DNA-based molecular classifiers for the profiling of gene expression signatures.

Although gene expression signatures offer tremendous potential in diseases diagnostic and prognostic, but massive gene expression signatures caused challenges for experimental detection and computational analysis in clinical setting. Here, we introduce a universal DNA-based molecular classifier for profiling gene expression signatures and generating immediate diagnostic outcomes. The molecular classifier begins with feature transformation, a modular and programmable strategy was used to capture relative relationships of low-concentration RNAs and convert them to general coding inputs. Then, competitive inhibition of the DNA catalytic reaction enables strict weight assignment for different inputs according to their importance, followed by summation, annihilation and reporting to accurately implement the mathematical model of the classifier. We validated the entire workflow by utilizing miRNA expression levels for the diagnosis of hepatocellular carcinoma (HCC) in clinical samples with an accuracy 85.7%. The results demonstrate the molecular classifier provides a universal solution to explore the correlation between gene expression patterns and disease diagnostics, monitoring, and prognosis, and supports personalized healthcare in primary care.

Programmable DNA barcode-encoded exponential amplification reaction for the multiplex detection of miRNAs.

Multiple analysis of miRNAs is essential for the early diagnosis and monitoring of diseases. Here, a programmable, multiplex, and sensitive approach was developed for one-pot detection of miRNAs by melting temperature encoded sequences and exponential isothermal amplification (E-EXPAR). In the presence of target miRNAs, the corresponding templates initiate the cycles of nicking and polymerization/displacement, generating numerous barcode strands with unique encoding sequences. Subsequently, generated barcode strands hybridize with fluorescent probes and quench the fluorophore by a triplet of G base through a photo-induced electron transfer mechanism. Finally, a melting curve analysis is performed to quantify miRNAs by calculating the rate of fluorescence change at the corresponding melting temperature. Based on this, miRNA-21, miRNA-9, and miRNA-122 were detected with the detection limits of 3.3 fM, 2.9 fM, and 1.7 fM, respectively. This E-EXPAR was also employed to simultaneously detect three miRNAs in biological samples, showing consistent results with RT-qPCR. Overall, this study provides a programmable and universal platform for multiplex analysis of miRNAs, and holds great promise as an alternative to the multiplex analysis in clinical diagnostics and prognostics for nucleic acid detection.

Dynamic DNA Assembly by Programmable Hybridization Chain Reaction Mimicking Tubulin.

Dynamic biological structures involve the continual turnover of molecules within supramolecular assemblies such as tubulin. Inspired by dynamic biology self-organizing systems, we build an artificial dynamic structure based on DNA nanotechnology through a nonequilibrium chemical system. Herein, a metastable domain (MD), essentially a stem-loop structure, was introduced into DNA hairpins within hybridization chain reaction (HCR), thereby imparting dynamic activity to the DNA polymers. Hairpins with MD thermodynamically assemble to a high-energy polymer in the presence of trigger strands. The polymer can relax back to the stable unassembled state once the invader is added and finally relax to the activated hairpin by an anti-invader. Reversible assembly/disassembly of the HCR is achieved through invader/anti-invader cycles. We accomplished kinetic modulation, reversible conformational switching, cascading regulation, and enzyme activity control through the MD-HCR. We believe that the design of the MD-HCR could inspire the development of autonomous biological functions within artificial systems.

LINC01480 as a Prognostic Biomarker Associated With Proliferation, Migration, and Invasion of Clear Cell Renal Cell Carcinoma.

