Multiarmed DNA jumper and metal-organic frameworks-functionalized paper-based bioplatform for small extracellular vesicle-derived miRNAs assay.

Small extracellular vesicle-derived microRNAs (sEV-miRNAs) have emerged as promising noninvasive biomarkers for early cancer diagnosis. Herein, we developed a molecular probe based on three-dimensional (3D) multiarmed DNA tetrahedral jumpers (mDNA-Js)-assisted DNAzyme activated by Na+, combined with a disposable paper-based electrode modified with a Zr-MOF-rGO-Au NP nanocomplex (ZrGA) to fabricate a novel biosensor for sEV-miRNAs Assay. Zr-MOF tightly wrapped by rGO was prepared via a one-step method, and it effectively aids electron transfer and maximizes the effective reaction area. In addition, the mechanically rigid, and nanoscale-addressable mDNA-Js assembled from the bottom up ensure the distance and orientation between fixed biological probes as well as avoid probe entanglement, considerably improving the efficiency of molecular hybridization. The fabricated bioplatform achieved the sensitive detection of sEV-miR-21 with a detection limit of 34.6 aM and a dynamic range from100 aM to 0.2 µM. In clinical blood sample tests, the proposed bioplatform showed results highly consistent with those of qRT-PCRs and the signal increased proportionally with the NSCLC staging. The proposed biosensor with a portable wireless USB-type analyzer is promising for the fast, easy, low-cost, and highly sensitive detection of various nucleic acids and their mutation derivatives, making it ideal for POC biosensing.

A Universal Strategy for Enhancing the Circulating miRNAs' Detection Performance of Rolling Circle Amplification by Using a Dual-Terminal Stem-Loop Padlock.

Rolling circle amplification (RCA) is one of the most promising nucleic acid detection technologies and has been widely used in the molecular diagnosis of disease. Padlock probes are often used to form circular templates, which are the core of RCA. However, RCA often suffers from insufficient specificity and sensitivity. Here we report a reconstruction strategy for conventional padlock probes to promote their overall performance in nucleic acid detection while maintaining probe functions uncompromised. When two rationally designed stem-loops were strategically placed at the two terminals of linear padlock probes, the specificity of target recognition was enhanced and the negative signal was significantly delayed. Our design achieved the best single-base discrimination compared with other structures and over a 1000-fold higher sensitivity than that of the conventional padlock probe, validating the effectiveness of this reconstruction. In addition, the underlying mechanisms of our design were elucidated through molecular dynamics simulations, and the versatility was validated with longer and shorter padlocks targeting the same target, as well as five additional targets (four miRNAs and dengue virus - 2 RNA mimic (DENV-2)). Finally, clinical applicability in multiplex detection was demonstrated by testing real plasma samples. Our exploration of the structures of nucleic acids provided another perspective for developing high-performance detection systems, improving the efficacy of practical detection strategies, and advancing clinical diagnostic research.

Triquinane Sesquiterpene Glycosides from the Basidiomycete Antrodiella zonata.

In our ongoing study of fungal bioactive natural products, 12 previously undescribed triquinane sesquiterpene glycosides, namely, antrodizonatins A-L (1-12), and four known compounds (13-16) have been obtained from the fermentation of the basidiomycete Antrodiella zonata. The structures were established unambiguously via extensive spectroscopic analysis and theoretical calculations of electronic circular dichroism spectra. This is the first report of triquinane sesquiterpene glycosides. Compounds 1, 5, and 12 displayed antibacterial activity against Staphylococcus aureus with MIC50 values of 35, 34, and 69 µM, respectively.

Polyketides from the fungus Pochonia chlamydosporia and their bioactivities.

Three previously undescribed griseofulvin derivatives, namely pochonichlamydins A-C, one small polyketide, namely pochonichlamydin D, together with nine known compounds, have been isolated from cultures of the fungus Pochonia chlamydosporia. Their structures with absolute configurations were elucidated on the basis of extensive spectrometric methods and single-crystal X-ray diffraction. Dechlorogriseofulvin and griseofulvin exhibited inhibitory activities against Candida albicans at the concentration of 100 µM, with inhibition rates of 69.1% and 56.3%, respectively. Meanwhile, pochonichlamydin C showed mild cytotoxicity against the human cancer MCF-7 cell line with an IC50 value of 33.1 µM.

