HERC3 promotes YAP/TAZ stability and tumorigenesis independently of its ubiquitin ligase activity.

YAP/TAZ transcriptional co-activators play pivotal roles in tumorigenesis. In the Hippo pathway, diverse signals activate the MST-LATS kinase cascade that leads to YAP/TAZ phosphorylation, and subsequent ubiquitination and proteasomal degradation by SCFß-TrCP . When the MST-LATS kinase cascade is inactive, unphosphorylated or dephosphorylated YAP/TAZ translocate into the nucleus to mediate TEAD-dependent gene transcription. Hippo signaling-independent YAP/TAZ activation in human malignancies has also been observed, yet the mechanism remains largely elusive. Here, we report that the ubiquitin E3 ligase HERC3 can promote YAP/TAZ activation independently of its enzymatic activity. HERC3 directly binds to ß-TrCP, blocks its interaction with YAP/TAZ, and thus prevents YAP/TAZ ubiquitination and degradation. Expression levels of HERC3 correlate with YAP/TAZ protein levels and expression of YAP/TAZ target genes in breast tumor cells and tissues. Accordingly, knockdown of HERC3 expression ameliorates tumorigenesis of breast cancer cells. Our results establish HERC3 as a critical regulator of the YAP/TAZ stability and a potential therapeutic target for breast cancer.

To Ub or not to Ub: a regulatory question in TGF-ß signaling.

The transforming growth factor ß (TGF-ß) superfamily controls a wide spectrum of biological processes in metazoans, including cell proliferation, apoptosis, differentiation, cell-fate determination, and embryonic development. Deregulation of TGF-ß-Smad signaling contributes to developmental anomalies and a variety of disorders and diseases such as tumorigenesis, fibrotic disorders, and immune diseases. In cancer, TGF-ß has dual effects through its antiproliferative and prometastatic actions. At the cellular level, TGF-ß functions mainly through the canonical Smad-dependent pathway in a cell type-specific and context-dependent manner. Accumulating evidence has demonstrated that ubiquitination plays a vital role in regulating TGF-ß-Smad signaling. We summarize current progress on ubiquitination (Ub) and the ubiquitin ligases that regulate TGF-ß-Smad signaling.

A Dual-Signal Sensing for the Visual and Luminescent Detection of p-Phenylenediamine Based on Cerium-Nitrogen-Co-Doped Carbon Dots.

Herein, a visual and luminescent dual-mode (colorimetric and fluorometric) method for the detection of P-phenylenediamine (PPD) in hair dye was successfully established based on cerium-nitrogen co-doped carbon dots (Ce, N-CDs) that displayed remarkable luminescence and peroxidase activity. Ce, N-CDs catalyzed H2O2 to produce superoxide anion, which then oxidized the colorless 3,3,5,5-tetramethylbenzidine (TMB) into blue oxidized TMB (oxTMB), capable of quenching the fluorescence through fluorescence resonance energy transfer (FRET) between Ce, N-CDs and oxTMB. The reducing properties of PPD could reduce oxTMB back to TMB, leading to a decrease in the absorption intensity of oxTMB and a fluorescence recovery of Ce, N-CDs. As a result, the quantitative detection of PPD could be achieved by measuring the absorption values of oxTMB and the fluorescence signal of Ce, N-CDs. The detection limits for PPD were calculated as 0.36 µM and 0.10 µM for colorimetry and fluorimetry, respectively. Furthermore, smartphone application (ColorPicker) capable of measuring the RGB value of the color was utilized in the detection system, facilitating on-site quantitative detection. This approach effectively shortens the detection time and simplifies the operation, offering a powerful and convenient tool for real-time monitoring of PPD.

A Ratiometric Fluorescence Assay for Detection of Ascorbic Acid Based on N,S Co-Doped Carbon Dots Combined With Ce4.

