MicroRNA and Protein Cargos of Human Limbal Epithelial Cell-Derived Exosomes and Their Regulatory Roles in Limbal Stromal Cells of Diabetic and Non-Diabetic Corneas.

Epithelial and stromal/mesenchymal limbal stem cells contribute to corneal homeostasis and cell renewal. Extracellular vesicles (EVs), including exosomes (Exos), can be paracrine mediators of intercellular communication. Previously, we described cargos and regulatory roles of limbal stromal cell (LSC)-derived Exos in non-diabetic (N) and diabetic (DM) limbal epithelial cells (LECs). Presently, we quantify the miRNA and proteome profiles of human LEC-derived Exos and their regulatory roles in N- and DM-LSC. We revealed some miRNA and protein differences in DM vs. N-LEC-derived Exos' cargos, including proteins involved in Exo biogenesis and packaging that may affect Exo production and ultimately cellular crosstalk and corneal function. Treatment by N-Exos, but not by DM-Exos, enhanced wound healing in cultured N-LSCs and increased proliferation rates in N and DM LSCs vs. corresponding untreated (control) cells. N-Exos-treated LSCs reduced the keratocyte markers ALDH3A1 and lumican and increased the MSC markers CD73, CD90, and CD105 vs. control LSCs. These being opposite to the changes quantified in wounded LSCs. Overall, N-LEC Exos have a more pronounced effect on LSC wound healing, proliferation, and stem cell marker expression than DM-LEC Exos. This suggests that regulatory miRNA and protein cargo differences in DM- vs. N-LEC-derived Exos could contribute to the disease state.

ß-Amyloid targeting nanodrug for neuron-specific delivery of nucleic acids in Alzheimer's disease mouse models.

Delivery of therapeutic substances into the brain poses a significant challenge in the treatment of neurological disorders. This is primarily due to the blood-brain barrier (BBB), which restricts access, alongside the limited stability and distribution of these agents within the brain tissue. Here we demonstrate an efficient delivery of microRNA (miRNA) and antisense RNA preferentially to neurons compared to astroglia in the brain of healthy and Alzheimer's disease mice, via disulfide-linked conjugation with poly(ß-L-malic acid-trileucine)-copolymer a biodegradable, amphiphilic, and multivalent platform. By conjugating a D-configured (D3)-peptide (vector) for specific targeting, highly efficient delivery across the BBB is achieved through the Low-Density Lipoprotein Receptor-Related Protein-1 (LRP-1) transcytosis pathway, amyloid beta (Aß) peptides. Nanodrug distribution was determined by fluorescent labeling and analyzed by microscopy in neurons, astroglia, and in extracellular amyloid plaques typical for Alzheimer's disease. Whereas D-configured BBB-vectors can efficiently target neurons, L-configured (e.g., AP2-peptide) guided vector can only cross BBB but not seem to bind neurons. An analysis of post-injection fluorescence distribution, and RNA-seq followed by real-time PCR validation, confirmed a successful in vivo delivery of morpholino-miRNA-186 nanoconjugates into mouse brain. The size and fluorescence intensity of the intracellular nanodrug particulates were analyzed and verified by a competition with non-fluorescent conjugates. Differentially expressed genes (DEGs) from RNA-seq were identified in the nanodrug injected mice, and the changes of selected DEGs related to Alzheimer's disease were further validated by western blot and real-time PCR. Collectively, these results demonstrated that D3-peptide-conjugated nanopolymer drug is able to achieve neuron-selective delivery of miRNA and can serve as an efficient brain delivery vehicle in Alzheimer's disease (AD) mouse models.

Reversal of dual epigenetic repression of non-canonical Wnt-5a normalises diabetic corneal epithelial wound healing and stem cells.

