Genetically encoded sensors for measuring histamine release both in vitro and in vivo.

Histamine (HA) is a key biogenic monoamine involved in a wide range of physiological and pathological processes in both the central and peripheral nervous systems. Because the ability to directly measure extracellular HA in real time will provide important insights into the functional role of HA in complex circuits under a variety of conditions, we developed a series of genetically encoded G-protein-coupled receptor-activation-based (GRAB) HA (GRABHA) sensors with good photostability, sub-second kinetics, nanomolar affinity, and high specificity. Using these GRABHA sensors, we measured electrical-stimulation-evoked HA release in acute brain slices with high spatiotemporal resolution. Moreover, we recorded HA release in the preoptic area of the hypothalamus and prefrontal cortex during the sleep-wake cycle in freely moving mice, finding distinct patterns of HA dynamics between these specific brain regions. Thus, GRABHA sensors are robust tools for measuring extracellular HA transmission in both physiological and pathological processes.

SiNiSan alleviates liver injury by promoting hepatic stem cell differentiation via Wnt/ß-catenin signaling pathway.

BACKGROUND: SiNiSan, a Traditional Chinese Medicine containing Radix Bupleuri, Radix Paeoniae Alba, Fructus Aurantii Immaturus, and Radix Glycyrrhizae, has been shown to be clinically effective in treating liver damage, its underlying molecular mechanisms however remains unclear. PURPOSE: The aim of the current study was to understand the molecular mechanisms of SiNiSan in the treatment of liver damage utilizing mice and cell culture models. METHODS: Here, mice were gavaged with 0.2% CCl4 to obtain acute liver injury model and with alcohol to obtain chronic liver injury model. H&E staining was performed to detect liver histomorphology. HPLC-MS was performed to analyze the composition of SiNiSan decoction and SiNiSan-medicated serum (SMS). In addition, western blots were done to analyze the representative protein expression in Wnt/ß-catenin signaling. Immunofluorescence staining was done to analyze the protein levels in WB-F344 cells. Finally, in an attempt to measure the influence of SiNiSan on liver regeneration in rats, we constructed a rats partial hepatectomy models. RESULTS: We demonstrated that SiNiSan treatment mitigated liver damage in mice, as evidenced by the decrease in serum AST and ALT levels, as well as improved liver tissue morphology. HPLC-MS results showed that SMS contained a variety of components from the SiNiSan decoction. Next, our results showed that SMS reduced the expression of α-fetoprotein (AFP) and enhanced the expression of albumin (ALB) and cytokeratin 19 (CK19) in WB-F344 cells. Further, SMS treatment induced the accumulation of ß-catenin. After 14 days of SMS treatment, ß-catenin protein underwent nuclear translocation and bound to the LEF1 receptor in the nucleus, which regulated c-Myc and Cyclin D1 factors to activate Wnt/ß-catenin signaling and promoted differentiation of WB-F344 cells. In addition, we demonstrated that SiNiSan increased liver regeneration in rat hepatectomy. CONCLUSION: Collectively, the current study revealed that SiNiSan alleviated the acute liver injury induced by CCl4 as well as the chronic liver damage triggered by alcohol and sucrose in vitro. Concurrently, SMS treatment induced hepatic stem cell differentiation by activating Wnt/ß-catenin signaling in vivo. Further study showed that SiNiSan promoted the regeneration of rats liver. The current study provides a theoretical basis for the clinical treatment of liver-related diseases with SiNiSan.

Water extract of Cayratia albifolia C.L.Li root relieves zymosan A-induced inflammation by restraining M1 macrophage polarization.

