A quantitative method for aquaporin-1 protein using magnetic preconcentration and probe-based immunoassay coupling to inductively coupled plasma mass spectrometry in urine analysis.

BACKGROUND: Aquaporin-1 (AQP1) protein plays a crucial role in intracellular and extracellular water homeostasis and fluid transport in organs and tissues associated with diverse life activities and is extremely abundant in the kidney. Accurate detection of AQP1 in urine can be applied as screening of early-stage disease. Application of magnetic preconcentration and probe-based signal amplification strategy coupling to inductively coupled plasma mass spectrometry (ICP-MS) is a more accurate, sensitive and specific detection method for AQP1 in complex biological samples compared to conventional methods. RESULTS: We described an element-labelling strategy based on magnetic preconcentration and probe-based immunoassay coupling to ICP-MS detection. The magnetic beads (MBs) modified with epoxy groups were capable of enriching AQP1 proteins and separating them from complex matrices. The probe constructed by conjugating anti-AQP1 antibody molecules on the surface of gold nanoparticles could specifically recognize AQP1 proteins attached on MBs and be analyzed by ICP-MS. The concentration of AQP1 protein could be precisely quantified and amplified by 14,000 times through the corresponding signal of Au atoms. This assay for AQP1 protein quantification achieved a detection limit down to 0.023 ng mL-1, a broad linear calibration curve between 0.3 ng mL-1 and 30 ng mL-1, as well as outstanding specificity. SIGNIFICANCE: The proposed method was successfully applied to detect AQP1 protein in human urine samples, showing the potential for its applications concerning accurate AQP1 quantification. It can also screen a wide range of proteins provided the antibodies specific to these target proteins are available.

Comparative analysis of characteristics of antibiotic resistomes between Arctic soils and representative contaminated samples using metagenomic approaches.

Antibiotic resistance is one of the most concerned global health issues. However, comprehensive profiles of antibiotic resistance genes (ARGs) in various environmental settings are still needed to address modern antibiotic resistome. Here, Arctic soils and representative contaminated samples from ARG pollution sources were analyzed using metagenomic approaches. The diversity and abundance of ARGs in Arctic soils were significantly lower than those in contaminated samples (p < 0.01). ARG profiles in Arctic soils were featured with the dominance of vanF, ceoB, and bacA related to multidrug and bacitracin, whereas those from ARG pollution sources were characterized by prevalent resistance to anthropogenic antibiotics such as sulfonamides, tetracyclines, and beta-lactams. Mobile genetic elements (MGEs) were found in all samples, and their abundance and relatedness to ARGs were both lower in Arctic soils than in polluted samples. Significant relationships between bacterial communities and ARGs were observed (p < 0.01). Cultural bacteria in Arctic soils had clinically-concerned resistance to erythromycin, vancomycin, ampicillin, etc., but ARGs relevant to those antibiotics were undetectable in their genomes. Our results suggested that Arctic environment could be an important reservoir of novel ARGs, and antibiotic stresses could cause ARG pollution via horizontal gene transfer and enrichment of resistant bacteria.

Characterization of metal resistance genes carried by waterborne free-living and particle-attached bacteria in the Pearl River Estuary.

