Metatranscriptomic Characterization of Coronavirus Disease 2019 Identified a Host Transcriptional Classifier Associated With Immune Signaling.

BACKGROUND: The recent identification of a novel coronavirus, also known as severe acute respiratory syndrome coronavirus 2, has caused a global outbreak of respiratory illnesses. The rapidly developing pandemic has posed great challenges to diagnosis of this novel infection. However, little is known about the metatranscriptomic characteristics of patients with coronavirus disease 2019 (COVID-19). METHODS: We analyzed metatranscriptomics in 187 patients (62 cases with COVID-19 and 125 with non-COVID-19 pneumonia). Transcriptional aspects of 3 core elements, pathogens, the microbiome, and host responses, were evaluated. Based on the host transcriptional signature, we built a host gene classifier and examined its potential for diagnosing COVID-19 and indicating disease severity. RESULTS: The airway microbiome in COVID-19 patients had reduced alpha diversity, with 18 taxa of differential abundance. Potentially pathogenic microbes were also detected in 47% of the COVID-19 cases, 58% of which were respiratory viruses. Host gene analysis revealed a transcriptional signature of 36 differentially expressed genes significantly associated with immune pathways, such as cytokine signaling. The host gene classifier built on such a signature exhibited the potential for diagnosing COVID-19 (area under the curve of 0.75-0.89) and indicating disease severity. CONCLUSIONS: Compared with those with non-COVID-19 pneumonias, COVID-19 patients appeared to have a more disrupted airway microbiome with frequent potential concurrent infections and a special trigger host immune response in certain pathways, such as interferon-gamma signaling. The immune-associated host transcriptional signatures of COVID-19 hold promise as a tool for improving COVID-19 diagnosis and indicating disease severity.

Clinical metagenomics assessments improve diagnosis and outcomes in community-acquired pneumonia.

BACKGROUND: Identifying the causes of community-acquired pneumonia (CAP) is challenging due to the disease's complex etiology and the limitations of traditional microbiological diagnostic methods. Recent advances in next generation sequencing (NGS)-based metagenomics allow pan-pathogen detection in a single assay, and may have significant advantages over culture-based techniques. RESULTS: We conducted a cohort study of 159 CAP patients to assess the diagnostic performance of a clinical metagenomics assay and its impact on clinical management and patient outcomes. When compared to other techniques, clinical metagenomics detected more pathogens in more CAP cases, and identified a substantial number of polymicrobial infections. Moreover, metagenomics results led to changes in or confirmation of clinical management in 35 of 59 cases; these 35 cases also had significantly improved patient outcomes. CONCLUSIONS: Clinical metagenomics could be a valuable tool for the diagnosis and treatment of CAP. TRIAL REGISTRATION: Trial registration number with the Chinese Clinical Trial Registry: ChiCTR2100043628 .

Identification, expression, and innate immune responses of two insulin-like peptide genes in the razor clam Sinonovacula constricta.

Insulin-like peptide (ILP) has emerged as a cell regulatory factor with multiple functions in vertebrates and invertebrates. In the present study, we identified and characterized two ILP genes, ILP1 and ILP2, in the razor clam Sinonovacula constricta. Both ILPs have a signal peptide and a mature domain consisting of six strictly conserved cysteines. The tertiary structure is divided into three main α-helices with a C-domain loop that separates helix 1 from helix 2. Both of ILPs were found to be regulated according to tissue type and developmental stage. After challenge with Vibrio anguillarum, Vibrio parahaemolyticus and Micrococcus lysodeikticus, the expression of two ILP genes was significantly up-regulated in the liver, hemocytes and mantle tissues, suggesting that the ILPs may play roles in the innate immunity in the razor clam Sinonovacula constricta.

A novel NIR fluorescent probe for copper(ii) imaging in Parkinson's disease mouse brain.

