A triggered DNA nanomachine with enzyme-free for the rapid detection of telomerase activity in a one-step method.

BACKGROUND: Telomerase is considered a biomarker for the early diagnosis and clinical treatment of cancer. The rapid and sensitive detection of telomerase activity is crucial to biological research, clinical diagnosis, and drug development. However, the main obstacles facing the current telomerase activity assay are the cumbersome and time-consuming procedure, the easy degradation of the telomerase RNA template and the need for additional proteases. Therefore, it is necessary to construct a new method for the detection of telomerase activity with easy steps, efficient reaction and strong anti-interference ability. RESULTS: Herein, an efficient, enzyme-free, economical, sensitive, fluorometric detection method for telomerase activity in one-step, named triggered-DNA (T-DNA) nanomachine, was created based on target-triggered DNAzyme-cleavage activity and catalytic molecular beacon (CMB). Telomerase served as a switch and extended few numbers of (TTAGGG)n repeat sequences to initiate the signal amplification in the T-DNA nanomachine, resulting in a strong fluorescent signal. The reaction was a one-step method with a shortened time of 1 h and a constant temperature of 37 °C, without the addition of any protease. It also sensitively distinguished telomerase activity in various cell lines. The T-DNA nanomachine offered a detection limit of 12 HeLa cells µL-1, 9 SK-Hep-1 cells µL-1 and 3 HuH-7 cells µL-1 with a linear correlation detection range of 0.39 × 102-6.25 × 102 HeLa cells µL-1 for telomerase activity. SIGNIFICANCE: In conclusion, our study demonstrated that the triggered-DNA nanomachine fulfills the requirements for rapid detection of telomerase activity in one-step under isothermal and enzyme-free conditions with excellent specificity, and its simple and stable structure makes it ideal for complex systems. These findings indicated the application prospect of DNA nanomachines in clinical diagnostics and provided new insights into the field of DNA nanomachine-based bioanalysis.

Effects of hydroxy methionine zinc on growth performance, immune response, antioxidant capacity, and intestinal microbiota of red claw crayfish (Procambarus clarkii).

This study aimed to evaluate the effects of varying zinc (Zn) levels on the growth performance, non-specific immune response, antioxidant capacity, and intestinal microbiota of red claw crayfish (Procambarus clarkii (P. clarkii)). Adopting hydroxy methionine zinc (Zn-MHA) as the Zn source, 180 healthy crayfish with an initial body mass of 6.50 ± 0.05 g were randomly divided into the following five groups: X1 (control group) and groups X2, X3, X4, and X5, which were fed the basal feed supplemented with Zn-MHA with 0, 15, 30, 60, and 90 mg kg-1, respectively. The results indicated that following the addition of various concentrations of Zn-MHA to the diet, the following was observed: Specific growth rate (SGR), weight gain rate (WGR), total protein (TP), total cholesterol (TC), the activities of alkaline phosphatase (AKP), phenoloxidase (PO), total antioxidant capacity (T-AOC), total superoxide dismutase (T-SOD) and catalase (CAT), the expression of CTL, GPX, and CuZn-SOD genes demonstrated a trend of rising and then declining-with a maximum value in group X4-which was significantly higher than that in group X1 (P < 0.05). Zn deposition in the intestine and hepatopancreas, the activity of GSH-PX, and the expression of GSH-PX were increased, exhibiting the highest value in group X5. The malonaldehyde (MDA) content was significantly reduced, with the lowest value in group X4, and the MDA content of the Zn-MHA addition groups were significantly lower than the control group (P < 0.05). In the analysis of the intestinal microbiota of P. clarkii, the number of operational taxonomic units in group X4 was the highest, and the richness and diversity indexes of groups X3 and X4 were significantly higher than those in group X1 (P < 0.05). Meanwhile, the dietary addition of Zn-MHA decreased and increased the relative abundance of Proteobacteria and Tenericutes, respectively. These findings indicate that supplementation of dietary Zn-MHA at an optimum dose of 60 mg kg-1 may effectively improve growth performance, immune response, antioxidant capacity, and intestinal microbiota richness and species diversity in crayfish.

Protonation-mediated DNA tile self-assembly with nuclease resistance characteristic for signal-amplified detection of microRNAs.

