New G-Triplex DNA Dramatically Activates the Fluorescence of Thioflavin T and Acts as a Turn-On Fluorescent Sensor for Uracil-DNA Glycosylase Activity Detection.

G-triplexes are G-rich oligonucleotides composed of three G-tracts and have absorbed much attention due to their potential biological functions and attractive performance in biosensing. Through the optimization of loop compositions, DNA lengths, and 5'-flanking bases of G-rich sequences, a new stable G-triplex sequence with 14 bases (G3-F15) was discovered to dramatically activate the fluorescence of Thioflavin T (ThT), a water-soluble fluorogenic dye. The fluorescence enhancement of ThT after binding with G3-F15 reached 3200 times, which was the strongest one by far among all of the G-rich sequences. The conformations of G3-F15 and G3-F15/ThT were studied by circular dichroism. The thermal stability measurements indicated that G3-F15 was a highly stable G-triplex structure. The conformations of G3-F15 and G3-F15/ThT in the presence of different metal cations were studied thoroughly by fluorescent spectroscopy, circular dichroism, and nuclear magnetic resonance. Furthermore, using the G3-F15/ThT complex as a fluorescent probe, a robust and simple turn-on fluorescent sensor for uracil-DNA glycosylase activity was developed. This study proposes a new systematic strategy to explore new functional G-rich sequences and their ligands, which will promote their applications in diagnosis, therapy, and biosensing.

Pubertal exposure to Microcystin-LR arrests spermatogonia proliferation by inducing DSB and inhibiting SIRT6 dependent DNA repair in vivo and in vitro.

The reproduction toxicity of pubertal exposure to Microcystin-LR (MC-LR) and the underlying mechanism needs to be further investigated. In the current study, pubertal male ICR mice were intraperitoneally injected with 2 µg/kg MC-LR for four weeks. Pubertal exposure to MC-LR decreased epididymal sperm concentration and blocked spermatogonia proliferation. In-vitro studies found MC-LR inhibited cell proliferation of GC-1 cells and arrested cell cycle in G2/M phase. Mechanistically, MC-LR exposure evoked excessive reactive oxygen species (ROS) and induced DNA double-strand break in GC-1 cells. Besides, MC-LR inhibited DNA repair by reducing PolyADP-ribosylation (PARylation) activity of PARP1. Further study found MC-LR caused proteasomal degradation of SIRT6, a monoADP-ribosylation enzyme which is essential for PARP1 PARylation activity, due to destruction of SIRT6-USP10 interaction. Additionally, MG132 pretreatment alleviated MC-LR-induced SIRT6 degradation and promoted DNA repair, leading to the restoration of cell proliferation inhibition. Correspondingly, N-Acetylcysteine (NAC) pre-treatment mitigated the disturbed SIRT6-USP10 interaction and SIRT6 degradation, causing recovered DNA repair and subsequently restoration of cell proliferation inhibition in MC-LR treated GC-1 cells. Together, pubertal exposure to MC-LR induced spermatogonia cell cycle arrest and sperm count reduction by oxidative DNA damage and simultaneous SIRT6-mediated DNA repair failing. This study reports the effect of pubertal exposure to MC-LR on spermatogenesis and complex mechanism how MC-LR induces spermatogonia cell proliferation inhibition.

Role of sleep in asthenospermia induced by di (2-ethyl-hexyl) phthalate.