Dear-DIAXMBD: Deep Autoencoder Enables Deconvolution of Data-Independent Acquisition Proteomics.

Data-independent acquisition (DIA) technology for protein identification from mass spectrometry and related algorithms is developing rapidly. The spectrum-centric analysis of DIA data without the use of spectra library from data-dependent acquisition data represents a promising direction. In this paper, we proposed an untargeted analysis method, Dear-DIAXMBD, for direct analysis of DIA data. Dear-DIAXMBD first integrates the deep variational autoencoder and triplet loss to learn the representations of the extracted fragment ion chromatograms, then uses the k-means clustering algorithm to aggregate fragments with similar representations into the same classes, and finally establishes the inverted index tables to determine the precursors of fragment clusters between precursors and peptides and between fragments and peptides. We show that Dear-DIAXMBD performs superiorly with the highly complicated DIA data of different species obtained by different instrument platforms. Dear-DIAXMBD is publicly available at https://github.com/jianweishuai/Dear-DIA-XMBD.

A Smart Nano-Theranostic Platform Based on Dual-microRNAs Guided Self-Feedback Tetrahedral Entropy-Driven DNA Circuit.

MicroRNAs (miRNAs) can act as oncogenes or tumor suppressors, capable of up or down-regulating gene expression during tumorigenesis; they are diagnostic biomarkers or therapeutic targets for tumors. To detect low abundance of intracellular oncogenic miRNAs (onco-miRNAs) and realize synergistic gene therapy of onco-miRNAs and tumor suppressors, a smart nano-theranostic platform based on dual-miRNAs guided self-feedback tetrahedral entropy-driven DNA circuit is created. The platform as a delivery vehicle is a DNA tetrahedral framework, in which the entropy-driven DNA circuit achieves a dual-miRNAs guided self-feedback, between an in situ amplification of the onco-miRNAs and activation of suppressor miRNAs release. To test this platform, dual-miRNAs are selected, miRNA-155, an up-regulated miRNA, as cancer indicators, and miRNA-122, a down-regulated miRNA as therapy targets in hepatocellular carcinoma, respectively. Through the circuit, the platform to detect onco-miRNAs at femtomolar level as well as visualized miRNAs inside cells, fixed tissues, and mice is programmed. Furthermore, triggered by miRNA-155, preloaded miRNA-122 is amplified via the self-feedback and released into target cells; the sudden increase of miRNA-122 and simultaneous decrease of miRNA-155 synergistically served as therapeutic drugs for gene regulation with enhanced antitumor efficacy and superior biosafety. It is envisioned that this nano-theranostic platform will initiate an essential step toward tumor theranostics in personalized/precise medicine.

HOXA-AS2 may be a potential prognostic biomarker in human cancers: A meta-analysis and bioinformatics analysis.

