Ligase detection reaction amplification-activated CRISPR-Cas12a for single-molecule counting of FEN1 in breast cancer tissues.

We construct a simple fluorescent biosensor for single-molecule counting of flap endonuclease 1 (FEN1) based on ligase detection reaction (LDR) amplification-activated CRISPR-Cas12a. This biosensor exhibits excellent selectivity and high sensitivity with a detection limit (LOD) of 1.31 × 10-8 U. Moreover, it can be employed to screen the FEN1 inhibitors and quantitatively measure the FEN1 activity in human cells and breast cancer tissues, holding great promise in clinical diagnosis and drug discovery.

Rapamycin protects mouse skin from ultraviolet B-induced photodamage by modulating Hspb2-mediated autophagy and apoptosis.

BACKGROUND: Continuous exposure to UVB is the main extrinsic cause of skin photodamage, which is associated with oxidative stress, DNA damage, apoptosis and degradation of collagen. Rapamycin, a mechanistic target inhibitor of rapamycin complex 1 (mTORC1), has been shown to play a crucial role anti-tumor and aging retardation, but its mechanism of action in UVB-induced photodamage still remains unknown. In this study, we investigated the role of rapamycin and Hspb2 (also known as Hsp27) in UVB-induced photodamage in mice. METHODS AND RESULTS: We constructed skin acute photodamage models on the ears of WT and Hspb2 KO mice, respectively, and administered rapamycin treatment. Histological results showed that knockout of the hspb2 exacerbated the skin damage, as evidenced by thickening of the epidermis, breakage and disruption of collagen fibers and reduction in their number, which is reversed by rapamycin treatment. In addition, hspb2 knockout promoted UVB-induced apoptosis and reduced autophagy levels, with a significant increase in p53 levels and Bax/Bcl-2 ratio, a reduction in LC3II/I ratio and an increase in p62 levels in the KO mice compared to those in WT mice after the same dose of UVB irradiation. Rapamycin was also found to inhibit collagen degradation induced by hspb2 knockdown through activation of the TGF-ß/Smad signaling pathway. CONCLUSIONS: Rapamycin can alleviate skin photodamage from Hspb2 knockout to some extent. It may be a potential therapeutic drug for skin photodamage. In this study, we investigated the role of rapamycin and Hspb2 in UVB-induced photodamage in mice. Histological results showed that knockout of the hspb2 exacerbated the skin damage, as evidenced by thickening of the epidermis, breakage and disruption of collagen fibers and reduction in their number, which is reversed by rapamycin treatment. In addition, hspb2 knockout promoted UVB-induced apoptosis and reduced autophagy levels. Rapamycin was also found to inhibit collagen degradation induced by hspb2 knockdown through activation of the TGF-ß/Smad signaling pathway. We conclude that rapamycin and Hspb2 exert a synergistic protective effect in skin photodamage.

Whole-Exome Sequencing Identifies Mutation Profile and Mutation Signature-Based Clustering Associated with Prognosis in Appendiceal Pseudomyxoma Peritonei.

Pseudomyxoma peritonei (PMP) is a rare malignant clinical syndrome with little known about the global mutation profile. In this study, whole-exome sequencing (WES) was performed in 49 appendiceal PMP to investigate mutation profiles and mutation signatures. A total of 4,020 somatic mutations were detected, with a median mutation number of 56 (1-402). Tumor mutation burden (TMB) was generally low (median 1.55 mutations/Mb, 0.12-11.26 mutations/Mb). Mutations were mainly enriched in the function of cancer-related axonogenesis, extracellular matrix-related processes, calcium signaling pathway, and cAMP signaling pathway. Mutations in FCGBP, RBFOX1, SPEG, RTK-RAS, PI3K-AKT, and focal adhesion pathways were associated with high-grade mucinous carcinoma peritonei. These findings revealed distinct mutation profile in appendiceal PMP. Ten mutation signatures were identified, dividing patients into mutation signature cluster (MSC) 1 (N = 28, 57.1%) and MSC 2 (N = 21, 42.9%) groups. MSC (P = 0.007) was one of the four independent factors associated with 3-year survival. TMB (P = 0.003) and microsatellite instability (P = 0.002) were independent factors associated with MSC 2 grouping. Taken together, our findings provided a broader view in the understanding of molecular pathologic mechanism in appendiceal PMP and may be critical to developing an individualized approach to appendiceal PMP treatment. IMPLICATIONS: This work describes exhaustive mutation profile of PMP based on WES data and derives ten mutation signatures, which divides patients into two clusters and serve as an independent prognostic factor associated with 3-year survival.