BACKGROUND/AIM: The present study aimed to identify key long noncoding RNAs (lncRNAs) involved in survival and metastasis of clear cell renal cell carcinoma (ccRCC). MATERIALS AND METHODS: A systemic screening for genes with differential expression in ccRCC was performed using publicly available databases. Cox regression analysis was used to identify lncRNAs associated with survival. A competing endogenous RNAs (ceRNA) regulation network of metastasis-related lncRNAs was constructed and hub lncRNAs were identified. Functional and pathway enrichment analyses were performed to investigate the role of lncRNA in ccRCC. Cell Counting Kit-8 and Transwell assays were used to determine the levels of cell proliferation, migration, and invasion. RESULTS: A total of 732 lncRNAs were found to be differentially expressed between ccRCC tumors and healthy samples. Among them, 139 lncRNAs were differentially expressed between metastasis and non-metastasis ccRCC samples and 75 lncRNAs were associated with overall survival and curated metastasis-related genes. Notably, LINC01480 was identified as the hub lncRNA involved in regulation of ccRCC metastasis. Clinically, LINC01480 may act as an independent factor for poor overall survival of ccRCC patients (log-rank p<0.05). Reverse transcription-quantitative PCR analysis validated that LINC01480 was significantly up-regulated in ccRCC compared to paired normal samples (n=20). Moreover, LINC01480 silencing inhibited the proliferation, migration, and invasion of ccRCC cells in vitro. Gene set enrichment analysis showed that high LINC01480 expression may promote ccRCC metastasis through enhancing immunodeficiency and amino acid metabolism. CONCLUSION: LINC01480 may act as a novel biomarker for overall prognosis in ccRCC and exhibit potential as a therapeutic target for the treatment of metastatic ccRCC.

Exploring the value of microorganisms in the appendix for inferring postmortem interval in Sprague-Dawley rats using high-throughput sequencing.

Various microorganisms play an important role in daily functions in the body and continue to flourish after death. Our prior investigation using frozen cadavers revealed that the appendix, rather than the transverse colon, was a superior sampling site for intestinal bacteria because the appendiceal flora had higher diversity than that in the transverse colon in the majority of experimental periods after death. We sought to explore out more about whether the appendicular flora is significantly related to postmortem interval (PMI) at natural temperatures following the host's death. In this work, we employed high-throughput sequencing to evaluate the contents of rats' appendices within 2 weeks after death and then utilized the random forest algorithm to build a PMI prediction model after completing basic visual analyses on the sequencing data. The findings revealed that Firmicutes was the absolute dominant species of appendicular flora; alpha-diversity of appendix flora first increased and then decreased, with the highest point appearing at 36 h after death; and the primary metabolic functions were carbohydrate metabolism, amino acid metabolism, as well as cofactors and vitamin metabolism. Finally, a random forest regression model for PMI prediction was built by the training data at the family level, with the mean absolute error of 10.27 h for prediction within 14 days postmortem, and the test set data subsequently proved the model's reliability. Changes in appendicular flora were strongly related to the PMI following rats' deaths, so we have reason to believe that the appendicular flora is valuable in predicting PMI.

Quantitative analysis of DNA methylation using sequence-specific, real-time loop-mediated isothermal amplification.

A number of genetic diseases and in the majority of tumor diseases have been reported to be associated with anomalous methylation levels of DNA. Accurate determine of the methylation level is an essential prerequisite for epigenetic diagnostics. Although some studies have used probe with a quencher enable real-time monitor of loop-mediated isothermal amplification (LAMP), most of them lack systematic investigations of design principle and applicable ambient when applied in LAMP based methylation detection. Herein, a sequence-specific, real-time loop-mediated isothermal amplification was developed for the quantitative analysis of DNA methylation. An oligonucleotide dimer was designed to implement three functions at the same time, including primer, sense probe and signal probe (PSS probe). Systematic investigations were performed to optimize the performance of PSS probe. After that, our method can accurately determine as low as 0.1% synthetic methylated DNA in the presence of numerous unmethylated DNA. Moreover, a higher methylated level of kazrin periplakin interacting protein (KAZN) gene was detected in colorectal cancer cells with a stable housekeeping gene ß-actin used as an endogenous control. The developed PSS probe for LAMP provide an option to analyze DNA methylation levels in resource-limited environments, and hold great potential for the diagnosis of methylation-related diseases.

