A novel photoelectrochemical strategy for sequence-spot bispecific analysis of N6-methyladenosine modification based on proximity ligation-triggered cascade amplification.

N6-methyladenosine (m6A) modification as the most prevalent mammalian RNA internal modification has been considered as the invasive biomarkers in clinical diagnosis and biological mechanism researches. It is still challenged to explore m6A functions due to technical limitations on base- and location-resolved m6A modification. Herein, we firstly proposed a sequence-spot bispecific photoelectrochemical (PEC) strategy based on in situ hybridization mediated proximity ligation assay for m6A RNA characterization with high sensitivity and accuracy. Firstly, the target m6A methylated RNA could be transferred to the exposed cohesive terminus of H1 based on the special self-designed auxiliary proximity ligation assay (PLA) with sequence-spot bispecific recognition. The exposed cohesive terminus of H1 could furtherly trigger the next catalytic hairpin assembly (CHA) amplification and in situ exponential nonlinear hyperbranched hybridization chain reaction for highly sensitive monitoring of m6A methylated RNA. Compared with conventional technologies, the proposed sequence-spot bispecific PEC strategy for m6A methylation of special RNA based on proximity ligation-triggered in situ nHCR performed improved sensitivity and selectivity with a detection limit of 53 fM, providing new insights into highly sensitive monitoring m6A methylation of RNA in bioassay, disease diagnosis and RNA mechanism.

DNA methylation detection and site analysis by using an electrochemical biosensor constructed based on toehold-mediated strand displacement reaction.

DNA methylation has become a novel target for early diagnosis and prognosis of cancer as well as other related diseases. The accurate detection of the methylation sites of specific genes proved to be of great significance. However, the complex biological nature of clinical samples and the detection of low-abundance targets led to higher requirements for the testing technology. It has been found that by virtue of high sensitivity, rapid response, low cost, facile operation and applicability to microanalysis, electrochemical sensors have greatly contributed to the process of clinical diagnosis. In this study, a facile, rapid and highly sensitive electrochemical biosensor based on the peak current change was developed on the basis of high selectivity of toehold and greater efficiency of PNA strand displacement and used for the detection and site analysis of DNA methylation. Moreover, compared with non-methylated DNA sequences, methylated DNA sequences could be readily invaded by PNA probes, thereby resulting in the strand displacement and significant electrical signals. Therefore, methylation of cytosine sites was primarily analyzed based on electrical signals. Strand displacement by the target DNA sequences with different methylated sites can lead to substantial changes of strand displacement efficiency. As a result, the methylation sites can be analyzed on the basis of corresponding peak current response relation. This method has a detection limit of 0.075 pM and does not involve various complicated steps such as bisulfite treatment, enzyme digestion and PCR amplification. Indeed, one detection cycle can be completed in 60 min. The proposed technology might exhibit great potential in early clinical diagnosis and risk assessment of cancers and related diseases.

Calreticulin enhances gastric cancer metastasis by dimethylating H3K9 in the E-cadherin promoter region mediating by G9a.

The latest study shows that gastric cancer (GC) ranked the fifth most common cancer (5.6%) with over 1 million estimated new cases annually and the fourth most common cause of cancer death (7.7%) globally in 2020. Metastasis is the leading cause of GC treatment failure. Therefore, clarifying the regulatory mechanisms for GC metastatic process is necessary. In the current study, we discovered that calreticulin (CALR) was highly expressed in GC tissues and related to lymph node metastasis and patient's terrible prognosis. The introduction of CALR dramatically promoted GC cell migration in vitro and in vivo, while the repression of CALR got the opposite effects. Cell migration is a functional consequence of the epithelial-mesenchymal transition (EMT) and is related to adhesion of cells. Additionally, we observed that CALR inhibition or overexpression regulated the expression of EMT markers (E-cadherin, ZO-1, Snail, N-cadherin, and ZEB1) and cellular adhesive moleculars (Fibronectin, integrin ß1and MMP2). Mechanistically, our data indicated that CALR could mediate DNA methylation of E-cadherin promoter by interacting with G9a, a major euchromatin methyltransferase responsible for methylation of histone H3 on lysine 9(H3K9me2) and recruiting G9a to the E-cadherin promoter. Knockdown of G9a in CALR overexpressing models restored E-cadherin expression and blocked the stimulatory effects of CALR on GC cell migration. Taken together, these findings not only reveal critical roles of CALR medicated GC metastasis but also provide novel treatment strategies for GC.

Electrochemical and Optical Biosensing Strategies for DNA Methylation Analysis.