Di (2-ethyl-hexyl) phthalate (DEHP) mainly enters the human body through the digestive tract, respiratory tract, and skin. At the same time, it has reproductive and developmental toxicity, neurotoxicity, and so on, which can cause the decrease of sperm motility. Asthenospermia is also known as low sperm motility, and the semen quality of men in some areas of China is declining year by year. Interestingly, previous studies have shown that sleep disorders can also lead to asthenospermia. However, the relationship between sleep, DEHP, and asthenospermia is still unclear. Analysis of the National Health and Nutrition Examination Survey (NHANES) population database showed that DEHP was associated with sleep disorders, and subsequent experiments in mice and Drosophila indicated that DEHP exposure had certain effects on sleep and asthenospermia. Furthermore, we analyzed the Comparative Toxicogenomics Database (CTD) to find out the common signaling pathway among the three: hypoxia-inducible factor 1(HIF-1). Then Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) was used to screen out the proteins that DEHP affected the HIF-1 pathway: glyceraldehyde-3-phosphate dehydrogenase (GAPDH), serine/threonine-protein kinase (AKT1), epidermal growth factor receptor (EGFR), and finally Western blot analysis was used to detect the expression levels of the three proteins. Compared with the control group, DEHP decreased the protein expression levels of GAPDH and AKT1 in the HIF-1 pathway, and caused sleep disorders and decreased sperm motility. This study provides preliminary evidence for exploring the mechanism among DEHP, sleep disorders, and asthenospermia.

Identification and Quantification of Locus-Specific 8-Oxo-7,8-dihydroguanine in DNA at Ultrahigh Resolution Based on G-Triplex-Assisted Rolling Circle Amplification.

Damage of reactive oxygen species to various molecules such as DNA has been related to many chronic and degenerative human diseases, aging, and even cancer. 8-Oxo-7,8-dihydroguanine (OG), the most significant oxidation product of guanine (G), has become a biomarker of oxidative stress as well as gene regulation. The positive effect of OG in activating transcription and the negative effect in inducing mutation are a double-edged sword; thus, site-specific quantification is helpful to quickly reveal the functional mechanism of OG at hotspots. Due to the possible biological effects of OG at extremely low abundance in the genome, the monitoring of OG is vulnerable to signal interference from a large amount of G. Herein, based on rolling circle amplification-induced G-triplex formation and Thioflavin T fluorescence enhancement, an ultrasensitive strategy for locus-specific OG quantification was constructed. Owing to the difference in the hydrogen-bonding pattern between OG and G, the nonspecific background signal of G sites was completely suppressed through enzymatic ligation of DNA probes and the triggered specificity of rolling circle amplification. After the signal amplification strategy was optimized, the high detection sensitivity of OG sites with an ultralow detection limit of 0.18 amol was achieved. Under the interference of G sites, as little as 0.05% of OG-containing DNA was first distinguished. This method was further used for qualitative and quantitative monitoring of locus-specific OG in genomic DNA under oxidative stress and identification of key OG sites with biological function.

Evaluating the Effect of Gestational Exposure to Perfluorohexane Sulfonate on Placental Development in Mice Combining Alternative Splicing and Gene Expression Analyses.

BACKGROUND: Perfluorohexane sulfonate (PFHxS) is a frequently detected per- and polyfluoroalkyl substance in most populations, including in individuals who are pregnant, a period critical for early life development. Despite epidemiological evidence of exposure, developmental toxicity, particularly at realistic human exposures, remains understudied. OBJECTIVES: We evaluated the effect of gestational exposure to human-relevant body burden of PFHxS on fetal and placental development and explored mechanisms of action combining alternative splicing (AS) and gene expression (GE) analyses. METHODS: Pregnant ICR mice were exposed to 0, 0.03, and 0.3µg/kg/day from gestational day 7 to day 17 via oral gavage. Upon euthanasia, PFHxS distribution was measured using liquid chromatography-tandem mass spectrometry. Maternal and fetal phenotypes were recorded, and histopathology was examined for placenta impairment. Multiomics was adopted by combining AS and GE analyses to unveil disruptions in mRNA quality and quantity. The key metabolite transporters were validated by quantitative real-time PCR (qRT-PCR) for quantification and three-dimensional (3D) structural simulation by AlphaFold2. Targeted metabolomics based on liquid chromatography-tandem mass spectrometry was used to detect amino acid and amides levels in the placenta. RESULTS: Pups developmentally exposed to PFHxS exhibited signs of intrauterine growth restriction (IUGR), characterized by smaller fetal weight and body length (p<0.01) compared to control mice. PFHxS concentration in maternal plasma was 5.01±0.54 ng/mL. PFHxS trans-placenta distribution suggested dose-dependent transfer through placental barrier. Histopathology of placenta of exposed dams showed placental dysplasia, manifested with an attenuated labyrinthine layer area and deescalated blood sinus counts and placental vascular development index marker CD34. Combined GE and AS analyses pinpointed differences in genes associated with key biological processes of placental development, proliferation, metabolism, and transport in placenta of exposed dams compared to that of control dams. Further detection of placental key transporter gene expression, protein structure simulation, and amino acid and amide metabolites levels suggested that PFHxS exposure during pregnancy led to impairment of placental amino acid transportation. DISCUSSION: The findings from this study suggest that exposure to human-relevant very-low-dose PFHxS during pregnancy in mice caused IUGR, likely via downregulating of placental amino acid transporters, thereby impairing placental amino acid transportation, resulting in impairment of placental development. Our findings confirm epidemiological findings and call for future attention on the health risk of this persistent yet ubiquitous chemical in the early developmental stage and provide a new approach for understanding gene expression from both quantitative and qualitative omics approaches in toxicological studies. https://doi.org/10.1289/EHP13217.