Toxic metals can substantially change the bacterial community and functions thereof in aquatic environments. Herein, metal resistance genes (MRGs) are the core genetic foundation for microbial responses to the threats of toxic metals. In this study, waterborne bacteria collected from the Pearl River Estuary (PRE) were separated into the free-living bacteria (FLB) and particle-attached bacteria (PAB), and analyzed using metagenomic approaches. MRGs were ubiquitous in the PRE water and mainly related to Cu, Cr, Zn, Cd and Hg. The levels of PAB MRGs in the PRE water ranged from 8.11 × 109 to 9.93 × 1012 copies/kg, which were significantly higher than those of the FLB (p < 0.01). It could be attributed to a large bacterial population attached on the suspended particulate matters (SPMs), which was evidenced by a significant correlation between the PAB MRGs and 16S rRNA gene levels in the PRE water (p < 0.05). Moreover, the total levels of PAB MRGs were also significantly correlated with those of FLB MRGs in the PRE water. The spatial pattern of MRGs of both FLB and PAB exhibited a declining trend from the low reaches of the PR to the PRE and on to the coastal areas, which was closely related to metal pollution degree. MRGs likely carried by plasmids were also enriched on the SPMs with a range from to 3.85 × 108 to 3.08 × 1012 copies/kg. MRG profiles and taxonomic composition of the predicted MRG hosts were significantly different between the FLB and PAB in the PRE water. Our results suggested that FLB and PAB could behave differential response to heavy metals in the aquatic environments from the perspective of MRGs.

Astragalus polysaccharides combined with Codonopsis pilosula polysaccharides modulates the physiological characteristics of trophoblasts via miR-92a-1-5p/CCR7 axis.

BACKGROUND: When the abnormality occurs in proliferation, differentiation and apoptosis of trophoblasts, the invasion ability of placental trophoblast is weakened, which is prone to trigger the occurrence of various pregnancy diseases such as repeated spontaneous abortion (RSA). Clinically, Astragalus and Codonopsis pilosula polysaccharides (APS and CPPS) are used for the treatment of unexplained recurrent spontaneous abortion (URSA). Therefore, we aimed to probe into the roles of APS and CPPS in biological behaviors of placental trophoblasts. METHODS: The trophoblasts were treated with APS and CPPS, and transfected with miR-92a-1-5p mimic and CCR7 plasmid to explore the roles of APS and CPPS. Cell viability and apoptosis were determined by CCK-8 and flow cytometry, respectively. The levels of miRNA/mRNA and protein were measured by qRT-PCR and western blot, respectively. The interaction between miR-92a-1-5p and CCR7 was analyzed by TargetScan and dual-luciferase reporter assay. Invasion and migration rates were assessed by Transwell and wound healing assays, respectively. RESULTS: APS combined with CPPS enhanced viability, Bcl-2 expression, and migration and invasion capabilities, while suppressing apoptosis, and expressions of Bax, Bim and miR-92a-1-5p in trophoblasts. Nevertheless, miR-92a-1-5p mimic produced the inverse modulations in trophoblasts, and partially reversed the effects of APS and CPPS. Furthermore, overexpression of CCR7, the target of miR-92a-1-5p, partially offset the effect of miR-92a-1-5p mimic in trophoblasts. CONCLUSION: Astragalus combined with Codonopsis pilosula polysaccharides modulates the biological behaviors of trophoblasts via miR-92a-1-5p/CCR7 axis. The regulatory axis we studied will be helpful for the treatment of URSA in the future.

Identification of suspended particulate matters as the hotspot of polycyclic aromatic hydrocarbon degradation-related bacteria and genes in the Pearl River Estuary using metagenomic approaches.

Bacterial degradation is unequivocally considered as an important way for the cleanup of polycyclic aromatic hydrocarbon (PAHs) in the aquatic environment. However, the diversity and distribution of PAH-degrading bacterial communities and PAH degradation-related genes (PAHDGs) in ambient environment need to be investigated. In this study, bacteria in the water of the Pearl River Estuary (PRE) were initially separated as the particle-attached bacteria (PAB) and free-living bacteria (FLB), and were further characterized using metagenomic approaches. Proteobacteria (80.1 %) was identified as the most abundant PAH-degrading phylum in the PRE water, followed by Bacteroidetes, Actinobacteria, and Firmicutes. A substantial difference in the community structure was observed between PAH-degrading PAB and FLB. Both of PAH-degrading bacteria and PAHDGs were enriched on the suspended particulate matters (SPMs), with the range of enrichment factor (EF) from 7.84 × 104 to 6.64 × 106 (PAH-degrading bacteria) and from 1.14 × 103 to 1.76 × 105 (PAHDGs). The levels of PAH-degrading bacteria 16 S rRNA genes and PAHDGs on the SPMs were both significantly correlated with those in the aqueous phase (AP) in the PRE water (p < 0.05), indicating a dynamic distribution of PAH-degrading bacteria between these two phases. The total PAH concentrations on the SPMs of the PRE water were also significantly correlated with the total PAHDG levels in the PAB (p < 0.05). Our results suggested that the SPMs could be the important compartment for the elimination of PAHs from the aquatic environment.