Hence, N,S-CDs with photoluminescent property were simply synthesized via a one-step hydrothermal method. Combined with the commercial reagent Ce4+, a ratiometric fluorescence assay for ascorbic acid (AA) detection was established. Ce4+, possessing oxidization, could directly oxidize o-phenylenediamine (OPD) to form the yellow fluorescent product oxOPD. Under the excitation wavelength of 370 nm, oxOPD had a maximum fluorescence emission at 562 nm. Meanwhile, due to the occurrence of the inner filter effect (IFE), oxOPD quenched the fluorescence of N,S-CDs. However, ascorbic acid (AA) inhibited the oxidation of Ce4+, causing the fluorescence of oxOPD at 562 nm to decrease, accompanied by an increase in the fluorescence belonging to N,S-CDs at 450 nm. Thus, a Ce4+-assisted ratiometric fluorescence method was established for AA detection. The two fluorescence output signals in this method had opposite changing trends, which could reduce system errors and improve the accuracy. This method was successfully applied to the determination of AA in drugs and fruits.

Microwave synthesized novel biomass carbon dots applied in the fluorescent detection of crystal violet.

To establish a new method for detecting crystal violet (CV), a harmful dye, herein, a genre of novel biomass carbon dots (CDs) was synthesized via a microwave method and employed as a fluorescent probe, in which water spinach and polyethylene glycol (PEG) performed as raw materials. Based on the inner filter effect (IFE) between the luminescent CDs and CV, the blue emission of this probe at 430 nm could be quenched by CV. Hence, a new strategy was proposed to selectively determine CV in aquaculture ambient. Moreover, under the optimal experiment conditions, this method showed a good linearity between the concentration of CV (c) and fluorescence quenching rate (ΔF/F0) in the concentration range of 4-200 µmol/L with the corresponding correlation coefficient (r) and the detection limit of 0.997 and 710 nmol/L, respectively. With advantages of environmental protectivity, sensitivity, affordability, and user-friendliness, the facilely fabricated CDs could be successfully applied in detecting CV in aquaculture samples, providing a technical foundation for monitoring the pollution of CV and ensuring the quality and safety of aquatic products.

PTPN3 acts as a tumor suppressor and boosts TGF-ß signaling independent of its phosphatase activity.

TGF-ß controls a variety of cellular functions during development. Abnormal TGF-ß responses are commonly found in human diseases such as cancer, suggesting that TGF-ß signaling must be tightly regulated. Here, we report that protein tyrosine phosphatase non-receptor 3 (PTPN3) profoundly potentiates TGF-ß signaling independent of its phosphatase activity. PTPN3 stabilizes TGF-ß type I receptor (TßRI) through attenuating the interaction between Smurf2 and TßRI. Consequently, PTPN3 facilitates TGF-ß-induced R-Smad phosphorylation, transcriptional responses, and subsequent physiological responses. Importantly, the leucine-to-arginine substitution at amino acid residue 232 (L232R) of PTPN3, a frequent mutation found in intrahepatic cholangiocarcinoma (ICC), disables its role in enhancing TGF-ß signaling and abolishes its tumor-suppressive function. Our findings have revealed a vital role of PTPN3 in regulating TGF-ß signaling during normal physiology and pathogenesis.

Comparative study of trimming and resection of diseased finger superficial flexor tendons in gouty carpal tunnel syndrome.

BACKGROUND: Hyperuricemia can lead to synovial hyperplasia in the wrist. In severe cases, it can lead to the deposition of gouty stone in the carpal tunnel, resulting in increased pressure in the carpal tunnel and compression of the median nerve to cause carpal tunnel syndrome (CTS), which is called gouty carpal tunnel syndrome (GCTS). As for the surgical treatment of gouty carpal tunnel syndrome, scholars have different opinions on whether it is necessary to remove the superficial flexor tendon. The purpose of this study was to compare the clinical efficacy of trimming and resection of the diseased superficial flexor tendon in the treatment of gouty carpal tunnel syndrome. METHODS: Clinical data were collected from May 2016 to July 2021 from 10 patients (13 affected wrists) diagnosed with gouty carpal tunnel syndrome and classified into two groups according to the surgical modality: the diseased portion of the gout-eroded superficial finger tendon was trimmed in 9 wrists, and the diseased superficial finger flexor tendon was excised in 4 wrists. Values related to flexion and extension functions, 2-PD, DASH, BCTQ, VAS and recurrence in the affected fingers were compared between the two groups as well as before and after surgery in each group. RESULTS: All affected limbs used were cleared of gouty stones, finger numbness improved, no skin necrosis occurred, and all incisions healed at stage I. At follow-up (13.58 ± 5.53 months), there was no significant difference between groups in flexion and extension function, 2-PD, DASH, BCTQ, and VAS with respect to the affected fingers, and patients in both groups improved significantly before and after surgery. Treatment of only one wrist involved trimming to remove lesion-affected portions of tendon, which reappeared 1 year after surgery, and there was one case of poor recovery from greater piriformis muscle atrophy in both procedures. CONCLUSION: Regarding surgical treatment of patients with gouty carpal tunnel syndrome in which the gouty stone has invaded the superficial flexor tendons of the fingers, the diseased superficial flexor tendons can be selectively excised, and the postoperative mobility of the affected fingers may not be impaired.

