Objective To investigate the effects of transient receptor potential cation channel 6 (TRPC6) on the expression of nephrin, desmin and caspase 9 and on the apoptosis of podocytes in a mouse model of podocyte injury induced by TGF-β1.Methods Conditionally immortalized mouse podocytes were cultured in vitro and divided into four groups: control, TGF-β1 treatment, TGF-β1+PGPU6/GFP/Neo-TRPC6-mus-581 (TRPC6 knockdown) and TGF-β1+PGPU6/GFP/Neo-NC (negative control).Real-time RT-PCR and Western blot analysis were performed to detect the expression of nephrin, desmin and caspase 9 at mRNA and protein levels, respectively.Flow cytometry was used to analyze the apoptotic rate of podocytes.DAPI fluorescent staining was used to observe the morphological changes of apoptotic podocytes.Results Green fluorescent protein (GFP)-expressing podocytes at 48 hours after transfection were significantly more than those at 24 hours after transfection.The level of TRPC6 in mouse podocytes transfected with PGPU6/GFP/Neo-TRPC6-mus-581 was significantly decreased as compared with that of the control group (P<0.05).No significant difference in the expression of TRPC6 was observed between the negative control group and the control group.Compared with the control group, the TGF-β1 treatment group showed increased expression of desmin and caspase 9 at both mRNA and protein levels (P<0.01), but decreased expression of nephrin at mRNA and protein levels at 48 hours after TGF-β1 intervention (P<0.05).The up-regulated desmin and caspase 9 and the down-regulated nephrin induced by TGF-β1 could be inhibited by the means of TRPC6 knockdown.The apoptosis rate of podocytes in TGF-β1 treatment group was (14.0±2.1)%, while that in TRPC6 knockdown was (10.90±0.56)% (P<0.05).The apoptosis rate of podocytes in negative control group was higher than that in TGF-β1 treatment group (P>0.05).More apoptotic cells with typical morphological features of apoptosis were observed after exposure to TGF-β1 for 48 hours.Conclusion TGF-β1 could induce the apoptosis of podocytes, inhibit the expression of nephrin and enhance the expression of caspase 9 and desmin, the possible mechanisms of which may be related to TRPC6 signal pathway.These changes in TGF-β1-treated podocytes could be alleviated by inhibiting the expression of TRPC6, which might have a protective effect on podocyte injury.

Biological sample pretreatment is an important step in biological sample analysis. Due to the diversity of biological matrices, the analysis of target substances in these samples presents significant challenges to sample processing. To meet these emerging demands on biopharmaceutical analysis, this paper summarizes several new techniques of on-line biological sample processing: solid phase extraction, solid phase micro-extraction, column switching, limited intake filler, molecularly imprinted solid phase extraction, tubular column, and micro-dialysis. We describe new developments, principles, and characteristics of these techniques, and the application of liquid chromatography-mass spectrometry (LC-MS) in biopharmaceutical analysis with these new techniques in on-line biological sample processing.

Zidovudine (AZT), the first drug approved by the US Food and Drug Administration for the treatment of human immunodeficiency virus (HIV) infection, is metabolized in the host cells to 5'-AZT triphosphate (AZT-TP) which inhibits HIV reverse transcriptase. As the pharmacokinetics of AZT and its phosphorylated metabolites in human peripheral blood mononuclear cells (hPBMCs) is limited, the aim of this study was to determine the pharmacokinetic parameters of AZT and its phosphorylated metabolites in hPBMCs from 12 healthy Chinese male subjects after a single oral dose of 600 mg of AZT. Blood samples were collected prior to drug administration, then at 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 5, 6, 8 and 10 h after drug administration. Mononuclear cells collected by Ficoll-Hypaque density gradient centrifugation were used for determination of AZT and metabolites [AZT monophosphate (AZT-MP), AZT diphosphate (AZT-DP) and AZT-TP] and the plasma was used to evaluate the pharmacokinetics of AZT. Plasma concentration of AZT peaked within 0.583 h and intracellular concentrations of AZT, AZT-MP, AZT-DP and AZT-TP peaked within 1.083, 1.500, 1.417 and 1.583 h, respectively. AZT in plasma was eliminated rapidly with t 1/2 of 2.022 h, and AZT-MP, AZT-DP and AZT-TP were eliminated with t 1/2 of 13.428, 8.285 and 4.240 h, respectively. The plasma concentration of the phosphorylated metabolites was not quantifiable.