DNA methylation is considered as a crucial part of epigenetic modifications and a popular research topic in recent decades. It usually occurs with a methyl group adding to the fifth carbon atom of cytosine while the base sequence of DNA remains unchanged. DNA methylation has significant influences on maintaining cell functions, genetic imprinting, embryonic development and tumorigenesis procedures and hence the analysis of DNA methylation is of great medical significance. With the development of analytical techniques and further research on DNA methylation, numerous DNA methylation detection strategies based on biosensing technology have been developed to fulfill various study requirements. This article reviewed the development of electrochemistry and optical biosensing analysis of DNA methylation in recent years; in addition, we also reviewed some recent advances in the detection of DNA methylation using new techniques, such as nanopore biosensors, and highlighted the key technical and biological challenges involved in these methods. We hope this paper will provide useful information for the selection and establishment of analysis of DNA methylation.

A novel fluorescent biosensor based on dendritic DNA nanostructure in combination with ligase reaction for ultrasensitive detection of DNA methylation.

BACKGROUND: DNA methylation detection is indispensable for the diagnosis and prognosis of various diseases including malignancies. Hence, it is crucial to develop a simple, sensitive, and specific detection strategy. METHODS: A novel fluorescent biosensor was developed based on a simple dual signal amplification strategy using functional dendritic DNA nanostructure and signal-enriching polystyrene microbeads in combination with ligase detection reaction (LDR). Dendritic DNA self-assembled from Y-DNA and X-DNA through enzyme-free DNA catalysis of a hairpin structure, which was prevented from unwinding at high temperature by adding psoralen. Then dendritic DNA polymer labeled with fluorescent dye Cy5 was ligated with reporter probe into a conjugate. Avidin-labeled polystyrene microbeads were specifically bound to biotin-labeled capture probe, and hybridized with target sequence and dendritic DNA. LDR was triggered by adding Taq ligase. When methylated cytosine existed, the capture probe and reporter probe labeled with fluorescent dye perfectly matched the target sequence, forming a stable duplex to generate a fluorescence signal. However, after bisulfite treatment, unmethylated cytosine was converted into uracil, resulting in a single base mismatch. No fluorescence signal was detected due to the absence of duplex. RESULTS: The obtained dendritic DNA polymer had a large volume. This method was time-saving and low-cost. Under the optimal experimental conditions using avidin-labeled polystyrene microbeads, the fluorescence signal was amplified more obviously, and DNA methylation was quantified ultrasensitively and selectively. The detection range of this sensor was 10-15 to 10-7 M, and the limit of detection reached as low as 0.4 fM. The constructed biosensor was also successfully used to analyze actual samples. CONCLUSION: This strategy has ultrasensitivity and high specificity for DNA methylation quantification, without requiring complex processes such as PCR and enzymatic digestion, which is thus of great value in tumor diagnosis and biomedical research.

Electrochemical Biosensor for DNA Methylation Detection through Hybridization Chain-Amplified Reaction Coupled with a Tetrahedral DNA Nanostructure.

DNA methylation is a key factor in the pathogenesis of gene expression diseases or malignancies. Thus, it has become a significant biomarker for the diagnosis and prognosis of these diseases. In this paper, we designed an ultrasensitive and specific electrochemical biosensor for DNA methylation detection. The platform consisted of stem-loop-tetrahedron composite DNA probes anchoring at a Au nanoparticle-coated gold electrode, a restriction enzyme digestion of HpaII, and signal amplification procedures including electrodeposition of Au nanoparticles, hybridization chain reaction, and horseradish peroxidase enzymatic catalysis. Under optimal conditions, the design showed a broad dynamic range from 1 aM to 1 pM and a detection limit of about 0.93 aM. The approach also showed ideal specificity, repeatability, and stability. The recovery test demonstrated that the design is a promising platform for DNA methylation detection under clinical circumstances and could meet the need for cancer diagnosis.

Mixed lineage leukaemia histone methylases 1 collaborate with ERα to regulate HOXA10 expression in AML.