Single-cell RNA sequencing uncovers the cell type-dependent transcriptomic changes in the retrosplenial cortex after peripheral nerve injury.

The retrosplenial cortex (RSC) is a vital area for storing remote memory and has recently been found to undergo broad changes after peripheral nerve injury. However, little is known about the role of RSC in pain regulation. Here, we examine the involvement of RSC in the pain of mice with nerve injury. Notably, reducing the activities of calcium-/calmodulin-dependent protein kinase type II-positive splenial neurons chemogenetically increases paw withdrawal threshold and extends thermal withdrawal latency in mice with nerve injury. The single-cell or single-nucleus RNA-sequencing results predict enhanced excitatory synaptic transmissions in RSC induced by nerve injury. Local infusion of 1-naphthyl acetyl spermine into RSC to decrease the excitatory synaptic transmissions relieves pain and induces conditioned place preference. Our data indicate that RSC is critical for regulating physiological and neuropathic pain. The cell type-dependent transcriptomic information would help understand the molecular basis of neuropathic pain.

Bioorthogonal Reaction-Mediated Enzymatic Elongation-Driven Dendritic Nanoassembly for Genome-Wide Analysis of 5-Hydroxymethyluracil in Breast Tissues.

5-Hydroxymethyluracil (5hmU) is an oxidation derivative of thymine in the genomes of various organisms and may serve as both an epigenetic mark and a cancer biomarker. However, the current 5hmU assays usually have drawbacks of laborious procedures, low specificity, and unsatisfactory sensitivity. Herein, we demonstrate the click chemistry-mediated hyperbranched amplification-driven dendritic nanoassembly for genome-wide analysis of 5hmU in breast cell lines and human breast tissues. The proposed strategy possesses good selectivity, ultralow background, and high sensitivity with a detection limit of 83.28 aM. This method can accurately detect even a 0.001% 5hmU level in the mixture. Moreover, it can determine 5hmU at single-cell level and distinguish the expressions of 5hmU in tissues of normal persons and breast cancer patients, holding great promise in 5hmU-related biological research and clinical diagnosis.

Rolling circle transcription amplification-directed construction of tandem spinach-based fluorescent light-up biosensor for label-free sensing of ß-glucosyltransferase activity.

ß-glucosyltransferase (ß-GT) can specifically catalyze the conversion of 5-hydroxymethylcytosine (5-hmC) to 5-glucosylhydroxy methylcytosine (5-ghmC), and it is associated with the control of phage-specific gene expression by affecting transcription process in vivo and in vitro. The current strategies for ß-GT assay usually involve expensive equipment, laborious treatment, radioactive hazard, and poor sensitivity. Here, we report a Spinach-based fluorescent light-up biosensor for label-free measurement of ß-GT activity by utilizing 5-hmC glucosylation-initiated rolling circle transcription amplification (RCTA). We design a 5-hmC-modified multifunctional circular detection probe (5-hmC-MCDP) that integrates the functions of target-recognition, signal transduction, and transcription amplification in one probe. The introduction of ß-GT catalyzes 5-hmC glucosylation of 5-hmC-MCDP probe, protecting the glucosylated 5-mC-MCDP probe from the cleavage by MspI. The remaining 5-hmC-MCDP probe can initiate RCTA reaction with the aid of T7 RNA polymerase, generating tandem Spinach RNA aptamers. The tandem Spinach RNA aptamers can be lightened up by fluorophore 3,5-difluoro-4-hydroxybenzylidene imidazolinone, facilitating label-free measurement of ß-GT activity. Notably, the high specificity of MspI-catalyzed cleavage of nonglucosylated probe can efficiently inhibit nonspecific amplification, endowing this assay with a low background. Due to the higher efficiency of RCTA than the canonical promoter-initiated RNA synthesis, the signal-to-noise ratio of RCTA is 4.6-fold higher than that of linear template-based transcription amplification. This method is capable of sensitively detecting ß-GT activity with a limit of detection of 2.03 × 10-5 U/mL, and it can be used for the screening of inhibitors and determination of kinetic parameters, with great potential in epigenetic research and drug discovery.

Target-activated T7 transcription circuit-mediated multiple cycling signal amplification for monitoring of flap endonuclease 1 activity in cancer cells.