The optimal tension for the reconstruction of the distal radioulnar ligaments.

PURPOSE: This study aimed to investigate the optimal tension for the reconstruction of the distal radioulnar ligaments (DRULs) in the treatment of the distal radioulnar joint (DRUJ) instability. METHODS: A total of eight human cadaver upper extremities were used. First, the Tekscan sensor film system was used to measure the contact characteristics of the intact DRUJ. Following this, the DRULs were resected, and the measurement was repeated. The DRULs were then reconstructed according to Adams' procedure, and the contact forces under different initial tension were compared with that of the intact group to obtain the optimal tension. At that point, the contact force of the DRUJ was close to normal. The reliability of the obtained tension was verified by translational testing, which reflected the stability of the DRUJ. RESULTS: In the neutral position, the contact force, area, and pressure inside DRUJ were 0.51 ± 0.10 N, 64.08 ± 11.58 mm2, and 8.33 ± 2.42 kPa, respectively. After the DRULs were resected, they were 0.19 ± 0.02 N, 41.75 ± 5.01 mm2, and 4.86 ± 1.06 kPa, respectively. The relationship between the tension and contact force was linear regression (Y = 0.0496x + 0.229, R2 = 0.9575, P < 0.0001). According to the equation, when the tension was 3.64-7.68 N, the contact force was close to normal. There was no statistical difference in the stability of the reconstructed DRUJ under this tension compared with the intact group (P = 0.08). CONCLUSION: By comparing the contact forces under different reconstruction tensions with the normal value, we obtained the optimal tension, which can provide the theoretical basis for the clinical treatment of chronic DRUJ instability.

Hsa_circ_0102171 aggravates the progression of cervical cancer through targeting miR-4465/CREBRF axis.

Cervical cancer (CC) has caused numerous cancer-related deaths in women. Recent years, circular RNAs have been reported as vital factors in CC tumorigenesis. Our current study focused on the role of hsa_circ_0102171 (called circ_0102171 subsequently) in CC. At first, we applied reverse transcription polymerase chain reaction to detect the expression of circ_0102171 in CC tissues and cells. Subsequently, we silenced circ_0102171 to conduct loss-of-function assays, including cell counting kit-8 assay, 5-ethynyl-2'-deoxyuridine staining, Transwell assay, and flow cytometry analysis. Interestingly, we discovered that circ_0102171 expressed at a high level in CC tissues and cells. Functionally, silencing circ_0102171 prohibited cell proliferation, migration and invasion, and strengthened cell apoptosis in CC in vitro. Mechanistic investigations revealed that circ_0102171 could act as a sponge for miR-4465. Gain-of-function assays demonstrated that miR-4465 hindered the growth and migration of CC cells. Moreover, circ_0102171 enhanced the level of CREB3 regulatory factor (CREBRF) which was the downstream target of miR-4465. Rescue assays suggested that CREBRF and miR-4465 could involve in circ_0102171-mediated CC progression. Finally, in vivo data supported that silencing circ_0102171 hindered CC cell growth. In conclusion, circ_0102171 aggravates CC progression via targeting miR-4465/CREBRF axis.

Ascorbate uptake enables tubular mitophagy to prevent septic AKI by PINK1-PARK2 axis.