Background: Dysregulation of long non-coding (lncRNA) has been reported in various solid tumors. HOXA cluster antisense RNA 2 (HOXA-AS2) is a newly identified lncRNA with abnormal expression in several human malignancies. However, its prognostic value remains controversial. This meta-analysis synthesized available data to clarify the association between HOXA-AS2 expression levels and clinical prognosis in multiple cancers. Methods: Four public databases (Embase, PubMed, Web of Science, The Cochrane Library) were used to identify eligible studies. Hazard ratios (HRs) and odds ratios (ORs) with their 95% confidence intervals (CIs) were combined to assess the correlation of HOXA-AS2 expression with survival outcomes and clinicopathological features of cancer patients. Publication bias was measured using Begg's funnel plot and Egger's regression test, and the stability of the combined results was measured using sensitivity analysis. Additionally, multiple public databases were screened and extracted to validate the results of this meta-analysis. Results: The study included 20 studies, containing 1331 patients. The meta-analysis showed that the overexpression of HOXA-AS2 was associated with poor overall survival (HR = 2.06, 95% CI 1.58-2.69, p < 0.001). In addition, the high expression of HOXA-AS2 could forecast advanced tumor stage (OR = 3.89, 95% CI 2.90-5.21, p < 0.001), earlier lymph node metastasis (OR = 3.48, 95% CI 2.29-5.29, p < 0.001), larger tumor size (OR = 2.36, 95% CI 1.52-3.66, p < 0.001) and earlier distant metastasis (OR = 3.54, 95% CI 2.00-6.28, p < 0.001). However, other clinicopathological features, including age (OR = 1.09, 95% CI 0.86-1.38, p = 0.467), gender (OR = 0.92, 95% CI 0.72-1.18, p = 0.496), depth of invasion (OR = 2.13, 95% CI 0.77-5.90, p = 0.146) and differentiation (OR = 1.02, 95% CI 0.65-1.59, p = 0.945) were not significantly different from HOXA-AS2 expression. Conclusion: Our study showed that the overexpression of HOXA-AS2 was related to poor overall survival and clinicopathological features. HOXA-AS2 may serve as a potential prognostic indicator and therapeutic target for tumor treatment.

Modularized Enzymatic Tandem Reaction for tsRNA Detection.

tRNA-derived small RNA (tsRNA) has emerged as a new biomarker for early diagnosis and prognosis prediction of breast cancer. Like the detection of other small non-coding RNAs, the traditional DNA circuit could be used for the tsRNA detection. However, the highly coupling DNA strands in the circuit increase the difficulty of design and could raise a false-positive signal. Here, we demonstrated a versatile modularized enzymatic tandem reaction, namely, reverse-transcribed nicking exponential truncation (RT-NExT). This enzymatic reaction was constructed by cohesive modules, which can work independently or in assembly. Each module could amplify and initiate the downstream module. The RT-NExT reaction could detect 10-18 M ts-66 or ts-86 within 10 min and exhibited excellent consistency to the qRT-PCR when measuring the tsRNA expression level of breast cancer or healthy patients. RT-NExT provides an appealing detection strategy for further research on the clinical diagnosis with tsRNAs.

Programming a DNA tetrahedral nanomachine as an integrative tool for intracellular microRNA biosensing and stimulus-unlocked target regulation.

The synchronous detection and regulation of microRNAs (miRNAs) are essential for the early tumor diagnosis and treatment but remains a challenge. An integrative DNA tetrahedral nanomachine was self-assembled for sensitive detection and negative feedback regulation of intracellular miRNAs. This nanomachine comprised a DNA tetrahedron nanostructure as the framework, and a miRNA inhibitor-controlled allosteric DNAzyme as the core. The DNA tetrahedron brought the DNAzyme and the substrate in spatial proximity and facilitated the cellular uptake of DNAzyme. In allosteric regulation of DNAzyme, the locked tetrahedral DNAzyme (L-tetra-D) and active tetrahedral DNAzyme (A-Tetra-D) were controlled by miRNA inhibitor. The combination of miRNA inhibitor and target could trigger the conformational change from L-tetra-D to A-Tetra-D. A-Tetra-D cleaved the substrate and released fluorescence for intracellular miRNA biosensing. The DNA tetrahedral nanomachine showed excellent sensitivity (with detection limit down to 0.77 pM), specificity (with one-base mismatch discrimination), biocompatibility and stability. Simultaneously, miRNA stimulus-unlocked inhibitor introduced by our nanomachine exhibited the synchronous regulation of target cells, of which regulatory performance has been verified by the upregulated levels of downstream genes/proteins and the increased cellular apoptosis. Our study demonstrated that the DNA tetrahedral nanomachine is a promising biosense-and-treat tool for the synchronous detection and regulation of intracellular miRNA, and is expected to be applied in the early diagnosis and tailored management of cancers.

[Two different techniques combined with MIS-TLIF in the treatment of degenerative lumbar spondylolisthesis:a case-control study].

