Depletion-assisted multiplexed cell-free RNA sequencing reveals distinct human and microbial signatures in plasma versus extracellular vesicles.

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.

Circulating small extracellular vesicle-based miRNA classifier for follicular thyroid carcinoma: a diagnostic study.

BACKGROUND: The diagnosis of follicular thyroid carcinoma (FTC) prior to surgery remains a major challenge in the clinic. METHODS: This multicentre diagnostic study involved 41 and 150 age- and sex-matched patients in the training cohort and validation cohort, respectively. The diagnostic properties of circulating small extracellular vesicle (sEV)-associated and cell-free RNAs were compared by RNA sequencing in the training cohort. Subsequently, using a quantitative real-time polymerase chain reaction (qRT‒PCR) assay, high-quality candidates were identified to construct an RNA classifier for FTC and verified in the validation cohort. The parallel expression, stability and influence of the RNA classifier on surgical strategy were also investigated. RESULTS: The diagnostic properties of sEV long RNAs, cell-free long RNAs and sEV microRNAs (miRNAs) were comparable and superior to those of cell-free miRNAs in RNA sequencing. Given the clinical application, the circulating sEV miRNA (CirsEV-miR) classifier was developed from five miRNAs based on qRT‒PCR data, which could well identify FTC patients (area under curve [AUC] of 0.924 in the training cohort and 0.844 in the multicentre validation cohort). Further tests revealed that the CirsEV-miR score was significantly correlated with the tumour burden, and the levels of sEV miRNAs were also higher in sEVs from the FTC cell line, organoid and tissue. Additionally, circulating sEV miRNAs remained constant after different treatments, and the addition of the CirsEV-miR classifier as a biomarker improves the current surgical strategy. CONCLUSIONS: The CirsEV-miR classifier could serve as a noninvasive, convenient, specific and stable auxiliary test to help diagnose FTC following ultrasonography.

cfOmics: a cell-free multi-Omics database for diseases.

Liquid biopsy has emerged as a promising non-invasive approach for detecting, monitoring diseases, and predicting their recurrence. However, the effective utilization of liquid biopsy data to identify reliable biomarkers for various cancers and other diseases requires further exploration. Here, we present cfOmics, a web-accessible database (https://cfomics.ncRNAlab.org/) that integrates comprehensive multi-omics liquid biopsy data, including cfDNA, cfRNA based on next-generation sequencing, and proteome, metabolome based on mass-spectrometry data. As the first multi-omics database in the field, cfOmics encompasses a total of 17 distinct data types and 13 specimen variations across 69 disease conditions, with a collection of 11345 samples. Moreover, cfOmics includes reported potential biomarkers for reference. To facilitate effective analysis and visualization of multi-omics data, cfOmics offers powerful functionalities to its users. These functionalities include browsing, profile visualization, the Integrative Genomic Viewer, and correlation analysis, all centered around genes, microbes, or end-motifs. The primary objective of cfOmics is to assist researchers in the field of liquid biopsy by providing comprehensive multi-omics data. This enables them to explore cell-free data and extract profound insights that can significantly impact disease diagnosis, treatment monitoring, and management.

Cell-free RNA for the liquid biopsy of gastrointestinal cancer.

Gastrointestinal (GI) cancer includes many cancer types, such as esophageal, liver, gastric, pancreatic, and colorectal cancer. As the cornerstone of personalized medicine for GI cancer, liquid biopsy based on noninvasive biomarkers provides promising opportunities for early diagnosis and dynamic treatment management. Recently, a growing number of studies have demonstrated the potential of cell-free RNA (cfRNA) as a new type of noninvasive biomarker in body fluids, such as blood, saliva, and urine. Meanwhile, transcriptomes based on high-throughput RNA detection technologies keep discovering new cfRNA biomarkers. In this review, we introduce the origins and applications of cfRNA, describe its detection and qualification methods in liquid biopsy, and summarize a comprehensive list of cfRNA biomarkers in different GI cancer types. Moreover, we also discuss perspective studies of cfRNA to overcome its current limitations in clinical applications. This article is categorized under: RNA in Disease and Development > RNA in Disease.