Ascorbate (Vitamin C) has been proposed as a promising therapeutic agent against sepsis in clinical trials, but there is little experimental evidence on its anti-septic efficacy. We report that Toll-like receptor 4 (TLR4) activation by LPS stimuli augments ascorbate uptake in murine and human tubular cells through upregulation of two ascorbate transporters SVCT-1 and -2 mediated by Fn14/SCFFbxw7α cascade. Ascorbate restriction, or knockout of SVCT-1 and -2, the circumstance reminiscent to blockade of ascorbate uptake, endows tubular cells more vulnerable to the LPS-inducible apoptosis, whereas exogenous administration of ascorbate overrides the ruin execution, for which the PINK1-PARK2, rather than BNIP3-NIX axis is required. Ascorbate increases, while SVCT-1 and -2 knockout or ascorbate restriction dampens tubular mitophagy upon LPS stimuli. Treatment of endotoxemic mice with high-dose ascorbate confers mitophagy and substantial protection against mortality and septic acute kidney injury (AKI). Our work provides a rationale for clinical management of septic AKI with high doses of ascorbate.

A Novel tp0548 Gene Type of Treponema pallidum Identified in Nanjing, China: Case Report and Review of Literature.

BACKGROUND: The tp0548 gene, hypothesized to encode for an outer-membrane protein, was originally used in the enhanced Centers for Disease Control and Prevention typing for molecular typing of Treponema pallidum. It plays an important role in the molecular epidemiology of Treponema because it is not only an important locus of multiple typing approaches but also suitable for strain typing of multiple Treponema subspecies. METHODS: A 27-year-old Chinese man attended the Institute of Dermatology, Chinese Academy of Medical Sciences Sexually Transmitted Disease Clinic in Nanjing, China, because of a genital ulcer and inguinal lymphadenopathy for 1 week. Workup consisted of microbiological and hematological investigations, and sequences analysis. The aims of this study were to describe a novel tp0548 sequence type "Qn" of this syphilis strain and to review all previously reported novel tp0548 genotypes. RESULTS: We identified a novel tp0548 gene type in a genital ulcer in a patient with primary syphilis in Nanjing, China. Using sequence alignment, we further found that this novel sequence was closely similar to "Q." Following the nomenclature used in the enhanced Centers for Disease Control and Prevention typing methodology, the letters "Qn" was assigned to the new sequence type. CONCLUSION: The novel tp0548 sequence type of T. pallidum not only expands the database up to 27 different sequence types but also indicates the substantial genetic diversity of the tp0548 gene sequence.

Exosomal miR-146a-5p from Treponema pallidum-stimulated macrophages reduces endothelial cells permeability and monocyte transendothelial migration by targeting JAM-C.

Exosomal microRNAs (miRNAs) transferred between cells have been implicated in modulating the host immune response in microbial infections. In this study, we isolated exosomes from Treponema pallidum (T. pallidum)-stimulated macrophages and detected differential exosomal miRNA expression using both microarrays, and RT-qPCR. A total of 65 differentially expressed miRNAs (35 upregulated and 30 downregulated) were identified. Of all identified miRNAs, miR-146a-5p was one of the most significantly changed miRNAs with high expression in exosomes from T. pallidum-stimulated macrophages. Furthermore, we isolated plasma exosomes from early syphilis patients and healthy controls, and confirmed miR-146a-5p upregulation in the former group. We also show that exosomal miR-146a-5p is efficiently transported into endothelial cells, reducing monocyte transendothelial migration and endothelial permeability by targeting junctional adhesion molecule C (JAM-C). Luciferase reporter assays confirmed binding of exosomal miR-146a-5p to the 3'untranslated region (3'UTR) of JAM-C. We then demonstrated that also exosomes derived from macrophages stimulated by T. pallidum expressed high levels of miR-146a-5p which could be delivered to endothelial cells, and decreased monocyte transendothelial migration by targeting JAM-C. Overall, this work provides novel insights into the mechanism by which T. pallidum hampers inflammatory reactions of the host via a blockade of leukocytes transendothelial migration and endothelial permeability.

Roles of diencephalon/mesencephalon homeobox 1 in the development and prognosis of hepatocellular carcinoma.