DNA nanotechnology, developing rapidly in recent years, has unprecedented superiorities in biological application-oriented research including high programmability, convenient functionalization, reconfigurable structure, and intrinsic biocompatibility. However, the susceptibility to nucleases in the physiological environment has been an obstacle to applying DNA nanostructures in biological science research. In this study, a new DNA self-assembly strategy, mediated by double-protonated small molecules instead of classical metal ions, is developed to enhance the nuclease resistance of DNA nanostructures while retaining their integrality and functionality, and the relative application has been launched in the detection of microRNAs (miRNAs). Faced with low-abundance miRNAs, we integrate hybrid chain reaction (HCR) with DNA self-assembly in the presence of double-protonated small molecules to construct a chemiluminescence detection platform with nuclease resistance, which utilizes the significant difference of molecular weight between DNA arrays and false-positive products to effectively separate of reaction products and remove the detection background. This strategy attaches importance to the nucleic acid stability during the assay process via improving nuclease resistance while rendering the detection results for miRNAs more authentic and reliable, opening our eyes to more possibilities for the multiple applications of customized DNA nanostructures in biology, including bioassay, bioimaging, drug delivery, and cell modulation.

Construction of HPQ-based activatable fluorescent probe for peroxynitrite and its application in ferroptosis and mice model of LPS-induced inflammation.

As one of the important members of reactive oxygen species, ONOO- plays a crucial role in signal transduction, immune response, and other physiological activities. Aberrant changes in ONOO- levels in the living organism are usually associated with many diseases. Therefore, it is important to establish a highly selective and sensitive method for the determination of ONOO- in vivo. Herein, we designed a novel ratio near-infrared fluorescent probe for ONOO- by directly conjugating dicyanoisophorone (DCI) to hydroxyphenyl-quinazolinone (HPQ). Surprisingly, HPQD was unaffected by environmental viscosity and responded rapidly to ONOO- within 40 s. The linear range of ONOO- detection was from 0 µM to 35 µM. Impressively, HPQD did not react with reactive oxygen species and was sensitive to exogenous/endogenous ONOO- in live cells. We also investigated the relationship between ONOO- and ferroptosis and achieved in vivo diagnosis and efficacy evaluation of mice model of LPS-induced inflammation, which showed promising prospects of HPQD in ONOO--related studies.

Size-Controlled DNA Tile Self-Assembly Nanostructures Through Caveolae-Mediated Endocytosis for Signal-Amplified Imaging of MicroRNAs in Living Cells.

Signal-amplified imaging of microRNAs (miRNAs) is a promising strategy at the single-cell level because liquid biopsy fails to reflect real-time dynamic miRNA levels. However, the internalization pathways for available conventional vectors predominantly involve endo-lysosomes, showing nonideal cytoplasmic delivery efficiency. In this study, size-controlled 9-tile nanoarrays are designed and constructed by integrating catalytic hairpin assembly (CHA) with DNA tile self-assembly technology to achieve caveolae-mediated endocytosis for the amplified imaging of miRNAs in a complex intracellular environment. Compared with classical CHA, the 9-tile nanoarrays possess high sensitivity and specificity for miRNAs, achieve excellent internalization efficiency by caveolar endocytosis, bypassing lysosomal traps, and exhibit more powerful signal-amplified imaging of intracellular miRNAs. Because of their excellent safety, physiological stability, and highly efficient cytoplasmic delivery, the 9-tile nanoarrays can realize real-time amplified monitoring of miRNAs in various tumor and identical cells of different periods, and imaging effects are consistent with the actual expression levels of miRNAs, ultimately demonstrating their feasibility and capacity. This strategy provides a high-potential delivery pathway for cell imaging and targeted delivery, simultaneously offering a meaningful reference for the application of DNA tile self-assembly technology in relevant fundamental research and medical diagnostics.

A microfluidic immunosensor based on magnetic separation for rapid detection of okadaic acid in marine shellfish.