OBJECTIVE: To analyze the difference in clinical efficacy of minimally invasive transforaminal lumbar interbody fusion (MIS-TLIF) under Quadrant channel system combined with microscope and percutaneous pedicle screw in the treatment of degenerative lumbar spondylolisthesis. METHODS: A total of 114 patients with single-segment degenerative lumbar spondylolisthesis from June 2015 to February 2019, were divided into three groups according to the surgical methods, such as the MIS-TLIF under the microscope surgery group ( microscope group), MIS-TLIF combined with percutaneous pedicle screw technique surgery group(percutaneous group) and posterior lumbar interbody fusion surgery group (open group). In the microscope group, there were 12 males and 26 females, aged from 42 to 83 years with an average of (63.29±9.09) years. In the percutaneous group, there were 16 males and 22 females, aged from 45 to 82 years with an average of (63.37±7.50) years. In the open group, there were 12 males and 26 females, aged from 51 to 82 years with an average of (63.76±8.21) years. The general conditions of operation, such as operation time, intraoperative blood loss, postoperative drainage, length of surgical incision, frequency of intraoperative fluoroscopy and postoperative time of lying in bed were recorded to analyze the differences in surgical related indicators. Visual analogue scale (VAS) of waist and leg pain in preoperative and postoperative period (3 days, 3 months, 6 months and 12 months) were recorded to evaluate pain remission;Oswestry Disability Index(ODI), Japanese Orthopaedic Association (JOA) score were recorded to evaluate the recovery of waist and leg function on preoperative and postoperative 12 months. The lumbar spondylolisthesis rate and intervertebral height at 12 months after operation were recorded to evaluate the reduction of spondylolisthesis. The Siepe intervertebral fusion standard was used to analyze the intervertebral fusion rate at 12 months after operation. RESULTS: ①All 114 patients were followed up more than 1 year, and no complications related to incision infection occurred. In the microscope group, there was 1 case of subcutaneous effusion 8 days after operation. After percutaneous puncture and drainage, waist compression, and then the healing was delayed. In the percutaneous group, 2 cases of paravertebral muscle necrosis occurred on the side of decompression, and the healing was delayed after debridement. In open group, there was 1 case of intraoperative dural tear, which was packed with free adipose tissue during the operation. There was no postoperative cerebrospinal fluid leakage and other related complications.① Compared with microscope group, percutaneous group increased in operation time, intraoperative blood loss, postoperative wound drainage, surgical incision length, intraoperative fluoroscopy times, and postoperative bed rest time. In open group, intraoperative blood loss, postoperative wound drainage, surgical incision length, and postoperative bed rest time increased, but the intraoperative fluoroscopy time decreased. Compared with percutaneous group, the intraoperative blood loss, wound drainage, surgical incision length, and postoperative bed rest time in open group increased, but operative time and the intraoperative fluoroscopy time decreased(P<0.05). ②ODI and JOA scores of the three groups at 12 months after operation were improved compared with those before operation (P<0.05), but there was no significant difference between the three group(P>0.05). ③Compared with microscope group, the VAS of low back pain in percutaneous group increased at 3 days after operation, and VAS of low back pain in open group increased at 3 days, and 12 month after operation. Compared with percutaneous group, the VAS low back pain score of the open group increased at 3 months after operation (P<0.05). ④ The lumbar spondylolisthesis rate of the three groups of patients at 12 months afrer operation was decreased compared with that before operation(P<0.05), and the intervertebral heigh was increased compared with that before operation(P<0.05), however, there was no significant difference among three groups at 12 months afrer operation(P>0.05). ⑤ There was no significant difference between three groups in the lumbar fusion rate at 12 months afrer operation(P>0.05). CONCLUSION: The MIS-TLIF assisted by microscope and the MIS-TLIF combined with percutaneous pedicle screw are safe and effective to treat the degenerative lumbar spondylolisthesis with single-segment, and the MIS-TLIF assisted by microscope may be more invasive, cause less blood loss and achieve better clinical efficacy.

Multi-level attention graph neural network based on co-expression gene modules for disease diagnosis and prognosis.