INTRODUCTION AND OBJECTIVES: The oncogene diencephalon/mesencephalon homeobox 1 (DMBX1) is widely overexpressed in a variety of human cancers. The present study aimed to analyze the expression and clinical importance of DMBX1 in nonneoplastic tissues and tumor tissues from patients with hepatocellular carcinoma (HCC). MATERIALS AND METHODS: DMBX1 expression in HCC and adjacent nontumor tissues was analyzed using immunohistochemical staining. Chi-square tests were applied to compare DMBX1 expression between the tumors and the adjacent normal tissues. We explored the correlation of DMBX1 expression with clinicopathological factors and its effect on the prognosis of HCC. Finally, we investigated the role of DMBX1 in HCC via knockdown experiments, which analyzed changes in cell invasion, cell proliferation and epithelial-mesenchymal transition (EMT) biomarkers (E-cadherin, N-cadherin, vimentin). The mRNAs that were coexpressed with DMBX1 in HCC, based on the TCGA cohort (n = 366), were obtained from the cBioPortal database. RESULTS: The average score for DMBX1 expression was significantly different (P < 0.001) between HCC and paired adjacent nontumor tissues, and DMBX1 expression correlated with hepatitis B virus (HBV) infection, tumor size, metastasis, and tumor node metastasis (TNM) stage (P < 0.05). A multivariate Cox regression analysis identified significant correlations of DMBX1 expression with tumor metastasis, TNM stage, and tumor capsule. Moreover, Kaplan-Meier survival analysis revealed an association between DMBX1 overexpression and shorter overall survival of patients with HCC (P < 0.05). In HCC cell lines, silencing DMBX1 markedly inhibited migration, proliferation and EMT markers. The mRNAs that were negatively (R ≤ -0.25, n = 1094) or positively (R ≥ 0.25, n = 2906) coexpressed with DMBX1 mRNA were selected for further Gene Ontology enrichment analysis, and the results revealed that the predicted functions of DMBX1 in HCC support the in vitro experimental results. CONCLUSIONS: Our data provide evidence that DMBX1 overexpression is associated with HCC metastasis and poor prognosis, suggesting that DMBX1 represents a therapeutic target in HCC.

l-Cysteine modified silver nanoparticles-based colorimetric sensing for the sensitive determination of Hg2+ in aqueous solutions.

A simple and sensitive colorimetric sensing method was constructed for detection of Hg2+ in aqueous solutions and based on silver nanoparticles functionalized with l-cysteine (l-Cys-Ag NPs). In this method, adenosine triphosphate (ATP) induced aggregation of l-Cys-Ag NPs. Simultaneously, the solution colour changed from bright yellow to brown. In the presence of Hg2+ , Hg2+ chelated ATP to form a complex and reduce the degree of aggregation of l-Cys-Ag NPs and was accompanied by a colour change from brown to bright yellow. The changing values of absorbance at 390 nm were linearly correlated with concentration of Hg2+ over the 4.00 × 10-8 to 1.04 × 10-6 mol·L-1 range, with a detection limit of 8 nM. This method was used successfully for detection of Hg2+ in real water samples and performed good selectivity and sensitivity. The recovery range was 91.5-109.1%, indicating that the method has vast application potential for determination of Hg2+ in the environment.

LINC02535 co-functions with PCBP2 to regulate DNA damage repair in cervical cancer by stabilizing RRM1 mRNA.

Cervical cancer (CC) is one of the commonest malignant cancers among women with high morbidity and mortality. Despite encouraging advances had been found in diagnostic and therapeutic strategies, effective therapeutic strategy and further exploration of the mechanism underlying in CC is still needed. We searched The Cancer Genome Atlas database and found that long noncoding RNA LINC02535 was highly expressed in CC. LINC02535 has not been studied in CC, and its molecular regulation mechanism remains unknown. Based on starBase database, LINC02535 could potentially bind poly (rC) binding protein 2 (PCBP2). In the present study, we discovered a significant increase of the LINC02535 and PCBP2 expression in CC tissues and cells as compared with the adjacent normal tissues and normal cervical epithelial cells. LINC02535 and PCBP2 can bind with each other and were colocated in cytoplasm. LINC02535 and PCBP2 promoted cell proliferation, migration, invasion, and suppressed apoptosis in CC. LINC02535 and PCBP2 facilitated the repair of DNA damage to promote CC progression. LINC02535 cooperated with PCBP2 to enhance the stability of RRM1 messenger RNA (mRNA). RRM1 promoted the repair of DNA damage and epithelial-to-mesenchymal transition (EMT) process in CC cells. LINC02535 regulated tumorigenesis in vivo. In conclusion, LINC02535 cooperated with PCBP2, regulated stability of RRM1 mRNA to promote cell proliferation and EMT process in CC cells by facilitating the repair of DNA damage, providing a potential biomarker for CC.