AIMS/HYPOTHESIS: Diabetes is associated with epigenetic modifications including DNA methylation and miRNA changes. Diabetic complications in the cornea can cause persistent epithelial defects and impaired wound healing due to limbal epithelial stem cell (LESC) dysfunction. In this study, we aimed to uncover epigenetic alterations in diabetic vs non-diabetic human limbal epithelial cells (LEC) enriched in LESC and identify new diabetic markers that can be targeted for therapy to normalise corneal epithelial wound healing and stem cell expression. METHODS: Human LEC were isolated, or organ-cultured corneas were obtained, from autopsy eyes from non-diabetic (59.87±20.89 years) and diabetic (71.93±9.29 years) donors. The groups were not statistically different in age. DNA was extracted from LEC for methylation analysis using Illumina Infinium 850K MethylationEPIC BeadChip and protein was extracted for Wnt phospho array analysis. Wound healing was studied using a scratch assay in LEC or 1-heptanol wounds in organ-cultured corneas. Organ-cultured corneas and LEC were transfected with WNT5A siRNA, miR-203a mimic or miR-203a inhibitor or were treated with recombinant Wnt-5a (200 ng/ml), DNA methylation inhibitor zebularine (1-20 µmol/l) or biodegradable nanobioconjugates (NBCs) based on polymalic acid scaffold containing antisense oligonucleotide (AON) to miR-203a or a control scrambled AON (15-20 µmol/l). RESULTS: There was significant differential DNA methylation between diabetic and non-diabetic LEC. WNT5A promoter was hypermethylated in diabetic LEC accompanied with markedly decreased Wnt-5a protein. Treatment of diabetic LEC and organ-cultured corneas with exogenous Wnt-5a accelerated wound healing by 1.4-fold (p<0.05) and 37% (p<0.05), respectively, and increased LESC and diabetic marker expression. Wnt-5a treatment in diabetic LEC increased the phosphorylation of members of the Ca2+-dependent non-canonical pathway (phospholipase Cγ1 and protein kinase Cß; by 1.15-fold [p<0.05] and 1.36-fold [p<0.05], respectively). In diabetic LEC, zebularine treatment increased the levels of Wnt-5a by 1.37-fold (p<0.01)and stimulated wound healing in a dose-dependent manner with a 1.6-fold (p<0.01) increase by 24 h. Moreover, zebularine also improved wound healing by 30% (p<0.01) in diabetic organ-cultured corneas and increased LESC and diabetic marker expression. Transfection of these cells with WNT5A siRNA abrogated wound healing stimulation by zebularine, suggesting that its effect was primarily due to inhibition of WNT5A hypermethylation. Treatment of diabetic LEC and organ-cultured corneas with NBC enhanced wound healing by 1.4-fold (p<0.01) and 23.3% (p<0.05), respectively, with increased expression of LESC and diabetic markers. CONCLUSIONS/INTERPRETATION: We provide the first account of epigenetic changes in diabetic corneas including dual inhibition of WNT5A by DNA methylation and miRNA action. Overall, Wnt-5a is a new corneal epithelial wound healing stimulator that can be targeted to improve wound healing and stem cells in the diabetic cornea. DATA AVAILABILITY: The DNA methylation dataset is available from the public GEO repository under accession no. GSE229328 ( https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE229328 ).

IL-17A expression by both T cells and non-T cells contribute to HSV-IL-2-induced CNS demyelination.

Previously we reported that a recombinant HSV-1 expressing murine IL-2 (HSV-IL-2) causes CNS demyelination in different strains of mice and in a T cell-dependent manner. Since TH17 cells have been implicated in CNS pathology, in the present study, we looked into the effects of IL-17A-/- and three of its receptors on HSV-IL-2-induced CNS demyelination. IL-17A-/- mice did not develop CNS demyelination, while IL-17RA-/-, IL-17RC-/-, IL-17RD-/- and IL-17RA-/-RC-/- mice developed CNS demyelination. Adoptive transfer of T cells from wild-type (WT) mice to IL-17A-/- mice or T cells from IL-17A-/- mice to Rag-/- mice induced CNS demyelination in infected mice. Adoptive T cell experiments suggest that both T cells and non-T cells expressing IL-17A contribute to HSV-IL-2-induced CNS demyelination with no difference in the severity of demyelination between the two groups of IL-17A producing cells. IL-6, IL-10, or TGFß did not contribute to CNS demyelination in infected mice. Transcriptome analysis between IL-17A-/- brain and spinal cord of infected mice with and without T cell transfer from WT mice revealed that "neuron projection extension involved in neuron projection guidance" and "ensheathment of neurons" pathways were associated with CNS demyelination. Collectively, the results indicate the importance of IL-17A in CNS demyelination and the possible involvement of more than three of IL-17 receptors in CNS demyelination.