1-Nitropyrene disrupts testicular steroidogenesis via oxidative stress-evoked PERK-eIF2α pathway.

Our previous study showed 1-Nitropyrene (1-NP) exposure disrupted testicular testosterone synthesis in mouse, but the exact mechanism needs further investigation. The present research found 4-phenylbutyric acid (4-PBA), an endoplasmic reticulum (ER) stress inhibitor, recovered 1-NP-induced ER stress and testosterone synthases reduction in TM3 cells. GSK2606414, a protein kinase-like ER kinase (PERK) kinase inhibitor, attenuated 1-NP-induced PERK-eukaryotic translation initiation factor 2α (eIF2α) signaling activation and downregulation of steroidogenic proteins in TM3 cells. Both 4-PBA and GSK2606414 attenuated 1-NP-induced steroidogenesis disruption in TM3 cells. Further studies used N-Acetyl-L-cysteine (NAC) as a classical antioxidant to explore whether oxidative stress-activated ER stress mediated 1-NP-induced testosterone synthases reduction and steroidogenesis disruption in TM3 cells and mouse testes. The results showed NAC pretreatment mitigated oxidative stress, and subsequently attenuated ER stress, particularly PERK-eIF2α signaling activation, and downregulation of testosterone synthases in 1-NP-treated TM3 cells. More importantly, NAC extenuated 1-NP-induced testosterone synthesis in vitro and in vivo. The current work indicated that oxidative stress-caused ER stress, particularly PERK-eIF2α pathway activation, mediates 1-NP-downregulated steroidogenic proteins and steroidogenesis disruption in TM3 cells and mouse testes. Significantly, the current study provides a theoretical basis and demonstrates the experimental evidence for the potential application of antioxidant, such as NAC, in public health prevention, particularly in 1-NP-induced endocrine disorder.

Colonic mechanism of serum NAD+ depletion induced by DEHP during pregnancy.