Objective To investigate the activity of Acorus tatarinowii extracts against dust mites, and to isolate and characterize active ingredient of A. tatarinowii extracts. Methods The essential oil components were extracted from A. tatarinowii rhizome powder by rotary evaporation with methanol as solvents, followed by petroleum ether extraction and rotary evaporation. The essential oil was mixed with Tween-80 at a ratio of 1:1 and diluted into concentrations of 1.000 00%, 0.500 00%, 0.250 00%, 0.125 00%, 0.062 50% and 0.031 25%, while diluted Tween-80 served as controls. A. tatarinowii essential oil at each concentration (200 μL) was transferred evenly to filter papers containing 100 adult mites, with each test repeated in triplicate, and controls were assigned for each concentration. Following treatment at 25 °C and 75% relative humidity for 24 h, the mean corrected mortality of mites was calculated. The essential oil components were separated by silica gel column chromatography, and the essential oil was prepared in the positive column of medium pressure; and then, each component was collected. Silica gel column chromatography was run with the mobile phase that consisted of petroleum ether solution containing 10% ethyl acetate and pure ethyl acetate, detection wavelength of 254 nm, positive silica gel column as the chromatography column, and room temperature as the column temperature. Each component of the purified A. tatarinowii essential oil was diluted into 1.000 00% for acaricidal tests. The components with less than 100% acaricidal activity were discarded, and the remaining components were diluted into 50% of the previous-round tests for subsequent acaricidal tests. The components with acaricidal activity were subjected to high-performance liquid chromatography, liquid chromatography-mass spectrometry and pulsed-Fourier transform nuclear magnetic resonance spectroscopy. The structure of active monomer compounds was determined by standard spectral library retrieval and literature review. Results A. tatarinowii essential oil at concentrations of 1.000 00%, 0.500 00%, 0.250 00% and 0.125 00% killed all dust mites, and the corrected mortality was all 100%. Exposure to A. tatarinowii extracts at an effective concentration of 0.062 50% for 24 hours resulted in 94.33% mortality of dust mites. Six components (A to F) were separated using gel column chromatography, and components D and E both showed a 100% acaricidal activity against dust mites at a concentration of 0.50000%. In addition, Component D was identified as isoeugenol methyl ether, and Component E as β-asarinol. Conclusion The extract of A. tatarinowii essential oil has acaricidal activity, and the isoeugenol methyl ether shows a remarkable acaricidal activity against dust mites.

Objective To investigate the bacterial community diversity in human Demodex mites, so as to provide insights into unraveling the role of human Demodex mites in them caused infectious diseases. Methods From June to July 2023, Demodex mites were collected from the faces of college students in a university in Wuhu City using the adhesive tape method, and the V4 region of 16S ribosomal RNA (16S rRNA) gene and the internal transcribed spacer (ITS) gene of nuclear ribosomal DNA were amplified on an Illumina PE250 high-throughput sequencing platform. Sequencing data were spliced according to the overlapping relations and filtered to yield effective sequences, and operational taxonomic units (OTUs) was clustered. The diversity index of obtained OUTs was analyzed, and the structure of the bacterial community was analyzed at various taxonomic levels. Results A total of 57 483 valid sequences were obtained using 16S rRNA gene sequencing, and 159 OUTs were classified according to similarity. Then, OUTs at a 97% similarity were included for taxonomic analyses, and the bacteria in Demodex mites belonged to 14 phyla, 20 classes, 51 orders, 72 families, and 94 genera. Proteobacteria was the dominant phylum, and Vibrio, Bradyrhizobium and Variovorax were dominant genera. A total of 56 362 valid sequences were obtained using ITS gene sequencing, and 147 OTUs were obtained, which belonged to 5 phyla, 17 classes, 34 orders, 68 families, and 93 genera and were annotated to Ascomycota, Basidiomycota and Chytridiomycota, with Ascomycota as the dominant phylum, and Alternaria alternata, Epicoccum, Penicillium, and Sarocladium as dominant genera. Conclusions There is a high diversity in the composition of bacterial communities in human Demodex mites, with multiple types of microorganisms and high species abundance.