Single-cell sequencing resolves the landscape of immune cells and regulatory mechanisms in HIV-infected immune non-responders.

Immune non-responder after highly active antiretroviral therapy (HAART) is the main cause of opportunistic infections and high mortality in AIDS patients, but the mechanism underlying immune reconstitution failure is poorly understood. Here, we performed scRNA-seq, and scATAC-seq analysis of peripheral blood mononuclear cells (PBMCs) derived from immune non-responder (INR) and responder (IR) HIV-1-infected subjects. We found low expression of mucosal-associated invariant T (MAIT) cells in INRs, which exhibited transcriptional profiles associated with impaired mitochondrial function and apoptosis signaling. Single-cell assays for transposase-accessible chromatin (scATAC-seq) and flow cytometry revealed diminished mitochondrial fitness in MAIT cells from INRs, and MAIT had low expression of transcription factor A for mitochondria (TFAM) and peroxisomal proliferator-activated receptor alpha (PPARA). These findings demonstrate that restoring mitochondrial function could modulate the immune dysfunction characteristic of MAIT against bacterial co-infections in INRs subjects.

Controllable crRNA Self-Transcription Aided Dual-Amplified CRISPR-Cas12a Strategy for Highly Sensitive Biosensing of FEN1 Activity.

A controllable crRNA self-transcription aided dual-amplified CRISPR-Cas12a strategy (termed CST-Cas12a) was developed for highly sensitive and specific biosensing of flap endonuclease 1 (FEN1), a structure-selective nuclease in eukaryotic cells. In this strategy, a branched DNA probe with a 5' overhanging flap was designed to serve as a hydrolysis substrate of FEN1. The flap cut by FEN1 was annealed with a template probe and functioned as a primer for an extension reaction to produce a double-stranded DNA (dsDNA) containing a T7 promoter and crRNA transcription template. Assisting the T7 RNA polymerase, abundant crRNA was generated and assembled with Cas12a to form a Cas12a/crRNA complex, which can be activated by a dsDNA trigger and unlock the indiscriminate fluorophore-quencher reporter cleavage. The highly efficient dual signal amplification and near-zero background enabled CST-Cas12a with extraordinarily high sensitivity. Under optimized conditions, this method allowed highly sensitive biosensing of FEN1 activity in the range of 1 × 10-5 U µL-1 to 5 × 10-2 U µL-1 with a detection limit of 5.2 × 10-6 U µL-1 and achieved excellent specificity for FEN1 in the presence of other interfering enzymes. The inhibitory capabilities of chemicals on FEN1 were also investigated. Further, the newly established CST-Cas12a strategy was successfully applied to FEN1 biosensing in complex biological samples, which might be a reliable biosensing platform for highly sensitive and specific detection of FEN1 activity in clinical applications.

Long noncoding RNA H19 mediates neural stem/progenitor cells proliferation, differentiation and apoptosis through the p53 signaling pathway after ischemic stroke.

Long non-coding RNA (LncRNA) H19 plays an important role on the biological functions of endogenous neural stem/progenitor cells (NSPCs). Our study aimed to explore the functions of H19 in NSPCs induced by oxygen-glucose deprivation/reperfusion (OGD/R) in vitro and the underlying mechanisms. In this study, our results showed that knockdown of H19 significantly inhibited NSPCs proliferation. Additionally, the apoptosis of NSPCs after ODG/R injury was notably promoted by H19 knockdown. Cell cycle arrest was induced in NSPCs at G0/G1 phase after OGD/R, while knockdown of H19 decreased the percentage of cells at G2/S phase. The results of immunofluorescence analysis revealed that H19 knockdown reduced the staining intensity of Ki-67 and DCX. Furthermore, H19 knockdown enhanced the expression of p53, Bax and Cleaved Caspase-3, while Bcl-2 expression was decreased. Silencing of H19 suppressed the NSPCs proliferation, cell cycle progression and differentiation, whereas cell apoptosis was promoted. Upregulation of H19 abolished OGD/R-induced NSPCs apoptosis, while cell proliferation and differentiation were promoted. Furthermore, the effects of overexpressed H19 on NSPCs proliferation, differentiation and apoptosis were abrogated by the upregulation of p53. In summary, overexpressed H19 resulted in the inactivation of p53, which promoted NSPCs proliferation, differentiation, and inhibited cell apoptosis. These findings suggested that H19 could promote cell proliferation and differentiation after OGD/R through suppressing the p53 signaling.