Di (2-ethylhexyl) phthalate (DEHP) is a widely used plasticizer in polyvinyl chloride products such as feed piping, packing bag, and medical consumable. Our previous studies have demonstrated that DEHP exposure reduced the concentration of nicotinamide adenine dinucleotide (NAD+) in pregnant mice serum, which cuts off the source of NAD+ to placenta and results fetal growth restriction. However, the mechanism of serum NAD+ depletion by DEHP remains elusive. This study investigated the intestinal mechanism of NAD+ shortage-induced by DEHP in pregnant mice. The transcriptome results implicated that the mRNA level of oxidative response genes Cyp1a1, Gsto2, Trpv1 and Trpv3 were upregulated in colon. These changes induced intestinal inflammation. Transmission Electron Microscopy results displayed that DEHP destroyed the tight junctions and cell polarity of colonic epithelial cells. These dysfunctions diminished the expression of NAD+ precursor transporters SLC12A8, SLC5A8, SLC7A5, and the NAD+ biosynthetic key enzymes NAMPT, NMNAT1-3, and TDO2 in colonic epithelial cells. Analysis of the gut microbiota showed that DEHP led to the dysbiosis of gut microbiota, reducing the relative abundance of Prevotella copri which possesses the VB3 biosynthetic pathway. Therefore, maternal DEHP exposure during pregnancy decreased the transportation of NAD+ precursors from enteric cavity to colonic epithelial cells, and inhibited the synthesis of NAD+ in colonic epithelial cells. Meanwhile, DEHP reduced the NAD+ precursors provided by gut microbiota. Eventually, serum NAD+ content was lowered. Taken together, our findings provide a new insight for understanding the intestinal mechanisms by which DEHP affects serum NAD+ levels.

Compartmentalized regulation of NAD+ by Di (2-ethyl-hexyl) phthalate induces DNA damage in placental trophoblast.

Di (2-ethyl-hexyl) phthalate (DEHP) is a wildly used plasticizer. Maternal exposure to DEHP during pregnancy blocks the placental cell cycle at the G2/M phase by reducing the efficiency of the DNA repair pathways and affects the health of offsprings. However, the mechanism by which DEHP inhibits the repair of DNA damage remains unclear. In this study, we demonstrated that DEHP inhibits DNA damage repair by reducing the activity of the DNA repair factor recruitment molecule PARP1. NAD+ and ATP are two substrates necessary for PARP1 activity. DEHP abated NAD+ in the nucleus by reducing the level of NAD+ synthase NMNAT1 and elevated NAD+ in the mitochondrial by promoting synthesis. Furthermore, DEHP destroyed the mitochondrial respiratory chain, affected the structure and quantity of mitochondria, and decreased ATP production. Therefore, DEHP inhibits PARP1 activity by reducing the amount of NAD+ and ATP, which hinders the DNA damage repair pathways. The supplement of NAD+ precursor NAM can partially rescue the DNA and mitochondria damage. It provides a new idea for the prevention of health problems of offsprings caused by DEHP injury to the placenta.

1-Nitropyrene disrupts testosterone biogenesis via AKAP1 degradation promoted mitochondrial fission in mouse Leydig cell.

Previous study found 1-NP disrupted steroidogenesis in mouse testis, but the underlying mechanism remained elusive. The current work aims to explore the roles of ROS-promoted AKAP1 degradation and excessive mitochondrial fission in 1-NP-induced steroidogenesis disruption in MLTC-1 cells. Transmission electron microscope analysis found 1-NP promoted excessive mitochondrial fission. Further data showed 1-NP disrupted mitochondrial function. pDRP1 (Ser637), a negative regulator of mitochondrial fission, was reduced in 1-NP-treated MLTC-1 cells. Mechanistically, 1-NP caused degradation of AKAP1, an upstream regulator of pDRP1 (Ser637). MG132, a proteasome inhibitor, attenuated 1-NP-induced AKAP1 degradation and downstream pDRP1 (Ser637) reduction, thereby ameliorating 1-NP-downregulated steroidogenesis. Further analysis found that cellular ROS was elevated and NOX4, HO-1 and SOD2 were upregulated in 1-NP-exposed MLTC-1 cells. NAC, a well-known commercial antioxidant, alleviated 1-NP-induced excessive ROS and oxidative stress. 1-NP-induced AKAP1 degradation and subsequent downregulation of pDRP1 (Ser637) were prevented by NAC pretreatment. Moreover, NAC attenuated 1-NP-resulted T synthesis disturbance in MLTC-1 cells. The present study indicates that ROS mediated AKAP1 degradation and subsequent pDRP1 (Ser637) dependent mitochondrial fission is indispensable in 1-NP caused T synthesis disruption. This study provides a new insight into 1-NP-induced endocrine disruption, and offers theoretical basis in public health prevention.

