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RNA based mNGS approach identifies a novel human coronavirus from two individual pneumonia cases in 2019 Wuhan outbreak
1 December, 2020
From December 2019, an outbreak of unusual pneumonia was reported in Wuhan with many cases linked to Huanan Seafood Market that sells seafood as well as live exotic animals. We investigated two patients who developed acute respiratory syndromes after independent contact history with this market. The two patients shared common clinical features including fever, cough, and multiple ground-glass opacities in the bilateral lung field with patchy infiltration. Here, we highlight the use of a low-input metagenomic next-generation sequencing (mNGS) approach on RNA extracted from bronchoalveolar lavage fluid (BALF). It rapidly identified a novel coronavirus (named 2019-nCoV according to World Health Organization announcement) which was the sole pathogens in the sample with very high abundance level (1.5% and 0.62% of total RNA sequenced). The entire viral genome is 29,881 nt in length (GenBank MN988668 and MN988669, Sequence Read Archive database Bioproject accession PRJNA601736) and is classified into β-coronavirus genus. Phylogenetic analysis indicates that 2019-nCoV is close to coronaviruses (CoVs) circulating in Rhinolophus (Horseshoe bats), such as 98.7% nucleotide identity to partial RdRp gene of bat coronavirus strain BtCoV/4991 (GenBank KP876546, 370 nt sequence of RdRp and lack of other genome sequence) and 87.9% nucleotide identity to bat coronavirus strain bat-SL-CoVZC45 and bat-SL-CoVZXC21. Evolutionary analysis based on ORF1a/1b, S, and N genes also suggests 2019-nCoV is more likely a novel CoV independently introduced from animals to humans.
Pages: 313-319 doi: 10.1080/22221751.2020.1725399
Characterization of the phenotypic and genotypic tolerance to abiotic stresses of natural populations of Heterorhabditis bacteriophora
1 December, 2020
Differential expression analysis was completed using the DESeq. 2 R package. Genes that varied from the control more than twofold, with an adjusted P-value of no more than 0.05, were considered differentially expressed. Venn diagrams were calculated using “Venny” tool57. The transcriptome was used for a search of the NCBI non-redundant (nr) protein database, employing the DIAMOND program58. The results were exported to Blast2GO version 4.059 for gene ontology (GO) assignments. The eggNOG-mapper v2 tool) http://eggnog-mapper.embl.de/ (was used for functional annotation. The KAAS tool (Kegg Automatic Annotation Server; http://www.genome.jp/tools/kaas/) was used for KEGG ontology and KEGG pathway assignments. Gene ontology enrichment analyses was carried out using Blast2GO59 software based on Fisher’s Exact Test60. The ReviGO web server was used for visualization of the GO terms in a semantic similarity-based scatterplot [http://revigo.irb.hr]61. Genome sequencing Isolates of the specie H. bacteriophora with varying degrees of tolerance to each of the stress conditions were chosen for genome sequencing in order to search for polymorphism in the genome in relation to the tolerance capabilities. Two-hundred μl (~50 ng/μl) of genomic DNA of each isolate were prepared and sent for sequencing. Macrogen Inc. (South Korea) performed the genome sequencing using TruSeq DNA PCR free for library construction and NovaSeq. 6000 for the sequencing. SNPs calling on genomic sequences Homogenous inbreed line of EN-01, IL-3, was used as a reference genome in the present study (Unpublished genome). Reads were aligned to IL-3 genome using Tophat2 software (v2.1) using default parameters. Gene abundance estimation was performed using Cufflinks suite (v2.2) by the Cuffquant combined with gene annotations from previous study 57, and then Cuffnorm was used for the normalization. Paired-end reads of the 3 samples were mapped to the reference genome (IL-3) using the BWA mem program with default parameters62. The resulting mapping file was processed using Picard tool (http://broadinstitute.github.io/picard/; version 1.95) for adding read group information, sorting, marking duplicates, and indexing. Then, the local re-alignment process for locally re-aligning reads such that the number of mismatching bases is minimized across all the reads was performed using the Realigner Target Creator of the Genome Analysis Toolkit version 3.4–0 [GATK; version