Development of a DNAzyme-Driven Fluorescent Light-Up Aptasensor for Label-Free Detection of Multiple lncRNAs.

Long noncoding RNAs (lncRNAs) act as the dynamic regulatory molecules that control the expression of genes and affect numerous biological processes, and their dysregulation is associated with tumor progression. Herein, we develop a fluorescent light-up aptasensor to simultaneously measure multiple lncRNAs in living cells and breast tissue samples based on the DNAzyme-mediated cleavage reaction and transcription-driven synthesis of light-up aptamers. When target lncRNAs are present, they can be recognized by template probes to form the active DNAzyme structures, initiating the T4 PNK-catalyzed dephosphorylation-triggered extension reaction to generate double-strand DNAs with the T7 promoter sequences. The corresponding T7 promoters can initiate the transcription amplification catalyzed by the T7 RNA polymerase to generate abundant Broccoli aptamers and malachite green aptamers, which can bind DFHBI-1T and MG to generate strong fluorescence signals. Taking advantage of the good selectivity of DNAzyme-mediated cleavage of lncRNAs, high amplification efficiency of T7 transcription-driven amplification reaction, and bright fluorescence of the RNA aptamer-fluorophore complex, this method exhibits high sensitivity with a detection limit of 21.4 aM for lncRNA HOTAIR and 18.47 aM for lncRNA MALAT1, and it can accurately measure multiple lncRNAs in both tumor cell lines and breast tissue samples, providing a powerful paradigm for biomedical research and early clinic diagnostics.

Elongation and Ligation-Mediated Differential Coding for Label-Free and Locus-Specific Analysis of 8-Oxo-7,8-dihydroguanine in DNA.

Oxidative DNA damage is closely associated with the occurrence of numerous human diseases and cancers. 8-Oxo-7,8-dihydroguanine (8-oxoG) is the most prevalent form of DNA damage, and it has become not only an oxidative stress biomarker but also a new epigenetic-like biomarker. However, few approaches are available for the locus-specific detection of 8-oxoG because of the low abundance of 8-oxoG damage in DNA and the limited sensitivity of existing assays. Herein, we demonstrate the elongation and ligation-mediated differential coding for label-free and locus-specific analysis of 8-oxoG in DNA. This assay is very simple without the involvement of any specific labeled probes, complicated steps, and large sample consumption. The utilization of Bsu DNA polymerase can specifically initiate a single-base extension reaction to incorporate dATP into the opposite position of 8-oxoG, endowing this assay with excellent selectivity. The introduction of cascade amplification reaction significantly enhances the sensitivity. The proposed method can monitor 8-oxoG with a limit of detection of 8.21 × 10-19 M (0.82 aM), and it can identify as low as 0.001% 8-oxoG damage from a complex mixture with excessive undamaged DNAs. This method can be further applied to measure 8-oxoG levels in the genomic DNA of human cells under diverse oxidative stress, holding prospect potential in the dynamic monitoring of critical 8-oxoG sites, early clinical diagnosis, and gene damage-related biomedical research.

SARM1 participates in axonal degeneration and mitochondrial dysfunction in prion disease.

Prion disease represents a group of fatal neurogenerative diseases in humans and animals that are associated with energy loss, axonal degeneration, and mitochondrial dysfunction. Axonal degeneration is an early hallmark of neurodegeneration and is triggered by SARM1. We found that depletion or dysfunctional mutation of SARM1 protected against NAD+ loss, axonal degeneration, and mitochondrial functional disorder induced by the neurotoxic peptide PrP106-126. NAD+ supplementation rescued prion-triggered axonal degeneration and mitochondrial dysfunction and SARM1 overexpression suppressed this protective effect. NAD+ supplementation in PrP106-126-incubated N2a cells, SARM1 depletion, and SARM1 dysfunctional mutation each blocked neuronal apoptosis and increased cell survival. Our results indicate that the axonal degeneration and mitochondrial dysfunction triggered by PrP106-126 are partially dependent on SARM1 NADase activity. This pathway has potential as a therapeutic target in the early stages of prion disease.

[Clinical significance of changes of lipid in elderly patients with hepatic schistosomiasis].

OBJECTIVE: To explore the clinical significance of lipid levels including total cholesterol (TC), triglyceride (TG), high-density lipoprotein (HDL-C), low density lipoprotein (LDL-C), and apolipoprotein (APOA I and APOB) of elderly patients with hepatic schistosomiasis. METHODS: A total of 280 hospitalized elderly patients with hepatic schistosomiasis (198 cases of chronic liver fibrosis and 82 cases of hepatocirrhosis) were chosen as study objects, and their clinical data were collected and analyzed retrospectively. Meanwhile, the lipid levels between the patients with liver fibrosis and hepatocirrhosis, and those among the patients with A, B, C degrees of Child-pugh grading of liver function were compared. RESULTS: Among the 280 patients, the abnormality rates of the lipid levels were 34.8%(69/198)and 100% (82/82) in the liver fibrosis group and hepatocirrhosis group respectively, and the difference between them were statistically significant (χ2 = 5.74, P < 0.05). The levels of TC, HDL-C, LDL-C, APOA I of the patients in the latter group were significantly lower than those in the former group (all P <0.05). The levels of TC, TG, HDL-C, APOA I, APOB of the patients with C degree liver function were significantly lower than those of the patients with A degree liver function, and the levels of TC, TG, HDL-C of the former were also lower than those of the patients with B degree liver function (all P <0.05). CONCLUSIONS: The lipid levels of the elderly patients with hepatic schistosomiasis reduce obviously in the course of hepatocirrhosis, and it is correlated with the damage level of the liver. Lipid and apolipoprotein detections have certain values on the illness judgment and prognosis assessment.

[Influencing factors in elderly patients with schistosomiasis liver disease combined with gallbladder diseases].

OBJECTIVE: To understand the status of elderly patients with schistosomiasis liver diseases combined with gallbladder diseases, and explore the influencing factors. METHODS: A total of 280 elderly patients with schistosomiasis liver disease were divided into two groups, 198 cases of chronic liver fibrosis and 82 cases of liver cirrhosis, and the results of their gallbladder ultrasound and liver function examinations were analyzed statistically. RESULTS: Among the 280 cases, 157 patients were combined with gallbladder diseases (56.1%), including gallbladder wall thickening (28.2%, 79/280), cholecystolithiasis (13.6%, 38/ 280), cholecystitis (11.1%, 31/280), and gallbladder polyp (3.2%, 9/280). The incidence rates of gallbladder wall thickening, cholecystitis and cholecystolithiasis in the schistosomiasis patients with cirrhosis were significantly higher than those in the schistosomiasis patients with liver fibrosis (chi2 = 4.568, P < 0.05). CONCLUSION: The main influencing factors of schistosomiasis liver disease combined with gallbladder diseases are the age, the course of the disease, liver cirrhosis and the portal hypertension degree.

Biological activities of skin secretions of the salamander Tylototriton verrucosus.

Water-soluble skin secretions of salamander Tylototriton verrucosus, first described by Anderson in 1871, were studied for their biological and enzymatic activities. They were found to be toxic to mice with an intraperitoneal LD50 of 11.5 mg/kg. Using Sephadex G-75 gel filtration, it was proven that the toxic components of the secretions are proteins with molecular weights ranging from 30,000 to 50,000 Da. The secretions of T. verrucosus display a wide spectrum of antimicrobial activities and also contain both proteolytic activity and trypsin inhibitory activity. In contrast, neither hemolytic nor hemorrhagic activities were found. The secretions were determined to have phospholipase A2 activity; however, no acetylcholine esterase activity was detectable under the assay conditions.