The structure-specific endonuclease flap endonuclease 1 (FEN1) is an essential functional protein in DNA replication and genome stability, and it has been identified as a promising biomarker and drug target for multiple cancers. Herein, we develop a target-activated T7 transcription circuit-mediated multiple cycling signal amplification platform for monitoring FEN1 activity in cancer cells. In the presence of FEN1, the flapped dumbbell probe is cleaved to generate a free 5' flap single-stranded DNA (ssDNA) with the 3'-OH terminus. The ssDNA can hybridize with the T7 promoter-bearing template probe to trigger the extension with the aid of Klenow fragment (KF) DNA polymerase. Upon the addition of T7 RNA polymerase, an efficient T7 transcription amplification reaction is initiated to produce abundant single-stranded RNAs (ssRNAs). The ssRNA can hybridize with a molecular beacon to form an RNA/DNA heteroduplex that can be selectively digested by DSN to generate an enhanced fluorescence signal. This method exhibits good specificity and high sensitivity with a limit of detection (LOD) of 1.75 × 10-6 U µL-1. Moreover, it can be applied for the screening of FEN1 inhibitors and the monitoring of FEN1 activity in human cells, holding great potential in drug discovery and clinical diagnosis.

Construction of a Single-Molecule Biosensor for Antibody-Free Detection of Locus-Specific N6-Methyladenosine in Cancer Cells and Tissues.

N6-Methyladenosine (m6A) has emerged as a key post-transcriptional regulator in mRNA metabolism, and its dysregulation is associated with multiple human diseases. Herein, we construct a single-molecule fluorescent biosensor for antibody-free detection of locus-specific m6A in cancer cells and tissues. A 5'-biotinylated capture probe and a 3'-hydroxylated assistant probe are designed for the recognition of specific m6A-mRNA. The m6A-sensitive endoribonuclease MazF can identify and cleave the unmethylated mRNA, and the retained intact m6A-mRNA can hybridize with assistant probes and capture probes to achieve sandwich hybrids. The sandwich hybrids are immobilized on magnetic beads (MBs) to initiate the terminal deoxynucleotidyl transferase (TdT)-assisted polymerization, facilitating the continuous incorporation of Cy5-dATP to form long Cy5-polyA tails for the production of an on-bead amplified fluorescence signal. After magnetic separation and exonuclease digestion, numerous Cy5 fluorophores are released and subsequently measured by single-molecule detection. Especially, this biosensor is implemented simply and isothermally without the involvement of either radiolabeling or m6A-specific antibody. Moreover, this biosensor shows ultrahigh sensitivity with a detection limit of 2.24 × 10-17 M, and it can discriminate a 0.01% m6A level from a large pool of coexisting counterparts. Furthermore, this biosensor can be used for monitoring cellular m6A-mRNA expression and differentiating the m6A level in the breast cancer patient tissues from that in the healthy person tissues, providing a new avenue for clinical diagnosis and epitranscriptomic research.

Pole Skipping in Holographic Theories with Bosonic Fields.

We study pole skipping in holographic conformal field theories dual to diffeomorphism invariant theories containing an arbitrary number of bosonic fields in the large N limit. Defining a weight to organize the bulk equations of motion, a set of general pole skipping conditions are derived. In particular, the frequencies simply follow from general covariance and weight matching. In the presence of higher-spin fields, we find that the imaginary frequency for the highest-weight pole skipping point equals the higher-spin Lyapunov exponent which lies outside of the chaos bound. Without higher-spin fields, we show that the energy density Green's function has its highest-weight pole skipping happening at a location related to the out-of-time-order correlator for arbitrary higher-derivative gravity, with a Lyapunov exponent saturating the chaos bound and a butterfly velocity matching that extracted from a shockwave calculation. We also suggest an explanation for this matching at the metric level by obtaining the on-shell shockwave solution from a regularized limit of the metric perturbation at the skipped pole.

Construction of a phos-tag-directed self-assembled fluorescent magnetobiosensor for the simultaneous detection of multiple protein kinases.