Gestational exposure to environmental cadmium induces placental apoptosis and fetal growth restriction via Parkin-modulated MCL-1 degradation.

Heavy metal cadmium (Cd), a classical environmental pollutant, causes placental apoptosis and fetal growth restriction (FGR), whereby the mechanism remains unclear. Here, our human case-control study firstly showed that there was a positive association of Parkin mitochondrial translocation, MCL-1 reduction, placental apoptosis, and all-cause FGR. Subsequently, Cd was administered to establish in vitro and in vivo models of placental apoptosis or FGR. Our models demonstrated that Parkin mitochondrial translocation was observed in Cd-administrated placental trophoblasts. Meaningfully, Parkin siRNA (siR) dramatically mitigated Cd-triggered apoptosis in placental trophoblasts. Mdivi-1 (M-1), an inhibitor for Parkin mitochondrial translocation, mitigated Cd-induced apoptosis in placental trophoblasts, which further ameliorated the effect of attenuated placental sizes in Cd-exposed mice. Furthermore, the interaction of MCL-1 with Parkin or Ub in Cd-stimulated cells was stronger than that in controls. MG132, an inhibitor for proteasome, abolished MCL-1 degradation in Cd-stimulated cells. Importantly, Parkin siR and M-1 memorably abolished the ubiquitin-dependent degradation of MCL-1 in placental trophoblasts. Interestingly, mito-TEMPO and melatonin, two mitochondria-targeted antioxidants, obviously rescued Cd-caused mitochondrial membrane potential (MMP) decrease, Parkin mitochondrial translocation, MCL-1 degradation, and apoptosis in placental trophoblasts. In conclusion, cadmium induces placental apoptosis and FGR via mtROS-mediated Parkin-modulated degradation of MCL-1.

Di (2-ethyl-hexyl) phthalate disrupts placental growth in a dual blocking mode.

Di (2-ethyl-hexyl) phthalate (DEHP) is a widely used plasticizer. Maternal DEHP exposure inhibits cell proliferation and reduces placentas size, which associates with fetal growth restriction and adulthood diseases. However, the mechanism of placental cell proliferation inhibition by DEHP remains elusive. This study investigated the effect of DEHP on placental cell proliferation from cell cycle arrest. Utilizing in vitro and in vivo experiments, we investigated cell cycle arrest, DNA double-strand break (DSB) repair, genotoxic stress response, and micronuclei formation. Most DEHP metabolizes to mono (2-Ethylhexyl) phthalate (MEHP) and distributes to organs quickly, so MEHP and DEHP were used in cultured cell and animal experiments, respectively. Here, a double blocking mode for the proliferation inhibition of the placental cell was revealed. One is that the classical DSB repair pathways were suppressed, which arrested the cell cycle at the G2/M phase. The other is that DEHP stimulated an elevated level of progesterone, which blocked the cell cycle at metaphase by disrupting chromosome arrangement. These two sets of events facilitated micronuclei formation and resulted in cell proliferation inhibition. This findings provide a novel mechanistic understanding for DEHP to inhibit placental cell proliferation.

Environmental cadmium exposure during pregnancy causes diabetes-like phenotypes in mouse offspring: Association with oxidative stress in the fetal liver.

Cadmium (Cd), a noxious heavy metal, is widespread in the living environment. Gestational exposure to Cd at environmental dose has been shown to cause fetal growth restriction (FGR). However, the long-term effects and the mechanisms underlying environmental Cd exposure on glucose metabolism in offspring remain unclear. Here, we established a murine model to study the impacts of gestational exposure to environmental Cd on glucose metabolism at different life stages of offspring. Results demonstrated that the offspring mice developed hyperglycemia in puberty and impaired glucose tolerance in adulthood following maternal Cd exposure during gestation. Further mechanistic investigation showed that Cd exposure upregulated the expression of key proteins in hepatic gluconeogenesis, including p-CREB, PGC-1α and G6PC, in pubertal and adult offspring. In addition, we demonstrated that Cd exposure during pregnancy markedly elevated the level of oxidative stress-related proteins, including NOX2, NOX4 and HO-1, in the fetal liver. The effects of gestational exposure to N-acetylcysteine (NAC), a free-radical scavenging antioxidant, presented that NAC supplementation alleviated hepatic oxidative stress in fetuses, and thereby reversed hyperglycemia and glucose intolerance in mouse offspring. Collectively, our data suggested that gestational exposure to environmental Cd caused diabetes-like phenotypes via enhancing hepatic gluconeogenesis, which is associated with oxidative stress in fetal livers. This work provides new insights into the protective effects of antioxidants on fetal-originated diabetes triggered by environmental toxicants.

