Mitochondrial calcium plays a crucial role in mitochondrial metabolism, cell calcium handling, and cell death. However, some mechanisms concerning mitochondrial calcium regulation are still unknown, especially how mitochondrial calcium couples with cytosolic calcium. In this work, we constructed a novel mitochondrial calcium fluorescent indicator (mito-GCaMP2) by genetic manipulation. Mito-GCaMP2 was imported into mitochondria with high efficiency and the fluorescent signals co-localized with that of tetramethyl rhodamine methyl ester, a mitochondrial membrane potential indicator. The mitochondrial inhibitors specifically decreased the signals of mito-GCaMP2. The apparent K_d of mito-GCaMP2 was 195.0 nmol/L at pH 8.0 in adult rat cardiomyocytes. Furthermore, we observed that mito-GCaMP2 preferred the alkaline pH surrounding of mitochondria. In HeLa cells, we found that mitochondrial calcium ([Ca²⁺]_mito) responded to the changes of cytosolic calcium ([Ca²⁺]_cyto) induced by histamine or thapasigargin. Moreover, external Ca²⁺ (100 μmol/L) directly induced an increase of [Ca²⁺]_mito in permeabilized HeLa cells. However, in rat cardiomyocytes [Ca²⁺]_mito did not respond to cytosolic calcium transients stimulated by electric pacing or caffeine. In permeabilized cardiomyocytes, 600 nmol/L free Ca²⁺ repeatedly increased the fluorescent signals of mito-GCaMP2, which excluded the possibility that mito-GCaMP2 lost its function in cardiomyocytes mitochondria. These results showed that the response of mitochondrial calcium is diverse in different cell lineages and suggested that mitochondria in cardiomyocytes may have a special defense mechanism to control calcium flux.

An indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) was developed using a highly sensitive and specific monoclonal antibody against avilamycin. The immunising antigen synthesised through the carbonyldiimidazole (CDI) method was used to prepare antibodies. The established ic-ELISA, after optimisation of conditions, possessed a half maximum inhibitory concentration (IC₅₀) value of 7.44 ng mL−1 and the detection limit of 0.21 ng mL⁻¹ in the standard curve. The spiked experiments indicated that the limits of detection were 1.86 µg kg⁻¹ and 2.31 µg kg⁻¹ in swine feed and chicken feed, respectively. In addition, the average recoveries ranged from 74.7 to 105.4% with the coefficient of variation less than 11%. The good correlation (R² = 0.9818) between the result of ic-ELISA and HPLC demonstrated the reliability of the developed ic-ELISA, which showed that the ic-ELISA developed here achieves the aim of strengthening the monitoring of avilamycin residues and the inspection of import and export related products.

Currently, as an important raw material of Chinese traditional patent medicines, Paridis Rhizome is in great demand, which led to its price increases. In order to protect the wild resources and satisfy market demand of Paridis rhizome, the researches in various directions were conducted, involved its chemical composition, pharmacological action, clinical application, resource investigation, artificial cultivation, etc. Herein, the chemical studies of genus Paridis Rhizome, aerial parts of Paridis Rhizome gummy and starchy Paridis Rhizome, and the studies of endophyte in Paridis Rhizome were reviewed and analyzed in order to explore the substitutes of Paridis Rhizome, and provide the reference for the enlargement of medicinal resources of Paridis Rhizome. It manifests that the steroidal saponins, the important chemical compositions in Paridis Rhizome were tested in genus Paridis Rhizome, aerial parts of Paridis Rhizome, gummy Paridis Rhizome and the endophyte in Paridis Rhizome. However, the further experimental studies and clinical verification works should be carried out to confirm the final substitute.