A self-oriented beacon liquid crystal assay for kanamycin detection with AuNPs signal enhancement.

The authors report a self-oriented beacon liquid crystal (LC) biosensor for kanamycin (Kana) detection with gold nanoparticle (AuNPs) signal enhancement. In this study, an assay was proposed for Kana detection using the aptamer as a self-oriented beacon. Without an additional orientation agent, the Kana aptamer was used as a self-oriented beacon both as an orientation agent for the LCs and as a signal recognition probe for biological molecules. Gold nanoparticles are blended with desired concentrations of the target molecules, which can greatly improve the performance of the biosensor. In the presence of Kana, AuNPs-Kana-aptamer conjugates will form on the sensing interface of the biosensor, which can remarkably destroy the orientated arrangement of the LCs, resulting in changes in the corresponding polarized images of the LCs. The limit of Kana detection is as low as 0.1 pmol L-1. It is important to note that the self-oriented beacons are immobilized on the assembled film of the glass slides for the specific recognition of Kana, simultaneously allowing the homeotropic orientation of the LCs. This study also provides a mechanism for the self-orientation beacon and liquid crystal biosensing.

Self-enhanced luminol-based electrochemiluminescent hydrogels: An ultrasensitive biosensing platform for fusion gene analysis coupled with target-initiated DNAzyme motor.

BCR/ABL fusion gene has been discovered as an important and reliable biomarker for early diagnosis of chronic myeloid leukemia (CML). Herein, a novel and switching electrochemiluminescence (ECL) biosensor was developed for ultrasensitive determination of the fusion gene based on the self-enhanced polyethyleneimine-luminol (PEI-Lum) hydrogels coupled with target-initiated DNAzyme motor. The facilely prepared PEI-Lum hydrogels could not only immobilize enormous luminol but shorten the distance of binary system, thus facilitating the mass and electron transfer efficiency of the sensing interface, so that the enhanced ECL signal was achieved. Moreover, the engineering DNA motor was powered by Mg2+-dependent DNAzyme for isothermal DNA signal amplification. As a result, the fabricated ECL biosensor enabled highly sensitive detection of BCR/ABL fusion gene with a broad linear range from 10.0 fM to 10.0 nM and a low detection limit of 3.75 fM (S/N = 3). Significantly, the developed biosensing method provides a potential tool for nucleic acid analysis in clinical diagnosis and a new avenue to design high-efficient ECL nanomaterials.

Predicting the postmortem interval using human intestinal microbiome data and random forest algorithm.

Gradual changes in microbial communities in a human body after death can be used to determine postmortem interval (PMI). In this study, gut microflora samples were collected from the vermiform appendix and the transverse colon of human cadavers with PMIs between 5 and 192 h. The results revealed that the appendix might be an excellent intestinal sampling site and the appendix flora had an inferred succession rule during human body decomposition. Firmicutes, Bacteroidetes, and their respective subclasses showed a predictable successionrule in relative abundance over time. A Random Forest regression model was developed to correlate human gut microbiota with PMI. We believe that our findings have increased the knowledge of the composition and abundance of the gut microbiota in human corpses, and suggest that the use of the human appendix microbial succession may be a potential method for forensic estimation of the time of death.

LncRNA MEG3 inhibits the proliferation of neural stem cells after ischemic stroke via the miR-493-5P/MIF axis.