Okadaic acid (OA) is a marine biotoxin that accumulates in seafood and can cause diarrheic shellfish poisoning if consumed. Accordingly, many countries have established regulatory limits for the content of OA in shellfish. At present, methods used for the detection of marine toxins are time-consuming and labor-intensive. In order to realize rapid, simple, and accurate detection of OA, we developed a novel microfluidic immunosensor based on magnetic beads modified with a highly specific and sensitive monoclonal antibody (mAb) against OA that is used in conjunction with smartphone imaging to realize the rapid detection of OA in shellfish. The method achieves on-site detection results within 1 h with an IC50 value of 3.30 ng/mL for OA and a limit of detection (LOD) of 0.49 ng/mL. In addition, the analysis of real samples showed that the recoveries for spiked shellfish samples ranged from 84.91% to 95.18%, and the results were confirmed by indirect competitive enzyme-linked immunosorbent assay (icELISA), indicating that the method has good accuracy and precision. Furthermore, the results are reported in a specially designed smartphone app. The microfluidic immunosensor has the advantages of simple operation, rapid detection, and high sensitivity, providing a reliable technical solution for detecting OA residues in shellfish.

A Formal and Visual Data-Mining Model for Complex Ship Behaviors and Patterns.

The successful emergence of real-time positioning systems in the maritime domain has favored the development of data infrastructures that provide valuable monitoring and decision-aided systems. However, there is still a need for the development of data mining approaches oriented to the detection of specific patterns such as unusual ship behaviors and collision risks. This research introduces a CSBP (complex ship behavioral pattern) mining model aiming at the detection of ship patterns. The modeling approach first integrates ship trajectories from automatic identification system (AIS) historical data, then categorizes different vessels' navigation behaviors, and introduces a visual-oriented framework to characterize and highlight such patterns. The potential of the model is illustrated by a case study applied to the Jiangsu and Zhejiang waters in China. The results show that the CSBP mining model can highlight complex ships' behavioral patterns over long periods, thus providing a valuable environment for supporting ship traffic management and preventing maritime accidents.

Hyperconnectivity Associated with Anosognosia Accelerating Clinical Progression in Amnestic Mild Cognitive Impairment.

The incidence and prevalence of anosognosia are highly variable in amnestic mild cognitive impairment (aMCI) patients. The study aims to explore the neuropathological mechanism of anosognosia in aMCI patients using two different but complementary technologies, including 18F-flortaucipir positron emission tomography and resting state functional magnetic resonance imaging. The study found that anosognosia was related to higher tau accumulation in the left medial orbitofrontal cortex (OFC), left posterior cingulate cortex, and right precuneus in aMCI patients. Intrinsic functional connectivity analyses found significant correlations between anosognosia index and hypoconnectivity between the left medial OFC and left middle temporal gyrus (MTG), right precuneus and left lingual gyrus. Longitudinally, the connectivity of these brain regions as well as the right precuneus and right cuneus showed hyperconnectivity in aMCI patients with anosognosia. The anosognosia index was also correlated with AD pathological markers (i.e., Aß, t-tau, and p-tau) and brain glucose metabolism in aMCI patients. In conclusion, anosognosia in aMCI patients is associated with the dysfunction of medial OFC-MTG circuit and the precuneus-visual cortex circuit and accelerates clinical progression to AD dementia.

Waterborne zinc bioaccumulation influences glucose metabolism in orange-spotted grouper embryos.

Fish embryos, as an endogenous system, strictly regulate an energy metabolism that is particularly sensitive to environmental pressure. This study used orange-spotted grouper embryos and stable isotope 67Zn to test the hypothesis that waterborne Zn exposure had a significant effect on energy metabolism in embryos. The fish embryos were exposed to a gradient level of waterborne 67Zn, and then sampled to quantify 67Zn bioaccumulation and mRNA expressions of key genes involved glucose metabolism. The results indicated that the bioaccumulated 67Zn generally increased with increasing waterborne 67Zn concentrations, while it tended to be saturated at waterborne 67Zn > 0.7 mg L-1. As we hypothesized, the expression of PK and PFK gene involved glycolysis pathway was significantly up-regulated under waterborne 67Zn exposure >4 mg L-1. Waterborne 67Zn exposure >2 mg L-1 significantly suppressed PCK and G6PC gene expression involved gluconeogenesis pathway, and also inhibited the AKT2, GSK-3beta and GLUT4 genes involved Akt signaling pathway. Our findings first characterized developmental stage-dependent Zn uptake and genotoxicity in fish embryos. We suggest fish embryos, as a small-scale modeling biosystem, have a large potential and wide applicability for determining cytotoxicity/genotoxicity of waterborne metal in aquatic ecosystem.