[Degradation characteristics of 2,4,6-trichlorophenol by the anaerobic consortium XH-1]. / 厌氧微生物菌群XH-1对2,4,6-ä¸‰æ°¯è‹¯é šçš„é™è§£ç‰¹æ€§.

Organic pollutant 2,4,6-trichlorophenol (2,4,6-TCP) is commonly found in anaerobic environments such as sediments and groundwater aquifers. To investigate the ability of the anaerobic consortium XH-1 to degrade 2,4,6-TCP, we established anaerobic incubations using 2,4,6-TCP as the substrate and inoculated the incubations with XH-1. Additional subcultures were established by amending with intermediate product 4-chlorophenol (4-CP) or phenol as the substrate. The transformation products of 2,4,6-TCP were analyzed and determined using high-performance liquid chromatography (HPLC). Microbial community structure and key microbial groups involved in the degradation of 2,4,6-TCP were analyzed based on 16S rRNA gene high-throughput sequencing. The results showed that the initial 122 µmol·L-1 2,4,6-TCP was completely transformed after a 80-day incubation at a rate of 0.15 µmol·d-1. 2,4-dichlorophenol (2,4-DCP), 4-CP and phenol were identified as the intermediate products. All intermediate products generated from 2,4,6-TCP transformation were completely degraded after being incubated for 325 days. The main microbial groups responsible for the reductive dechlorination of 2,4,6-TCP might be the organohalide respiring Dehalobacter and Dehalococcoides. The subsequent reductive dechlorination of 4-CP to phenol was likely driven by Dehalococcoides. The cooperation between the organohalide respiring bacteria, Syntrophorhabdus and methanogens (e.g. Methanosaeta and Methanofolis) was responsible for the complete degradation of 2,4,6-TCP.

Sex chromosome degeneration, turnover, and sex-biased expression of sex-linked transcripts in African clawed frogs (Xenopus).

The tempo of sex chromosome evolution-how quickly, in what order, why and how their particular characteristics emerge during evolution-remains poorly understood. To understand this further, we studied three closely related species of African clawed frog (genus Xenopus), that each has independently evolved sex chromosomes. We identified population polymorphism in the extent of sex chromosome differentiation in wild-caught Xenopus borealis that corresponds to a large, previously identified region of recombination suppression. This large sex-linked region of X. borealis has an extreme concentration of genes that encode transcripts with sex-biased expression, and we recovered similar findings in the smaller sex-linked regions of Xenopus laevis and Xenopus tropicalis. In two of these species, strong skews in expression (mostly female-biased in X. borealis, mostly male-biased in X. tropicalis) are consistent with expectations associated with recombination suppression, and in X. borealis, we hypothesize that a degenerate ancestral Y-chromosome transitioned into its contemporary Z-chromosome. These findings indicate that Xenopus species are tolerant of differences between the sexes in dosage of the products of multiple genes, and offer insights into how evolutionary transformations of ancestral sex chromosomes carry forward to affect the function of new sex chromosomes. This article is part of the theme issue 'Challenging the paradigm in sex chromosome evolution: empirical and theoretical insights with a focus on vertebrates (Part I)'.

A frog with three sex chromosomes that co-mingle together in nature: Xenopus tropicalis has a degenerate W and a Y that evolved from a Z chromosome.

In many species, sexual differentiation is a vital prelude to reproduction, and disruption of this process can have severe fitness effects, including sterility. It is thus interesting that genetic systems governing sexual differentiation vary among-and even within-species. To understand these systems more, we investigated a rare example of a frog with three sex chromosomes: the Western clawed frog, Xenopus tropicalis. We demonstrate that natural populations from the western and eastern edges of Ghana have a young Y chromosome, and that a male-determining factor on this Y chromosome is in a very similar genomic location as a previously known female-determining factor on the W chromosome. Nucleotide polymorphism of expressed transcripts suggests genetic degeneration on the W chromosome, emergence of a new Y chromosome from an ancestral Z chromosome, and natural co-mingling of the W, Z, and Y chromosomes in the same population. Compared to the rest of the genome, a small sex-associated portion of the sex chromosomes has a 50-fold enrichment of transcripts with male-biased expression during early gonadal differentiation. Additionally, X. tropicalis has sex-differences in the rates and genomic locations of recombination events during gametogenesis that are similar to at least two other Xenopus species, which suggests that sex differences in recombination are genus-wide. These findings are consistent with theoretical expectations associated with recombination suppression on sex chromosomes, demonstrate that several characteristics of old and established sex chromosomes (e.g., nucleotide divergence, sex biased expression) can arise well before sex chromosomes become cytogenetically distinguished, and show how these characteristics can have lingering consequences that are carried forward through sex chromosome turnovers.