Vortioxetine induces apoptosis and autophagy of gastric cancer AGS cells via the PI3K/AKT pathway.

Vortioxetine is a potent antagonist of the 5-hydroxytryptamine receptor and serotonin transporter and has been reported to function as an antidepressant in the treatment of major depressive disorder. However, its antitumor effects remain unclear. Here, we examined whether vortioxetine affects the characteristics of GC cells. Cell viability was measured by a colony formation assay and, in addition, cell invasion, migration and apoptosis assays were performed with a transwell assay and a flow cytometry assay. Protein levels were measured by western blotting. We found that vortioxetine inhibited the proliferation, invasion and migration abilities of AGS cells. Additionally, vortioxetine could induce apoptosis and autophagy by increasing the levels of Bax, active caspase-3/-9, Beclin-1 and light chain 3, as well as by downregulating Bcl-2 and P62. Further investigations indicated that vortioxetine regulated apoptosis and autophagy via activation of the phosphoinositide 3-kinase/AKT pathway. Taken together, our data suggest that vortioxetine has cytotoxic effects against GC AGS cells as a result of inhibiting proliferation, invasion and migration, as well as by inducing apoptosis and autophagy through the phosphoinositide 3-kinase/AKT pathway.

In Vivo Photoacoustic Imaging of Brain Injury and Rehabilitation by High-Efficient Near-Infrared Dye Labeled Mesenchymal Stem Cells with Enhanced Brain Barrier Permeability.

Stem cell migration and interaction with pathology are critical to understand the complexity and status of disease recovery progress. However, the dynamic visualization still remains a great challenge due to imaging technical limitation, cell labeling difficulty, or blood-brain barrier (BBB). Herein, fast photoacoustic tomography (PAT) with optical molecular probes is applied to noninvasively monitor traumatic brain injury (TBI) and its rehabilitation. The vascular distribution and TBI hemorrhage are clearly imaged, longitudinally monitored, and quantified. Bone mesenchymal stem cells (BMSCs) labeled with modified Prussian blue particles (PBPs), excellent near-infrared dyes and photoacoustic contrasts, are intravenously injected to the mice for improved observation and efficient therapy. BMSCs are demonstrated to be capable of overcoming BBB with enhanced delivery of PBPs to the brain parenchyma. Notably, the versatile BMSCs are observed by PAT to home to the damage region and repair the ruptured vasculature. Moreover, the wound treated by BMSCs exhibits much faster recovery speed than that without treatment. These findings can potentially provide a new noninvasive and high-resolution approach to image TBI, monitor recovery process, and especially trace BMSCs. This study will stimulate extensive researches on brain diseases and provide promising strategies of dye labeled BMSCs in regenerative medicine.

Nonsymmetrical dithienylethenes bearing dithieno[3,2-b:2',3'-d]thiophene units with photochromic performance in the crystalline phase.

Four novel nonsymmetrical photochromic diarylethene compounds containing dithieno[3,2-b:2',3'-d]thiophene units were designed and synthesized to investigate their photochromic properties. All these molecules adopt a photoactive antiparallel conformation in single crystals, as revealed by X-ray crystallographic analysis, and exhibit excellent photochromism in solution as well as in the crystalline phase.

Hydrogen-bonding interactions in adrenaline-water complexes: DFT and QTAIM studies of structures, properties, and topologies.

ωB97XD/6-311++G(d,p) calculations were carried out to investigate the hydrogen-bonding interactions between adrenaline (Ad) and water. Six Ad-H(2)O complexes possessing various types of hydrogen bonds (H-bonds) were characterized in terms of their geometries, energies, vibrational frequencies, and electron-density topology. Natural bond orbital (NBO) and quantum theory of atoms in molecules (QTAIM) analyses were performed to elucidate the nature of the hydrogen-bonding interactions in these complexes. The intramolecular H-bond between the amino and carboxyl oxygen atom of Ad was retained in most of the complexes, and cooperativity between the intra- and intermolecular H-bonds was present in some of the complexes. H-bonds in which hydroxyls of Ad/water acted as proton donors were stronger than other H-bonds. Both hydrogen-bonding interactions and structural deformation play important roles in the relative stabilities of the complexes. The intramolecular H-bond was broken during the formation of the most stable complex, which indicates that Ad tends to break the intramolecular H-bond and form two new intermolecular H-bonds with the first water molecule.

