The utility of cell-free nucleic acids in monitoring cancer has been recognized by both scientists and clinicians. In addition to human transcripts, a fraction of cell-free nucleic acids in human ...plasma were proven to be derived from microbes and reported to have relevance to cancer. To obtain a better understanding of plasma cell-free RNAs (cfRNAs) in cancer patients, we profiled cfRNAs in ~300 plasma samples of 5 cancer types (colorectal cancer, stomach cancer, liver cancer, lung cancer, and esophageal cancer) and healthy donors (HDs) with RNA-seq. Microbe-derived cfRNAs were consistently detected by different computational methods when potential contaminations were carefully filtered. Clinically relevant signals were identified from human and microbial reads, and enriched Kyoto Encyclopedia of Genes and Genomes pathways of downregulated human genes and higher prevalence torque teno viruses both suggest that a fraction of cancer patients were immunosuppressed. Our data support the diagnostic value of human and microbe-derived plasma cfRNAs for cancer detection, as an area under the ROC curve of approximately 0.9 for distinguishing cancer patients from HDs was achieved. Moreover, human and microbial cfRNAs both have cancer type specificity, and combining two types of features could distinguish tumors of five different primary locations with an average recall of 60.4%. Compared to using human features alone, adding microbial features improved the average recall by approximately 8%. In summary, this work provides evidence for the clinical relevance of human and microbe-derived plasma cfRNAs and their potential utilities in cancer detection as well as the determination of tumor sites.
Bacteria-based biotechnology processes are constantly under threat from bacteriophage infection, with phage contamination being a non-neglectable problem for microbial fermentation. The essence of ...this problem is the complex co-evolutionary relationship between phages and bacteria. The development of phage control strategies requires further knowledge about phage-host interactions, while the widespread use of
strain BL21 (DE3) in biotechnological processes makes the study of phage receptors in this strain particularly important. Here, eight phages infecting
BL21 (DE3) via different receptors were isolated and subsequently identified as members of the genera
,
,
,
, and
. Phage receptors were identified by whole-genome sequencing of phage-resistant
strains and sequence comparison with wild-type BL21 (DE3). Results showed that the receptors for the isolated phages, designated vB_EcoS_IME18, vB_EcoS_IME253, vB_EcoM_IME281, vB_EcoM_IME338, vB_EcoM_IME339, vB_EcoM_IME340, vB_EcoM_IME341, and vB_EcoS_IME347 were FhuA, FepA, OmpF, lipopolysaccharide, Tsx, OmpA, FadL, and YncD, respectively. A polyvalent phage-resistant BL21 (DE3)-derived strain, designated PR8, was then identified by screening with a phage cocktail consisting of the eight phages. Strain PR8 is resistant to 23 of 32 tested phages including
and
phages. Strains BL21 (DE3) and PR8 showed similar expression levels of enhanced green fluorescent protein. Thus, PR8 may be used as a phage resistant strain for fermentation processes. The findings of this study contribute significantly to our knowledge of phage-host interactions and may help prevent phage contamination in fermentation.
Preterm birth (PTB) is the main driver of newborn deaths. The identification of pregnancies at risk of PTB remains challenging, as the incomplete understanding of molecular mechanisms associated with ...PTB. Although several transcriptome studies have been done on the placenta and plasma from PTB women, a comprehensive description of the RNA profiles from plasma and placenta associated with PTB remains lacking.
Candidate markers with consistent trends in the placenta and plasma were identified by implementing differential expression analysis using placental tissue and maternal plasma RNA-seq datasets, and then validated by RT-qPCR in an independent cohort. In combination with bioinformatics analysis tools, we set up two protein-protein interaction networks of the significant PTB-related modules. The support vector machine (SVM) model was used to verify the prediction potential of cell free RNAs (cfRNAs) in plasma for PTB and late PTB.
We identified 15 genes with consistent regulatory trends in placenta and plasma of PTB while the full term birth (FTB) acts as a control. Subsequently, we verified seven cfRNAs in an independent cohort by RT-qPCR in maternal plasma. The cfRNA ARHGEF28 showed consistence in the experimental validation and performed excellently in prediction of PTB in the model. The AUC achieved 0.990 for whole PTB and 0.986 for late PTB.
In a comparison of PTB versus FTB, the combined investigation of placental and plasma RNA profiles has shown a further understanding of the mechanism of PTB. Then, the cfRNA identified has the capacity of predicting whole PTB and late PTB.