Transcriptional response of Mycobacterium sp. strain A1-PYR to multiple polycyclic aromatic hydrocarbon contaminations.

Cometabolism mechanisms of organic pollutants in environmental microbes have not been fully understood. In this study, a global analysis of Mycobacterium sp. strain A1-PYR transcriptomes on different PAH substrates (single or binary of pyrene (PYR) and phenanthrene (PHE)) was conducted. Comparative results demonstrated that expression levels of 23 PAH degradation enzymes were significantly higher in the binary substrate than in the PYR-only one. These enzymes constituted an integrated enzymatic system to actualize all transformation steps of PYR, and most of their encoded genes formed a novel gene cascade in the genome of strain A1-PYR. The roles of different genotypes of enzymes in PYR cometabolism were also discriminated even though all of their gene sequences were presented in the genome of this strain. NidAB and PdoA2B2 instead of NidA3B3 served the initial oxidization of PAHs, and PcaL replaced PcaCD to catalyze the formation of 3-oxoadipate. Novel genes associated with PYR cometabolism was also predicted by the relationships between their transcription profiles and PYR removal. The results showed that ABC-type transporters probably played important roles in the transport of PAHs and their metabolites through cell membrane, and [4Fe-4S] ferredoxin might be essential for dioxygenases (NidAB and PdoA2B2) to achieve oxidative activities. This study provided molecular insight in that microbial degrader subtly cometabolized recalcitrant PAHs with relatively more degradable ones.

[Effect of estradiol and progesterone on production of macrophage colony-stimulating factor by human granulosa cells in vitro].

OBJECTIVE: To investigate the effect of estradiol (E(2)) and progesterone (P) on macrophage colony-stimulating factor (M-CSF) production by human luteinized granulosa cells in vitro. METHODS: Human luteinized granulosa cells were isolated from follicular fluid of superovulated infertile patients undergoing intracytoplasmatic sperm injection (ICSI). Luteinized granulosa cells were cultured with HAM's F-10 medium with various concentrations of E(2) or P (0, 1 x 10(-7), 1 x 10(-6), 1 x 10(-5), 1 x 10(-4), 1 x 10(-3) mol/L). Media were removed at 72 h after culture. M-CSF in media was measured by a solid phase enzyme-linked immunosorbant assay (ELISA) and estradiol and progesterone in media were measured by enzyme immunoassays (EIA). RESULTS: The basic concentration of M-CSF in cultivated granulosa cells without E(2) stimulation was low (47 +/- 15 ng/L). However, E(2) at concentrations of 1 x 10(-6) - 1 x 10(-3) mol/L caused a significant increase of M-CSF secretion by luteinized granulosa cells in a dose-dependent mode (2.3, 4.5, 6.9, 7.9 times higher than the basic level, respectively) (P < 0.05). E(2) at a concentration of 1 x 10(-7) mol/L, however, did not stimulate the production of M-CSF (P > 0.05). P at concentrations of 1 x 10(-7) - 1 x 10(-3) mol/L showed no effect on M-CSF production by luteinized granulosa cells (P > 0.05). CONCLUSIONS: Estradiol can stimulate M-CSF production by luteinized granulosa cells in vitro in a dose-dependent mode, on the other hand P cannot induce M-CSF production by luteinized granulosa cells. The mechanism of estradiol regulating follicular development may partially via M-CSF/receptor pathway.