OBJECTIVE: The proliferation of neural stem cells (NSCs1), or lack thereof, can have profound effects on brain tissue remodeling for ischemic stroke (IS2). In this study, we aimed to reveal the influence of the lncRNA MEG3/miR-493-5p/MIF axis on NSC proliferation after IS. METHODS: We established an oxygen glucose-deprivation/reoxygenation (OGD/R3) in vitro model of IS in NSCs. We evaluated NSC isolation efficiency and proliferation by NESTIN, SOX2, and PCNA immunofluorescence staining. MEG3 and miR-493-5P levels were assessed by quantitative real-time polymerase chain reaction (qRT-PCR4). Changes in MIF protein expression levels were analyzed using Western blotting. We then evaluated the role of MEG3 and miR-493-5p by transfection of si-MEG3, a miR-493-5p mimic, or miR-493-5p inhibitor. NSC proliferation was quantified using Cell Counting Kit-8 analysis. RESULTS: NESTIN and SOX2 were co-expressed in endogenous NSCs. Following OGD/R, MEG3 and miR-493-5P were significantly upregulated in NSCs, while MIF levels decreased and proliferation was inhibited. Knockdown of MEG3 inhibited miR-493-5p and rescued expression of MIF and PCNA, restoring cellular proliferation levels. In NSCs transfected with a miR-493-5p mimic or inhibitor, MIF levels were down- or upregulated, respectively. Consistently, transfection of a miR-493-5p mimic reduced NSC proliferation, while transfection with a miR-493-5p inhibitor or si-MEG3 rescued the inhibitory effect of OGD/R on NSC proliferation. After co-transfection of si-MEG3 and a miR-493-5p mimic of OGD/R-induced NSCs, levels of PCNA, an indicator of cellular proliferation, were significantly reduced. Conclusion MEG3 inhibits NSC proliferation of after IS via positive regulation of miR-493-5p and potential subsequent downregulation of MIF.

Theoretical and in silico Analyses Reveal MYC as a Dynamic Network Biomarker in Colon and Rectal Cancer.

In this article, we make a theoretical and in silico study for uncovering and evaluating biomarkers in colon and rectal cancer (CRC) by the dynamic network biomarker (DNB) theory. We propose a strategy to employ the theoretical concept of UICC TNM classification in CRC. To reveal the critical transition of CRC, the DNB algorithm was implemented to analyze the genome-wide dynamic network through temporal gene expression data. The relationship between gene sets and clinical features was evaluated by weighted gene co-expression network analysis. The results show that MYC was significantly associated with tumor amplification, tumor immune cells, and survival times. The candidate tumor suppressor genes were ZBTB16, MAL, LIFR, and SLIT2. Protein-protein interaction (PPI) analysis shows that these candidate tumor suppressor genes were significant in immune cells. Data from the Human Protein Atlas showed that a high expression of these candidate tumor suppressor genes was associated with favorable prognosis in TNM stages I-IV. In conclusion, this work provides significant and novel information regarding the TNM stage, cause, and consequences of elevated MYC expression in CRC. MYC expression levels had significant negative correlations with tumor suppressor genes and immune cells.

The Regulatory Functions of lncRNAs on Angiogenesis Following Ischemic Stroke.

Ischemic stroke is one of the leading causes of global mortality and disability. It is a multi-factorial disease involving multiple factors, and gene dysregulation is considered as the major molecular mechanisms underlying disease progression. Angiogenesis can promote collateral circulation, which helps the restoration of blood supply in the ischemic area and reduces ischemic necrosis following ischemic injury. Aberrant expression of long non-coding RNAs (lncRNAs) in ischemic stroke is associated with various biological functions of endothelial cells and serves essential roles on the angiogenesis of ischemic stroke. The key roles of lncRNAs on angiogenesis suggest their potential as novel therapeutic targets for future diagnosis and treatment. This review elucidates the detailed regulatory functions of lncRNAs on angiogenesis following ischemic stroke through numerous mechanisms, such as interaction with target microRNAs, downstream signaling pathways and target molecules.