Background
Cell‐free long RNAs in human plasma and extracellular vesicles (EVs) have shown promise as biomarkers in liquid biopsy, despite their fragmented nature.
Methods
To investigate these ...fragmented cell‐free RNAs (cfRNAs), we developed a cost‐effective cfRNA sequencing method called DETECTOR‐seq (depletion‐assisted multiplexed cell‐free total RNA sequencing). DETECTOR‐seq utilised a meticulously tailored set of customised guide RNAs to remove large amounts of unwanted RNAs (i.e., fragmented ribosomal and mitochondrial RNAs) in human plasma. Early barcoding strategy was implemented to reduce costs and minimise plasma requirements.
Results
Using DETECTOR‐seq, we conducted a comprehensive analysis of cell‐free transcriptomes in both whole human plasma and EVs. Our analysis revealed discernible distributions of RNA types in plasma and EVs. Plasma exhibited pronounced enrichment in structured circular RNAs, tRNAs, Y RNAs and viral RNAs, while EVs showed enrichment in messenger RNAs (mRNAs) and signal recognition particle RNAs (srpRNAs). Functional pathway analysis highlighted RNA splicing‐related ribonucleoproteins (RNPs) and antimicrobial humoral response genes in plasma, while EVs demonstrated enrichment in transcriptional activity, cell migration and antigen receptor‐mediated immune signals. Our study indicates the comparable potential of cfRNAs from whole plasma and EVs in distinguishing cancer patients (i.e., colorectal and lung cancer) from healthy donors. And microbial cfRNAs in plasma showed potential in classifying specific cancer types.
Conclusions
Our comprehensive analysis of total and EV cfRNAs in paired plasma samples provides valuable insights for determining the need for EV purification in cfRNA‐based studies. We envision the cost effectiveness and efficiency of DETECTOR‐seq will empower transcriptome‐wide investigations in the fields of cfRNAs and liquid biopsy.
Keypoints
DETECTOR‐seq (depletion‐assisted multiplexed cell‐free total RNA sequencing) enabled efficient and specific depletion of sequences derived from fragmented ribosomal and mitochondrial RNAs in plasma.
Distinct human and microbial cell‐free RNA (cfRNA) signatures in whole Plasma versus extracellular vesicles (EVs) were revealed.
Both Plasma and EV cfRNAs were capable of distinguishing cancer patients from normal individuals, while microbial RNAs in Plasma cfRNAs enabled better classification of cancer types than EV cfRNAs.
DETECTOR‐seq (depletion‐assisted multiplexed cell‐free total RNA sequencing) enabled efficient and specific depletion of sequences derived from fragmented ribosomal and mitochondrial RNAs in plasma.
Distinct human and microbial cell‐free RNA (cfRNA) signatures in whole Plasma versus extracellular vesicles (EVs) were revealed.
Both Plasma and EV cfRNAs were capable of distinguishing cancer patients from normal individuals, while microbial RNAs in Plasma cfRNAs enabled better classification of cancer types than EV cfRNAs.
is the most common clinically important opportunistic bacterial pathogen and its infection is often iatrogenic. Its drug resistance poses a grave threat to public health. The genomic data reported ...here comprise an important resource for research on phage therapy in the control of drug-resistant bacteria.
We report here the whole-genome sequence of a new Enterococcus faecalis phage, vB_EfaS_IME197, which has a linear double-stranded DNA genome of 41,307 bp with 34% G+C content. We describe the main ...features of the genome of vB_EfaS_IME197.
As an opportunist pathogen,
Vibrio alginolyticus
(
V. alginolyticus
), causes disease in marine animals. Bacterial contamination of seafood is not uncommon, and phage therapy is considered a safe way ...to decontaminate such foods to control the emergence of vibriosis. Here, we report on the isolation of a new, virulent phage called vB_ValP_IME271 (designated phage IME271), which infects
V. alginolyticus
and was isolated from seawater. Phage IME271 displayed good pH (7–9) and temperature tolerance (< 40 °C) and had a broad host range against Vibrio isolates, including 7 strains of
V. alginolyticus
and11 strains of
V. parahaemolyticus
. The IME271 genome was sequenced and annotated, the results of which showed that this phage is a
Podoviridae
family member with a genome length of 50,345 base pairs. The complete genome is double-stranded DNA with a G+C content of 41.4%. Encoded within the genome are 67 putative proteins, of which only 22 coding sequences have known functions, and no tRNAs are present. The BLASTn results for IME271 showed that it only shares similarity with the Vibrio phage VPp1 (sequence identity score of 96% over 87% of the genome) whose host is
V. parahaemolyticus
. Comparative analysis showed that IME271 and VPp1 share a similar genomic structure, and the structural proteins are highly similar (> 95% similarity score). In summary, our work identified a new lytic
Podoviridae
bacteriophage, which is infective to
V. alginolytic
us and
V. parahaemolyticus
. This bacteriophage could potentially be used to control
V. alginolyticus
and
V. parahaemolyticus
infections in marine animals.