http://www.broadinstitute.org/gatk/]63. Finally, the variant calling procedure was performed using Haplotype Caller of the GATK toolkit, for the detection of SNPs between the variants and the reference IL-3 genome. Only homozygous SNPs were further analyzed. An in-house Perl script was used to define SNPs in genes or upstream/downstream to genes (1000 bp) and the define stop codon or non-synonymous substitutions. Statistical analysis All the statistical analysis was done using JMP, Version 14. SAS Institute Inc., Cary, NC, 1989–2019. Results are presented as mean ± SE of replicate analysis and are either representative of or include at least three independent experiments. Means of replicates were subjected to statistical analysis and considered significant when P
doi: 10.1038/s41598-020-67097-0
Distinct fibroblast functional states drive clinical outcomes in ovarian cancer and are regulated by TCF21
3 August, 2020
Recent studies indicate that cancer-associated fibroblasts (CAFs) are phenotypically and functionally heterogeneous. However, little is known about CAF subtypes, the roles they play in cancer progression, and molecular mediators of the CAF "state." Here, we identify a novel cell surface pan-CAF marker, CD49e, and demonstrate that two distinct CAF states, distinguished by expression of fibroblast activation protein (FAP), coexist within the CD49e+ CAF compartment in high-grade serous ovarian cancers. We show for the first time that CAF state influences patient outcomes and that this is mediated by the ability of FAP-high, but not FAP-low, CAFs to aggressively promote proliferation, invasion and therapy resistance of cancer cells. Overexpression of the FAP-low-specific transcription factor TCF21 in FAP-high CAFs decreases their ability to promote invasion, chemoresistance, and in vivo tumor growth, indicating that it acts as a master regulator of the CAF state. Understanding CAF states in more detail could lead to better patient stratification and novel therapeutic strategies.
doi: 10.1084/jem.20191094
Textured nanofibrils drive microglial phenotype
1 July, 2020
Microglia are highly plastic cells that change their properties in response to their microenvironment. By using immunofluorescence, live-cell imaging, electrophysiological recordings and RNA sequencing, we investigated the regulation of modified bacterial cellulose (mBC) nanofibril substrates on microglial properties. We demonstrate that mBC substrates induces ramified microglia with constantly extending and retracting processes, reminiscent of what is observed in vivo. Patch-clamp recordings showed that microglia acquire a more negative resting membrane potential and have increased inward rectifier K+ currents, caused by an upregulation of Kir2.1 channels. Transcriptome analysis shows upregulation of genes involved in the immune response and downregulation of genes linked to cell adhesion and cell motion. Furthermore, Arp2/3 complex activation and integrin-mediated signaling modulate microglial morphology and motility. Our studies demonstrate that mBC nanofibril substrates modulate microglial phenotype, paving the way for a microglia-material interface that may be very valuable for anti-neuroinflammatory drug screening.
Pages: 120177 doi: 10.1016/j.biomaterials.2020.120177
The PD-1 Pathway Regulates Development and Function of Memory CD8+ T Cells following Respiratory Viral Infection
30 June, 2020
The PD-1 pathway regulates dysfunctional T cells in chronic infection and cancer, but the role of this pathway during acute infection remains less clear. Here, we demonstrate that PD-1 signals are needed for optimal memory. Mice deficient in the PD-1 pathway exhibit impaired CD8+ T cell memory following acute influenza infection, including reduced virus-specific CD8+ T cell numbers and compromised recall responses. PD-1 blockade during priming leads to similar differences early post-infection but without the defect in memory formation, suggesting that timing and/or duration of PD-1 blockade could be tailored to modulate host responses. Our studies reveal a role for PD-1 as an integrator of CD8+ T cell signals that promotes CD8+ T cell memory formation and suggest PD-1 continues to fine-tune CD8+ T cells after they migrate into non-lymphoid tissues. These findings have important implications for PD-1-based immunotherapy, in which PD-1 inhibition may influence memory responses in patients.
Pages: 107827 doi: 10.1016/j.celrep.2020.107827