Synthesis and Characterization of a Mg2+-Selective Probe Based on Benzoyl Hydrazine Derivative and Its Application in Cell Imaging.

A simple benzoyl hydrazine derivative P was successfully synthesized and characterized as Mg2+-selective fluorescent probe. The binding of P with Mg2+ caused an obvious fluorescence enhancement at 482 nm. The fluorescent, UV-vis spectra, 1H-NMR, and IR spectra confirmed the formation of P-Mg2+ complex, and the formation of a 1:1 stoichiometry complex was proved by Job's plot and mass spectrometry. The recognition mechanism of P to Mg2+ was owing to the photoinduced electron transfer effect (PET). The fluorescent response was linear in the range of 0.9-4.0 µM with the detection limit of 0.3 µM Mg2+ in water-ethanol solution (1:9, v:v, pH10.0, 20 mM HEPES). In addition, the results of cell imaging of Mg2+ in Hl-7701 cells was satisfying.

A pH tuning single fluorescent probe based on naphthalene for dual-analytes (Mg2+ and Al3+) and its application in cell imaging.

In this study, a naphthalene Schiff-base P which serves as a dual-analyte probe for the quantitative detection of Al3+ and Mg2+ has been designed. The proposed probe showed an ''off-on'' fluorescent response toward Al3+ in ethanol-water solution (1 : 9, v/v, pH 6.3, 20 mM HEPES) over other metal ions and anions, while the detection by the probe could be switched to Mg2+ by regulating the pH from 6.3 to 9.4. The sensing mechanisms of P to Al3+/Mg2+ are attributed to inhibition of the photo-induced electron transfer (PET) process by the formation of 1 : 1 ligand-metal complexes. More importantly, the probe was applied successfully in living cells for the fluorescent cell-imaging of Al3+ and Mg2+.

Feasibility of Using Seaweed (Gracilaria coronopifolia) Synbiotic as a Bioactive Material for Intestinal Health.

The market contains only limited health care products that combine prebiotics and probiotics. In this study, we developed a seaweed-based Gracilaria coronopifolia synbiotic and verified the efficacy by small intestinal cells (Caco-2). We also developed a functional material that promotes intestinal health and prevents intestinal inflammation. G. coronopifolia was used as a red seaweed prebiotic, and Bifidobacterium bifidums, B. longum subsp. infantis, B. longum subsp. longum, Lactobacillus acidophilus, and L. delbrueckii subsp. bulgaricus were mixed for the seaweed's synbiotics. G. coronopifolia synbiotics were nontoxic to Caco-2 cells, and the survival rate was 101% to 117% for a multiplicative effect on cell survival. After cells were induced by H2O2, the levels of reactive oxygen species (ROS) increased to 151.5%, but after G. coronopifolia synbiotic treatment, decreased to a range between 101.8% and 109.6%. After cells were induced by tumor necrosis factor α, the ROS levels increased to 124.5%, but decreased to 57.7% with G. coronopifolia symbiotic treatment. G. coronopifolia synbiotics could effectively inhibit the production of ROS intestinal cells under oxidative stress (induced by H2O2 and tumor necrosis factor α (TNF-α)), which can reduce the damage of cells under oxidative stress. Functioning of intestinal cells could be improved by inhibiting the production of inflammatory factor substances (interleukin 8) with G. coronopifolia symbiotic treatment. Also, gastrointestinal diseases may be retarded by a synbiotic developed from G. coronopifolia to promote intestinal health and prevent intestinal inflammation.

Al(iii)-responsive "off-on" chemosensor based on rhodamine derivative and its application in cell imaging.

In this work, a new rhodamine chemosensor (P) with excellent photochromic properties upon vis irradiation was designed and synthesized. The fabricated chemosensor P could detect Al3+ via the opening of the spirolactam ring of the rhodamine unit with high selectivity and sensitivity. The spirolactam ring opening was confirmed by NMR and infrared spectroscopy. Upon binding with Al3+, the generated 1 : 1 P-Al3+ complex, confirmed by Job's plot titrations and mass spectrometry analysis, could exhibit a remarkable fluorescence enhancement with a limit of detection (LOD) of 0.16 µM. Furthermore, the sensing of P to Al3+ in vivo was also studied quantitatively and qualitatively in detail, and the results showed that the coordination between P with Al3+ was reversible in living cells.