Gold nanoparticle-based signal enhancement of an aptasensor for ractopamine using liquid crystal based optical imaging.

The authors report on a method for the determination of ractopamine (RAC) via liquid crystal (LC) optical imaging and gold nanoparticle-induced signal enhancement. The gold nanoparticles (AuNPs) were blended with the desired concentrations of RAC, and this is found to strongly improve the performance of the assay. The RAC aptamers were immobilized on the self-assembled film of a glass slide for specific recognition of RAC. This causes a homeotropic re-orientation of the LCs. Notably, the aptamers need not be immobilized on the nanoparticles like in other methods. The addition of RAC causes the formation of an AuNP-RAC-aptamer conjugate on the sensing interface. This disrupts the orientation of LCs and results in a change of the polarized images of the LCs. The method has a detection limit as low as 1 pM of RAC. Graphical abstract Schematic presentation of a method for the determination of ractopamine (RAC) using liquid crystal (LC) optical imaging and gold nanoparticle-induced signal enhancement. The aptamers need not be immobilized on the nanoparticles like in other methods.

Combined analysis and validation for DNA methylation and gene expression profiles associated with prostate cancer.

BACKGROUND: Prostate cancer (PCa) is a malignancy cause of cancer deaths and frequently diagnosed in male. This study aimed to identify tumor suppressor genes, hub genes and their pathways by combined bioinformatics analysis. METHODS: A combined analysis method was used for two types of microarray datasets (DNA methylation and gene expression profiles) from the Gene Expression Omnibus (GEO). Differentially methylated genes (DMGs) were identified by the R package minfi and differentially expressed genes (DEGs) were screened out via the R package limma. A total of 4451 DMGs and 1509 DEGs, identified with nine overlaps between DMGs, DEGs and tumor suppressor genes, were screened for candidate tumor suppressor genes. All these nine candidate tumor suppressor genes were validated by TCGA (The Cancer Genome Atlas) database and Oncomine database. And then, the gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes pathway (KEGG) enrichment analyses were performed by DAVID (Database for Annotation, Visualization and Integrated Discovery) database. Protein-protein interaction (PPI) network was constructed by STRING and visualized in Cytoscape. At last, Kaplan-Meier analysis was performed to validate these genes. RESULTS: The candidate tumor suppressor genes were IKZF1, PPM1A, FBP1, SMCHD1, ALPL, CASP5, PYHIN1, DAPK1 and CASP8. By validation in TCGA database, PPM1A, DAPK1, FBP1, PYHIN1, ALPL and SMCHD1 were significant. The hub genes were FGFR1, FGF13 and CCND1. These hub genes were identified from the PPI network, and sub-networks revealed by these genes were involved in significant pathways. CONCLUSION: In summary, the study indicated that the combined analysis for identifying target genes with PCa by bioinformatics tools promote our understanding of the molecular mechanisms and underlying the development of PCa. And the hub genes might serve as molecular targets and diagnostic biomarkers for precise diagnosis and treatment of PCa.

Similar bowtie structures and distinct largest strong components are identified in the transcriptional regulatory networks of Arabidopsis thaliana during photomorphogenesis and heat shock.

Photomorphogenesis and heat shock are critical biological processes of plants. A recent research constructed the transcriptional regulatory networks (TRNs) of Arabidopsis thaliana during these processes using DNase-seq. In this study, by strong decomposition, we revealed that each of these TRNs can be represented as a similar bowtie structure with only one non-trivial and distinct strong component. We further identified distinct patterns of variation of a few light-related genes in these bowtie structures during photomorphogenesis. These results suggest that bowtie structure may be a common property of TRNs of plants, and distinct variation patterns of genes in bowtie structures of TRNs during biological processes may reflect distinct functions. Overall, our study provides an insight into the molecular mechanisms underlying photomorphogenesis and heat shock, and emphasizes the necessity to investigate the strong connectivity structures while studying TRNs.