[Effects of Different Land Use Typess on the Molecular Ecological Network of Soil Bacteria].

The bacterial community composition in four land-use types was determined and the visualized bacterial network was constructed by 16S rDNA Illumina MiSeq high-throughput sequencing technology and a molecular ecological network method. The results show that Proteobacteria, Acidobacteria, Bacteroidetes, Chloroflexi, Actinobacteria, Planctomycetes, Verrucomicrobia, Cyanobacteria, Gemmatimonadetes, Firmicutes, Nitrospirae, and Chlorobi are the main bacteria in this area. The number of nodes of urban green land, paddy field, and dry field bacteria networks is higher, and that of natural forest land is lower. The number of connections and average connectivity of dry fields are the highest; following are those of urban green land and paddy field, and those of natural forest land are the lowest. The four bacterial networks are dominated by positive correlation, and the ratio of competition relationship is TL > LD > HT > ST. The average network path and modularity of the soil bacteria networks of paddy field and dry land are small, while the average connectivity and clustering coefficient are higher. Some flora of Acidobacteria, Firmicutes, and Proteobacteria play an important role in the soil bacterial network in this area. The classification of operational taxonomic units is different among the key nodes of different bacterial molecular ecological networks, and there is almost no overlap. The relative abundance of bacteria of some key nodes in the four bacterial networks is low (<1%), and these are not the main bacteria in this area. The soil microflora in dry land are mainly affected by TP (P<0.05), the soil microflora in paddy field were mainly affected by clay, silt, and water content (P<0.05), and that in natural forest land and urban green land were mainly affected by C/N (P<0.05). The above results show that different land-use patterns lead to changes in soil physical and chemical properties and the interaction between soil bacteria species. The bacterial network of dry land soil is larger and the relationship between species is more complex. The bacteria in different land-use types are mainly cooperative, and the competition is weak. Compared with other land-use types, there is stronger competition between the bacteria in natural forest soil. The soil bacteria in paddy field and dry land are the most sensitive to the external environment, respond more quickly, and the community structure is easier to change. The response of soil bacteria in natural forest land and urban green land is slower, and the disturbance of environmental factors does not affect the whole bacterial ecological network in a short time, and thus the community structure is more stable. Some bacteria have the phenomenon of species role transformation between networks. The abundance and community distribution of microorganisms cannot indicate the strength of their connectivity between network nodes; low-abundance bacteria in soil play an important role in the construction of bacterial networks.

Developmental Systems Drift and the Drivers of Sex Chromosome Evolution.

Phenotypic invariance-the outcome of purifying selection-is a hallmark of biological importance. However, invariant phenotypes might be controlled by diverged genetic systems in different species. Here, we explore how an important and invariant phenotype-the development of sexually differentiated individuals-is controlled in over two dozen species in the frog family Pipidae. We uncovered evidence in different species for 1) an ancestral W chromosome that is not found in many females and is found in some males, 2) independent losses and 3) autosomal segregation of this W chromosome, 4) changes in male versus female heterogamy, and 5) substantial variation among species in recombination suppression on sex chromosomes. We further provide evidence of, and evolutionary context for, the origins of at least seven distinct systems for regulating sex determination among three closely related genera. These systems are distinct in their genomic locations, evolutionary origins, and/or male versus female heterogamy. Our findings demonstrate that the developmental control of sexual differentiation changed via loss, sidelining, and empowerment of a mechanistically influential gene, and offer insights into novel factors that impinge on the diverse evolutionary fates of sex chromosomes.

[Pollution Characteristics and Source Apportionment of Polycyclic Aromatic Hydrocarbons in Soils of Shenyang North New Area].