Protein kinases play important roles in regulating various cellular processes and may function as potential diagnostic and therapeutic targets for various diseases including cancers. Herein, we construct a phos-tag-directed self-assembled fluorescent magnetobiosensor to simultaneously detect multiple protein kinases with good selectivity and high sensitivity. In the presence of protein kinases (i.e., PKA and Akt1), their substrate peptides (i.e., a FITC-labeled substrate peptide and a Cy5-labeled substrate peptide) are phosphorylated, and are then specifically recognized and captured by a biotinylated phos-tag to generate biotinylated substrate peptides for the assembly of magnetic bead (MB)-peptides-FITC/Cy5 nanostructures. After magnetic separation, the phosphorylated substrate peptides are disassembled from the MB-peptides-FITC/Cy5 nanostructures using deionized water at 80 °C, releasing FITC and Cy5 molecules. The released FITC and Cy5 molecules are detected by steady-state fluorescence measurements, with FITC indicating PKA and Cy5 indicating Akt1. This magnetobiosensor only involves one phos-tag without the requirement of radiolabeling, antibody screening, carboxypeptidase Y (CPY) cleavage, and cumbersome chemical/enzyme reactions. The introduction of magnetic separation can effectively eliminate the interference from complex real samples, generating an extremely low background signal. Moreover, this magnetobiosensor can accurately measure cellular protein kinase activities and screen inhibitors, with great potential for kinase-related biomedical research and therapeutic applications.

Single-Molecule Counting of FTO in Human Breast Tissues Based on a Rolling Circle Transcription Amplification-Driven Clustered Regularly Interspaced Short Palindromic Repeat─Cas12a.

N6-methyladenosine modification as an mRNA modification in mammalian cells is dynamically reversible, regulated by RNA demethylase [e.g., fat mass and obesity-associated protein (FTO)]. The abnormal expression of FTO is closely related to numerous diseases (e.g., various cancers and obesity). Herein, we demonstrate the single-molecule counting of FTO in human cancer cells and breast tissues based on a T7 RNA polymerase-mediated rolling circle transcription (RCT) amplification-driven clustered regularly interspaced short palindromic repeat (CRISPR)─Cas12a. When FTO is present, it demethylates the DNA substrate, initiating the DpnII-mediated cleavage reaction. After magnetic separation, the cleaved DNA fragments trigger the T7 RNA polymerase-mediated RCT amplification, activating CRISPR-/Cas12a-mediated cleavage of signal probes and releasing abundant FAM molecules that are simply counted via single-molecule detection. In this assay, only target FTO can generate CRISPR RNAs, efficiently improving detection specificity. Moreover, the integration of single-molecule detection with magnetic separation achieves zero background and effectively enhances detection sensitivity. This method can specifically and sensitively monitor FTO activity with a limit of detection of 1.20 × 10-13 M, and it may measure FTO at the single-cell level. Furthermore, it may accurately discriminate the FTO expression level in breast tissues between healthy persons and breast cancer patients and screen the FTO inhibitors as well, with great potential in clinical diagnosis and drug discovery.

Development of a single-molecule biosensor with an ultra-low background for the simultaneous detection of multiple retroviral DNAs.

Human T-cell lymphotropic virus type I and type II (HTLV-I and HTLV-II) are the two most prevalent subtypes of HTLVs, and they usually infect individuals asymptomatically and may induce various diseases. Herein, we develop a single-molecule biosensor with an ultra-low background for the simultaneous detection of multiple retroviral DNAs. This biosensor is constructed by immobilizing two types of signal probes (i.e., signal probes 1 and 2) onto the surface of magnetic beads (MBs) through specific biotin-streptavidin interactions. The presence of HTLV-I DNA and HTLV-II DNA will initiate the RNase H-assisted cyclic cleavage of signal probes, inducing the release of Cy3 and Cy5 fluorophores from the MBs. After magnetic separation, the Cy3 and Cy5 fluorophores can be directly quantified by single-molecule detection, with the Cy3 signal indicating HTLV-I DNA and the Cy5 signal indicating HTLV-II DNA. This biosensor enables the all-in-one and simultaneous detection of HTLV-I DNA and HTLV-II DNA under isothermal conditions, greatly simplifying the operation procedures and reducing the assay time. Due to the high amplification efficiency of RNase H-assisted target recycling, the ultra-low background resulting from magnetic separation, and the intrinsic high signal-to-noise ratio of single-molecule detection, this biosensor exhibits high sensitivity with a detection limit of 66.1 aM for HTLV-I DNA and 82.8 aM for HTLV-II DNA. Moreover, it can be applied for the discrimination of HTLV-positive cells from HTLV-negative cells, and even simultaneously quantify endogenous HTLV-I DNA and HTLV-II DNA at the single-cell level. Furthermore, this biosensor can be extended to detect other nucleotide molecules by rationally designing signal probes, providing a universal and powerful tool for clinical diagnosis and biomedical research.