HOXA10, a homeobox-containing gene involved in definitive haematopoiesis, which implicated in the pathogenesis of AML (acute myeloid leukaemia), has been studied extensively. But the regulatory mechanism that drives HOXA10 expression is still unclear. In the present paper, HOXA10 regulated by MLL1 (mixed lineage leukaemia histone methylase 1) with an epigenetic way has been demonstrated. The HOXA10 promoter contains several EREs (oestrogen response elements), including ERE1 and ERE2, which are close to the transcription start site, and are associated with E2-mediated activation of HOXA10. It has been shown that knockdown of the ERα (oestrogen receptor α) suppresses E2-mediated activation of HOXA10. Similarly, knockdown of MLL1 suppresses activation of HOXA10 and is bound to the ERE of HOXA10 promoter in an E2-dependent manner by forming complex with ERα. Knockdown of ERα affects the E2-dependent binding of MLL1 into HOXA10 EREs, suggesting critical roles of ERα in recruiting MLL on the HOXA10 promoter. More interestingly, the methylation status of histone protein H3K4 (H3 at lysine 4) with E2 is much higher than without E2 treatment in leukaemia cell. On the contrary, the methylation status of HOXA10 promoter with E2 treatment is much lower, which elevate the HOXA10 expression. Moreover, with ERα knockdown, the H3K4 methylation level is also decrease in myeloid cell. Overall, it has been clearly demonstrated that HOXA10 is transcriptionally regulated by MLL1, which, in coordination with ERα, plays a critical role in this process with epigenetic way and suggests a potential anti-E2 treatment of AML.

The role of NO in macrophage dysfunction at early stage after burn injury.

AIM: To explore the role of nitric oxide (NO) in macrophage dysfunction at early stage after burn injury. METHOD: Peritoneal macrophages were isolated and cultured from early stage burnt mice. NO production and inducible NO synthase (iNOS) expression in the macrophages were checked by the Greiss method and real-time PCR (TaqMan), respectively. l-Arginine, the substrate of NO producing, or N-monomethyl-l-arginine (l-NMMA), a competing blocker of NOS was administered to the culture, the changes of NO, TNF-alpha and PGE2 productions were measured, additionally the changes of the iNOS, TNF-alpha and COX-2 expression were assayed by real-time PCR. After that, the effects of l-arginine and l-NMMA were determined on burnt macrophage influencing the proliferation of normal splenic lymphocytes. RESULT: A large amount of NO was produced by macrophages from post burn hour 6 (6PBH) with a high level of iNOS expression. l-Arginine could increase NO production in a dosage-dependent manner, while l-NMMA attenuated NO production, but neither could affect iNOS expression. Moreover, l-arginine enhanced productions of both the latter produced TNF-alpha and PGE2 from burnt macrophages, and the expressions of TNF-alpha and COX-2 were improved significantly, while l-NMMA did reverse ways. It was found that macrophages from post burn hour 24 mice could inhibit Con A-stimulated normal splenic lymphocytes dramatically, l-NMMA could decrease this function significantly, but l-arginine could not influence the suppression. CONCLUSION: Our experiment indicated NO derived from burnt macrophage played a vital role in macrophage producing excessive TNF-alpha and PGE2, and suppressing lymphocyte function at early stage after burn injury.

[The influence of the application of cytotoxic lymphocyte antigen 4-Ig adenovirus on the burn wounds with alloskin grafting on the murine immune function].

OBJECTIVE: To investigate the influence of local application of cytotoxic lymphocyte antigen 4-Ig (CTLA4-Ig) adenovirus on the burn wound with alloskin grafting upon the murine immune function. METHODS: Sixty BALB/c mice were randomly divided into A (operation control), B (CTLA4-Ig transfection) and C (normal control) groups, with 20 mice in each group. Skin wounds (full-thickness loss) sized 1.5 cm x 1.5 cm were created on the backs of mice in A and B groups. Then the skin grafts of the same size obtained from C57BL mice were grafted into the skin wounds. 0.1 g of cross-linking polyacrylic resin (carbomer cream) without adenovirus was daubed onto the wounds in A group, and the same amount of carbomer cream with adenovirus in titers of 5 x 10(9)/L was daubed onto the wounds in B group, while no treatment was given in C group. 1 ml of 10% SRBC (sheep red blood cell) was injected intraperitoneally to all the mice of the three groups on the 1st post injury day (PID). Splenocytes from BALB/c, C57BL and Kunming mice were harvested for mixed lymphocyte culture on 7, 14, 21 and 28 PIDs. Agglutination assay was used in the same time to detect the SRBC antibody titers. RESULTS: The reaction of murine splenocytes in B group to the donor (C57BL) splenocytes was suppressed in a specific way (P < 0.05) within 14 PIDs. There was no difference in the titers of anti-SRBC antibody among the 3 groups (P > 0.05). CONCLUSION: Local application of CTLA4-Ig recombinant adenovirus exhibited no influence on the murine humoral immunity, but might induce systemic and specific T cell tolerance in immunity system.