MOTIVATION: Advanced deep learning techniques have been widely applied in disease diagnosis and prognosis with clinical omics, especially gene expression data. In the regulation of biological processes and disease progression, genes often work interactively rather than individually. Therefore, investigating gene association information and co-functional gene modules can facilitate disease state prediction. RESULTS: To explore the gene modules and inter-gene relational information contained in the omics data, we propose a novel multi-level attention graph neural network (MLA-GNN) for disease diagnosis and prognosis. Specifically, we format omics data into co-expression graphs via weighted correlation network analysis, and then construct multi-level graph features, finally fuse them through a well-designed multi-level graph feature fully fusion module to conduct predictions. For model interpretation, a novel full-gradient graph saliency mechanism is developed to identify the disease-relevant genes. MLA-GNN achieves state-of-the-art performance on transcriptomic data from TCGA-LGG/TCGA-GBM and proteomic data from coronavirus disease 2019 (COVID-19)/non-COVID-19 patient sera. More importantly, the relevant genes selected by our model are interpretable and are consistent with the clinical understanding. AVAILABILITYAND IMPLEMENTATION: The codes are available at https://github.com/TencentAILabHealthcare/MLA-GNN.

Enzyme-free and copper-free strategy based on cyclic click chemical-triggered hairpin stacking circuit for accurate detection of circulating microRNAs.

Accurate detection of circulating microRNAs (miRNAs) plays a vital role in the diagnosis of various diseases. However, enzyme-free amplification detection remains challenging. Here, we report an enzyme-free fluorescence resonance energy transfer assay termed "3C-TASK" (cyclic click chemical-triggered hairpin stacking kit) for the detection of circulating miRNA. In this strategy, the miRNA could initiate copper-free click chemical ligation reactions and the ligated products then trigger another hairpin stacking circuit. The first signal amplification was achieved through the recycling of the target miRNA in the click chemical ligation circuit, and the second signal amplification was realized through the recycling of ligated probes in a hairpin stacking circuit driven by thermodynamics. The two-step chain reaction event triggered by miRNAs was quantified by the fluorescence signal value so that accurate detection of target miRNA could be achieved. The 3C-TASK was easily controlled because no enzyme was involved in the entire procedure. Although simple, this strategy showed sensitivity with a detection limit of 8.63 pM and specificity for distinguishing miRNA sequences with single-base variations. In addition, the applicability of this method in complex biological samples was verified by detecting target miRNA in diluted plasma samples. Hence, our method achieved sensitive and specific detection of miRNA and may offer a new perspective for the broader application of enzyme-free chemical reaction and DNA circuits in biosensing.

A multiplexed circulating tumor DNA detection platform engineered from 3D-coded interlocked DNA rings.

Circulating tumor DNA (ctDNA) is a critical biomarker not only important for the early detection of tumors but also invaluable for personalized treatments. Currently ctDNA detection relies on sequencing. Here, a platform termed three-dimensional-coded interlocked DNA rings (3D-coded ID rings) was created for multiplexed ctDNA identification. The ID rings provide a ctDNA recognition ring that is physically interlocked with a reporter ring. The specific binding of ctDNA to the recognition ring initiates target-responsive cutting via a restriction endonuclease; the cutting then triggers rolling circle amplification on the reporter ring. The signals are further integrated with internal 3D codes for multiplexed readouts. ctDNAs from non-invasive clinical specimens including plasma, feces, and urine were detected and validated at a sensitivity much higher than those obtained through sequencing. This 3D-coded ID ring platform can detect any multiple DNA fragments simultaneously without sequencing. We envision that our platform will facilitate the implementation of future personalized/precision medicine.

Deep representation features from DreamDIAXMBD improve the analysis of data-independent acquisition proteomics.

We developed DreamDIAXMBD (denoted as DreamDIA), a software suite based on a deep representation model for data-independent acquisition (DIA) data analysis. DreamDIA adopts a data-driven strategy to capture comprehensive information from elution patterns of peptides in DIA data and achieves considerable improvements on both identification and quantification performance compared with other state-of-the-art methods such as OpenSWATH, Skyline and DIA-NN. Specifically, in contrast to existing methods which use only 6 to 10 selected fragment ions from spectral libraries, DreamDIA extracts additional features from hundreds of theoretical elution profiles originated from different ions of each precursor using a deep representation network. To achieve higher coverage of target peptides without sacrificing specificity, the extracted features are further processed by nonlinear discriminative models under the framework of positive-unlabeled learning with decoy peptides as affirmative negative controls. DreamDIA is publicly available at https://github.com/xmuyulab/DreamDIA-XMBD for high coverage and accuracy DIA data analysis.