Abnormal copper ion (Cu2+) levels are considered to be one of the pathological factors of Parkinson's disease (PD), but the internal relationship between Cu2+ and PD progression remains elusive. Visualizing Cu2+ in the brain will be pivotal for comprehending the underlying pathophysiological processes of PD. In this work, a near-infrared (NIR) fluorescent probe, DDAO-Cu, capable of detecting Cu2+ with exceptional sensitivity (about 1.8 nM of detection limit) and selectivity, rapid response (<3 min), and deep tissue penetration, was designed for quantification and visualization of the Cu2+ level. It could detect not only Cu2+ in cells but also the changes in the Cu2+ level in the rotenone-induced cell and zebrafish PD models. Moreover, DDAO-Cu can cross the blood-brain barrier to image Cu2+ in the brain of PD model mice. The imaging result showed a significant increase in Cu2+ levels in brain regions of PD model mice, including the cerebral cortex, hippocampus, and striatum. Meanwhile, Cu2+ levels in the substantia nigra region were significantly reduced in PD model mice. It revealed the nuanced relationship of Cu2+ levels in different brain regions in the disease and indicated the pathological complexity of PD. Overall, DDAO-Cu represents a novel and practical tool for investigating Cu2+-related physiological and pathological processes underlying Parkinson's disease.

Multicenter assessment of shotgun metagenomics for pathogen detection.

BACKGROUND: Shotgun metagenomics has been used clinically for diagnosing infectious diseases. However, most technical assessments have been limited to individual sets of reference standards, experimental workflows, and laboratories. METHODS: A reference panel and performance metrics were designed and used to examine the performance of shotgun metagenomics at 17 laboratories in a coordinated collaborative study. We comprehensively assessed the reliability, key performance determinants, reproducibility, and quantitative potential. FINDINGS: Assay performance varied significantly across sites and microbial classes, with a read depth of 20 millions as a generally cost-efficient assay setting. Results of mapped reads by shotgun metagenomics could indicate relative and intra-site (but not absolute or inter-site) microbial abundance. INTERPRETATION: Assay performance was significantly impacted by the microbial type, the host context, and read depth, which emphasizes the importance of these factors when designing reference reagents and benchmarking studies. Across sites, workflows and platforms, false positive reporting and considerable site/library effects were common challenges to the assay's accuracy and quantifiability. Our study also suggested that laboratory-developed shotgun metagenomics tests for pathogen detection should aim to detect microbes at 500 CFU/mL (or copies/mL) in a clinically relevant host context (10^5 human cells/mL) within a 24h turn-around time, and with an efficient read depth of 20M. FUNDING: This work was supported by National Science and Technology Major Project of China (2018ZX10102001).

Integrated mRNA and Small RNA Sequencing Reveals Regulatory Expression of Larval Metamorphosis of the Razor Clam.

The razor clam, Sinonovacula constricta, is an important economic marine shellfish, and its larval development involves obvious morphological and physiological changes. MicroRNA plays a key role in the physiological changes of the organism through regulating targeted mRNA. This study performed miRNA-mRNA sequencing for eight different developmental stages of S. constricta using Illumina sequencing. A total of 2156 miRNAs were obtained, including 2069 known miRNAs and 87 novel miRNAs. In addition, target genes were predicted for key miRNAs differentially expressed between adjacent development samples by integrating the mRNA transcriptome. Further analysis revealed that the differentially expressed genes were enriched in complement activation, alternative pathways, translation, and negative regulation of monocyte molecular protein-1 production. KEGG pathway annotation showed significant enrichment in the regulation of the ribosome, phagosome, tuberculosis and fluid shear stress, and atherosclerosis. Ten mRNAs and ten miRNAs that are related to larval metamorphosis were identified using real-time PCR. Furthermore, the double luciferase experiment validated the negative regulatory relationship between miR-133 and peroxisome proliferator-activated receptor-γ (PPAR-γ). These results indicated that the target genes regulated by these differentially expressed miRNAs may play an important regulatory role in the metamorphosis development of S. constricta.

Construction of the First High-Density Genetic Linkage Map and Analysis of Quantitative Trait Loci for Growth-Related Traits in Sinonovacula constricta.