Hydroxymethylation-Specific Ligation-Mediated Single Quantum Dot-Based Nanosensors for Sensitive Detection of 5-Hydroxymethylcytosine in Cancer Cells.

5-Hydroxymethylcytosine (5hmC) modification is a key epigenetic regulator of cellular processes in mammalian cells, and its misregulation may lead to various diseases. Herein, we develop a hydroxymethylation-specific ligation-mediated single quantum dot (QD)-based fluorescence resonance energy transfer (FRET) nanosensor for sensitive quantification of 5hmC modification in cancer cells. We design a Cy5-modified signal probe and a biotinylated capture probe for the recognition of specific 5hmC-containing genes. 5hmC in target DNA can be selectively converted by T4 ß-glucosyltransferase to produce a glycosyl-modified 5hmC, which cannot be cleaved by methylation-insensitive restriction enzyme MspI. The glycosylated 5hmC DNA may act as a template to ligate a signal probe and a capture probe, initiating hydroxymethylation-specific ligation to generate large amounts of biotin-/Cy5-modified single-stranded DNAs (ssDNAs). The assembly of biotin-/Cy5-modified ssDNAs onto a single QD through streptavidin-biotin interaction results in FRET and consequently the generation of a Cy5 signal. The nanosensor is very simple without the need for bisulfite treatment, radioactive reagents, and 5hmC-specific antibodies. Owing to excellent specificity and high amplification efficiency of hydroxymethylation-specific ligation and near-zero background of a single QD-based FRET, this nanosensor can quantify 5hmC DNA with a limit of detection of 33.61 aM and a wider linear range of 7 orders of magnitude, and it may discriminate the single-nucleotide difference among 5hmC, 5-methylcytosine, and unmodified cytosine. Moreover, this nanosensor can distinguish as low as a 0.001% 5hmC DNA in complex mixtures, and it can monitor the cellular 5hmC level and discriminate cancer cells from normal cells, holding great potential in biomedical research and clinical diagnostics.

HSP27 Protects Skin From Ultraviolet B -Induced Photodamage by Regulating Autophagy and Reactive Oxygen Species Production.

Ultraviolet (UV) irradiation has been well documented to be linked with almost all skin problems we know, and both dermis and epidermis may be affected to varying degrees by UV irradiation. Every time when exposed to sunlight without protection, our skin will step closer to photoaging, leading to irreversible consequences ultimately. Heat shock protein 27 (HSP27) is a vital protein involved in cell growth, autophagy, apoptosis, drug resistance, tumor genesis and metastasis. Evidence suggests that the organism is subjected to various internal and external environmental stresses (heat, oxidative stress, organic toxicants, etc.), and HSP27 with high expression has protective function. However, the expression of HSP27 in coping with UV irradiation have not been examined thoroughly. In this study, photodamage models were developed through different doses of UVB irradiation in human epidermal keratinocytes (HEKs) (30 mJ/cm2), human dermal fibroblasts (HDFs) (150 mJ/cm2) and mouse skin (2,700 mJ/cm2). HSP27 knockdown decreased cell viability and increased the incidence of UVB-induced reactive oxygen species (ROS) production. We got consistent results in vivo and vitro. Compared with that in the UVB group, the expression of LC3B was significantly lower, while the expression of p62 was significantly higher in the UVB + si-HSP27 group. It was also revealed that HSP27 knockdown reduced the expressions of some antioxidants, such as superoxide dismutase (SOD) and catalase (CAT), which accelerated UVB-induced ROS release. Moreover, histological results showed that epidermis was thickened and collagen fibers were disorganized in the UVB + si-HSP27 group. These findings have demonstrated that HSP27 might play a photoprotective role in the UVB-induced skin damage process by maintaining the normal autophagy and antioxidant level. It is implied that HSP27 could be a potential therapeutic target of photodamage. However, determination of the definitive mechanism requires further exploration.

Bsu polymerase-mediated fluorescence coding for rapid and sensitive detection of 8-oxo-7,8-dihydroguanine in telomeres of cancer cells.

