loss of repeat-induced point mutation resulting in unconstrained tandem gene duplication coinciding with increased host range potential and speciation. Alterations of genomic architecture and plasticity can substantially influence and shape the evolutionary trajectory of fungal pathogens and their adaptability. Furthermore, our study provides a large increase in available genomic resources to propel future studies of Claviceps in pharmacology and agricultural research, as well as, research into deeper understanding of the evolution of adaptable plant pathogens.
ISG15 drives immune pathology and respiratory failure during viral infection
Authors: Shaabani Namir , Zak Jaroslav , Johnson Jennifer L. , Huang Zhe , Nguyen Nhan , Lazar Daniel C. , Vartabedian Vincent F. , Honke Nadine , Prinz Marco , Knobeloch Klaus-Peter , Arimoto Kei-ichiro , Zhang Dong-Er , Catz Sergio D. , Teijaro John R. 
Published: April 2020

AbstractCytokine storm during respiratory viral infection is an indicator of disease severity and poor prognosis. Type 1 interferon (IFN-I) production and signaling has been reported to be causal in cytokine storm-associated pathology in several respiratory viral infections, however, the mechanisms by which IFN-I promotes disease pathogenesis remain poorly understood. Here, usingUsp18-deficient, USP18 enzymatic-inactive andIsg15-deficient mouse models, we report that lack of deISGylation during persistent viral infection leads to severe immune pathology characterized by hematological disruptions, cytokine amplification, lung vascular leakage and death. This pathology requires T cells but not T cell-intrinsic deletion ofUsp18. However, lack ofUsp18in myeloid cells mimicked the pathological manifestations observed inUsp18-/-orUsp18C61Amice which were dependent onIsg15. We further mechanistically demonstrate that interrupting the ISGylation/deISGylation circuit increases extracellular levels of ISG15 which is accompanied by inflammatory neutrophil accumulation to the lung. Importantly, neutrophil depletion reversed morbidity and mortality inUsp18C61Amice. In summary, we reveal that the enzymatic function ofUsp18is crucial for regulating extracellular release of ISG15. This is accompanied by altered neutrophil differentiation, cytokine amplification and mortality following persistent viral infection. Moreover, our results suggest that extracellular ISG15 may drive the inflammatory pathology observed and could be both a prospective predictor of disease outcome and a therapeutic target during severe respiratory viral infections.
Investigate the relevance of major signaling pathways in cancer survival using a biologically meaningful deep learning model
Authors: Feng Jiarui , Zhang Heming , Li Fuhai 
Published: April 2020

AbstractSurvival analysis and prediction are important in cancer studies. In addition to the Cox proportional hazards model, recently deep learning models have been proposed to integrate the multi-omics data for survival prediction. Cancer signaling pathways are important and interpretable concepts that define the signaling cascades regulating cancer development and drug resistance. Thus, it is interesting and important to investigate the relevance to patients’ survival of individual signaling pathways. In this exploratory study, we propose to investigate the relevance and difference of a small set of core cancer signaling pathways in the survival analysis of cancer patients. Specifically, we built a biologically meaningful and simplified deep neural network, DeepSigSurvNet, for survival prediction. In the model, the gene expression and copy number data of 1648 genes from 46 major signaling pathways are used. We applied the model on 4 types of cancer and investigated the relevance and difference of the 46 signaling pathways among the 4 types of cancer. Interestingly, the interpretable analysis identified the distinct patterns of these signaling pathways, which are helpful to understand the relevance of the signaling pathways in terms of their association with cancer survival time. These highly relevant signaling pathways can be novel targets, combined with other essential signaling pathways inhibitors, for drug and drug combination prediction to improve cancer patients’ survival time.