Environmental cadmium exposure induces fetal growth restriction via triggering PERK-regulated mitophagy in placental trophoblasts.

Cadmium (Cd), an environmental toxicant, is positively associated with fetal growth restriction (FGR). However, the mechanism by which gestational exposure to Cd induces FGR remains unclear. This study designed in vitro and in vivo experiments to explore the role of placental mitophagy in Cd-impaired fetal growth. Based on our case-control study, we also investigated the association of placental mitophagy with reduced progesterone (P4) level and all-cause FGR. We firstly found environmental Cd exposure lowered the P4 content in maternal sera, placentae and amnioticfluids of mice. The level of three mitochondrial P4 synthases, including StAR, CYP11A1 and 3ß-HSD, was also reduced in Cd-treated placentae. Furthermore, Cd triggered mitophagy, as determined by the degradation of two mitochondrial proteins HSP60 and COX IV, and the accumulation of co-localizations of TOM20 with LC3B or Parkin in placental trophoblasts. Correspondingly, Cd elevated mitochondrial Parkin level in placental trophoblasts. Mdivi-1, a mitophagy inhibitor, obviously attenuated Cd-induced reduction of placental P4 and FGR in mice. Moreover, mdivi-1 and Parkin siRNA (siR) markedly reversed Cd-caused P4 synthesis inhibition in human placental trophoblasts. Interestedly, the PERK/ATF4 signaling was activated in Cd-stimulated placental trophoblasts. PERK siR inhibited mitochondrial proteins degradation in Cd-stimulated placental trophoblasts. In particularly, mitophagy activation and P4 synthesis suppression occurred in small-for-gestational-age placentae based on our case-control study. Environmental Cd exposure induced FGR via activating PERK-regulated mitophagy and inhibiting P4 synthesis in placentaltrophoblasts. Furthermore, placental mitophagy was related to the reduced progesterone level and all-cause fetal growth restriction based on our case-control study. As above, placental mitophagy maybe the common mechanism of environmental toxicants-impaired fetal growth.

Environmental exposure to cadmium impairs fetal growth and placental angiogenesis via GCN-2-mediated mitochondrial stress.

Cadmium (Cd), a well-known environmental pollutant, can lead to placental insufficiency and fetal growth restriction. However, the underlying mechanism is unknown. The purpose of our study is to explore the effect of Cd on placental angiogenesis and its mechanism using in vitro and in vivo models. Results found that gestational Cd exposure obviously decreased placental weight and impaired placental vascular development in mice. Correspondingly, Cd exposure evidently downregulated the expression of VEGF-A protein (a key indicator of angiogenesis) and progesterone receptor (PR) in placental trophoblasts. Further experiment showed that lentivirus PR overexpression reversed Cd-caused the reduction of VEGF-A level in human placental trophoblasts. In addition, Cd significantly reduced progesterone level, down-regulated the expression of key progesterone synthase (StAR, CYP11A1), and activated mitochondrial stress response and GCN-2/p-eIF2α signaling in placental trophoblasts. Additional experiment showed that GCN-2 siRNA pretreatment markedly alleviated Cd-activated mitochondrial stress response, restored Cd-downregulated the expression of CYP11A1, reversed Cd-reduced the level of progesterone and VEGF-A in human placental trophoblasts. Finally, our case-control study confirmed that impaired placental angiogenesis and reduced progesterone level occurred in all-cause small for gestational age placenta. Taken together, environmental exposure to Cd impairs fetal growth and placental angiogenesis via GCN-2-mediated mitochondrial stress.