Topsoil (0-20 cm) samples (n=101) in 5 different land use types in Shenyang North New Area (SNNA), Shenyang, China were collected using the uniform grid layout method to investigate the spatial distribution characteristics, composition spectrum, and source analysis of 16 polycyclic aromatic hydrocarbons (PAHs) listed as priority pollutants by the Environmental Protection Agency of the United States. Results showed that the total concentration of the 16 PAHs (ΣPAHs) in soils of SNNA ranged from 123.7 µg·kg-1 to 932.5 µg·kg-1. The PAH components were mainly dominated by 3-ring and 4-ring PAHs, of which the proportion of 3-ring PAHs was the highest. The spatial distribution of the ΣPAHs concentration was obvious, showing a decreasing tendency from south to north and from east to west. In the five soil types, the average concentrations of the ΣPAHs were relatively higher in the urban green space and the artificial forest, followed by the vegetable land, while the total PAH concentrations in paddy fields and corn fields were relatively lower and had no obvious spatial distribution differences. Source apportionment results studied using characteristic ratio analysis and factor analysis/multivariate linear regression showed that the main sources of PAHs in the topsoil of SNNA were mixed sources. Industrial coal combustion and motor vehicle exhaust were the main PAH contributors, with a combined contribution rate of 79.6%. The oil spill and coke oven contribution rate was about 16.2%, and the biomass fuel combustion was about 4.2%.

[Genetic basis of immune response of lymphocyte-like cells in the mucosal immune system of Lampetra japonica].

In recent years, the antigen recognition mechanism based on variable lymphocyte receptors (VLRs) was found in agnathan lamprey. To illuminate the genetic basis of immune response of lymphocyte-like cells in the mucosal immune system of lamprey and explore the evolutionary relationship of adaptive immune responses between the jawless and jawed vertebrates, we constructed cDNA libraries of lamprey (Lampetra japonica) gills before and after stimulation, and then performed high-throughput transcriptome sequencing and analysis. Through functional annotation of 88 525 assembled unigenes, 21 704 and 9769 unigenes were annotated in Gene Ontology (GO) and Kyto Encyclopedia of Genes and Genomes (KEGG) databases, respectively. Among 999 unigenes involved in multiple pathways of immune system, 184 unigenes were highly homologous to 51 TCR (T cell receptor) and BCR (B cell receptor) signalling molecules in higher vertebrates, indicating that molecules involved in adaptive immune signalling pathways in higher vertebrates also exist in lampreys. In addition, identification of five VLRA, seven VLRB and four VLRC molecules suggest that at least three types of lymphocyte subsets are distributed in lamprey gill mucosal immune tissues. The results of real-time fluorescence quantitative PCR showed that the expression levels of Lck, Fyn and Zap70 were up-regulated after immune stimulation while those of Syk, Btk and Blnk were not changed significantly, indicating the activation of TCR-like signal transduction pathway after antigen stimulation in lamprey gill tissues. Our studies preliminaryly proved that two parallel adaptive immune systems in jawless and jawed vertebrates have common genetic basis, and also provided valuable clues to the exploration of signalling processes of VLRA⁺, VLRB⁺, and VLRC⁺ lymphocyte-like cells in response to antigens.

[Petroleum pollution and the microbial community structure in the soil of Shengli Oilfield].

Soils around a new oil well (2011- ) and an old oil well (1966-2003) were sampled to investigate the characteristics of petroleum pollution in the oilfield. The structure of soil microbial community was analyzed by PCR-DGGE and clone sequencing techniques. Results showed that the soils around the two oil wells were generally contaminated with petroleum, and the concentrations of total petroleum hydrocarbons mostly exceeded the threshold of the environmental quality standards of soil (500 mg x kg(-1)). The total petroleum hydrocarbons concentration of the polluted soil was significantly positively correlated with the contents of organic carbon, total nitrogen and available potassium, respectively. The microbial diversity index in the soil around the old oil well decreased with the increasing total petroleum hydrocarbons concentration, however, it was reversed for the soil around the new oil well. Sequence analysis of the prominent bands in DGGE profiles showed that some dominant species existed in the petroleum-contaminated soils around the oil wells and mostly were oil-associated and hydrocarbon degrading bacteria, including Microbacterium, Streptomyces, Dietzia, Flavobacterium, alpha-Proteobacteria, and gamma-Proteobacteria.