The expression of lncRNA-MALAT1 in breast cancer patients and its influences on prognosis.

This study aimed to explore the expression of lncRNA-metastasis associated lung adenocarcinoma transcript 1 (lncRNA-MALAT1) in breast cancer (BC) patients and its influences on the prognosis of the patients. A total of 120 BC patients admitted to our hospital were enrolled as a BC group, of which 58 patients at I/II stage were treated with breast-conserving surgery as an operation group, and the other 62 patients at III/IV stage were treated with neoadjuvant chemotherapy combined with breast-conserving surgery as a combination group. Meantime, 100 healthy people in physical examination during the same period were enrolled as a normal group. The expression of serum lncRNA-MALAT1 in the subjects was determined, and the expression in BC patients and its influences on the patients were analyzed. LncRNA-MALAT1 was over-expressed in patients from the BC group, and the area-under-the-curve (AUC) of it for diagnosing BC was 0.911. After treatment, the expression of lncRNA-MALAT1 in the operation group and the combination group significantly decreased, and the expression of it in patients with good prognosis was greatly lower than that in patients with poor prognosis. The AUC of lncRNA-MALAT1 for predicting poor prognosis was 0.838, and TNM staging, pathological differentiation, tumor diameter, and lncRNA-MALAT1 were independent prognostic factors for poor prognosis of the patients. Furthermore, low expression of lncRNA-MALAT1 was associated with a relatively high 5-year overall survival (OS) of BC patients. The expression of lncRNA-MALAT1 was up-regulated in BC patients, while it was down-regulated in BC patients treated with breast-conserving surgery combined with neo-adjuvant chemotherapy, so lncRNA-MALAT1 can be used as a potential indicator for early diagnosis and prognosis prediction of BC patients.

Amplified electrochemical determination of UO22+ based on the cleavage of the DNAzyme and DNA-modified gold nanoparticle network structure.

A superior electrochemical biosensor was designed for the determination of UO22+ in aqueous solution by integration of DNAzyme and DNA-modified gold nanoparticle (DNA-AuNP) network structure. Key features of this method include UO22+ inducing the cleavage of the DNAzyme and signal amplification of DNA-AuNP network structure. In this electrochemical method, the DNA-AuNP network structure can be effectively modified on the surface of gold electrode and then employed as an ideal signal amplification unit to generate amplified electrochemical response by inserting a large amount of electrochemically active indicator methylene blue (MB). In the presence of UO22+, the specific sites on DNA-AuNP network structure can be cleaved by UO22+, releasing the DNA-AuNP network structure with detectable reduction of electrochemical response intensity. The electrochemical response intensity is related to the concentration of UO22+. The logarithm of electrochemical response intensity and UO22+ concentration showed a wide linear range of 10~100 pM, and the detection limit reached 8.1 pM (S/N = 3). This method is successfully used for determination of UO22+ in water samples. Graphical abstract Fabricated DNAzyme network structure for enhanced electrical signal. Numerical experiments show that the current signal decreases as the concentration of UO22+ increases. It can be seen that the biosensors could be used to detect UO22+ in aqueous solution effectively.

Temperature Gradient Method for Alleviating Bonding-Induced Warpage in a High-Precision Capacitive MEMS Accelerometer.