Guanine is the most susceptible to oxidation among all the DNA bases, and 8-oxo-7,8-dihydroguanine (OG) is one of main oxidation products that can occur in any part of chromosomal DNA. OG in the telomere sequence is associated with telomere shortening, cell aging, and dysfunction, and it may induce cancers. The accurate detection of OG in telomeres is important to early clinical diagnosis and molecular research. Herein, we develop a simple and rapid method to sensitively measure 8-oxo-7,8-dihydroguanine (OG) in telomeres of cancer cells by using Bsu polymerase-mediated fluorescence coding. This method is very simple without the requirement for any nucleic acid amplification or specific restriction enzyme recognition reaction, and Bsu polymerase can selectively incorporate Cy5-dATP into the opposite site of OG, endowing this method with good specificity. Moreover, the introduction of single-molecule detection significantly improves the sensitivity. This method can detect OG within 70 min with a limit of detection (LOD) of 2.45 × 10-18 M, and it can detect OG in genomic DNA extracted from H2O2-treated HeLa cells with a LOD of 0.0094 ng, holding great potential in disease-specific gene damage research and early clinic diagnosis.

High throughput and very specific screening of anabolic-androgenic steroid adulterants in healthy foods based on stable isotope labelling and flow injection analysis-tandem mass spectrometry with simultaneous monitoring proton adduct ions and chloride adduct ions.

In this work, a stable isotope labelling-flow injection analysis-tandem mass spectrometry (SIL-FIA-MS/MS) with simultaneous monitoring [M+H]+ and [M+Cl]- method was developed for very specific and high throughput screening of anabolic-androgenic steroids (AAS) illegally added to healthy foods. Initially, a simple centrifugation step was carried out for liquid samples, and for solid samples, a solid-liquid extraction step was conducted. Afterwards, batch chemical derivatization was carried out. After adding a certain amount of 13C6-3-NPH labelled AAS standards as the internal standards, it can be directly transferred for FIA-MS/MS analysis based on the no MS response characteristics of 3-NPH. The 3-NPH labelled AAS showed dual-polarity property, observing chloride adduct ion ([M+Cl]-) in negative ion mode and proton adduct ion ([M+H]+) in positive ion mode. The average time cost for pretreatment of each sample was less than 1 min by carrying out batch processing. The subsequent FIA-MS/MS detection enabled rapid and high throughput detection. The addition of 13C6-3-NPH-labelled AAS as internal standards can correct the matrix effect to achieve accurate quantitative analysis. The detection sensitivity was also improved by 2-5 folds after 3-NPH labelling. The limits of detection (LODs) in positive MRM mode were in ranges of 0.1-0.3 ng/mL. The validated method with simultaneous monitoring [M+H]+ and [M+Cl]- was validated in the range of 6.0-1000 ng/mL with the linear coefficient (R2) greater than 0.997. Satisfactory recoveries were found to be in ranges of 93.0-108.7%. The intra-day and inter-day RSDs were in the range of 3.5-9.9% and 5.1-14.1%, respectively. No changes in detection sensitivity of the mass spectrometry and no carry-over effects were found after numerous consecutive injections of AAS derivates. Compared with previously reported methods, the proposed method proved accurate, very specific, high throughput with good sensitivity.

Label-free and sensitive detection of RNA demethylase FTO with primer generation rolling circle amplification.

We develop for the first time a label-free fluorescent method for sensitive detection of fat mass and obesity-associated protein (FTO) activity using MazF-mediated primer generation rolling circle amplification. This method is very simple with ultrahigh sensitivity and good specificity, and it can detect FTO activity at the single-cell level. Moreover, this method can be applied for the measurement of kinetic parameters and the screening of FTO inhibitors.

A copper-free and enzyme-free click chemistry-mediated single quantum dot nanosensor for accurate detection of microRNAs in cancer cells and tissues.

MicroRNAs (miRNAs) play key roles in the post-transcriptional regulation of genes, and their aberrant expression may disturb the normal gene regulation network to induce various diseases, and thus accurate detection of miRNAs is essential to early clinical diagnosis. Herein, we develop for the first time a single-quantum dot (QD)-based Förster resonance energy transfer (FRET) nanosensor to accurately detect miRNAs based on copper-free and enzyme-free cycling click chemistry-mediated tricyclic ligase chain reaction (LCR) amplification. We design four DNA probes namely DNA probes 1-4, with DNA probes 1 and 3 being modified with azide (N3) and DNA probes 2 and 4 being modified with dibenzocyclooctyne (DBCO). When target miRNA is present, DNA probes 1 and 2 can proceed via copper-free and enzyme-free click chemistry to generate the probes 1-2 ligation product. Subsequently, DNA probes 3 and 4 can hybridize with the probes 1-2 ligation product to generate the probes 3-4 ligation product. Both the probes 1-2 ligation product and probes 3-4 ligation product can act as the templates to initiate cycling click chemistry-mediated tricyclic LCR amplification whose products can be easily measured by the single-QD-based FRET nanosensor. This assay does not involve any enzymatic reverse transcription, copper catalyst, and ligase enzyme, and it exhibits excellent selectivity, high sensitivity, and the capability of differentiating even single-base mismatches. Moreover, this nanosensor can accurately quantify miRNA-155 even at the single-cell level, and it can distinguish the miRNA-155 expression in tissues of healthy persons and nonsmall cell lung cancer (NSCLC) patients.