Design and high-level expression of a hybrid antimicrobial peptide LF15-CA8 in Escherichia coli.

Antimicrobial peptides (AMPs) have been paid considerable attention owing to their broad-spectrum antimicrobial activity and have great potential as novel antimicrobials. In this study, a novel hybrid peptide LF15-CA8 was designed on the basis of bovine lactoferricin (LfcinB) and cecropin A. The gene segment encoding LF15-CA8 was synthesized and cloned into pGEX-4T-BH to form pGEX-4T-LC1 containing one copy of the LF15-CA8 coding region. A series of recombinant vectors containing up to six multiple-copy LF15-CA8 coding regions, i.e., pGEX-4T-LCn (n = 1-6), were subsequently constructed, and used for transformation in Escherichia coli BL21(DE3). After induction with IPTG, pGEX-4T-LC1 and pGEX-4T-LC2 transformants successfully expressed fusion proteins GST-LF15-CA8 and GST-(LF15-CA8)2 in the form of inclusion bodies, respectively. The inclusion bodies were dissolved and the peptide was successfully released in 70 % formic acid in a single step. After purification, about 10.0 mg of the recombinant peptide LF15-CA8 with purity more than 97 % was obtained from 1 l of bacteria culture of pGEX-4T-LC2 transformants. LF15-CA8 caused an increase in antibacterial activity against Gram-positive bacterium (Staphylococcus aureus ATCC 25923) compared with the parent peptides and did not show obvious hemolytic activity against human erythrocytes in the range of effective antibacterial concentration. These results suggest that the peptide LF15-CA8 could be a promising candidate for therapeutic applications, and may lead to a cost-effective solution for the large-scale production of AMPs.

Inhibition of cytochrome P450 enzymes by rhein in rat liver microsomes.

Rhein, an active ingredient extensively found in plants such as Aloe, Cassitora L., rhubarb and so on, has been used for a long time in China. Pharmacological tests revealed that rhein not only had a strong antibacterial action, but also may be useful in cancer chemotherapy as a biochemical modulator. Its therapeutic action and toxicity is still the subject of considerable research. With microsome incubation assays in vitro and HPLC methods, the inhibition of rat liver CYP1A2, CYP2C9, CYP2D6, CYP2E1 and CYP3A enzymes by rhein were studied kinetically. The results showed the most inhibition of CYP2E1 by rhein (K(i) = 10 microm, mixed); CYP3A and CYP2C9 were also inhibited by rhein, K(i) = 30 microm (mixed) and K(i) = 38 microm (mixed), respectively; rhein revealed some inhibition of CYP1A2 (K(i) = 62 microm, uncompetitive) and CYP2D6 (K(i) = 74 microm, mixed). Drug-drug interactions, especially cytochrome P450 (CYP)-mediated interactions, cause an enhancement or attenuation in the efficacy of co-administered drugs. Inhibition of the five major CYP enzymes observed for rhein suggested that changes in pharmacokinetics of co-administered drugs were likely to occur. Therefore, caution should be paid to the possible drug interaction of medicinal plants containing rhein and CYP substrates.

[Hemodynamic performance of newly developed composite stentless porcine aortic valve: in vitro testing and in vivo experiment with sheep].