Capacitive MEMS accelerometers with area-variable periodic-electrode displacementtransducers found wide applications in disaster monitoring, resource exploration and inertialnavigation. The bonding-induced warpage, due to the difference in the coefficients of thermalexpansion of the bonded slices, has a negative influence on the precise control of the interelectrodespacing that is essential to the sensitivity of accelerometers. In this work, we propose the theory,simulation and experiment of a method that can alleviate both the stress and the warpage byapplying different bonding temperature on the bonded slices. A quasi-zero warpage is achievedexperimentally, proving the feasibility of the method. As a benefit of the flat surface, the spacing ofthe capacitive displacement transducer can be precisely controlled, improving the self-noise of theaccelerometer to 6 ng/√Hz @0.07 Hz, which is about two times lower than that of the accelerometerusing a uniform-temperature bonding process.

Mesoporous Silica Containers and Programmed Catalytic Hairpin Assembly/Hybridization Chain Reaction Based Electrochemical Sensing Platform for MicroRNA Ultrasensitive Detection with Low Background.

In this work, based on mesoporous silica containers (MSNs) with the programmed enzyme-free DNA assembly amplification of catalytic hairpin assembly (CHA) and hybridization chain reaction (HCR), an ultrasensitive electrochemical sensing platform with low background is developed for the detection of microRNA (miRNA). Herein, the electrochemical reporter methylene blue (MB) was sealed in the pores of MSNs by the double-stranded DNA (dsDNA) gate of hairpin DNA H1 and anchor DNA. In the absence of target, neither the CHA nor the HCR process happened, which enabled a low background. After target was added, DNA H1 was displaced from the MSNs surface and participated in the CHA process with the assistance of hairpin DNA H2, which accelerated the release of MB from the MSNs pore. Meanwhile, the CHA products H1-H2 were hybridized with the capture probes (SH-CP) on the electrode surface, which further initiated the HCR process. The released MB from the MSNs will effectively intercalate into long dsDNA polymers of HCR products, resulting in a significant electrochemical response. Taking miRNA-21 as the model target, the proposed sensing platform achieves a satisfactory detection limit down to 0.037 fM, which is lower than that of electrochemical assay with amplification methods. In addition, the strategy shows good selectivity against other miRNAs and is capable in practical analytes. Benefitting from the features of being label-free and enzyme-free and having low background, high sensitivity, and selectivity, this strategy shows great potential in bioanalysis and clinical diagnostics.

Low Background Cascade Signal Amplification Electrochemical Sensing Platform for Tumor-Related mRNA Quantification by Target-Activated Hybridization Chain Reaction and Electroactive Cargo Release.

Herein a low background cascade signal amplification electrochemical sensing platform has been proposed for the ultrasensitive detection of mRNA (mRNA) by coupling the target-activated hybridization chain reaction and electroactive cargo release from mesoporous silica nanocontainers (MSNs). In this sensing platform, the 5'-phosphate-terminated DNA (5'-PO4 cDNA) complement to target mRNA is hybridized with the trigger DNA and anchor DNA on the surface of the MSNs, aiming at forming a double-stranded DNA gate molecule and sealing the methylene blue (MB) in the inner pores of the MSNs. In the presence of target mRNA, the 5'-PO4 cDNA is displaced from the MSNs and competitively hybridizes with mRNA, which led to the liberation of the trigger DNA and the opening of the MSNs pore. The liberated trigger DNA can be then immobilized onto the electrode surface through hybridization with the capture DNA, triggering HCR on the electrode surface. At the same time, the MB released from the MSNs will selectively intercalate into the HCR long dsDNA polymers, giving rise to significant electrochemical response. In addition, due to the λ-exonuclease (λ-Exo) cleavage reaction-assisted target recycling, more amounts of trigger DNA will be liberated and trigger HCR, and numerous MB are uncapped and intercalate into the HCR products. As proof of concept, thymidine kinase 1 (TK1) mRNA was used as a model target. Featured with amplification efficiency, label-free capability, and low background signal, the strategy could quantitatively detect TK1 mRNA down to 2.0 aM with a linear calibration range from 0.1 fM to 1 pM. We have also demonstrated the practical application of our proposed sensing platform for detecting TK1 mRNA in real samples, opening up new avenues for highly sensitive quantification of biomarkers in bioanalysis and clinical diagnosis.