Prenatal di-(2-ethylhexyl) phthalate maternal exposure impairs the spatial memory of adult mouse offspring in a phase- and gender-dependent manner.

DEHP is a wildly used plasticizer. Maternal DEHP exposure induced fetal growth restriction (FGR) and behavioral abnormalities in adolescence and adulthood mouse. The effect of low birth weight induced by DEHP on behaviors after growing up is not certain. In this study, the ICR pregnant mice were exposed to 200 mg/kg DEHP during gestation 6-12 days or 13-17 days, which can create FGR model. The F1 offspring were performed three ethological experiments at puberty (6 weeks postpartum) and adult period (8 weeks postpartum). The open field test was performed to detect the locomotor activity and anxiety, the elevated plus maze to test anxiety-like behavior, and the Morris water maze assay to measure the spatial learning and memory capability of male and female offspring. The results showed that spatial memory ability was dramatically impaired for male rather than female offspring in gestation 13-17 days' group. Other behaviors had no statistically different between groups. These findings suggest that prenatal DEHP exposure disturbed mouse offspring spatial memory ability in a phase- and gender-dependent manner.

Ultrasensitive detection of microRNA based on a homogeneous label-free electrochemical platform using G-triplex/methylene blue as a signal generator.

The electrochemical methods for microRNA (miRNA) detection have received increasing attention because high portability and affordability of electrochemical biosensors may facilitate point-of-care quantitative detection of miRNAs. Among these biosensors, the homogenous label-free electrochemical biosensors for miRNAs are rarely reported due to the lack of a universal and efficient signal read-out-mode. A newly discovered G-triplex, 5'-CTGGGAGGGAGGGA-3' (denoted as G3), can specifically bind with methylene blue (MB), leading to a significant decrease of the diffusion current of MB. By using miRNAs as a driving force, a two-stage isothermal exponential amplification reaction was proposed to generate G3 through miRNAs. The generated G3 can combine with MB and produce observable current changes, which depend on the concentration of miRNAs. Therefore, a novel homogeneous label-free electrochemical biosensor for miRNA detection was successfully constructed. By choosing let-7a, the down-regulation of which is possibly associated with the over-expression of RAS and HMGA2 oncogenes, as a model, we discovered that this biosensor demonstrated excellent analytical performance in detecting let-7a, with an ultralow limit of detection (0.45 fM) and high specificity (discriminating one nucleotide variation). Moreover, the proposed biosensor was successfully applied in monitoring the expression levels of the low-abundant miRNAs in the human lung adenocarcinoma cell lines. This assay successfully verified the feasibility of G-triplex/MB as an efficient and sensitive probe for immobilization-free and label-free electrochemical detection of nucleic acids, which would greatly promote the rapid development of homogeneous label-free electrochemical biosensors.

Ganoderma Lucidum Polysaccharides Ameliorates Hepatic Steatosis and Oxidative Stress in db/db Mice via Targeting Nuclear Factor E2 (Erythroid-Derived 2)-Related Factor-2/Heme Oxygenase-1 (HO-1) Pathway.