OBJECTIVE: To investigate the effects of a newly developed composite stentless porcine aortic valve constructed from noncoronary leaflets of three porcine aortic valves. METHODS: Fresh porcine hearts with ascending aorta were obtained from a slaughterhouse. The porcine aortic roots with ascending aorta and anterior leaflet of mitral valve and partial ventricular septum were dissected out and were pressurized to maintain their natural anatomical shapes with the leaflets floating freely at zero-pressure. Three noncoronary leaflets fixed in 0.6% glutaraldehyde were carefully matched for size and symmetry to construct a novel composite stentless porcine aortic valve. The lower margin and outside of the valve was covered with a piece of bovine pericardium. The novel stentless valves were tested in vitro pulsatile flow to detect the effective orifice area (EOA) and mean pressure difference (DeltaP) of the valve compared with the stented bovine pericardial bioprostheses of the same size. Sixteen male juvenile sheep underwent implantation of the novel valves in the supra-annular position in under cardio-pulmonary bypass. The intraoperative and postoperative echocardiography and pathological specimen were given to evaluate the hemodynamic performance and observed in the respects of a long-term survival, function of valve and pathological specimen. RESULTS: Since novel valves had the unfavorable muscle-based right coronary leaflet characteristic of porcine valve removed it had larger EOA. Pulsatile flow testing indicated that the EOA of the 3 novel valves was (3.47 +/- 0.15), (3.69 +/- 0.11), and (3.92 +/- 0.18) cm2 respectively, all significantly greater than those of the stented bovine pericardial bioprosthesis [(2.00 +/- 0.16), (2.21 +/- 0.26), and (2.37 +/- 0.42) cm2 respectively] at each integral simulated cardiac output between 3 - 6 L/min (all P < 0.05), while the DeltaP levels of the novel valves were (2.35 +/- 0.41), (3.10 +/- 0.20), and (3.56 +/- 0.16) mm Hg respectively, all significantly lower than those of the stented bovine pericardial bioprosthesis [(4.98 +/- 0.46), (6.82 +/- 1.27), and (8.40 +/- 1.83) mm Hg respectively, all P < 0.05]. Twelve of the sixteen sheep survived after operation. Five of them had lived for more than 90 days, 3 more than 180 days, and 2 more than 360 days. The intra-operative echocardiographic analyses showed low DeltaP [(3.90 +/- 0.78) mm Hg] and no regurgitation in all sheep. After 15 to 360 days, all valves performed excellently. The sheep were postoperatively sacrificed in 5 d, 15 d, 45 d, 90 d, 180 d, or 300 d respectively. Necropsy revealed the valves had a low to mild level of calcification, without periprosthetic leakage and overgrowth of fibrous tissue. CONCLUSION: The newly developed composite stentless porcine aortic valves show excellent hemodynamic performance with lower transvalvular pressure gradient and are relatively easy to implant.

[Determination of 1, 2, 3, 4, 6-penta-O-galloyl-D-glucose in forty four kinds of Chinese traditional medicines by HPLC].

OBJECTIVE: To determine 1, 2, 3, 4, 6-penta-O-galloyl-D-glucose (PGG) in forty four kinds of Chinese traditional medicines by HPLC. METHOD: The PGG was extracted with a solvent consisting of ethanol and water (50:50) in ultrasonic bath at 50-60 degrees C for 1 hour. A Diamonsil C18 column (4.6 mm x 250 mm, 5 microm) was used for the separation and analysis of PGG with a mobile phase consisting of acetonitrile and 2. 5 % acetic acid solution (18:82). The wavelength of detection was at 280 nm, and flow rate was set at 1.0 mL min(-1). RESULT: The calibration curve for PGG is linear over the range of 1-150 microg mL(-1) (r =0.9994), and PGG was found in seventeen kinds of Chinese traditional medicines, such as gall tree peony bark, red peony root, white peony root, and so on. CONCLUSION: The contents of PGG were determined in forty four kinds of Chinese traditional medicines by a rapid and precision HPLC method.

[Effect of temperature on kinetic of soil urease inhibited by Hg].

Urease kinetics inhibited by Hg in two meadow burozem, fertilized by different ways and two phaeozem soils with different organic matter content was investigated at different temperatures. The results showed that the urease activity and kinetic parameter V(max) and V(max)/K(m) were higher in soils with high organic matter content than that in soils with low organic matter among the same soil type. It indicated that organic matter had great adsorption capacity to urease. Soil urease V(max) and V(max)/K(m) in phaeozem were generally higher than that in meadow organic matter had great adsorption capacity to urease. Soil urease V(max) and V(max)/K(m) in phaeozem were generally higher than that in meadow burozem, but urease activity and K(m) were not comparable among different soil types. K(m) depended on not only the organic matter content of soils, but also fertilization ways. As incubating temperature increased, urease activity, V(max) and V(max)/K(m) value enhanced under the optical catalysis temperature. Hg behaved as uncompetitive inhibitor to soil urease in this experiment. The negative effect that Hg inhibited urease activity, K(m), V(max) and V(max)/K(m) increased with temperature increasing, indicated that the protective capacity of soil on urease decreased with the temperature increasing.