The razor clam (Sinonovacula constricta) is an important aquaculture species, for which a high-density genetic linkage map would play an important role in marker-assisted selection (MAS). In this study, we constructed a high-density genetic map and detected quantitative trait loci (QTLs) for Sinonovacula constricta with an F1 cross population by using the specific locus amplified fragment sequencing (SLAF-seq) method. A total of 315,553 SLAF markers out of 467.71 Mreads were developed. The final linkage map was composed of 7516 SLAFs (156.60-fold in the parents and 20.80-fold in each F1 population on average). The total distance of the linkage map was 2383.85 cM, covering 19 linkage groups with an average inter-marker distance of 0.32 cM. The proportion of gaps less than 5.0 cM was on average 96.90%. A total of 16 suggestive QTLs for five growth-related traits (five QTLs for shell height, six QTLs for shell length, three QTLs for shell width, one QTL for total body weight, and one QTL for soft body weight) were identified. These QTLs were distributed on five linkage groups, and the regions showed overlapping on LG9 and LG13. In conclusion, the high-density genetic map and QTLs for S. constricta provide a valuable genetic resource and a basis for MAS.

Developmental Transcriptome Analysis and Identification of Genes Involved in Larval Metamorphosis of the Razor Clam, Sinonovacula constricta.

The razor clam Sinonovacula constricta is an important commercial species. The deficiency of developmental transcriptomic data is becoming the bottleneck of further researches on the mechanisms underlying settlement and metamorphosis in early development. In this study, de novo transcriptome sequencing was performed for S. constricta at different early developmental stages by using Illumina HiSeq 2000 paired-end (PE) sequencing technology. A total of 112,209,077 PE clean reads were generated. De novo assembly generated 249,795 contigs with an average length of 585 bp. Gene annotation resulted in the identification of 22,870 unigene hits against the NCBI database. Eight unique sequences related to metamorphosis were identified and analyzed using real-time PCR. The razor clam reference transcriptome would provide useful information on early developmental and metamorphosis mechanisms and could be used in the genetic breeding of shellfish.

Identification and expression characterization of the myostatin (MSTN) gene and association analysis with growth traits in the razor clam Sinonovacula constricta.

Myostatin (MSTN) is a member of the transforming growth factor-ß superfamily (TGF-ß) and is an important negative regulator of muscle growth in vertebrates. In this study, we cloned and analyzed the MSTN gene (Sc-MSTN) from razor clam (Sinonovacula constricta). The full length of Sc-MSTN cDNA sequence consists of 4226 base pairs (bp), comprising a 522-bp 5' untranslated region (UTR), a 2342-bp 3'UTR, and an open reading frame (ORF) that is 1362 in length. The ORF encodes 453 amino acids with a RXXR proteolytic site and nine conserved cysteines. Quantitative real-time PCR analysis revealed that the Sc-MSTN transcript was expressed in a wide range of tissues but appeared to exhibit the greatest level of expression in the foot. The transcript was widely detected in early developmental stages, showing the highest expression in the trochophore stage. Furthermore, six SNPs were identified in the coding region of the Sc-MSTN gene using direct sequencing. SNP-1 is non-synonymous and involves an amino acid change from Leu to Ser. Association analysis showed that SNP-1 and SNP-6 had significant influences on shell length (SL). The results suggested that MSTN could be selected as a candidate gene for the future molecular breeding of razor clam strains.

Identification, expression, and responses to bacterial challenge of the cathepsin C gene from the razor clam Sinonovacula constricta.

Cathepsin C (dipeptidyl-peptidaseI, DPPI) is a lysosomal cysteine proteinase that belongs to the papain superfamily, and it is involved in protein degradation and proenzyme activation. However, very little is known about the function of cathepsin C in bivalves. In the present study, we identified the cathepsin C gene in the razor clam Sinonovacula constricta (Sc-CTSC). The full-length Sc-CTSC cDNA contained a complete open reading frame (ORF) of 1371 nt encoding 456 amino acids, a 98 bp 5' UTR, and a 1043 bp 3' UTR. The ORF of Sc-CTSC consisted of a putative signal peptide of 22 aa, a propeptide of 229 aa, and a mature peptide of 205 aa containing the active site triad of Cys, His, and Asn. The Sc-CTSC transcript was expressed in a wide range of tissues but exhibited the greatest level of expression in the digestive gland. During the early developmental stages, the transcript was detected widely. Upon injection with Vibrio anguillarum, the Sc-CTSC transcript was significantly up-regulated in digestive gland, mantle, and gill tissues. The results provided important information for further exploring the roles of cathepsin C in the innate immune responses.