Microsolvation effect and hydrogen-bonding pattern of taurine-water TA-(H2O)n (n = 1-3) complexes.

The microsolvation of taurine (TA) with one, two or three water molecules was investigated by a density functional theory (DFT) approach. Quantum theory of atoms in molecules (QTAIM) analyses were employed to elucidate the hydrogen bond (H-bond) interaction characteristics in TA-(H(2)O)(n) (n = 1-3) complexes. The results showed that the intramolecular H-bond formed between the hydroxyl and the N atom of TA are retained in most TA-(H(2)O)(n) (n = 1-3) complexes, and are strengthened via cooperative effects among multiple H-bonds from n = 1-3. A trend of proton transformation exists from the hydroxyl to the N atom, which finally results in the cleavage of the origin intramolecular H-bond and the formation of a new intramolecular H-bond between the amino and the O atom of TA. Therefore, the most stable TA-(H(2)O)(3) complex becomes a zwitterionic complex rather than a neutral type. A many-body interaction analysis showed that the major contributors to the binding energies for complexes are the two-body energies, while three-body energies and relaxation energies make significant contributions to the binding energies for some complexes, whereas the four-body energies are too small to be significant.

Microsolvation of aminoethanol: a study using DFT combined with QTAIM.

The microsolvation of aminoethanol (AE) with one, two, three or four water molecules was investigated using a density functional theory (DFT) approach. Quantum theory of atoms in molecules (QTAIM) analyses were employed to elucidate the hydrogen-bonding characteristics of AE-(H(2)O)( n ) (n = 1-4) complexes. The results showed that AE tends to break its intramolecular OH(AE)···N(AE) hydrogen bond (H-bond) upon microsolvation and form intermolecular H-bonds with water molecules, while complexes that retain the intramolecular OH(AE)···N(AE) H-bond show reduced stabilities. The intermolecular H-bond that forms between the nitrogen atom of AE and the hydroxyl of a water molecule is the strongest one for the most stable AE-(H(2)O)( n ) (n = 1-4) complexes, and as n increases from 1 to 4 they grow stronger. The partial covalent character of this H-bond was confirmed by QTAIM analyses. Many-body interaction analysis showed that the relaxation energies and two- and three-body energies make significant contributions to the binding energies of the complexes.

Hydrogen bonding interactions in noradrenaline-DMSO complexes: DFT and QTAIM studies of structure, properties and topology.

The hydrogen bonding interactions between noradrenaline (NA) and DMSO were studied with density functional theory (DFT) regarding their geometries, energies, vibrational frequencies, and topological features of the electron density. The quantum theory of atoms in molecules (QTAIM) and the natural bond orbital (NBO) analyses were employed to elucidate the hydrogen bonding interaction characteristics in noradrenaline-DMSO complexes. The H-bonds involving the hydroxyls hydrogen in NA and the O atom in DMSO are dominant intermolecular H-bonds and are stronger than other H-bonds involving the methyl hydrogen of DMSO as a H-donor. The weak H-bonds also include a π H-bond which involves the benzene ring as a H-donor or H-acceptor. QTAIM identified the weak H-bonds formed between the methyl hydrogen of DMSO and the N atom in NA in some complexes (AB5, AB6 and AB7), which cannot be further confirmed by NBO and other methods, so there are probably no interactions between hydrogen and nitrogen atoms among these complexes. A good linear relationship between logarithmic electron density (lnρ ( b )) at the bond critical point (BCP) and structural parameter (δR (H···Y)) was found. The formations of new H-bonds in some complexes are helpful to strengthen the original intramolecular H-bond, this is attributed to the cooperativity of H-bonds in complexes and can be learned from the structure results and the NBO and QTAIM analyses. Analysis of various physically meaningful contributions arising from the energy decomposition procedures show that the orbital interactions of H-bond is predominant during the formation of the complex, moreover, both the hydrogen bonding interaction and the structural deformation are responsible for the stability of the complexes.