BACKGROUND Type 2 diabetes mellitus (T2DM) and its comorbidities, including obesity, hypertension, and hyperlipidemia, are commonly associated with non-alcoholic fatty liver disease (NAFLD). Ganoderma lucidum polysaccharide (GDLP) is one of the central bioactive components in Ganoderma lucidum with anti-inflammatory, antioxidant, and hepatoprotective properties. However, the effect and mechanisms of GDLP in hepatic steatosis remain largely unknown. In the present study, we aimed to investigate the function of GDLP in hepatic steatosis and the underlying mechanism. MATERIAL AND METHODS In this study, male db/db mice were received with a high-fat diet (HFD) to investigate the effect of GDLP in T2DM-induced hepatic steatosis. The biological characteristics of the hepatic steatosis were evaluated through the detection of clinical indicators, including biochemical parameters, histopathology, and related cytokine levels. Additionally, the protein expression levels of Nrf2 (nuclear factor E2 (erythroid-derived 2)-related factor-2) signaling pathway were investigated by using western blotting and immunohistochemical staining. RESULTS The levels of food/water intake, body weight, fasting blood glucose, plasma lipids, urinary biomarkers, hepatic lipid accumulation, and tumor necrosis factor (TNF)-alpha were observably decreased in GDLP-treated db/db mice. Additionally, administration of GDLP increased the expression of various antioxidases, including superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px), whereas it reduced the level of malonaldehyde (MDA). Furthermore, GDLP was significantly promoted protein expression level of Nrf2 and its downstream target gene HO-1 (heme oxygenase-1) while decreased TNF-alpha expression. CONCLUSIONS These results indicate that GDLP against T2DM-induced hepatic steatosis, oxidative stress, and inflammation by improving the Nrf2/HO-1 signaling pathway in db/db mice, suggesting the GDLP may serve as an effective strategy for in fatty liver treatment.

Paternal cadmium exposure increases the susceptibility to diet-induced testicular injury and spermatogenic disorders in mouse offspring.

The impairments of gestational cadmium (Cd) exposure on testicular development and male fertility in offspring have been reported. Here, we investigated the effect of paternal low-concentration cadmium exposure on testicular development and spermatogenesis in offspring. Five-week-old male mice were exposed to cadmium chloride (100 mg/L) in drinking water for 20 weeks. Results presented that Cd did not affect the testicular histology and sperm count in mice. After mating with untreated females, pregnant mice and pups were then evaluated. No significant difference in the rate for successful pregnancy and the body weight of pups was observed in Cd-exposed mice compared to the controls. Male offspring were given with a chow and high-fat diet from postnatal day (PND) 35 to PND70. Our data indicated that high-fat diet obviously decreased No. of sperm in epididymides of adult offspring due to paternal Cd exposure. Testicular histology revealed that the percentage of seminiferous tubules in stages IX-XII and the atypical residual bodies positive tubules in CdH (paternal cadmium exposure and pubertal high-fat diet) group were higher than these in CdC (paternal cadmium exposure and pubertal chow diet) group. Further analysis demonstrated that high-fat diet markedly accelerated testicular apoptosis, as determined by TUNEL assay and immunostaining for cleaved caspase-3, in male offspring due to paternal Cd exposure. Collectively, high-fat diet exacerbates the damage of testicular development and spermatogenesis in offspring due to paternal cadmium exposure.

High-throughput ultra-sensitive discrimination of single nucleotide polymorphism via click chemical ligation.

Single nucleotide polymorphisms (SNPs) have been proven to be important biomarkers for disease diagnosis, prognosis and disease pathogenesis. Here, taking the advantages of a self-assembled oligonucleotide sandwich structure and robust chemical reactions, we have developed a simple, high-throughput and effective colorimetric analytical technique termed CuAAC-based ligation-assisted assays (CuAAC-LA) for SNP detection using a DNA-BIND 96-well plate. With the 5'-azide and 3'-alkyne groups labelled on two oligonucleotide probes, the target DNA can direct a Cu(i)-catalyzed alkyne-azide cycloaddition (CuAAC) click reaction. Since the small difference in duplex stability caused by a single-nucleotide mismatch was amplified by the steric effects of these reactive groups for the ligation reaction of an unstable duplex, CuAAC-LA exhibited an ultra-sensitive discrimination ability for a mutant type target in the presence of large amounts of wild type targets. As low as 0.05% SNP could be clearly detected, which was better than most previously reported methods by various DNA ligases, indicating that a simple and rapid synthetic method i.e., the DNA template-directed click reaction held the potential to replace the ligase for SNP detection.