[Soil-based eco-toxicity of petroleum to terrestrial higher plant after phytoremediation].

Petroleum-contaminated soil after five-year phytoremediation was taken as tested soil initially spiked with a serial diesel concentration of 5 000, 15 000 and 30 000 mg/kg (dry weight). Residual concentrations of mineral oil by chemical analysis of gravimetry, as well as the soil-based eco-toxicity to wheat (Triticum aestivum L.), the terrestrial higher plant by several ecotoxicological bioassays including seed germination and root elongation test, early seedling growth test, contents of cytochrome P450 monooxygenases, activities of antioxidant enzymes (superoxide dismutase, SOD, and peroxidase, POD), and lipid peroxide (malondialdehyde, MDA content in wheat seedling leaves, etc. were evaluated. Results showed that mineral oil was well removed in each treatment by chemical analysis, with residual concentrations ranging from 199 to 877 mg/kg (dry weight) and with total removal rates between 90.1% and 97.2%. The evaluating results by eco-toxicological assays differed to some extent from those by chemical analysis, meanwhile, eco-toxicity of each treatment differed depending on endpoints by different bioassays. Among the ecotoxicological indexes, root length (48 h), root fresh weight (7 d), contents of P450, activities of SOD, and contents of MDA, etc. exhibited better indication to the soil toxicity. The general evaluation by combining the two analyses chemical and eco-toxicological indicated that the ecological risk was higher in most intermediately- and heavily-contaminated treatments (initially spiked with diesel concentration of 15,000 mg/kg and 30,000 mg/kg).

[Establishment of method for cytochrome P450 of earthworms (Eisenia fetida) as a biomarker].

Earthworm (Eisenia fetida) was chosen as test soil fauna to establish the method for determination of cytochrome P450 content. By means of thoroughly washing with salt solution, changing centrifugal acceleration and dissolving microsomal membranes of earthworms with sodium cholate, the determination of cytochrome P450 was performed. Base on the method, dynamic relationships of dose-response for cytochrome P450 contents in earthworm and phenanthrene concentrations were conducted by contact filter paper and soil contaminated with phenanthrene. Results indicate that cytochrome P450 are covered up by interferential material in earthworm, which makes the classic peak lag and appear at 455 - 457nm in the CO difference spectrum with a spectrophotometer. Through increasing centrifugal acceleration and adding reagent of solubilization, the interference is removed mostly and the classic peak of P450 returns to appear at 450nm +/- 1nm. The exposure tests of phenanthrene with contact filter paper (10(-6), 10(-5), 10(-4), 10(-3) and 10(-2) mg x mL(-1)) and soil (1, 2, 4 and 8 mg x kg(-1)) reveal that the dose-response relationships exist between the pollutant and cytochrome P450. Soil tests of different exposure durations (1, 3, 7, 14 and 28d) show the effects of different phenanthrene concentration on earthworm cytochrome P450 contents are to activate (7d) and to inhibit (14d and 28d), and the content of P450 is 0.99 - 1.41 and 0.77 - 0.88 (p < 0.05) times of control respectively. From this study, cytochrome P450 content of earthworm has the merit of simpleness, speediness and economy for determination, and it could be used as a sensitive biomarker for monitoring the exposure of sublethal pollution in terrestrial ecosystem.

Effects of magnesium ions on thermal inactivation of alkaline phosphatase.

The effect of Mg2+ on the thermal inactivation and unfolding of calf intestinal alkaline phosphatase has been studied at different temperatures and Mg2+ concentrations. Increasing the Mg2+ concentration in the denatured system significantly enhanced the inactivation and unfolding of the enzyme during thermal inactivation. The analysis of the kinetic course of substrate reaction during thermal inactivation showed that at 47 degrees C the increased free Mg2+ concentration caused the inactivation rate to increase. Increasing the temperature strengthened the effect of Mg2+ on the thermal inactivation. Control experiment showed that this is not due to salt effect. The time course of fluorescence emission spectra showed that the emission maximum for Mg2+-containing system was always higher than that of Mg2+-free system, and the higher temperature enhanced this difference. In addition, Mg2+ also enhanced the unfolding rate of the enzyme at 47 degrees C. The potential biological significance of these results are discussed.