The effectiveness of E-learning in continuing medical education for tuberculosis health workers: a quasi-experiment from China.

BACKGROUND: Given the context of rapid technological change and COIVD-19 pandemics, E-learning may provide a unique opportunity for addressing the challenges in traditional face-to-face continuing medical education (CME). However, the effectiveness of E-learning in CME interventions remains unclear. This study aims to evaluate whether E-learning training program can improve TB health personnel's knowledge and behaviour in China. METHODS: This study used a convergent mixed method research design to evaluate the impact of E-learning programs for tuberculosis (TB) health workers in terms of knowledge improvement and behaviour change during the China-Gates TB Project (add the time span). Quantitative data was collected by staff surveys (baseline n = 555; final n = 757) and management information systems to measure the demographic characteristics, training participation, and TB knowledge. Difference-in-difference (DID) and multiple linear regression models were employed to capture the effectiveness of knowledge improvement. Qualitative data was collected by interviews (n = 30) and focus group discussions (n = 44) with managers, teachers, and learners to explore their learning experience. RESULTS: Synchronous E-learning improved the knowledge of TB clinicians (average treatment effect, ATE: 7.3 scores/100, P = 0.026). Asynchronous E-learning has a significant impact on knowledge among primary care workers (ATE: 10.9/100, P < 0.001), but not in clinicians or public health physicians. Traditional face-to-face training has no significant impact on all medical staff. Most of the learners (57.3%) agreed that they could apply what they learned to their practice. Qualitative data revealed that high quality content is the key facilitator of the behaviour change, while of learning content difficulty, relevancy, and hardware constraints are key barriers. CONCLUSIONS: The effectiveness of E-learning in CME varies across different types of training formats, organizational environment, and target audience. Although clinicians and primary care workers improved their knowledge by E-learning activities, public health physicians didn't benefit from the interventions.

Process evaluation of E-learning in continuing medical education: evidence from the China-Gates Foundation Tuberculosis Control Program.

BACKGROUND: E-learning is a growing phenomenon which provides a unique opportunity to address the challenges in continuing medical education (CME). The China-Gates Foundation Tuberculosis (TB) Control Program implemented online training for TB health workers in three provinces of China. We aim to evaluate the implementation of E-learning CME programs, analyse the barriers and facilitators during the implementation process, and to provide policy recommendations. METHODS: Routine monitoring data were collected through the project office from December 2017 to June 2019. In-depth interviews, focus group discussion with project management personnel, teachers, and trainees (n = 78), and staff survey (baseline n = 555, final n = 757) were conducted in selected pilot areas at the provincial, municipal, and county/district levels in the three project provinces (Zhejiang, Jilin, and Ningxia). Descriptive analysis of quantitative data summarized the participation, registration, and certification rates for training activities. Thematic approach was used for qualitative data analysis. RESULTS: By the end of June 2019, the national and provincial remote training platforms had organized 98 synchronous learning activities, with an average of 173.2 people [standard deviation (SD) = 49.8] per online training session, 163.3 people (SD = 41.2) per online case discussion. In the pilot area, 64.5% of TB health workforce registered the asynchronous learning platform, and 50.1% obtained their professional certifications. Participants agreed that E-learning CME was more economical, has better content as well as more flexible work schedules. However, the project still faced challenges in terms of unmet learning needs, disorganized governance, insufficient hardware and software, unsupported environment, and lack of incentive mechanisms. CONCLUSIONS: Our results suggested that it's feasible to conduct large scale E-learning CME activities in the three project provinces of China. Training content and format are key facilitators of the program implementation, while the matching of training supply and demand, organizational coordination, internet technology, motivations, and sustainability are key barriers.