Comparison of high-throughput single-cell RNA sequencing data processing pipelines.

With the development of single-cell RNA sequencing (scRNA-seq) technology, it has become possible to perform large-scale transcript profiling for tens of thousands of cells in a single experiment. Many analysis pipelines have been developed for data generated from different high-throughput scRNA-seq platforms, bringing a new challenge to users to choose a proper workflow that is efficient, robust and reliable for a specific sequencing platform. Moreover, as the amount of public scRNA-seq data has increased rapidly, integrated analysis of scRNA-seq data from different sources has become increasingly popular. However, it remains unclear whether such integrated analysis would be biassed if the data were processed by different upstream pipelines. In this study, we encapsulated seven existing high-throughput scRNA-seq data processing pipelines with Nextflow, a general integrative workflow management framework, and evaluated their performance in terms of running time, computational resource consumption and data analysis consistency using eight public datasets generated from five different high-throughput scRNA-seq platforms. Our work provides a useful guideline for the selection of scRNA-seq data processing pipelines based on their performance on different real datasets. In addition, these guidelines can serve as a performance evaluation framework for future developments in high-throughput scRNA-seq data processing.

Diamond: a multi-modal DIA mass spectrometry data processing pipeline.

SUMMARY: Currently, various software tools are used to support two mainstream workflows for data-independent acquisition (DIA) mass spectrometry (MS) data processing, namely, spectrum-centric scoring (SCS) and peptide-centric scoring (PCS). However, a fully automatic, easily reproducible and freely accessible pipeline that simultaneously integrates SCS and PCS strategies and supports both library-free and library-based modes is absent. We developed Diamond, a Nextflow-based, containerized, multi-modal DIA-MS data processing pipeline for peptide identification and quantification. Diamond integrated two mainstream workflows for DIA data analysis, namely, SCS and PCS, for use cases both with and without assay libraries. This multi-modal pipeline serves as a versatile, easy-to-use and easily extendable toolbox for large-scale DIA data processing. AVAILABILITY: Diamond is hosted on GitHub (https://github.com/xmuyulab/Diamond) and is released under the highly permissive MIT license to encourage further customization and modification. The Docker image for Diamond is freely accessible at https://hub.docker.com/r/zeroli/diamond.

An assay for DNA polymerase ß lyase inhibitors that engage the catalytic nucleophile for binding.

DNA polymerase ß (Pol ß) repairs cellular DNA damage. When such damage is inflicted upon the DNA in tumor cells treated with DNA targeted antitumor agents, Pol ß thus diminishes their efficacy. Accordingly, this enzyme has long been a target for antitumor therapy. Although numerous inhibitors of the lyase activity of the enzyme have been reported, none has yet proven adequate for development as a therapeutic agent. In the present study, we developed a new strategy to identify lyase inhibitors that critically engage the lyase active site primary nucleophile Lys72 as part of the binding interface. This involves a parallel evaluation of the effect of the inhibitors on the wild-type DNA polymerase ß (Pol ß) and Pol ß modified with a lysine analogue at position 72. A model panel of five structurally diverse lyase inhibitors identified in our previous studies (only one of which has been published) with unknown modes of binding were used for testing, and one compound, cis-9,10-epoxyoctadecanoic acid, was found to have the desired characteristics. This finding was further corroborated by in silico docking, demonstrating that the predominant mode of binding of the inhibitor involves an important electrostatic interaction between the oxygen atom of the epoxy group and Nε of the main catalytic nucleophile, Lys72. The strategy, which is designed to identify compounds that engage certain structural elements of the target enzyme, could find broader application for identification of ligands with predetermined sites of binding.