BACKGROUND: Cayratia albifolia C.L.Li (CAC) is a traditional Chinese herbal medicine used to treat inflammatory diseases. Our laboratory has firstly reported that the water extract from CAC relieved lipopolysaccharide (LPS)-induced inflammation, however stronger evidence is still needed to prove its anti-inflammatory effects and the mechanisms involved are also ambiguous. PURPOSE: This study sought to provide more evidence for the application of CAC in alleviating infectious inflammation and disclose novel pharmacological mechanisms. METHODS: Mice were injected with zymA into their paws or peritoneal cavities, and then treated with CAC. ELISA, immunofluorescence and flow cytometry were performed to detect the cytokines (IL-1ß, IL-6, TNF-α and IL-10) generation, the cell infiltration, and the CD86 or CD206 expression of macrophages. Then in vitro assays were performed on bone marrow-derived macrophages (BMDMs) and peritoneal macrophages (PMs) to detect their expression of iNOS, arg-1 and the cytokines above. On mechanisms, western blotting (WB), electrophoretic mobility shift assay (EMSA) and flow cytometry were carried out to measure NF-κB transcriptional activity, mitochondrial bioactivity and the mTORC1 activation when BMDMs were stimulated by zymA and treated with CAC. Finally, the chemical components consisted in the extract were analyzed by LC-MS. RESULTS: 200 mg/kg CAC clearly inhibited zymA induced mouse paw edema and reduced the contents of IL-1ß, IL-6 and TNF-α rather than IL-10 in local tissues. CAC also reduced CD86 but not CD206 in macrophages in situ. Through in vitro experiments, it was discovered that CAC reduced the protein and mRNA levels of IL-1ß, IL-6 and TNF-α, and also inhibited iNOS expression, but showed no influence on IL-10 and arg-1 in macrophages. We found CAC reduced NF-κB transcriptional activity, down-regulated mitochondrial membrane potential and ROS levels, and inhibited mTORC1 activity. Finally, we identified 15 major compounds in the extract, among which 4-guanidinobutyric acid and kynurenic acid were the most abundant. CONCLUSION: This study provides further evidence that CAC significantly reduces zymA induced infectious inflammation. In addition, this novel data revealed that CAC restrained M1 rather than promoting M2 macrophages polarization via multi-target inhibitory effects, based on its potentially active components.

Nanogapped Au(core) @ Au-Ag(shell) structures coupled with Fe3O4 magnetic nanoparticles for the detection of Ochratoxin A.

Contamination of mycotoxins has been a general problem of food safety and afflicts people all around the world. Ochratoxin A (OTA), an abundant and representative food-contaminating mycotoxin, has a detrimental effect on our health. Herein, an ultrasensitive surface-enhanced Raman scattering (SERS) aptasensor was fabricated for OTA detection. Au(core)@Au-Ag(shell) nanogapped nanostructures (Au@Au-Ag NNSs) were coupled with Fe3O4 magnetic nanoparticles (Fe3O4 MNPs) by OTA aptamer and its complementary DNA sequence. The distance between two nanoparticles become far away in presence of the OTA due to the affinity of aptamer. This effect can obviously bring about the change of Raman intensity which can be used for the detection of OTA. Compared with pure metal nanoparticles, Au@Au-Ag NNSs can effectively enhance the Raman intensity of Raman signal molecule (4-MBA) via the ultrasmall nanogap, which contribute to the sensitivity of the sensor system. Additionally, the utilization of Fe3O4 MNPs offers a green, economical and facile technology to capture targets from solution. The specially modified aptamer and its complementary strand establish a bridge connecting between Au@Au-Ag NNSs and Fe3O4 MNPs. In this study, OTA concentration with a linear range of 0.01-50 ng mL-1 and the limit of detection (LOD) was as low as 0.004 ng mL-1. The proposed SERS aptasensor can combine both of these above type of materials and provide a rapid, sensitive, and low-cost methodology for numerous future applications of point-of-care diagnostics.

Ultrasensitive SERS aptasensor for the detection of oxytetracycline based on a gold-enhanced nano-assembly.

This paper investigated a new detection method of oxytetracycline (OTC) in aquatic products with ultrasensitive detection limit. The method was constructed on the basis of raman hot spot between gold nanoparticles (AuNPs) (13nm and 80nm diameter respectively) linked by an DNA sequence. The DNA sequence combined with the OTC aptamer including its complementary sequence as well as a stem-loop structure. The raman signal molecule (4-MBA) was modified at the surface of 13nm AuNPs. After the exposure of OTC, the aptamer sequence was preferentially combined with OTC and partially dehybridized with its complementary sequence which led the 13nm AuNPs to get more closer to the 80nm AuNPs. The raman intensity was thus increased for the more enhanced hot spot generated. Under the optimal experimental conditions, the SERS signal was positively related to the OTC concentration with a wide working range of 4.60×10-2-4.60×102fg/mL and the limit of detection (LOD) was as low as 4.35×10-3fg/mL. The recovery rates of fishmeal ranged from 91.29-110.98%. The specificity of this method was further examined, and the results showed that the AuNPs based aptasensor was highly selective. This developed ultrasensitive aptamer-based SERS detection platform suggested that it may be a promising strategy for a variety of sensing applications.