Enterococcus faecalis
is one of the main bacteria in the human and animal intestine but is also classed as an opportunistic pathogen. During normal growth,
E. faecalis
produces natural antibiotics ...and is conducive to human health. As ectopic parasites,
E. faecalis
is capable of causing infective endocarditis, neonatal sepsis, bloodstream infections, bacteremia, and intraabdominal infections. With the incidence of antibiotic resistance reaching crisis point, it is imperative to find alternative treatments for multidrug-resistant infections. Using phage for pathogen control is a promising treatment option to combat bacterial resistance. In this study, a lytic phage, designated vB_EfaP_IME195, was isolated from hospital sewage using a clinical multidrug-resistant
Enterococcus faecalis
strain as an indicator. The one-step growth curve with the optimal multiplicity of infection of (MOI) 0.01 revealed a latent period of ~ 30 min and a burst size of ~ 120 plaque-forming units (pfu) per cell. Transmission electron microscopy showed that the phage belongs to the family
Podoviridae
. Phage vB_EfaP_IME195 has a linear, double-stranded DNA genome of 18,607 bp with a G + C content of 33% and 27 coding sequences (GenBank accession no. KT932700). Run-off sequencing experiments showed that the phage has a unique 59-bp inverted repeat sequences at the terminal ends. BLASTn analysis revealed that vB_EfaP_IME195 shares 92% identity (93% genome coverage) with unpublished
E. faecalis
phage Idefix. This study reported a novel
E. faecalis
phage with unique genome termini containing inverted repeats. The isolation and characterization of this novel lytic
E. faecalis
phage provides the basis for the development of new therapeutic agents like phage cocktails for multidrug-resistant
E. faecalis
infection, and its unique genomic feature would also provide valuable knowledge and insight for further phage genome analysis.
Long extracellular RNAs (exRNAs) in plasma can be profiled by new sequencing technologies, even with low abundance. However, cancer-related exRNAs and their variations remain understudied.
We ...investigated different variations (i.e. differential expression, alternative splicing, alternative polyadenylation, and differential editing) in diverse long exRNA species (e.g. long noncoding RNAs and circular RNAs) using 79 plasma exosomal RNA-seq (exoRNA-seq) datasets of multiple cancer types. We then integrated 53 exoRNA-seq datasets and 65 self-profiled cell-free RNA-seq (cfRNA-seq) datasets to identify recurrent variations in liver cancer patients. We further combined TCGA tissue RNA-seq datasets and validated biomarker candidates by RT-qPCR in an individual cohort of more than 100 plasma samples. Finally, we used machine learning models to identify a signature of 3 noncoding RNAs for the detection of liver cancer.
We found that different types of RNA variations identified from exoRNA-seq data were enriched in pathways related to tumorigenesis and metastasis, immune, and metabolism, suggesting that cancer signals can be detected from long exRNAs. Subsequently, we identified more than 100 recurrent variations in plasma from liver cancer patients by integrating exoRNA-seq and cfRNA-seq datasets. From these datasets, 5 significantly up-regulated long exRNAs were confirmed by TCGA data and validated by RT-qPCR in an independent cohort. When using machine learning models to combine two of these validated circular and structured RNAs (
) with a miRNA (
) as a panel to classify liver cancer patients from healthy donors, the average AUROC of the cross-validation was 89.4%. The selected 3-RNA panel successfully detected 79.2% AFP-negative samples and 77.1% early-stage liver cancer samples in the testing and validation sets.
Our study revealed that different types of RNA variations related to cancer can be detected in plasma and identified a 3-RNA detection panel for liver cancer, especially for AFP-negative and early-stage patients.
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